U.S. patent application number 16/420609 was filed with the patent office on 2019-10-17 for gh61 polypeptide variants and polynucleotides encoding same.
This patent application is currently assigned to NOVOZYMES, INC.. The applicant listed for this patent is NOVOZYMES A/S, NOVOZYMES, INC.. Invention is credited to Leslie Beresford, Doreen Bohan, Douglas J. Boyle III, Bjarne Gram Hansen, Michael Lamsa, Janine Lin, Michelle Maranta, Frank Winther Rasmussen, Matt Sweeney.
Application Number | 20190316103 16/420609 |
Document ID | / |
Family ID | 48539370 |
Filed Date | 2019-10-17 |
United States Patent
Application |
20190316103 |
Kind Code |
A1 |
Lin; Janine ; et
al. |
October 17, 2019 |
GH61 Polypeptide Variants and Polynucleotides Encoding Same
Abstract
The present invention relates to GH61 polypeptide variants. The
present invention also relates to polynucleotides encoding the
variants; nucleic acid constructs, vectors, and host cells
comprising the polynucleotides; and methods of using the
variants.
Inventors: |
Lin; Janine; (Davis, CA)
; Bohan; Doreen; (Fairfield, CA) ; Maranta;
Michelle; (Davis, CA) ; Beresford; Leslie;
(Woodland, CA) ; Lamsa; Michael; (Woodland,
CA) ; Hansen; Bjarne Gram; (Frederiksberg, DK)
; Rasmussen; Frank Winther; (Roskilde, DK) ;
Sweeney; Matt; (Sacramento, CA) ; Boyle III; Douglas
J.; (Davis, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES, INC.
NOVOZYMES A/S |
Davis
Bagsvaerd |
CA |
US
DK |
|
|
Assignee: |
NOVOZYMES, INC.
Davis
CA
Novozymes A/S
Bagsvaerd
|
Family ID: |
48539370 |
Appl. No.: |
16/420609 |
Filed: |
May 23, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14358642 |
May 15, 2014 |
10351834 |
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PCT/US2012/066278 |
Nov 21, 2012 |
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16420609 |
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61562277 |
Nov 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C11D 3/38636 20130101;
D21H 17/005 20130101; D21C 5/005 20130101; C11D 3/38645 20130101;
C12N 9/0004 20130101; C12N 9/2402 20130101; C11D 3/38681 20130101;
C12N 9/0071 20130101; C12P 19/02 20130101; C11D 3/386 20130101 |
International
Class: |
C12N 9/24 20060101
C12N009/24; D21H 17/00 20060101 D21H017/00; C12N 9/02 20060101
C12N009/02; C12P 19/02 20060101 C12P019/02; D21C 5/00 20060101
D21C005/00; C11D 3/386 20060101 C11D003/386 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under
Cooperative Agreement DE-FC36-08GO18080 awarded by the Department
of Energy. The government has certain rights in this invention.
Claims
1-21. (canceled)
22. A variant GH61 polypeptide having cellulolytic enhancing
activity, comprising a substitution at one or more positions
corresponding to positions 105, 154, 188, 189, 216, and 229 of the
mature polypeptide of SEQ ID NO: 30, wherein the substitution at
corresponding position 105 is with Pro or Lys, the substitution at
corresponding position 154 is with Ile or Leu, the substitution at
corresponding position 188 is with Ala, Phe, Met, or Trp, the
substitution at corresponding position 189 is with His or Lys, the
substitution at corresponding position 216 is with Leu or Tyr, and
the substitution at corresponding position 229 is with Trp, His,
Ile, or Tyr, wherein the variant has cellulolytic enhancing
activity, wherein the variant has increased thermostability
relative to a GH61 polypeptide without the substitution at the one
or more positions corresponding to positions 105, 154, 188, 189,
216, and 229 of the mature polypeptide of SEQ ID NO: 30, and
wherein the variant has at least 95% sequence identity, but less
than 100% sequence identity, to the mature polypeptide of SEQ ID
NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,
106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,
184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,
210, 212, 214, or 216.
23. The variant of claim 22, wherein the variant has at least 96%
sequence identity, but less than 100% sequence identity, to the
mature polypeptide of the GH61 polypeptide of SEQ ID NO: 2, 4, 6,
8, 10, 12, 16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,
136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,
162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,
188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, or 216.
24. The variant of claim 22, wherein the variant has at least 97%
sequence identity, but less than 100% sequence identity, to the
mature polypeptide of the GH61 polypeptide of SEQ ID NO: 2, 4, 6,
8, 10, 12, 16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,
136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,
162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,
188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, or 216.
25. The variant of claim 22, wherein the variant has at least 98%
sequence identity, but less than 100% sequence identity, to the
mature polypeptide of the GH61 polypeptide of SEQ ID NO: 2, 4, 6,
8, 10, 12, 16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,
136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,
162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,
188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, or 216.
26. The variant of claim 22, wherein the variant has at least 99%
sequence identity, but less than 100% sequence identity, to the
mature polypeptide of the GH61 polypeptide of SEQ ID NO: 2, 4, 6,
8, 10, 12, 16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,
136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,
162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,
188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, or 216.
27. The variant of claim 22, which further comprises a substitution
at one or more positions corresponding to positions 111, 152, 155,
and 162 of the mature polypeptide of SEQ ID NO: 30, wherein the
substitution at corresponding position 111 is with Val, the
substitution at corresponding position 152 is with Ser, the
substitution at corresponding position 155 is with Leu, and the
substitution at corresponding position 162 is with Trp, and wherein
the variant has cellulolytic enhancing activity.
28. The variant of claim 27, wherein said variant comprises one or
more substitutions or corresponding substitutions selected from the
group consisting of a substitution of Leu at position 111 with Val,
a substitution of Asp at position 152 with Ser, a substitution of
Met at position 155 with Leu, and a substitution of Ala at position
162 with Trp.
29. The variant of claim 22, wherein the thermostability of the
variant is increased at least 1.01-fold compared to the parent.
30. An isolated polynucleotide encoding the GH61 polypeptide
variant of claim 22.
31. A recombinant host cell transformed with the polynucleotide of
claim 30.
32. A method of producing a GH61 polypeptide variant, comprising:
(a) cultivating the recombinant host cell of claim 31 under
conditions suitable for expression of the variant; and optionally
(b) recovering the variant.
33. A transgenic plant, plant part or plant cell transformed with
the polynucleotide of claim 30.
34. A method of producing a GH61 polypeptide variant, comprising:
(a) cultivating the transgenic plant or a plant cell of claim 33
under conditions conducive for production of the variant; and
optionally (b) recovering the variant.
35. A process for degrading or converting a cellulosic material,
comprising: treating the cellulosic material with an enzyme
composition comprising the GH61 polypeptide variant having
cellulolytic enhancing activity of claim 22.
36. A process for producing a fermentation product, comprising: (a)
saccharifying a cellulosic material with an enzyme composition
comprising the GH61 polypeptide variant having cellulolytic
enhancing activity of claim 22; (b) fermenting the saccharified
cellulosic material with one or more fermenting microorganisms to
produce the fermentation product; and (c) recovering the
fermentation product from the fermentation.
37. A process of fermenting a cellulosic material, comprising:
fermenting the cellulosic material with one or more fermenting
microorganisms, wherein the cellulosic material is saccharified
with an enzyme composition comprising the GH61 polypeptide variant
having cellulolytic enhancing activity of claim 22.
38. A whole broth formulation or cell culture composition,
comprising the GH61 polypeptide variant of claim 22.
39. A detergent composition, comprising a surfactant and the GH61
polypeptide variant of claim 22.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 14/358,642 filed May 15, 2014, which is a 35
U.S.C. .sctn. 371 national application of PCT/US2012/066278 filed
Nov. 21, 2012, which claims priority or the benefit under 35 U.S.C.
.sctn. 119 of U.S. Provisional Application No. 61/562,277 filed
Nov. 21, 2011, the contents of which are fully incorporated herein
by reference.
REFERENCE TO A SEQUENCE LISTING
[0003] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] The present invention relates to GH61 polypeptide variants,
polynucleotides encoding the variants, methods of producing the
variants, and methods of using the variants.
Description of the Related Art
[0005] Cellulose is a polymer of the simple sugar glucose
covalently linked by beta-1,4-bonds. Many microorganisms produce
enzymes that hydrolyze beta-linked glucans. These enzymes include
endoglucanases, cellobiohydrolases, and beta-glucosidases.
Endoglucanases digest the cellulose polymer at random locations,
opening it to attack by cellobiohydrolases. Cellobiohydrolases
sequentially release molecules of cellobiose from the ends of the
cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked
dimer of glucose. Beta-glucosidases hydrolyze cellobiose to
glucose.
[0006] The conversion of lignocellulosic feedstocks into ethanol
has the advantages of the ready availability of large amounts of
feedstock, the desirability of avoiding burning or land filling the
materials, and the cleanliness of the ethanol fuel. Wood,
agricultural residues, herbaceous crops, and municipal solid wastes
have been considered as feedstocks for ethanol production. These
materials primarily consist of cellulose, hemicellulose, and
lignin. Once the lignocellulose is converted to fermentable sugars,
e.g., glucose, the fermentable sugars can easily be fermented by
yeast into ethanol.
[0007] WO 2005/074647, WO 2008/148131, and WO 2011/035027 disclose
isolated GH61 polypeptides having cellulolytic enhancing activity
and the polynucleotides thereof from Thielavia terrestris. WO
2005/074656 and WO 2010/065830 disclose isolated GH61 polypeptides
having cellulolytic enhancing activity and the polynucleotides
thereof from Thermoascus aurantiacus. WO 2007/089290 and WO
2012/149344 disclose isolated GH61 polypeptides having cellulolytic
enhancing activity and the polynucleotides thereof from Trichoderma
reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864, and WO
2009/085868 disclose isolated GH61 polypeptides having cellulolytic
enhancing activity and the polynucleotides thereof from
Myceliophthora thermophila. WO 2010/138754 discloses an isolated
GH61 polypeptide having cellulolytic enhancing activity and the
polynucleotide thereof from Aspergillus fumigatus. WO 2011/005867
discloses an isolated GH61 polypeptide having cellulolytic
enhancing activity and the polynucleotide thereof from Penicillium
pinophilum. WO 2011/039319 discloses an isolated GH61 polypeptide
having cellulolytic enhancing activity and the polynucleotide
thereof from Thermoascus sp. WO 2011/041397 discloses an isolated
GH61 polypeptide having cellulolytic enhancing activity and the
polynucleotide thereof from Penicillium sp. (emersonii). WO
2011/041504 discloses isolated GH61 polypeptides having
cellulolytic enhancing activity and the polynucleotides thereof
from Thermoascus crustaceus. WO 2012/030799 discloses GH61
polypeptides having cellulolytic enhancing activity and the
polynucleotides thereof from Aspergillus aculeatus. WO 2012/113340
discloses GH61 polypeptides having cellulolytic enhancing activity
and the polynucleotides thereof from Thermomyces lanuginosus. WO
2012/146171 discloses GH61 polypeptides having cellulolytic
enhancing activity and the polynucleotides thereof from Humicola
insolens. WO 2008/151043 discloses methods of increasing the
activity of a GH61 polypeptide having cellulolytic enhancing
activity by adding a soluble activating divalent metal cation to a
composition comprising the polypeptide.
[0008] WO 2012/044835 and WO 2012/044836 disclose GH61 polypeptide
variants having cellulolytic enhancing activity with improved
thermal activity and thermostability.
[0009] The present invention provides GH61 polypeptide variants
with increased thermostability.
SUMMARY OF THE INVENTION
[0010] The present invention relates to isolated GH61 polypeptide
variants, comprising a substitution at one or more (e.g., several)
positions corresponding to positions 105, 154, 188, 189, 216, and
229 of the mature polypeptide of SEQ ID NO: 30, wherein the
variants have cellulolytic enhancing activity.
[0011] The present invention also relates to isolated
polynucleotides encoding the variants; nucleic acid constructs,
vectors, and host cells comprising the polynucleotides; and methods
of producing the variants.
[0012] The present invention also relates to processes for
degrading or converting a cellulosic material, comprising: treating
the cellulosic material with an enzyme composition in the presence
of a GH61 polypeptide variant of the present invention. In one
aspect, the processes further comprise recovering the degraded or
converted cellulosic material.
[0013] The present invention also relates to processes of producing
a fermentation product, comprising: (a) saccharifying a cellulosic
material with an enzyme composition in the presence of a GH61
polypeptide variant of the present invention; (b) fermenting the
saccharified cellulosic material with one or more (e.g., several)
fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
[0014] The present invention also relates to processes of
fermenting a cellulosic material, comprising: fermenting the
cellulosic material with one or more (e.g., several) fermenting
microorganisms, wherein the cellulosic material is saccharified
with an enzyme composition in the presence of a GH61 polypeptide
variant of the present invention. In one aspect, the fermenting of
the cellulosic material produces a fermentation product. In another
aspect, the processes further comprise recovering the fermentation
product from the fermentation.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 shows the genomic DNA sequence (SEQ ID NO: 29) and
the deduced amino acid sequence (SEQ ID NO: 30) of an Aspergillus
fumigatus gene encoding a GH61B polypeptide having cellulolytic
enhancing activity.
[0016] FIG. 2 shows a restriction map of plasmid pMMar44.
[0017] FIG. 3 shows a restriction map of plasmid pMMar49.
[0018] FIG. 4 shows a restriction map of plasmid pMMar45.
[0019] FIG. 5 shows a restriction map of plasmid pDFng113.
[0020] FIG. 6 shows the effect of addition of Aspergillus fumigatus
GH61B polypeptide variants in the conversion of PCS by a
high-temperature cellulase composition at 50.degree. C., 55.degree.
C., and 60.degree. C.
[0021] FIG. 7 shows a restriction map of plasmid pDFng153-4.
[0022] FIG. 8 shows a restriction map of plasmid pDFng154-17.
[0023] FIG. 9 shows a restriction map of plasmid pDFng155-33.
[0024] FIG. 10 shows a restriction map of plasmid pBGMH16.
DEFINITIONS
[0025] Acetylxylan esterase: The term "acetylxylan esterase" means
a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of
acetyl groups from polymeric xylan, acetylated xylose, acetylated
glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For
purposes of the present invention, acetylxylan esterase activity is
determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM
sodium acetate pH 5.0 containing 0.01% TWEEN.TM. 20
(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan
esterase is defined as the amount of enzyme capable of releasing 1
.mu.mole of p-nitrophenolate anion per minute at pH 5, 25.degree.
C.
[0026] Allelic variant: The term "allelic variant" means any of two
or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. An allelic
variant of a polypeptide is a polypeptide encoded by an allelic
variant of a gene.
[0027] Alpha-L-arabinofuranosidase: The term
"alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside
arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis
of terminal non-reducing alpha-L-arabinofuranoside residues in
alpha-L-arabinosides. The enzyme acts on
alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-
and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.
Alpha-L-arabinofuranosidase is also known as arabinosidase,
alpha-arabinosidase, alpha-L-arabinosidase,
alpha-arabinofuranosidase, polysaccharide
alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,
L-arabinosidase, or alpha-L-arabinanase. For purposes of the
present invention, alpha-L-arabinofuranosidase activity is
determined using 5 mg of medium viscosity wheat arabinoxylan
(Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland)
per ml of 100 mM sodium acetate pH 5 in a total volume of 200 .mu.l
for 30 minutes at 40.degree. C. followed by arabinose analysis by
AMINEX.RTM. HPX-87H column chromatography (Bio-Rad Laboratories,
Inc., Hercules, Calif., USA).
[0028] Alpha-glucuronidase: The term "alpha-glucuronidase" means an
alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that
catalyzes the hydrolysis of an alpha-D-glucuronoside to
D-glucuronate and an alcohol. For purposes of the present
invention, alpha-glucuronidase activity is determined according to
de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of
alpha-glucuronidase equals the amount of enzyme capable of
releasing 1 .mu.mole of glucuronic or 4-O-methylglucuronic acid per
minute at pH 5, 40.degree. C.
[0029] Beta-glucosidase: The term "beta-glucosidase" means a
beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the
hydrolysis of terminal non-reducing beta-D-glucose residues with
the release of beta-D-glucose. For purposes of the present
invention, beta-glucosidase activity is determined using
p-nitrophenyl-beta-D-glucopyranoside as substrate according to the
procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase
from Chaetomium thermophilum var. coprophilum: production,
purification and some biochemical properties, J. Basic Microbiol.
42: 55-66. One unit of beta-glucosidase is defined as 1.0 .mu.mole
of p-nitrophenolate anion produced per minute at 25.degree. C., pH
4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in
50 mM sodium citrate containing 0.01% TWEEN.RTM. 20.
[0030] Beta-xylosidase: The term "beta-xylosidase" means a
beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the
exo-hydrolysis of short beta (1.fwdarw.4)-xylooligosaccharides to
remove successive D-xylose residues from non-reducing termini. For
purposes of the present invention, one unit of beta-xylosidase is
defined as 1.0 .mu.mole of p-nitrophenolate anion produced per
minute at 40.degree. C., pH 5 from 1 mM
p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate
containing 0.01% TWEEN.RTM. 20.
[0031] cDNA: The term "cDNA" means a DNA molecule that can be
prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks
intron sequences that may be present in the corresponding genomic
DNA. The initial, primary RNA transcript is a precursor to mRNA
that is processed through a series of steps, including splicing,
before appearing as mature spliced mRNA.
[0032] Cellobiohydrolase: The term "cellobiohydrolase" means a
1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C.
3.2.1.176) that catalyzes the hydrolysis of 1,4-beta-D-glucosidic
linkages in cellulose, cellooligosaccharides, or any
beta-1,4-linked glucose containing polymer, releasing cellobiose
from the reducing end (cellobiohydrolase I) or non-reducing end
(cellobiohydrolase II) of the chain (Teeri, 1997, Crystalline
cellulose degradation: New insight into the function of
cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et
al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient
on crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178).
Cellobiohydrolase activity is determined according to the
procedures described by Lever et al., 1972, Anal. Biochem. 47:
273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156;
van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and
Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.
[0033] Cellulolytic enzyme or cellulase: The term "cellulolytic
enzyme" or "cellulase" means one or more (e.g., several) enzymes
that hydrolyze a cellulosic material. Such enzymes include
endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or
combinations thereof. The two basic approaches for measuring
cellulolytic activity include: (1) measuring the total cellulolytic
activity, and (2) measuring the individual cellulolytic activities
(endoglucanases, cellobiohydrolases, and beta-glucosidases) as
reviewed in Zhang et al., Outlook for cellulase improvement:
Screening and selection strategies, 2006, Biotechnology Advances
24: 452-481. Total cellulolytic activity is usually measured using
insoluble substrates, including Whatman No1 filter paper,
microcrystalline cellulose, bacterial cellulose, algal cellulose,
cotton, pretreated lignocellulose, etc. The most common total
cellulolytic activity assay is the filter paper assay using Whatman
No1 filter paper as the substrate. The assay was established by the
International Union of Pure and Applied Chemistry (IUPAC) (Ghose,
1987, Measurement of cellulase activities, Pure Appl. Chem. 59:
257-68).
[0034] For purposes of the present invention, cellulolytic enzyme
activity is determined by measuring the increase in hydrolysis of a
cellulosic material by cellulolytic enzyme(s) under the following
conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose
in PCS (or other pretreated cellulosic material) for 3-7 days at a
suitable temperature such as 40.degree. C.-80.degree. C., e.g.,
50.degree. C., 55.degree. C., 60.degree. C., 65.degree. C., or
70.degree. C., and a suitable pH such as 4-9, e.g., 5.0, 5.5, 6.0,
6.5, or 7.0, compared to a control hydrolysis without addition of
cellulolytic enzyme protein. Typical conditions are 1 ml reactions,
washed or unwashed pretreated corn stover (PCS), 5% insoluble
solids, 50 mM sodium acetate pH 5, 1 mM MnSO.sub.4, 50.degree. C.,
55.degree. C., or 60.degree. C., 72 hours, sugar analysis by
AMINEX.RTM. HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,
Calif., USA). 40.degree. C.-80.degree. C., e.g., 50.degree. C.,
55.degree. C., 60.degree. C., 65.degree. C., or 70.degree. C.
[0035] Cellulosic material: The term "cellulosic material" means
any material containing cellulose. The predominant polysaccharide
in the primary cell wall of biomass is cellulose, the second most
abundant is hemicellulose, and the third is pectin. The secondary
cell wall, produced after the cell has stopped growing, also
contains polysaccharides and is strengthened by polymeric lignin
covalently cross-linked to hemicellulose. Cellulose is a
homopolymer of anhydrocellobiose and thus a linear
beta-(1-4)-D-glucan, while hemicelluloses include a variety of
compounds, such as xylans, xyloglucans, arabinoxylans, and mannans
in complex branched structures with a spectrum of substituents.
Although generally polymorphous, cellulose is found in plant tissue
primarily as an insoluble crystalline matrix of parallel glucan
chains. Hemicelluloses usually hydrogen bond to cellulose, as well
as to other hemicelluloses, which help stabilize the cell wall
matrix.
[0036] Cellulose is generally found, for example, in the stems,
leaves, hulls, husks, and cobs of plants or leaves, branches, and
wood of trees. The cellulosic material can be, but is not limited
to, agricultural residue, herbaceous material (including energy
crops), municipal solid waste, pulp and paper mill residue, waste
paper, and wood (including forestry residue) (see, for example,
Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E.
Wyman, editor), pp. 105-118, Taylor & Francis, Washington D.C.;
Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied
Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999,
Recent Progress in Bioconversion of Lignocellulosics, in Advances
in Biochemical Engineering/Biotechnology, T. Scheper, managing
editor, Volume 65, pp. 23-40, Springer-Verlag, New York). It is
understood herein that the cellulose may be in the form of
lignocellulose, a plant cell wall material containing lignin,
cellulose, and hemicellulose in a mixed matrix. In a preferred
aspect, the cellulosic material is any biomass material. In another
preferred aspect, the cellulosic material is lignocellulose, which
comprises cellulose, hemicelluloses, and lignin.
[0037] In one aspect, the cellulosic material is agricultural
residue. In another aspect, the cellulosic material is herbaceous
material (including energy crops). In another aspect, the
cellulosic material is municipal solid waste. In another aspect,
the cellulosic material is pulp and paper mill residue. In another
aspect, the cellulosic material is waste paper. In another aspect,
the cellulosic material is wood (including forestry residue).
[0038] In another aspect, the cellulosic material is arundo. In
another aspect, the cellulosic material is bagasse. In another
aspect, the cellulosic material is bamboo. In another aspect, the
cellulosic material is corn cob. In another aspect, the cellulosic
material is corn fiber. In another aspect, the cellulosic material
is corn stover. In another aspect, the cellulosic material is
miscanthus. In another aspect, the cellulosic material is orange
peel. In another aspect, the cellulosic material is rice straw. In
another aspect, the cellulosic material is switchgrass. In another
aspect, the cellulosic material is wheat straw.
[0039] In another aspect, the cellulosic material is aspen. In
another aspect, the cellulosic material is eucalyptus. In another
aspect, the cellulosic material is fir. In another aspect, the
cellulosic material is pine. In another aspect, the cellulosic
material is poplar. In another aspect, the cellulosic material is
spruce. In another aspect, the cellulosic material is willow.
[0040] In another aspect, the cellulosic material is algal
cellulose. In another aspect, the cellulosic material is bacterial
cellulose. In another aspect, the cellulosic material is cotton
linter. In another aspect, the cellulosic material is filter paper.
In another aspect, the cellulosic material is microcrystalline
cellulose. In another aspect, the cellulosic material is
phosphoric-acid treated cellulose.
[0041] In another aspect, the cellulosic material is an aquatic
biomass. As used herein the term "aquatic biomass" means biomass
produced in an aquatic environment by a photosynthesis process. The
aquatic biomass can be algae, emergent plants, floating-leaf
plants, or submerged plants.
[0042] The cellulosic material may be used as is or may be
subjected to pretreatment, using conventional methods known in the
art, as described herein. In a preferred aspect, the cellulosic
material is pretreated.
[0043] Coding sequence: The term "coding sequence" means a
polynucleotide, which directly specifies the amino acid sequence of
a variant. The boundaries of the coding sequence are generally
determined by an open reading frame, which begins with a start
codon such as ATG, GTG or TTG and ends with a stop codon such as
TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA,
synthetic DNA, or a combination thereof.
[0044] Control sequences: The term "control sequences" means
nucleic acid sequences necessary for expression of a polynucleotide
encoding a variant of the present invention. Each control sequence
may be native (i.e., from the same gene) or foreign (i.e., from a
different gene) to the polynucleotide encoding the variant or
native or foreign to each other. Such control sequences include,
but are not limited to, a leader, polyadenylation sequence,
propeptide sequence, promoter, signal peptide sequence, and
transcription terminator. At a minimum, the control sequences
include a promoter, and transcriptional and translational stop
signals. The control sequences may be provided with linkers for the
purpose of introducing specific restriction sites facilitating
ligation of the control sequences with the coding region of the
polynucleotide encoding a variant.
[0045] Endoglucanase: The term "endoglucanase" means an
endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4)
that catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in
cellulose, cellulose derivatives (such as carboxymethyl cellulose
and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed
beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and
other plant material containing cellulosic components.
Endoglucanase activity can be determined by measuring reduction in
substrate viscosity or increase in reducing ends determined by a
reducing sugar assay (Zhang et al., 2006, Biotechnology Advances
24: 452-481). For purposes of the present invention, endoglucanase
activity is determined using carboxymethyl cellulose (CMC) as
substrate according to the procedure of Ghose, 1987, Pure and Appl.
Chem. 59: 257-268, at pH 5, 40.degree. C.
[0046] Expression: The term "expression" includes any step involved
in the production of a variant including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0047] Expression vector: The term "expression vector" means a
linear or circular DNA molecule that comprises a polynucleotide
encoding a variant and is operably linked to control sequences that
provide for its expression.
[0048] Family 61 glycoside hydrolase: The term "Family 61 glycoside
hydrolase" or "Family GH61" or "GH61" means a polypeptide falling
into the glycoside hydrolase Family 61 according to Henrissat B.,
1991, A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat B.,
and Bairoch A., 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this
family were originally classified as a glycoside hydrolase family
based on measurement of very weak endo-1,4-beta-D-glucanase
activity in one family member. The structure and mode of action of
these enzymes are non-canonical and they cannot be considered as
bona fide glycosidases. However, they are kept in the CAZy
classification on the basis of their capacity to enhance the
breakdown of lignocellulose when used in conjunction with a
cellulase or a mixture of cellulases.
[0049] Feruloyl esterase: The term "feruloyl esterase" means a
4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that
catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)
groups from esterified sugar, which is usually arabinose in natural
biomass substrates, to produce ferulate
(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as
ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III,
cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For
purposes of the present invention, feruloyl esterase activity is
determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM
sodium acetate pH 5.0. One unit of feruloyl esterase equals the
amount of enzyme capable of releasing 1 .mu.mole of
p-nitrophenolate anion per minute at pH 5, 25.degree. C.
[0050] Fragment: The term "fragment" means a polypeptide having one
or more (e.g., several) amino acids absent from the amino and/or
carboxyl terminus of the mature polypeptide thereof, wherein the
fragment has cellulolytic enhancing activity. In one aspect, a
fragment contains at least 85% of the amino acid residues, e.g., at
least 90% of the amino acid residues or at least 95% of the amino
acid residues of the mature polypeptide of a GH61 polypeptide.
[0051] Hemicellulolytic enzyme or hemicellulase: The term
"hemicellulolytic enzyme" or "hemicellulase" means one or more
(e.g., several) enzymes that hydrolyze a hemicellulosic material.
See, for example, Shallom, D. and Shoham, Y. Microbial
hemicellulases. Current Opinion In Microbiology, 2003, 6(3):
219-228). Hemicellulases are key components in the degradation of
plant biomass. Examples of hemicellulases include, but are not
limited to, an acetylmannan esterase, an acetylxylan esterase, an
arabinanase, an arabinofuranosidase, a coumaric acid esterase, a
feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl
esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
The substrates of these enzymes, the hemicelluloses, are a
heterogeneous group of branched and linear polysaccharides that are
bound via hydrogen bonds to the cellulose microfibrils in the plant
cell wall, crosslinking them into a robust network. Hemicelluloses
are also covalently attached to lignin, forming together with
cellulose a highly complex structure. The variable structure and
organization of hemicelluloses require the concerted action of many
enzymes for its complete degradation. The catalytic modules of
hemicellulases are either glycoside hydrolases (GHs) that hydrolyze
glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze
ester linkages of acetate or ferulic acid side groups. These
catalytic modules, based on homology of their primary sequence, can
be assigned into GH and CE families. Some families, with an overall
similar fold, can be further grouped into clans, marked
alphabetically (e.g., GH-A). A most informative and updated
classification of these and other carbohydrate active enzymes is
available in the Carbohydrate-Active Enzymes (CAZy) database.
Hemicellulolytic enzyme activities can be measured according to
Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a
suitable temperature, e.g., 50.degree. C., 55.degree. C., or
60.degree. C., and pH, e.g., 5.0 or 5.5.
[0052] High stringency conditions: The term "high stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 50% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 65.degree. C.
[0053] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, or the
like with a nucleic acid construct or expression vector comprising
a polynucleotide of the present invention. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication.
[0054] Improved property: The term "improved property" means a
characteristic associated with a variant that is improved compared
to the parent. Such an improved property includes, but is not
limited to, increased thermostability.
[0055] Increased thermostability: The term "increased
thermostability" means a higher retention of cellulolytic enhancing
activity of a GH61 polypeptide variant after a period of incubation
at a temperature relative to the parent. The increased
thermostability of the variant relative to the parent can be
assessed, for example, under conditions of one or more (e.g.,
several) temperatures. For example, the one or more (e.g., several)
temperatures can be any temperature or temperatures in the range of
45.degree. C. to 95.degree. C., e.g., 45, 50, 55, 60, 65, 70, 75,
80, 85, or 95.degree. C. (or in between, e.g., 62.degree. C.,
68.degree. C., 72.degree. C., etc.) at one or more (e.g., several)
pHs in the range of 3 to 9, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 (or in between) for a suitable
period (time) of incubation, e.g., 1 minute, 5 minutes, 10 minutes,
15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, or 60
minutes (or in between, e.g., 23 minutes, 37 minutes, etc.), such
that the variant retains residual activity. However, longer periods
of incubation can also be used. The term "increased
thermostability" can be used interchangeably with "improved
thermostability".
[0056] The increased thermostability of the variant relative to the
parent can be determined by differential scanning calorimetry (DSC)
using methods standard in the art (see, for example, Sturtevant,
1987, Annual Review of Physical Chemistry 38: 463-488; Examples 9
and 17). The increased thermostability of the variant relative to
the parent can also be determined using protein thermal unfolding
analysis (see, for example, Examples 10, 18, and 23 herein). The
increased thermostability of the variant relative to the parent can
also be determined using any enzyme assay known in the art for GH61
polypeptides having cellulolytic enhancing activity to measure
residual activity after a temperature treatment. See for example,
WO 2005/074647, WO 2008/148131 WO 2005/074656, WO 2010/065830, WO
2007/089290, WO 2009/085935, WO 2009/085859, WO 2009/085864, WO
2009/085868, and WO 2008/151043, which are incorporated herein by
reference. Alternatively, the increased thermostability of the
variant relative to the parent can be determined using any
application assay for the variant where the performance of the
variant is compared to the parent. For example, the application
assays described in Examples 5, 12, and 13 can be used.
[0057] Isolated: The term "isolated" means a substance in a form or
environment that does not occur in nature. Non-limiting examples of
isolated substances include (1) any non-naturally occurring
substance, (2) any substance including, but not limited to, any
enzyme, variant, nucleic acid, protein, peptide or cofactor, that
is at least partially removed from one or more or all of the
naturally occurring constituents with which it is associated in
nature; (3) any substance modified by the hand of man relative to
that substance found in nature; or (4) any substance modified by
increasing the amount of the substance relative to other components
with which it is naturally associated (e.g., recombinant production
in a host cell; multiple copies of a gene encoding the substance;
and use of a stronger promoter than the promoter naturally
associated with the gene encoding the substance).
[0058] Low stringency conditions: The term "low stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 25% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 50.degree. C.
[0059] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. In one
aspect, the mature polypeptide is amino acids 20 to 326 of SEQ ID
NO: 2 based on the SignalP program (Nielsen et al., 1997, Protein
Engineering 10: 1-6) that predicts amino acids 1 to 19 of SEQ ID
NO: 2 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 18 to 239 of SEQ ID NO: 4 based on the
SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 4
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 20 to 258 of SEQ ID NO: 6 based on the SignalP program
that predicts amino acids 1 to 19 of SEQ ID NO: 6 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
19 to 226 of SEQ ID NO: 8 based on the SignalP program that
predicts amino acids 1 to 18 of SEQ ID NO: 8 are a signal peptide.
In another aspect, the mature polypeptide is amino acids 20 to 304
of SEQ ID NO: 10 based on the SignalP program that predicts amino
acids 1 to 19 of SEQ ID NO: 10 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 16 to 317 of SEQ ID
NO: 12 based on the SignalP program that predicts amino acids 1 to
15 of SEQ ID NO: 12 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 22 to 249 of SEQ ID NO: 14 based
on the SignalP program that predicts amino acids 1 to 21 of SEQ ID
NO: 14 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 20 to 249 of SEQ ID NO: 16 based on the
SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 16
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 18 to 232 of SEQ ID NO: 18 based on the SignalP program
that predicts amino acids 1 to 17 of SEQ ID NO: 18 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
16 to 235 of SEQ ID NO: 20 based on the SignalP program that
predicts amino acids 1 to 15 of SEQ ID NO: 20 are a signal peptide.
In another aspect, the mature polypeptide is amino acids 19 to 323
of SEQ ID NO: 22 based on the SignalP program that predicts amino
acids 1 to 18 of SEQ ID NO: 22 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 16 to 310 of SEQ ID
NO: 24 based on the SignalP program that predicts amino acids 1 to
15 of SEQ ID NO: 24 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 20 to 246 of SEQ ID NO: 26 based
on the SignalP program that predicts amino acids 1 to 19 of SEQ ID
NO: 26 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 22 to 354 of SEQ ID NO: 28 based on the
SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 28
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 22 to 250 of SEQ ID NO: 30 based on the SignalP program
that predicts amino acids 1 to 21 of SEQ ID NO: 30 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
22 to 322 of SEQ ID NO: 32 based on the SignalP program that
predicts amino acids 1 to 21 of SEQ ID NO: 32 are a signal peptide.
In another aspect, the mature polypeptide is amino acids 24 to 444
of SEQ ID NO: 34 based on the SignalP program that predicts amino
acids 1 to 23 of SEQ ID NO: 34 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 26 to 253 of SEQ ID
NO: 36 based on the SignalP program that predicts amino acids 1 to
25 of SEQ ID NO: 36 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 18 to 246 of SEQ ID NO: 38 based
on the SignalP program that predicts amino acids 1 to 17 of SEQ ID
NO: 38 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 20 to 334 of SEQ ID NO: 40 based on the
SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 40
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 18 to 227 of SEQ ID NO: 42 based on the SignalP program
that predicts amino acids 1 to 17 of SEQ ID NO: 42 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
20 to 223 of SEQ ID NO: 44 based on the SignalP program that
predicts amino acids 1 to 19 of SEQ ID NO: 44 are a signal peptide.
In another aspect, the mature polypeptide is amino acids 22 to 368
of SEQ ID NO: 46 based on the SignalP program that predicts amino
acids 1 to 21 of SEQ ID NO: 46 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 25 to 330 of SEQ ID
NO: 48 based on the SignalP program that predicts amino acids 1 to
24 of SEQ ID NO: 48 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 17 to 236 of SEQ ID NO: 50 based
on the SignalP program that predicts amino acids 1 to 16 of SEQ ID
NO: 50 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 19 to 250 of SEQ ID NO: 52 based on the
SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 52
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 23 to 478 of SEQ ID NO: 54 based on the SignalP program
that predicts amino acids 1 to 22 of SEQ ID NO: 54 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
17 to 230 of SEQ ID NO: 56 based on the SignalP program that
predicts amino acids 1 to 16 of SEQ ID NO: 56 are a signal peptide.
In another aspect, the mature polypeptide is amino acids 20 to 257
of SEQ ID NO: 58 based on the SignalP program that predicts amino
acids 1 to 19 of SEQ ID NO: 58 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 23 to 251 of SEQ ID
NO: 60 based on the SignalP program that predicts amino acids 1 to
22 of SEQ ID NO: 60 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 19 to 349 of SEQ ID NO: 62 based
on the SignalP program that predicts amino acids 1 to 18 of SEQ ID
NO: 62 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 24 to 436 of SEQ ID NO: 64 based on the
SignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 64
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 21 to 344 of SEQ ID NO: 66 based on the SignalP program
that predicts amino acids 1 to 23 of SEQ ID NO: 66 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
26 to 400 of SEQ ID NO: 68 based on the SignalP program that
predicts amino acids 1 to 25 of SEQ ID NO: 68 are a signal peptide.
In another aspect, the mature polypeptide is amino acids 21 to 389
of SEQ ID NO: 70 based on the SignalP program that predicts amino
acids 1 to 20 of SEQ ID NO: 70 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 22 to 406 of SEQ ID
NO: 72 based on the SignalP program that predicts amino acids 1 to
21 of SEQ ID NO: 72 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 20 to 427 of SEQ ID NO: 74 based
on the SignalP program that predicts amino acids 1 to 19 of SEQ ID
NO: 74 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 18 to 267 of SEQ ID NO: 76 based on the
SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 76
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 21 to 273 of SEQ ID NO: 78 based on the SignalP program
that predicts amino acids 1 to 20 of SEQ ID NO: 78 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
23 to 272 of SEQ ID NO: 80 based on the SignalP program that
predicts amino acids 1 to 22 of SEQ ID NO: 80 are a signal peptide.
In another aspect, the mature polypeptide is amino acids 22 to 327
of SEQ ID NO: 82 based on the SignalP program that predicts amino
acids 1 to 21 of SEQ ID NO: 82 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 23 to 274 of SEQ ID
NO: 84 based on the SignalP program that predicts amino acids 1 to
22 of SEQ ID NO: 84 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 21 to 322 of SEQ ID NO: 86 based
on the SignalP program that predicts amino acids 1 to 20 of SEQ ID
NO: 86 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 18 to 234 of SEQ ID NO: 88 based on the
SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 88
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 24 to 233 of SEQ ID NO: 90 based on the SignalP program
that predicts amino acids 1 to 23 of SEQ ID NO: 90 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
17 to 237 of SEQ ID NO: 92 based on the SignalP program that
predicts amino acids 1 to 16 of SEQ ID NO: 92 are a signal peptide.
In another aspect, the mature polypeptide is amino acids 20 to 484
of SEQ ID NO: 94 based on the SignalP program that predicts amino
acids 1 to 19 of SEQ ID NO: 94 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 22 to 329 of SEQ ID
NO: 96 based on the SignalP program that predicts amino acids 1 to
21 of SEQ ID NO: 96 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 18 to 227 of SEQ ID NO: 98 based
on the SignalP program that predicts amino acids 1 to 17 of SEQ ID
NO: 98 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 17 to 257 of SEQ ID NO: 100 based on the
SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 100
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 20 to 246 of SEQ ID NO: 102 based on the SignalP
program that predicts amino acids 1 to 19 of SEQ ID NO: 102 are a
signal peptide. In another aspect, the mature polypeptide is amino
acids 28 to 265 of SEQ ID NO: 104 based on the SignalP program that
predicts amino acids 1 to 27 of SEQ ID NO: 104 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
16 to 310 of SEQ ID NO: 106 based on the SignalP program that
predicts amino acids 1 to 15 of SEQ ID NO: 106 are a signal
peptide. In one aspect, the mature polypeptide is amino acids 21 to
354 of SEQ ID NO: 108 based on the SignalP program that predicts
amino acids 1 to 20 of SEQ ID NO: 108 are a signal peptide. In
another aspect, the mature polypeptide is amino acids 22 to 267 of
SEQ ID NO: 110 based on the SignalP program that predicts amino
acids 1 to 21 of SEQ ID NO: 110 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 16 to 237 of SEQ ID
NO: 112 based on the SignalP program that predicts amino acids 1 to
15 of SEQ ID NO: 112 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 20 to 234 of SEQ ID NO: 114 based
on the SignalP program that predicts amino acids 1 to 19 of SEQ ID
NO: 114 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 18 to 226 of SEQ ID NO: 116 based on the
SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 116
are a signal peptide. In one aspect, the mature polypeptide is
amino acids 17 to 231 of SEQ ID NO: 118 based on the SignalP
program that predicts amino acids 1 to 16 of SEQ ID NO: 118 are a
signal peptide. In another aspect, the mature polypeptide is amino
acids 22 to 248 of SEQ ID NO: 120 based on the SignalP program that
predicts amino acids 1 to 21 of SEQ ID NO: 120 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
18 to 233 of SEQ ID NO: 122 based on the SignalP program that
predicts amino acids 1 to 17 of SEQ ID NO: 122 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
21 to 243 of SEQ ID NO: 124 based on the SignalP program that
predicts amino acids 1 to 20 of SEQ ID NO: 124 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
21 to 363 of SEQ ID NO: 126 based on the SignalP program that
predicts amino acids 1 to 20 of SEQ ID NO: 126 are a signal
peptide. In one aspect, the mature polypeptide is amino acids 20 to
296 of SEQ ID NO: 128 based on the SignalP program that predicts
amino acids 1 to 19 of SEQ ID NO: 128 are a signal peptide. In
another aspect, the mature polypeptide is amino acids 16 to 318 of
SEQ ID NO: 130 based on the SignalP program that predicts amino
acids 1 to 15 of SEQ ID NO: 130 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 19 to 259 of SEQ ID
NO: 132 based on the SignalP program that predicts amino acids 1 to
18 of SEQ ID NO: 132 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 20 to 325 of SEQ ID NO: 134 based
on the SignalP program that predicts amino acids 1 to 19 of SEQ ID
NO: 134 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 19 to 298 of SEQ ID NO: 136 based on the
SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 136
are a signal peptide. In one aspect, the mature polypeptide is
amino acids 20 to 298 of SEQ ID NO: 138 based on the SignalP
program that predicts amino acids 1 to 19 of SEQ ID NO: 138 are a
signal peptide. In another aspect, the mature polypeptide is amino
acids 22 to 344 of SEQ ID NO: 140 based on the SignalP program that
predicts amino acids 1 to 21 of SEQ ID NO: 140 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
20 to 330 of SEQ ID NO: 142 based on the SignalP program that
predicts amino acids 1 to 19 of SEQ ID NO: 142 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
19 to 216 of SEQ ID NO: 144 based on the SignalP program that
predicts amino acids 1 to 18 of SEQ ID NO: 144 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
18 to 490 of SEQ ID NO: 146 based on the SignalP program that
predicts amino acids 1 to 17 of SEQ ID NO: 146 are a signal
peptide. In one aspect, the mature polypeptide is amino acids 21 to
306 of SEQ ID NO: 148 based on the SignalP program) that predicts
amino acids 1 to 20 of SEQ ID NO: 148 are a signal peptide. In
another aspect, the mature polypeptide is amino acids 22 to 339 of
SEQ ID NO: 150 based on the SignalP program that predicts amino
acids 1 to 21 of SEQ ID NO: 150 are a signal peptide. In another
aspect, the mature polypeptide is amino acids 22 to 344 of SEQ ID
NO: 152 based on the SignalP program that predicts amino acids 1 to
21 of SEQ ID NO: 152 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 23 to 408 of SEQ ID NO: 154 based
on the SignalP program that predicts amino acids 1 to 22 of SEQ ID
NO: 154 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 19 to 234 of SEQ ID NO: 156 based on the
SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 156
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 22 to 248 of SEQ ID NO: 158 based on the SignalP
program that predicts amino acids 1 to 21 of SEQ ID NO: 158 are a
signal peptide. In another aspect, the mature polypeptide is amino
acids 21 to 242 of SEQ ID NO: 160 based on the SignalP program that
predicts amino acids 1 to 20 of SEQ ID NO: 160 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
23 to 334 of SEQ ID NO: 162 based on the SignalP program that
predicts amino acids 1 to 22 of SEQ ID NO: 162 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
18 to 230 of SEQ ID NO: 164 based on the SignalP program that
predicts amino acids 1 to 17 of SEQ ID NO: 164 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
19 to 397 of SEQ ID NO: 166 based on the SignalP program that
predicts amino acids 1 to 18 of SEQ ID NO: 166 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
23 to 410 of SEQ ID NO: 168 based on the SignalP program that
predicts amino acids 1 to 22 of SEQ ID NO: 168 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
20 to 232 of SEQ ID NO: 170 based on the SignalP program that
predicts amino acids 1 to 19 of SEQ ID NO: 170 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
21 to 266 of SEQ ID NO: 172 based on the SignalP program that
predicts amino acids 1 to 20 of SEQ ID NO: 172 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
24 to 324 of SEQ ID NO: 174 based on the SignalP program that
predicts amino acids 1 to 23 of SEQ ID NO: 174 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
21 to 240 of SEQ ID NO: 176 based on the SignalP program that
predicts amino acids 1 to 20 of SEQ ID NO: 176 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
21 to 225 of SEQ ID NO: 178 based on the SignalP program that
predicts amino acids 1 to 20 of SEQ ID NO: 178 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
16 to 235 of SEQ ID NO: 180 based on the SignalP program that
predicts amino acids 1 to 15 of SEQ ID NO: 180 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
20 to 336 of SEQ ID NO: 182 based on the SignalP program that
predicts amino acids 1 to 19 of SEQ ID NO: 182 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
17 to 253 of SEQ ID NO: 184 based on the SignalP program that
predicts amino acids 1 to 16 of SEQ ID NO: 184 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
18 to 255 of SEQ ID NO: 186 based on the SignalP program that
predicts amino acids 1 to 17 of SEQ ID NO: 186 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
18 to 225 of SEQ ID NO: 188 based on the SignalP program that
predicts amino acids 1 to 17 of SEQ ID NO: 188 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
16 to 237 of SEQ ID NO: 190 based on the SignalP program that
predicts amino acids 1 to 15
of SEQ ID NO: 190 are a signal peptide. In another aspect, the
mature polypeptide is amino acids 18 to 227 of SEQ ID NO: 192 based
on the SignalP program that predicts amino acids 1 to 17 of SEQ ID
NO: 192 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 22 to 315 of SEQ ID NO: 194 based on the
SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 194
are a signal peptide. In another aspect, the mature polypeptide is
amino acids 21 to 439 of SEQ ID NO: 196 based on the SignalP
program that predicts amino acids 1 to 20 of SEQ ID NO: 196 are a
signal peptide. In another aspect, the mature polypeptide is amino
acids 18 to 246 of SEQ ID NO: 198 based on the SignalP program that
predicts amino acids 1 to 17 of SEQ ID NO: 198 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
19 to 324 of SEQ ID NO: 200 based on the SignalP program that
predicts amino acids 1 to 18 of SEQ ID NO: 200 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
21 to 242 of SEQ ID NO: 202 based on the SignalP program that
predicts amino acids 1 to 20 of SEQ ID NO: 202 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
16 to 306 of SEQ ID NO: 204 based on the SignalP program that
predicts amino acids 1 to 15 of SEQ ID NO: 204 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
20 to 252 of SEQ ID NO: 206 based on the SignalP program that
predicts amino acids 1 to 19 of SEQ ID NO: 206 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
20 to 344 of SEQ ID NO: 208 based on the SignalP program that
predicts amino acids 1 to 19 of SEQ ID NO: 208 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
22 to 347 of SEQ ID NO: 210 based on the SignalP program that
predicts amino acids 1 to 21 of SEQ ID NO: 210 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
23 to 334 of SEQ ID NO: 212 based on the SignalP program that
predicts amino acids 1 to 22 of SEQ ID NO: 212 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
24 to 366 of SEQ ID NO: 214 based on the SignalP program that
predicts amino acids 1 to 23 of SEQ ID NO: 214 are a signal
peptide. In another aspect, the mature polypeptide is amino acids
21 to 364 of SEQ ID NO: 216 based on the SignalP program that
predicts amino acids 1 to 20 of SEQ ID NO: 216 are a signal
peptide. It is known in the art that a host cell may produce a
mixture of two of more different mature polypeptides (i.e., with a
different C-terminal and/or N-terminal amino acid) expressed by the
same polynucleotide.
[0060] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means a polynucleotide that encodes a
mature polypeptide having cellulolytic enhancing activity. In one
aspect, the mature polypeptide coding sequence is nucleotides 388
to 1332 of SEQ ID NO: 1 based on the SignalP program (Nielsen et
al., 1997, supra) that predicts nucleotides 330 to 387 of SEQ ID
NO: 1 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 98 to 821 of SEQ ID NO:
3 based on the SignalP program that predicts nucleotides 47 to 97
of SEQ ID NO: 3 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 126 to 978 of SEQ
ID NO: 5 based on the SignalP program that predicts nucleotides 69
to 125 of SEQ ID NO: 5 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 55 to 678 of
SEQ ID NO: 7 based on the SignalP program that predicts nucleotides
1 to 54 of SEQ ID NO: 7 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 58 to 912 of
SEQ ID NO: 9 based on the SignalP program that predicts nucleotides
1 to 57 of SEQ ID NO: 9 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 46 to 951 of
SEQ ID NO: 11 based on the SignalP program that predicts
nucleotides 1 to 45 of SEQ ID NO: 11 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 64 to 796 of SEQ ID NO: 13 based on the SignalP program
that predicts nucleotides 1 to 63 of SEQ ID NO: 13 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 77 to 766 of SEQ ID NO: 15 based on the SignalP
program that predicts nucleotides 20 to 76 of SEQ ID NO: 15 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 52 to 921 of SEQ ID NO: 17 based on the
SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 17
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 46 to 851 of SEQ ID NO: 19 based on
the SignalP program that predicts nucleotides 1 to 45 of SEQ ID NO:
19 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 55 to 1239 of SEQ ID NO:
21 based on the SignalP program that predicts nucleotides 1 to 54
of SEQ ID NO: 21 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 46 to 1250 of SEQ
ID NO: 23 based on the SignalP program that predicts nucleotides 1
to 45 of SEQ ID NO: 23 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 58 to 811 of
SEQ ID NO: 25 based on the SignalP program that predicts
nucleotides 1 to 57 of SEQ ID NO: 25 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 64 to 1112 of SEQ ID NO: 27 based on the SignalP
program that predicts nucleotides 1 to 63 of SEQ ID NO: 27 encode a
signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 64 to 859 of SEQ ID NO: 29 based on the
SignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 29
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 64 to 1018 of SEQ ID NO: 31 based on
the SignalP program that predicts nucleotides 1 to 63 of SEQ ID NO:
31 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 70 to 1483 of SEQ ID NO:
33 based on the SignalP program that predicts nucleotides 1 to 69
of SEQ ID NO: 33 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 76 to 832 of SEQ
ID NO: 35 based on the SignalP program that predicts nucleotides 1
to 75 of SEQ ID NO: 35 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 52 to 875 of
SEQ ID NO: 37 based on the SignalP program that predicts
nucleotides 1 to 51 of SEQ ID NO: 37 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 58 to 1250 of SEQ ID NO: 39 based on the SignalP
program that predicts nucleotides 1 to 57 of SEQ ID NO: 39 encode a
signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 52 to 795 of SEQ ID NO: 41 based on the
SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 41
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 58 to 974 of SEQ ID NO: 43 based on
the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO:
43 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 64 to 1104 of SEQ ID NO:
45 based on the SignalP program that predicts nucleotides 1 to 63
of SEQ ID NO: 45 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 73 to 990 of SEQ
ID NO: 47 based on the SignalP program that predicts nucleotides 1
to 72 of SEQ ID NO: 47 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 49 to 1218 of
SEQ ID NO: 49 based on the SignalP program that predicts
nucleotides 1 to 48 of SEQ ID NO: 49 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 55 to 930 of SEQ ID NO: 51 based on the SignalP program
that predicts nucleotides 1 to 54 of SEQ ID NO: 51 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 67 to 1581 of SEQ ID NO: 53 based on the SignalP
program that predicts nucleotides 1 to 66 of SEQ ID NO: 53 encode a
signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 49 to 865 of SEQ ID NO: 55 based on the
SignalP program that predicts nucleotides 1 to 48 of SEQ ID NO: 55
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 58 to 1065 of SEQ ID NO: 57 based on
the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO:
57 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 67 to 868 of SEQ ID NO:
59 based on the SignalP program that predicts nucleotides 1 to 66
of SEQ ID NO: 59 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 55 to 1099 of SEQ
ID NO: 61 based on the SignalP program that predicts nucleotides 1
to 54 of SEQ ID NO: 61 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 70 to 1490 of
SEQ ID NO: 63 based on the SignalP program that predicts
nucleotides 1 to 69 of SEQ ID NO: 63 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 61 to 1032 of SEQ ID NO: 65 based on the SignalP
program that predicts nucleotides 1 to 60 of SEQ ID NO: 65 encode a
signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 76 to 1200 of SEQ ID NO: 67 based on the
SignalP program that predicts nucleotides 1 to 75 of SEQ ID NO: 67
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 61 to 1167 of SEQ ID NO: 69 based on
the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO:
69 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 64 to 1218 of SEQ ID NO:
71 based on the SignalP program that predicts nucleotides 1 to 63
of SEQ ID NO: 71 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 58 to 1281 of SEQ
ID NO: 73 based on the SignalP program that predicts nucleotides 1
to 57 of SEQ ID NO: 73 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 52 to 801 of
SEQ ID NO: 75 based on the SignalP program that predicts
nucleotides 1 to 51 of SEQ ID NO: 75 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 61 to 819 of SEQ ID NO: 77 based on the SignalP program
that predicts nucleotides 1 to 60 of SEQ ID NO: 77 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 67 to 869 of SEQ ID NO: 79 based on the SignalP
program that predicts nucleotides 1 to 66 of SEQ ID NO: 79 encode a
signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 64 to 1036 of SEQ ID NO: 81 based on the
SignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 81
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 67 to 878 of SEQ ID NO: 83 based on
the SignalP program that predicts nucleotides 1 to 66 of SEQ ID NO:
83 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 61 to 966 of SEQ ID NO:
85 based on the SignalP program that predicts nucleotides 1 to 60
of SEQ ID NO: 85 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 52 to 702 of SEQ
ID NO: 87 based on the SignalP program that predicts nucleotides 1
to 51 of SEQ ID NO: 87 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 70 to 699 of
SEQ ID NO: 89 based on the SignalP program that predicts
nucleotides 1 to 69 of SEQ ID NO: 89 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 49 to 711 of SEQ ID NO: 91 based on the SignalP program
that predicts nucleotides 1 to 48 of SEQ ID NO: 91 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 58 to 1452 of SEQ ID NO: 93 based on the SignalP
program that predicts nucleotides 1 to 57 of SEQ ID NO: 93 encode a
signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 64 to 1018 of SEQ ID NO: 95 based on the
SignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 95
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 52 to 818 of SEQ ID NO: 97 based on
the SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO:
97 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 49 to 1117 of SEQ ID NO:
99 based on the SignalP program that predicts nucleotides 1 to 48
of SEQ ID NO: 99 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 58 to 875 of SEQ
ID NO: 101 based on the SignalP program that predicts nucleotides 1
to 57 of SEQ ID NO: 101 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 82 to 1064 of
SEQ ID NO: 103 based on the SignalP program that predicts
nucleotides 1 to 81 of SEQ ID NO: 103 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 46 to 1032 of SEQ ID NO: 105 based on the SignalP
program that predicts nucleotides 1 to 45 of SEQ ID NO: 105 encode
a signal peptide. In one aspect, the mature polypeptide coding
sequence is nucleotides 61 to 1062 of SEQ ID NO: 107 based on the
SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 107
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 64 to 801 of SEQ ID NO: 109 based on
the SignalP program that predicts nucleotides 1 to 63 of SEQ ID NO:
109 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 46 to 840 of SEQ ID NO:
111 based on the SignalP program that predicts nucleotides 1 to 45
of SEQ ID NO: 111 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 58 to 702 of SEQ
ID NO: 113 based on the SignalP program that predicts nucleotides 1
to 57 of SEQ ID NO: 113 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 52 to 750 of
SEQ ID NO: 115 based on the SignalP program that predicts
nucleotides 1 to 51 of SEQ ID NO: 115 encode a signal peptide. In
one aspect, the mature polypeptide coding sequence is nucleotides
49 to 851 of SEQ ID NO: 117 based on the SignalP program that
predicts nucleotides 1 to 48 of SEQ ID NO: 117 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 64 to 860 of SEQ ID NO: 119 based on the SignalP
program that predicts nucleotides 1 to 63 of SEQ ID NO: 119 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 52 to 830 of SEQ ID NO: 121 based on the
SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 121
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 61 to 925 of SEQ ID NO: 123 based on
the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO:
123 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 61 to 1089 of SEQ ID NO:
125 based on the SignalP program that predicts nucleotides 1 to 60
of SEQ ID NO: 125 encode a signal peptide. In one aspect, the
mature polypeptide coding sequence is nucleotides 58 to 1083 of SEQ
ID NO: 127 based on the SignalP program that predicts nucleotides 1
to 57 of SEQ ID NO: 127 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 46 to 1029 of
SEQ ID NO: 129 based on the SignalP program that predicts
nucleotides 1 to 45 of SEQ ID NO: 129 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 55 to 1110 of SEQ ID NO: 131 based on the SignalP
program that predicts nucleotides 1 to 54 of SEQ ID NO: 131 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 58 to 1100 of SEQ ID NO: 133 based on the
SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 133
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 55 to 1036 of SEQ ID NO: 135 based
on the SignalP program that predicts nucleotides 1 to 54 of SEQ ID
NO: 135 encode a signal peptide. In one aspect, the mature
polypeptide coding sequence is nucleotides 58 to 1022 of SEQ ID NO:
137 based on the SignalP program that predicts nucleotides 1 to 57
of SEQ ID NO: 137 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 64 to 1032 of SEQ
ID NO: 139 based on the SignalP program that predicts nucleotides 1
to 63 of SEQ ID NO: 139 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 58 to 1054 of
SEQ ID NO: 141 based on the SignalP program that predicts
nucleotides 1 to 57 of SEQ ID NO: 141 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 55 to 769 of SEQ ID NO: 143 based on the SignalP
program that predicts nucleotides 1 to 54 of SEQ ID NO: 143 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 52 to 1533 of SEQ ID NO: 145 based on the
SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 145
encode a signal peptide. In one aspect, the mature polypeptide
coding sequence is nucleotides 61 to 918 of SEQ ID NO: 147 based on
the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO:
147 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 64 to 1089 of SEQ ID NO:
149 based on the SignalP program that predicts nucleotides 1 to 63
of SEQ ID NO: 149 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 64 to 1086 of SEQ
ID NO: 151 based on the SignalP program that predicts nucleotides 1
to 63 of SEQ ID NO: 151 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 67 to 1395 of
SEQ ID NO: 153 based on the SignalP program that predicts
nucleotides 1 to 66 of SEQ ID NO: 155 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 55 to 899 of SEQ ID NO: 155 based on the SignalP
program that predicts nucleotides 1 to 54 of SEQ ID NO: 155 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 64 to 807 of SEQ ID NO: 157 based on the
SignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 157
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 61 to 726 of SEQ ID NO: 159 based on
the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO:
159 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 67 to 1078 of SEQ ID NO:
161 based on the SignalP program that predicts nucleotides 1 to 66
of SEQ ID NO: 161 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 52 to 872 of SEQ
ID NO: 163 based on the SignalP program that predicts nucleotides 1
to 51 of SEQ ID NO: 163 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 55 to 1191 of
SEQ ID NO: 165 based on the SignalP program that predicts
nucleotides 1 to 54 of SEQ ID NO: 165 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 67 to 1230 of SEQ ID NO: 167 based on the SignalP
program that predicts nucleotides 1 to 66 of SEQ ID NO: 167 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 58 to 696 of SEQ ID NO: 169 based on the
SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 169
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 61 to 798 of SEQ ID NO: 171
based on the SignalP program that predicts nucleotides 1 to 60 of
SEQ ID NO: 171 encode a signal peptide. In one aspect, the mature
polypeptide coding sequence is nucleotides 70 to 972 of SEQ ID NO:
173 based on the SignalP program that predicts nucleotides 1 to 69
of SEQ ID NO: 173 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 61 to 1112 of SEQ
ID NO: 175 based on the SignalP program that predicts nucleotides 1
to 60 of SEQ ID NO: 175 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 61 to 985 of
SEQ ID NO: 177 based on the SignalP program that predicts
nucleotides 1 to 60 of SEQ ID NO: 177 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 46 to 856 of SEQ ID NO: 179 based on the SignalP
program that predicts nucleotides 1 to 45 of SEQ ID NO: 179 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 58 to 1008 of SEQ ID NO: 181 based on the
SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 181
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 49 to 1312 of SEQ ID NO: 183 based
on the SignalP program that predicts nucleotides 1 to 48 of SEQ ID
NO: 183 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 52 to 921 of SEQ ID NO:
185 based on the SignalP program that predicts nucleotides 1 to 51
of SEQ ID NO: 185 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 52 to 739 of SEQ
ID NO: 187 based on the SignalP program that predicts nucleotides 1
to 51 of SEQ ID NO: 187 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 46 to 898 of
SEQ ID NO: 189 based on the SignalP program that predicts
nucleotides 1 to 45 of SEQ ID NO: 189 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 52 to 941 of SEQ ID NO: 191 based on the SignalP
program that predicts nucleotides 1 to 51 of SEQ ID NO: 191 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 64 to 945 of SEQ ID NO: 193 based on the
SignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 193
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 61 to 1377 of SEQ ID NO: 195 based
on the SignalP program that predicts nucleotides 1 to 60 of SEQ ID
NO: 195 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 52 to 818 of SEQ ID NO:
197 based on the SignalP program that predicts nucleotides 1 to 51
of SEQ ID NO: 197 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 55 to 1122 of SEQ
ID NO: 199 based on the SignalP program that predicts nucleotides 1
to 54 of SEQ ID NO: 199 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 60 to 1034 of
SEQ ID NO: 201 based on the SignalP program that predicts
nucleotides 1 to 61 of SEQ ID NO: 201 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 46 to 1197 of SEQ ID NO: 203 based on the SignalP
program that predicts nucleotides 1 to 45 of SEQ ID NO: 203 encode
a signal peptide. In another aspect, the mature polypeptide coding
sequence is nucleotides 58 to 756 of SEQ ID NO: 205 based on the
SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 205
encode a signal peptide. In another aspect, the mature polypeptide
coding sequence is nucleotides 58 to 1032 of SEQ ID NO: 207 based
on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID
NO: 207 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 64 to 1041 of SEQ ID NO:
209 based on the SignalP program that predicts nucleotides 1 to 63
of SEQ ID NO: 209 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 67 to 1002 of SEQ
ID NO: 211 based on the SignalP program that predicts nucleotides 1
to 66 of SEQ ID NO: 211 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 70 to 1098 of
SEQ ID NO: 213 based on the SignalP program that predicts
nucleotides 1 to 69 of SEQ ID NO: 213 encode a signal peptide. In
another aspect, the mature polypeptide coding sequence is
nucleotides 61 to 1088 of SEQ ID NO: 215 based on the SignalP
program that predicts nucleotides 1 to 60 of SEQ ID NO: 215 encode
a signal peptide. In each of the aspects above, the term "mature
polypeptide coding sequence" shall be understood to include the
cDNA sequence of the genomic DNA sequence or the genomic DNA
sequence of the cDNA sequence.
[0061] Medium stringency conditions: The term "medium stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 35% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 55.degree. C.
[0062] Medium-high stringency conditions: The term "medium-high
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 35% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 60.degree. C.
[0063] Mutant: The term "mutant" means a polynucleotide encoding a
variant.
[0064] Nucleic acid construct: The term "nucleic acid construct"
means a nucleic acid molecule, either single- or double-stranded,
which is isolated from a naturally occurring gene or is modified to
contain segments of nucleic acids in a manner that would not
otherwise exist in nature or which is synthetic, which comprises
one or more control sequences.
[0065] Operably linked: The term "operably linked" means a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of a
polynucleotide such that the control sequence directs expression of
the coding sequence.
[0066] Parent or parent GH61 polypeptide: The term "parent" or
"parent GH61 polypeptide" means a GH61 polypeptide to which an
alteration is made to produce the GH61 polypeptide variants of the
present invention. The parent may be a naturally occurring
(wild-type) polypeptide or a variant or fragment thereof.
[0067] Polypeptide having cellulolytic enhancing activity: The term
"polypeptide having cellulolytic enhancing activity" means a GH61
polypeptide or variant thereof that catalyzes the enhancement of
the hydrolysis of a cellulosic material by enzyme having
cellulolytic activity. For purposes of the present invention,
cellulolytic enhancing activity is determined by measuring the
increase in reducing sugars or the increase of the total of
cellobiose and glucose from the hydrolysis of a cellulosic material
by cellulolytic enzyme under the following conditions: 1-50 mg of
total protein/g of cellulose in pretreated corn stover (PCS),
wherein total protein is comprised of 50-99.5% w/w cellulolytic
enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide or
variant thereof for 1-7 days at a suitable temperature such as
40.degree. C.-80.degree. C., e.g., 50.degree. C., 55.degree. C.,
60.degree. C., 65.degree. C., or 70.degree. C., and a suitable pH
such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0, compared to a
control hydrolysis with equal total protein loading without
cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g
of cellulose in PCS). In a preferred aspect, a mixture of
CELLUCLAST.RTM. 1.5 L (Novozymes A/S, Bagsvrd, Denmark) in the
presence of 2-3% of total protein weight Aspergillus oryzae
beta-glucosidase (recombinantly produced in Aspergillus oryzae
according to WO 02/095014) or 2-3% of total protein weight
Aspergillus fumigatus beta-glucosidase (recombinantly produced in
Aspergillus oryzae as described in WO 2002/095014) of cellulase
protein loading is used as the source of the cellulolytic activity.
Another assay for determining the cellulolytic enhancing activity
of a GH61 polypeptide or variant thereof is to incubate the GH61
polypeptide or variant with 0.5% phosphoric acid swollen cellulose
(PASC), 100 mM sodium acetate pH 5, 1 mM MnSO.sub.4, 0.1% gallic
acid, 0.025 mg/ml of Aspergillus fumigatus beta-glucosidase, and
0.01% TRITON.RTM. X100 for 24-96 hours at 40.degree. C. followed by
determination of the glucose released from the PASC.
[0068] The GH61 polypeptides or variants thereof having
cellulolytic enhancing activity enhance the hydrolysis of a
cellulosic material catalyzed by enzyme having cellulolytic
activity by reducing the amount of cellulolytic enzyme required to
reach the same degree of hydrolysis preferably at least 1.01-fold,
e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold,
at least 1.5-fold, at least 2-fold, at least 3-fold, at least
4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.
[0069] Pretreated corn stover: The term "PCS" or "Pretreated Corn
Stover" means a cellulosic material derived from corn stover by
treatment with heat and dilute sulfuric acid, alkaline
pretreatment, or neutral pretreatment.
[0070] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0071] For purposes of the present invention, the sequence identity
between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 5.0.0 or later. The parameters used are gap open
penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version of BLOSUM62) substitution matrix. The output of
Needle labeled "longest identity" (obtained using the -nobrief
option) is used as the percent identity and is calculated as
follows: (Identical Residues.times.100)/(Length of Alignment-Total
Number of Gaps in Alignment) For purposes of the present invention,
the sequence identity between two deoxyribonucleotide sequences is
determined using the Needleman-Wunsch algorithm (Needleman and
Wunsch, 1970, supra) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, supra), preferably version 5.0.0
or later. The parameters used are gap open penalty of 10, gap
extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI
NUC4.4) substitution matrix. The output of Needle labeled "longest
identity" (obtained using the -nobrief option) is used as the
percent identity and is calculated as follows: (Identical
Deoxyribonucleotides.times.100)/(Length of Alignment-Total Number
of Gaps in Alignment)
[0072] Subsequence: The term "subsequence" means a polynucleotide
having one or more (e.g., several) nucleotides absent from the 5'
and/or 3' end of a mature polypeptide coding sequence, wherein the
subsequence encodes a fragment having cellulolytic enhancing
activity. In one aspect, a subsequence contains at least 85% of the
nucleotides, e.g., at least 90% of the nucleotides or at least 95%
of the nucleotides of the mature polypeptide coding sequence of a
GH61 polypeptide.
[0073] Variant: The term "variant" means a polypeptide having
cellulolytic enhancing activity comprising an alteration, i.e., a
substitution, insertion, and/or deletion, at one or more (e.g.,
several) positions. A substitution means replacement of the amino
acid occupying a position with a different amino acid; a deletion
means removal of the amino acid occupying a position; and an
insertion means adding an amino acid adjacent to and immediately
following the amino acid occupying a position. The variants of the
present invention have at least 20%, e.g., at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, or at least 100% of the cellulolytic enhancing activity
of their parent GH61 polypeptides.
[0074] Very high stringency conditions: The term "very high
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 50% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 70.degree. C.
[0075] Very low stringency conditions: The term "very low
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 25% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 2.times.SSC,
0.2% SDS at 45.degree. C.
[0076] Wild-type GH61 polypeptide: The term "wild-type" GH61
polypeptide means a GH61 polypeptide expressed by a naturally
occurring microorganism, such as a bacterium, yeast, or filamentous
fungus found in nature.
[0077] Xylan-containing material: The term "xylan-containing
material" means any material comprising a plant cell wall
polysaccharide containing a backbone of beta-(1-4)-linked xylose
residues. Xylans of terrestrial plants are heteropolymers
possessing a beta-(1-4)-D-xylopyranose backbone, which is branched
by short carbohydrate chains. They comprise D-glucuronic acid or
its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides,
composed of D-xylose, L-arabinose, D- or L-galactose, and
D-glucose. Xylan-type polysaccharides can be divided into
homoxylans and heteroxylans, which include glucuronoxylans,
(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans,
and complex heteroxylans. See, for example, Ebringerova et al.,
2005, Adv. Polym. Sci. 186: 1-67.
[0078] In the processes of the present invention, any material
containing xylan may be used. In a preferred aspect, the
xylan-containing material is lignocellulose.
[0079] Xylan degrading activity or xylanolytic activity: The term
"xylan degrading activity" or "xylanolytic activity" means a
biological activity that hydrolyzes xylan-containing material. The
two basic approaches for measuring xylanolytic activity include:
(1) measuring the total xylanolytic activity, and (2) measuring the
individual xylanolytic activities (e.g., endoxylanases,
beta-xylosidases, arabinofuranosidases, alpha-glucuronidases,
acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl
esterases). Recent progress in assays of xylanolytic enzymes was
summarized in several publications including Biely and Puchard,
2006, Recent progress in the assays of xylanolytic enzymes, Journal
of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova
and Biely, 2006, Glucuronoyl esterase--Novel carbohydrate esterase
produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601;
Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997,
The beta-D-xylosidase of Trichoderma reesei is a multifunctional
beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
[0080] Total xylan degrading activity can be measured by
determining the reducing sugars formed from various types of xylan,
including, for example, oat spelt, beechwood, and larchwood xylans,
or by photometric determination of dyed xylan fragments released
from various covalently dyed xylans. The most common total
xylanolytic activity assay is based on production of reducing
sugars from polymeric 4-O-methyl glucuronoxylan as described in
Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods
for assay of xylanase activity, Journal of Biotechnology 23(3):
257-270. Xylanase activity can also be determined with 0.2%
AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100
(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM
sodium phosphate buffer pH 6 at 37.degree. C. One unit of xylanase
activity is defined as 1.0 .mu.mole of azurine produced per minute
at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in
200 mM sodium phosphate pH 6 buffer.
[0081] For purposes of the present invention, xylan degrading
activity is determined by measuring the increase in hydrolysis of
birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by
xylan-degrading enzyme(s) under the following typical conditions: 1
ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic
protein/g of substrate, 50 mM sodium acetate pH 5, 50.degree. C.,
24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide
(PHBAH) assay as described by Lever, 1972, A new reaction for
colorimetric determination of carbohydrates, Anal. Biochem 47:
273-279.
[0082] Xylanase: The term "xylanase" means a
1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the
endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For
purposes of the present invention, xylanase activity is determined
with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100
and 200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit
of xylanase activity is defined as 1.0 .mu.mole of azurine produced
per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as
substrate in 200 mM sodium phosphate pH 6 buffer.
Conventions for Designation of Variants
[0083] For purposes of the present invention, the mature
polypeptide disclosed in SEQ ID NO: 30 is used to determine the
corresponding amino acid residue in another GH61 polypeptide. The
amino acid sequence of another GH61 polypeptide is aligned with the
mature polypeptide disclosed in SEQ ID NO: 30, and based on the
alignment, the amino acid position number corresponding to any
amino acid residue in the mature polypeptide disclosed in SEQ ID
NO: 30 is determined using the Needleman-Wunsch algorithm
(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as
implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or
later. The parameters used are gap open penalty of 10, gap
extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of
BLOSUM62) substitution matrix. Numbering of the amino acid
positions is based on the full-length polypeptide (e.g., including
the signal peptide) of SEQ ID NO: 30 wherein position 1 is the
first amino acid of the signal peptide (e.g., Met).
[0084] Identification of the corresponding amino acid residue in
another GH61 polypeptide can be determined by alignment of multiple
polypeptide sequences using several computer programs including,
but not limited to MUSCLE (multiple sequence comparison by
log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids
Research 32: 1792-1797); MAFFT (version 6.857 or later; Katoh and
Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al.,
2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007,
Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in
Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics
26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;
Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using
their respective default parameters.
[0085] For example, the position corresponding to position 105 of
the Aspergillus fumigatus GH61 polypeptide (SEQ ID NO: 30) is
position 109 in the Penicillium emersonii GH61 polypeptide (SEQ ID
NO: 36), position 105 in the Thermoascus aurantiacus GH61
polypeptide (SEQ ID NO: 14), and position 103 in the Aspergillus
aculeatus GH61 polypeptide (SEQ ID NO: 68); the position
corresponding to position 188 of the Aspergillus fumigatus GH61
polypeptide is position 192 in the Penicillium emersonii GH61
polypeptide, position 188 in the Thermoascus aurantiacus GH61
polypeptide, and position 186 in the Aspergillus aculeatus GH61
polypeptide; the position corresponding to position 154 of the
Aspergillus fumigatus GH61 polypeptide is position 152 in the
Aspergillus aculeatus GH61 polypeptide; and the position
corresponding to position 189 of the Aspergillus fumigatus GH61
polypeptide is position 193 in the Penicillium emersonii GH61
polypeptide and position 187 in the Aspergillus aculeatus GH61
polypeptide.
[0086] When another GH61 polypeptide has diverged from the mature
polypeptide of SEQ ID NO: 30 such that traditional sequence-based
comparison fails to detect their relationship (Lindahl and
Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise
sequence comparison algorithms can be used. Greater sensitivity in
sequence-based searching can be attained using search programs that
utilize probabilistic representations of polypeptide families
(profiles) to search databases. For example, the PSI-BLAST program
generates profiles through an iterative database search process and
is capable of detecting remote homologs (Atschul et al., 1997,
Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be
achieved if the family or superfamily for the polypeptide has one
or more representatives in the protein structure databases.
Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:
797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881)
utilize information from a variety of sources (PSI-BLAST, secondary
structure prediction, structural alignment profiles, and solvation
potentials) as input to a neural network that predicts the
structural fold for a query sequence. Similarly, the method of
Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to
align a sequence of unknown structure with the superfamily models
present in the SCOP database. These alignments can in turn be used
to generate homology models for the polypeptide, and such models
can be assessed for accuracy using a variety of tools developed for
that purpose.
[0087] For proteins of known structure, several tools and resources
are available for retrieving and generating structural alignments.
For example the SCOP superfamilies of proteins have been
structurally aligned, and those alignments are accessible and
downloadable. Two or more protein structures can be aligned using a
variety of algorithms such as the distance alignment matrix (Holm
and Sander, 1998, Proteins 33: 88-96) or combinatorial extension
(Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), and
implementation of these algorithms can additionally be utilized to
query structure databases with a structure of interest in order to
discover possible structural homologs (e.g., Holm and Park, 2000,
Bioinformatics 16: 566-567).
[0088] In describing the GH61 polypeptide variants of the present
invention, the nomenclature described below is adapted for ease of
reference. The accepted IUPAC single letter or three letter amino
acid abbreviation is employed.
[0089] Substitutions.
[0090] For an amino acid substitution, the following nomenclature
is used: Original amino acid, position, substituted amino acid.
Accordingly, the substitution of threonine at position 226 with
alanine is designated as "Thr226Ala" or "T226A". Multiple mutations
are separated by addition marks ("+"), e.g., "Gly205Arg+Ser411Phe"
or "G205R+S411F", representing substitutions at positions 205 and
411 of glycine (G) with arginine (R) and serine (S) with
phenylalanine (F), respectively.
[0091] Deletions.
[0092] For an amino acid deletion, the following nomenclature is
used: Original amino acid, position, *. Accordingly, the deletion
of glycine at position 195 is designated as "Gly195*" or "G195*".
Multiple deletions are separated by addition marks ("+"), e.g.,
"Gly195*+Ser411*" or "G195*+S411*".
[0093] Insertions.
[0094] For an amino acid insertion, the following nomenclature is
used: Original amino acid, position, original amino acid, inserted
amino acid. Accordingly the insertion of lysine after glycine at
position 195 is designated "Gly195GlyLys" or "G195GK". An insertion
of multiple amino acids is designated [Original amino acid,
position, original amino acid, inserted amino acid #1, inserted
amino acid #2; etc.]. For example, the insertion of lysine and
alanine after glycine at position 195 is indicated as
"Gly195GlyLysAla" or "G195GKA".
[0095] In such cases the inserted amino acid residue(s) are
numbered by the addition of lower case letters to the position
number of the amino acid residue preceding the inserted amino acid
residue(s). In the above example, the sequence would thus be:
TABLE-US-00001 Parent: Variant: 195 195 195a 195b G G - K - A
[0096] Multiple Substitutions.
[0097] Variants comprising multiple substitutions are separated by
addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E"
representing a substitution of arginine and glycine at positions
170 and 195 with tyrosine and glutamic acid, respectively.
[0098] Different Substitutions.
[0099] Where different substitutions can be introduced at a
position, the different substitutions are separated by a comma,
e.g., "Arg170Tyr,Glu" represents a substitution of arginine at
position 170 with tyrosine or glutamic acid. Thus,
"Tyr167Gly,Ala+Arg170Gly,Ala" designates the following variants:
"Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala",
"Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".
DETAILED DESCRIPTION OF THE INVENTION
[0100] The present invention relates to isolated GH61 polypeptide
variants, comprising a substitution at one or more (e.g., several)
positions corresponding to positions 105, 154, 188, 189, 216, and
229 of the mature polypeptide of SEQ ID NO: 30, wherein the
variants have cellulolytic enhancing activity.
Variants
[0101] In an embodiment, the variant has a sequence identity of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99%, but less than 100%, to the amino acid sequence of the
parent GH61 polypeptide.
[0102] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,
126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,
152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
178, or 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,
204, 206, 208, 210, 212, 214, or 216.
[0103] In one aspect, the number of substitutions in the variants
of the present invention is 1-6, e.g., 1, 2, 3, 4, 5, or 6
substitutions.
[0104] In another aspect, a variant comprises a substitution at one
or more (e.g., several) positions corresponding to positions 105,
154, 188, 189, 216, and 229. In another aspect, a variant comprises
a substitution at two positions corresponding to any of positions
105, 154, 188, 189, 216, and 229. In another aspect, a variant
comprises a substitution at three positions corresponding to any of
positions 105, 154, 188, 189, 216, and 229. In another aspect, a
variant comprises a substitution at four positions corresponding to
any of positions 105, 154, 188, 189, 216, and 229. In another
aspect, a variant comprises a substitution at five positions
corresponding to any of positions 105, 154, 188, 189, 216, and 229.
In another aspect, a variant comprises a substitution at each
position corresponding to positions 105, 154, 188, 189, 216, and
229.
[0105] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 105. In
another aspect, the amino acid at a position corresponding to
position 105 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Pro or Lys. In another aspect, the variant
comprises or consists of the substitution E105P or E105K of the
mature polypeptide of SEQ ID NO: 30.
[0106] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 154. In
another aspect, the amino acid at a position corresponding to
position 154 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Ile or Leu. In another aspect, the variant
comprises or consists of the substitution E154I or E154L of the
mature polypeptide of SEQ ID NO: 30.
[0107] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 188. In
another aspect, the amino acid at a position corresponding to
position 188 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Ala, Met, Phe, or Trp. In another aspect, the
variant comprises or consists of the substitution G188A, G188F,
G188M, or G188W of the mature polypeptide of SEQ ID NO: 30.
[0108] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 189. In
another aspect, the amino acid at a position corresponding to
position 189 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with His or Lys. In another aspect, the variant
comprises or consists of the substitution N189H or N189K of the
mature polypeptide of SEQ ID NO: 30.
[0109] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 216. In
another aspect, the amino acid at a position corresponding to
position 216 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Leu or Tyr. In another aspect, the variant
comprises or consists of the substitution A216L or A216Y of the
mature polypeptide of SEQ ID NO: 30.
[0110] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 229. In
another aspect, the amino acid at a position corresponding to
position 229 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Trp, His, Ile, or Tyr. In another aspect, the
variant comprises or consists of the substitution K229W, K229H,
K229I, or K229Y of the mature polypeptide of SEQ ID NO: 30.
[0111] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105 and 154,
such as those described above.
[0112] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105 and 188,
such as those described above.
[0113] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105 and 189,
such as those described above.
[0114] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105 and 216,
such as those described above.
[0115] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105 and 229,
such as those described above.
[0116] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154 and 188,
such as those described above.
[0117] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154 and 189,
such as those described above.
[0118] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154 and 216,
such as those described above.
[0119] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154 and 229,
such as those described above.
[0120] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 188 and 189,
such as those described above.
[0121] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 188 and 216,
such as those described above.
[0122] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 188 and 229,
such as those described above.
[0123] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 189 and 216,
such as those described above.
[0124] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 189 and 229,
such as those described above.
[0125] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 216 and 229,
such as those described above.
[0126] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154, and
188, such as those described above.
[0127] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154, and
189, such as those described above.
[0128] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154, and
216, such as those described above.
[0129] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154, and
229, such as those described above.
[0130] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 188, and
189, such as those described above.
[0131] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 188, and
216, such as those described above.
[0132] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 188, and
229, such as those described above.
[0133] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 189, and
216, such as those described above.
[0134] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 189, and
229, such as those described above.
[0135] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 216, and
229, such as those described above.
[0136] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 188, and
189, such as those described above.
[0137] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 188, and
216, such as those described above.
[0138] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 188, and
229, such as those described above.
[0139] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 189, and
216, such as those described above.
[0140] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 189, and
229, such as those described above.
[0141] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 216, and
229, such as those described above.
[0142] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 188, 189, and
216, such as those described above.
[0143] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 188, 189, and
229, such as those described above.
[0144] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 188, 216, and
229, such as those described above.
[0145] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 189, 216, and
229, such as those described above.
[0146] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
188, and 189, such as those described above.
[0147] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
188, and 216, such as those described above.
[0148] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
188, and 229, such as those described above.
[0149] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
189, and 216, such as those described above.
[0150] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
189, and 229, such as those described above. In another aspect, the
variant comprises or consists of substitutions at positions
corresponding to positions 105, 154, 216, and 229, such as those
described above.
[0151] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 188,
189, and 216, such as those described above.
[0152] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 188,
189, and 229, such as those described above. In another aspect, the
variant comprises or consists of substitutions at positions
corresponding to positions 105, 188, 216, and 229, such as those
described above.
[0153] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 189,
216, and 229, such as those described above.
[0154] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 188,
189, and 216, such as those described above.
[0155] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 188,
189, and 229, such as those described above.
[0156] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 188,
216, and 229, such as those described above. In another aspect, the
variant comprises or consists of substitutions at positions
corresponding to positions 154, 189, 216, and 229, such as those
described above.
[0157] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 188, 189,
216, and 229, such as those described above.
[0158] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
188, 189, and 216, such as those described above.
[0159] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
188, 189, and 229, such as those described above.
[0160] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
188, 216, and 229, such as those described above. In another
aspect, the variant comprises or consists of substitutions at
positions corresponding to positions 105, 154, 189, 216, and 229,
such as those described above.
[0161] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 188,
189, 216, and 229, such as those described above.
[0162] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 154, 188,
189, 216, and 229, such as those described above.
[0163] In another aspect, the variant comprises or consists of
substitutions at positions corresponding to positions 105, 154,
188, 189, 216, and 229, such as those described above.
[0164] In another aspect, the variant comprises or consists of one
or more (e.g., several) substitutions selected from the group
consisting of E105P,K; E154I,L; G188A,F,M,W; A216L,Y; and
K229W,H,I,Y, or the one or more (e.g., several) substitutions
selected from the group consisting of E105P,K; E154I,L;
G188A,F,M,W; A216L,Y; and K229W,H,I,Y at positions corresponding to
SEQ ID NO: 30 in other GH61 polypeptides described herein.
[0165] In each of the aspects below, the variant comprises or
consists of the one or more (e.g., several) substitutions described
below at positions corresponding to SEQ ID NO: 30 in other GH61
polypeptides described herein.
[0166] In another aspect, the variant comprises or consists of the
substitutions E105P,K and E154I,L of the mature polypeptide of SEQ
ID NO: 30.
[0167] In another aspect, the variant comprises or consists of the
substitutions E105P,K and G188A,F,M,W of the mature polypeptide of
SEQ ID NO: 30.
[0168] In another aspect, the variant comprises or consists of the
substitutions E105P,K and N189H,K of the mature polypeptide of SEQ
ID NO: 30.
[0169] In another aspect, the variant comprises or consists of the
substitutions E105P,K and A216L,Y of the mature polypeptide of SEQ
ID NO: 30.
[0170] In another aspect, the variant comprises or consists of the
substitutions E105P,K and K229W,H,I,Y of the mature polypeptide of
SEQ ID NO: 30.
[0171] In another aspect, the variant comprises or consists of the
substitutions E154I,L and G188A,F,M,W of the mature polypeptide of
SEQ ID NO: 30.
[0172] In another aspect, the variant comprises or consists of the
substitutions E154I,L and N189H,K of the mature polypeptide of SEQ
ID NO: 30.
[0173] In another aspect, the variant comprises or consists of the
substitutions E154I,L and A216L,Y of the mature polypeptide of SEQ
ID NO: 30.
[0174] In another aspect, the variant comprises or consists of the
substitutions E154I,L and K229W,H,I,Y of the mature polypeptide of
SEQ ID NO: 30.
[0175] In another aspect, the variant comprises or consists of the
substitutions G188A,F,M,W and N189H,K of the mature polypeptide of
SEQ ID NO: 30.
[0176] In another aspect, the variant comprises or consists of the
substitutions G188A,F,M,W and A216L,Y of the mature polypeptide of
SEQ ID NO: 30.
[0177] In another aspect, the variant comprises or consists of the
substitutions G188A,F,M,W and K229W,H,I,Y of the mature polypeptide
of SEQ ID NO: 30.
[0178] In another aspect, the variant comprises or consists of the
substitutions N189H,K and A216L,Y of the mature polypeptide of SEQ
ID NO: 30.
[0179] In another aspect, the variant comprises or consists of the
substitutions N189H,K and K229W,H,I,Y of the mature polypeptide of
SEQ ID NO: 30.
[0180] In another aspect, the variant comprises or consists of the
substitutions A216L,Y and K229W,H,I,Y of the mature polypeptide of
SEQ ID NO: 30.
[0181] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; and G188A,F,M,W of the mature
polypeptide of SEQ ID NO: 30.
[0182] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; and N189H,K of the mature
polypeptide of SEQ ID NO: 30.
[0183] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; and A216L,Y of the mature
polypeptide of SEQ ID NO: 30.
[0184] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0185] In another aspect, the variant comprises or consists of the
substitutions E105P,K; G188A,F,M,W; and N189H,K of the mature
polypeptide of SEQ ID NO: 30.
[0186] In another aspect, the variant comprises or consists of the
substitutions E105P,K; G188A,F,M,W; and A216L,Y of the mature
polypeptide of SEQ ID NO: 30.
[0187] In another aspect, the variant comprises or consists of the
substitutions E105P,K; G188A,F,M,W; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0188] In another aspect, the variant comprises or consists of the
substitutions E105P,K; N189H,K; and A216L,Y of the mature
polypeptide of SEQ ID NO: 30.
[0189] In another aspect, the variant comprises or consists of the
substitutions E105P,K; N189H,K; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0190] In another aspect, the variant comprises or consists of the
substitutions E105P,K; A216L,Y; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0191] In another aspect, the variant comprises or consists of the
substitutions E154I,L; G188A,F,M,W; and N189H,K of the mature
polypeptide of SEQ ID NO: 30.
[0192] In another aspect, the variant comprises or consists of the
substitutions E154I,L; G188A,F,M,W; and A216L,Y of the mature
polypeptide of SEQ ID NO: 30.
[0193] In another aspect, the variant comprises or consists of the
substitutions E154I,L; G188A,F,M,W; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0194] In another aspect, the variant comprises or consists of the
substitutions E154I,L; N189H,K; and A216L,Y of the mature
polypeptide of SEQ ID NO: 30.
[0195] In another aspect, the variant comprises or consists of the
substitutions E154I,L; N189H,K; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0196] In another aspect, the variant comprises or consists of the
substitutions E154I,L; A216L,Y; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0197] In another aspect, the variant comprises or consists of the
substitutions G188A,F,M,W; N189H,K; and A216L,Y of the mature
polypeptide of SEQ ID NO: 30.
[0198] In another aspect, the variant comprises or consists of the
substitutions G188A,F,M,W; N189H,K; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0199] In another aspect, the variant comprises or consists of the
substitutions G188A,F,M,W; A216L,Y; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0200] In another aspect, the variant comprises or consists of the
substitutions N189H,K; A216L,Y; and K229W,H,I,Y of the mature
polypeptide of SEQ ID NO: 30.
[0201] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; G188A,F,M,W; and N189H,K of the
mature polypeptide of SEQ ID NO: 30.
[0202] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; G188A,F,M,W; and A216L,Y of the
mature polypeptide of SEQ ID NO: 30.
[0203] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; G188A,F,M,W; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0204] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; N189H,K; and A216L,Y of the mature
polypeptide of SEQ ID NO: 30.
[0205] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; N189H,K; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0206] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; A216L,Y; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0207] In another aspect, the variant comprises or consists of the
substitutions E105P,K; G188A,F,M,W; N189H,K; and A216L,Y of the
mature polypeptide of SEQ ID NO: 30.
[0208] In another aspect, the variant comprises or consists of the
substitutions E105P,K; G188A,F,M,W; N189H,K; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0209] In another aspect, the variant comprises or consists of the
substitutions E105P,K; G188A,F,M,W; A216L,Y; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0210] In another aspect, the variant comprises or consists of the
substitutions E105P,K; N189H,K; A216L,Y; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0211] In another aspect, the variant comprises or consists of the
substitutions E154I,L; G188A,F,M,W; N189H,K; and A216L,Y of the
mature polypeptide of SEQ ID NO: 30.
[0212] In another aspect, the variant comprises or consists of the
substitutions E154I,L; G188A,F,M,W; N189H,K; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0213] In another aspect, the variant comprises or consists of the
substitutions E154I,L; G188A,F,M,W; A216L,Y; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0214] In another aspect, the variant comprises or consists of the
substitutions E154I,L; N189H,K; A216L,Y; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0215] In another aspect, the variant comprises or consists of the
substitutions G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y of the
mature polypeptide of SEQ ID NO: 30.
[0216] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; and A216L,Y
of the mature polypeptide of SEQ ID NO: 30.
[0217] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; and
K229W,H,I,Y of the mature polypeptide of SEQ ID NO: 30.
[0218] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; G188A,F,M,W; A216L,Y; and
K229W,H,I,Y of the mature polypeptide of SEQ ID NO: 30.
[0219] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; N189H,K; A216L,Y; and K229W,H,I,Y
of the mature polypeptide of SEQ ID NO: 30.
[0220] In another aspect, the variant comprises or consists of the
substitutions E105P,K; G188A,F,M,W; N189H,K; A216L,Y; and
K229W,H,I,Y of the mature polypeptide of SEQ ID NO: 30.
[0221] In another aspect, the variant comprises or consists of the
substitutions E154I,L; G188A,F,M,W; N189H,K; A216L,Y; and
K229W,H,I,Y of the mature polypeptide of SEQ ID NO: 30.
[0222] In another aspect, the variant comprises or consists of the
substitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; A216L,Y; and
K229W,H,I,Y of the mature polypeptide of SEQ ID NO: 30.
[0223] In another aspect, the variant comprises or consists of the
substitutions D105K,P of the mature polypeptide of SEQ ID NO: 14.
In another aspect, the variant comprises or consists of the
substitutions Q188W,F,M of the mature polypeptide of SEQ ID NO:
14.
[0224] In another aspect, the variant comprises or consists of the
substitution D109P,K of the mature polypeptide of SEQ ID NO: 36. In
another aspect, the variant comprises or consists of the
substitution N192A,W,M of the mature polypeptide of SEQ ID NO: 36.
In another aspect the variant comprises or consists of the
substitution N193K,H of the mature polypeptide of SEQ ID NO: 36. In
another aspect, the variant comprises or consists of the
substitution D109P,K of the mature polypeptide of SEQ ID NO: 36. In
another aspect, the substitution is N192A,W,M of the mature
polypeptide of SEQ ID NO: 36. In another aspect, the variant
comprises or consists of the substitution N193K,H of the mature
polypeptide of SEQ ID NO: 36.
[0225] In another aspect, the variant comprises or consists of the
substitution D103K,P of the mature polypeptide of SEQ ID NO: 68. In
another aspect, the variant comprises or consists of the
substitution N1521,L of the mature polypeptide of SEQ ID NO: 68. In
another aspect the variant comprises or consists of the
substitution G186A,F,M,W of the mature polypeptide of SEQ ID NO:
68. In another aspect, the variant comprises or consists of the
substitution N187H,K of the mature polypeptide of SEQ ID NO:
68.
[0226] The variants may further comprise one or more additional
alterations, e.g., substitutions, insertions, or deletions at one
or more (e.g., several) other positions.
[0227] The amino acid changes may be of a minor nature, that is
conservative amino acid substitutions or insertions that do not
significantly affect the folding and/or activity of the protein;
small deletions, typically of 1-30 amino acids; small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue; a small linker peptide of up to 20-25 residues; or a small
extension that facilitates purification by changing net charge or
another function, such as a poly-histidine tract, an antigenic
epitope or a binding domain.
[0228] Examples of conservative substitutions are within the groups
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter specific activity are known in the art and are
described, for example, by H. Neurath and R. L. Hill, 1979, In, The
Proteins, Academic Press, New York. Common substitutions are
Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,
Leu/Val, Ala/Glu, and Asp/Gly.
[0229] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0230] The variants of the present invention may further comprise a
substitution at one or more (e.g., several) positions corresponding
to positions 111, 152, 155, and 162 of the mature polypeptide of
SEQ ID NO: 30, wherein the variants have cellulolytic enhancing
activity (WO 2012/044835).
[0231] In one aspect, the number of additional substitutions in the
variants of the present invention is 1-4, such as 1, 2, 3, or 4
substitutions.
[0232] In another aspect, the variant further comprises a
substitution at one or more (e.g., several) positions corresponding
to positions 111, 152, 155, and 162. In another aspect, the variant
further comprises a substitution at two positions corresponding to
any of positions 111, 152, 155, and 162. In another aspect, the
variant further comprises a substitution at three positions
corresponding to any of positions 111, 152, 155, and 162. In
another aspect, the variant further comprises a substitution at
each position corresponding to positions 111, 152, 155, and
162.
[0233] In another aspect, the variant further comprises a
substitution at a position corresponding to position 111. In
another aspect, the amino acid at a position corresponding to
position 111 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Val. In another aspect, the variant further
comprises the substitution L111V of the mature polypeptide of SEQ
ID NO: 30.
[0234] In another aspect, the variant further comprises a
substitution at a position corresponding to position 152. In
another aspect, the amino acid at a position corresponding to
position 152 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Ser. In another aspect, the variant further
comprises the substitution D152S of the mature polypeptide of SEQ
ID NO: 30.
[0235] In another aspect, the variant further comprises a
substitution at a position corresponding to position 155. In
another aspect, the amino acid at a position corresponding to
position 155 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Leu. In another aspect, the variant further
comprises the substitution M155L of the mature polypeptide of SEQ
ID NO: 30.
[0236] In another aspect, the variant further comprises a
substitution at a position corresponding to position 162. In
another aspect, the amino acid at a position corresponding to
position 162 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Trp. In another aspect, the variant further
comprises the substitution A162W of the mature polypeptide of SEQ
ID NO: 30.
[0237] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 111 and 152,
such as those described above.
[0238] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 111 and 155,
such as those described above.
[0239] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 111 and 162,
such as those described above.
[0240] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 152 and 155,
such as those described above.
[0241] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 152 and 162,
such as those described above.
[0242] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 155 and 162,
such as those described above.
[0243] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 111, 152, and
155, such as those described above.
[0244] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 111, 152, and
162, such as those described above.
[0245] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 111, 155, and
162, such as those described above.
[0246] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 152, 155, and
162, such as those described above.
[0247] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 111, 152,
155, and 162, such as those described above. In another aspect, the
variant further comprises one or more (e.g., several) substitutions
selected from the group consisting of L111V, D152S, M155L, and
A162W, or the one or more (e.g., several) substitutions selected
from the group consisting of L111V, D152S, M155L, and A162W at
positions corresponding to SEQ ID NO: 30 in other GH61 polypeptides
described herein.
[0248] In another aspect, the variant comprises substitutions
L111V, D152S, M155L, A162W, and G188A, or the same substitutions at
corresponding positions thereof. In another aspect, the variant
comprises substitutions L111V, D152S, M155L, A162W, G188F, and
K229W, or the same substitutions at corresponding positions
thereof. In another aspect, the variant comprises substitutions
L111V, D152S, M155L, A162W, and K229W, or corresponding
substitutions thereof. In another aspect, the variant comprises
substitutions L111V, D152S, M155L, A162W, A216Y, and K229W, or the
same substitutions at corresponding positions thereof. In another
aspect, the variant comprises substitutions L111V, D152S, M155L,
A162W, N189K, and K229W, or the same substitutions at corresponding
positions thereof. In another aspect, the variant comprises
substitutions L111V, D152S, M155L, A162W, and N189K, or the same
substitutions at corresponding positions thereof. In another
aspect, the variant comprises substitutions L111V, D152S, M155L,
A162W, and G188W, or the same substitutions at corresponding
positions thereof.
[0249] In each of the aspects below, the variant further comprises
the one or more (e.g., several) substitutions described below at
positions corresponding to SEQ ID NO: 30 in other GH61 polypeptides
described herein.
[0250] In another aspect, the variant further comprises the
substitutions L111V+D152S of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0251] In another aspect, the variant further comprises the
substitutions L111V+M155L of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0252] In another aspect, the variant further comprises the
substitutions L111V+A162W of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0253] In another aspect, the variant further comprises the
substitutions D152S+M155L of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0254] In another aspect, the variant further comprises the
substitutions D152S+A162W of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0255] In another aspect, the variant further comprises the
substitutions M155L+A162W of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0256] In another aspect, the variant further comprises the
substitutions L111V+D152S+M155L of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0257] In another aspect, the variant further comprises the
substitutions L111V+D152S+A162W of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0258] In another aspect, the variant further comprises the
substitutions L111V+M155L+A162W of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0259] In another aspect, the variant further comprises the
substitutions D152S+M155L+A162W of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0260] In another aspect, the variant further comprises the
substitutions L111V+D152S+M155L+A162W of the mature polypeptide of
SEQ ID NO: 30, or corresponding substitutions thereof.
[0261] In each of the aspects above, the variants of the present
invention may further comprise a substitution at one or more (e.g.,
several) positions corresponding to positions 96, 98, 200, 202, and
204 of the mature polypeptide of SEQ ID NO: 30, wherein the
variants have cellulolytic enhancing activity (WO 2012/044836).
[0262] In one aspect, the number of additional substitutions in the
variants of the present invention is 1-5, such as 1, 2, 3, 4, or 5
substitutions.
[0263] In another aspect, the variant further comprises a
substitution at one or more (e.g., several) positions corresponding
to positions 96, 98, 200, 202, and 204. In another aspect, the
variant further comprises a substitution at two positions
corresponding to any of positions 96, 98, 200, 202, and 204. In
another aspect, the variant further comprises a substitution at
three positions corresponding to any of positions 96, 98, 200, 202,
and 204. In another aspect, the variant further comprises a
substitution at four positions corresponding to any of positions
96, 98, 200, 202, and 204. In another aspect, the variant further
comprises a substitution at each position corresponding to
positions 96, 98, 200, 202, and 204.
[0264] In another aspect, the variant further comprises a
substitution at a position corresponding to position 96. In another
aspect, the amino acid at a position corresponding to position 96
is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Val. In another aspect, the variant further
comprises the substitution 196V of the mature polypeptide of SEQ ID
NO: 30.
[0265] In another aspect, the variant further comprises a
substitution at a position corresponding to position 98. In another
aspect, the amino acid at a position corresponding to position 98
is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Leu. In another aspect, the variant further
comprises the substitution F98L of the mature polypeptide of SEQ ID
NO: 30.
[0266] In another aspect, the variant further comprises a
substitution at a position corresponding to position 200. In
another aspect, the amino acid at a position corresponding to
position 200 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Ile. In another aspect, the variant further
comprises the substitution F200I of the mature polypeptide of SEQ
ID NO: 30.
[0267] In another aspect, the variant further comprises a
substitution at a position corresponding to position 202. In
another aspect, the amino acid at a position corresponding to
position 202 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Leu. In another aspect, the variant further
comprises the substitution I202L of the mature polypeptide of SEQ
ID NO: 30.
[0268] In another aspect, the variant further comprises a
substitution at a position corresponding to position 204. In
another aspect, the amino acid at a position corresponding to
position 204 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
preferably with Val. In another aspect, the variant further
comprises the substitution I204V of the mature polypeptide of SEQ
ID NO: 30.
[0269] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96 and 98,
such as those described above.
[0270] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96 and 200,
such as those described above.
[0271] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96 and 202,
such as those described above.
[0272] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96 and 204,
such as those described above.
[0273] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 98 and 200,
such as those described above.
[0274] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 98 and 202,
such as those described above.
[0275] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 98 and 204,
such as those described above.
[0276] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 200 and 202,
such as those described above.
[0277] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 200 and 204,
such as those described above.
[0278] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 202 and 204,
such as those described above.
[0279] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 98, and
200, such as those described above.
[0280] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 98, and
202, such as those described above.
[0281] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 98, and
204, such as those described above.
[0282] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 200, and
202, such as those described above.
[0283] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 200, and
204, such as those described above.
[0284] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 202, and
204, such as those described above.
[0285] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 98, 200, and
202, such as those described above.
[0286] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 98, 200, and
204, such as those described above.
[0287] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 200, 202, and
204, such as those described above.
[0288] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 98, 202, and
204, such as those described above.
[0289] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 98, 200,
and 202, such as those described above.
[0290] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 200, 202,
and 204, such as those described above. In another aspect, the
variant further comprises substitutions at positions corresponding
to positions 96, 98, 202, and 204, such as those described
above.
[0291] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 98, 200,
and 204, such as those described above.
[0292] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 98, 200, 202,
and 204, such as those described above.
[0293] In another aspect, the variant further comprises
substitutions at positions corresponding to positions 96, 98, 200,
202, and 204, such as those described above.
[0294] In another aspect, the variant further comprises one or more
(e.g., several) substitutions selected from the group consisting of
I196V, F98L, F200I, I202L, and I204V, or the one or more (e.g.,
several) substitutions selected from the group consisting of I196V,
F98L, F200I, I202L, and I204V at positions corresponding to SEQ ID
NO: 30 in other GH61 polypeptides described herein.
[0295] In each of the aspects below, the variant further comprises
the one or more (e.g., several) substitutions described below at
positions corresponding to SEQ ID NO: 30 in other GH61 polypeptides
described herein.
[0296] In another aspect, the variant further comprises the
substitutions I96V+F98L of the mature polypeptide of SEQ ID NO: 30,
or corresponding substitutions thereof.
[0297] In another aspect, the variant further comprises the
substitutions I96V+F200I of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0298] In another aspect, the variant further comprises the
substitutions I96V+I202L of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0299] In another aspect, the variant further comprises the
substitutions I96V+I204V of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0300] In another aspect, the variant further comprises the
substitutions F98L+F200I of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0301] In another aspect, the variant further comprises the
substitutions F98L+I202L of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0302] In another aspect, the variant further comprises the
substitutions F98L+I204V of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0303] In another aspect, the variant further comprises the
substitutions F200I+I202L of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0304] In another aspect, the variant further comprises the
substitutions F200I+I204V of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0305] In another aspect, the variant further comprises the
substitutions I202L+I204V of the mature polypeptide of SEQ ID NO:
30, or corresponding substitutions thereof.
[0306] In another aspect, the variant further comprises the
substitutions I96V+F98L+F200I of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0307] In another aspect, the variant further comprises the
substitutions I96V+F98L+I202L of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0308] In another aspect, the variant further comprises the
substitutions I96V+F98L+I204V of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0309] In another aspect, the variant further comprises the
substitutions I96V+F200I+I202L of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0310] In another aspect, the variant further comprises the
substitutions I96V+F200I+I204V of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0311] In another aspect, the variant further comprises the
substitutions I96V+I202L+I204V of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0312] In another aspect, the variant further comprises the
substitutions F98L+F200I+I202L of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0313] In another aspect, the variant further comprises the
substitutions F98L+F200I+I204V of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0314] In another aspect, the variant further comprises the
substitutions F200I+I202L+I204V of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0315] In another aspect, the variant further comprises the
substitutions F98L+I202L+I204V of the mature polypeptide of SEQ ID
NO: 30, or corresponding substitutions thereof.
[0316] In another aspect, the variant further comprises the
substitutions I96V+F98L+F200I+I202L of the mature polypeptide of
SEQ ID NO: 30, or corresponding substitutions thereof.
[0317] In another aspect, the variant further comprises the
substitutions I96V+F200I+I202L+I204V of the mature polypeptide of
SEQ ID NO: 30, or corresponding substitutions thereof.
[0318] In another aspect, the variant further comprises the
substitutions I96V+F98L+I202L+I204V of the mature polypeptide of
SEQ ID NO: 30, or corresponding substitutions thereof.
[0319] In another aspect, the variant further comprises the
substitutions I96V+F98L+F200I+I204V of the mature polypeptide of
SEQ ID NO: 30, or corresponding substitutions thereof.
[0320] In another aspect, the variant further comprises the
substitutions F98L+F200I+I202L+I204V of the mature polypeptide of
SEQ ID NO: 30, or corresponding substitutions thereof.
[0321] In another aspect, the variant further comprises the
substitutions I96V+F98L+F200I+I202L+I204V of the mature polypeptide
of SEQ ID NO: 30, or corresponding substitutions thereof.
[0322] The variants may consist of at least 85% of the amino acid
residues, e.g., at least 90% of the amino acid residues or at least
95% of the amino acid residues of the mature polypeptides of the
corresponding parent GH61 polypeptides.
[0323] Essential amino acids in a polypeptide can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,
1989, Science 244: 1081-1085). In the latter technique, single
alanine mutations are introduced at every residue in the molecule,
and the resultant mutant molecules are tested for cellulolytic
enhancing activity to identify amino acid residues that are
critical to the activity of the molecule. See also, Hilton et al.,
1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme
or other biological interaction can also be determined by physical
analysis of structure, as determined by such techniques as nuclear
magnetic resonance, crystallography, electron diffraction, or
photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids. See, for example, de Vos et al., 1992,
Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:
899-904; Wlodaver et al., 1992, FEBS Lett.
[0324] 309: 59-64. The identity of essential amino acids can also
be inferred from an alignment with a related polypeptide. Essential
amino acids in GH61 polypeptides correspond to positions 22, 107,
194, and/or 196 of the mature polypeptide of SEQ ID NO: 30.
[0325] In an embodiment, the variants have increased
thermostability compared to their parent GH61 polypeptides.
[0326] In one aspect, the thermostability of the variant relative
to the parent is determined at pH 3.0 and 45.degree. C. In another
aspect, the thermostability of the variant relative to the parent
is determined at pH 3.0 and 50.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 3.0 and 55.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 3.0 and
60.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 3.0 and 62.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 3.0 and 65.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 3.0 and 68.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 3.0 and 70.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 3.0 and
72.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 3.0 and 75.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 3.0 and 80.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 3.0 and 85.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 3.0 and 90.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 3.0 and
95.degree. C.
[0327] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 3.5 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 3.5 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 3.5 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 3.5 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 3.5 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 3.5 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 3.5 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 3.5 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 3.5 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 3.5 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 3.5 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 3.5 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 3.5 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 3.5 and 95.degree. C.
[0328] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 4.0 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 4.0 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 4.0 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 4.0 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 4.0 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 4.0 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 4.0 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 4.0 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 4.0 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 4.0 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 4.0 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 4.0 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 4.0 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 4.0 and 95.degree. C.
[0329] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 4.5 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 4.5 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 4.5 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 4.5 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 4.5 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 4.5 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 4.5 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 4.5 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 4.5 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 4.5 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 4.5 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 4.5 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 4.5 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 4.5 and 95.degree. C.
[0330] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 5.0 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 5.0 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 5.0 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 5.0 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 5.0 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 5.0 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 5.0 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 5.0 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 5.0 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 5.0 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 5.0 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 5.0 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 5.0 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 5.0 and 95.degree. C.
[0331] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 5.5 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 5.5 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 5.5 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 5.5 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 5.5 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 5.5 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 5.5 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 5.5 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 5.5 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 5.5 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 5.5 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 5.5 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 5.5 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 5.5 and 95.degree. C.
[0332] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 6.0 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 6.0 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 6.0 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 6.0 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 6.0 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 6.0 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 6.0 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 6.0 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 6.0 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 6.0 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 6.0 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 6.0 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 6.0 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 6.0 and 95.degree. C.
[0333] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 6.5 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 6.5 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 6.5 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 6.5 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 6.5 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 6.5 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 6.5 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 6.5 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 6.5 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 6.5 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 6.5 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 6.5 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 6.5 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 6.5 and 95.degree. C.
[0334] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 7.0 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 7.0 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 7.0 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 7.0 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 7.0 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 7.0 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 7.0 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 7.0 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 7.0 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 7.0 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 7.0 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 7.0 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 7.0 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 7.0 and 95.degree. C.
[0335] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 7.5 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 7.5 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 7.5 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 7.5 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 7.5 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 7.5 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 7.5 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 7.5 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 7.5 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 7.5 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 7.5 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 7.5 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 7.5 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 7.5 and 95.degree. C.
[0336] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 8.0 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 8.0 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 8.0 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 8.0 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 8.0 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 8.0 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 8.0 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 8.0 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 8.0 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 8.0 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 8.0 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 8.0 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 8.0 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 8.0 and 95.degree. C.
[0337] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 8.5 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 8.5 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 8.5 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 8.5 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 8.5 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 8.5 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 8.5 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 8.5 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 8.5 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 8.5 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 8.5 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 8.5 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 8.5 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 8.5 and 95.degree. C.
[0338] In another aspect, the thermostability of the variant
relative to the parent is determined at pH 9.0 and 45.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 9.0 and 50.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 9.0 and 55.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 9.0 and 60.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 9.0 and
62.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 9.0 and 65.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 9.0 and 68.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 9.0 and 70.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 9.0 and 72.degree. C. In another aspect, the thermostability
of the variant relative to the parent is determined at pH 9.0 and
75.degree. C. In another aspect, the thermostability of the variant
relative to the parent is determined at pH 9.0 and 80.degree. C. In
another aspect, the thermostability of the variant relative to the
parent is determined at pH 9.0 and 85.degree. C. In another aspect,
the thermostability of the variant relative to the parent is
determined at pH 9.0 and 90.degree. C. In another aspect, the
thermostability of the variant relative to the parent is determined
at pH 9.0 and 95.degree. C.
[0339] In each of the aspects above, the thermostability of the
variant relative to the parent can be determined by incubating the
variant and parent for 1 minute. In each of the aspects above, the
thermostability of the variant relative to the parent can be
determined by incubating the variant and parent for 5 minutes. In
each of the aspects above, the thermostability of the variant
relative to the parent can be determined by incubating the variant
and parent for 10 minutes. In each of the aspects above, the
thermostability of the variant relative to the parent can be
determined by incubating the variant and parent for 15 minutes. In
each of the aspects above, the thermostability of the variant
relative to the parent can be determined by incubating the variant
and parent for 20 minutes. In each of the aspects above, the
thermostability of the variant relative to the parent can be
determined by incubating the variant and parent for 25 minutes. In
each of the aspects above, the thermostability of the variant
relative to the parent can be determined by incubating the variant
and parent for 30 minutes. In each of the aspects above, the
thermostability of the variant relative to the parent can be
determined by incubating the variant and parent for 45 minutes. In
each of the aspects above, the thermostability of the variant
relative to the parent can be determined by incubating the variant
and parent for 60 minutes. A time period longer than 60 minutes can
also be used.
[0340] In one aspect, the thermostability of the variant having
cellulolytic enhancing activity is increased at least 1.01-fold,
e.g., at least 1.05-fold, at least 1.1-fold, at least 1.2-fold, at
least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least
1.8-fold, at least 2-fold, at least 5-fold, at least 10-fold, at
least 15-fold, at least 20-fold, at least 25-fold, at least
50-fold, at least 75-fold, or at least 100-fold compared to the
parent.
Parent GH61 Polypeptides
[0341] The parent GH61 polypeptide may be any GH61 polypeptide
having cellulolytic enhancing activity.
[0342] The parent GH61 polypeptide may be (a) a polypeptide having
at least 60% sequence identity to the mature polypeptide of SEQ ID
NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, 78, 80, 82, 84, 86, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,
127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,
153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,
179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,
205, 207, 209, 211, 213, or 215; (b) a polypeptide encoded by a
polynucleotide that hybridizes under at least low stringency
conditions with (i) the mature polypeptide coding sequence of 1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,
105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,
131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,
157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,
183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,
209, 211, 213, or 215, or (ii) the full-length complement of (i);
or (c) a polypeptide encoded by a polynucleotide having at least
60% sequence identity to the mature polypeptide coding sequence of
SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,
65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,
99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,
125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,
151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,
177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,
203, 205, 207, 209, 211, 213, or 215.
[0343] In one aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216 of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%, which have cellulolytic enhancing activity.
[0344] In another aspect, the amino acid sequence of the parent
differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 from the mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,
82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,
138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,
164, 166, 168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188,
190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or
216.
[0345] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216.
[0346] In another aspect, the parent comprises or consists of the
mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216.
[0347] In another aspect, the parent is a fragment containing at
least 85% of the amino acid residues, e.g., at least 90% of the
amino acid residues or at least 95% of the amino acid residues of
the mature polypeptide of a GH61 polypeptide.
[0348] In another embodiment, the parent is an allelic variant of
the mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,
86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216.
[0349] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under very low stringency conditions, low
stringency conditions, medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,
93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,
121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,
147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,
173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,
199, 201, 203, 205, 207, 209, 211, 213, or 215, or the full-length
complements thereof (Sambrook et al., 1989, Molecular Cloning, A
Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).
[0350] The polynucleotide of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,
85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,
115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,
141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,
167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,
193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215, or
subsequences thereof, as well as the polypeptide of SEQ ID NO: 2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,
104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,
130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,
156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180,
182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,
208, 210, 212, 214, or 216, or fragments thereof, may be used to
design nucleic acid probes to identify and clone DNA encoding a
parent from strains of different genera or species according to
methods well known in the art. In particular, such probes can be
used for hybridization with the genomic DNA or cDNA of a cell of
interest, following standard Southern blotting procedures, in order
to identify and isolate the corresponding gene therein. Such probes
can be considerably shorter than the entire sequence, but should be
at least 15, e.g., at least 25, at least 35, or at least 70
nucleotides in length. Preferably, the nucleic acid probe is at
least 100 nucleotides in length, e.g., at least 200 nucleotides, at
least 300 nucleotides, at least 400 nucleotides, at least 500
nucleotides, at least 600 nucleotides, at least 700 nucleotides, at
least 800 nucleotides, or at least 900 nucleotides in length. Both
DNA and RNA probes can be used. The probes are typically labeled
for detecting the corresponding gene (for example, with .sup.32P,
.sup.3H, .sup.35S, biotin, or avidin). Such probes are encompassed
by the present invention.
[0351] A genomic DNA or cDNA library prepared from such other
strains may be screened for DNA that hybridizes with the probes
described above and encodes a parent. Genomic or other DNA from
such other strains may be separated by agarose or polyacrylamide
gel electrophoresis, or other separation techniques. DNA from the
libraries or the separated DNA may be transferred to and
immobilized on nitrocellulose or other suitable carrier material.
In order to identify a clone or DNA that hybridizes with SEQ ID NO:
1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,
71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,
103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,
129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,
155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179,
181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,
207, 209, 211, 213, or 215, or subsequences thereof, the carrier
material is used in a Southern blot.
[0352] For purposes of the present invention, hybridization
indicates that the polynucleotide hybridizes to a labeled nucleic
acid probe corresponding to (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,
113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,
139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,
165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,
191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215;
(ii) the mature polypeptide coding sequence thereof; (iii) the
full-length complement thereof; or (iv) a subsequence thereof;
under very low to very high stringency conditions. Molecules to
which the nucleic acid probe hybridizes under these conditions can
be detected using, for example, X-ray film or any other detection
means known in the art.
[0353] In one aspect, the nucleic acid probe is the mature
polypeptide coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,
113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,
139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,
165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,
191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or
215.
[0354] In another aspect, the nucleic acid probe is a
polynucleotide that encodes the polypeptide of SEQ ID NO: 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,
76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,
108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,
134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,
160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180, 182, 184,
186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,
212, 214, or 216; the mature polypeptide thereof; or a fragment
thereof.
[0355] In another aspect, the nucleic acid probe is SEQ ID NO: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,
105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,
131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,
157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,
183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,
209, 211, 213, or 215.
[0356] In another embodiment, the parent is encoded by a
polynucleotide having a sequence identity to the mature polypeptide
coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,
89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,
117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,
143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,
169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,
195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215 of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.
[0357] The parent may be a hybrid polypeptide in which a region of
one polypeptide is fused at the N-terminus or the C-terminus of a
region of another polypeptide.
[0358] The parent may be a fusion polypeptide or cleavable fusion
polypeptide in which another polypeptide is fused at the N-terminus
or the C-terminus of the polypeptide of the present invention. A
fusion polypeptide is produced by fusing a polynucleotide encoding
another polypeptide to a polynucleotide of the present invention.
Techniques for producing fusion polypeptides are known in the art,
and include ligating the coding sequences encoding the polypeptides
so that they are in frame and that expression of the fusion
polypeptide is under control of the same promoter(s) and
terminator. Fusion polypeptides may also be constructed using
intein technology in which fusion polypeptides are created
post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583;
Dawson et al., 1994, Science 266: 776-779).
[0359] A fusion polypeptide can further comprise a cleavage site
between the two polypeptides. Upon secretion of the fusion protein,
the site is cleaved releasing the two polypeptides. Examples of
cleavage sites include, but are not limited to, the sites disclosed
in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576;
Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson
et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al.,
1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:
505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987;
Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:
240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
[0360] The parent may be obtained from microorganisms of any genus.
For purposes of the present invention, the term "obtained from" as
used herein in connection with a given source shall mean that the
parent encoded by a polynucleotide is produced by the source or by
a strain in which the polynucleotide from the source has been
inserted. In one aspect, the parent is secreted
extracellularly.
[0361] The parent may be a bacterial GH61 polypeptide. For example,
the parent may be a Gram-positive bacterial polypeptide such as a
Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,
Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or
Streptomyces GH61 polypeptide, or a Gram-negative bacterial
polypeptide such as a Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, or Ureaplasma GH61 polypeptide.
[0362] In one aspect, the parent is a Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis GH61 polypeptide.
[0363] In another aspect, the parent is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus GH61 polypeptide.
[0364] In another aspect, the parent is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans GH61
polypeptide.
[0365] The parent may be a fungal GH61 polypeptide. For example,
the parent may be a yeast GH61 polypeptide such as a Candida,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia GH61 polypeptide; or a filamentous fungal GH61 polypeptide
such as an Acremonium, Agaricus, Alternaria, Aspergillus,
Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,
Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,
Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,
Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola,
Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,
Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora,
Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,
Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum,
Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria
GH61 polypeptide.
[0366] In another aspect, the parent is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis GH61
polypeptide.
[0367] In another aspect, the parent is an Acremonium
cellulolyticus, Aspergillus aculeatus, Aspergillus awamori,
Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus,
Aspergillus lentulus, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Aspergillus terreus, Chrysosporium inops,
Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium
queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum,
Fennellia nivea, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium emersonii, Penicillium
funiculosum, Penicillium pinophilum, Penicillium purpurogenum,
Phanerochaete chrysosporium, Talaromyces leycettanus, Thermoascus
aurantiacus, Thielavia achromatica, Thielavia albomyces, Thielavia
albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia
microspora, Thielavia ovispora, Thielavia peruviana, Thielavia
setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia
terrestris, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride GH61 polypeptide.
[0368] It will be understood that for the aforementioned species,
the invention encompasses both the perfect and imperfect states,
and other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
[0369] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0370] The parent may be identified and obtained from other sources
including microorganisms isolated from nature (e.g., soil,
composts, water, etc.) or DNA samples obtained directly from
natural materials (e.g., soil, composts, water, etc.) using the
above-mentioned probes. Techniques for isolating microorganisms and
DNA directly from natural habitats are well known in the art. A
polynucleotide encoding a parent may then be obtained by similarly
screening a genomic DNA or cDNA library of another microorganism or
mixed DNA sample. Once a polynucleotide encoding a parent has been
detected with the probe(s), the polynucleotide can be isolated or
cloned by utilizing techniques that are known to those of ordinary
skill in the art (see, e.g., Sambrook et al., 1989, supra).
Preparation of Variants
[0371] The present invention also relates to methods for obtaining
a GH61 polypeptide variant having cellulolytic enhancing activity,
comprising: (a) introducing into a parent GH61 polypeptide a
substitution at one or more (e.g., several) positions corresponding
to positions 105, 154, 188, 189, 216, and 229 of the mature
polypeptide of SEQ ID NO: 30, wherein the variant has cellulolytic
enhancing activity; and optionally (b) recovering the variant. In
one aspect, the methods further comprise introducing into the
parent GH61 polypeptide a substitution at one or more (e.g.,
several) positions corresponding to positions 111, 152, 155, and
162 of the mature polypeptide of SEQ ID NO: 30, wherein the variant
has cellulolytic enhancing activity. In another aspect, the methods
further or even further comprise introducing into the parent GH61
polypeptide a substitution at one or more (e.g., several) positions
corresponding to positions 96, 98, 200, 202, and 204 of the mature
polypeptide of SEQ ID NO: 30, wherein the variant has cellulolytic
enhancing activity.
[0372] The variants can be prepared using any mutagenesis procedure
known in the art, such as site-directed mutagenesis, synthetic gene
construction, semi-synthetic gene construction, random mutagenesis,
shuffling, etc.
[0373] Site-directed mutagenesis is a technique in which one or
more (e.g., several) mutations are introduced at one or more
defined sites in a polynucleotide encoding the parent.
[0374] Site-directed mutagenesis can be accomplished in vitro by
PCR involving the use of oligonucleotide primers containing the
desired mutation. Site-directed mutagenesis can also be performed
in vitro by cassette mutagenesis involving the cleavage by a
restriction enzyme at a site in the plasmid comprising a
polynucleotide encoding the parent and subsequent ligation of an
oligonucleotide containing the mutation in the polynucleotide.
Usually the restriction enzyme that digests the plasmid and the
oligonucleotide is the same, permitting sticky ends of the plasmid
and the insert to ligate to one another. See, e.g., Scherer and
Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton
et al., 1990, Nucleic Acids Res. 18: 7349-4966.
[0375] Site-directed mutagenesis can also be accomplished in vivo
by methods known in the art. See, e.g., U.S. Patent Application
Publication No. 2004/0171154; Storici et al., 2001, Nature
Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;
and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:
15-16.
[0376] Site-saturation mutagenesis systematically replaces a
polypeptide coding sequence with sequences encoding all 19 amino
acids at one or more (e.g., several) specific positions (Parikh and
Matsumura, 2005, J. Mol. Biol. 352: 621-628).
[0377] Any site-directed mutagenesis procedure can be used in the
present invention. There are many commercial kits available that
can be used to prepare variants.
[0378] Synthetic gene construction entails in vitro synthesis of a
designed polynucleotide molecule to encode a polypeptide of
interest. Gene synthesis can be performed utilizing a number of
techniques, such as the multiplex microchip-based technology
described by Tian et al. (2004, Nature 432: 1050-1054) and similar
technologies wherein oligonucleotides are synthesized and assembled
upon photo-programmable microfluidic chips.
[0379] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0380] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
[0381] Semi-synthetic gene construction is accomplished by
combining aspects of synthetic gene construction, and/or
site-directed mutagenesis, and/or random mutagenesis, and/or
shuffling. Semi-synthetic construction is typified by a process
utilizing polynucleotide fragments that are synthesized, in
combination with PCR techniques. Defined regions of genes may thus
be synthesized de novo, while other regions may be amplified using
site-specific mutagenic primers, while yet other regions may be
subjected to error-prone PCR or non-error prone PCR amplification.
Polynucleotide subsequences may then be shuffled.
Polynucleotides
[0382] The present invention also relates to isolated
polynucleotides encoding GH61 polypeptide variants of the present
invention.
Nucleic Acid Constructs
[0383] The present invention also relates to nucleic acid
constructs comprising a polynucleotide encoding a GH61 polypeptide
variant of the present invention operably linked to one or more
control sequences that direct the expression of the coding sequence
in a suitable host cell under conditions compatible with the
control sequences.
[0384] The polynucleotide may be manipulated in a variety of ways
to provide for expression of a GH61 polypeptide variant.
Manipulation of the polynucleotide prior to its insertion into a
vector may be desirable or necessary depending on the expression
vector. The techniques for modifying polynucleotides utilizing
recombinant DNA methods are well known in the art.
[0385] The control sequence may be a promoter, a polynucleotide
recognized by a host cell for expression of a polynucleotide
encoding a variant of the present invention. The promoter contains
transcriptional control sequences that mediate the expression of
the GH61 polypeptide variant. The promoter may be any
polynucleotide that shows transcriptional activity in the host cell
including mutant, truncated, and hybrid promoters, and may be
obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host
cell.
[0386] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
bacterial host cell are the promoters obtained from the Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
alpha-amylase gene (amyL), Bacillus licheniformis penicillinase
gene (penP), Bacillus stearothermophilus maltogenic amylase gene
(amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus
subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene
(Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.
coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69:
301-315), Streptomyces coelicolor agarase gene (dagA), and
prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc.
Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter
(DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25).
Further promoters are described in "Useful proteins from
recombinant bacteria" in Gilbert et al., 1980, Scientific American
242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem
promoters are disclosed in WO 99/43835.
[0387] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
filamentous fungal host cell are promoters obtained from the genes
for Aspergillus nidulans acetamidase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline
protease, Aspergillus oryzae triose phosphate isomerase, Fusarium
oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum
amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO
00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor
miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma
reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I,
Trichoderma reesei cellobiohydrolase II, Trichoderma reesei
endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma
reesei endoglucanase III, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei xylanase III, Trichoderma reesei
beta-xylosidase, and Trichoderma reesei translation elongation
factor, as well as the NA2-tpi promoter (a modified promoter from
an Aspergillus neutral alpha-amylase gene in which the untranslated
leader has been replaced by an untranslated leader from an
Aspergillus triose phosphate isomerase gene; non-limiting examples
include modified promoters from an Aspergillus niger neutral
alpha-amylase gene in which the untranslated leader has been
replaced by an untranslated leader from an Aspergillus nidulans or
Aspergillus oryzae triose phosphate isomerase gene); and mutant,
truncated, and hybrid promoters thereof. Other promoters are
described in U.S. Pat. No. 6,011,147.
[0388] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0389] The control sequence may also be a transcription terminator,
which is recognized by a host cell to terminate transcription. The
terminator is operably linked to the 3'-terminus of the
polynucleotide encoding the GH61 polypeptide variant. Any
terminator that is functional in the host cell may be used.
[0390] Preferred terminators for bacterial host cells are obtained
from the genes for Bacillus clausii alkaline protease (aprH),
Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli
ribosomal RNA (rrnB).
[0391] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans acetamidase,
Aspergillus nidulans anthranilate synthase, Aspergillus niger
glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus
oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease,
Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma
reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma
reesei beta-xylosidase, and Trichoderma reesei translation
elongation factor.
[0392] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0393] The control sequence may also be an mRNA stabilizer region
downstream of a promoter and upstream of the coding sequence of a
gene which increases expression of the gene.
[0394] Examples of suitable mRNA stabilizer regions are obtained
from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a
Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of
Bacteriology 177: 3465-3471).
[0395] The control sequence may also be a leader, a nontranslated
region of an mRNA that is important for translation by the host
cell. The leader is operably linked to the 5'-terminus of the
polynucleotide encoding the GH61 polypeptide variant. Any leader
that is functional in the host cell may be used.
[0396] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0397] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0398] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3'-terminus of the GH61
polypeptide variant-encoding sequence and, when transcribed, is
recognized by the host cell as a signal to add polyadenosine
residues to transcribed mRNA. Any polyadenylation sequence that is
functional in the host cell may be used.
[0399] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus nidulans
anthranilate synthase, Aspergillus nigerglucoamylase, Aspergillus
niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and
Fusarium oxysporum trypsin-like protease.
[0400] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0401] The control sequence may also be a signal peptide coding
region that encodes a signal peptide linked to the N-terminus of a
GH61 polypeptide variant and directs the variant into the cell's
secretory pathway. The 5'-end of the coding sequence of the
polynucleotide may inherently contain a signal peptide coding
sequence naturally linked in translation reading frame with the
segment of the coding sequence that encodes the variant.
Alternatively, the 5'-end of the coding sequence may contain a
signal peptide coding sequence that is foreign to the coding
sequence. A foreign signal peptide coding sequence may be required
where the coding sequence does not naturally contain a signal
peptide coding sequence. Alternatively, a foreign signal peptide
coding sequence may simply replace the natural signal peptide
coding sequence in order to enhance secretion of the variant.
However, any signal peptide coding sequence that directs the
expressed variant into the secretory pathway of a host cell may be
used.
[0402] Effective signal peptide coding sequences for bacterial host
cells are the signal peptide coding sequences obtained from the
genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus
licheniformis subtilisin, Bacillus licheniformis beta-lactamase,
Bacillus stearothermophilus alpha-amylase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0403] Effective signal peptide coding sequences for filamentous
fungal host cells are the signal peptide coding sequences obtained
from the genes for Aspergillus niger neutral amylase, Aspergillus
niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola
insolens cellulase, Humicola insolens endoglucanase V, Humicola
lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
[0404] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding sequences are described by Romanos et al., 1992, supra.
[0405] The control sequence may also be a propeptide coding
sequence that encodes a propeptide positioned at the N-terminus of
a GH61 polypeptide variant. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to an
active polypeptide by catalytic or autocatalytic cleavage of the
propeptide from the propolypeptide. The propeptide coding sequence
may be obtained from the genes for Bacillus subtilis alkaline
protease (aprE), Bacillus subtilis neutral protease (nprT),
Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei
aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
[0406] Where both signal peptide and propeptide sequences are
present, the propeptide sequence is positioned next to the
N-terminus of the GH61 polypeptide variant and the signal peptide
sequence is positioned next to the N-terminus of the propeptide
sequence.
[0407] It may also be desirable to add regulatory sequences that
regulate expression of the GH61 polypeptide variant relative to the
growth of the host cell. Examples of regulatory sequences are those
that cause expression of the gene to be turned on or off in
response to a chemical or physical stimulus, including the presence
of a regulatory compound. Regulatory systems in prokaryotic systems
include the lac, tac, and trp operator systems. In yeast, the ADH2
system or GAL1 system may be used. In filamentous fungi, the
Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA
alpha-amylase promoter, and Aspergillus oryzae glucoamylase
promoter, Trichoderma reesei cellobiohydrolase I promoter, and
Trichoderma reesei cellobiohydrolase II promoter may be used. Other
examples of regulatory sequences are those that allow for gene
amplification. In eukaryotic systems, these regulatory sequences
include the dihydrofolate reductase gene that is amplified in the
presence of methotrexate, and the metallothionein genes that are
amplified with heavy metals. In these cases, the polynucleotide
encoding the variant would be operably linked to the regulatory
sequence.
Expression Vectors
[0408] The present invention also relates to recombinant expression
vectors comprising a polynucleotide encoding a GH61 polypeptide
variant of the present invention, a promoter, and transcriptional
and translational stop signals. The various nucleotide and control
sequences may be joined together to produce a recombinant
expression vector that may include one or more convenient
restriction sites to allow for insertion or substitution of the
polynucleotide encoding the variant at such sites. Alternatively,
the polynucleotide may be expressed by inserting the polynucleotide
or a nucleic acid construct comprising the polynucleotide into an
appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the
coding sequence is operably linked with the appropriate control
sequences for expression.
[0409] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) that can be conveniently subjected to recombinant
DNA procedures and can bring about expression of the
polynucleotide. The choice of the vector will typically depend on
the compatibility of the vector with the host cell into which the
vector is to be introduced. The vector may be a linear or closed
circular plasmid.
[0410] The vector may be an autonomously replicating vector, i.e.,
a vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a
plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
that, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids that together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
[0411] The vector preferably contains one or more selectable
markers that permit easy selection of transformed, transfected,
transduced, or the like cells. A selectable marker is a gene the
product of which provides for biocide or viral resistance,
resistance to heavy metals, prototrophy to auxotrophs, and the
like.
[0412] Examples of bacterial selectable markers are Bacillus
licheniformis or Bacillus subtilis dal genes, or markers that
confer antibiotic resistance such as ampicillin, chloramphenicol,
kanamycin, neomycin, spectinomycin, or tetracycline resistance.
Suitable markers for yeast host cells include, but are not limited
to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable
markers for use in a filamentous fungal host cell include, but are
not limited to, adeA (phosphoribosylaminoim idazole-succinocarboxam
ide synthase), adeB (phosphoribosyl-aminoimidazole synthase), amdS
(acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hph (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), and trpC (anthranilate synthase), as well as
equivalents thereof. Preferred for use in an Aspergillus cell are
Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and
a Streptomyces hygroscopicus bar gene. Preferred for use in a
Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.
[0413] The selectable marker may be a dual selectable marker system
as described in WO 2010/039889. In one aspect, the dual selectable
marker is a hph-tk dual selectable marker system.
[0414] The vector preferably contains an element(s) that permits
integration of the vector into the host cell's genome or autonomous
replication of the vector in the cell independent of the
genome.
[0415] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the GH61 polypeptide
variant or any other element of the vector for integration into the
genome by homologous or non-homologous recombination.
Alternatively, the vector may contain additional polynucleotides
for directing integration by homologous recombination into the
genome of the host cell at a precise location(s) in the
chromosome(s). To increase the likelihood of integration at a
precise location, the integrational elements should contain a
sufficient number of nucleic acids, such as 100 to 10,000 base
pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs,
which have a high degree of sequence identity to the corresponding
target sequence to enhance the probability of homologous
recombination. The integrational elements may be any sequence that
is homologous with the target sequence in the genome of the host
cell. Furthermore, the integrational elements may be non-encoding
or encoding polynucleotides. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0416] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication that functions in a cell. The term "origin of
replication" or "plasmid replicator" means a polynucleotide that
enables a plasmid or vector to replicate in vivo.
[0417] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAM.beta.1 permitting replication in Bacillus.
[0418] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and
CEN6.
[0419] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67;
Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0420] More than one copy of a polynucleotide of the present
invention may be inserted into a host cell to increase production
of a GH61 polypeptide variant. An increase in the copy number of
the polynucleotide can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the
polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0421] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0422] The present invention also relates to recombinant host
cells, comprising a polynucleotide encoding a GH61 polypeptide
variant of the present invention operably linked to one or more
control sequences that direct the production of a variant of the
present invention. A construct or vector comprising a
polynucleotide is introduced into a host cell so that the construct
or vector is maintained as a chromosomal integrant or as a
self-replicating extra-chromosomal vector as described earlier. The
term "host cell" encompasses any progeny of a parent cell that is
not identical to the parent cell due to mutations that occur during
replication. The choice of a host cell will to a large extent
depend upon the gene encoding the variant and its source.
[0423] The host cell may be any cell useful in the recombinant
production of a GH61 polypeptide variant, e.g., a prokaryote or a
eukaryote.
[0424] The prokaryotic host cell may be any Gram-positive or
Gram-negative bacterium. Gram-positive bacteria include, but are
not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, and Streptomyces. Gram-negative bacteria include,
but are not limited to, Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, and Ureaplasma.
[0425] The bacterial host cell may be any Bacillus cell including,
but not limited to, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,
Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis,
and Bacillus thuringiensis cells.
[0426] The bacterial host cell may also be any Streptococcus cell
including, but not limited to, Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, and Streptococcus
equi subsp. Zooepidemicus cells.
[0427] The bacterial host cell may also be any Streptomyces cell,
including, but not limited to, Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, and Streptomyces lividans cells.
[0428] The introduction of DNA into a Bacillus cell may be effected
by protoplast transformation (see, e.g., Chang and Cohen, 1979,
Mol. Gen. Genet. 168: 111-115), competent cell transformation (see,
e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or
Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221),
electroporation (see, e.g., Shigekawa and Dower, 1988,
Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and
Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of
DNA into an E. coli cell may be effected by protoplast
transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:
557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic
Acids Res. 16: 6127-6145). The introduction of DNA into a
Streptomyces cell may be effected by protoplast transformation,
electroporation (see, e.g., Gong et al., 2004, Folia Microbiol.
(Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al.,
1989, J. Bacteriol. 171: 3583-3585), or transduction (see, e.g.,
Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The
introduction of DNA into a Pseudomonas cell may be effected by
electroporation (see, e.g., Choi et al., 2006, J. Microbiol.
Methods 64: 391-397), or conjugation (see, e.g., Pinedo and Smets,
2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA
into a Streptococcus cell may be effected by natural competence
(see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:
1295-1297), protoplast transformation (see, e.g., Catt and Jollick,
1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley
et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or
conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:
409-436). However, any method known in the art for introducing DNA
into a host cell can be used.
[0429] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0430] The host cell may be a fungal cell. "Fungi" as used herein
includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and
Zygomycota as well as the Oomycota and all mitosporic fungi (as
defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary
of The Fungi, 8th edition, 1995, CAB International, University
Press, Cambridge, UK).
[0431] The fungal host cell may be a yeast cell. "Yeast" as used
herein includes ascosporogenous yeast (Endomycetales),
basidiosporogenous yeast, and yeast belonging to the Fungi
Imperfecti (Blastomycetes). Since the classification of yeast may
change in the future, for the purposes of this invention, yeast
shall be defined as described in Biology and Activities of Yeast
(Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol.
Symposium Series No. 9, 1980).
[0432] The yeast host cell may be a Candida, Hansenula,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia
lipolytica cell.
[0433] The fungal host cell may be a filamentous fungal cell.
"Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
1995, supra). The filamentous fungi are generally characterized by
a mycelial wall composed of chitin, cellulose, glucan, chitosan,
mannan, and other complex polysaccharides. Vegetative growth is by
hyphal elongation and carbon catabolism is obligately aerobic. In
contrast, vegetative growth by yeasts such as Saccharomyces
cerevisiae is by budding of a unicellular thallus and carbon
catabolism may be fermentative.
[0434] The filamentous fungal host cell may be an Acremonium,
Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Phiebia, Piromyces, Pleurotus, Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or
Trichoderma cell.
[0435] For example, the filamentous fungal host cell may be an
Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,
Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium
keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,
Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus,
Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor
miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phiebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0436] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238023, Yelton et al., 1984, Proc. Natl.
Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988,
Bio/Technology 6: 1419-1422. Suitable methods for transforming
Fusarium species are described by Malardier et al., 1989, Gene 78:
147-156, and WO 96/00787. Yeast may be transformed using the
procedures described by Becker and Guarente, In Abelson, J. N. and
Simon, M. I., editors, Guide to Yeast Genetics and Molecular
Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic
Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163;
and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
Methods of Production
[0437] The present invention also relates to methods of producing a
GH61 polypeptide variant, comprising: (a) cultivating a host cell
of the present invention under conditions suitable for expression
of the variant; and optionally (b) recovering the variant.
[0438] The host cells are cultivated in a nutrient medium suitable
for production of the GH61 polypeptide variant using methods known
in the art. For example, the cells may be cultivated by shake flask
cultivation, or small-scale or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in
laboratory or industrial fermentors in a suitable medium and under
conditions allowing the variant to be expressed and/or isolated.
The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the variant is secreted into the nutrient medium,
the variant can be recovered directly from the medium. If the
variant is not secreted, it can be recovered from cell lysates.
[0439] The GH61 polypeptide variant may be detected using methods
known in the art that are specific for the variant. These detection
methods include, but are not limited to, use of specific
antibodies, formation of an enzyme product, or disappearance of an
enzyme substrate. For example, an enzyme assay may be used to
determine the activity of the variant. See, for example, the assay
described in Example 5.
[0440] The GH61 polypeptide variant may be recovered using methods
known in the art. For example, the variant may be recovered from
the nutrient medium by conventional procedures including, but not
limited to, collection, centrifugation, filtration, extraction,
spray-drying, evaporation, or precipitation. In one aspect, a whole
fermentation broth comprising a variant of the present invention is
recovered.
[0441] The GH61 polypeptide variant may be purified by a variety of
procedures known in the art including, but not limited to,
chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction
(see, e.g., Protein Purification, Janson and Ryden, editors, VCH
Publishers, New York, 1989) to obtain substantially pure
variants.
[0442] In an alternative aspect, the GH61 polypeptide variant is
not recovered, but rather a host cell of the present invention
expressing the variant is used as a source of the variant.
Fermentation Broth Formulations or Cell Compositions
[0443] The present invention also relates to a fermentation broth
formulation or a cell composition comprising a variant of the
present invention. The fermentation broth product further comprises
additional ingredients used in the fermentation process, such as,
for example, cells (including, the host cells containing the gene
encoding the polypeptide of the present invention which are used to
produce the polypeptide of interest), cell debris, biomass,
fermentation media and/or fermentation products. In some
embodiments, the composition is a cell-killed whole broth
containing organic acid(s), killed cells and/or cell debris, and
culture medium.
[0444] The term "fermentation broth" as used herein refers to a
preparation produced by cellular fermentation that undergoes no or
minimal recovery and/or purification. For example, fermentation
broths are produced when microbial cultures are grown to
saturation, incubated under carbon-limiting conditions to allow
protein synthesis (e.g., expression of enzymes by host cells) and
secretion into cell culture medium. The fermentation broth can
contain unfractionated or fractionated contents of the fermentation
materials derived at the end of the fermentation. Typically, the
fermentation broth is unfractionated and comprises the spent
culture medium and cell debris present after the microbial cells
(e.g., filamentous fungal cells) are removed, e.g., by
centrifugation. In some embodiments, the fermentation broth
contains spent cell culture medium, extracellular enzymes, and
viable and/or nonviable microbial cells.
[0445] In an embodiment, the fermentation broth formulation and
cell compositions comprise a first organic acid component
comprising at least one 1-5 carbon organic acid and/or a salt
thereof and a second organic acid component comprising at least one
6 or more carbon organic acid and/or a salt thereof. In a specific
embodiment, the first organic acid component is acetic acid, formic
acid, propionic acid, a salt thereof, or a mixture of two or more
of the foregoing and the second organic acid component is benzoic
acid, cyclohexanecarboxylic acid, 4-methylvaleric acid,
phenylacetic acid, a salt thereof, or a mixture of two or more of
the foregoing.
[0446] In one aspect, the composition contains an organic acid(s),
and optionally further contains killed cells and/or cell debris. In
one embodiment, the killed cells and/or cell debris are removed
from a cell-killed whole broth to provide a composition that is
free of these components.
[0447] The fermentation broth formulations or cell compositions may
further comprise a preservative and/or anti-microbial (e.g.,
bacteriostatic) agent, including, but not limited to, sorbitol,
sodium chloride, potassium sorbate, and others known in the
art.
[0448] The fermentation broth formulations or cell compositions may
further comprise multiple enzymatic activities, such as one or more
(e.g., several) enzymes selected from the group consisting of a
cellulase, a hemicellulase, an esterase, an expansin, a laccase, a
ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a
swollenin. The fermentation broth formulations or cell compositions
may also comprise one or more (e.g., several) enzymes selected from
the group consisting of a hydrolase, an isomerase, a ligase, a
lyase, an oxidoreductase, or a transferase, e.g., an
alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,
beta-galactosidase, beta-glucosidase, beta-xylosidase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, glucoamylase,
invertase, laccase, lipase, mannosidase, mutanase, oxidase,
pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or
xylanase.
[0449] The cell-killed whole broth or composition may contain the
unfractionated contents of the fermentation materials derived at
the end of the fermentation. Typically, the cell-killed whole broth
or composition contains the spent culture medium and cell debris
present after the microbial cells (e.g., filamentous fungal cells)
are grown to saturation, incubated under carbon-limiting conditions
to allow protein synthesis (e.g., expression of cellulase and/or
glucosidase enzyme(s)). In some embodiments, the cell-killed whole
broth or composition contains the spent cell culture medium,
extracellular enzymes, and killed filamentous fungal cells. In some
embodiments, the microbial cells present in the cell-killed whole
broth or composition can be permeabilized and/or lysed using
methods known in the art.
[0450] A whole broth or cell composition as described herein is
typically a liquid, but may contain insoluble components, such as
killed cells, cell debris, culture media components, and/or
insoluble enzyme(s). In some embodiments, insoluble components may
be removed to provide a clarified liquid composition.
[0451] The whole broth formulations and cell compositions of the
present invention may be produced by a method described in WO
90/15861 or WO 2010/096673.
[0452] Examples are given below of preferred uses of the
compositions of the present invention. The dosage of the
composition and other conditions under which the composition is
used may be determined on the basis of methods known in the
art.
Enzyme Compositions
[0453] The present invention also relates to compositions
comprising a variant of the present invention. Preferably, the
compositions are enriched in such a variant. The term "enriched"
indicates that the cellulolytic enhancing activity of the
composition has been increased, e.g., with an enrichment factor of
at least 1.1.
[0454] The compositions may comprise a variant of the present
invention as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the compositions may comprise multiple
enzymatic activities, such as one or more (e.g., several) enzymes
selected from the group consisting of a cellulase, a hemicellulase,
an esterase, an expansin, a laccase, a ligninolytic enzyme, a
pectinase, a peroxidase, a protease, and a swollenin. The
compositions may also comprise one or more (e.g., several) enzymes
selected from the group consisting of a hydrolase, an isomerase, a
ligase, a lyase, an oxidoreductase, or a transferase, e.g., an
alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,
beta-galactosidase, beta-glucosidase, beta-xylosidase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, glucoamylase,
invertase, laccase, lipase, mannosidase, mutanase, oxidase,
pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The compositions may be prepared in accordance with methods known
in the art and may be in the form of a liquid or a dry composition.
The compositions may be stabilized in accordance with methods known
in the art.
[0455] Examples are given below of preferred uses of the
compositions of the present invention. The dosage of the
composition and other conditions under which the composition is
used may be determined on the basis of methods known in the
art.
Uses
[0456] The present invention is also directed to the following
processes for using the GH61 polypeptide variants having
cellulolytic enhancing activity, or compositions thereof.
[0457] The present invention also relates to processes for
degrading or converting a cellulosic material, comprising: treating
the cellulosic material with an enzyme composition in the presence
of a GH61 polypeptide variant of the present invention. In one
aspect, the processes further comprise recovering the degraded or
converted cellulosic material. Soluble products of degradation or
conversion of the cellulosic material can be separated from
insoluble cellulosic material using a method known in the art such
as, for example, centrifugation, filtration, or gravity
settling.
[0458] The present invention also relates to processes of producing
a fermentation product, comprising: (a) saccharifying a cellulosic
material with an enzyme composition in the presence of a GH61
polypeptide variant of the present invention; (b) fermenting the
saccharified cellulosic material with one or more (e.g., several)
fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
[0459] The present invention also relates to processes of
fermenting a cellulosic material, comprising: fermenting the
cellulosic material with one or more (e.g., several) fermenting
microorganisms, wherein the cellulosic material is saccharified
with an enzyme composition in the presence of a GH61 polypeptide
variant of the present invention. In one aspect, the fermenting of
the cellulosic material produces a fermentation product. In another
aspect, the processes further comprise recovering the fermentation
product from the fermentation.
[0460] The processes of the present invention can be used to
saccharify the cellulosic material to fermentable sugars and to
convert the fermentable sugars to many useful fermentation
products, e.g., fuel, potable ethanol, and/or platform chemicals
(e.g., acids, alcohols, ketones, gases, and the like). The
production of a desired fermentation product from the cellulosic
material typically involves pretreatment, enzymatic hydrolysis
(saccharification), and fermentation.
[0461] The processing of the cellulosic material according to the
present invention can be accomplished using methods conventional in
the art. Moreover, the processes of the present invention can be
implemented using any conventional biomass processing apparatus
configured to operate in accordance with the invention.
[0462] Hydrolysis (saccharification) and fermentation, separate or
simultaneous, include, but are not limited to, separate hydrolysis
and fermentation (SHF); simultaneous saccharification and
fermentation (SSF); simultaneous saccharification and
co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF);
separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis
and co-fermentation (HHCF); and direct microbial conversion (DMC),
also sometimes called consolidated bioprocessing (CBP). SHF uses
separate process steps to first enzymatically hydrolyze the
cellulosic material to fermentable sugars, e.g., glucose,
cellobiose, and pentose monomers, and then ferment the fermentable
sugars to ethanol. In SSF, the enzymatic hydrolysis of the
cellulosic material and the fermentation of sugars to ethanol are
combined in one step (Philippidis, G. P., 1996, Cellulose
bioconversion technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
D.C., 179-212). SSCF involves the co-fermentation of multiple
sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the
environment: A strategic perspective on the U.S. Department of
Energy's research and development activities for bioethanol,
Biotechnol. Prog. 15: 817-827). HHF involves a separate hydrolysis
step, and in addition a simultaneous saccharification and
hydrolysis step, which can be carried out in the same reactor. The
steps in an HHF process can be carried out at different
temperatures, i.e., high temperature enzymatic saccharification
followed by SSF at a lower temperature that the fermentation strain
can tolerate. DMC combines all three processes (enzyme production,
hydrolysis, and fermentation) in one or more (e.g., several) steps
where the same organism is used to produce the enzymes for
conversion of the cellulosic material to fermentable sugars and to
convert the fermentable sugars into a final product (Lynd, L. R.,
Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002,
Microbial cellulose utilization: Fundamentals and biotechnology,
Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein
that any method known in the art comprising pretreatment, enzymatic
hydrolysis (saccharification), fermentation, or a combination
thereof, can be used in the practicing the processes of the present
invention.
[0463] A conventional apparatus can include a fed-batch stirred
reactor, a batch stirred reactor, a continuous flow stirred reactor
with ultrafiltration, and/or a continuous plug-flow column reactor
(Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella
Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch
reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology
25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of
the enzymatic hydrolysis of cellulose: 1. A mathematical model for
a batch reactor process, Enz. Microb. Technol. 7: 346-352), an
attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion
of waste cellulose by using an attrition bioreactor, Biotechnol.
Bioeng. 25: 53-65), or a reactor with intensive stirring induced by
an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P.,
Davydkin, I. Y., Davydkin, V. Y., Protas, 0. V., 1996, Enhancement
of enzymatic cellulose hydrolysis using a novel type of bioreactor
with intensive stirring induced by electromagnetic field, Appl.
Biochem. Biotechnol. 56: 141-153). Additional reactor types include
fluidized bed, upflow blanket, immobilized, and extruder type
reactors for hydrolysis and/or fermentation.
[0464] Pretreatment.
[0465] In practicing the processes of the present invention, any
pretreatment process known in the art can be used to disrupt plant
cell wall components of the cellulosic material (Chandra et al.,
2007, Substrate pretreatment: The key to effective enzymatic
hydrolysis of lignocellulosics?, Adv. Biochem. Engin./Biotechnol.
108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic
materials for efficient bioethanol production, Adv. Biochem.
Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,
Pretreatments to enhance the digestibility of lignocellulosic
biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005,
Features of promising technologies for pretreatment of
lignocellulosic biomass, Bioresource Technol. 96: 673-686;
Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes
to improve ethanol and biogas production: A review, Int. J. of Mol.
Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to
unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and
Biorefining-Biofpr. 2: 26-40).
[0466] The cellulosic material can also be subjected to particle
size reduction, sieving, pre-soaking, wetting, washing, and/or
conditioning prior to pretreatment using methods known in the
art.
[0467] Conventional pretreatments include, but are not limited to,
steam pretreatment (with or without explosion), dilute acid
pretreatment, hot water pretreatment, alkaline pretreatment, lime
pretreatment, wet oxidation, wet explosion, ammonia fiber
explosion, organosolv pretreatment, and biological pretreatment.
Additional pretreatments include ammonia percolation, ultrasound,
electroporation, microwave, supercritical CO.sub.2, supercritical
H.sub.2O, ozone, ionic liquid, and gamma irradiation
pretreatments.
[0468] The cellulosic material can be pretreated before hydrolysis
and/or fermentation. Pretreatment is preferably performed prior to
the hydrolysis. Alternatively, the pretreatment can be carried out
simultaneously with enzyme hydrolysis to release fermentable
sugars, such as glucose, xylose, and/or cellobiose. In most cases
the pretreatment step itself results in some conversion of biomass
to fermentable sugars (even in absence of enzymes).
[0469] Steam Pretreatment. In steam pretreatment, the cellulosic
material is heated to disrupt the plant cell wall components,
including lignin, hemicellulose, and cellulose to make the
cellulose and other fractions, e.g., hemicellulose, accessible to
enzymes. The cellulosic material is passed to or through a reaction
vessel where steam is injected to increase the temperature to the
required temperature and pressure and is retained therein for the
desired reaction time. Steam pretreatment is preferably performed
at 140-250.degree. C., e.g., 160-200.degree. C. or 170-190.degree.
C., where the optimal temperature range depends on addition of a
chemical catalyst. Residence time for the steam pretreatment is
preferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12
minutes, or 4-10 minutes, where the optimal residence time depends
on temperature range and addition of a chemical catalyst. Steam
pretreatment allows for relatively high solids loadings, so that
the cellulosic material is generally only moist during the
pretreatment. The steam pretreatment is often combined with an
explosive discharge of the material after the pretreatment, which
is known as steam explosion, that is, rapid flashing to atmospheric
pressure and turbulent flow of the material to increase the
accessible surface area by fragmentation (Duff and Murray, 1996,
Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.
Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.
20020164730). During steam pretreatment, hemicellulose acetyl
groups are cleaved and the resulting acid autocatalyzes partial
hydrolysis of the hemicellulose to monosaccharides and
oligosaccharides. Lignin is removed to only a limited extent.
[0470] Chemical Pretreatment: The term "chemical treatment" refers
to any chemical pretreatment that promotes the separation and/or
release of cellulose, hemicellulose, and/or lignin. Such a
pretreatment can convert crystalline cellulose to amorphous
cellulose. Examples of suitable chemical pretreatment processes
include, for example, dilute acid pretreatment, lime pretreatment,
wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia
percolation (APR), ionic liquid, and organosolv pretreatments.
[0471] A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically
0.3 to 5% w/w) is often added prior to steam pretreatment, which
decreases the time and temperature, increases the recovery, and
improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl.
Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl.
Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme
Microb. Technol. 39: 756-762). In dilute acid pretreatment, the
cellulosic material is mixed with dilute acid, typically
H.sub.2SO.sub.4, and water to form a slurry, heated by steam to the
desired temperature, and after a residence time flashed to
atmospheric pressure. The dilute acid pretreatment can be performed
with a number of reactor designs, e.g., plug-flow reactors,
counter-current reactors, or continuous counter-current shrinking
bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004,
Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv. Biochem.
Eng. Biotechnol. 65: 93-115).
[0472] Several methods of pretreatment under alkaline conditions
can also be used. These alkaline pretreatments include, but are not
limited to, sodium hydroxide, lime, wet oxidation, ammonia
percolation (APR), and ammonia fiber/freeze explosion (AFEX).
[0473] Lime pretreatment is performed with calcium oxide or calcium
hydroxide at temperatures of 85-150.degree. C. and residence times
from 1 hour to several days (Wyman et al., 2005, Bioresource
Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol.
96: 673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900, and
WO 2006/110901 disclose pretreatment methods using ammonia.
[0474] Wet oxidation is a thermal pretreatment performed typically
at 180-200.degree. C. for 5-15 minutes with addition of an
oxidative agent such as hydrogen peroxide or over-pressure of
oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64:
139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117:
1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin
et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The
pretreatment is performed preferably at 1-40% dry matter, e.g.,
2-30% dry matter or 5-20% dry matter, and often the initial pH is
increased by the addition of alkali such as sodium carbonate.
[0475] A modification of the wet oxidation pretreatment method,
known as wet explosion (combination of wet oxidation and steam
explosion) can handle dry matter up to 30%. In wet explosion, the
oxidizing agent is introduced during pretreatment after a certain
residence time. The pretreatment is then ended by flashing to
atmospheric pressure (WO 2006/032282).
[0476] Ammonia fiber explosion (AFEX) involves treating the
cellulosic material with liquid or gaseous ammonia at moderate
temperatures such as 90-150.degree. C. and high pressure such as
17-20 bar for 5-10 minutes, where the dry matter content can be as
high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol.
98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231;
Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141;
Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018). During
AFEX pretreatment cellulose and hemicelluloses remain relatively
intact. Lignin-carbohydrate complexes are cleaved.
[0477] Organosolv pretreatment delignifies the cellulosic material
by extraction using aqueous ethanol (40-60% ethanol) at
160-200.degree. C. for 30-60 minutes (Pan et al., 2005, Biotechnol.
Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94:
851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121:
219-230). Sulphuric acid is usually added as a catalyst. In
organosolv pretreatment, the majority of hemicellulose and lignin
is removed.
[0478] Other examples of suitable pretreatment methods are
described by Schell et al., 2003, Appl. Biochem. and Biotechnol.
Vol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource
Technology 96: 673-686, and U.S. Published Application
2002/0164730.
[0479] In one aspect, the chemical pretreatment is preferably
carried out as a dilute acid treatment, and more preferably as a
continuous dilute acid treatment. The acid is typically sulfuric
acid, but other acids can also be used, such as acetic acid, citric
acid, nitric acid, phosphoric acid, tartaric acid, succinic acid,
hydrogen chloride, or mixtures thereof. Mild acid treatment is
conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5. In
one aspect, the acid concentration is in the range from preferably
0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or 0.1 to 2 wt %
acid. The acid is contacted with the cellulosic material and held
at a temperature in the range of preferably 140-200.degree. C.,
e.g., 165-190.degree. C., for periods ranging from 1 to 60
minutes.
[0480] In another aspect, pretreatment takes place in an aqueous
slurry. In preferred aspects, the cellulosic material is present
during pretreatment in amounts preferably between 10-80 wt %, e.g.,
20-70 wt % or 30-60 wt %, such as around 40 wt %. The pretreated
cellulosic material can be unwashed or washed using any method
known in the art, e.g., washed with water.
[0481] Mechanical Pretreatment or Physical Pretreatment: The term
"mechanical pretreatment" or "physical pretreatment" refers to any
pretreatment that promotes size reduction of particles. For
example, such pretreatment can involve various types of grinding or
milling (e.g., dry milling, wet milling, or vibratory ball
milling).
[0482] The cellulosic material can be pretreated both physically
(mechanically) and chemically. Mechanical or physical pretreatment
can be coupled with steaming/steam explosion, hydrothermolysis,
dilute or mild acid treatment, high temperature, high pressure
treatment, irradiation (e.g., microwave irradiation), or
combinations thereof. In one aspect, high pressure means pressure
in the range of preferably about 100 to about 400 psi, e.g., about
150 to about 250 psi. In another aspect, high temperature means
temperatures in the range of about 100 to about 300.degree. C.,
e.g., about 140 to about 200.degree. C. In a preferred aspect,
mechanical or physical pretreatment is performed in a batch-process
using a steam gun hydrolyzer system that uses high pressure and
high temperature as defined above, e.g., a Sunds Hydrolyzer
available from Sunds Defibrator AB, Sweden. The physical and
chemical pretreatments can be carried out sequentially or
simultaneously, as desired.
[0483] Accordingly, in a preferred aspect, the cellulosic material
is subjected to physical (mechanical) or chemical pretreatment, or
any combination thereof, to promote the separation and/or release
of cellulose, hemicellulose, and/or lignin.
[0484] Biological Pretreatment: The term "biological pretreatment"
refers to any biological pretreatment that promotes the separation
and/or release of cellulose, hemicellulose, and/or lignin from the
cellulosic material. Biological pretreatment techniques can involve
applying lignin-solubilizing microorganisms and/or enzymes (see,
for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook
on Bioethanol: Production and Utilization, Wyman, C. E., ed.,
Taylor & Francis, Washington, D.C., 179-212; Ghosh and Singh,
1993, Physicochemical and biological treatments for
enzymatic/microbial conversion of cellulosic biomass, Adv. Appl.
Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating
lignocellulosic biomass: a review, in Enzymatic Conversion of
Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and
Overend, R. P., eds., ACS Symposium Series 566, American Chemical
Society, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du,
J., and Tsao, G. T., 1999, Ethanol production from renewable
resources, in Advances in Biochemical Engineering/Biotechnology,
Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65:
207-241; Olsson and Hahn-Hagerdal, 1996, Fermentation of
lignocellulosic hydrolysates for ethanol production, Enz. Microb.
Tech. 18: 312-331; and Vallander and Eriksson, 1990, Production of
ethanol from lignocellulosic materials: State of the art, Adv.
Biochem. Eng./Biotechnol. 42: 63-95).
[0485] Saccharification.
[0486] In the hydrolysis step, also known as saccharification, the
cellulosic material, e.g., pretreated, is hydrolyzed to break down
cellulose and/or hemicellulose to fermentable sugars, such as
glucose, cellobiose, xylose, xylulose, arabinose, mannose,
galactose, and/or soluble oligosaccharides. The hydrolysis is
performed enzymatically by an enzyme composition in the presence of
a GH61 polypeptide variant of the present invention. The enzymes of
the compositions can be added simultaneously or sequentially.
[0487] Enzymatic hydrolysis is preferably carried out in a suitable
aqueous environment under conditions that can be readily determined
by one skilled in the art. In one aspect, hydrolysis is performed
under conditions suitable for the activity of the enzyme(s), i.e.,
optimal for the enzyme(s). The hydrolysis can be carried out as a
fed batch or continuous process where the cellulosic material is
fed gradually to, for example, an enzyme containing hydrolysis
solution.
[0488] The saccharification is generally performed in stirred-tank
reactors or fermentors under controlled pH, temperature, and mixing
conditions. Suitable process time, temperature and pH conditions
can readily be determined by one skilled in the art. For example,
the saccharification can last up to 200 hours, but is typically
performed for preferably about 12 to about 120 hours, e.g., about
16 to about 72 hours or about 24 to about 48 hours. The temperature
is in the range of preferably about 25.degree. C. to about
80.degree. C., e.g., about 30.degree. C. to about 65.degree. C.,
about 40.degree. C. to about 60.degree. C., or about 50.degree. C.
to about 55.degree. C. The pH is in the range of preferably about 3
to about 9, e.g., about 3.5 to about 7, about 4 to about 6, or
about 5.0 to about 5.5. The dry solids content is in the range of
preferably about 5 to about 50 wt %, e.g., about 10 to about 40 wt
% or about 20 to about 30 wt %.
[0489] The enzyme compositions can comprise any protein useful in
degrading the cellulosic material.
[0490] In one aspect, the enzyme composition comprises or further
comprises one or more (e.g., several) proteins selected from the
group consisting of a cellulase, a polypeptide having cellulolytic
enhancing activity, a hemicellulase, an esterase, an expansin, a
laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a
protease, and a swollenin. In another aspect, the cellulase is
preferably one or more (e.g., several) enzymes selected from the
group consisting of an endoglucanase, a cellobiohydrolase, and a
beta-glucosidase. In another aspect, the hemicellulase is
preferably one or more (e.g., several) enzymes selected from the
group consisting of an acetylmannan esterase, an acetylxylan
esterase, an arabinanase, an arabinofuranosidase, a coumaric acid
esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a
glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a
xylosidase.
[0491] In another aspect, the enzyme composition comprises one or
more (e.g., several) cellulolytic enzymes. In another aspect, the
enzyme composition comprises or further comprises one or more
(e.g., several) hemicellulolytic enzymes. In another aspect, the
enzyme composition comprises one or more (e.g., several)
cellulolytic enzymes and one or more (e.g., several)
hemicellulolytic enzymes. In another aspect, the enzyme composition
comprises one or more (e.g., several) enzymes selected from the
group of cellulolytic enzymes and hemicellulolytic enzymes. In
another aspect, the enzyme composition comprises an endoglucanase.
In another aspect, the enzyme composition comprises a
cellobiohydrolase. In another aspect, the enzyme composition
comprises a beta-glucosidase. In another aspect, the enzyme
composition comprises a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises an
endoglucanase and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises a
cellobiohydrolase and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises a
beta-glucosidase and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises an
endoglucanase and a cellobiohydrolase. In another aspect, the
enzyme composition comprises an endoglucanase and a
beta-glucosidase. In another aspect, the enzyme composition
comprises a cellobiohydrolase and a beta-glucosidase. In another
aspect, the enzyme composition comprises an endoglucanase, a
cellobiohydrolase, and a polypeptide having cellulolytic enhancing
activity. In another aspect, the enzyme composition comprises an
endoglucanase, a beta-glucosidase, and a polypeptide having
cellulolytic enhancing activity. In another aspect, the enzyme
composition comprises a cellobiohydrolase, a beta-glucosidase, and
a polypeptide having cellulolytic enhancing activity. In another
aspect, the enzyme composition comprises an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase. In another aspect, the
enzyme composition comprises an endoglucanase, a cellobiohydrolase,
a beta-glucosidase, and a polypeptide having cellulolytic enhancing
activity.
[0492] In another aspect, the enzyme composition comprises an
acetylmannan esterase. In another aspect, the enzyme composition
comprises an acetylxylan esterase. In another aspect, the enzyme
composition comprises an arabinanase (e.g., alpha-L-arabinanase).
In another aspect, the enzyme composition comprises an
arabinofuranosidase (e.g., alpha-L-arabinofuranosidase). In another
aspect, the enzyme composition comprises a coumaric acid esterase.
In another aspect, the enzyme composition comprises a feruloyl
esterase. In another aspect, the enzyme composition comprises a
galactosidase (e.g., alpha-galactosidase and/or
beta-galactosidase). In another aspect, the enzyme composition
comprises a glucuronidase (e.g., alpha-D-glucuronidase). In another
aspect, the enzyme composition comprises a glucuronoyl esterase. In
another aspect, the enzyme composition comprises a mannanase. In
another aspect, the enzyme composition comprises a mannosidase
(e.g., beta-mannosidase).
[0493] In another aspect, the enzyme composition comprises a
xylanase. In a preferred aspect, the xylanase is a Family 10
xylanase. In another preferred aspect, the xylanase is a Family 11
xylanase. In another aspect, the enzyme composition comprises a
xylosidase (e.g., beta-xylosidase).
[0494] In another aspect, the enzyme composition comprises an
esterase. In another aspect, the enzyme composition comprises an
expansin. In another aspect, the enzyme composition comprises a
laccase. In another aspect, the enzyme composition comprises a
ligninolytic enzyme. In a preferred aspect, the ligninolytic enzyme
is a manganese peroxidase. In another preferred aspect, the
ligninolytic enzyme is a lignin peroxidase. In another preferred
aspect, the ligninolytic enzyme is a H.sub.2O.sub.2-producing
enzyme. In another aspect, the enzyme composition comprises a
pectinase. In another aspect, the enzyme composition comprises a
peroxidase. In another aspect, the enzyme composition comprises a
protease. In another aspect, the enzyme composition comprises a
swollenin.
[0495] In the processes of the present invention, the enzyme(s) can
be added prior to or during saccharification, saccharification and
fermentation, or fermentation.
[0496] One or more (e.g., several) components of the enzyme
composition may be wild-type proteins, recombinant proteins, or a
combination of wild-type proteins and recombinant proteins. For
example, one or more (e.g., several) components may be native
proteins of a cell, which is used as a host cell to express
recombinantly one or more (e.g., several) other components of the
enzyme composition. One or more (e.g., several) components of the
enzyme composition may be produced as monocomponents, which are
then combined to form the enzyme composition. The enzyme
composition may be a combination of multicomponent and
monocomponent protein preparations.
[0497] The enzymes used in the processes of the present invention
may be in any form suitable for use, such as, for example, a
fermentation broth formulation or a cell composition, a cell lysate
with or without cellular debris, a semi-purified or purified enzyme
preparation, or a host cell as a source of the enzymes. The enzyme
composition may be a dry powder or granulate, a non-dusting
granulate, a liquid, a stabilized liquid, or a stabilized protected
enzyme. Liquid enzyme preparations may, for instance, be stabilized
by adding stabilizers such as a sugar, a sugar alcohol or another
polyol, and/or lactic acid or another organic acid according to
established processes.
[0498] The optimum amounts of the enzymes and the GH61 polypeptide
variants depend on several factors including, but not limited to,
the mixture of component cellulolytic enzymes and/or
hemicellulolytic enzymes, the cellulosic material, the
concentration of cellulosic material, the pretreatment(s) of the
cellulosic material, temperature, time, pH, and inclusion of
fermenting organism (e.g., yeast for Simultaneous Saccharification
and Fermentation).
[0499] In one aspect, an effective amount of cellulolytic or
hemicellulolytic enzyme to the cellulosic material is about 0.5 to
about 50 mg, e.g., about 0.5 to about 40 mg, about 0.5 to about 25
mg, about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5
to about 10 mg, or about 2.5 to about 10 mg per g of the cellulosic
material.
[0500] In another aspect, an effective amount of a GH61 polypeptide
variant to the cellulosic material is about 0.01 to about 50.0 mg,
e.g., about 0.01 to about 40 mg, about 0.01 to about 30 mg, about
0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about
5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,
about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about
0.15 to about 1.25 mg, or about 0.25 to about 1.0 mg per g of the
cellulosic material.
[0501] In another aspect, an effective amount of a GH61 polypeptide
variant to cellulolytic or hemicellulolytic enzyme is about 0.005
to about 1.0 g, e.g., about 0.01 to about 1.0 g, about 0.15 to
about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g,
about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g per g of
cellulolytic or hemicellulolytic enzyme.
[0502] The polypeptides having cellulolytic enzyme activity or
hemicellulolytic enzyme activity as well as other
proteins/polypeptides useful in the degradation of the cellulosic
material, e.g., GH61 polypeptides having cellulolytic enhancing
activity (collectively hereinafter "polypeptides having enzyme
activity") can be derived or obtained from any suitable origin,
including, bacterial, fungal, yeast, plant, or mammalian origin.
The term "obtained" also means herein that the enzyme may have been
produced recombinantly in a host organism employing methods
described herein, wherein the recombinantly produced enzyme is
either native or foreign to the host organism or has a modified
amino acid sequence, e.g., having one or more (e.g., several) amino
acids that are deleted, inserted and/or substituted, i.e., a
recombinantly produced enzyme that is a mutant and/or a fragment of
a native amino acid sequence or an enzyme produced by nucleic acid
shuffling processes known in the art. Encompassed within the
meaning of a native enzyme are natural variants and within the
meaning of a foreign enzyme are variants obtained recombinantly,
such as by site-directed mutagenesis or shuffling.
[0503] A polypeptide having enzyme activity may be a bacterial
polypeptide. For example, the polypeptide may be a Gram-positive
bacterial polypeptide such as a Bacillus, Streptococcus,
Streptomyces, Staphylococcus, Enterococcus, Lactobacillus,
Lactococcus, Clostridium, Geobacillus, Caldicellulosiruptor,
Acidothermus, Thermobifidia, or Oceanobacillus polypeptide having
enzyme activity, or a Gram-negative bacterial polypeptide such as
an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,
Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma
polypeptide having enzyme activity.
[0504] In one aspect, the polypeptide is a Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis polypeptide having enzyme
activity.
[0505] In another aspect, the polypeptide is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus polypeptide having enzyme
activity.
[0506] In another aspect, the polypeptide is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans polypeptide having
enzyme activity.
[0507] The polypeptide having enzyme activity may also be a fungal
polypeptide, and more preferably a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide having enzyme activity; or more preferably
a filamentous fungal polypeptide such as an Acremonium, Agaricus,
Alternaria, Aspergillus, Aureobasidium, Botryosphaeria,
Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,
Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,
Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,
Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,
Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide
having enzyme activity.
[0508] In one aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having enzyme activity.
[0509] In another aspect, the polypeptide is an Acremonium
cellulolyticus, Aspergillus aculeatus, Aspergillus awamori,
Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium
inops, Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium funiculosum,
Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia
setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, Trichoderma viride, or Trichophaea saccata
polypeptide having enzyme activity.
[0510] Chemically modified or protein engineered mutants of
polypeptides having enzyme activity may also be used.
[0511] One or more (e.g., several) components of the enzyme
composition may be a recombinant component, i.e., produced by
cloning of a DNA sequence encoding the single component and
subsequent cell transformed with the DNA sequence and expressed in
a host (see, for example, WO 91/17243 and WO 91/17244). The host is
preferably a heterologous host (enzyme is foreign to host), but the
host may under certain conditions also be a homologous host (enzyme
is native to host). Monocomponent cellulolytic proteins may also be
prepared by purifying such a protein from a fermentation broth.
[0512] In one aspect, the one or more (e.g., several) cellulolytic
enzymes comprise a commercial cellulolytic enzyme preparation.
Examples of commercial cellulolytic enzyme preparations suitable
for use in the present invention include, for example, CELLIC.RTM.
CTec (Novozymes A/S), CELLIC.RTM. CTec2 (Novozymes A/S),
CELLIC.RTM. CTec3 (Novozymes A/S), CELLUCLAST.TM. (Novozymes A/S),
NOVOZYM.TM. 188 (Novozymes A/S), CELLUZYME.TM. (Novozymes A/S),
CEREFLO.TM. (Novozymes A/S), and ULTRAFLO.TM. (Novozymes A/S),
ACCELERASE.TM. (Genencor Int.), LAMINEX.TM. (Genencor Int.),
SPEZYME.TM. CP (Genencor Int.), FILTRASE.RTM. NL (DSM);
METHAPLUS.RTM. S/L 100 (DSM), ROHAMENT.TM. 7069 W (Rohm GmbH),
FIBREZYME.RTM. LDI (Dyadic International, Inc.), FIBREZYME.RTM. LBR
(Dyadic International, Inc.), or VISCOSTAR.RTM. 150 L (Dyadic
International, Inc.). The cellulase enzymes are added in amounts
effective from about 0.001 to about 5.0 wt % of solids, e.g., about
0.025 to about 4.0 wt % of solids or about 0.005 to about 2.0 wt %
of solids.
[0513] Examples of bacterial endoglucanases that can be used in the
processes of the present invention, include, but are not limited
to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO
93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No.
5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca
endoglucanase III (WO 05/093050); and Thermobifida fusca
endoglucanase V (WO 05/093050).
[0514] Examples of fungal endoglucanases that can be used in the
present invention, include, but are not limited to, a Trichoderma
reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263,
Trichoderma reesei CeI7B endoglucanase I (GENBANK.TM. accession no.
M15665), Trichoderma reesei endoglucanase II (Saloheimo, et al.,
1988, Gene 63:11-22), Trichoderma reesei CeI5A endoglucanase II
(GENBANK.TM. accession no. M19373), Trichoderma reesei
endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol.
64: 555-563, GENBANK.TM. accession no. AB003694), Trichoderma
reesei endoglucanase V (Saloheimo et al., 1994, Molecular
Microbiology 13: 219-228, GENBANK.TM. accession no. Z33381),
Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic
Acids Research 18: 5884), Aspergillus kawachii endoglucanase
(Sakamoto et al., 1995, Current Genetics 27: 435-439), Erwinia
carotovara endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14),
Fusarium oxysporum endoglucanase (GENBANK.TM. accession no.
L29381), Humicola grisea var. thermoidea endoglucanase (GENBANK.TM.
accession no. AB003107), Melanocarpus albomyces endoglucanase
(GENBANK.TM. accession no. MAL515703), Neurospora crassa
endoglucanase (GENBANK.TM. accession no. XM_324477), Humicola
insolens endoglucanase V, Myceliophthora thermophila CBS 117.65
endoglucanase, basidiomycete CBS 495.95 endoglucanase,
basidiomycete CBS 494.95 endoglucanase, Thielavia terrestris NRRL
8126 CEL6B endoglucanase, Thielavia terrestris NRRL 8126 CEL6C
endoglucanase, Thielavia terrestris NRRL 8126 CEL7C endoglucanase,
Thielavia terrestris NRRL 8126 CEL7E endoglucanase, Thielavia
terrestris NRRL 8126 CEL7F endoglucanase, Cladorrhinum
foecundissimum ATCC 62373 CEL7A endoglucanase, and Trichoderma
reesei strain No. VTT-D-80133 endoglucanase (GENBANK.TM. accession
no. M15665).
[0515] Examples of cellobiohydrolases useful in the present
invention include, but are not limited to, Aspergillus aculeatus
cellobiohydrolase II (WO 2011/059740), Chaetomium thermophilum
cellobiohydrolase I, Chaetomium thermophilum cellobiohydrolase II,
Humicola insolens cellobiohydrolase I, Myceliophthora thermophila
cellobiohydrolase II (WO 2009/042871), Thielavia hyrcanie
cellobiohydrolase II (WO 2010/141325), Thielavia terrestris
cellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and
Trichophaea saccata cellobiohydrolase II (WO 2010/057086).
[0516] Examples of beta-glucosidases useful in the present
invention include, but are not limited to, beta-glucosidases from
Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173: 287-288),
Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et
al., 2000, J. Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO
2002/095014), Penicillium brasilianum IBT 20888 (WO 2007/019442 and
WO 2010/088387), Thielavia terrestris (WO 2011/035029), and
Trichophaea saccata (WO 2007/019442).
[0517] The beta-glucosidase may be a fusion protein. In one aspect,
the beta-glucosidase is an Aspergillus oryzae beta-glucosidase
variant BG fusion protein (WO 2008/057637) or an Aspergillus oryzae
beta-glucosidase fusion protein (WO 2008/057637).
[0518] Other useful endoglucanases, cellobiohydrolases, and
beta-glucosidases are disclosed in numerous Glycosyl Hydrolase
families using the classification according to Henrissat B., 1991,
A classification of glycosyl hydrolases based on amino-acid
sequence similarities, Biochem. J. 280: 309-316, and Henrissat B.,
and Bairoch A., 1996, Updating the sequence-based classification of
glycosyl hydrolases, Biochem. J. 316: 695-696.
[0519] Other cellulolytic enzymes that may be used in the present
invention are described in WO 98/13465, WO 98/015619, WO 98/015633,
WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO
2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO
2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO
2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO
2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO
2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S.
Pat. Nos. 5,457,046, 5,648,263, and 5,686,593.
[0520] In the processes of the present invention, any GH61
polypeptide having cellulolytic enhancing activity can be used as a
component of the enzyme composition.
[0521] Examples of GH61 polypeptides having cellulolytic enhancing
activity useful in the processes of the present invention include,
but are not limited to, GH61 polypeptides from Thielavia terrestris
(WO 2005/074647, WO 2008/148131, and WO 2011/035027), Thermoascus
aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei
(WO 2007/089290), Myceliophthora thermophila (WO 2009/085935, WO
2009/085859, WO 2009/085864, WO 2009/085868), Aspergillus fumigatus
(WO 2010/138754), GH61 polypeptides from Penicillium pinophilum (WO
2011/005867), Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO
2011/041397), Thermoascus crustaceus (WO 2011/041504), Aspergillus
aculeatus (WO 2012/0307990, and Thermomyces lanuginosus (WO
2012/113340). WO 2012/146171 discloses GH61 polypeptides having
cellulolytic enhancing activity and the polynucleotides thereof
from Humicola insolens.
[0522] In one aspect, the GH61 polypeptide variant and GH61
polypeptide having cellulolytic enhancing activity is used in the
presence of a soluble activating divalent metal cation according to
WO 2008/151043, e.g., manganese or copper.
[0523] In another aspect, the GH61 polypeptide variant and GH61
polypeptide having cellulolytic enhancing activity is used in the
presence of a dioxy compound, a bicylic compound, a heterocyclic
compound, a nitrogen-containing compound, a quinone compound, a
sulfur-containing compound, or a liquor obtained from a pretreated
cellulosic material such as pretreated corn stover (PCS).
[0524] The dioxy compound may include any suitable compound
containing two or more oxygen atoms. In some aspects, the dioxy
compounds contain a substituted aryl moiety as described herein.
The dioxy compounds may comprise one or more (e.g., several)
hydroxyl and/or hydroxyl derivatives, but also include substituted
aryl moieties lacking hydroxyl and hydroxyl derivatives.
Non-limiting examples of the dioxy compounds include pyrocatechol
or catechol; caffeic acid; 3,4-dihydroxybenzoic acid;
4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;
methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;
2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;
4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid;
ethyl gallate; methyl glycolate; dihydroxyfumaric acid;
2-butyne-1,4-diol; (croconic acid; 1,3-propanediol; tartaric acid;
2,4-pentanediol; 3-ethyoxy-1,2-propanediol;
2,4,4'-trihydroxybenzophenone; cis-2-butene-1,4-diol;
3,4-dihydroxy-3-cyclobutene-1,2-dione; dihydroxyacetone; acrolein
acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid; and
methyl-3,5-dimethoxy-4-hydroxybenzoate; or a salt or solvate
thereof.
[0525] The bicyclic compound may include any suitable substituted
fused ring system as described herein. The compounds may comprise
one or more (e.g., several) additional rings, and are not limited
to a specific number of rings unless otherwise stated. In one
aspect, the bicyclic compound is a flavonoid. In another aspect,
the bicyclic compound is an optionally substituted isoflavonoid. In
another aspect, the bicyclic compound is an optionally substituted
flavylium ion, such as an optionally substituted anthocyanidin or
optionally substituted anthocyanin, or derivative thereof.
Non-limiting examples of the bicyclic compounds include
epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin;
acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin;
kuromanin; keracyanin; or a salt or solvate thereof.
[0526] The heterocyclic compound may be any suitable compound, such
as an optionally substituted aromatic or non-aromatic ring
comprising a heteroatom, as described herein. In one aspect, the
heterocyclic is a compound comprising an optionally substituted
heterocycloalkyl moiety or an optionally substituted heteroaryl
moiety. In another aspect, the optionally substituted
heterocycloalkyl moiety or optionally substituted heteroaryl moiety
is an optionally substituted 5-membered heterocycloalkyl or an
optionally substituted 5-membered heteroaryl moiety. In another
aspect, the optionally substituted heterocycloalkyl or optionally
substituted heteroaryl moiety is an optionally substituted moiety
selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,
oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,
thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl,
thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl,
benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl,
benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl,
acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl,
tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl,
diazepinyl, azepinyl, thiepinyl, piperidinyl, and oxepinyl. In
another aspect, the optionally substituted heterocycloalkyl moiety
or optionally substituted heteroaryl moiety is an optionally
substituted furanyl. Non-limiting examples of the heterocyclic
compounds include
(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;
4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;
[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione;
.alpha.-hydroxy-.gamma.-butyrolactone; ribonic .gamma.-lactone;
aldohexuronicaldohexuronic acid .gamma.-lactone; gluconic acid
.delta.-lactone; 4-hydroxycoumarin; dihydrobenzofuran;
5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;
5,6-dihydro-2H-pyran-2-one; and
5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate
thereof.
[0527] The nitrogen-containing compound may be any suitable
compound with one or more nitrogen atoms. In one aspect, the
nitrogen-containing compound comprises an amine, imine,
hydroxylamine, or nitroxide moiety. Non-limiting examples of the
nitrogen-containing compounds include acetone oxime; violuric acid;
pyridine-2-aldoxime; 2-aminophenol; 1,2-benzenediamine;
2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8-tetrahydrobiopterin;
6,7-dimethyl-5,6,7,8-tetrahydropterine; and maleamic acid; or a
salt or solvate thereof.
[0528] The quinone compound may be any suitable compound comprising
a quinone moiety as described herein. Non-limiting examples of the
quinone compounds include 1,4-benzoquinone; 1,4-naphthoquinone;
2-hydroxy-1,4-naphthoquinone;
2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q.sub.0;
2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;
1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or
adrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone;
pyrroloquinoline quinone; or a salt or solvate thereof.
[0529] The sulfur-containing compound may be any suitable compound
comprising one or more sulfur atoms. In one aspect, the
sulfur-containing comprises a moiety selected from thionyl,
thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic
acid, and sulfonic ester. Non-limiting examples of the
sulfur-containing compounds include ethanethiol; 2-propanethiol;
2-propene-1-thiol; 2-mercaptoethanesulfonic acid; benzenethiol;
benzene-1,2-dithiol; cysteine; methionine; glutathione; cystine; or
a salt or solvate thereof.
[0530] In one aspect, an effective amount of such a compound
described above to cellulosic material as a molar ratio to glucosyl
units of cellulose is about 10.sup.-6 to about 10, e.g., about
10.sup.-6 to about 7.5, about 10.sup.-6 to about 5, about 10.sup.-6
to about 2.5, about 10.sup.-6 to about 1, about 10.sup.-5 to about
1, about 10.sup.-5 to about 10.sup.-1, about 10.sup.-4 to about
10.sup.-1, about 10.sup.-3 to about 10.sup.-1, or about 10.sup.-3
to about 10.sup.-2. In another aspect, an effective amount of such
a compound described above is about 0.1 .mu.M to about 1 M, e.g.,
about 0.5 .mu.M to about 0.75 M, about 0.75 .mu.M to about 0.5 M,
about 1 .mu.M to about 0.25 M, about 1 .mu.M to about 0.1 M, about
5 .mu.M to about 50 mM, about 10 .mu.M to about 25 mM, about 50
.mu.M to about 25 mM, about 10 .mu.M to about 10 mM, about 5 .mu.M
to about 5 mM, or about 0.1 mM to about 1 mM.
[0531] The term "liquor" means the solution phase, either aqueous,
organic, or a combination thereof, arising from treatment of a
lignocellulose and/or hemicellulose material in a slurry, or
monosaccharides thereof, e.g., xylose, arabinose, mannose, etc.,
under conditions as described herein, and the soluble contents
thereof. A liquor for cellulolytic enhancement of a GH61
polypeptide or variant can be produced by treating a lignocellulose
or hemicellulose material (or feedstock) by applying heat and/or
pressure, optionally in the presence of a catalyst, e.g., acid,
optionally in the presence of an organic solvent, and optionally in
combination with physical disruption of the material, and then
separating the solution from the residual solids. Such conditions
determine the degree of cellulolytic enhancement obtainable through
the combination of liquor and a GH61 polypeptide or variant during
hydrolysis of a cellulosic substrate by a cellulase preparation.
The liquor can be separated from the treated material using a
method standard in the art, such as filtration, sedimentation, or
centrifugation.
[0532] In one aspect, an effective amount of the liquor to
cellulose is about 10.sup.-6 to about 10 g per g of cellulose,
e.g., about 10.sup.-6 to about 7.5 g, about 10.sup.-6 to about 5,
about 10.sup.-6 to about 2.5 g, about 10.sup.-6 to about 1 g, about
10.sup.-5 to about 1 g, about 10.sup.-5 to about 10.sup.-1 g, about
10.sup.-4 to about 10.sup.-1g, about 10.sup.-3 to about 10.sup.-1g,
or about 10.sup.-3 to about 10.sup.-2 g per g of cellulose.
[0533] In one aspect, the one or more (e.g., several)
hemicellulolytic enzymes comprise a commercial hemicellulolytic
enzyme preparation. Examples of commercial hemicellulolytic enzyme
preparations suitable for use in the present invention include, for
example, SHEARZYME.TM. (Novozymes A/S), CELLIC.RTM. HTec (Novozymes
A/S), CELLIC.RTM. HTec2 (Novozymes A/S), CELLIC.RTM. HTec3
(Novozymes A/S), VISCOZYME.RTM. (Novozymes A/S), ULTRAFLO.RTM.
(Novozymes A/S), PULPZYME.RTM. HC (Novozymes A/S), MULTIFECT.RTM.
Xylanase (Genencor), ACCELLERASE.RTM. XY (Genencor),
ACCELLERASE.RTM. XC (Genencor), ECOPULP.RTM. TX-200A (AB Enzymes),
HSP 6000 Xylanase (DSM), DEPOL.TM. 333P (Biocatalysts Limit, Wales,
UK), DEPOL.TM. 740 L. (Biocatalysts Limit, Wales, UK), and
DEPOL.TM. 762P (Biocatalysts Limit, Wales, UK).
[0534] Examples of xylanases useful in the processes of the present
invention include, but are not limited to, xylanases from
Aspergillus aculeatus (GeneSeqP:AAR63790; WO 94/21785), Aspergillus
fumigatus (WO 2006/078256), Penicillium pinophilum (WO
2011/041405), Penicillium sp. (WO 2010/126772), Thielavia
terrestris NRRL 8126 (WO 2009/079210), and Trichophaea saccata GH10
(WO 2011/057083).
[0535] Examples of beta-xylosidases useful in the processes of the
present invention include, but are not limited to, beta-xylosidases
from Neurospora crassa (SwissProt accession number Q7SOW4),
Trichoderma reesei (UniProtKB/TrEMBL accession number Q92458), and
Talaromyces emersonii (SwissProt accession number Q8X212).
[0536] Examples of acetylxylan esterases useful in the processes of
the present invention include, but are not limited to, acetylxylan
esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium
globosum (Uniprot accession number Q2GWX4), Chaetomium gracile
(GeneSeqP accession number AAB82124), Humicola insolens DSM 1800
(WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera
thermophila (WO 2010/014880), Neurospora crassa (UniProt accession
number q7s259), Phaeosphaeria nodorum (Uniprot accession number
Q0UHJ1), and Thielavia terrestris NRRL 8126 (WO 2009/042846).
[0537] Examples of feruloyl esterases (ferulic acid esterases)
useful in the processes of the present invention include, but are
not limited to, feruloyl esterases form Humicola insolens DSM 1800
(WO 2009/076122), Neosartorya fischeri (UniProt Accession number
A1D9T4), Neurospora crassa (UniProt accession number Q9HGR3),
Penicillium aurantiogriseum (WO 2009/127729), and Thielavia
terrestris (WO 2010/053838 and WO 2010/065448).
[0538] Examples of arabinofuranosidases useful in the processes of
the present invention include, but are not limited to,
arabinofuranosidases from Aspergillus niger (GeneSeqP accession
number AAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO
2009/073383), and M. giganteus (WO 2006/114094).
[0539] Examples of alpha-glucuronidases useful in the processes of
the present invention include, but are not limited to,
alpha-glucuronidases from Aspergillus clavatus (UniProt accession
number alcc12), Aspergillus fumigatus (SwissProt accession number
Q4WW45), Aspergillus niger (Uniprot accession number Q96WX9),
Aspergillus terreus (SwissProt accession number Q0CJP9), Humicola
insolens (WO 2010/014706), Penicillium aurantiogriseum (WO
2009/068565), Talaromyces emersonii (UniProt accession number
Q8X211), and Trichoderma reesei (Uniprot accession number
Q99024).
[0540] The polypeptides having enzyme activity used in the
processes of the present invention may be produced by fermentation
of the above-noted microbial strains on a nutrient medium
containing suitable carbon and nitrogen sources and inorganic
salts, using procedures known in the art (see, e.g., Bennett, J. W.
and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic
Press, C A, 1991). Suitable media are available from commercial
suppliers or may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection).
Temperature ranges and other conditions suitable for growth and
enzyme production are known in the art (see, e.g., Bailey, J. E.,
and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill
Book Company, N Y, 1986).
[0541] The fermentation can be any method of cultivation of a cell
resulting in the expression or isolation of an enzyme or protein.
Fermentation may, therefore, be understood as comprising shake
flask cultivation, or small- or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in
laboratory or industrial fermentors performed in a suitable medium
and under conditions allowing the enzyme to be expressed or
isolated. The resulting enzymes produced by the methods described
above may be recovered from the fermentation medium and purified by
conventional procedures.
[0542] Fermentation.
[0543] The fermentable sugars obtained from the hydrolyzed
cellulosic material can be fermented by one or more (e.g., several)
fermenting microorganisms capable of fermenting the sugars directly
or indirectly into a desired fermentation product. "Fermentation"
or "fermentation process" refers to any fermentation process or any
process comprising a fermentation step. Fermentation processes also
include fermentation processes used in the consumable alcohol
industry (e.g., beer and wine), dairy industry (e.g., fermented
dairy products), leather industry, and tobacco industry. The
fermentation conditions depend on the desired fermentation product
and fermenting organism and can easily be determined by one skilled
in the art.
[0544] In the fermentation step, sugars, released from the
cellulosic material as a result of the pretreatment and enzymatic
hydrolysis steps, are fermented to a product, e.g., ethanol, by a
fermenting organism, such as yeast. Hydrolysis (saccharification)
and fermentation can be separate or simultaneous, as described
herein.
[0545] Any suitable hydrolyzed cellulosic material can be used in
the fermentation step in practicing the present invention. The
material is generally selected based on the desired fermentation
product, i.e., the substance to be obtained from the fermentation,
and the process employed, as is well known in the art.
[0546] The term "fermentation medium" is understood herein to refer
to a medium before the fermenting microorganism(s) is(are) added,
such as, a medium resulting from a saccharification process, as
well as a medium used in a simultaneous saccharification and
fermentation process (SSF).
[0547] "Fermenting microorganism" refers to any microorganism,
including bacterial and fungal organisms, suitable for use in a
desired fermentation process to produce a fermentation product. The
fermenting organism can be hexose and/or pentose fermenting
organisms, or a combination thereof. Both hexose and pentose
fermenting organisms are well known in the art. Suitable fermenting
microorganisms are able to ferment, i.e., convert, sugars, such as
glucose, xylose, xylulose, arabinose, maltose, mannose, galactose,
and/or oligosaccharides, directly or indirectly into the desired
fermentation product. Examples of bacterial and fungal fermenting
organisms producing ethanol are described by Lin et al., 2006,
Appl. Microbiol. Biotechnol. 69: 627-642.
[0548] Examples of fermenting microorganisms that can ferment
hexose sugars include bacterial and fungal organisms, such as
yeast. Preferred yeast includes strains of Candida, Kluyveromyces,
and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces
marxianus, and Saccharomyces cerevisiae.
[0549] Examples of fermenting organisms that can ferment pentose
sugars in their native state include bacterial and fungal
organisms, such as some yeast. Preferred xylose fermenting yeast
include strains of Candida, preferably C. sheatae or C. sonorensis;
and strains of Pichia, preferably P. stipitis, such as P. stipitis
CBS 5773. Preferred pentose fermenting yeast include strains of
Pachysolen, preferably P. tannophilus. Organisms not capable of
fermenting pentose sugars, such as xylose and arabinose, may be
genetically modified to do so by methods known in the art.
[0550] Examples of bacteria that can efficiently ferment hexose and
pentose to ethanol include, for example, Bacillus coagulans,
Clostridium acetobutylicum, Clostridium thermocellum, Clostridium
phytofermentans, Geobacillus sp., Thermoanaerobacter
saccharolyticum, and Zymomonas mobilis (Philippidis, 1996,
supra).
[0551] Other fermenting organisms include strains of Bacillus, such
as Bacillus coagulans; Candida, such as C. sonorensis, C.
methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C.
blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C.
boidinii, C. utilis, and C. scehatae; Clostridium, such as C.
acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli,
especially E. coli strains that have been genetically modified to
improve the yield of ethanol; Geobacillus sp.; Hansenula, such as
Hansenula anomala; Klebsiella, such as K. oxytoca; Kluyveromyces,
such as K. marxianus, K. lactis, K. thermotolerans, and K.
fragilis; Schizosaccharomyces, such as S. pombe;
Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum; and
Zymomonas, such as Zymomonas mobilis.
[0552] In a preferred aspect, the yeast is a Bretannomyces. In a
more preferred aspect, the yeast is Bretannomyces clausenii. In
another preferred aspect, the yeast is a Candida. In another more
preferred aspect, the yeast is Candida sonorensis. In another more
preferred aspect, the yeast is Candida boidinii. In another more
preferred aspect, the yeast is Candida blankii. In another more
preferred aspect, the yeast is Candida brassicae. In another more
preferred aspect, the yeast is Candida diddensii. In another more
preferred aspect, the yeast is Candida entomophiliia. In another
more preferred aspect, the yeast is Candida pseudotropicalis. In
another more preferred aspect, the yeast is Candida scehatae. In
another more preferred aspect, the yeast is Candida utilis. In
another preferred aspect, the yeast is a Clavispora. In another
more preferred aspect, the yeast is Clavispora lusitaniae. In
another more preferred aspect, the yeast is Clavispora opuntiae. In
another preferred aspect, the yeast is a Kluyveromyces. In another
more preferred aspect, the yeast is Kluyveromyces fragilis. In
another more preferred aspect, the yeast is Kluyveromyces
marxianus. In another more preferred aspect, the yeast is
Kluyveromyces thermotolerans. In another preferred aspect, the
yeast is a Pachysolen. In another more preferred aspect, the yeast
is Pachysolen tannophilus. In another preferred aspect, the yeast
is a Pichia. In another more preferred aspect, the yeast is a
Pichia stipitis. In another preferred aspect, the yeast is a
Saccharomyces spp. In another more preferred aspect, the yeast is
Saccharomyces cerevisiae. In another more preferred aspect, the
yeast is Saccharomyces distaticus. In another more preferred
aspect, the yeast is Saccharomyces uvarum.
[0553] In a preferred aspect, the bacterium is a Bacillus. In a
more preferred aspect, the bacterium is Bacillus coagulans. In
another preferred aspect, the bacterium is a Clostridium. In
another more preferred aspect, the bacterium is Clostridium
acetobutylicum. In another more preferred aspect, the bacterium is
Clostridium phytofermentans. In another more preferred aspect, the
bacterium is Clostridium thermocellum. In another more preferred
aspect, the bacterium is Geobacillus sp. In another more preferred
aspect, the bacterium is a Thermoanaerobacter. In another more
preferred aspect, the bacterium is Thermoanaerobacter
saccharolyticum. In another preferred aspect, the bacterium is a
Zymomonas. In another more preferred aspect, the bacterium is
Zymomonas mobilis.
[0554] Commercially available yeast suitable for ethanol production
include, e.g., BIOFERM.TM. AFT and XR (NABC--North American
Bioproducts Corporation, GA, USA), ETHANOL RED.TM. yeast
(Fermentis/Lesaffre, USA), FALI.TM. (Fleischmann's Yeast, USA),
FERMIOL.TM. (DSM Specialties), GERT STRAND.TM. (Gert Strand AB,
Sweden), and SUPERSTART.TM. and THERMOSACC.TM. fresh yeast (Ethanol
Technology, WI, USA).
[0555] In a preferred aspect, the fermenting microorganism has been
genetically modified to provide the ability to ferment pentose
sugars, such as xylose utilizing, arabinose utilizing, and xylose
and arabinose co-utilizing microorganisms.
[0556] The cloning of heterologous genes into various fermenting
microorganisms has led to the construction of organisms capable of
converting hexoses and pentoses to ethanol (co-fermentation) (Chen
and Ho, 1993, Cloning and improving the expression of Pichia
stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.
Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically
engineered Saccharomyces yeast capable of effectively cofermenting
glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter
and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae,
Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995,
Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing
the TKL1 and TALI genes encoding the pentose phosphate pathway
enzymes transketolase and transaldolase, Appl. Environ. Microbiol.
61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering
of Saccharomyces cerevisiae for efficient anaerobic xylose
fermentation: a proof of principle, FEMS Yeast Research 4: 655-664;
Beall et al., 1991, Parametric studies of ethanol production from
xylose and other sugars by recombinant Escherichia coli, Biotech.
Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of
bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214;
Zhang et al., 1995, Metabolic engineering of a pentose metabolism
pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243;
Deanda et al., 1996, Development of an arabinose-fermenting
Zymomonas mobilis strain by metabolic pathway engineering, Appl.
Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose
isomerase).
[0557] In a preferred aspect, the genetically modified fermenting
microorganism is Candida sonorensis. In another preferred aspect,
the genetically modified fermenting microorganism is Escherichia
coli. In another preferred aspect, the genetically modified
fermenting microorganism is Klebsiella oxytoca. In another
preferred aspect, the genetically modified fermenting microorganism
is Kluyveromyces marxianus. In another preferred aspect, the
genetically modified fermenting microorganism is Saccharomyces
cerevisiae. In another preferred aspect, the genetically modified
fermenting microorganism is Zymomonas mobilis.
[0558] It is well known in the art that the organisms described
above can also be used to produce other substances, as described
herein.
[0559] The fermenting microorganism is typically added to the
degraded cellulosic material or hydrolysate and the fermentation is
performed for about 8 to about 96 hours, e.g., about 24 to about 60
hours. The temperature is typically between about 26.degree. C. to
about 60.degree. C., e.g., about 32.degree. C. or 50.degree. C.,
and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
[0560] In one aspect, the yeast and/or another microorganism are
applied to the degraded cellulosic material and the fermentation is
performed for about 12 to about 96 hours, such as typically 24-60
hours. In another aspect, the temperature is preferably between
about 20.degree. C. to about 60.degree. C., e.g., about 25.degree.
C. to about 50.degree. C., about 32.degree. C. to about 50.degree.
C., or about 32.degree. C. to about 50.degree. C., and the pH is
generally from about pH 3 to about pH 7, e.g., about pH 4 to about
pH 7. However, some fermenting organisms, e.g., bacteria, have
higher fermentation temperature optima. Yeast or another
microorganism is preferably applied in amounts of approximately
10.sup.5 to 10.sup.12, preferably from approximately 10.sup.7 to
10.sup.10, especially approximately 2.times.10.sup.8 viable cell
count per ml of fermentation broth. Further guidance in respect of
using yeast for fermentation can be found in, e.g., "The Alcohol
Textbook" (Editors K. Jacques, T. P. Lyons and D. R. Kelsall,
Nottingham University Press, United Kingdom 1999), which is hereby
incorporated by reference.
[0561] A fermentation stimulator can be used in combination with
any of the processes described herein to further improve the
fermentation process, and in particular, the performance of the
fermenting microorganism, such as, rate enhancement and ethanol
yield. A "fermentation stimulator" refers to stimulators for growth
of the fermenting microorganisms, in particular, yeast. Preferred
fermentation stimulators for growth include vitamins and minerals.
Examples of vitamins include multivitamins, biotin, pantothenate,
nicotinic acid, meso-inositol, thiamine, pyridoxine,
para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See, for example, Alfenore et al., Improving ethanol
production and viability of Saccharomyces cerevisiae by a vitamin
feeding strategy during fed-batch process, Springer-Verlag (2002),
which is hereby incorporated by reference. Examples of minerals
include minerals and mineral salts that can supply nutrients
comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
[0562] Fermentation Products:
[0563] A fermentation product can be any substance derived from the
fermentation. The fermentation product can be, without limitation,
an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol,
glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene
glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane
(e.g., pentane, hexane, heptane, octane, nonane, decane, undecane,
and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane,
cycloheptane, and cyclooctane), an alkene (e.g. pentene, hexene,
heptene, and octene); an amino acid (e.g., aspartic acid, glutamic
acid, glycine, lysine, serine, and threonine); a gas (e.g.,
methane, hydrogen (H.sub.2), carbon dioxide (CO.sub.2), and carbon
monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid
(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid,
citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid,
glucaric acid, gluconic acid, glucuronic acid, glutaric acid,
3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,
malonic acid, oxalic acid, oxaloacetic acid, propionic acid,
succinic acid, and xylonic acid); and polyketide. The fermentation
product can also be protein as a high value product.
[0564] In a preferred aspect, the fermentation product is an
alcohol. It will be understood that the term "alcohol" encompasses
a substance that contains one or more hydroxyl moieties. In a more
preferred aspect, the alcohol is n-butanol. In another more
preferred aspect, the alcohol is isobutanol. In another more
preferred aspect, the alcohol is ethanol. In another more preferred
aspect, the alcohol is methanol. In another more preferred aspect,
the alcohol is arabinitol. In another more preferred aspect, the
alcohol is butanediol. In another more preferred aspect, the
alcohol is ethylene glycol. In another more preferred aspect, the
alcohol is glycerin. In another more preferred aspect, the alcohol
is glycerol. In another more preferred aspect, the alcohol is
1,3-propanediol. In another more preferred aspect, the alcohol is
sorbitol. In another more preferred aspect, the alcohol is xylitol.
See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,
1999, Ethanol production from renewable resources, in Advances in
Biochemical Engineering/Biotechnology, Scheper, T., ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira,
M. M., and Jonas, R., 2002, The biotechnological production of
sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and
Singh, D., 1995, Processes for fermentative production of
xylitol--a sugar substitute, Process Biochemistry 30 (2): 117-124;
Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production of
acetone, butanol and ethanol by Clostridium beijerinckii BA101 and
in situ recovery by gas stripping, World Journal of Microbiology
and Biotechnology 19 (6): 595-603.
[0565] In another preferred aspect, the fermentation product is an
alkane. The alkane can be an unbranched or a branched alkane. In
another more preferred aspect, the alkane is pentane. In another
more preferred aspect, the alkane is hexane. In another more
preferred aspect, the alkane is heptane. In another more preferred
aspect, the alkane is octane. In another more preferred aspect, the
alkane is nonane. In another more preferred aspect, the alkane is
decane. In another more preferred aspect, the alkane is undecane.
In another more preferred aspect, the alkane is dodecane.
[0566] In another preferred aspect, the fermentation product is a
cycloalkane. In another more preferred aspect, the cycloalkane is
cyclopentane. In another more preferred aspect, the cycloalkane is
cyclohexane. In another more preferred aspect, the cycloalkane is
cycloheptane. In another more preferred aspect, the cycloalkane is
cyclooctane.
[0567] In another preferred aspect, the fermentation product is an
alkene. The alkene can be an unbranched or a branched alkene. In
another more preferred aspect, the alkene is pentene. In another
more preferred aspect, the alkene is hexene. In another more
preferred aspect, the alkene is heptene. In another more preferred
aspect, the alkene is octene.
[0568] In another preferred aspect, the fermentation product is an
amino acid. In another more preferred aspect, the organic acid is
aspartic acid. In another more preferred aspect, the amino acid is
glutamic acid. In another more preferred aspect, the amino acid is
glycine. In another more preferred aspect, the amino acid is
lysine. In another more preferred aspect, the amino acid is serine.
In another more preferred aspect, the amino acid is threonine. See,
for example, Richard, A., and Margaritis, A., 2004, Empirical
modeling of batch fermentation kinetics for poly(glutamic acid)
production and other microbial biopolymers, Biotechnology and
Bioengineering 87 (4): 501-515.
[0569] In another preferred aspect, the fermentation product is a
gas. In another more preferred aspect, the gas is methane. In
another more preferred aspect, the gas is H.sub.2. In another more
preferred aspect, the gas is CO.sub.2. In another more preferred
aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and
K. Kiriyama, 1997, Studies on hydrogen production by continuous
culture system of hydrogen-producing anaerobic bacteria, Water
Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. in
Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic
digestion of biomass for methane production: A review.
[0570] In another preferred aspect, the fermentation product is
isoprene.
[0571] In another preferred aspect, the fermentation product is a
ketone. It will be understood that the term "ketone" encompasses a
substance that contains one or more ketone moieties. In another
more preferred aspect, the ketone is acetone. See, for example,
Qureshi and Blaschek, 2003, supra.
[0572] In another preferred aspect, the fermentation product is an
organic acid. In another more preferred aspect, the organic acid is
acetic acid. In another more preferred aspect, the organic acid is
acetonic acid. In another more preferred aspect, the organic acid
is adipic acid. In another more preferred aspect, the organic acid
is ascorbic acid. In another more preferred aspect, the organic
acid is citric acid. In another more preferred aspect, the organic
acid is 2,5-diketo-D-gluconic acid. In another more preferred
aspect, the organic acid is formic acid. In another more preferred
aspect, the organic acid is fumaric acid. In another more preferred
aspect, the organic acid is glucaric acid. In another more
preferred aspect, the organic acid is gluconic acid. In another
more preferred aspect, the organic acid is glucuronic acid. In
another more preferred aspect, the organic acid is glutaric acid.
In another preferred aspect, the organic acid is 3-hydroxypropionic
acid. In another more preferred aspect, the organic acid is
itaconic acid. In another more preferred aspect, the organic acid
is lactic acid. In another more preferred aspect, the organic acid
is malic acid. In another more preferred aspect, the organic acid
is malonic acid. In another more preferred aspect, the organic acid
is oxalic acid. In another more preferred aspect, the organic acid
is propionic acid. In another more preferred aspect, the organic
acid is succinic acid. In another more preferred aspect, the
organic acid is xylonic acid. See, for example, Chen, R., and Lee,
Y. Y., 1997, Membrane-mediated extractive fermentation for lactic
acid production from cellulosic biomass, Appl. Biochem. Biotechnol.
63-65: 435-448.
[0573] In another preferred aspect, the fermentation product is
polyketide.
[0574] Recovery.
[0575] The fermentation product(s) can be optionally recovered from
the fermentation medium using any method known in the art
including, but not limited to, chromatography, electrophoretic
procedures, differential solubility, distillation, or extraction.
For example, alcohol is separated from the fermented cellulosic
material and purified by conventional methods of distillation.
Ethanol with a purity of up to about 96 vol. % can be obtained,
which can be used as, for example, fuel ethanol, drinking ethanol,
i.e., potable neutral spirits, or industrial ethanol.
Detergent Compositions
[0576] The present invention also relates to detergent compositions
comprising a GH61 polypeptide variant of the present invention and
a surfactant. A GH61 polypeptide variant of the present invention
may be added to and thus become a component of a detergent
composition.
[0577] The detergent composition of the present invention may be
formulated, for example, as a hand or machine laundry detergent
composition including a laundry additive composition suitable for
pre-treatment of stained fabrics and a rinse added fabric softener
composition, or be formulated as a detergent composition for use in
general household hard surface cleaning operations, or be
formulated for hand or machine dishwashing operations. In one
aspect, the present invention also relates to methods for cleaning
or washing a hard surface or laundry, the method comprising
contacting the hard surface or the laundry with a detergent
composition of the present invention.
[0578] In a specific aspect, the present invention provides a
detergent additive comprising a GH61 polypeptide variant of the
invention. The detergent additive as well as the detergent
composition may comprise one or more (e.g., several) enzymes
selected from the group consisting of an amylase, arabinase,
cutinase, carbohydrase, cellulase, galactanase, laccase, lipase,
mannanase, oxidase, pectinase, peroxidase, protease, and
xylanase.
[0579] In general the properties of the selected enzyme(s) should
be compatible with the selected detergent, (i.e., pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective
amounts.
[0580] Cellulases:
[0581] Suitable cellulases include those of bacterial or fungal
origin. Chemically modified or protein engineered mutants are
included. Suitable cellulases include cellulases from the genera
Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,
e.g., the fungal cellulases produced from Humicola insolens,
Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S.
Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757 and WO
89/09259.
[0582] Especially suitable cellulases are the alkaline or neutral
cellulases having color care benefits. Examples of such cellulases
are cellulases described in EP 0 495 257, EP 0 531 372, WO
96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase
variants such as those described in WO 94/07998, EP 0 531 315, U.S.
Pat. Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307
and PCT/DK98/00299.
[0583] Commercially available cellulases include CELLUZYME.TM., and
CAREZYME.TM. (Novozymes A/S), CLAZINASE.TM., and PURADAX HA.TM.
(Genencor International Inc.), and KAC-500(B).TM. (Kao
Corporation).
[0584] Proteases:
[0585] Suitable proteases include those of animal, vegetable or
microbial origin. Microbial origin is preferred. Chemically
modified or protein engineered mutants are included. The protease
may be a serine protease or a metalloprotease, preferably an
alkaline microbial protease or a trypsin-like protease. Examples of
alkaline proteases are subtilisins, especially those derived from
Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin
309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
Examples of trypsin-like proteases are trypsin (e.g., of porcine or
bovine origin) and the Fusarium protease described in WO 89/06270
and WO 94/25583.
[0586] Examples of useful proteases are the variants described in
WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially
the variants with substitutions in one or more of the following
positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170,
194, 206, 218, 222, 224, 235, and 274.
[0587] Preferred commercially available protease enzymes include
ALCALASE.TM. SAVINASE.TM., PRIMASE.TM., DURALASE.TM., ESPERASE.TM.,
and KANNASE.TM. (Novozymes A/S), MAXATASE.TM., MAXACAL.TM.,
MAXAPEM.TM., PROPERASE.TM., PURAFECT.TM., PURAFECT OXP.TM.,
FN2.TM., and FN3.TM. (Genencor International Inc.).
[0588] Lipases:
[0589] Suitable lipases include those of bacterial or fungal
origin. Chemically modified or protein engineered mutants are
included. Examples of useful lipases include lipases from Humicola
(synonym Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) as
described in EP 258 068 and EP 305 216 or from H. insolens as
described in WO 96/13580, a Pseudomonas lipase, e.g., from P.
alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP
331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas
sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis
(WO 96/12012), a Bacillus lipase, e.g., from B. subtilis (Dartois
et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B.
stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
[0590] Other examples are lipase variants such as those described
in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381,
WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO
97/04079 and WO 97/07202.
[0591] Preferred commercially available lipase enzymes include
LIPOLASE.TM. and LIPOLASE ULTRA.TM. (Novozymes A/S).
[0592] Amylases:
[0593] Suitable amylases (.alpha. and/or .beta.) include those of
bacterial or fungal origin.
[0594] Chemically modified or protein engineered mutants are
included. Amylases include, for example, .alpha.-amylases obtained
from Bacillus, e.g., a special strain of Bacillus licheniformis,
described in more detail in GB 1,296,839.
[0595] Examples of useful amylases are the variants described in WO
94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the
variants with substitutions in one or more of the following
positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188,
190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.
[0596] Commercially available amylases are DURAMYL.TM.,
TERMAMYL.TM., FUNGAMYL.TM. and BAN.TM. (Novozymes A/S),
RAPIDASE.TM. and PURASTAR.TM. (from Genencor International
Inc.).
[0597] Peroxidases/Oxidases:
[0598] Suitable peroxidases/oxidases include those of plant,
bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Examples of useful peroxidases
include peroxidases from Coprinus, e.g., from C. cinereus, and
variants thereof as those described in WO 93/24618, WO 95/10602,
and WO 98/15257.
[0599] Commercially available peroxidases include GUARDZYME.TM.
(Novozymes A/S).
[0600] The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
(e.g., several) enzymes, or by adding a combined additive
comprising all of these enzymes. A detergent additive of the
invention, i.e., a separate additive or a combined additive, can be
formulated, for example, as a granulate, liquid, slurry, etc.
Preferred detergent additive formulations are granulates, in
particular non-dusting granulates, liquids, in particular
stabilized liquids, or slurries.
[0601] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (polyethyleneglycol,
PEG) with mean molar weights of 1000 to 20000; ethoxylated
nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated
fatty alcohols in which the alcohol contains from 12 to 20 carbon
atoms and in which there are 15 to 80 ethylene oxide units; fatty
alcohols; fatty acids; and mono- and di- and triglycerides of fatty
acids. Examples of film-forming coating materials suitable for
application by fluid bed techniques are given in GB 1483591. Liquid
enzyme preparations may, for instance, be stabilized by adding a
polyol such as propylene glycol, a sugar or sugar alcohol, lactic
acid or boric acid according to established methods. Protected
enzymes may be prepared according to the method disclosed in EP
238,216.
[0602] The detergent composition of the invention may be in any
convenient form, e.g., a bar, a tablet, a powder, a granule, a
paste, or a liquid. A liquid detergent may be aqueous, typically
containing up to 70% water and 0-30% organic solvent, or
non-aqueous.
[0603] The detergent composition comprises one or more (e.g.,
several) surfactants, which may be non-ionic including semi-polar
and/or anionic and/or cationic and/or zwitterionic. The surfactants
are typically present at a level of from 0.1% to 60% by weight.
[0604] When included therein the detergent will usually contain
from about 1% to about 40% of an anionic surfactant such as linear
alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty
alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic
acid, or soap.
[0605] When included therein the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl N-alkyl derivatives of glucosamine ("glucamides").
[0606] The detergent may contain 0-65% of a detergent builder or
complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, carbonate, citrate, nitrilotriacetic acid,
ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic
acid, alkyl- or alkenylsuccinic acid, soluble silicates, or layered
silicates (e.g., SKS-6 from Hoechst).
[0607] The detergent may comprise one or more (e.g., several)
polymers. Examples are carboxymethylcellulose,
poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl
alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),
polycarboxylates such as polyacrylates, maleic/acrylic acid
copolymers, and lauryl methacrylate/acrylic acid copolymers.
[0608] The detergent may contain a bleaching system which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
Alternatively, the bleaching system may comprise peroxyacids of,
for example, the amide, imide, or sulfone type.
[0609] The enzyme(s) of the detergent composition of the invention
may be stabilized using conventional stabilizing agents, e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, boric acid, or a boric acid derivative, e.g.,
an aromatic borate ester, or a phenyl boronic acid derivative such
as 4-formylphenyl boronic acid, and the composition may be
formulated as described in, for example, WO 92/19709 and WO
92/19708.
[0610] The detergent may also contain other conventional detergent
ingredients such as, e.g., fabric conditioners including clays,
foam boosters, suds suppressors, anti-corrosion agents,
soil-suspending agents, anti-soil redeposition agents, dyes,
bactericides, optical brighteners, hydrotropes, tarnish inhibitors,
or perfumes.
[0611] In the detergent compositions, any enzyme may be added in an
amount corresponding to 0.01-100 mg of enzyme protein per liter of
wash liquor, preferably 0.05-5 mg of enzyme protein per liter of
wash liquor, in particular 0.1-1 mg of enzyme protein per liter of
wash liquor. In the detergent compositions, a GH61 polypeptide
variant of the present invention having cellulolytic enhancing
activity may be added in an amount corresponding to 0.001-100 mg of
protein, preferably 0.005-50 mg of protein, more preferably 0.01-25
mg of protein, even more preferably 0.05-10 mg of protein, most
preferably 0.05-5 mg of protein, and even most preferably 0.01-1 mg
of protein per liter of wash liquor.
[0612] A GH61 polypeptide variant of the present invention having
cellulolytic enhancing activity may also be incorporated in the
detergent formulations disclosed in WO 97/07202, which is hereby
incorporated by reference.
Plants
[0613] The present invention also relates to plants, e.g., a
transgenic plant, plant part, or plant cell, comprising a
polynucleotide of the present invention so as to express and
produce a GH61 polypeptide variant in recoverable quantities. The
variant may be recovered from the plant or plant part.
Alternatively, the plant or plant part containing the variant may
be used as such for improving the quality of a food or feed, e.g.,
improving nutritional value, palatability, and rheological
properties, or to destroy an antinutritive factor.
[0614] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0615] Examples of dicot plants are tobacco, legumes, such as
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0616] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers as well as the individual tissues
comprising these parts, e.g., epidermis, mesophyll, parenchyme,
vascular tissues, meristems. Specific plant cell compartments, such
as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilization of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seed coats.
[0617] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0618] The transgenic plant or plant cell expressing a variant may
be constructed in accordance with methods known in the art. In
short, the plant or plant cell is constructed by incorporating one
or more expression constructs encoding a variant into the plant
host genome or chloroplast genome and propagating the resulting
modified plant or plant cell into a transgenic plant or plant
cell.
[0619] The expression construct is conveniently a nucleic acid
construct that comprises a polynucleotide encoding a variant
operably linked with appropriate regulatory sequences required for
expression of the polynucleotide in the plant or plant part of
choice. Furthermore, the expression construct may comprise a
selectable marker useful for identifying plant cells into which the
expression construct has been integrated and DNA sequences
necessary for introduction of the construct into the plant in
question (the latter depends on the DNA introduction method to be
used).
[0620] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences, is
determined, for example, on the basis of when, where, and how the
variant is desired to be expressed. For instance, the expression of
the gene encoding a variant may be constitutive or inducible, or
may be developmental, stage or tissue specific, and the gene
product may be targeted to a specific tissue or plant part such as
seeds or leaves. Regulatory sequences are, for example, described
by Tague et al., 1988, Plant Physiology 86: 506.
[0621] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, or the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol.
Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from
metabolic sink tissues such as meristems (Ito et al., 1994, Plant
Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter
from the legumin B4 and the unknown seed protein gene from Vicia
faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a
promoter from a seed oil body protein (Chen et al., 1998, Plant
Cell Physiol. 39: 935-941), the storage protein napA promoter from
Brassica napus, or any other seed specific promoter known in the
art, e.g., as described in WO 91/14772. Furthermore, the promoter
may be a leaf specific promoter such as the rbcs promoter from rice
or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the
chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter
from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or
a wound inducible promoter such as the potato pin2 promoter (Xu et
al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter
may be induced by abiotic treatments such as temperature, drought,
or alterations in salinity or induced by exogenously applied
substances that activate the promoter, e.g., ethanol, oestrogens,
plant hormones such as ethylene, abscisic acid, and gibberellic
acid, and heavy metals.
[0622] A promoter enhancer element may also be used to achieve
higher expression of a variant in the plant. For instance, the
promoter enhancer element may be an intron that is placed between
the promoter and the polynucleotide encoding a variant. For
instance, Xu et al., 1993, supra, disclose the use of the first
intron of the rice actin 1 gene to enhance expression.
[0623] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0624] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0625] Agrobacterium tumefaciens-mediated gene transfer is a method
for generating transgenic dicots (for a review, see Hooykas and
Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for
transforming monocots, although other transformation methods may be
used for these plants. A method for generating transgenic monocots
is particle bombardment (microscopic gold or tungsten particles
coated with the transforming DNA) of embryonic calli or developing
embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994,
Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,
Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Mol. Biol. 21: 415-428.
Additional transformation methods include those described in U.S.
Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein
incorporated by reference in their entirety).
[0626] Following transformation, the transformants having
incorporated the expression construct are selected and regenerated
into whole plants according to methods well known in the art. Often
the transformation procedure is designed for the selective
elimination of selection genes either during regeneration or in the
following generations by using, for example, co-transformation with
two separate T-DNA constructs or site specific excision of the
selection gene by a specific recombinase.
[0627] In addition to direct transformation of a particular plant
genotype with a construct of the present invention, transgenic
plants may be made by crossing a plant having the construct to a
second plant lacking the construct. For example, a construct
encoding a variant can be introduced into a particular plant
variety by crossing, without the need for ever directly
transforming a plant of that given variety. Therefore, the present
invention encompasses not only a plant directly regenerated from
cells which have been transformed in accordance with the present
invention, but also the progeny of such plants. As used herein,
progeny may refer to the offspring of any generation of a parent
plant prepared in accordance with the present invention. Such
progeny may include a DNA construct prepared in accordance with the
present invention. Crossing results in the introduction of a
transgene into a plant line by cross pollinating a starting line
with a donor plant line. Non-limiting examples of such steps are
described in U.S. Pat. No. 7,151,204.
[0628] Plants may be generated through a process of backcross
conversion. For example, plants include plants referred to as a
backcross converted genotype, line, inbred, or hybrid.
[0629] Genetic markers may be used to assist in the introgression
of one or more transgenes of the invention from one genetic
background into another. Marker assisted selection offers
advantages relative to conventional breeding in that it can be used
to avoid errors caused by phenotypic variations. Further, genetic
markers may provide data regarding the relative degree of elite
germplasm in the individual progeny of a particular cross. For
example, when a plant with a desired trait which otherwise has a
non-agronomically desirable genetic background is crossed to an
elite parent, genetic markers may be used to select progeny which
not only possess the trait of interest, but also have a relatively
large proportion of the desired germplasm. In this way, the number
of generations required to introgress one or more traits into a
particular genetic background is minimized.
[0630] The present invention also relates to methods of producing a
variant of the present invention comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding the variant under conditions conducive for production of
the variant; and optionally (b) recovering the variant.
[0631] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
[0632] Strains Aspergillus oryzae strain PFJO218 (amy.sup.-,
alp.sup.-, Npl.sup.-, CPA.sup.-, KA.sup.-, pyrG.sup.-, ku70.sup.-;
U.S. Patent Application 20100221783) was used as an expression host
for the GH61 polypeptide variants.
[0633] Aspergillus oryzae strain COLs1300 was also used as an
expression host for GH61 polypeptide variants. A. niger COLs1300
(amyA, amyB, amyC, alpA, nprA, kusA, niaD, amdS+) was created from
A. oryzae PFJ0220 (EP 2 147 107 B1) by deleting the promoter and 5'
part of both the nitrite reductase (niiA) gene and nitrate
reductase (niaD) gene.
Media and Reagents
[0634] AMG trace metals solution was composed of 14.3 g of
ZnSO.sub.4.7H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, 0.5 g of
NiCl.sub.2.6H.sub.2O, 13.8 g of FeSO.sub.4.7H.sub.2O, 8.5 g of
MnSO.sub.4.H.sub.2O, 3 g of citric acid, and deionized water to 1
liter.
[0635] COLs1300 cultivating medium was composed of 100 ml of
sucrose medium and 1 ml of 1 M urea.
[0636] COLs1300 protoplasting solution was composed of 80 mg of
GLUCANEX.RTM. (Novozymes A/S, Bagsvaerd, Denmark), 0.5 mg/ml of
chitinase (Sigma Chemical Co., Inc., St. Louis, Mo., USA), 10 ml of
1.2 M MgSO.sub.4, and 100 .mu.l of 1 M NaH.sub.2PO.sub.4 pH
5.8.
[0637] COVE-N-Gly plates were composed of 50 ml of COVE salt
solution, 218 g of sorbitol, 10 g of glycerol, 2.02 g of KNO.sub.3,
25 g of Noble agar, and deionized water to 1 liter.
[0638] COVE-N-Gly plates with 10 mM uridine were composed of 50 ml
of COVE salt solution, 218 g of sorbitol, 10 g of glycerol, 2.02 g
of KNO.sub.3, 25 g of Noble agar, and deionized water to 1 liter;
uridine was then added at a concentration of 10 mM to individual
plates.
[0639] COVE salt solution was composed of 26 g of KCl, 26 g of
MgSO.sub.4.7H.sub.2O, 76 g of KH.sub.2PO.sub.4, 50 ml of COVE trace
elements solution, and deionized water to 1 liter.
[0640] COVE trace elements solution was composed of 40 mg of
Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 0.4 g of CuSO.sub.4.5H.sub.2O,
1.2 g of FeSO.sub.4.7H.sub.2O, 0.7 g of MnSO.sub.4. H.sub.2O, 0.8 g
of Na.sub.2MoO.sub.2.2H.sub.2O, 10 g of ZnSO.sub.4.7H.sub.2O, and
deionized water to 1 liter.
[0641] LB medium was composed of 10 g of tryptone, 5 g of yeast
extract, 5 g of NaCl, and deionized water to 1 liter.
[0642] LB+Amp medium was composed of LB medium supplemented with
100 .mu.g of ampicillin per ml.
[0643] M400 medium was composed of 50 g of maltodextrin, 2 g of
MgSO.sub.4.7H.sub.2O, 2 g of KH.sub.2PO.sub.4, 4 g of citric acid,
8 g of yeast extract, 2 g of urea, 0.5 ml of AMG trace metals
solution, 0.5 g of CaCl.sub.2, and deionized water to 1 liter;
adjusted with NaOH to pH 6. After pH adjustment 0.7 ml of antifoam
was added.
[0644] Magnificent Broth was composed of 50 g of Magnificent Broth
powder (MacConnell Research Corp. San Diego, Calif., USA) and
deionized water to 1 liter.
[0645] MaltV1 medium was composed of 20 g of maltose, 10 g of Bacto
Peptone, 1 g of yeast extract, 1.45 g of (NH.sub.4).sub.2SO.sub.4,
2.08 g of KH.sub.2PO.sub.4, 0.28 g of CaCl.sub.2, 0.42 g of
MgSO.sub.4.7H.sub.2O, 0.42 ml of Trichoderma trace metals solution,
0.48 g of citric acid, 19.52 g of 2-(N-morpholino)ethanesulfonic
acid (MES), and deionized water to 1 liter; adjusted with NaOH to
pH 5.5.
[0646] MDU2BP medium (pH 5.0) was composed of 135 g of maltose, 3 g
of MgSO.sub.4.7H.sub.2O, 3 g of NaCl, 6 g of K.sub.2SO.sub.4, 36 g
of KH.sub.2PO.sub.4, 21 g of yeast extract, 6 g of urea, 1.5 ml of
AMG trace metals solution, and deionized water up to 1 liter.
[0647] PEG solution was composed of 6 g of polyethylene glycol 4000
(PEG 4000), 100 .mu.l of 1 M Tris pH 7.5, 100 .mu.l of 1 M
CaCl.sub.2, and deionized water to 10 ml.
[0648] Protoplasting cultivation medium was composed of 92 ml of
transformation sucrose medium, 2 ml of 1 M uridine, 1 ml of 1 M
NaNO.sub.3, and 10 ml of YP medium.
[0649] Protoplasting solution was composed of 15 ml of 1.2 M
MgSO.sub.4, 150 .mu.l of 1 M NaH.sub.2PO.sub.4 (pH 5.8), 100 mg of
GLUCANEX.RTM. (Novozymes A/S, Bagsvaerd, Denmark), and 10 mg of
chitinase (Sigma Chemical Co., Inc., St. Louis, Mo., USA).
[0650] ST solution was composed of 1.5 ml of 2 M sorbitol, 500
.mu.l of 1 M Tris pH 7.5, and deionized water to 5 ml.
[0651] STC solution was composed of 60 ml of 2 M sorbitol, 1 ml of
1 M Tris pH 7.5, 1 ml of 1 M CaCl.sub.2, and deionized water to 100
ml.
[0652] Sucrose medium was composed of 20 ml of COVE salt solution,
342 g of sucrose, and deionized water to 1 liter.
[0653] Sucrose agar plate was composed of 20 ml of Trichoderma
trace element solution, 20 g of Noble agar, 342 g of sucrose, and
deionized water to 1 liter.
[0654] TAE buffer was composed of 40 mM
2-amino-2-hydroxymethyl-propane-1,3-diol, 20 mM Glacial acetic
acid, and 2 mM ethylenediaminetetraacetic acid at pH 8.0.
[0655] TBE buffer was composed of 10.8 g of Tris base, 5.5 g of
boric acid, and 0.74 g of EDTA (pH 8) in deionized water to 1
liter.
[0656] TE buffer was composed of 10 mM Tris-0.1 mM EDTA pH 8.
[0657] Top agar was composed of 500 ml of sucrose medium, 5 g of
low melting agarose, and 10 ml of 20 mM Tris pH 7.5.
[0658] Transformation sucrose medium was composed of 70 ml of 1 M
sucrose and 20 ml of COVE salt solution.
[0659] Trichoderma trace metals solution was composed of 216 g of
FeCl.sub.3.6H.sub.2O, 58 g of ZnSO.sub.4.7H.sub.2O, 27 g of
MnSO.sub.4.H.sub.2O, 10 g of CuSO.sub.4.5H.sub.2O, 2.4 g of
H.sub.3BO.sub.3, 336 g of citric acid, and deionized water to 1
liter.
[0660] 2XYT agar plates were composed of 16 g of tryptone, 10 g of
yeast extract, 5 g of NaCl, 15 g of Bacto agar, and deionized water
to 1 liter.
[0661] 2XYT+Amp agar plates were composed of 2XYT agar supplemented
with 100 .mu.g of ampicillin per ml.
[0662] YP medium was composed of 10 g of Bacto yeast extract, 20 g
of Bacto peptone, and deionized water to 1 liter.
Example 1: Construction of Expression Vectors pMMar44, pMMar49,
pMMar45, and pDFng113
[0663] Plasmid pMMar44 was constructed as described below for
expression of the Aspergillus fumigatus GH61B polypeptide, and
generation of mutant gene libraries. Additionally, plasmids
pMMar49, pMMar45, and pDFng113 were constructed as described below
for expression of the Aspergillus fumigatus GH61B polypeptide
mutant (WO 2012/044835), Penicillium sp. (emersonii) GH61A
polypeptide (hereinafter Penicillium emersonii GH61A polypeptide),
and Thermoascus aurantiacus GH61A polypeptide, respectively, and
generation of variants.
[0664] Plasmid pENI2376 (U.S. Patent Application 20060234340)
containing the AMA sequence for autonomous maintenance in
Aspergillus was digested with Barn HI and Not I to linearize the
plasmid and remove an 8 bp fragment. The digested plasmid was
purified using a PCR Purification Kit (QIAGEN Inc., Valencia,
Calif., USA) according to the manufacturer's instructions.
[0665] The Aspergillus fumigatus GH61B polypeptide coding sequence
(FIG. 1; SEQ ID NO: 29 [genomic DNA sequence] and SEQ ID NO: 30
[deduced amino acid sequence]), mutated Aspergillus fumigatus GH61B
polypeptide coding sequence (WO 2012/044835), Penicillium emersonii
GH61A polypeptide coding sequence (SEQ ID NO: 35 [genomic DNA
sequence] and SEQ ID NO: 36 [deduced amino acid sequence]), and
Thermoascus aurantiacus GH61A polypeptide coding sequence (SEQ ID
NO: 13 [genomic DNA sequence] and SEQ ID NO: 14 [deduced amino acid
sequence]) were amplified from source plasmids described below
using the primers shown in Table 1. Bold letters represent coding
sequence. The remaining sequences are homologous to insertion sites
of pENI2376 for expression of the GH61 polypeptide coding
sequences.
TABLE-US-00002 TABLE 1 GH61 Polypeptide Source origin Template
Plasmid Primer ID Primer Sequence Aspergillus pAG43 (WO pMMar44
AspfuGH61Bp CACAACTGGGGATCCATGACT fumigatus 2010/138754) ENI2376F_2
TTGTCCAAGATCACTTCCA GH61B (SEQ ID NO: 217) AspfuGH61Bp
GGCCTCCGCGGCCGCTTAAG ENI2376R_2 CGTTGAACAGTGCAGGACCA (SEQ ID NO:
218) Mutated pTH230 (WO pMMar49 AfumGH61SD CACAACpATGGGGATCCATGACT
Aspergillus 2012/044835) MB3pENI3376F TTGCCAAGATCACTTCCA fumigatus
(SEQ ID NO: 219) GH61B AfumGH61SD GGCCTCCGCGGCCGCTTAAG MB3pENI3376R
CGTTGAACAGTGCAGGACCA (SEQ ID NO: 220) Penicillium pDM286 pMMar45
PenemGH61pE CACAACTGGGGATCCATGCTG emersonii NI2376F
TCTTCGACGACTCGCACCC GH61A (SEQ ID NO: 221) PenemGH61pE
GGCCTCCGCGGCCGCCTAGA NI2376R ACGTCGGCTCAGGCGGCCCC (SEQ ID NO: 222)
Thermoascus pDZA2 pDFng113 TaGH61aBaM CTGGGGATCCATGTCCTTTTC
aurantiacus (WO HltagF CAAGAT (SEQ ID NO: 223) GH61A 2005/074656)
TaGH61aNcolt CTCCGCGGCCGCTTAACCAGT agR ATACAGAG (SEQ ID NO:
224)
[0666] Construction of plasmid pMMar44 containing the Aspergillus
fumigatus GH61B polypeptide coding sequence is described below. The
Aspergillus fumigatus GH61B polypeptide coding sequence was
amplified from plasmid pAG43 (WO 2010/138754) using the primers
shown in Table 1 with overhangs designed for cloning into plasmid
pENI2376.
[0667] Fifty picomoles of each of the primers listed in Table 1
were used in a PCR reaction composed of 90 ng of pAG43, 1.times.
ADVANTAGE.RTM. 2 PCR Buffer (Clontech Laboratories, Inc., Mountain
View, Calif., USA), 1 .mu.l of a blend of dATP, dTTP, dGTP, and
dCTP, each at 10 mM, and 1.times. ADVANTAGE.RTM. 2 DNA Polymerase
Mix (Clontech Laboratories, Inc., Mountain View, Calif., USA), in a
final volume of 50 .mu.l. The amplification was performed using an
EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 (Eppendorf Scientific, Inc.,
Westbury, N.Y., USA) programmed for 1 cycle at 95.degree. C. for 1
minute; 30 cycles each at 95.degree. C. for 30 seconds, 60.degree.
C. for 30 seconds, and 72.degree. C. for 1 minute; and a final
elongation at 72.degree. C. for 10 minutes. The heat block then
went to a 4.degree. C. soak cycle.
[0668] The reaction product was isolated by 1.0% agarose gel
electrophoresis using TBE buffer where an approximately 862 bp PCR
product band was excised from the gel and extracted using a
QIAQUICK.RTM. Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.,
USA).
[0669] The homologous ends of the 862 bp PCR product and the
digested pENI2376 were joined together using an IN-FUSION.TM.
ADVANTAGE.RTM. PCR Cloning Kit (Clontech Laboratories, Inc.,
Mountain View, Calif., USA). A total of 63 ng of the 862 bp PCR
product and 200 ng of the Barn HI/Not I digested pENI2376 were used
in a reaction composed of 4 .mu.l of 5.times. IN-FUSION.TM.
reaction buffer (Clontech Laboratories, Inc., Mountain View,
Calif., USA) and 2 .mu.l of IN-FUSION.TM. enzyme (Clontech
Laboratories, Inc., Mountain View, Calif., USA), in a final volume
of 20 .mu.l. The reaction was incubated for 15 minutes at
37.degree. C., followed by 15 minutes at 50.degree. C., and then
placed on ice. The reaction volume was increased to 100 .mu.l with
TE buffer and 2 .mu.l of the reaction were transformed into E. coli
XL10-GOLD.RTM. Super Competent Cells (Stratagene, La Jolla, Calif.,
USA) according to the manufacturer's instructions. E. coli
transformants were selected on 2XYT+Amp agar plates. Plasmid DNA
from several of the resulting E. coli transformants was prepared
using a BIOROBOT.RTM. 9600 (QIAGEN Inc., Valencia, Calif., USA).
The Aspergillus fumigatus GH61B polypeptide coding sequence insert
was confirmed by DNA sequencing with a Model 377 XL Automated DNA
Sequencer (Applied Biosystems Inc., Foster City, Calif., USA) using
dye-terminator chemistry (Giesecke et al., 1992, J. Virol. Methods
38: 47-60). Sequencing primers used for verification of the gene
insert and sequence are shown below.
TABLE-US-00003 Primer 996271: (SEQ ID NO: 225)
ACTCAATTTACCTCTATCCACACTT Primer pALLO2 3': (SEQ ID NO: 226)
GAATTGTGAGCGGATAACAATTTCA
[0670] A plasmid containing the correct A. fumigatus GH61B
polypeptide coding sequence was selected and designated pMMar44
(FIG. 2).
[0671] Construction of plasmid pMMar49 containing eight base-pair
changes resulted in four amino acid mutations of the Aspergillus
fumigatus GH61B polypeptide (WO 2012/044835) is described below.
The mutated Aspergillus fumigatus GH61B polypeptide coding sequence
(WO 2012/044835) was amplified from plasmid pTH230 using the
primers shown in Table 1 with overhangs designed for cloning into
plasmid pENI2376.
[0672] Fifty picomoles of each of the primers listed in Table 1
were used in a PCR reaction composed of 100 ng of pTH230, 1.times.
ADVANTAGE.RTM. 2 PCR Buffer (Clontech Laboratories, Inc., Mountain
View, Calif., USA), 1 .mu.l of a blend of dATP, dTTP, dGTP, and
dCTP, each at 10 mM, and 1.times. ADVANTAGE.RTM. 2 DNA Polymerase
Mix (Clontech Laboratories, Inc., Mountain View, Calif., USA), in a
final volume of 50 .mu.l. The amplification was performed using an
EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 1 cycle at
95.degree. C. for 1 minute; 30 cycles each at 95.degree. C. for 30
seconds, 60.degree. C. for 30 seconds, and 72.degree. C. for 1
minute; and a final elongation at 72.degree. C. for 7 minutes. The
heat block then went to a 4.degree. C. soak cycle.
[0673] The reaction product was isolated by 1.0% agarose gel
electrophoresis using TBE buffer where an approximately 862 bp PCR
product band was excised from the gel and extracted using a
QIAQUICK.RTM. Gel Extraction Kit.
[0674] The homologous ends of the 862 bp PCR product and the
digested pENI2376 were joined together using an IN-FUSION.TM.
ADVANTAGE.RTM. PCR Cloning Kit. A total of 90 ng of the 862 bp PCR
product and 220 ng of the Barn HI/Not I digested pENI2376 were used
in a reaction composed of 4 .mu.l of 5.times. IN-FUSION.TM.
reaction buffer and 2 .mu.l of IN-FUSION.TM. enzyme in a final
volume of 20 .mu.l. The reaction was incubated for 15 minutes at
37.degree. C., followed by 15 minutes at 50.degree. C., and then
placed on ice. The reaction volume was increased to 100 .mu.l with
TE buffer and 2 .mu.l of the reaction were transformed into E. coli
XL10-GOLD.RTM. Super Competent Cells according to the
manufacturer's instructions. E. coli transformants were selected on
2XYT+Amp agar plates. Plasmid DNA from several of the resulting E.
coli transformants was prepared using a BIOROBOT.RTM. 9600. The
mutated Aspergillus fumigatus GH61B polypeptide coding sequence
insert was confirmed by DNA sequencing with a Model 377 XL
Automated DNA Sequencer using dye-terminator chemistry (Giesecke et
al., 1992, supra). The sequencing primers 996271 and pALLO2 3' were
used for verification of the gene insert and sequence.
[0675] A plasmid containing the correct mutated A. fumigatus GH61B
polypeptide coding sequence was selected and designated pMMar49
(FIG. 3).
[0676] Construction of plasmid pMMar45 containing the Penicillium
emersonii GH61A polypeptide coding sequence is described below. The
Penicillium emersonii GH61A polypeptide coding sequence was
amplified from plasmid pDM286 containing the Penicillium emersonii
GH61A polypeptide coding sequence using the primers shown in Table
1 with overhangs designed for cloning into plasmid pENI2376.
[0677] Plasmid pDM286 was constructed according to the following
protocol. The P. emersonii GH61A polypeptide gene was amplified
from plasmid pGH61D23Y4 (WO 2011/041397) using PHUSION.TM.
High-Fidelity Hot Start DNA Polymerase (Finnzymes Oy, Espoo,
Finland) and gene-specific forward and reverse primers shown below.
The region in italics represents vector homology to the site of
insertion.
TABLE-US-00004 Forward primer: (SEQ ID NO: 227)
5'-CGGACTGCGCACCATGCTGTCTTCGACGACTCGCAC-3' Reverse primer: (SEQ ID
NO: 228) 5'-TCGCCACGGAGCTTATCGACTTCTTCTAGAACGTC-3'
[0678] The amplification reaction contained 30 ng of plasmid
pGH61D23Y4, 50 pmoles of each of the primers listed above, 1 .mu.l
of a 10 mM blend of dATP, dTTP, dGTP, and dCTP, 1.times.
PHUSION.TM. High-Fidelity Hot Start DNA Polymerase buffer
(Finnzymes Oy, Espoo, Finland) and 1 unit of PHUSION.TM.
High-Fidelity Hot Start DNA Polymerase buffer (Finnzymes Oy, Espoo,
Finland) in a final volume of 50 .mu.l.
[0679] The amplification reaction was incubated in an
EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 epgradient S (Eppendorf
Scientific, Inc., Westbury, N.Y., USA) programmed for 1 cycle at
98.degree. C. for 30 seconds; 35 cycles each at 98.degree. C. for
10 seconds, 60.degree. C. for 30 seconds, and 72.degree. C. for 30
seconds, and 1 cycle at 72.degree. C. for 10 minutes.
[0680] PCR products were separated by 1% agarose gel
electrophoresis using TAE buffer. A 0.87 kb fragment was excised
from the gel and extracted using a NUCLEOSPIN.RTM. Extract II Kit
(Macherey-Nagel, Inc., Bethlehem, Pa., USA) according to the
manufacturer's protocol.
[0681] Plasmid pMJ09 (US 2005/0214920 A1) was digested with Nco I
and Pac I, and after digestion, the digested vector was isolated by
1.0% agarose gel electrophoresis using TBE buffer where an
approximately 7.1 kb fragment was excised from the gel and
extracted using a QIAQUICK.RTM. Gel Extraction Kit. The 0.87 kb PCR
product was inserted into Nco I/Pac I-digested pMJ09 using an
IN-FUSION.TM. ADVANTAGE.RTM. PCR Cloning Kit according to the
manufacturer's protocol. The IN-FUSION.TM. reaction was composed of
1.times. IN-FUSION.TM. Reaction buffer, 180 ng of Not I/Pac I
digested plasmid pMJ09, 108 ng of the 0.87 kb PCR product, and 1
.mu.l of IN-FUSION.TM. Enzyme in a 10 .mu.l reaction volume. The
reaction was incubated for 15 minutes at 37.degree. C. and then for
15 minutes at 50.degree. C. To the reaction 40 .mu.l of TE were
added and 2 .mu.l were used to transform ONE SHOT.RTM. TOP10
competent cells (Invitrogen, Carlsbad, Calif., USA) according to
the manufacturer's protocol. Transformants were screened by
sequencing and one clone containing the insert with no PCR errors
was identified and designated plasmid pDM286. Plasmid pDM286 can be
digested with Pme I to generate an approximately 5.4 kb fragment
for T. reesei transformation. This 5.4 kb fragment contains the
expression cassette [T. reesei Cel7A cellobiohydrolase (CBHI)
promoter, P. emersonii glycosyl hydrolase 61A (GH61A) gene, T.
reesei Cel7A cellobiohydrolase (CBHI) terminator], and Aspergillus
nidulans acetamidase (amdS)gene.
[0682] For construction of pMMar45, 50 picomoles of each of the
primers listed in Table 1 were used in a PCR reaction composed of
120 ng of pDM286, 1.times. EXPAND.RTM. PCR Buffer (Roche
Diagnostics, Inc., Indianapolis, Ind., USA), 1 .mu.l of a blend of
dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 1.times. EXPAND.RTM.
DNA Polymerase Mix (Roche Diagnostics, Inc., Indianapolis, Ind.,
USA), in a final volume of 50 .mu.l. The amplification was
performed using an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed
for 1 cycle at 95.degree. C. for 1 minute; 30 cycles each at
95.degree. C. for 30 seconds, 60.degree. C. for 30 seconds, and
72.degree. C. for 1 minute; and a final elongation at 72.degree. C.
for 10 minutes. The heat block then went to a 4.degree. C. soak
cycle.
[0683] The reaction product was isolated by 1.0% agarose gel
electrophoresis using TBE buffer where an approximately 762 bp PCR
product band was excised from the gel and extracted using a
QIAQUICK.RTM. Gel Extraction Kit.
[0684] The homologous ends of the 762 bp PCR product and the Barn
HI/Not I digested pENI2376 were joined together using an
IN-FUSION.TM. ADVANTAGE.RTM. PCR Cloning Kit. A total of 90 ng of
the 762 bp PCR product and 200 ng of the digested pENI2376 were
used in a reaction composed of 4 .mu.l of 5.times. IN-FUSION.TM.
reaction buffer and 2 .mu.l of IN-FUSION.TM. enzyme, in a final
volume of 20 .mu.l. The reaction was incubated for 15 minutes at
37.degree. C., followed by 15 minutes at 50.degree. C., and then
placed on ice. The reaction volume was increased to 100 .mu.l with
TE buffer and 2 .mu.l of the reaction were transformed into E. coli
XL10-GOLD.RTM. Super Competent Cells according to the
manufacturer's instructions. E. coli transformants were selected on
2XYT+Amp agar plates. Plasmid DNA from several of the resulting E.
coli transformants was prepared using a BIOROBOT.RTM. 9600. The P.
emersonii GH61A polypeptide coding sequence insert was confirmed by
DNA sequencing with a Model 377 XL Automated DNA Sequencer using
dye-terminator chemistry (Giesecke et al., 1992, supra). The
sequencing primers 996271 and pALLO2 3' were used for verification
of the gene insert and sequence.
[0685] A plasmid containing the correct P. emersonii GH61A
polypeptide coding sequence was selected and designated pMMar45
(FIG. 4).
[0686] Construction of plasmid pDFng113 containing the Thermoascus
aurantiacus GH61A polypeptide coding sequence is described below.
The Thermoascus aurantiacus GH61A polypeptide coding sequence was
amplified from plasmid pDZA2 (WO 2005/074656) using the primers
shown in Table 1 with overhangs designed for cloning into plasmid
pENI2376.
[0687] Fifty picomoles of each of the primers listed in Table 1
were used in a PCR reaction composed of 100 ng of pDZA2, 1.times.
EXPAND.RTM. PCR Buffer, 1 .mu.l of a blend of dATP, dTTP, dGTP, and
dCTP, each at 10 mM, and 1.times. EXPAND.RTM. DNA Polymerase Mix,
in a final volume of 50 .mu.l. The amplification was performed
using an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 1
cycle at 94.degree. C. for 2 minutes; 30 cycles each at 94.degree.
C. for 15 seconds, 59.9.degree. C. for 30 seconds, and 72.degree.
C. for 1 minute; and a final elongation at 72.degree. C. for 7
minutes. The heat block then went to a 4.degree. C. soak cycle.
[0688] The reaction product was isolated by 1.0% agarose gel
electrophoresis using TBE buffer where an approximately 822 bp PCR
product band was excised from the gel and extracted using a
QIAQUICK.RTM. Gel Extraction Kit.
[0689] The homologous ends of the 822 bp PCR product and the Barn
HI/Not I digested pENI2376 were joined together using an
IN-FUSION.TM. ADVANTAGE.RTM. PCR Cloning Kit. A total of 37 ng of
the 799 bp PCR product and 200 ng of the digested pENI2376 were
used in a reaction composed of 4 .mu.l of 5.times. IN-FUSION.TM.
reaction buffer and 2 .mu.l of IN-FUSION.TM. enzyme in a final
volume of 20 .mu.l. The reaction was incubated for 15 minutes at
37.degree. C., followed by 15 minutes at 50.degree. C., and then
placed on ice. The reaction volume was increased to 50 .mu.l with
TE buffer and 2 .mu.l of the reaction were transformed into E. coli
XL10-GOLD.RTM. Ultra Competent Cells according to the
manufacturer's instructions. E. coli transformants were selected on
2XYT+Amp agar plates. Plasmid DNA from several of the resulting E.
coli transformants was prepared using a BIOROBOT.RTM. 9600. The T.
aurantiacus GH61A polypeptide coding sequence insert was confirmed
by DNA sequencing with a Model 377 XL Automated DNA Sequencer using
dye-terminator chemistry (Giesecke et al., 1992, supra). The
sequencing primers 996271 and pALLO2 3' were used for verification
of the gene insert and sequence.
[0690] A plasmid containing the correct T. aurantiacus GH61A
polypeptide coding sequence was selected and designated pDFng113
(FIG. 5).
Example 2: Construction of an Aspergillus fumigatus GH61B
Polypeptide Site Saturation Library
[0691] A site saturation library of the Aspergillus fumigatus GH61B
polypeptide coding sequence was synthesized by GeneArt AG
(Regensburg, Germany). An average of 16.8 mutations per position
was synthesized for a total of 165 residues, excluding the most
conserved residues, resulting in a total of 2768 mutants. E. coli
DH10B (Invitrogen, Carlsbad, Calif., USA) strains containing mutant
plasmids with known mutations were arrayed in 96 well plates as 50
.mu.l glycerol stocks, and stored at -80.degree. C.
[0692] DNA was generated from a thawed GeneArt plate by using a
sterile 96 well replicator to stamp the GeneArt plate onto a
2XYT+Amp agar plate. The agar plate was incubated overnight at
37.degree. C. Resulting colonies from the agar plate were used to
inoculate a 96 deep well block with each well containing 1 ml of
Magnificent broth supplemented with 400 .mu.g of ampicillin per ml.
The block was covered with an airpore breathable lid and then
incubated in a humidified box at 37.degree. C. overnight at 350
rpm. The block was centrifuged at 1100.times.g for 10 minutes and
the supernatant discarded. Plasmids were extracted from the cell
pellets using a BIOROBOT.RTM. 9600.
Example 3: Expression of the A. fumigatus GH61B, P. emersonii
GH61A, and T. aurantiacus GH61A Polypeptide Variants in Aspergillus
oryzae PFJO218
[0693] Aspergillus oryzae PFJO218 was inoculated onto a COVE-N-Gly
plate with 10 mM uridine and incubated at 34.degree. C. until
confluent. Spores were collected from the plate by washing with 8
ml of 0.01% TWEEN.RTM. 20. One ml of the spore suspension was used
to inoculate 103 ml of the Protoplasting cultivation medium in a
500 ml polycarbonate shake flask. The shake flask was incubated at
30.degree. C. with agitation at 180 rpm for 17-20 hours. Mycelia
were filtered through a funnel lined with MIRACLOTH.RTM.
(Calbiochem, La Jolla, Calif., USA) and washed with 200 ml of 0.6 M
MgSO.sub.4. Washed mycelia were resuspended in 15 ml of
Protoplasting solution in a 125 ml sterile polycarbonate shake
flask and incubated on ice for 5 minutes. One ml of a solution of
12 mg of bovine serum albumin per ml of deionized water was added
to the shake flask and the shake flask was then incubated at
37.degree. C. with mixing at 70 rpm for 1-3 hours until
protoplasting was complete. The mycelia/protoplast mixture was
filtered through a funnel lined with MIRACLOTH.RTM. into a 50 ml
conical tube and overlayed with 5 ml of ST. The 50 ml conical tube
was centrifuged at 1050.times.g for 15 minutes with slow
acceleration/deceleration. After centrifugation, the liquid was
separated into 3 phases. The interphase which contained the
protoplasts was transferred to a new 50 ml conical tube. Two
volumes of STC were added to the protoplasts followed by a brief
centrifugation at 1050.times.g for 5 minutes. The supernatant was
discarded. The protoplasts were washed twice with 20 ml of STC with
resuspension of the protoplast pellet, centrifugation at
1050.times.g for 10 minutes, and decanting of the supernatant each
time. After the final decanting, the protoplast pellet was
resuspended in STC at a concentration of 1.times.10.sup.8/ml.
Protoplasts were frozen at -80.degree. C. until transformation.
[0694] A 1.3 .mu.l volume of each mutant plasmid was used to
transform 3.5 .mu.l of A. oryzae PFJO218 protoplasts with 3.5 .mu.l
of PEG solution per well in a 24 well plate. Plasmid pMMar44,
pMMar45, or pDFng113 (Table 1) was also transformed as above into
A. oryzae PFJO218 protoplasts to provide broth comprising the A.
fumigatus, P. emersonii, or T. aurantiacus wild-type GH61
polypeptides. The 24 well plate was incubated at 37.degree. C.
stationary for 30 minutes followed by addition of 28.6 .mu.l of
Transformation sucrose medium containing 10 mM NaNO.sub.3 and 14.3
.mu.l of STC. The 24 well plate was then placed in a humidified box
at 37.degree. C. stationary for 7 days. On day 7, 1 ml of MaltV1
medium was added to each well. The plate was returned to the
humidified box at 39.degree. C. stationary and incubated for an
additional 5 days. At least 550 .mu.l of broth for each variant or
the wild-type A. fumigatus, P. emersonii, or T. aurantiacus GH61
polypeptide were harvested using a pipette to remove the mycelia
mat and aspirate the liquid, for assay using PASC as a substrate.
Mutant plasmids resulting in variants with improved thermostability
using a PASC assay (Example 5) were transformed again and retested
using the protocols described above.
[0695] Some of the variants were spore-purified for further
characterization. After a 7 day incubation of the transformation
and prior to the addition of 1 ml of MaltV1 expression medium, a
loop was swiped over the initial growth from the transformation to
collect spores in the well. The spores were then streaked onto a
COVE-N-Gly plate and incubated at 37.degree. C. for approximately
36 hours. Single individual transformants were excised from the
plate and transferred onto fresh COVE-N-Gly plates. The plates were
stored at 34.degree. C. until confluent. Once confluent, a loop
dipped in 0.01% TWEEN.RTM. 20 was swiped over the spores which was
then used to inoculate a 24 well plate with each well containing 1
ml of MaltV1 expression medium. The 24 well plate was placed in a
humidified box at 39.degree. C. Samples were harvested on the fifth
day by removing the mycelia mat and pipetting up the broth.
Example 4: Preparation of Aspergillus fumigatus
Beta-Glucosidase
[0696] Aspergillus fumigatus NN055679 Cel3A beta-glucosidase (SEQ
ID NO: 243 [DNA sequence] and SEQ ID NO: 244 [deduced amino acid
sequence]) was recombinantly prepared according to WO 2005/047499
using Aspergillus oryzae as a host.
[0697] The filtered broth was adjusted to pH 8.0 with 20% sodium
acetate, which made the solution turbid. To remove the turbidity,
the solution was centrifuged at 20,000.times.g for 20 minutes, and
the supernatant was filtered through a 0.2 .mu.m filtration unit
(Nalgene, Rochester, N.Y., USA). The filtrate was diluted with
deionized water to reach the same conductivity as 50 mM Tris/HCl pH
8.0. The adjusted enzyme solution was applied to a Q SEPHAROSE.RTM.
Fast Flow column (GE Healthcare, Piscataway, N.J., USA)
equilibrated with 50 mM Tris-HCl pH 8.0 and eluted with a linear
gradient from 0 to 500 mM sodium chloride. Fractions were pooled
and treated with 1% (w/v) activated charcoal to remove color from
the beta-glucosidase pool. The charcoal was removed by filtration
of the suspension through a 0.2 .mu.m filtration unit (Nalgene,
Rochester, N.Y., USA). The filtrate was adjusted to pH 5.0 with 20%
acetic acid and diluted 10 times with deionized water. The adjusted
filtrate was applied to a SP SEPHAROSE.RTM. Fast Flow column (GE
Healthcare, Piscataway, N.J., USA) equilibrated with 10 mM succinic
acid pH 5.0 and eluted with a linear gradient from 0 to 500 mM
sodium chloride. Protein concentration was determined using a
Microplate BCA.TM. Protein Assay Kit (Thermo Fischer Scientific,
Waltham, Mass., USA) in which bovine serum albumin was used as a
protein standard.
Example 5: Screening of Aspergillus fumigatus GH61B Polypeptide
Variant Libraries
[0698] Using a BIOMEK.RTM. FX Laboratory Automation Workstation
(Beckman Coulter, Fullerton, Calif., USA) with a DYAD.RTM. Thermal
Cycler (Bio-Rad Laboratories, Inc., Richmond, Calif., USA), 80
.mu.l of each broth sample from the library plates of the
Aspergillus fumigatus GH61B variants and parent (wild-type)
polypeptide grown in MaltV1 medium (Example 3) were mixed with 20
.mu.l of 1 M sodium acetate-10 mM MnSO.sub.4 pH 5.0 buffer. The
samples were then heat challenged at 62.degree. C., 65.degree. C.,
and 68.degree. C. for 20 minutes and compared to ambient
temperature controls. After the heat challenge, the broth samples
were diluted 1.25, 2.5, 6.25, and 15.625-fold in 2 mM
MnSO.sub.4-200 mM sodium acetate pH 5 and 12.5 .mu.l of the
dilutions were then transferred to 384-well polypropylene assay
plates containing 25 .mu.l of 1% phosphoric acid swollen cellulose
(PASC) and 12.5 .mu.l of a cofactor solution (400 mM sodium acetate
pH 5, 4 mM MnSO.sub.4, 0.4% gallic acid, 0.1 mg/ml of Aspergillus
fumigatus beta-glucosidase, and 0.04% TRITON.RTM. X100). The plates
were heat-sealed using an ALPS-300.TM. (Abgene, Epsom, United
Kingdom) with a plastic sheet and incubated at 40.degree. C. for 4
days.
[0699] Background glucose concentration of the buffer-treated broth
samples was determined prior to incubation by performing a glucose
assay using the following reagents per liter: 0.9951 g of ATP,
0.5176 g of NAD, 0.5511 g of MgSO.sub.4.7H.sub.2O, 20.9 g of MOPS,
1000 units of hexokinase, 1000 units of glucose-6-phosphate
dehydrogenase, and 0.01% TRITON.RTM. X-100, pH 7.5. The BIOMEK.RTM.
FX Laboratory Automation Workstation was used for this assay. Four
2-fold serial dilutions were performed in 384-well polystyrene
plates using water as diluent. Five .mu.l of the dilutions were
added to a new 384-well polystyrene plate, followed by addition of
60 .mu.l of the above reagents. The plate was incubated at ambient
temperature (22.degree. C..+-.2.degree. C.) for 30 to 45 minutes.
Relative fluorescent units (RFU) were determined using a DTX 880
plate reader (Beckman Coulter, Fullerton, Calif., USA) with
excitation at 360 nm and emission at 465 nm and compared to glucose
standards (1 mg/ml and 0.125 mg/ml) diluted in the same plate as
the samples. At the end of four days, the 40.degree. C. incubated
PASC plates were analyzed for glucose concentration using the
glucose assay described above. Any background glucose was
subtracted from the appropriate samples and then residual activity
was calculated by comparing the glucose released in the PASC assay
of the ambient sample treatment to the glucose released in the PASC
assay of the heat challenge sample treatment. Only data that fits
in the linear part of the curve (defined as less than or equal to 1
mg/ml glucose produced in an assay containing 5 mg/ml PASC) was
used in the calculation. The formula for calculating the residual
activity of the heat treatment was as follows: (mg/ml glucose
produced for heat treated sample/mg/ml glucose produced for ambient
treated sample).times.100%. Improved variants were those having a
higher % residual activity as compared to wild-type A. fumigatus
GH61A polypeptide broth from MaltV1 medium in at least one heat
treatment condition. MICROSOFT.RTM. EXCEL.RTM. (Microsoft
Corporation, Redmond, Wash., USA) was used for all
calculations.
Example 6: Thermostability of Aspergillus fumigatus GH61B
Polypeptide Variants Measured by Residual Activity after Heat
Treatment
[0700] Based on the residual activity ratios as described in
Example 5, screening of libraries constructed in the previous
Examples generated the results listed in Tables 2 and 3.
[0701] Table 2 shows average % Residual Activity (from 3-5 samples
of each variant and the wild type control) after treatment at 62,
65, or 68.degree. C. The parent Aspergillus fumigatus GH61B
polypeptide showed decreased residual activity of 70%, 45%, and 22%
when the temperature was increased from 62.degree. C. to 65.degree.
C. to 68.degree. C., respectively. The increase in thermostability
of the Aspergillus fumigatus GH61B polypeptide variants ranged from
1.03- to 1.1-fold increase at 62.degree. C., 1.09- to 1.4-fold
increase at 65.degree. C., and 1.5- to 2.5-fold increase at
68.degree. C. treatment compared to the wild-type A. fumigatus GH61
polypeptide. The results showed that improvements were most
significant at 68.degree. C. treatment.
TABLE-US-00005 TABLE 2 Variants with improved thermostability at
62.degree. C., 65.degree. C., and 68.degree. C. treatment Avg %
Res. Avg % Res. Avg % Res. Act. 62.degree. C. Standard Act.
65.degree. C. Standard Act. 68.degree. C. Standard Variant
treatment Deviation treatment Deviation treatment Deviation Parent
(Wild-Type) 70% 9% 45% 4% 22% 6% E105K 72% 6% 54% 3% 39% 4% E105P
80% 11% 54% 3% 33% 4% E154L 78% 8% 64% 3% 47% 4% G188A 77% 7% 62%
8% 55% 8% G188W 75% 13% 63% 8% 46% 7% N189K 73% 1% 61% 6% 46% 8%
A216L 75% 8% 62% 4% 42% 3% A216Y 77% 6% 59% 3% 42% 3% K229H 76% 13%
59% 2% 40% 3% K229I 76% 3% 56% 2% 36% 6% K229W 66% 8% 49% 9% 43%
10% K229Y 75% 3% 54% 4% 31% 2%
[0702] Table 3 shows average % Residual Activity (from 3-5 samples
of each variant and 110 samples of the wild type control) after
treatment at 62.degree. C., 65.degree. C., or 68.degree. C. The
parent Aspergillus fumigatus GH61B polypeptide showed decreased
residual activity of 56%, 35%, and 12% when the temperature was
increased from 62.degree. C. to 65.degree. C. to 68.degree. C.,
respectively. The increase in thermostability of the Aspergillus
fumigatus GH61B polypeptide variants ranged from 1.02-fold to
1.2-fold increase at 62.degree. C., 1.14-fold to 1.6-fold increase
at 65.degree. C., and 2.08-fold to 3.25-fold increase at 68.degree.
C. treatment compared to the wild-type A. fumigatus GH61
polypeptide. The results showed that improvements were most
significant at 68.degree. C. treatment.
TABLE-US-00006 TABLE 3 Aspergillus fumigatus GH61B polypeptide
variants with improved thermostability at 62.degree. C., 65.degree.
C., and 68.degree. C. treatment Avg % Res. Avg % Res. Avg % Res.
Act. 62.degree. C. Standard Act. 65.degree. C. Standard Act.
68.degree. C. Standard Variant treatment Deviation treatment
Deviation treatment Deviation Parent (Wild-Type) 56% 13% 35% 16%
12% 10% E105K 61% 24% 50% 23% 38% 21% E105P 57% 23% 44% 21% 32% 21%
E154I 68% 13% 57% 16% 34% 9% E154L 56% 26% 41% 23% 25% 20% G188F
56% 21% 45% 22% 33% 27% G188M 61% 13% 51% 13% 34% 15% G188A 57% 24%
44% 25% 39% 24% G188W 52% 24% 40% 25% 31% 26% N189H 60% 18% 44% 19%
26% 18% N189K 51% 23% 41% 24% 30% 21% A216Y 56% 27% 43% 27% 32% 23%
A216L 56% 24% 46% 25% 33% 21% K229W 51% 22% 41% 21% 39% 26% K229H
58% 24% 46% 22% 31% 21% K229I 58% 25% 45% 24% 30% 21% K229Y 55% 23%
43% 22% 28% 18%
Example 7: Thermostability of Aspergillus fumigatus GH61B
Combinatorial Variants
[0703] Four variants of the Aspergillus fumigatus GH61B polypeptide
were constructed by performing site-directed mutagenesis on pMMar49
(Example 1) using a QUIKCHANGE.RTM. II XL Site-Directed Mutagenesis
Kit (Stratagene, La Jolla, Calif., USA). Two mutagenic primers were
designed for each construct to insert the desired mutation. 125 ng
of each primer (Table 4) was used in a PCR reaction containing
approximately 25 ng of template plasmid, lx QUIKCHANGE.RTM.
reaction buffer, 3 .mu.l of QUIKSOLUTION.RTM., 1 .mu.l of XL dNTP
mix, and 1 .mu.l of 2.5 U/.mu.l Pfu Ultra enzyme in a final volume
of 50 .mu.l. An EPPENDORF.RTM. MASTERCYCLER.RTM. thermocycler was
used with the following settings: 95.degree. C. hot start, one
cycle at 95.degree. C. for 1 minute; 18 cycles each at 95.degree.
C. for 50 seconds, 60.degree. C. for 50 seconds, and 68.degree. C.
for 10 minutes; and 4.degree. C. hold. One microliter of Dpn I was
directly added to the amplification reaction and incubated at
37.degree. C. for 1 hour. A 2 .mu.l volume of the Dpnl digested
reaction was used to transform E. coli XL10-Gold Ultracompetent
Cells (Stratagene, La Jolla, Calif.) according to the
manufacturer's instructions. E. coli transformants were selected on
2XYT+Amp agar plates. One of the clones with the desired mutation
was designated as each plasmid listed below.
[0704] Mutations G188A, G188W, K229W, and N189K were added
individually on top of the A. fumigatus GH61B polypeptide variant
containing mutations L111V, D152S, M155L, and A162W (pMMar49,
Example 1), resulting in pLSBF09-1, pLSBF09-2, pLSBF09-3, and
pLSBF09-4, respectively. A summary of the oligos used for the
site-directed mutagenesis reactions are shown below in Table 4.
[0705] Three additional variants of Aspergillus fumigatus GH61B
were constructed by performing site-directed mutagenesis on
pLSBF09-3 using a QUIKCHANGE.RTM. II XL Site-Directed Mutagenesis
Kit as described above. Mutations G188F, N 189K, and A216Y were
individually added as described, resulting in pDFNG146, pDFNG147,
and pLSBF21. A summary of the oligos used for the site-directed
mutagenesis reactions are shown below in Table 4.
[0706] The seven variant plasmids above were prepared using a
BIOROBOT.RTM. 9600. The variant plasmid constructs were then
sequenced using a 3130xl Genetic Analyzer (Applied Biosystems,
Foster City, Calif., USA) to verify the changes.
TABLE-US-00007 TABLE 4 Plasmid Mutation Oligo ID # Sequence
pLSBF09-1 L111V, 615626 ATCATCGCCCTTCACTCTGCGGCCAACCTGA D152S,
ACGGCGCGCAGAAC (SEQ ID NO: 229) M155L, 615630
GTTCTGCGCGCCGTTCAGGTTGGCCGCAGA A162W, GTGAAGGGCGATGAT (SEQ ID NO:
230) G188A pLSBF09-2 L111V, 615627 ATCATCGCCCTTCACTCTGCGTGGAACCTGA
D152S, ACGGCGCGCAGAAC (SEQ ID NO: 231) M155L, 615631
GTTCTGCGCGCCGTTCAGGTTCCACGCAGA A162W, GTGAAGGGCGATGAT (SEQ ID NO:
232) G188W pLSBF09-3 L111V, 615628 ACAAGAATACTGATCCTGGCATCTGGTTTGA
D152S, CATCTACTCGGATCTGAG (SEQ ID NO: 233) M155L, 615632
CTCAGATCCGAGTAGATGTCAAACCAGATGC A162W, CAGGATCAGTATTCTTGT (SEQ ID
NO: 234) K229W pLSBF09-4 L111V, 615629
TCGCCCTTCACTCTGCGGGTAAGCTGAACGG D152S, CGCGCAGAACTAC (SEQ ID NO:
235) M155L, 615633 GTAGTTCTGCGCGCCGTTCAGCTTACCCGCA A162W,
GAGTGAAGGGCGA (SEQ ID NO: 236) N189K pDFng146 L111V, 1200378
ATCATCGCCCTTCACTCTGCGTTTAACCTGAA D152S, CGGCGCGCAGAAC (SEQ ID NO:
237) M155L, 1200379 GTTCTGCGCGCCGTTCAGGTTAAACGCAGAG A162W,
TGAAGGGCGATGAT (SEQ ID NO: 238) G188F, K229W pDFng147 L111V,
1200380 TCGCCCTTCACTCTGCGGGTAAGCTGAACGG D152S, CGCGCAGAACTAC (SEQ
ID NO: 239) M155L, 1200381 GTAGTTCTGCGCGCCGTTCAGCTTACCCGCA A162W,
GAGTGAAGGGCGA (SEQ ID NO: 240) N189K K229W pLSBF21 L111V, 1200277
GTGCTCAGGGATCTGGCACCTACGGCACGT D152S, CCCTGTACAAGAATA (SEQ ID NO:
241) M155L, 1200278 TATTCTTGTACAGGGACGTGCCGTAGGTGCC A162W,
AGATCCCTGAGCAC (SEQ ID NO: 242) A216Y K229W
[0707] Based on the residual activity ratios determined according
to Example 5, screening of libraries constructed in the previous
Examples generated the results listed in Table 5. Table 5 shows an
average % Residual Activity (from 1-14 samples each for the
combinatorial variants and 23 samples of the wild type) after
treatment at 65.degree. C., 68.degree. C., or 72.degree. C.
[0708] The parent Aspergillus fumigatus GH61 B polypeptide showed
decreased residual activity of 33%, 3%, and 1% when the temperature
of treatment was increased from 65.degree. C. to 68.degree. C. to
72.degree. C., respectively. The increase in thermostability of the
Aspergillus fumigatus GH61B polypeptide combinatorial variants
ranged from 1.66-fold to 2.42-fold increase at 65.degree. C.,
14.36-fold to 19.57-fold increase at 68.degree. C., and 31.45-fold
to 80.07-fold increase at 72.degree. C. compared to the wild-type
A. fumigatus GH61 polypeptide. The results showed that improvements
were most significant at 72.degree. C. treatment.
TABLE-US-00008 TABLE 5 Aspergillus fumigatus GH61B polypeptide
variants with improved thermostability at 65.degree. C., 68.degree.
C., and 72.degree. C. treatment Avg % Res. Standard Avg % Res.
Standard Avg % Res. Standard Mutations Act. 65.degree. C. Deviation
Act. 68.degree. C. Deviation Act. 72.degree. C. Deviation L111V,
D152S, M155L, 55% 10% 56% 2% 51% 7% A162W, G188F, K229W L111V,
D152S, M155L, 77% 8% 56% 6% 30% 5% A162W, G188A L111V, D152S,
M155L, 78% 15% 64% 16% 30% 6% A162W, A216Y, K229W L111V, D152S,
M155L, 66% 16% 54% 18% 25% 10% A162W, K229W L111V, D152S, M155L,
58% ND 47% ND 24% ND A162W, N189K, K229W L111V, D152S, M155L, 67%
8% 52% 4% 20% 18% A162W, N189K L111V, D152S, M155L, 80% 10% 57% 10%
20% 11% A162W, G188W L111V, D152S, M155L, 53% 11% 34% 7% 7% 6%
A162W Wild-Type 33% 12% 3% 9% 0.6% 3%
Example 8: Purification of Aspergillus fumigatus GH61B Polypeptide
Variants
[0709] Expression and purification of the wild-type Aspergillus
fumigatus GH61B polypeptide was conducted as previously described
in WO 2012/044835.
[0710] The Aspergillus fumigatus GH61B polypeptide variant strains
were grown to recover culture broths for purification. Following
isolation of single colonies, Aspergillus oryzae PFJO218
transformants were cultured for 4 days at 34.degree. C. on
COVE-N-GLY plates in preparation for larger scale fermentation.
Spores were recovered from each plate using 0.01% TWEEN.RTM. 20.
Each spore suspension (500 .mu.l) was inoculated into 25 ml of M400
medium in 125 ml plastic shake flasks. The transformants were
fermented for 3 days at 39.degree. C. with agitation at 150 rpm and
the broths were collected and filtered using 0.22 .mu.m filters.
The filtered culture broths were then concentrated by centrifugal
ultrafiltration using VIVACELL.RTM. 100 5 kDa MWCO centrifugal
concentration devices (Sartorius Stedim, Goettingen, Germany) and
then buffer exchanged into 20 mM Tris-HCl pH 8.5.
[0711] The concentrated and buffer exchanged Aspergillus fumigatus
GH61B polypeptide variants were further purified by one of two
chromatographic methods. In one method, the concentrated and buffer
exchanged broths were then each applied to a MONO Q.RTM. HR 16/10
column (GE Healthcare, Piscataway, N.J., USA) equilibrated with 20
mM Tris-HCl pH 8.0. Bound proteins were eluted with a linear
gradient of 0-600 mM sodium chloride in 20 mM Tris-HCl pH 8.0.
Fractions were analyzed by SDS-PAGE using a CRITERION.RTM.
Stain-Free Tris-HCl 8-16% SDS-PAGE gel (Bio-Rad Laboratories, Inc.,
Hercules, Calif., USA), pooled based on the abundance of an
approximately 25 kDa band, and concentrated using VIVASPIN.RTM. 5
kDa MWCO centrifugal concentration devices (GE Healthcare,
Buckinghamshire, United Kingdom). Alternatively, the concentrated
and desalted broths were then each applied to a HILOAD.RTM. 26/60
SUPERDEX.RTM. 75 (GE Healthcare, Piscataway, N.J., USA) size
exclusion column which had been equilibrated with 20 mM Tris-HCl pH
8.0 and 150 mM NaCl. Applied proteins were eluted isocraticly using
20 mM Tris-HCl pH 8.0 and 150 mM NaCl as the mobile phase.
Fractions were analyzed by SDS-PAGE using a CRITERION.RTM.
Stain-Free Tris-HCl 8-16% SDS-PAGE gel, pooled based on the
abundance of an approximately 25 kDa band, and concentrated using
VIVASPIN.RTM. 5 kDa MWCO centrifugal concentration devices.
[0712] Protein concentrations were determined using a Microplate
BCA.TM. Protein Assay Kit in which bovine serum albumin was used as
a protein standard.
Example 9: Determination of Tm (Melting Temperature) of the
Aspergillus fumigatus Wild-Type GH61B Polypeptide and Aspergillus
fumigatus GH61B Polypeptide Variants by Differential Scanning
Calorimetry
[0713] The thermostabilities of the A. fumigatus wild-type GH61B
polypeptide and the Aspergillus fumigatus GH61B polypeptide
variants, which were purified as described in Example 8, were
determined by Differential Scanning calorimetry (DSC) using a VP
Differential Scanning calorimeter (MicroCal Inc., GE Healthcare,
Piscataway, N.J., USA). The melting temperature, Tm (.degree. C.),
was taken as the top of denaturation peak (major endothermic peak)
in thermograms (Cp vs. T) obtained after heating a 1 mg protein per
ml solution of the enzyme in 50 mM sodium acetate pH 5.0, 100 .mu.M
CuSO.sub.4, or a 1 mg protein per ml solution of the enzyme in 50
mM sodium acetate pH 5.0, 10 mM EDTA pH 5.0, at a constant
programmed heating rate. One ml of sample and reference-solutions
were degassed at 25.degree. C. using a ThermoVac (MicroCal Inc., GE
Healthcare, Piscataway, N.J., USA) prior to loading of sample and
reference cells of the calorimeter. Sample and reference
(reference: degassed water) solutions were manually loaded into the
DSC and thermally pre-equilibrated to 25.degree. C. before the DSC
scan was performed from 25.degree. C. to 95.degree. C. at a scan
rate of 90 K/hour. Denaturation temperatures were determined at an
accuracy of approximately +/-1.degree. C. The results of the
thermostability determination of the A. fumigatus GH61 B
polypeptide variants are shown in Table 6.
TABLE-US-00009 TABLE 6 Melting temperatures (.degree. C.) of the A.
fumigatus GH61B polypeptide and variants of the A. fumigatus GH61B
polypeptide, as determined by differential scanning calorimetry Tm
+ 100 .mu.m Tm + 10 mM Mutations CuSO.sub.4 EDTA pH 5 Wild-Type 69
59 G188A 75 n.d. G188W 75 63 N189K 74 63 K229W 74 63 L111V + D152S
+ M155L + A162W 76 66 L111V + D152S + M155L + A162W + n.d. 70 K229W
L111V + D152S + M155L + A162W + 83 73 G188F + K229W
Example 10: Determination of Tm (Melting Temperature) of the
Aspergillus fumigatus Wild-Type GH61B Polypeptide and Aspergillus
fumigatus GH61B Polypeptide Variants by Protein Thermal Unfolding
Analysis
[0714] Protein thermal unfolding of the Aspergillus fumigatus GH61B
polypeptide variants was monitored using SYPRO.RTM. Orange Protein
Stain (Invitrogen, Naerum, Denmark) using a StepOnePlus.TM.
Real-Time PCR System (Applied Biosystems Inc., Foster City, Calif.,
USA). In a 96-well white PCR-plate, 15 .mu.l of a protein sample
(prepared as described in Example 8) in 100 mM sodium acetate pH
5.0 was mixed (1:1) with Sypro Orange (resulting
concentration=10.times.; stock solution=5000.times. in DMSO) in 20
mM EDTA. The plate was sealed with an optical PCR seal. The PCR
instrument was set at a scan-rate of 76.degree. C. per hour,
starting at 25.degree. C. and finishing at 96.degree. C.
Fluorescence was monitored every 20 seconds using a built-in LED
blue light for excitation and ROX-filter (610 nm, emission).
Tm-values were calculated as the maximum value of the first
derivative (dF/dK) (Gregory et al., 2009, J. Biomol. Screen. 14:
700). The results of the thermostability determinations are shown
in Table 7.
TABLE-US-00010 TABLE 7 Melting temperatures (.degree. C.) of the A.
fumigatus GH61B polypeptide and variants determined by thermal
unfolding analysis Mutations Tm Wild-Type 59 E105P 62 E154L 61
A216Y 60 A216L 63 K229H 61 K229I 60
Example 11: Preparation of a High-Temperature Cellulase
Composition
[0715] Aspergillus fumigatus GH7A cellobiohydrolase I (SEQ ID NO:
245 [genomic DNA sequence] and SEQ ID NO: 246 [deduced amino acid
sequence]) was prepared recombinantly in Aspergillus oryzae as
described in WO 2011/057140. The filtered broth of the Aspergillus
fumigatus GH7A cellobiohydrolase I was concentrated and buffer
exchanged with 20 mM Tris-HCl pH 8.0 using a tangential flow
concentrator (Pall Filtron, Northborough, Mass., USA) equipped with
a 10 kDa polyethersulfone membrane (Pall Filtron, Northborough,
Mass., USA).
[0716] Aspergillus fumigatus GH6A cellobiohydrolase II (SEQ ID NO:
247 [genomic DNA sequence] and SEQ ID NO: 248 [deduced amino acid
sequence]) was prepared recombinantly in Aspergillus oryzae as
described in WO 2011/057140. The filtered broth of the Aspergillus
fumigatus GH6A cellobiohydrolase II was concentrated and buffer
exchanged with 20 mM Tris-HCl pH 8.0 using a tangential flow
concentrator equipped with a 10 kDa polyethersulfone membrane.
[0717] Trichoderma reesei GH5 endoglucanase II (SEQ ID NO: 249
[genomic DNA sequence] and SEQ ID NO: 250 [deduced amino acid
sequence]) was prepared recombinantly in Aspergillus oryzae as
described in WO 2011/057140. Filtered broth of the Trichoderma
reesei GH5 endoglucanase II was concentrated and buffer exchanged
with 20 mM Tris-HCl pH 8.0 using a tangential flow concentrator
equipped with a 10 kDa polyethersulfone membrane. Aspergillus
fumigatus GH10 xylanase (xyn3) (SEQ ID NO: 251 [genomic DNA
sequence] and SEQ ID NO: 252 [deduced amino acid sequence]) was
prepared recombinantly according to WO 2006/078256 using
Aspergillus oryzae BECh2 (WO 2000/39322) as a host. Filtered broth
of the Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) was
desalted and buffer-exchanged with 50 mM sodium acetate pH 5.0
using a HIPREP.RTM. 26/10 Desalting column (GE Healthcare,
Piscataway, N.J., USA) according to the manufacturer's
instructions.
[0718] Aspergillus fumigatus Cel3A beta-glucosidase was prepared as
described in Example 4.
[0719] Aspergillus fumigatus GH3 beta-xylosidase (SEQ ID NO: 253
[genomic DNA sequence] and SEQ ID NO: 254 [deduced amino acid
sequence]) was prepared recombinantly in Aspergillus oryzae as
described in WO 2011/057140 and purified according to WO
2011/057140.
[0720] The protein concentration for each of the monocomponents
described above was determined using a Microplate BCA.TM. Protein
Assay Kit in which bovine serum albumin was used as a protein
standard. The cellulase composition was composed of 44.7%
Aspergillus fumigatus Cel7A cellobiohydrolase I, 29.4% Aspergillus
fumigatus Cel6A cellobiohydrolase II, 11.8% Trichoderma reesei GH5
endoglucanase II, 5.9% Aspergillus fumigatus GH10 xylanase (xyn3),
5.9% Aspergillus fumigatus beta-glucosidase, and 2.3% Aspergillus
fumigatus beta-xylosidase. The cellulase composition is designated
herein as a "high-temperature cellulase composition".
Example 12: Pretreated Corn Stover Hydrolysis Assay
[0721] Corn stover was pretreated at the U.S. Department of Energy
National Renewable Energy Laboratory (NREL) using 1.4 wt % sulfuric
acid at 165.degree. C. and 107 psi for 8 minutes. The
water-insoluble solids in the pretreated corn stover (PCS)
contained approximately 59% cellulose, 5% hemicelluloses, and 28%
lignin. Cellulose and hemicellulose were determined by a two-stage
sulfuric acid hydrolysis with subsequent analysis of sugars by high
performance liquid chromatography using NREL Standard Analytical
Procedure #002. Lignin was determined gravimetrically after
hydrolyzing the cellulose and hemicellulose fractions with sulfuric
acid using NREL Standard Analytical Procedure #003.
[0722] The hydrolysis of PCS was conducted using 2.2 ml deep-well
plates (Axygen, Union City, Calif., USA) in a total reaction volume
of 1.0 ml. The hydrolysis was performed with 50 mg of PCS per ml of
50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese
sulfate and a protein loading of the high-temperature cellulase
composition (expressed as mg protein per gram of cellulose). Enzyme
mixtures were prepared and then added simultaneously to all wells
in a volume of 100 .mu.l, for a final volume of 1 ml in each
reaction. The plate was then sealed using an ALPS-300.TM. plate
heat sealer (Abgene, Epsom, United Kingdom), mixed thoroughly, and
incubated at 50.degree. C., 55.degree. C., and 60.degree. C. for 72
hours. All experiments were performed in triplicate.
[0723] Following hydrolysis, samples were filtered with a 0.45
.mu.m MULTISCREEN.RTM. 96-well filter plate (Millipore, Bedford,
Mass., USA) and filtrates analyzed for sugar content as described
below. When not used immediately, filtered sugary aliquots were
frozen at -20.degree. C. The sugar concentrations of samples
diluted in 0.005 M H.sub.2SO.sub.4 were measured using a
4.6.times.250 mm AMINEX.RTM. HPX-87H column (Bio-Rad Laboratories,
Inc., Hercules, Calif., USA) by elution with 0.05% w/w benzoic
acid-0.005 M H.sub.2SO.sub.4 at a flow rate of 0.6 ml per minute at
65.degree. C., and quantitation by integration of glucose and
cellobiose signals using a refractive index detector
(CHEMSTATION.RTM., AGILENT.RTM. 1100 HPLC, Agilent Technologies,
Santa Clara, Calif., USA) calibrated by pure sugar samples. The
resultant equivalents were used to calculate the percentage of
cellulose conversion for each reaction.
[0724] All HPLC data processing was performed using MICROSOFT
EXCEL.TM. software (Microsoft, Richland, Wash., USA). Measured
sugar concentrations were adjusted for the appropriate dilution
factor. Glucose and cellobiose were measured individually. However,
to calculate total conversion the glucose and cellobiose values
were combined. Cellobiose concentration was multiplied by 1.053 in
order to convert to glucose equivalents and added to the glucose
concentration. The degree of cellulose conversion was calculated
using the following equation:
% conversion=([sample glucose concentration]/[glucose concentration
in a limit digest]).times.100
[0725] In order to calculate % conversion, a 100% conversion point
was set based on a cellulase control of 50 mg of the cellulase
composition per gram cellulose (CELLUCLAST PLUS.TM., Novozymes A/S,
Bagsvaerd, Denmark), and all values were divided by this number and
then multiplied by 100. Triplicate data points were averaged and
standard deviation was calculated.
Example 13: Effect of Addition of Aspergillus fumigatus GH61B
Polypeptide Variants in the Conversion of PCS by a High-Temperature
Cellulase Composition at 50.degree. C., 55.degree. C., and
60.degree. C.
[0726] A. fumigatus GH61B wild-type polypeptide and Aspergillus
fumigatus GH61B polypeptide variants N189K, G188W, K229W, and G188A
were evaluated for their ability to enhance the hydrolysis of PCS
by the high temperature cellulase composition (Example 11) at
50.degree. C., 55.degree. C., or 60.degree. C. The pretreated corn
stover hydrolysis assay was performed as described in Example 12.
The high temperature composition when loaded at 3 mg total protein
per gram cellulose in the assay had the following enzyme loadings
per gram cellulose: 1.34 mg of A. fumigatus cellobiohydrolase I per
gram cellulose, 0.88 mg of A. fumigatus cellobiohydrolase II per
gram cellulose, 0.18 mg of A. fumigatus beta-glucosidase per gram
cellulose, 0.18 mg of Aspergillus fumigatus GH10 xylanase (Xyl3)
per gram cellulose, 0.18 mg of A. fumigatus beta-xylosidase per
gram cellulose, and 0.35 mg of T. reesei CELSA endoglucanase II per
gram cellulose.
[0727] The conversion of pretreated corn stover by the high
temperature cellulase composition (3 mg protein per gram
cellulose); the combination of the high temperature cellulase
composition (3 mg protein per gram cellulose) and A. fumigatus
GH61B wild-type polypeptide (0.25 mg protein per gram cellulose);
the combination of the high temperature cellulase composition (3 mg
protein per gram cellulose) and Aspergillus fumigatus GH61B variant
N168K polypeptide (0.25 mg protein per gram cellulose); the
combination of the high temperature cellulase composition (3 mg
protein per gram cellulose) and Aspergillus fumigatus GH61B variant
G167W polypeptide (0.25 mg protein per gram cellulose); the
combination of the high temperature cellulase composition (3 mg
protein per gram cellulose) and Aspergillus fumigatus GH61B variant
K208W polypeptide (0.25 mg protein per gram cellulose); and the
combination of the high temperature cellulase composition (3 mg
protein per gram cellulose) and Aspergillus fumigatus GH61B variant
G167A polypeptide (0.25 mg protein per gram cellulose) were assayed
as described in Example 12. Data were collected and analyzed, as
described in Example 12, after 72 hours of incubation at 50.degree.
C., 55.degree. C., or 60.degree. C. These results are shown in FIG.
6.
[0728] The high temperature cellulase composition (3 mg protein per
gram cellulose) resulted in conversions of 46.8.+-.0.3%,
47.9.+-.1.0%, and 45.2.+-.0.2% at 50.degree. C., 55.degree. C., or
60.degree. C., respectively, of the pretreated corn stover.
[0729] The combination of the high temperature cellulase
composition (3 mg protein per gram cellulose) and A. fumigatus
GH61B wild-type polypeptide (0.25 mg protein per gram cellulose)
resulted in conversions of 55.3.+-.0.3%, 56.2.+-.0.7%, and
51.3.+-.0.4% at 50.degree. C., 55.degree. C., or 60.degree. C.,
respectively, of the pretreated corn stover.
[0730] The combination of the high temperature cellulase
composition (3 mg protein per gram cellulose) and A. fumigatus
GH61B variant N189K polypeptide (0.25 mg protein per gram
cellulose) resulted in conversions of the pretreated corn stover of
56.3.+-.0.5%, 57.5.+-.0.3%, and 51.3.+-.0.3% at 50.degree. C.,
55.degree. C., or 60.degree. C., respectively, of the pretreated
corn stover.
[0731] The combination of the high temperature cellulase
composition (3 mg protein per gram cellulose) and A. fumigatus
GH61B variant G188W polypeptide (0.25 mg protein per gram
cellulose) resulted in conversions of 55.8.+-.0.4%, 57.5.+-.0.4%,
and 52.4.+-.0.04% at 50.degree. C., 55.degree. C., or 60.degree.
C., respectively, of the pretreated corn stover.
[0732] The combination of the high temperature cellulase
composition (3 mg protein per gram cellulose) and A. fumigatus
GH61B variant K229W polypeptide (0.25 mg protein per gram
cellulose) resulted in conversions of 56.8.+-.0.7%, 57.6.+-.0.1%,
and 53.0.+-.0.3% at 50.degree. C., 55.degree. C., or 60.degree. C.,
respectively, of the pretreated corn stover.
[0733] The combination of the high temperature cellulase
composition (3 mg protein per gram cellulose) and A. fumigatus
GH61B variant G188A polypeptide (0.25 mg protein per gram
cellulose) resulted in conversions of 55.9.+-.0.3%, 56.9.+-.0.5%,
and 51.3.+-.0.3% at 50.degree. C., 55.degree. C., or 60.degree. C.,
respectively, of the pretreated corn stover.
Example 14: Construction of Penicillium emersonii GH61A and
Thermoascus aurantiacus GH61A Polypeptide Variants
[0734] Variants of the Penicillium emersonii GH61A polypeptide (SEQ
ID NO: 35 [DNA sequence] and SEQ ID NO: 36 [amino acid sequence]),
and Thermoascus aurantiacus GH61A polypeptide (SEQ ID NO: 13 [DNA
sequence] and SEQ ID NO: 14 [amino acid sequence]) were constructed
by performing site-directed mutagenesis on plasmids pMMar45 and
pDFng113, respectively, using a QUIKCHANGE.RTM. Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, Calif., USA) as described in
Example 7. A summary of the primers used for the site-directed
mutagenesis and the variants obtained are shown in Table 8. The
same protocol described in Example 7 was used.
[0735] The resulting mutant plasmid DNAs were prepared using a
BIOROBOT.RTM. 9600. Each mutant plasmid was sequenced using a
3130xl Genetic Analyzer to verify the substitutions. The sequencing
primers 996271 and pALLO2 3' were used for verification.
TABLE-US-00011 TABLE 8 Variant Variant Template Amino Acid Primer
Plasmid Backbone Substitution ID Primer Sequence Name Penicillium
D109P 615190 GCAGTGGACGCCGTGGCCGCCGAG pLSBF07-1 emersonii
CCACCACGGACCCGTCAT (SEQ ID GH61A NO: 255) 615201
ATGACGGGTCCGTGGTGGCTCGGC GGCCACGGCGTCCACTGC (SEQ ID NO: 256)
Penicillium D109K 615191 GCAGTGGACGCCGTGGCCGAAGAG pLSBF07-2
emersonii CCACCACGGACCCGTCAT (SEQ ID GH61A NO: 257) 615202
ATGACGGGTCCGTGGTGGCTCTTCG GCCACGGCGTCCACTGC (SEQ ID NO: 258)
Penicillium N192A 615193 CATCGCCCTGCACTCGGCCGCCAAC pLSBF07-4
emersonii AAGGACGGCGCCCAGAAC (SEQ ID GH61A NO: 259) 615204
GTTCTGGGCGCCGTCCTTGTTGGCG GCCGAGTGCAGGGCGATG (SEQ ID NO: 260)
Penicillium N192W 615194 CATCGCCCTGCACTCGGCCTGGAAC pLSBF07-5
emersonii AAGGACGGCGCCCAGAAC (SEQ ID GH61A NO: 261) 615205
GTTCTGGGCGCCGTCCTTGTTCCAG GCCGAGTGCAGGGCGATG (SEQ ID NO: 262)
Penicillium N193K 615195 CGCCCTGCACTCGGCCAACAAGAAG pLSBF07-6
emersonii GACGGCGCCCAGAACTAC (SEQ ID GH61A NO: 263) 615206
GTAGTTCTGGGCGCCGTCCTTCTTG TTGGCCGAGTGCAGGGCG (SEQ ID NO: 264)
Thermoascus D105K 615253 GCTTCAATGGACTCCATGGCCTAAA pDFng113-1
aurantiacus TCTCACCATGGCCCAGTTATCA GH61A (SEQ ID NO: 265) 615254
TGATAACTGGGCCATGGTGAGATTT AGGCCATGGAGTCCATTGAAGC (SEQ ID NO: 266)
Thermoascus D105P 615255 GCTTCAATGGACTCCATGGCCTCCT pDFng113-3
aurantiacus TCTCACCATGGCCCAGTTATCA GH61A (SEQ ID NO: 267) 615256
TGATAACTGGGCCATGGTGAGAAGG AGGCCATGGAGTCCATTGAAGC (SEQ ID NO: 268)
Thermoascus Q188W 615273 GAGATTATTGCTCTTCACTCAGCTTG pDFng113-
aurantiacus GAACCAGGATGGTGCCCAGAAC 28 GH61A (SEQ ID NO: 269) 615275
GTTCTGGGCACCATCCTGGTTCCAA GCTGAGTGAAGAGCAATAATCTC (SEQ ID NO:
270)
[0736] The P. emersonii GH61A polypeptide variants and T.
aurantiacus GH61A polypeptide variants were expressed using
Aspergillus oryzae PFJO218 as host was performed according to the
procedure described in Example 3.
Example 15: Preparation of Penicillium emersonii Wild-Type GH61A
Polypeptide and P. emersonii GH61A Polypeptide Variants
[0737] The P. emersonii GH61A polypeptide wild-type and variant
strains were grown as described in Example 8 to recover culture
broths for purification.
[0738] The filtered culture broths were then concentrated by
centrifugal ultrafiltration using VIVACELL.RTM. 20 5 kDa MWCO
centrifugal concentration devices (Sartorius Stedim, Goettingen,
Germany) and then buffer exchanged into 20 mM Tris-HCl pH 8.0.
Protein concentrations were determined using a Microplate BCA.TM.
Protein Assay Kit in which bovine serum albumin was used as a
protein standard.
[0739] In the cases of P. emersonii GH61A wild-type polypeptide, P.
emersonii GH61A polypeptide variant N192W, and P. emersonii GH61A
polypeptide variant N193K, further purification was conducted by
application of the concentrated and buffer exchanged broths to
HITRAP.RTM. Q SEPHAROSE.RTM. Fast Flow columns (GE Healthcare,
Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl pH 8.0.
Bound proteins were eluted with a linear gradient of 0-500 mM
sodium chloride in 20 mM Tris-HCl pH 8.0. Fractions were analyzed
by SDS-PAGE using a CRITERION.RTM. Tris-HCl 8-16% SDS-PAGE gel
(Bio-Rad Laboratories, Inc., Hercules, Calif., USA), pooled based
on the abundance of an approximately 25 kDa band, and concentrated
using VIVASPIN.RTM. 5 kDa MWCO centrifugal concentration devices.
Protein concentrations were determined using a Microplate BCA.TM.
Protein Assay Kit in which bovine serum albumin was used as a
protein standard.
Example 16: Preparation of Thermoascus aurantiacus GH61A
Polypeptide Variants
[0740] The T. aurantiacus GH61A polypeptide wild-type and variant
strains were grown as described in Example 8 to recover culture
broths for purification.
[0741] The filtered culture broths were then concentrated by
centrifugal ultrafiltration using VIVACELL.RTM. 20 5 kDa MWCO
centrifugal concentration devices (Sartorius Stedim, Goettingen,
Germany) and then buffer exchanged into 20 mM Tris-HCl pH 8.0.
Protein concentrations were determined using a Microplate BCA.TM.
Protein Assay Kit in which bovine serum albumin was used as a
protein standard.
[0742] The purification was conducted by application of the
concentrated and buffer exchanged broths to HITRAP.RTM. Q
SEPHAROSE.RTM. Fast Flow columns equilibrated with 20 mM Tris-HCl
pH 8.0. Bound proteins were eluted with a linear gradient of 0-500
mM sodium chloride in 20 mM Tris-HCl pH 8.0. Fractions were
analyzed by SDS-PAGE using a CRITERION.RTM. Tris-HCl 8-16% SDS-PAGE
gel, pooled based on the abundance of an approximately 25 kDa band,
and concentrated using VIVASPIN.RTM. 5 kDa MWCO centrifugal
concentration devices. Protein concentrations were determined using
a Microplate BCA.TM. Protein Assay Kit in which bovine serum
albumin was used as a protein standard.
Example 17: Determination of Tm (Melting Temperature) of the P.
emersonii Wild-Type GH61A Polypeptide and P. emersonii GH61A
Polypeptide Variants by Differential Scanning Calorimetry
[0743] The thermostabilities of the P. emersonii wild-type GH61A
polypeptide and the P. emersonii GH61A polypeptide variants,
purified as described in Example 15, were determined by
Differential Scanning calorimetry (DSC) using a VP Differential
Scanning calorimeter. The melting temperature, Tm (.degree. C.),
was taken as the top of denaturation peak (major endothermic peak)
in thermograms (Cp vs. T) obtained after heating a 1 mg protein per
ml solution of the enzyme in 50 mM sodium acetate pH 5.0, 100 .mu.M
CuSO.sub.4, or a 1 mg protein per ml solution of the enzyme in 50
mM sodium acetate pH 5.0, 10 mM EDTA pH 5.0, at a constant
programmed heating rate. One ml of sample and reference-solutions
were degassed at 25.degree. C. using a ThermoVac prior to loading
of sample and reference cells of the calorimeter. Sample and
reference (reference: degassed water) solutions were manually
loaded into the DSC and thermally pre-equilibrated to 25.degree. C.
before the DSC scan was performed from 25.degree. C. to 95.degree.
C. at a scan rate of 90 K/hour. Denaturation temperatures were
determined at an accuracy of approximately +/-1.degree. C.
[0744] The results of the thermostability determination of the P.
emersonii GH61A polypeptide variants are shown in Table 9.
TABLE-US-00012 TABLE 9 Melting temperatures (.degree. C.) of P.
emersonii GH61A polypeptide and variants of P. emersonii GH61A
polypeptide, as determined by differential scanning calorimetry
Enzyme sample Tm + 100 .mu.M CuSO.sub.4 P. emersonii GH61A 82 P.
emersonii GH61A N192W 84 P. emersonii GH61A N193K 83
Example 18: Determination of Tm (Melting Temperature) of
Penicillium emersonii GH61A and Thermoascus aurantiacus GH61A
Polypeptide Variants by Protein Thermal Unfolding Analysis
[0745] Protein thermal unfolding of the Penicillium emersonii GH61A
and Thermoascus aurantiacus GH61A polypeptide variants was
monitored using SYPRO.RTM. Orange Protein Stain and was performed
using a StepOnePlus.TM. Real-Time PCR System as described as
Example 10. P. emersonii GH61A wild-type polypeptide and P.
emersonii GH61A polypeptide variants were concentrated and buffer
exchanged as described in Example 15. T. aurantiacus GH61A
wild-type polypeptide and T. aurantiacus GH61A polypeptides
variants were purified as described in Example 16. The results of
the thermostability determination are shown in Table 10.
TABLE-US-00013 TABLE 10 Melting temperature (.degree. C.) of
Penicillium emersonii GH61A and Thermoascus aurantiacus GH61A
polypeptide variants by protein thermal unfolding analysis Protein
backbone Sample type Mutations Tm P. emersonii GH61A Concentrated
Wild-Type 69 P. emersonii GH61A Concentrated D109P 71 P. emersonii
GH61A Concentrated D109K 70 P. emersonii GH61A Concentrated N192A
70 P. emersonii GH61A Concentrated N192W 71 P. emersonii GH61A
Concentrated N193K 71 T. aurantiacus GH61A Purified WT 72 T.
aurantiacus GH61A Purified D105K 74 T. aurantiacus GH61A Purified
D105P 74 T. aurantiacus GH61A Purified Q188W 74
Example 19: Construction of Expression Vectors pDFng153-4,
pDFng154-17, and pDFng155-33
[0746] Plasmids pDFng153-4 (FIG. 7), pDFng154-17 (FIG. 8), and
pDFng155-33 (FIG. 9) were constructed as described below for
expression of the Thermoascus aurantiacus GH61A polypeptide,
Penicillium emersonii GH61A polypeptide, and Aspergillus aculeatus
GH61 polypeptide, respectively, and generation of the variants
listed in Table 11. The plasmids were constructed using plasmid
pBGMH16 (FIG. 10).
[0747] Plasmid pBGMH16 was constructed according to the protocol
described below. A Nb.Btsl recognition site in pUC19 was removed by
PCR amplifying pUC19 with primer pair BGMH24/BGMH25 followed by the
uracil-specific excision reagent USER.TM. based cloning (New
England BioLabs, Ipswich, Mass., USA). Plasmid pUC19 is described
in Yanisch-Perron et al., 1985, Gene 33(1):103-19.
TABLE-US-00014 BGMH 24 ATGCAGCGCUGCCATAACCATGAGTGA (SEQ ID NO: 271)
BGMH 25 AGCGCTGCAUAATTCTCTTACTGTCATG (SEQ ID NO: 272)
Underlined sequence is used in the USER.TM. assisted fusion of the
PCR fragments creating pBGMH13. USER.TM. (Uracil-Specific Excision
Reagent) Enzyme (New England Biolabs, Ipswich, Mass., USA)
generates a single nucleotide gap at the location of a uracil.
USER.TM. Enzyme is a mixture of Uracil DNA glycosylase (UDG) and
the DNA glycosylase-lyase Endonuclease VIII. UDG catalyzes the
excision of a uracil base, forming an abasic (apyrimidinic) site
while leaving the phosphodiester backbone intact. The lyase
activity of Endonuclease VIII breaks the phosphodiester backbone at
the 3' and 5' sides of the basic site so that base-free deoxyribose
is released.
[0748] The amplification reaction was composed of 100 ng of each
primer, 10 ng of pUC19, 1.times. PfuTurbo.RTM. C.sub.x Reaction
Buffer (Stratagene, La Jolla, Calif., USA), 2.5 .mu.l of a blend of
dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 2.5 units of
PfuTurbo.RTM. C.sub.x Hot Start DNA Polymerase (Stratagene, La
Jolla, Calif., USA), in a final volume of 50 .mu.l. The reaction
was performed using a EPPENDORF.RTM. MASTERCYCLER.RTM. 5333
programmed for 1 cycle at 95.degree. C. for 2 minutes; 32 cycles
each at 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds,
and 72.degree. C. for 3 minutes; and a final elongation at
72.degree. C. for 7 minutes. Five .mu.l of 10.times. NEBuffer 4
(New England Biolabs, Inc., Ipswich, Mass., USA), and 20 units of
Dpn I were added and incubated 1 hour at 37.degree. C. The Dpn I
was inactivated at 80.degree. C. for 20 minutes. A total of 100 ng
of the PCR product and 1 unit of USER.TM. enzyme in a total volume
of 10 .mu.l were incubated 20 minutes at 37.degree. C. followed by
20 minutes at 25.degree. C. Ten .mu.l were transformed into ONE
SHOT.RTM. TOP10 competent cells. This resulted in plasmid
pBHMG13.
[0749] Plasmid pBGMH14 contains part of pBGMH13 as vector backbone
and a Pac I/Nt.BbvCI USER.TM. cassette (Hansen et al., 2011, Appl.
Environ. Microbiol. 77(9): 3044-51) which is flanked by part of the
A. oryzae niaD gene on one side and part of the A. oryzae niiA gene
on the other side. The Pac I/Nt.BbvCI USER.TM. cassette can be
linearized with Pac I and Nt.BbvCI and a PCR product with
compatible overhangs can be cloned into this site (New England
Biolabs, Ipswich, Mass., USA).
TABLE-US-00015 BGMH 27 AATTAAGUCCTCAGCGTGATTTAAAACGCCATTGCT (SEQ ID
NO: 273) BGMH 28 ACTTAATUAAACCCTCAGCGCAGTTAGGTTGGTGTTCTTCT (SEQ ID
NO: 274) BGMH 29 AGCTCAAGGAUACCTACAGTTATTCGAAA (SEQ ID NO: 275)
BGMH 30 ATCCTTGAGCUGTTTCCTGTGTGAAATTGTTATCC (SEQ ID NO: 276) BGMH
31 ATCTCCTCUGCTGGTCTGGTTAAGCCAGCCCCGACAC (SEQ ID NO: 277) BGMH 32
AGAGGAGAUAATACTCTGCGCTCCGCC (SEQ ID NO: 278)
[0750] Underlined sequence was used in the USER.TM. assisted fusion
of the three fragments. The sequence marked in bold was used to
introduce a PacI/Nt.BbvCI USER.TM. cassette (Hansen et al., 2011,
supra) between the niiA and niaD fragments.
[0751] An Aspergillus oryzae niiA fragment was generated using
primers BGMH27 and BGMH29. The primer pair BGMH28/BGMH32 was used
to amplify the Aspergillus oryzae niaD gene region and primer-pair
BGMH30/BGMH31 was used to amplify the plasmid backbone region.
[0752] Genomic DNA from A. oryzae BECH2 (WO 00/39322) was purified
using a FASTDNA.TM. 2 ml SPIN Kit for Soil (MP Biomedicals, Santa
Ana, Calif., USA).
[0753] The amplification reaction was composed of 100 ng of each
primer, template DNA (pBGMH13 or A. oryzae BECH2 genomic DNA),
1.times. PfuTurbo.RTM. C, Reaction Buffer, 2.5 .mu.l of a blend of
dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 2.5 units of
PfuTurbo.RTM. C, Hot Start DNA Polymerase, in a final volume of 50
.mu.l. The reaction was performed using a EPPENDORF.RTM.
MASTERCYCLER.RTM. 5333 programmed for 1 cycle at 95.degree. C. for
2 minutes; 32 cycles each at 95.degree. C. for 30 seconds,
55.degree. C. for 30 seconds, and 72.degree. C. for 4 minutes; and
a final elongation at 72.degree. C. for 10 minutes. For PCR tubes
where template DNA was a plasmid, 5 .mu.l of 10.times. NEBuffer 4
and 20 units of Dpn I were added and incubated 1 hour at 37.degree.
C. The Dpn I was inactivated at 80.degree. C. for 20 minutes. Fifty
ng of each of the PCR products and 1 unit of USER.TM. enzyme in a
total volume of 10 .mu.l were incubated for 20 minutes at
37.degree. C. followed by 20 minutes at 25.degree. C. Then 10 .mu.l
were transformed into ONE SHOT.RTM. TOP10 competent cells. The
three fragments were fused by uracil-specific excision reagent
based cloning resulting in pBGMH14.
[0754] The promoter P13amy is a derivative of the NA-2 tpi promoter
from pJaL676 (WO 2003/008575). The A. niger AMG terminator used is
described by Christensen et al., 1988, Nature Biotechnology 6:
141-1422.
[0755] The P13amy promoter and AMG terminator were cloned into the
PacI/Nt.BbvCI USER.TM. cassette in pBGMH14. The primers were
designed so that an AsiSI/Nb.Btsl USER.TM. cassette (Hansen et al.,
2011, supra) was introduced between the promoter and
terminator.
TABLE-US-00016 BGMH 49 GGGTTTAAUCCTCACACAGGAAACAGCTATGA (SEQ ID NO:
279) BGMH 50 AGTGTCTGCGAUCGCTCTCACTGCCCCCAGTTGTGTATATAG AGGA (SEQ
ID NO: 280) BGMH 51 ATCGCAGACACUGCTGGCGGTAGACAATCAATCCAT (SEQ ID
NO: 281) BGMH 52 GGACTTAAUGGATCTAAGATGAGCTCATGGCT (SEQ ID NO:
282)
[0756] Underlined sequence was used in the USER.TM. assisted fusion
of the two fragments into a PacI/Nt.BbvCI digested pBGMH14. The
sequence marked in bold was used to introduce a AsiSI/Nb.Btsl
USER.TM. cassette (Hansen et al., 2011, supra) between the promoter
and terminator.
[0757] Promoter P13amy and AMG terminator was PCR amplified using
the primer pair BGMH49/BGMH50 to amplify promoter P13amy and the
primer pair BGMH51/BGMH52 to amplify the AMG terminator. The
amplification reaction was composed of 100 ng of each primer,
template DNA, 1.times. PfuTurbo.RTM. C.sub.x Reaction Buffer, 2.5
.mu.l of a blend of dATP, dTTP, dGTP, and dCTP, each at 10 mM, and
2.5 units of PfuTurbo.RTM. C.sub.x Hot Start DNA Polymerase, in a
final volume of 50 .mu.l. The reaction was performed using a
EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 1 cycle at
95.degree. C. for 2 minutes; 32 cycles each at 95.degree. C. for 30
seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 45
seconds; and a final elongation at 72.degree. C. for 3 minutes.
Then 5 .mu.l of 10.times. NEBuffer 4 and 20 units of Dpn I were
added and incubated 1 hour at 37.degree. C. The Dpn I was
inactivated at 80.degree. C. for 20 minutes.
[0758] The two fragments were fused into PacI/Nt.BbvCI digested
pBGMH14 by USER.TM. based cloning method in a reaction composed of
10 ng of PacI/Nt.BbvCI digested pBGMH14, 50 ng of each of the two
PCR products, and 1 unit of USER.TM. enzyme in a total volume of 10
.mu.l. The reaction was incubated for 20 minutes at 37.degree. C.
followed by 20 minutes at 25.degree. C. Then 10 .mu.l were
transformed into ONE SHOT.RTM. TOP10 competent cells. E. coli
transformants were selected on 2XYT+Amp agar plates and plasmid DNA
was prepared using QIAPREP.RTM. Spin Miniprep Kit (QIAGEN Inc.,
Valencia, Calif., USA). Plasmid pBGMH16 was confirmed by sequencing
analysis.
[0759] DNA sequencing was performed using a Model 377 XL Automated
DNA Sequencer and dye-terminator chemistry (Giesecke et al., 1992,
supra). Sequencing primers used for verification of niiA, niaD, the
P13amy promoter, AsiSI/Nb.Btsl USER.TM. cassette, and AMG
terminator sequence in BGMH16 are shown below.
TABLE-US-00017 BGMH 36 ACGCCATTGCTATGATGCTTGAAG (SEQ ID NO: 283)
BGMH 37 TGGTGAGGTGCTATCGTCCTT (SEQ ID NO: 284) BGMH 38
CTTCCTGTAGGTGCACCGAAG (SEQ ID NO: 285) BGMH 39
ACAGAACGATATCGGACCTCG (SEQ ID NO: 286) BGMH 40
TCGTTATGTTAAGTCTTCTATCA (SEQ ID NO: 287) BGMH 41
AGAGCTCGAAGTTCCTCCGAG (SEQ ID NO: 288) BGMH 42
TATCACGAGGCCCTTTCGTCTC (SEQ ID NO: 289) BGMH 43
TCCGTCGGCTCCTCTCCTTCGT (SEQ ID NO: 290) BGMH 44
TGCATATCCTCTGACAGTATATGA (SEQ ID NO: 291) BGMH 45
CAGTGAAGAGGGCAGTCGATAGT (SEQ ID NO: 292) BGMH 46
ACGAGGAACATGGCTATCTGGA (SEQ ID NO: 293) BGMH 47
TCAGCTCATTCTGGGAGGTGGGA (SEQ ID NO: 294) BGMH 48
ACTCCAGGATCCTTTAAATCCA (SEQ ID NO: 295) BGMH 53
ACTGGCAAGGGATGCCATGCT (SEQ ID NO: 296) BGMH 54
TGATCATATAACCAATTGCCCT (SEQ ID NO: 297) BGMH 55
AGTTGTGTATATAGAGGATTGA (SEQ ID NO: 298) BGMH 56
TGGTCCTTCGCTCGTGATGTGGA (SEQ ID NO: 299) BGMH 57
AGTCCTCAGCGTTACCGGCA (SEQ ID NO: 300) BGMH 58 ACCCTCAGCTGTGTCCGGGA
(SEQ ID NO: 301) BGMH 59 TGGTATGTGAACGCCAGTCTG (SEQ ID NO: 302)
[0760] Plasmid pBGMH16 contains flanking regions designed to repair
the niiA gene and niaD gene in Aspergillus oryzae COLs1300. Plasmid
pBGMH16 was digested with Asi Si and Nb. Bts I to linearize the
plasmid and create single stranded overhangs so that a PCR product
with compatible overhangs can be cloned into this site by USER.TM.
cloning (New England Biolabs, Inc., Ipswich, Mass., USA). The
digested plasmid was purified using a DNA Purification Kit (QIAGEN
Inc., Valencia, Calif., USA) according to the manufacturer's
instructions.
[0761] The T. aurantiacus GH61A polypeptide coding sequence (SEQ ID
NO: 13 [genomic DNA sequence] and SEQ ID NO: 14 [deduced amino acid
sequence]), P. emersonii GH61A polypeptide coding sequence (SEQ ID
NO: 35 [genomic DNA sequence] and SEQ ID NO: 36 [deduced amino acid
sequence]), and A. aculeatus GH61 polypeptide coding sequence (SEQ
ID NO: 67 [genomic DNA sequence] and SEQ ID NO: 68 [deduced amino
acid sequence]) were amplified from source plasmids described below
using the primers shown in Table 11. Bold letters represent coding
sequence. The single deoxyuridine (U) residue inserted into each
primer is the U that is excised from the PCR products using the
USER.TM. enzyme (New England Biolabs, Inc., Ipswich, Mass., USA) to
obtain overhangs for the insertion site. The underline letters
represent a His tag. The remaining sequences are homologous to
insertion sites of pBGMH16 for expression of the GH61
polypeptides.
TABLE-US-00018 TABLE 11 GH61 Source origin Template Plasmid Primer
ID Primer Sequence Thermoascus pDFng113 pDFng153-4 TaGH61_USE
AGAGCGA(U)ATGTCCTTTTCC aurantiacus Example 1 RtagF AAGATAAT (SEQ ID
NO: 303) GH61A TaGH61_USE TCTGCGA(U)TTAGTGATGGTG R_HIStagR
GTGATGATGACCAGTATACAG AGGAGGAC (SEQ ID NO: 304) Penicillium pMMar45
pDFng154-17 PeGH61_USE AGAGCGA(U)ATGCTGTCTTCG emersonii Example 1
RtagF ACGACTCG (SEQ ID NO: 305) GH61A PeGH61_USE
TCTGCGA(U)CTAGTGATGGTG R_HIStagR GTGATGATGGAACGTCGGCT CAGGCGGCC
(SEQ ID NO: 306) Aspergillus Xyz1566 pDFng155-33 AaGH61_USE
AGAGCGA(U)ATGTCTGTTGCT aculeatus (WO 2012/ RtagF AAGTTTGCTGGTG (SEQ
ID GH61 030799) NO: 307) AaGH61_USE TCTGCGA(U)TTAGTGATGGTG
R_HIStagR GTGATGATGGGCGGAGAGGT CACGGGCGT (SEQ ID NO: 308)
[0762] Construction of plasmid pDFng153-4 containing the
Thermoascus aurantiacus GH61A polypeptide coding sequence is
described below. The T. aurantiacus GH61A polypeptide coding
sequence was amplified from plasmid pDFng113 using the primers
shown in Table 11 with overhangs designed for cloning into plasmid
pBGMH16. The amplification was composed of 100 ng of each primer
listed in Table 11, 30 ng of pDFng113, 1.times. PfuTurbo.RTM.
C.sub.x Reaction Buffer, 2.5 .mu.l of a blend of dATP, dTTP, dGTP,
and dCTP, each at 10 mM, and 2.5 units of PfuTurbo.RTM. C, Hot
Start DNA Polymerase, in a final volume of 50 .mu.l. The
amplification was performed using an EPPENDORF.RTM.
MASTERCYCLER.RTM. 5333 programmed for 1 cycle at 95.degree. C. for
2 minutes; 30 cycles each at 95.degree. C. for 30 seconds,
57.7.degree. C. for 30 seconds, and 72.degree. C. for 1.5 minutes;
and a final elongation at 72.degree. C. for 10 minutes. The heat
block then went to a 10.degree. C. soak cycle.
[0763] The PCR reaction was analyzed by 0.7% agarose gel
electrophoresis using TBE buffer where an approximately 894 bp PCR
product band was observed. The PCR reaction was then digested with
1 .mu.l of Dpn I and 4.5 .mu.l of NEBuffer 4 at 37.degree. C.
overnight and purified using a QIAGEN.RTM. Purification Kit
according to the manufacturer's instructions.
[0764] The homologous ends of the 894 bp PCR reaction and the AsiSI
and Nb.Btsl digested pBGMH16 were joined together in a reaction
composed of 10 .mu.l of the PCR containing the 894 bp PCR product,
1 .mu.l of the AsiSI and Nb.Btsl digested plasmid pBGMH16, and 1
.mu.l of USER.TM. enzyme (New England Biolabs, Inc., Ipswich,
Mass., USA). The reaction was incubated for 15 minutes at
37.degree. C., followed by 15 minutes at 25.degree. C. Ten .mu.l of
the reaction were transformed into E. coli XL10-GOLD.RTM. Super
Competent Cells according to the manufacturer's instructions. E.
coli transformants were selected on 2XYT+Amp agar plates. Plasmid
DNA from several of the resulting E. coli transformants was
prepared using a BIOROBOT.RTM. 9600. The T. aurantiacus GH61A
polypeptide coding sequence insert was confirmed by DNA sequencing
using a Model 377 XL Automated DNA Sequencer and dye-terminator
chemistry (Giesecke et al., 1992, supra). The sequencing primers
shown below were used for verification of the gene insert and
sequence.
TABLE-US-00019 Primer TaGH61seqF: (SEQ ID NO: 309)
CCCAGTTATCAACTACCTTG Primer pBGMH16seqF: (SEQ ID NO: 310)
CTCAATTTACCTCTATCCAC Primer pBGMH16seqR: (SEQ ID NO: 311)
TATAACCAATTGCCCTCATC
[0765] A plasmid containing the correct T. aurantiacus GH61A
polypeptide coding sequence was selected and designated
pDFng153-4.
[0766] Construction of plasmid pDFng154-17 containing the
Penicillium emersonii GH61A polypeptide coding sequence is
described below. The P. emersonii GH61A polypeptide coding sequence
was amplified from plasmid pMMar45 using the primers shown in Table
11 with overhangs designed for cloning into plasmid pBGMH16. The
amplification was composed of 100 ng of each primer listed in Table
11, 30 ng of pMMar45, 1.times. PfuTurbo.RTM. C.sub.x Reaction
Buffer, 2.5 .mu.l of a blend of dATP, dTTP, dGTP, and dCTP, each at
10 mM, and 2.5 units of PfuTurbo.RTM. C.sub.x Hot Start DNA
Polymerase, in a final volume of 50 .mu.l. The amplification was
performed using an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed
for 1 cycle at 95.degree. C. for 2 minutes; 30 cycles each at
95.degree. C. for 30 seconds, 64.1.degree. C. for 30 seconds, and
72.degree. C. for 1.5 minutes; and a final elongation at 72.degree.
C. for 10 minutes. The heat block then went to a 10.degree. C. soak
cycle.
[0767] The PCR reaction was analyzed by 0.7% agarose gel
electrophoresis using TBE buffer where an approximately 930 bp PCR
product band was observed. The PCR reaction was then digested with
1 .mu.l of Dpn I and 4.5 .mu.l of NEBuffer 4 at 37.degree. C.
overnight and purified using a QIAGEN.RTM. Purification Kit
according to the manufacturer's instructions.
[0768] The homologous ends of the 930 bp PCR reaction and the AsiSI
and Nb.Btsl digested pBGMH16 were joined together in a reaction
composed of 10 .mu.l of the PCR containing the 930 bp PCR product,
1 .mu.l of the AsiSI and Nb.Btsl digested pBGMH16, and 1 .mu.l of
USER.TM. enzyme. The reaction was incubated for 15 minutes at
37.degree. C., followed by 15 minutes at 25.degree. C. Ten .mu.l of
the reaction were transformed into E. coli XL10-GOLD.RTM. Super
Competent Cells according to the manufacturer's instructions. E.
coli transformants were selected on 2XYT+Amp agar plates. Plasmid
DNA from several of the resulting E. coli transformants was
prepared using a BIOROBOT.RTM. 9600. The P. emersonii GH61A
polypeptide coding sequence insert was confirmed by DNA sequencing
using a Model 377 XL Automated DNA Sequencer and dye-terminator
chemistry (Giesecke et al., 1992, supra). The sequencing primers
pBGMH16seqF and pBGMH16seqR and primer PeGH61seqF shown below were
used for verification of the gene insert and sequence.
TABLE-US-00020 PeGH61seqF: (SEQ ID NO: 312)
GCACCGTCGAGCTGCAGTGG
[0769] A plasmid containing the correct P. emersonii GH61A
polypeptide coding sequence was selected and designated
pDFng154-17.
[0770] Construction of plasmid pDFng155-33 containing the
Aspergillus aculeatus GH61A polypeptide coding sequence is
described below. The A. aculeatus GH61A polypeptide coding sequence
was amplified from plasmid Xyz1566 (WO 2012/030799 Example 3,
P23NJ4 gene) using primers shown in Table 11 with overhangs
designed for cloning into plasmid pBGMH16. The amplification
reaction was composed of 100 ng of each primer listed in Table 11,
30 ng of plasmid Xyz1566, 1.times. PfuTurbo.RTM. C, Reaction
Buffer, 2.5 .mu.l of a blend of dATP, dTTP, dGTP, and dCTP, each at
10 mM, and 2.5 units of PfuTurbo.RTM. C, Hot Start DNA Polymerase,
in a final volume of 50 .mu.l. The amplification was performed
using an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 1
cycle at 95.degree. C. for 2 minutes; 30 cycles each at 95.degree.
C. for 30 seconds, 63.4.degree. C. for 30 seconds, and 72.degree.
C. for 1.5 minutes; and a final elongation at 72.degree. C. for 10
minutes. The heat block then went to a 10.degree. C. soak
cycle.
[0771] The PCR reaction was analyzed by 0.7% agarose gel
electrophoresis using TBE buffer where an approximately 1.3 kb PCR
product band was observed. The PCR reaction was then digested with
1 .mu.l of Dpn I and 4.5 .mu.l of NEBuffer 4 at 37.degree. C.
overnight and purified using a QIAGEN.RTM. Purification Kit
according to the manufacturer's instructions.
[0772] The homologous ends of the 1.3 kb PCR reaction and the
digested pBGMH16 were joined together in a reaction composed of 10
.mu.l of the PCR containing the 1.3 kb PCR product, 1 .mu.l of the
digested pBGMH16, and 1 .mu.l of USER.TM. enzyme. The reaction was
incubated for 15 minutes at 37.degree. C., followed by 15 minutes
at 25.degree. C. Ten .mu.l of the reaction were transformed into E.
coli XL10-GOLD.RTM. Super Competent Cells according to the
manufacturer's instructions. E. coli transformants were selected on
2XYT+Amp agar plates. Plasmid DNA from several of the resulting E.
coli transformants was prepared using a BIOROBOT.RTM. 9600. The A.
aculeatus GH61A polypeptide coding sequence insert was confirmed by
DNA sequencing using a Model 377 XL Automated DNA Sequencer and
dye-terminator chemistry (Giesecke et al., 1992, supra). The
sequencing primers pBGMH16seqF and pBGMH16seqR and primer
AaGH61seqF shown below were used for verification of the gene
insert and sequence.
TABLE-US-00021 Primer AaGH61seqF: (SEQ ID NO: 313)
CCTTGCCAACTGCAATGGTG
[0773] A plasmid containing the correct A. aculeatus GH61A
polypeptide coding sequence was selected and designated
pDFng155-33.
Example 20: Construction of the Thermoascus aurantiacus GH61A and
Penicillium emersonii GH61A Polypeptide Variants
[0774] Variants of the T. aurantiacus GH61A and P. emersonii GH61A
polypeptides were constructed by performing site-directed
mutagenesis on plasmids pDFng153-4 and pDFng154-17, respectively,
according to the procedure described in Example 7 using the primers
described in Table 12.
[0775] The sequencing primers pBGMH16seqF, pBGMH16seqR, and
TaGH61seqF (used only for T. aurantiacus GH61A variants), and
primer PeGH61seqR shown below (used only for P. emersonii GH61A
variants) were used for verification.
TABLE-US-00022 PeGH61seqR (only used for P. emersonii GH61A
variants): (SEQ ID NO: 314) GCACCGTCGAGCTGCAGTGG
TABLE-US-00023 TABLE 12 Variant Amino Variant Template Acid Primer
Plasmid Backbone Substitution ID Primer Sequence Name Thermoascus
Q188F 1202295 ATTATTGCTCTTCACTCAGCTTTCAACCAGGA TaSDM2 aurantiacus
TGGTGCCCAGAAC (SEQ ID NO: 315) GH61A 1202296
GTTCTGGGCACCATCCTGGTTGAAAGCTGAG (pDFng153-4) TGAAGAGCAATAAT (SEQ ID
NO: 316) Q188M 1202297 ATTATTGCTCTTCACTCAGCTATGAACCAGGA TaSDM3
TGGTGCCCAGAAC (SEQ ID NO: 317) 1202298
GTTCTGGGCACCATCCTGGTTCATAGCTGAG TGAAGAGCAATAAT (SEQ ID NO: 318)
Penicillium N192M 1202305 CCCTGCACTCGGCCATGAACAAGGACGGCG PeSDM6
emersonii C (SEQ ID NO: 319) GH61A 1202306
GCGCCGTCCTTGTTCATGGCCGAGTGCAGG (pDFng154- G (SEQ ID NO: 320) 17)
N193H 1202307 GCACTCGGCCAACCACAAGGACGGCGCCC PeSDM7 (SEQ ID NO: 321)
1202308 GGGCGCCGTCCTTGTGGTTGGCCGAGTGC (SEQ ID NO: 322)
[0776] PCR fragments were amplified from the mutant plasmids, the
T. aurantiacus GH61A polypeptide plasmid pDFng153-4, and the P.
emersonii GH61A polypeptide plasmid pDFng154-17 for A. oryzae
COLs1300 transformation. The amplification was composed of 10 .mu.M
each of primers 1201513 and 1201514 (see below), 10 ng of either
pDFng153-4, pDFng154-17, or one of the mutant plasmids, 5.times.
PHUSION.RTM. High-Fidelity Buffer (New England Biolabs, Inc.,
Ipswich, Mass., USA), 1 .mu.l of a blend of dATP, dTTP, dGTP, and
dCTP, each at 10 mM, and 0.5 .mu.l of PHUSION.RTM. High-Fidelity
DNA polymerase (New England Biolabs, Inc., Ipswich, Mass., USA), in
a final volume of 50 .mu.l. For pDFng154-17 and the P. emersonii
GH61 polypeptide mutant plasmids, 1.5 .mu.l of DMSO were also
added. The amplification was performed using an EPPENDORF.RTM.
MASTERCYCLER.RTM. 5333 programmed for 1 cycle at 98.degree. C. for
30 seconds; 10 cycles each at 98.degree. C. for 10 seconds,
65.degree. C. minus 1.degree. C. per cycle for 30 seconds, and
72.degree. C. for 3 minutes; 25 cycles each at 98.degree. C. for 10
seconds, 55.degree. C. at 30 seconds, and 72.degree. C. for 3
minutes; and a final elongation at 72.degree. C. for 10 minutes.
The heat block then went to a 10.degree. C. soak cycle.
TABLE-US-00024 Primer 1201513: (SEQ ID NO: 323)
CCAGACCAGCAGAGGAGATAATACT Primer 1201514: (SEQ ID NO: 324)
CAAGGATACCTACAGTTATTCGA
[0777] Each PCR reaction was analyzed by 0.7% agarose gel
electrophoresis using TBE buffer where either a 7718 bp PCR product
from T. aurantiacus or a 7754 bp PCR product band from P. emersonii
was observed. The PCR reaction was then digested with 1 .mu.l of
Dpn I and 4.5 .mu.l of NEBuffer 4 at 37.degree. C. overnight and
purified using a QIAGEN.RTM. Purification Kit according to the
manufacturer's instructions.
Example 21: Construction of Aspergillus aculeatus GH61 Polypeptide
Variants
[0778] The Aspergillus aculeatus GH61 polypeptide variants were
constructed by SOE-PCR (Splicing by Overhang Extension Polymerase
Chain Reaction) with plasmid pDFng155-33. In brief, the first PCR
reaction used forward primer BGMH110V2F and a mutation specific
reverse primer (Table 13). The second PCR reaction used a reverse
primer BGMH109V2R and a mutation specific forward primer (Table 13)
containing the sequence coding for the altered amino acid. The
mutation specific forward and reverse primers contained 15-20
overlapping nucleotides. The third PCR reaction used the
overlapping nucleotides to splice together the fragments produced
in the first and second reaction. Finally, using forward primer
BGMH110V2F and reverse primer BGMH109V2R, the spliced fragment was
amplified by PCR.
TABLE-US-00025 Primer BGMH110V2F: (SEQ ID NO: 325)
5'-CCAGACCAGCAGAGGAGATAATACTCTGCG-3' Primer BGMH109V2R: (SEQ ID NO:
326) 5'-CAAGGATACCTACAGTTATTCGAAACCTCCTG-3'
[0779] The first SOE-PCR reactions for the A. aculeatus GH61
polypeptide variants contained 0.5 picomole of the BGMH110V2F
primer, 0.5 picomole of the reverse primer listed in Table 13, 50
ng of template (pDFng155-33), 5 nanomoles each dATP, dTTP, dGTP,
and dCTP, PHUSION.RTM. High-Fidelity Buffer, and 0.7 unit of
PHUSION.RTM. High-Fidelity DNA Polymerase, in a final reaction
volume of 50 .mu.l. The amplification was performed using an
EPPENDORF.RTM. MASTERCYCLER.RTM. Gradient (Eppendorf Scientific,
Inc., Westbury, N.Y., USA) programmed for 1 cycle at 98.degree. C.
for 2 minutes; 35 cycles each at 98.degree. C. for 25 seconds,
66.degree. C. for 30 seconds, and 72.degree. C. for 5 minute; and a
final elongation at 72.degree. C. for 10 minutes. The heat block
then went to a 10.degree. C. hold stage.
[0780] The second SOE-PCR reactions for the A. aculeatus GH61
variants contained 0.5 picomole of the forward primer listed in
Table 13, 0.5 picomole of the BGMH109V2R primer, 50 ng of template
(pDFng155-33), 5 nanomoles each dATP, dTTP, dGTP, and dCTP,
PHUSION.RTM. High-Fidelity Buffer, and 0.7 unit of PHUSION.RTM.
High-Fidelity DNA Polymerase, in a final reaction volume of 50
.mu.l. The amplification was performed using an EPPENDORF.RTM.
MASTERCYCLER.RTM. Gradient programmed for 1 cycle at 98.degree. C.
for 2 minutes; 35 cycles each at 98.degree. C. for 25 seconds,
66.degree. C. for 30 seconds, and 72.degree. C. for 5 minutes; and
a final elongation at 72.degree. C. for 10 minutes. The heat block
then went to a 10.degree. C. hold stage.
[0781] Each PCR reaction was analyzed by 1.0% agarose
electrophoresis using TAE buffer where a 3.9 to 6.5 kb (as
specified in Table 13) PCR product band was observed indicating
proper amplification. The remaining 45 microliters were then
treated with 10 units of Dpn I and 1.times.NEB4 to remove the
remaining wild-type template. The reaction was incubated for 1 hour
at 37.degree. C. and then purified using a MINELUTE.RTM. 96 UF
Purification Kit (QIAGEN Inc., Valencia, Calif., USA). The purified
PCR products were resuspended in deionized water to a final volume
equal to 20 .mu.l. The concentration of each fragment was measured
using a NanoDrop 2000 (Thermo Scientific, Wilmington, Del.,
USA).
[0782] The third PCR reaction for the A. aculeatus GH61 variants
contained 100 to 200 ng of each fragment produced in the first and
second SOE-PCR reactions, 5 nanomoles each dATP, dTTP, dGTP, and
dCTP, 1.times. PHUSION.RTM. High-Fidelity Buffer, and 0.7 units
PHUSION.RTM. High-Fidelity DNA Polymerase, in a final reaction
volume of 50 .mu.l. The amplification was performed using an
EPPENDORF.RTM. MASTERCYCLER.RTM. Gradient programmed for 1 cycle at
98.degree. C. for 2 minutes; 35 cycles each at 98.degree. C. for 15
seconds, 68.degree. C. for 30 seconds, and 72.degree. C. for 10
minutes; and a final elongation at 72.degree. C. for 10 minutes.
The heat block then went to a 10.degree. C. hold stage. Primer
BGMH110V2F primer (0.5 picomole) and primer BGMH109V2R (0.5
picomole) were added during the annealing/elongation step of the
fifth cycle to allow for the overlapping nucleotides to splice.
[0783] The wild-type fragment was produced using conditions similar
to the third PCR reaction. The reaction was composed of 50 ng of
template (pDFng155-33), 0.5 picomole of primer BGMH110V2F, 0.5
picomole of primer BGMH109V2R, 5 nanomoles each dATP, dTTP, dGTP,
and dCTP, 1.times. PHUSION.RTM. High-Fidelity Buffer, and 0.7 units
PHUSION.RTM. High-Fidelity DNA Polymerase, in a final reaction
volume of 50 .mu.l. The amplification was performed using an
EPPENDORF.RTM. MASTERCYCLER.RTM. Gradient programmed for 1 cycle at
98.degree. C. for 2 minutes; 35 cycles each at 98.degree. C. for 15
seconds, 68.degree. C. for 30 seconds, and 72.degree. C. for 10
minutes; and a final elongation at 72.degree. C. for 10 minutes.
The heat block then went to a 10.degree. C. hold stage.
[0784] Each PCR reaction was analyzed by 1.0% agarose
electrophoresis using TAE buffer where an approximately 8 kb PCR
product band was observed indicating proper amplification. The
remaining 45 .mu.l of each PCR reaction were then purified using a
MINELUTE.RTM. 96 UF Purification Kit. The purified PCR products
were resuspended in deionized water to a final volume equal to 20
.mu.l. The concentration of each fragment was measured using a
NanoDrop 2000. The entire volume was then transformed into the
Aspergillus oryzae COLs1300 strain as described in Example 22.
TABLE-US-00026 TABLE 13 PCR Amino fragment Template Acid Primer
Primer size Backbone Substitution ID Direction Primer Sequence (kb)
Aspergillus D103K 1202768 Fwd TCCAGTGGACTACCTGGCCCAAGAGCCACCA 4.1
aculeatus CGGCCCTGTCC (SEQ ID NO: 327) GH61 1202769 Rev
GGGCCAGGTAGTCCACTGGAGCTCAACAGTA 6.5 C (SEQ ID NO: 328) Aspergillus
D103P 1202770 Fwd TCCAGTGGACTACCTGGCCCCCCAGCCACCA 4.1 aculeatus
CGGCCCTGTCC (SEQ ID NO: 329) GH61 1202769 Rev
GGGCCAGGTAGTCCACTGGAGCTCAACAGTA 6.5 C (SEQ ID NO: 330) Aspergillus
N152I 1202771 Fwd CCGGTACCTGGGCCAGTGATATCTTGATCGC 4.0 aculeatus
CAACAACAACAGCTG (SEQ ID NO: 331) GH61 1202772 Rev
ATCACTGGCCCAGGTACCGGGGACGTCGTC 6.4 (SEQ ID NO: 332) Aspergillus
N152L 1202773 Fwd CCGGTACCTGGGCCAGTGATCTCTTGATCGC 4.0 aculeatus
CAACAACAACAGCTG (SEQ ID NO: 333) GH61 1202772 Rev
ATCACTGGCCCAGGTACCGGGGACGTCGTC 6.4 (SEQ ID NO: 334) Aspergillus
G186F 1202774 Fwd AAATCATTGCCCTTCACTCTGCTTTCAACAAG 3.9 aculeatus
GATGGTGCTCAGAACTA (SEQ ID NO: 335) GH61 1202775 Rev
AGCAGAGTGAAGGGCAATGATTTCGTGACGG 6.3 AG (SEQ ID NO: 336) Aspergillus
G186M 1202776 Fwd AAATCATTGCCCTTCACTCTGCTATGAACAAG 3.9 aculeatus
GATGGTGCTCAGAACTA (SEQ ID NO: 337) GH61 1202775 Rev
AGCAGAGTGAAGGGCAATGATTTCGTGACGG 6.3 AG (SEQ ID NO: 338) Aspergillus
G186A 1202777 Fwd AAATCATTGCCCTTCACTCTGCTGCCAACAAG 3.9 aculeatus
GATGGTGCTCAGAACTA (SEQ ID NO: 339) GH61 1202775 Rev
AGCAGAGTGAAGGGCAATGATTTCGTGACGG 6.3 AG (SEQ ID NO: 340) Aspergillus
G186W 1202778 Fwd AAATCATTGCCCTTCACTCTGCTTGGAACAAG 3.9 aculeatus
GATGGTGCTCAGAACTA (SEQ ID NO: 341) GH61 1202775 Rev
AGCAGAGTGAAGGGCAATGATTTCGTGACGG 6.3 AG (SEQ ID NO: 342) Aspergillus
N187H 1202779 Fwd CATTGCCCTTCACTCTGCTGGTCACAAGGATG 3.9 aculeatus
GTGCTCAGAACTACC (SEQ ID NO: 343) GH61 1202780 Rev
ACCAGCAGAGTGAAGGGCAATGATTTCGTGA 6.3 CGG (SEQ ID NO: 344)
Aspergillus N187K 1202781 Fwd CATTGCCCTTCACTCTGCTGGTAAGAAGGATG 3.9
aculeatus GTGCTCAGAACTACC (SEQ ID NO: 345) GH61 1202780 Rev
ACCAGCAGAGTGAAGGGCAATGATTTCGTGA 6.3 CGG (SEQ ID NO: 346)
Example 22: Expression of the T. aurantiacus GH61A, P. emersonii
GH61A, and A. aculeatus GH61 Polypeptides Variants in Aspergillus
oryzae COLs1300
[0785] Aspergillus oryzae COLs1300 was inoculated onto a COVE-N-Gly
plate containing 10 mM urea and incubated at 34.degree. C. until
confluent. Spores were collected from the plate by washing with 10
ml of YP medium. The whole spore suspension was used to inoculate
101 ml of COL1300 protoplasting cultivation medium in a 500 ml
polycarbonate shake flask. The shake flask was incubated at
30.degree. C. with agitation at 200 rpm for 18-24 hours. Mycelia
were filtered through a funnel lined with MIRACLOTH.RTM. and washed
with 200 ml of 0.6 M MgSO.sub.4. Washed mycelia were resuspended in
10 ml of COLs1300 protoplasting solution in a 125 ml sterile
polycarbonate shake flask and incubated at room temperature for 3
minutes. One ml of a solution of 12 mg of BSA per ml of deionized
water was added to the shake flask and the shake flask was then
incubated at 37.degree. C. with mixing at 65 rpm for 45-90 minutes
until protoplasting was complete. The mycelia/protoplast mixture
was filtered through a funnel lined with MIRACLOTH.RTM. in a 50 ml
conical tube and overlayed with 5 ml of ST. The 50 ml conical tube
was centrifuged at 1050.times.g for 15 minutes with slow
acceleration/deceleration. After centrifugation, the liquid was
separated in 3 phases. The interphase which contained the
protoplasts was transferred to a new 50 ml conical tube. Two
volumes of STC were added to the protoplasts followed by a brief
centrifugation at 1050.times.g for 5 minutes. The supernatant was
discarded and the protoplasts were washed twice with 5 ml of STC
with resuspension of the protoplast pellet, centrifugation at
1050.times.g for 5 minutes, and decanting of the supernatant each
time. After the final decanting, the protoplast pellet was
resuspended in STC at a concentration of 5.times.10.sup.7/ml.
Protoplasts were frozen at -80.degree. C. until transformation.
[0786] A 15 .mu.l volume of each mutant fragment, as described in
Example 21, was used to transform 100 .mu.l of A. oryzae COLs1300
protoplasts in a 15 ml round bottom tube. After an initial
incubation at room temperature for 15 minutes, 300 .mu.l of PEG
solution was added to the 15 ml round bottom tube containing the
transformation mixture. The reaction was incubated for an
additional 15 minutes at room temperature. Six ml of melted top
agar were added to the reaction and the whole mixture was poured
evenly onto a sucrose agar plate supplemented with 10 mM NaNO.sub.3
and left at room temperature until the top agar was set. The plates
were incubated at 37.degree. C. for 4-6 days. Resulting
transformants were picked using sterile inoculating loops and
inoculated into a 96 well flat bottom plate contain 200 .mu.l of
MDU2BP per well. The plate was incubated at 34.degree. C.,
stationary in a humidified box. Samples were harvested on the third
day by removing the mycelia mat.
Example 23: Determination of Tm (Melting Temperature) of
Thermoascus aurantiacus GH61A, Penicillium emersonii GH61A, and
Aspergillus aculeatus GH61 Polypeptide Variants by Protein Thermal
Unfolding Analysis
[0787] Protein thermal unfolding of the Thermoascus aurantiacus
GH61A, Penicillium emersonii GH61A, and Aspergillus aculeatus GH61
polypeptide variants was determined by protein thermal unfolding
analysis described according to Example 10. The Thermoascus
aurantiacus GH61A, Penicillium emersonii GH61A, and Aspergillus
aculeatus GH61 polypeptide variants and wild type polypeptides
thereof were prepared as described in Example 22. The results of
the thermostability determinations are shown in Table 14.
TABLE-US-00027 TABLE 14 Melting temperatures (.degree. C.) of
Thermoascus aurantiacus GH61A, Penicillium emersonii GH61A,
Aspergillus aculeatus GH61 polypeptide variants determined by
protein thermal unfolding analysis Protein backbone Mutations Tm T.
aurantiacus GH61 Wild-Type 75 T. aurantiacus GH61 Q188F 78 T.
aurantiacus GH61 Q188M 76 P. emersonii GH61 Wild-Type 71 P.
emersonii GH61 N192M 74 P. emersonii GH61 N193H 73 A. aculeatus
GH61 Wild-Type 46 A. aculeatus GH61 D103K 48 A. aculeatus GH61
D103P 48 A. aculeatus GH61 N152I 48 A. aculeatus GH61 N152L 49 A.
aculeatus GH61 G186F 51 A. aculeatus GH61 G186M 51 A. aculeatus
GH61 G186A 48 A. aculeatus GH61 G186W 49 A. aculeatus GH61 N187H 48
A. aculeatus GH61 N187K 48
[0788] The present invention is further described by the following
numbered paragraphs:
[1] A GH61 polypeptide variant, comprising a substitution at one or
more positions corresponding to positions 105, 154, 188, 189, 216,
and 229 of the mature polypeptide of 30, wherein the variant has
cellulolytic enhancing activity. [2] The variant of paragraph 1,
which has at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to the amino acid sequence of a parent GH61 polypeptide.
[3] The variant of any of paragraphs 1 or 2, which is a variant of
a parent GH61 polypeptide selected from the group consisting of:
(a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216; (b)
a polypeptide encoded by a polynucleotide that hybridizes under at
least low stringency conditions with (i) the mature polypeptide
coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,
89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,
117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,
143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,
169, 171, 173, 175, 177, or 179, 181, 183, 185, 187, 189, 191, 193,
195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215, or (ii)
the full-length complement of (i); (c) a polypeptide encoded by a
polynucleotide having at least 60% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,
113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,
139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,
165, 167, 169, 171, 173, 175, 177, or 179, 181, 183, 185, 187, 189,
191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215;
and (d) a fragment of the mature polypeptide of SEQ ID NO: 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,
76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,
108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,
134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,
160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180, 182, 184,
186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,
212, 214, or 216, which has cellulolytic enhancing activity. [4]
The variant of paragraph 3, wherein the parent GH61 polypeptide has
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% or 100% sequence identity to the mature
polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,
92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,
120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,
146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,
172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192, 194, 196,
198, 200, 202, 204, 206, 208, 210, 212, 214, or 216. [5] The
variant of paragraph 3, wherein the parent GH61 polypeptide is
encoded by a polynucleotide that hybridizes under low stringency
conditions, medium stringency conditions, medium-high stringency
conditions, high stringency conditions, or very high stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,
127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,
153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, or
179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,
205, 207, 209, 211, 213, or 215, or (ii) the full-length complement
of (i). [6] The variant of paragraph 3, wherein the parent GH61
polypeptide is encoded by a polynucleotide having at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% sequence identity to the mature polypeptide coding sequence
of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,
29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,
63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,
97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,
125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,
151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,
177, or 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,
203, 205, 207, 209, 211, 213, or 215. [7] The variant of paragraph
3, wherein the parent GH61 polypeptide comprises or consists of the
mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216. [8]
The variant of paragraph 3, wherein the parent GH61 polypeptide is
a fragment of the mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,
109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,
135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,
161, 163, 165, 167, 169, 171, 173, 175, 177, or 179, 181, 183, 185,
187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,
213, or 215, wherein the fragment has cellulolytic enhancing
activity. [9] The variant of any of paragraphs 1-8, which has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99%, but less than 100%, sequence identity to the
mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216. [10]
The variant of any of paragraphs 2-9, wherein the variant consists
of at least 85% of the amino acid residues, e.g., at least 90% of
the amino acid residues or at least 95% of the amino acid residues
of the mature polypeptide of the parent GH61 polypeptide. [11] The
variant of any of paragraphs 1-10, wherein the number of
substitutions is 1-6, e.g., 1, 2, 3, 4, 5, or 6 substitutions. [12]
The variant of any of paragraphs 1-11, which comprises a
substitution at a position corresponding to position 105. [13] The
variant of paragraph 12, wherein the substitution is Pro or Lys.
[14] The variant of any of paragraphs 1-13, which comprises a
substitution at a position corresponding to position 154. [15] The
variant of paragraph 14, wherein the substitution is Ile or Leu.
[16] The variant of any of paragraphs 1-15, which comprises a
substitution at a position corresponding to position 188. [17] The
variant of paragraph 16, wherein the substitution is Ala, Met, Phe,
or Trp. [18] The variant of any of paragraphs 1-17, which comprises
a substitution at a position corresponding to position 189. [19]
The variant of paragraph 18, wherein the substitution is His or
Lys. [20] The variant of any of paragraphs 1-19, which comprises a
substitution at a position corresponding to position 216. [21] The
variant of paragraph 20, wherein the substitution is Leu or Tyr.
[22] The variant of any of paragraphs 1-21, which comprises a
substitution at a position corresponding to position 229. [23] The
variant of paragraph 22, wherein the substitution is Trp, His, Ile,
or Tyr. [24] The variant of any of paragraphs 1-23, which comprises
a substitution at two positions corresponding to any of positions
105, 154, 188, 189, 216, and 229. [25] The variant of any of
paragraphs 1-23, which comprises a substitution at three positions
corresponding to any of positions 105, 154, 188, 189, 216, and 229.
[26] The variant of any of paragraphs 1-23, which comprises a
substitution at four positions corresponding to any of positions
105, 154, 188, 189, 216, and 229. [27] The variant of any of
paragraphs 1-23, which comprises a substitution at five positions
corresponding to any of positions 105, 154, 188, 189, 216, and 229.
[28] The variant of any of paragraphs 1-23, which comprises a
substitution at each position corresponding to positions 105, 154,
188, 189, 216, and 229. [29] The variant of any of paragraphs 1-28,
which comprises one or more substitutions or corresponding
substitutions selected from the group consisting of E105P,K;
E154I,L; G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y. [30] The
variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K and E154I,L; or corresponding substitutions
thereof. [31] The variant of any of paragraphs 1-29, which
comprises the substitutions E105P,K and G188A,F,M,W; or
corresponding substitutions thereof. [32] The variant of any of
paragraphs 1-29, which comprises the substitutions E105P,K and
N189H,K; or corresponding substitutions thereof. [33] The variant
of any of paragraphs 1-29, which comprises the substitutions
E105P,K and A216L,Y; or corresponding substitutions thereof. [34]
The variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K and
[0789] K229W,H,I,Y; or corresponding substitutions thereof.
[35] The variant of any of paragraphs 1-29, which comprises the
substitutions E154I,L and G188A,F,M,W; or corresponding
substitutions thereof. [36] The variant of any of paragraphs 1-29,
which comprises the substitutions E154I,L and N189H,K; or
corresponding substitutions thereof. [37] The variant of any of
paragraphs 1-29, which comprises the substitutions E154I,L and
A216L,Y; or corresponding substitutions thereof. [38] The variant
of any of paragraphs 1-29, which comprises the substitutions
E154I,L and K229W,H,I,Y; or corresponding substitutions thereof.
[39] The variant of any of paragraphs 1-29, which comprises the
substitutions G188A,F,M,W and N189H,K; or corresponding
substitutions thereof. [40] The variant of any of paragraphs 1-29,
which comprises the substitutions G188A,F,M,W and A216L,Y; or
corresponding substitutions thereof. [41] The variant of any of
paragraphs 1-29, which comprises the substitutions G188A,F,M,W and
K229W,H,I,Y; or corresponding substitutions thereof. [42] The
variant of any of paragraphs 1-29, which comprises the
substitutions N189H,K and A216L,Y; or corresponding substitutions
thereof. [43] The variant of any of paragraphs 1-29, which
comprises the substitutions N189H,K and K229W,H,I,Y; or
corresponding substitutions thereof. [44] The variant of any of
paragraphs 1-29, which comprises the substitutions A216L,Y and
[0790] K229W,H,I,Y; or corresponding substitutions thereof.
[45] The variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; E154I,L; and G188A,F,M,W; or corresponding
substitutions thereof. [46] The variant of any of paragraphs 1-29,
which comprises the substitutions E105P,K; E154I,L; and N189H,K; or
corresponding substitutions thereof. [47] The variant of any of
paragraphs 1-29, which comprises the substitutions E105P,K;
E154I,L; and A216L,Y; or corresponding substitutions thereof. [48]
The variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; E154I,L; and K229W,H,I,Y; or corresponding
substitutions thereof. [49] The variant of any of paragraphs 1-29,
which comprises the substitutions E105P,K; G188A,F,M,W; and
N189H,K; or corresponding substitutions thereof. [50] The variant
of any of paragraphs 1-29, which comprises the substitutions
E105P,K; G188A,F,M,W; and A216L,Y; or corresponding substitutions
thereof. [51] The variant of any of paragraphs 1-29, which
comprises the substitutions E105P,K; G188A,F,M,W; and K229W,H,I,Y;
or corresponding substitutions thereof. [52] The variant of any of
paragraphs 1-29, which comprises the substitutions E105P,K;
N189H,K; and A216L,Y; or corresponding substitutions thereof. [53]
The variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; N189H,K; and K229W,H,I,Y; or corresponding
substitutions thereof. [54] The variant of any of paragraphs 1-29,
which comprises the substitutions E105P,K; A216L,Y; and
K229W,H,I,Y; or corresponding substitutions thereof. [55] The
variant of any of paragraphs 1-29, which comprises the
substitutions E154I,L; G188A,F,M,W; and N189H,K; or corresponding
substitutions thereof. [56] The variant of any of paragraphs 1-29,
which comprises the substitutions E154I,L; G188A,F,M,W; and
A216L,Y; or corresponding substitutions thereof. [57] The variant
of any of paragraphs 1-29, which comprises the substitutions
E154I,L; G188A,F,M,W; and K229W,H,I,Y; or corresponding
substitutions thereof. [58] The variant of any of paragraphs 1-29,
which comprises the substitutions E154I,L; N189H,K; and A216L,Y; or
corresponding substitutions thereof. [59] The variant of any of
paragraphs 1-29, which comprises the substitutions E154I,L;
N189H,K; and K229W,H,I,Y; or corresponding substitutions thereof.
[60] The variant of any of paragraphs 1-29, which comprises the
substitutions E154I,L; A216L,Y; and K229W,H,I,Y; or corresponding
substitutions thereof. [61] The variant of any of paragraphs 1-29,
which comprises the substitutions G188A,F,M,W; N189H,K; and
A216L,Y; or corresponding substitutions thereof. [62] The variant
of any of paragraphs 1-29, which comprises the substitutions
G188A,F,M,W; N189H,K; and K229W,H,I,Y; or corresponding
substitutions thereof. [63] The variant of any of paragraphs 1-29,
which comprises the substitutions G188A,F,M,W; A216L,Y; and
K229W,H,I,Y; or corresponding substitutions thereof. [64] The
variant of any of paragraphs 1-29, which comprises the
substitutions N189H,K; A216L,Y; and K229W,H,I,Y; or corresponding
substitutions thereof. [65] The variant of any of paragraphs 1-29,
which comprises the substitutions E105P,K; E154I,L; G188A,F,M,W;
and N189H,K; or corresponding substitutions thereof. [66] The
variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; E154I,L; G188A,F,M,W; and A216L,Y; or
corresponding substitutions thereof. [67] The variant of any of
paragraphs 1-29, which comprises the substitutions E105P,K;
E154I,L; G188A,F,M,W; and K229W,H,I,Y; or corresponding
substitutions thereof. [68] The variant of any of paragraphs 1-29,
which comprises the substitutions E105P,K; E154I,L; N189H,K; and
A216L,Y; or corresponding substitutions thereof. [69] The variant
of any of paragraphs 1-29, which comprises the substitutions
E105P,K; E154I,L; N189H,K; and K229W,H,I,Y; or corresponding
substitutions thereof. [70] The variant of any of paragraphs 1-29,
which comprises the substitutions E105P,K; E154I,L; A216L,Y; and
K229W,H,I,Y; or corresponding substitutions thereof. [71] The
variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; G188A,F,M,W; N189H,K; and A216L,Y; or
corresponding substitutions thereof. [72] The variant of any of
paragraphs 1-29, which comprises the substitutions E105P,K;
G188A,F,M,W; N189H,K; and K229W,H,I,Y; or corresponding
substitutions thereof. [73] The variant of any of paragraphs 1-29,
which comprises the substitutions E105P,K; G188A,F,M,W; A216L,Y;
and K229W,H,I,Y; or corresponding substitutions thereof. [74] The
variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; N189H,K; A216L,Y; and K229W,H,I,Y; or
corresponding substitutions thereof. [75] The variant of any of
paragraphs 1-29, which comprises the substitutions E154I,L;
G188A,F,M,W; N189H,K; and A216L,Y; or corresponding substitutions
thereof. [76] The variant of any of paragraphs 1-29, which
comprises the substitutions E154I,L; G188A,F,M,W; N189H,K; and
K229W,H,I,Y; or corresponding substitutions thereof. [77] The
variant of any of paragraphs 1-29, which comprises the
substitutions E154I,L; G188A,F,M,W; A216L,Y; and K229W,H,I,Y; or
corresponding substitutions thereof. [78] The variant of any of
paragraphs 1-29, which comprises the substitutions E154I,L;
N189H,K; A216L,Y; and K229W,H,I,Y; or corresponding substitutions
thereof. [79] The variant of any of paragraphs 1-29, which
comprises the substitutions G188A,F,M,W; N189H,K; A216L,Y; and
K229W,H,I,Y; or corresponding substitutions thereof. [80] The
variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; and A216L,Y;
or corresponding substitutions thereof. [81] The variant of any of
paragraphs 1-29, which comprises the substitutions E105P,K;
E154I,L; G188A,F,M,W; N189H,K; and K229W,H,I,Y; or corresponding
substitutions thereof. [82] The variant of any of paragraphs 1-29,
which comprises the substitutions E105P,K; E154I,L; G188A,F,M,W;
A216L,Y; and K229W,H,I,Y; or corresponding substitutions thereof.
[83] The variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; E154I,L; N189H,K; A216L,Y; and K229W,H,I,Y;
or corresponding substitutions thereof. [84] The variant of any of
paragraphs 1-29, which comprises the substitutions E105P,K;
G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y; or corresponding
substitutions thereof. [85] The variant of any of paragraphs 1-29,
which comprises the substitutions E154I,L; G188A,F,M,W; N189H,K;
A216L,Y; and K229W,H,I,Y; or corresponding substitutions thereof.
[86] The variant of any of paragraphs 1-29, which comprises the
substitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; A216L,Y; and
K229W,H,I,Y; or corresponding substitutions thereof. [87] The
variant of any of paragraphs 1-86, which further comprises a
substitution at one or more positions corresponding to positions
111, 152, 155, and 162 of the mature polypeptide of 30, wherein the
variant has cellulolytic enhancing activity. [88] The variant of
paragraph 87, wherein the number of substitutions is 1-4, e.g.,
such as 1, 2, 3, or 4 substitutions. [89] The variant of paragraph
87 or 88, which comprises a substitution at a position
corresponding to position 111. [90] The variant of paragraph 89,
wherein the substitution is Val. [91] The variant of any of
paragraphs 87-90, which comprises a substitution at a position
corresponding to position 152. [92] The variant of paragraph 91,
wherein the substitution is Ser. [93] The variant of any of
paragraphs 87-92, which comprises a substitution at a position
corresponding to position 155. [94] The variant of paragraph 93,
wherein the substitution is Leu. [95] The variant of any of
paragraphs 87-94, which comprises a substitution at a position
corresponding to position 162. [96] The variant of paragraph 95,
wherein the substitution is Trp. [97] The variant of any of
paragraphs 87-96, which comprises a substitution at two positions
corresponding to any of positions 111, 152, 155, and 162. [98] The
variant of any of paragraphs 87-96, which comprises a substitution
at three positions corresponding to any of positions 111, 152, 155,
and 162. [99] The variant of any of paragraphs 87-96, which
comprises a substitution at each position corresponding to
positions 111, 152, 155, and 162. [100] The variant of any of
paragraphs 87-99, which comprises one or more substitutions or
corresponding substitutions selected from the group consisting of
L111V, D152S, M155L, and A162W. [101] The variant of any of
paragraphs 87-100, which comprises the substitutions L111V+D152S;
or corresponding substitutions thereof. [102] The variant of any of
paragraphs 87-100, which comprises the substitutions L111V+M155L;
or corresponding substitutions thereof. [103] The variant of any of
paragraphs 87-100, which comprises the substitutions L111V+A162W;
or corresponding substitutions thereof. [104] The variant of any of
paragraphs 87-100, which comprises the substitutions D152S+M155L;
or corresponding substitutions thereof. [105] The variant of any of
paragraphs 87-100, which comprises the substitutions D152S+A162W;
or corresponding substitutions thereof. [106] The variant of any of
paragraphs 87-100, which comprises the substitutions M155L+A162W;
or corresponding substitutions thereof. [107] The variant of any of
paragraphs 87-100, which comprises the substitutions
L111V+D152S+M155L; or corresponding substitutions thereof. [108]
The variant of any of paragraphs 87-100, which comprises the
substitutions L111V+D152S+A162W; or corresponding substitutions
thereof. [109] The variant of any of paragraphs 87-100, which
comprises the substitutions L111V+M155L+A162W; or corresponding
substitutions thereof. [110] The variant of any of paragraphs
87-100, which comprises the substitutions D152S+M155L+A162W; or
corresponding substitutions thereof. [111] The variant of any of
paragraphs 87-100, which comprises the substitutions
L111V+D152S+M155L+A162W; or corresponding substitutions thereof.
[112] The variant of any of paragraphs 1-111, which further
comprises a substitution at one or more positions corresponding to
positions 96, 98, 200, 202, and 204 of the mature polypeptide of
30, wherein the variant has cellulolytic enhancing activity. [113]
The variant of paragraph 112, wherein the number of substitutions
is 1-5, e.g., such as 1, 2, 3, 4, or 5 substitutions. [114] The
variant of paragraph 112 or 113, which comprises a substitution at
a position corresponding to position 96. [115] The variant of
paragraph 114, wherein the substitution is Val. [116] The variant
of any of paragraphs 112-115, which comprises a substitution at a
position corresponding to position 98. [117] The variant of
paragraph 116 wherein the substitution is Leu. [118] The variant of
any of paragraphs 112-117, which comprises a substitution at a
position corresponding to position 200. [119] The variant of
paragraph 118, wherein the substitution is Ile. [120] The variant
of any of paragraphs 112-119, which comprises a substitution at a
position corresponding to position 202. [121] The variant of
paragraph 120, wherein the substitution is Leu. [122] The variant
of any of paragraphs 112-121, which comprises a substitution at a
position corresponding to position 204. [123] The variant of
paragraph 120, wherein the substitution is Val. [124] The variant
of any of paragraphs 112-123, which comprises a substitution at two
positions corresponding to any of positions 96, 98, 200, 202, and
204. [125] The variant of any of paragraphs 112-123, which
comprises a substitution at three positions corresponding to any of
positions 96, 98, 200, 202, and 204. [126] The variant of any of
paragraphs 112-123, which comprises a substitution at four
positions corresponding to any of positions 96, 98, 200, 202, and
204. [127] The variant of any of paragraphs 112-123, which
comprises a substitution at each position corresponding to
positions 96, 98, 200, 202, and 204. [128] The variant of any of
paragraphs 112-127, which comprises one or more substitutions or
corresponding substitutions selected from the group consisting of
I96V, F98L, F200I, I202L, and I204V. [129] The variant of any of
paragraphs 112-128, which comprises the substitutions I96V+F98L; or
corresponding substitutions thereof. [130] The variant of any of
paragraphs 112-128, which comprises the substitutions I96V+F200I;
or corresponding substitutions thereof. [131] The variant of any of
paragraphs 112-128, which comprises the substitutions I96V+I202L;
or corresponding substitutions thereof. [132] The variant of any of
paragraphs 112-128, which comprises the substitutions I96V+I204V;
or corresponding substitutions thereof. [133] The variant of any of
paragraphs 112-128, which comprises the substitutions F98L+F200I;
or corresponding substitutions thereof. [134] The variant of any of
paragraphs 112-128, which comprises the substitutions F98L+I202L;
or corresponding substitutions thereof. [135] The variant of any of
paragraphs 112-128, which comprises the substitutions F98L+I204V;
or corresponding substitutions thereof. [136] The variant of any of
paragraphs 112-128, which comprises the substitutions F200I+I202L;
or corresponding substitutions thereof. [137] The variant of any of
paragraphs 112-128, which comprises the substitutions F200I+I204V;
or corresponding substitutions thereof. [138] The variant of any of
paragraphs 112-128, which comprises the substitutions I202L+I204V;
or corresponding substitutions thereof. [139] The variant of any of
paragraphs 112-128, which comprises the substitutions
I96V+F98L+F200I; or corresponding substitutions thereof. [140] The
variant of any of paragraphs 112-128, which comprises the
substitutions I96V+F98L+I202L; or corresponding substitutions
thereof. [141] The variant of any of paragraphs 112-128, which
comprises the substitutions I96V+F98L+I204V; or corresponding
substitutions thereof. [142] The variant of any of paragraphs
112-128, which comprises the substitutions I96V+F200I+I202L; or
corresponding substitutions thereof. [143] The variant of any of
paragraphs 112-128, which comprises the substitutions
I96V+F200I+I204V; or corresponding substitutions thereof. [144] The
variant of any of paragraphs 112-128, which comprises the
substitutions I96V+I202L+I204V; or corresponding substitutions
thereof. [145] The variant of any of paragraphs 112-128, which
comprises the substitutions F98L+F200I+I202L; or corresponding
substitutions thereof. [146] The variant of any of paragraphs
112-128, which comprises the substitutions F98L+F200I+I204V; or
corresponding substitutions thereof. [147] The variant of any of
paragraphs 112-128, which comprises the substitutions
F200I+I202L+I204V; or corresponding substitutions thereof. [148]
The variant of any of paragraphs 112-128, which comprises the
substitutions F98L+I202L+I204V; or corresponding substitutions
thereof. [149] The variant of any of paragraphs 112-128, which
comprises the substitutions I96V+F98L+F200I+I202L; or corresponding
substitutions thereof. [150] The variant of any of paragraphs
112-128, which comprises the substitutions I96V+F200I+I202L+I204V;
or corresponding substitutions thereof. [151] The variant of any of
paragraphs 112-128, which comprises the substitutions
I96V+F98L+I202L+I204V; or corresponding substitutions thereof.
[152] The variant of any of paragraphs 112-128, which comprises the
substitutions I96V+F98L+F200I+I204V; or corresponding substitutions
thereof. [153] The variant of any of paragraphs 112-128, which
comprises the substitutions F98L+F200I+I202L+I204V; or
corresponding substitutions thereof. [154] The variant of any of
paragraphs 112-128, which comprises the substitutions
I96V+F98L+F200I+I202L+I204V; or corresponding substitutions
thereof. [155] The variant of any of paragraphs 1-154, wherein the
thermostability of the variant is increased at least 1.01-fold,
e.g., at least 1.05-fold, at least 1.1-fold, at least 1.2-fold, at
least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least
1.8-fold, at least 2-fold, at least 5-fold, at least 10-fold, at
least 15-fold, at least 20-fold, at least 25-fold, at least
50-fold, at least 75-fold, or at least 100-fold compared to the
parent. [156] An isolated polynucleotide encoding the variant of
any of paragraphs 1-155. [157] A nucleic acid construct comprising
the polynucleotide of paragraph 156. [158] An expression vector
comprising the polynucleotide of paragraph 156. [159] A host cell
comprising the polynucleotide of paragraph 156. [160] A method of
producing a GH61 polypeptide variant, comprising: cultivating the
host cell of paragraph 159 under conditions suitable for expression
of the variant. [161] The method of paragraph 160, further
comprising recovering the variant. [162] A transgenic plant, plant
part or plant cell transformed with the polynucleotide of paragraph
156. [163] A method of producing a variant of any of paragraphs
1-155, comprising: cultivating a transgenic plant or a plant cell
comprising a polynucleotide encoding the variant under conditions
conducive for production of the variant. [164] The method of
paragraph 163, further comprising recovering the variant. [165] A
method for obtaining a GH61 polypeptide variant, comprising
introducing into a parent GH61 polypeptide a substitution at one or
more positions corresponding to positions 105, 154, 188, 189, 216,
and 229 of the mature polypeptide of
30, wherein the variant has cellulolytic enhancing activity; and
optionally recovering the variant. [166] The method of paragraph
165, further comprising introducing into the parent GH61
polypeptide a substitution at one or more (e.g., several) positions
corresponding to positions 111, 152, 155, and 162 of the mature
polypeptide of 30, wherein the variant has cellulolytic enhancing
activity. [167] The method of paragraph 165 or 166, further
comprising introducing into the parent GH61 polypeptide a
substitution at one or more (e.g., several) positions corresponding
to positions 96, 98, 200, 202, and 204 of the mature polypeptide of
30, wherein the variant has cellulolytic enhancing activity. [168]
A process for degrading or converting a cellulosic material,
comprising: treating the cellulosic material with an enzyme
composition in the presence of the GH61 polypeptide variant having
cellulolytic enhancing activity of any of paragraphs 1-155. [169]
The process of paragraph 168, wherein the cellulosic material is
pretreated. [170] The process of paragraph 168 or 169, further
comprising recovering the degraded cellulosic material. [171] The
process of any of paragraphs 168-170, wherein the enzyme
composition comprises one or more enzymes selected from the group
consisting of a cellulase, a polypeptide having cellulolytic
enhancing activity, a hemicellulase, an esterase, an expansin, a
laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a
protease, and a swollenin. [172] The process of paragraph 171,
wherein the cellulase is one or more enzymes selected from the
group consisting of an endoglucanase, a endoglucanase, and a
beta-glucosidase. [173] The process of paragraph 171, wherein the
hemicellulase is one or more enzymes selected from the group
consisting of a xylanase, an acetyxylan esterase, a feruloyl
esterase, an arabinofuranosidase, a xylosidase, and a
glucuronidase. [174] The process of any of paragraphs 168-173,
wherein the degraded cellulosic material is a sugar. [175] The
process of paragraph 174, wherein the sugar is selected from the
group consisting of glucose, xylose, mannose, galactose, and
arabinose. [176] A process for producing a fermentation product,
comprising: (a) saccharifying a cellulosic material with an enzyme
composition in the presence of the GH61 polypeptide variant having
cellulolytic enhancing activity of any of paragraphs 1-155; (b)
fermenting the saccharified cellulosic material with one or more
fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
[177] The process of paragraph 176, wherein the cellulosic material
is pretreated. [178] The process of paragraph 176 or 177, wherein
the enzyme composition comprises one or more enzymes selected from
the group consisting of a cellulase, a polypeptide having
cellulolytic enhancing activity, a hemicellulase, an esterase, an
expansin, a laccase, a ligninolytic enzyme, a pectinase, a
peroxidase, a protease, and a swollenin. [179] The process of
paragraph 178, wherein the cellulase is one or more enzymes
selected from the group consisting of an endoglucanase, a
endoglucanase, and a beta-glucosidase. [180] The process of
paragraph 178, wherein the hemicellulase is one or more enzymes
selected from the group consisting of a xylanase, an acetyxylan
esterase, a feruloyl esterase, an arabinofuranosidase, a
xylosidase, and a glucuronidase. [181] The process of any of
paragraphs 176-180, wherein steps (a) and (b) are performed
simultaneously in a simultaneous saccharification and fermentation.
[182] The process of any of paragraphs 176-181, wherein the
fermentation product is an alcohol, an alkane, a cycloalkane, an
alkene, an amino acid, a gas, isoprene, a ketone, an organic acid,
or polyketide. [183] A process of fermenting a cellulosic material,
comprising: fermenting the cellulosic material with one or more
fermenting microorganisms, wherein the cellulosic material is
saccharified with an enzyme composition in the presence of the GH61
polypeptide variant having cellulolytic enhancing activity of any
of paragraphs 1-155. [184] The process of paragraph 183, wherein
the cellulosic material is pretreated before saccharification.
[185] The process of paragraph 183 or 184, wherein the enzyme
composition comprises one or more enzymes selected from the group
consisting of a cellulase, a polypeptide having cellulolytic
enhancing activity, a hemicellulase, an esterase, an expansin, a
laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a
protease, and a swollenin. [186] The process of paragraph 185,
wherein the cellulase is one or more enzymes selected from the
group consisting of an endoglucanase, a endoglucanase, and a
beta-glucosidase. [187] The process of paragraph 185, wherein the
hemicellulase is one or more enzymes selected from the group
consisting of a xylanase, an acetyxylan esterase, a feruloyl
esterase, an arabinofuranosidase, a xylosidase, and a
glucuronidase. [188] The process of any of paragraphs 183-187,
wherein the fermenting of the cellulosic material produces a
fermentation product. [189] The process of paragraph 189, further
comprising recovering the fermentation product from the
fermentation. [190] The process of paragraph 188 or 189, wherein
the fermentation product is an alcohol, an alkane, a cycloalkane,
an alkene, an amino acid, a gas, isoprene, a ketone, an organic
acid, or polyketide. [191] A whole broth formulation or cell
culture composition, comprising the variant of any of paragraphs
1-155. [192] A detergent composition, comprising a surfactant and
the variant of any of paragraphs 1-155. [193] The composition of
paragraph 192, further comprising one or more (e.g., several)
enzymes selected from the group consisting of an amylase,
arabinase, cutinase, carbohydrase, cellulase, galactanase, laccase,
lipase, mannanase, oxidase, pectinase, peroxidase, protease, and
xylanase. [194] The composition of paragraph 192 or 193, which is
formulated as a bar, a tablet, a powder, a granule, a paste, or a
liquid. [195] A method for cleaning or washing a hard surface or
laundry, the method comprising contacting the hard surface or the
laundry with the composition of any of paragraphs 192-194.
[0791] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
Sequence CWU 1
1
34611846DNAThielavia terrestris 1aattgaagga gggagtggcg gagtggccac
caagtcaggc ggctgtcaac taaccaagga 60tgggaacagt tcggctcgcc ttgcccgagg
gcagcgttcc ctgatgggga cgaaccatgg 120gactggggtc agctgctgta
taaaagttca aatcgatgat ctctcagatg gcgctgctgg 180ggtgttctgc
gcttttccat cctcgcaacc tggtatccca ctagtccagc gttcggcacc
240atgaagtcgt tcaccattgc cgccttggca gccctatggg cccaggaggc
cgccgcccac 300gcgaccttcc aggacctctg gattgatgga gtcgactacg
gctcgcaatg tgtccgcctc 360ccggcgtcca actcccccgt caccaatgtt
gcgtccgacg atatccgatg caatgtcggc 420acctcgaggc ccaccgtcaa
gtgcccggtc aaggccggct ccacggtcac gatcgagatg 480caccaggttc
gcacgcctct ctgcgtaggc cccccagcta ctatatggca ctaacacgac
540ctccagcaac ctggcgaccg gtcttgcgcc aacgaggcta tcggcggcga
ccactacggc 600cccgtaatgg tgtacatgtc caaggtcgat gacgcggtga
cagccgacgg ttcatcgggc 660tggttcaagg tgttccagga cagctgggcc
aagaacccgt cgggttcgac gggcgacgac 720gactactggg gcaccaagga
cctcaactcg tgctgcggca agatgaacgt caagatcccc 780gaagacatcg
agccgggcga ctacctgctc cgcgccgagg ttatcgcgct gcacgtggcc
840gccagctcgg gcggcgcgca gttctacatg tcctgctacc agctgaccgt
gacgggctcc 900ggcagcgcca ccccctcgac cgtgaatttc ccgggcgcct
actcggccag cgacccgggc 960atcctgatca acatccacgc gcccatgtcg
acctacgtcg tcccgggccc gaccgtgtac 1020gcgggcggct cgaccaagtc
ggctggcagc tcctgctccg gctgcgaggc gacctgcacg 1080gttggttccg
gccccagcgc gacactgacg cagcccacct ccaccgcgac cgcgacctcc
1140gcccctggcg gcggcggctc cggctgcacg gcggccaagt accagcagtg
cggcggcacc 1200ggctacactg ggtgcaccac ctgcgctgta agttccctcg
tgatatgcag cggaacaccg 1260tctggactgt tttgctaact cgcgtcgtag
tccgggtcta cctgcagcgc cgtctcgcct 1320ccgtactact cgcagtgcct
ctaagccggg agcgcttgct cagcgggctg ctgtgaagga 1380gctccatgtc
cccatgccgc catggccgga gtaccgggct gagcgcccaa ttcttgtata
1440tagttgagtt ttcccaatca tgaatacata tgcatctgca tggactgttg
cgtcgtcagt 1500ctacatcctt tgctccactg aactgtgaga ccccatgtca
tccggaccat tcgatcggtg 1560ctcgctctac catctcggtt gatgggtctg
ggcttgagag tcactggcac gtcctcggcg 1620gtaatgaaat gtggaggaaa
gtgtgagctg tctgacgcac tcggcgctga tgagacgttg 1680agcgcggccc
acactggtgt tctgtaagcc agcacacaaa agaatactcc aggatggccc
1740atagcggcaa atatacagta tcagggatgc aaaaagtgca aaagtaaggg
gctcaatcgg 1800ggatcgaacc cgagacctcg cacatgactt atttcaagtc aggggt
18462326PRTThielavia terrestris 2Met Lys Ser Phe Thr Ile Ala Ala
Leu Ala Ala Leu Trp Ala Gln Glu1 5 10 15Ala Ala Ala His Ala Thr Phe
Gln Asp Leu Trp Ile Asp Gly Val Asp 20 25 30Tyr Gly Ser Gln Cys Val
Arg Leu Pro Ala Ser Asn Ser Pro Val Thr 35 40 45Asn Val Ala Ser Asp
Asp Ile Arg Cys Asn Val Gly Thr Ser Arg Pro 50 55 60Thr Val Lys Cys
Pro Val Lys Ala Gly Ser Thr Val Thr Ile Glu Met65 70 75 80His Gln
Gln Pro Gly Asp Arg Ser Cys Ala Asn Glu Ala Ile Gly Gly 85 90 95Asp
His Tyr Gly Pro Val Met Val Tyr Met Ser Lys Val Asp Asp Ala 100 105
110Val Thr Ala Asp Gly Ser Ser Gly Trp Phe Lys Val Phe Gln Asp Ser
115 120 125Trp Ala Lys Asn Pro Ser Gly Ser Thr Gly Asp Asp Asp Tyr
Trp Gly 130 135 140Thr Lys Asp Leu Asn Ser Cys Cys Gly Lys Met Asn
Val Lys Ile Pro145 150 155 160Glu Asp Ile Glu Pro Gly Asp Tyr Leu
Leu Arg Ala Glu Val Ile Ala 165 170 175Leu His Val Ala Ala Ser Ser
Gly Gly Ala Gln Phe Tyr Met Ser Cys 180 185 190Tyr Gln Leu Thr Val
Thr Gly Ser Gly Ser Ala Thr Pro Ser Thr Val 195 200 205Asn Phe Pro
Gly Ala Tyr Ser Ala Ser Asp Pro Gly Ile Leu Ile Asn 210 215 220Ile
His Ala Pro Met Ser Thr Tyr Val Val Pro Gly Pro Thr Val Tyr225 230
235 240Ala Gly Gly Ser Thr Lys Ser Ala Gly Ser Ser Cys Ser Gly Cys
Glu 245 250 255Ala Thr Cys Thr Val Gly Ser Gly Pro Ser Ala Thr Leu
Thr Gln Pro 260 265 270Thr Ser Thr Ala Thr Ala Thr Ser Ala Pro Gly
Gly Gly Gly Ser Gly 275 280 285Cys Thr Ala Ala Lys Tyr Gln Gln Cys
Gly Gly Thr Gly Tyr Thr Gly 290 295 300Cys Thr Thr Cys Ala Ser Gly
Ser Thr Cys Ser Ala Val Ser Pro Pro305 310 315 320Tyr Tyr Ser Gln
Cys Leu 3253880DNAThielavia terrestris 3accccgggat cactgcccct
aggaaccagc acacctcggt ccaatcatgc ggttcgacgc 60cctctccgcc ctcgctcttg
cgccgcttgt ggctggccac ggcgccgtga ccagctacat 120catcggcggc
aaaacctatc ccggctacga gggcttctcg cctgcctcga gcccgccgac
180gatccagtac cagtggcccg actacaaccc gaccctgagc gtgaccgacc
cgaagatgcg 240ctgcaacggc ggcacctcgg cagagctcag cgcgcccgtc
caggccggcg agaacgtgac 300ggccgtctgg aagcagtgga cccaccagca
aggccccgtc atggtctgga tgttcaagtg 360ccccggcgac ttctcgtcgt
gccacggcga cggcaagggc tggttcaaga tcgaccagct 420gggcctgtgg
ggcaacaacc tcaactcgaa caactggggc accgcgatcg tctacaagac
480cctccagtgg agcaacccga tccccaagaa cctcgcgccg ggcaactacc
tcatccgcca 540cgagctgctc gccctgcacc aggccaacac gccgcagttc
tacgccgagt gcgcccagct 600ggtcgtctcc ggcagcggct ccgccctgcc
cccgtccgac tacctctaca gcatccccgt 660ctacgcgccc cagaacgacc
ccggcatcac cgtgagtggg cttccgttcc gcggcgagct 720ctgtggaaat
cttgctgacg atgggctagg ttgacatcta caacggcggg cttacctcct
780acaccccgcc cggcggcccc gtctggtctg gcttcgagtt ttaggcgcat
tgagtcgggg 840gctacgaggg gaaggcatct gttcgcatga gcgtgggtac
8804239PRTThielavia terrestris 4Met Arg Phe Asp Ala Leu Ser Ala Leu
Ala Leu Ala Pro Leu Val Ala1 5 10 15Gly His Gly Ala Val Thr Ser Tyr
Ile Ile Gly Gly Lys Thr Tyr Pro 20 25 30Gly Tyr Glu Gly Phe Ser Pro
Ala Ser Ser Pro Pro Thr Ile Gln Tyr 35 40 45Gln Trp Pro Asp Tyr Asn
Pro Thr Leu Ser Val Thr Asp Pro Lys Met 50 55 60Arg Cys Asn Gly Gly
Thr Ser Ala Glu Leu Ser Ala Pro Val Gln Ala65 70 75 80Gly Glu Asn
Val Thr Ala Val Trp Lys Gln Trp Thr His Gln Gln Gly 85 90 95Pro Val
Met Val Trp Met Phe Lys Cys Pro Gly Asp Phe Ser Ser Ser 100 105
110His Gly Asp Gly Lys Gly Trp Phe Lys Ile Asp Gln Leu Gly Leu Trp
115 120 125Gly Asn Asn Leu Asn Ser Asn Asn Trp Gly Thr Ala Ile Val
Tyr Lys 130 135 140Thr Leu Gln Trp Ser Asn Pro Ile Pro Lys Asn Leu
Ala Pro Gly Asn145 150 155 160Tyr Leu Ile Arg His Glu Leu Leu Ala
Leu His Gln Ala Asn Thr Pro 165 170 175Gln Phe Tyr Ala Glu Cys Ala
Gln Leu Val Val Ser Gly Ser Gly Ser 180 185 190Ala Leu Pro Pro Ser
Asp Tyr Leu Tyr Ser Ile Pro Val Tyr Ala Pro 195 200 205Gln Asn Asp
Pro Gly Ile Thr Val Asp Ile Tyr Asn Gly Gly Leu Thr 210 215 220Ser
Tyr Thr Pro Pro Gly Gly Pro Val Trp Ser Gly Phe Glu Phe225 230
23551000DNAThielavia terrestris 5ctcctgttcc tgggccaccg cttgttgcct
gcactattgg tagagttggt ctattgctag 60agttggccat gcttctcaca tcagtcctcg
gctcggctgc cctgcttgct agcggcgctg 120cggcacacgg cgccgtgacc
agctacatca tcgccggcaa gaattacccg gggtgggtag 180ctgattattg
agggcgcatt caaggttcat accggtgtgc atggctgaca accggctggc
240agataccaag gcttttctcc tgcgaactcg ccgaacgtca tccaatggca
atggcatgac 300tacaaccccg tcttgtcgtg cagcgactcg aagcttcgct
gcaacggcgg cacgtcggcc 360accctgaacg ccacggccgc accgggcgac
accatcaccg ccatctgggc gcagtggacg 420cacagccagg gccccatcct
ggtgtggatg tacaagtgcc cgggctcctt cagctcctgt 480gacggctccg
gcgctggctg gttcaagatc gacgaggccg gcttccacgg cgacggcgtc
540aaggtcttcc tcgacaccga gaacccgtcc ggctgggaca tcgccaagct
cgtcggcggc 600aacaagcagt ggagcagcaa ggtccccgag ggcctcgccc
ccggcaacta cctcgtccgc 660cacgagttga tcgccctgca ccaggccaac
aacccgcagt tctacccgga gtgcgcccag 720gtcgtcatca ccggctccgg
caccgcgcag ccggatgcct catacaaggc ggctatcccc 780ggctactgca
accagaatga cccgaacatc aaggtgagat ccaggcgtaa tgcagtctac
840tgctggaaag aaagtggtcc aagctaaacc gcgctccagg tgcccatcaa
cgaccactcc 900atccctcaga cctacaagat tcccggccct cccgtcttca
agggcaccgc cagcaagaag 960gcccgggact tcaccgcctg aagttgttga
atcgatggag 10006258PRTThielavia terrestris 6Met Leu Leu Thr Ser Val
Leu Gly Ser Ala Ala Leu Leu Ala Ser Gly1 5 10 15Ala Ala Ala His Gly
Ala Val Thr Ser Tyr Ile Ile Ala Gly Lys Asn 20 25 30Tyr Pro Gly Tyr
Gln Gly Phe Ser Pro Ala Asn Ser Pro Asn Val Ile 35 40 45Gln Trp Gln
Trp His Asp Tyr Asn Pro Val Leu Ser Cys Ser Asp Ser 50 55 60Lys Leu
Arg Cys Asn Gly Gly Thr Ser Ala Thr Leu Asn Ala Thr Ala65 70 75
80Ala Pro Gly Asp Thr Ile Thr Ala Ile Trp Ala Gln Trp Thr His Ser
85 90 95Gln Gly Pro Ile Leu Val Trp Met Tyr Lys Cys Pro Gly Ser Phe
Ser 100 105 110Ser Cys Asp Gly Ser Gly Ala Gly Trp Phe Lys Ile Asp
Glu Ala Gly 115 120 125Phe His Gly Asp Gly Val Lys Val Phe Leu Asp
Thr Glu Asn Pro Ser 130 135 140Gly Trp Asp Ile Ala Lys Leu Val Gly
Gly Asn Lys Gln Trp Ser Ser145 150 155 160Lys Val Pro Glu Gly Leu
Ala Pro Gly Asn Tyr Leu Val Arg His Glu 165 170 175Leu Ile Ala Leu
His Gln Ala Asn Asn Pro Gln Phe Tyr Pro Glu Cys 180 185 190Ala Gln
Val Val Ile Thr Gly Ser Gly Thr Ala Gln Pro Asp Ala Ser 195 200
205Tyr Lys Ala Ala Ile Pro Gly Tyr Cys Asn Gln Asn Asp Pro Asn Ile
210 215 220Lys Val Pro Ile Asn Asp His Ser Ile Pro Gln Thr Tyr Lys
Ile Pro225 230 235 240Gly Pro Pro Val Phe Lys Gly Thr Ala Ser Lys
Lys Ala Arg Asp Phe 245 250 255Thr Ala7681DNAThielavia terrestris
7atgctcgcaa acggtgccat cgtcttcctg gccgccgccc tcggcgtcag tggccactac
60acctggccac gggttaacga cggcgccgac tggcaacagg tccgtaaggc ggacaactgg
120caggacaacg gctacgtcgg ggatgtcacg tcgccacaga tccgctgttt
ccaggcgacc 180ccgtccccgg ccccatccgt cctcaacacc acggccggct
cgaccgtgac ctactgggcc 240aaccccgacg tctaccaccc cgggcctgtg
cagttttaca tggcccgcgt gcccgatggc 300gaggacatca actcgtggaa
cggcgacggc gccgtgtggt tcaaggtgta cgaggaccat 360cctacctttg
gcgctcagct cacatggccc agcacgggca agagctcgtt cgcggttccc
420atccccccgt gcatcaagtc cggctactac ctcctccggg cggagcaaat
cggcctgcac 480gtcgcccaga gcgtaggcgg agcgcagttc tacatctcat
gcgcccagct cagcgtcacc 540ggcggcggca gcaccgagcc gccgaacaag
gtggccttcc ccggcgctta cagtgcgacg 600gacccgggca ttctgatcaa
catctactac cctgttccca cgtcctacca gaaccccggc 660ccggccgtct
tcagctgctg a 6818226PRTThielavia terrestris 8Met Leu Ala Asn Gly
Ala Ile Val Phe Leu Ala Ala Ala Leu Gly Val1 5 10 15Ser Gly His Tyr
Thr Trp Pro Arg Val Asn Asp Gly Ala Asp Trp Gln 20 25 30Gln Val Arg
Lys Ala Asp Asn Trp Gln Asp Asn Gly Tyr Val Gly Asp 35 40 45Val Thr
Ser Pro Gln Ile Arg Cys Phe Gln Ala Thr Pro Ser Pro Ala 50 55 60Pro
Ser Val Leu Asn Thr Thr Ala Gly Ser Thr Val Thr Tyr Trp Ala65 70 75
80Asn Pro Asp Val Tyr His Pro Gly Pro Val Gln Phe Tyr Met Ala Arg
85 90 95Val Pro Asp Gly Glu Asp Ile Asn Ser Trp Asn Gly Asp Gly Ala
Val 100 105 110Trp Phe Lys Val Tyr Glu Asp His Pro Thr Phe Gly Ala
Gln Leu Thr 115 120 125Trp Pro Ser Thr Gly Lys Ser Ser Phe Ala Val
Pro Ile Pro Pro Cys 130 135 140Ile Lys Ser Gly Tyr Tyr Leu Leu Arg
Ala Glu Gln Ile Gly Leu His145 150 155 160Val Ala Gln Ser Val Gly
Gly Ala Gln Phe Tyr Ile Ser Cys Ala Gln 165 170 175Leu Ser Val Thr
Gly Gly Gly Ser Thr Glu Pro Pro Asn Lys Val Ala 180 185 190Phe Pro
Gly Ala Tyr Ser Ala Thr Asp Pro Gly Ile Leu Ile Asn Ile 195 200
205Tyr Tyr Pro Val Pro Thr Ser Tyr Gln Asn Pro Gly Pro Ala Val Phe
210 215 220Ser Cys2259960DNAThielavia terrestris 9atgaagggac
ttttcagtgc cgccgccctc tccctggccg tcggccaggc ttcggcccat 60tacatcttcc
agcaactctc catcaacggg aaccagtttc cggtgtacca atatattcgc
120aagaacacca attataacag tcccgttacc gatctcacgt ccgacgatct
tcggtgcaat 180gtcggcgccc agggtgctgg gacagacacc gtcacggtga
aggccggcga ccagttcacc 240ttcacccttg acacccctgt ttaccaccag
gggcccatct ccatctacat gtccaaggcc 300ccgggcgcgg cgtcagacta
cgatggcagc ggcggctggt tcaagatcaa ggactggggc 360ccgactttca
acgccgacgg cacggccacc tgggacatgg ccggctcata cacctacaac
420atcccgacct gcattcccga cggcgactat ctgctccgca tccagtcgct
ggccatccac 480aacccctggc cggcgggcat cccgcagttc tacatctcct
gcgcccagat caccgtgacc 540ggcggcggca acggcaaccc tggcccgacg
gccctcatcc ccggcgcctt caaggacacc 600gacccgggct acacggtgaa
catctacacg aacttccaca actacacggt tcccggcccg 660gaggtcttca
gctgcaacgg cggcggctcg aacccgcccc cgccggtgag tagcagcacg
720cccgcgacca cgacgctggt cacgtcgacg cgcaccacgt cctccacgtc
ctccgcctcg 780acgccggcct cgaccggcgg ctgcaccgtc gccaagtggg
gccagtgcgg cggcaacggg 840tacaccggct gcacgacctg cgcggccggg
tccacctgca gcaagcagaa cgactactac 900tcgcagtgct tgtaagggag
gccgcaaagc atgaggtgtt tgaagaggag gagaggggtc 96010304PRTThielavia
terrestris 10Met Lys Gly Leu Phe Ser Ala Ala Ala Leu Ser Leu Ala
Val Gly Gln1 5 10 15Ala Ser Ala His Tyr Ile Phe Gln Gln Leu Ser Ile
Asn Gly Asn Gln 20 25 30Phe Pro Val Tyr Gln Tyr Ile Arg Lys Asn Thr
Asn Tyr Asn Ser Pro 35 40 45Val Thr Asp Leu Thr Ser Asp Asp Leu Arg
Cys Asn Val Gly Ala Gln 50 55 60Gly Ala Gly Thr Asp Thr Val Thr Val
Lys Ala Gly Asp Gln Phe Thr65 70 75 80Phe Thr Leu Asp Thr Pro Val
Tyr His Gln Gly Pro Ile Ser Ile Tyr 85 90 95Met Ser Lys Ala Pro Gly
Ala Ala Ser Asp Tyr Asp Gly Ser Gly Gly 100 105 110Trp Phe Lys Ile
Lys Asp Trp Gly Pro Thr Phe Asn Ala Asp Gly Thr 115 120 125Ala Thr
Trp Asp Met Ala Gly Ser Tyr Thr Tyr Asn Ile Pro Thr Cys 130 135
140Ile Pro Asp Gly Asp Tyr Leu Leu Arg Ile Gln Ser Leu Ala Ile
His145 150 155 160Asn Pro Trp Pro Ala Gly Ile Pro Gln Phe Tyr Ile
Ser Cys Ala Gln 165 170 175Ile Thr Val Thr Gly Gly Gly Asn Gly Asn
Pro Gly Pro Thr Ala Leu 180 185 190Ile Pro Gly Ala Phe Lys Asp Thr
Asp Pro Gly Tyr Thr Val Asn Ile 195 200 205Tyr Thr Asn Phe His Asn
Tyr Thr Val Pro Gly Pro Glu Val Phe Ser 210 215 220Cys Asn Gly Gly
Gly Ser Asn Pro Pro Pro Pro Val Ser Ser Ser Thr225 230 235 240Pro
Ala Thr Thr Thr Leu Val Thr Ser Thr Arg Thr Thr Ser Ser Thr 245 250
255Ser Ser Ala Ser Thr Pro Ala Ser Thr Gly Gly Cys Thr Val Ala Lys
260 265 270Trp Gly Gln Cys Gly Gly Asn Gly Tyr Thr Gly Cys Thr Thr
Cys Ala 275 280 285Ala Gly Ser Thr Cys Ser Lys Gln Asn Asp Tyr Tyr
Ser Gln Cys Leu 290 295 30011954DNAThielavia terrestris
11atgaagggcc tcagcctcct cgccgctgcg tcggcagcga ctgctcatac catcttcgtg
60cagctcgagt cagggggaac gacctatccg gtatcctacg gcatccggga ccctagctac
120gacggtccca tcaccgacgt cacctccgac tcactggctt gcaatggtcc
cccgaacccc 180acgacgccgt ccccgtacat catcaacgtc accgccggca
ccacggtcgc ggcgatctgg 240aggcacaccc tcacatccgg ccccgacgat
gtcatggacg ccagccacaa ggggccgacc 300ctggcctacc tcaagaaggt
cgatgatgcc ttgaccgaca cgggtatcgg cggcggctgg 360ttcaagatcc
aggaggccgg ttacgacaat ggcaattggg ctaccagcac ggtgatcacc
420aacggtggct tccaatatat tgacatcccc gcctgcattc ccaacggcca
gtatctgctc 480cgcgccgaga tgatcgcgct ccacgccgcc agcacgcagg
gtggtgccca gctctacatg 540gagtgcgcgc agatcaacgt ggtgggcggc
tccggcagcg ccagcccgca gacgtacagc 600atcccgggca tctaccaggc
aaccgacccg ggcctgctga tcaacatcta ctccatgacg 660ccgtccagcc
agtacaccat tccgggtccg cccctgttca cctgcagcgg cagcggcaac
720aacggcggcg gcagcaaccc gtcgggcggg cagaccacga cggcgaagcc
cacgacgacg 780acggcggcga cgaccacctc ctccgccgct cctaccagca
gccagggggg cagcagcggt 840tgcaccgttc cccagtggca gcagtgcggt
ggcatctcgt tcaccggctg caccacctgc 900gcggcgggct acacctgcaa
gtatctgaac gactattact cgcaatgcca gtaa 95412317PRTThielavia
terrestris 12Met Lys Gly Leu Ser Leu Leu Ala Ala Ala Ser Ala Ala
Thr Ala His1 5 10 15Thr Ile Phe Val Gln Leu Glu
Ser Gly Gly Thr Thr Tyr Pro Val Ser 20 25 30Tyr Gly Ile Arg Asp Pro
Ser Tyr Asp Gly Pro Ile Thr Asp Val Thr 35 40 45Ser Asp Ser Leu Ala
Cys Asn Gly Pro Pro Asn Pro Thr Thr Pro Ser 50 55 60Pro Tyr Ile Ile
Asn Val Thr Ala Gly Thr Thr Val Ala Ala Ile Trp65 70 75 80Arg His
Thr Leu Thr Ser Gly Pro Asp Asp Val Met Asp Ala Ser His 85 90 95Lys
Gly Pro Thr Leu Ala Tyr Leu Lys Lys Val Asp Asp Ala Leu Thr 100 105
110Asp Thr Gly Ile Gly Gly Gly Trp Phe Lys Ile Gln Glu Ala Gly Tyr
115 120 125Asp Asn Gly Asn Trp Ala Thr Ser Thr Val Ile Thr Asn Gly
Gly Phe 130 135 140Gln Tyr Ile Asp Ile Pro Ala Cys Ile Pro Asn Gly
Gln Tyr Leu Leu145 150 155 160Arg Ala Glu Met Ile Ala Leu His Ala
Ala Ser Thr Gln Gly Gly Ala 165 170 175Gln Leu Tyr Met Glu Cys Ala
Gln Ile Asn Val Val Gly Gly Ser Gly 180 185 190Ser Ala Ser Pro Gln
Thr Tyr Ser Ile Pro Gly Ile Tyr Gln Ala Thr 195 200 205Asp Pro Gly
Leu Leu Ile Asn Ile Tyr Ser Met Thr Pro Ser Ser Gln 210 215 220Tyr
Thr Ile Pro Gly Pro Pro Leu Phe Thr Cys Ser Gly Ser Gly Asn225 230
235 240Asn Gly Gly Gly Ser Asn Pro Ser Gly Gly Gln Thr Thr Thr Ala
Lys 245 250 255Pro Thr Thr Thr Thr Ala Ala Thr Thr Thr Ser Ser Ala
Ala Pro Thr 260 265 270Ser Ser Gln Gly Gly Ser Ser Gly Cys Thr Val
Pro Gln Trp Gln Gln 275 280 285Cys Gly Gly Ile Ser Phe Thr Gly Cys
Thr Thr Cys Ala Ala Gly Tyr 290 295 300Thr Cys Lys Tyr Leu Asn Asp
Tyr Tyr Ser Gln Cys Gln305 310 31513799DNAThermoascus aurantiacus
13atgtcctttt ccaagataat tgctactgcc ggcgttcttg cctctgcttc tctagtggct
60ggccatggct tcgttcagaa catcgtgatt gatggtaaaa agtatgtcat tgcaagacgc
120acataagcgg caacagctga caatcgacag ttatggcggg tatctagtga
accagtatcc 180atacatgtcc aatcctccag aggtcatcgc ctggtctact
acggcaactg atcttggatt 240tgtggacggt actggatacc aaaccccaga
tatcatctgc cataggggcg ccaagcctgg 300agccctgact gctccagtct
ctccaggagg aactgttgag cttcaatgga ctccatggcc 360tgattctcac
catggcccag ttatcaacta ccttgctccg tgcaatggtg attgttccac
420tgtggataag acccaattag aattcttcaa aattgccgag agcggtctca
tcaatgatga 480caatcctcct gggatctggg cttcagacaa tctgatagca
gccaacaaca gctggactgt 540caccattcca accacaattg cacctggaaa
ctatgttctg aggcatgaga ttattgctct 600tcactcagct cagaaccagg
atggtgccca gaactatccc cagtgcatca atctgcaggt 660cactggaggt
ggttctgata accctgctgg aactcttgga acggcactct accacgatac
720cgatcctgga attctgatca acatctatca gaaactttcc agctatatca
tccctggtcc 780tcctctgtat actggttaa 79914249PRTThermoascus
aurantiacus 14Met Ser Phe Ser Lys Ile Ile Ala Thr Ala Gly Val Leu
Ala Ser Ala1 5 10 15Ser Leu Val Ala Gly His Gly Phe Val Gln Asn Ile
Val Ile Asp Gly 20 25 30Lys Tyr Tyr Gly Gly Tyr Leu Val Asn Gln Tyr
Pro Tyr Met Ser Asn 35 40 45Pro Pro Glu Val Ile Ala Trp Ser Thr Thr
Ala Thr Asp Leu Gly Phe 50 55 60Val Asp Gly Thr Gly Tyr Gln Thr Pro
Asp Ile Ile Cys His Arg Gly65 70 75 80Ala Lys Pro Gly Ala Leu Thr
Ala Pro Val Ser Pro Gly Gly Thr Val 85 90 95Glu Leu Gln Trp Thr Pro
Trp Pro Asp Ser His His Gly Pro Val Ile 100 105 110Asn Tyr Leu Ala
Pro Cys Asn Gly Asp Cys Ser Thr Val Asp Lys Thr 115 120 125Gln Leu
Glu Phe Phe Lys Ile Ala Glu Ser Gly Leu Ile Asn Asp Asp 130 135
140Asn Pro Pro Gly Ile Trp Ala Ser Asp Asn Leu Ile Ala Ala Asn
Asn145 150 155 160Ser Trp Thr Val Thr Ile Pro Thr Thr Ile Ala Pro
Gly Asn Tyr Val 165 170 175Leu Arg His Glu Ile Ile Ala Leu His Ser
Ala Gln Asn Gln Asp Gly 180 185 190Ala Gln Asn Tyr Pro Gln Cys Ile
Asn Leu Gln Val Thr Gly Gly Gly 195 200 205Ser Asp Asn Pro Ala Gly
Thr Leu Gly Thr Ala Leu Tyr His Asp Thr 210 215 220Asp Pro Gly Ile
Leu Ile Asn Ile Tyr Gln Lys Leu Ser Ser Tyr Ile225 230 235 240Ile
Pro Gly Pro Pro Leu Tyr Thr Gly 245151172DNATrichoderma reesei
15ggatctaagc cccatcgata tgaagtcctg cgccattctt gcagcccttg gctgtcttgc
60cgggagcgtt ctcggccatg gacaagtcca aaacttcacg atcaatggac aatacaatca
120gggtttcatt ctcgattact actatcagaa gcagaatact ggtcacttcc
ccaacgttgc 180tggctggtac gccgaggacc tagacctggg cttcatctcc
cctgaccaat acaccacgcc 240cgacattgtc tgtcacaaga acgcggcccc
aggtgccatt tctgccactg cagcggccgg 300cagcaacatc gtcttccaat
ggggccctgg cgtctggcct cacccctacg gtcccatcgt 360tacctacgtg
gctgagtgca gcggatcgtg cacgaccgtg aacaagaaca acctgcgctg
420ggtcaagatt caggaggccg gcatcaacta taacacccaa gtctgggcgc
agcaggatct 480gatcaaccag ggcaacaagt ggactgtgaa gatcccgtcg
agcctcaggc ccggaaacta 540tgtcttccgc catgaacttc ttgctgccca
tggtgcctct agtgcgaacg gcatgcagaa 600ctatcctcag tgcgtgaaca
tcgccgtcac aggctcgggc acgaaagcgc tccctgccgg 660aactcctgca
actcagctct acaagcccac tgaccctggc atcttgttca acccttacac
720aacaatcacg agctacacca tccctggccc agccctgtgg caaggctaga
tccaggggta 780cggtgttggc gttcgtgaag tcggagctgt tgacaaggat
atctgatgat gaacggagag 840gactgatggg cgtgactgag tgtatatatt
tttgatgacc aaattgtata cgaaatccga 900acgcatggtg atcattgttt
atccctgtag tatattgtct ccaggctgct aagagcccac 960cgggtgtatt
acggcaacaa agtcaggaat ttgggtggca atgaacgcag gtctccatga
1020atgtatatgt gaagaggcat cggctggcat gggcattacc agatataggc
cctgtgaaac 1080atatagtact tgaacgtgct actggaacgg atcataagca
agtcatcaac atgtgaaaaa 1140acactacatg taaaaaaaaa aaaaaaaaaa aa
117216249PRTTrichoderma reesei 16Met Lys Ser Cys Ala Ile Leu Ala
Ala Leu Gly Cys Leu Ala Gly Ser1 5 10 15Val Leu Gly His Gly Gln Val
Gln Asn Phe Thr Ile Asn Gly Gln Tyr 20 25 30Asn Gln Gly Phe Ile Leu
Asp Tyr Tyr Tyr Gln Lys Gln Asn Thr Gly 35 40 45His Phe Pro Asn Val
Ala Gly Trp Tyr Ala Glu Asp Leu Asp Leu Gly 50 55 60Phe Ile Ser Pro
Asp Gln Tyr Thr Thr Pro Asp Ile Val Cys His Lys65 70 75 80Asn Ala
Ala Pro Gly Ala Ile Ser Ala Thr Ala Ala Ala Gly Ser Asn 85 90 95Ile
Val Phe Gln Trp Gly Pro Gly Val Trp Pro His Pro Tyr Gly Pro 100 105
110Ile Val Thr Tyr Val Val Glu Cys Ser Gly Ser Cys Thr Thr Val Asn
115 120 125Lys Asn Asn Leu Arg Trp Val Lys Ile Gln Glu Ala Gly Ile
Asn Tyr 130 135 140Asn Thr Gln Val Trp Ala Gln Gln Asp Leu Ile Asn
Gln Gly Asn Lys145 150 155 160Trp Thr Val Lys Ile Pro Ser Ser Leu
Arg Pro Gly Asn Tyr Val Phe 165 170 175Arg His Glu Leu Leu Ala Ala
His Gly Ala Ser Ser Ala Asn Gly Met 180 185 190Gln Asn Tyr Pro Gln
Cys Val Asn Ile Ala Val Thr Gly Ser Gly Thr 195 200 205Lys Ala Leu
Pro Ala Gly Thr Pro Ala Thr Gln Leu Tyr Lys Pro Thr 210 215 220Asp
Pro Gly Ile Leu Phe Asn Pro Tyr Thr Thr Ile Thr Ser Tyr Thr225 230
235 240Ile Pro Gly Pro Ala Leu Trp Gln Gly
24517924DNAMyceliophthora thermophila 17atgaagttca cctcgtccct
cgctgtcctg gccgctgccg gcgcccaggc tcactgttag 60tcgaccctcg aacccaacac
ccccctcccc ccttttctcc tccatctcct cggcctcact 120tagtagccgc
tgacaacgac tagatacctt ccctagggcc ggcactggtg gctcgctctc
180tggcgagtgg gaggtggtcc gcatgaccga gaaccattac tcgcacggcc
cggtcaccga 240tgtcaccagc cccgagatga cctgctatca gtccggcgtg
cagggtgcgc cccagaccgt 300ccaggtcaag gcgggctccc aattcacctt
cagcgtggat ccctcgatcg gccaccccgg 360ccctctccag ttctacatgg
ctaaggtgcc gtcgggccag acggccgcca cctttgacgg 420cacgggagcc
gtgtggttca agatctacca agacggcccg aacggcctcg gcaccgacag
480cattacctgg cccagcgccg gttcgtgact tcctccccac tcgctttttt
ttttttattt 540tttatttttt tttctttcgg aactcaagaa tctttctctc
tctctcccgt ctttggcctt 600gaacaacact aaaactcttc cttactgtat
taattaggca aaaccgaggt ctcggtcacc 660atccccagct gcatcgatga
tggcgagtac ctgctccggg tcgagcacat cgcgctccac 720agcgccagca
gcgtgggcgg cgctcagttc tacattgcct gcgcccagct ctccgtcacc
780ggcggctccg gcaccctcaa cacgggctcg ctcgtctccc tgcccggcgc
ctacaaggcc 840accgacccgg gcatcctctt ccagctctac tggcccatcc
cgaccgagta catcaacccc 900ggcccggccc ccgtctcttg ctaa
92418232PRTMyceliophthora thermophila 18Met Lys Phe Thr Ser Ser Leu
Ala Val Leu Ala Ala Ala Gly Ala Gln1 5 10 15Ala His Tyr Thr Phe Pro
Arg Ala Gly Thr Gly Gly Ser Leu Ser Gly 20 25 30Glu Trp Glu Val Val
Arg Met Thr Glu Asn His Tyr Ser His Gly Pro 35 40 45Val Thr Asp Val
Thr Ser Pro Glu Met Thr Cys Tyr Gln Ser Gly Val 50 55 60Gln Gly Ala
Pro Gln Thr Val Gln Val Lys Ala Gly Ser Gln Phe Thr65 70 75 80Phe
Ser Val Asp Pro Ser Ile Gly His Pro Gly Pro Leu Gln Phe Tyr 85 90
95Met Ala Lys Val Pro Ser Gly Gln Thr Ala Ala Thr Phe Asp Gly Thr
100 105 110Gly Ala Val Trp Phe Lys Ile Tyr Gln Asp Gly Pro Asn Gly
Leu Gly 115 120 125Thr Asp Ser Ile Thr Trp Pro Ser Ala Gly Lys Thr
Glu Val Ser Val 130 135 140Thr Ile Pro Ser Cys Ile Asp Asp Gly Glu
Tyr Leu Leu Arg Val Glu145 150 155 160His Ile Ala Leu His Ser Ala
Ser Ser Val Gly Gly Ala Gln Phe Tyr 165 170 175Ile Ala Cys Ala Gln
Leu Ser Val Thr Gly Gly Ser Gly Thr Leu Asn 180 185 190Thr Gly Ser
Leu Val Ser Leu Pro Gly Ala Tyr Lys Ala Thr Asp Pro 195 200 205Gly
Ile Leu Phe Gln Leu Tyr Trp Pro Ile Pro Thr Glu Tyr Ile Asn 210 215
220Pro Gly Pro Ala Pro Val Ser Cys225 23019854DNAMyceliophthora
thermophila 19atgaaggccc tctctctcct tgcggctgcc tcggcagtct
ctgcgcatac catcttcgtc 60cagctcgaag cagacggcac gaggtacccg gtctcgtacg
ggatccggga cccaagctac 120gacggcccca tcaccgacgt cacatccaac
gacgttgctt gcaacggcgg gccgaacccg 180acgaccccct ccagcgacgt
catcaccgtc accgcgggca ccacggtcaa ggccatctgg 240aggcacaccc
tccaatccgg cccggacgat gtcatggacg ccagccacaa gggcccgacc
300ctggcctacc tcaagaaggt cggcgatgcc accaaggact cgggcgtcgg
cggtggctgg 360ttcaagattc aggaggacgg ctacaacaac ggccagtggg
gcaccagcac cgttatctcc 420aacggcggcg agcactacat gtgagccatt
cctccgagag aagaccaaga ctcttgacga 480tctcgctgac ccgtgcaaca
agtgacatcc cggcctgcat ccccgagggt cagtacctcc 540tccgcgccga
gatgatcgcc ctccacgcgg ccgggtcccc cggcggtgcc cagctctacg
600taagcctctg cccttccccc cttcctcttg atcgaatcgg actgcccacc
ccccttttcg 660actccgacta acaccgttgc cagatggaat gtgcccagat
caacatcgtc ggcggctccg 720gctcggtgcc cagctcgacc gtcagcttcc
ccggcgcgta cagccccaac gacccgggtc 780tcctcatcaa catctattcc
atgtcgccct cgagctcgta caccatcccg ggcccgcccg 840tcttcaagtg ctag
85420235PRTMyceliophthora thermophila 20Met Lys Ala Leu Ser Leu Leu
Ala Ala Ala Ser Ala Val Ser Ala His1 5 10 15Thr Ile Phe Val Gln Leu
Glu Ala Asp Gly Thr Arg Tyr Pro Val Ser 20 25 30Tyr Gly Ile Arg Asp
Pro Ser Tyr Asp Gly Pro Ile Thr Asp Val Thr 35 40 45Ser Asn Asp Val
Ala Cys Asn Gly Gly Pro Asn Pro Thr Thr Pro Ser 50 55 60Ser Asp Val
Ile Thr Val Thr Ala Gly Thr Thr Val Lys Ala Ile Trp65 70 75 80Arg
His Thr Leu Gln Ser Gly Pro Asp Asp Val Met Asp Ala Ser His 85 90
95Lys Gly Pro Thr Leu Ala Tyr Leu Lys Lys Val Gly Asp Ala Thr Lys
100 105 110Asp Ser Gly Val Gly Gly Gly Trp Phe Lys Ile Gln Glu Asp
Gly Tyr 115 120 125Asn Asn Gly Gln Trp Gly Thr Ser Thr Val Ile Ser
Asn Gly Gly Glu 130 135 140His Tyr Ile Asp Ile Pro Ala Cys Ile Pro
Glu Gly Gln Tyr Leu Leu145 150 155 160Arg Ala Glu Met Ile Ala Leu
His Ala Ala Gly Ser Pro Gly Gly Ala 165 170 175Gln Leu Tyr Met Glu
Cys Ala Gln Ile Asn Ile Val Gly Gly Ser Gly 180 185 190Ser Val Pro
Ser Ser Thr Val Ser Phe Pro Gly Ala Tyr Ser Pro Asn 195 200 205Asp
Pro Gly Leu Leu Ile Asn Ile Tyr Ser Met Ser Pro Ser Ser Ser 210 215
220Tyr Thr Ile Pro Gly Pro Pro Val Phe Lys Cys225 230
235211242DNAMyceliophthora thermophila 21atgaagtcct tcgccctcac
cactctggcc gccctggccg gcaacgccgc cgctcacgcg 60accttccagg ccctctgggt
cgacggcgtc gactacggcg cgcagtgtgc ccgtctgccc 120gcgtccaact
ccccggtcac cgacgtgacc tccaacgcga tccgctgcaa cgccaacccg
180tcgcccgctc ggggcaagtg cccggtcaag gccggctcga ccgttacggt
cgagatgcat 240caggtacgtt ggatgaatga aaggggaaag gaagcagagg
cagaagggga aggcgaaggg 300aaagaaaaag aaaaagaaat ggaaaagaaa
aagaaatgga aaagaaaaag aaaaatgaaa 360aagaaagtgg aaaccgtcag
actaactggg gctcctcccc cccacccctc ctttgatatc 420agcaacccgg
tgaccggtcg tgcagcagcg aggcgatcgg cggggcgcac tacggccccg
480tcatggtgta catgtccaag gtgtcggacg cggcgtcggc ggacgggtcg
tcgggctggt 540tcaaggtgtt cgaggacggc tgggccaaga acccgtccgg
cgggtcgggc gacgacgact 600actggggcac caaggacctg aactcgtgct
gcgggaagat gaacgtcaag atccccgccg 660acctgccctc gggcgactac
ctgctccggg ccgaggccct cgcgctgcac acggcgggca 720gcgccggcgg
cgcccagttc tacatgacgt gctaccagct caccgtgacg ggctccggca
780gcgccagccc gcccaccgtc tccttcccgg gcgcctacaa ggccaccgac
ccgggcatcc 840tcgtcaacat ccacgccccg ctgtccggct acaccgtgcc
cggcccggcc gtctactccg 900gcggctccac caagaaggcc ggcagcgcct
gcaccggctg cgagtccacc tgcgccgtcg 960gctccggccc caccgccacc
gtctcccagt cgcccggttc caccgccacc tccgcccccg 1020gcggcggcgg
cggctgcacc gtccagaagt accagcagtg cggcggcgag ggctacaccg
1080gctgcaccaa ctgcgcggta cgtttttcaa ccccgttttt ttttttcctt
ccctacctta 1140tttggttacc taattaatta ctttccggct gctgactttt
tgctttagtc cggctctacc 1200tgcagcgccg tctcgccgcc ctactactcg
cagtgcgtct aa 124222323PRTMyceliophthora thermophila 22Met Lys Ser
Phe Ala Leu Thr Thr Leu Ala Ala Leu Ala Gly Asn Ala1 5 10 15Ala Ala
His Ala Thr Phe Gln Ala Leu Trp Val Asp Gly Val Asp Tyr 20 25 30Gly
Ala Gln Cys Ala Arg Leu Pro Ala Ser Asn Ser Pro Val Thr Asp 35 40
45Val Thr Ser Asn Ala Ile Arg Cys Asn Ala Asn Pro Ser Pro Ala Arg
50 55 60Gly Lys Cys Pro Val Lys Ala Gly Ser Thr Val Thr Val Glu Met
His65 70 75 80Gln Gln Pro Gly Asp Arg Ser Cys Ser Ser Glu Ala Ile
Gly Gly Ala 85 90 95His Tyr Gly Pro Val Met Val Tyr Met Ser Lys Val
Ser Asp Ala Ala 100 105 110Ser Ala Asp Gly Ser Ser Gly Trp Phe Lys
Val Phe Glu Asp Gly Trp 115 120 125Ala Lys Asn Pro Ser Gly Gly Ser
Gly Asp Asp Asp Tyr Trp Gly Thr 130 135 140Lys Asp Leu Asn Ser Cys
Cys Gly Lys Met Asn Val Lys Ile Pro Ala145 150 155 160Asp Leu Pro
Ser Gly Asp Tyr Leu Leu Arg Ala Glu Ala Leu Ala Leu 165 170 175His
Thr Ala Gly Ser Ala Gly Gly Ala Gln Phe Tyr Met Thr Cys Tyr 180 185
190Gln Leu Thr Val Thr Gly Ser Gly Ser Ala Ser Pro Pro Thr Val Ser
195 200 205Phe Pro Gly Ala Tyr Lys Ala Thr Asp Pro Gly Ile Leu Val
Asn Ile 210 215 220His Ala Pro Leu Ser Gly Tyr Thr Val Pro Gly Pro
Ala Val Tyr Ser225 230 235 240Gly Gly Ser Thr Lys Lys Ala Gly Ser
Ala Cys Thr Gly Cys Glu Ser 245 250 255Thr Cys Ala Val Gly Ser Gly
Pro Thr Ala Thr Val Ser Gln Ser Pro 260 265 270Gly Ser Thr Ala Thr
Ser Ala Pro Gly Gly Gly Gly Gly Cys Thr Val 275 280 285Gln Lys Tyr
Gln Gln Cys Gly Gly Glu Gly Tyr Thr Gly Cys Thr Asn 290 295 300Cys
Ala Ser Gly Ser Thr Cys Ser Ala Val Ser Pro Pro Tyr Tyr Ser305 310
315 320Gln Cys Val231253DNAMyceliophthora thermophila
23atgaagcctt ttagcctcgt cgccctggcg accgccgtga gcggccatgc catcttccag
60cgggtgtcgg tcaacgggca ggaccagggc cagctcaagg gggtgcgggc gccgtcgagc
120aactccccga tccagaacgt caacgatgcc aacatggcct gcaacgccaa
cattgtgtac 180cacgacagca ccatcatcaa ggtgcccgcg ggagcccgcg
tcggcgcgtg gtggcagcac 240gtcatcggcg ggccgcaggg cgccaacgac
ccggacaacc cgatcgcggc ctcccacaag 300ggtatgatga tcgatgatgc
ctctctcttc ccccgttctt gatggacagg cgatggctcc 360caggaacacg
cgtgactgac caccgaatcc aggccccatc caggtctacc tggccaaggt
420ggacaacgcg gcgacggcgt cgccgtcggg cctcaggtgg ttcaaggtgg
ccgagcgcgg 480cctgaacaac ggcgtgtggg ccgtcgatga gctcatcgcc
aacaacggct ggcactactt 540cgacctgccg tcgtgcgtgg cccccggcca
gtacctgatg cgcgtcgagc tgctcgccct 600gcacagcgcc tcaagccccg
gcggcgccca gttctacatg ggctgcgcac agatcgaagg 660tgcgtcgatc
tttgttctcc ttccgtgtcc tctctgatcc tttctctctt ctttttcttt
720cttttactcc ctttccttcc atcttcggag aagcaacgaa gggggaaagg
gatagaagag 780aggaatgaga gacgacgaaa gagaggattg gggaaagaca
agacagggaa aaaaagacaa 840gaaaaaaaaa aaaaaaaaaa aacagagtga
gctaacaaga acaatcagtc actggctccg 900gcaccaactc gggctccgac
tttgtctcgt tccccggcgc ctactcggcc aacgatccgg 960gcatcttgct
aagcatctac gacagctcgg gcaagcccac caacggcggg cgctcgtacc
1020cgatccccgg cccgcgcccc atctcctgct ccggcagcgg cgacggcggc
aacaacggcg 1080gcggcggcga cgacaacaac aataacaacg gtggtggcaa
caacggcggc ggcggcggcg 1140gcagcgtccc cctgtacggg cagtgcggcg
gcatcggcta cacgggcccg accacctgtg 1200cccagggaac ttgcaaggtg
tcgaacgaat actacagcca gtgcctcccc tag 125324310PRTMyceliophthora
thermophila 24Met Lys Pro Phe Ser Leu Val Ala Leu Ala Thr Ala Val
Ser Gly His1 5 10 15Ala Ile Phe Gln Arg Val Ser Val Asn Gly Gln Asp
Gln Gly Gln Leu 20 25 30Lys Gly Val Arg Ala Pro Ser Ser Asn Ser Pro
Ile Gln Asn Val Asn 35 40 45Asp Ala Asn Met Ala Cys Asn Ala Asn Ile
Val Tyr His Asp Ser Thr 50 55 60Ile Ile Lys Val Pro Ala Gly Ala Arg
Val Gly Ala Trp Trp Gln His65 70 75 80Val Ile Gly Gly Pro Gln Gly
Ala Asn Asp Pro Asp Asn Pro Ile Ala 85 90 95Ala Ser His Lys Gly Pro
Ile Gln Val Tyr Leu Ala Lys Val Asp Asn 100 105 110Ala Ala Thr Ala
Ser Pro Ser Gly Leu Arg Trp Phe Lys Val Ala Glu 115 120 125Arg Gly
Leu Asn Asn Gly Val Trp Ala Val Asp Glu Leu Ile Ala Asn 130 135
140Asn Gly Trp His Tyr Phe Asp Leu Pro Ser Cys Val Ala Pro Gly
Gln145 150 155 160Tyr Leu Met Arg Val Glu Leu Leu Ala Leu His Ser
Ala Ser Ser Pro 165 170 175Gly Gly Ala Gln Phe Tyr Met Gly Cys Ala
Gln Ile Glu Val Thr Gly 180 185 190Ser Gly Thr Asn Ser Gly Ser Asp
Phe Val Ser Phe Pro Gly Ala Tyr 195 200 205Ser Ala Asn Asp Pro Gly
Ile Leu Leu Ser Ile Tyr Asp Ser Ser Gly 210 215 220Lys Pro Thr Asn
Gly Gly Arg Ser Tyr Pro Ile Pro Gly Pro Arg Pro225 230 235 240Ile
Ser Cys Ser Gly Ser Gly Asp Gly Gly Asn Asn Gly Gly Gly Gly 245 250
255Asp Asp Asn Asn Asn Asn Asn Gly Gly Gly Asn Asn Gly Gly Gly Gly
260 265 270Gly Gly Ser Val Pro Leu Tyr Gly Gln Cys Gly Gly Ile Gly
Tyr Thr 275 280 285Gly Pro Thr Thr Cys Ala Gln Gly Thr Cys Lys Val
Ser Asn Glu Tyr 290 295 300Tyr Ser Gln Cys Leu Pro305
31025814DNAMyceliophthora thermophila 25atgaagctct ccctcttctc
cgtcctggcc actgccctca ccgtcgaggg gcatgccatc 60ttccagaagg tctccgtcaa
cggagcggac cagggctccc tcaccggcct ccgcgctccc 120aacaacaaca
accccgtgca ggatgtcaac agccaggaca tgatctgcgg ccagtcggga
180tcgacgtcga acactatcat cgaggtcaag gccggcgata ggatcggtgc
ctggtatcag 240catgtcatcg gcggtgccca gttccccaac gacccagaca
acccgattgc caagtcgcac 300aagggccccg tcatggccta cctcgccaag
gttgacaatg ccgcaaccgc cagcaagacg 360ggcctgaagt ggtatgtatt
cccgcggccc gagggacatc gggttgggca agtcgagact 420gacggagctc
gcttctccgt ataggttcaa gatttgggag gataccttta atcccagcac
480caagacctgg ggtgtcgaca acctcatcaa taacaacggc tgggtgtact
tcaacctccc 540gcagtgcatc gccgacggca actacctcct ccgcgtcgag
gtcctcgctc tgcactcggc 600ctactctcag ggccaggctc agttctacca
gtcctgcgcc cagatcaacg tatccggcgg 660cggctccttc acaccgccgt
cgactgtcag cttcccgggt gcctacagcg ccagcgaccc 720cggtatcctg
atcaacatct acggcgccac cggccagccc gacaacaacg gccagccgta
780cactgcccct gggcccgcgc ccatctcctg ctga 81426246PRTMyceliophthora
thermophila 26Met Lys Leu Ser Leu Phe Ser Val Leu Ala Thr Ala Leu
Thr Val Glu1 5 10 15Gly His Ala Ile Phe Gln Lys Val Ser Val Asn Gly
Ala Asp Gln Gly 20 25 30Ser Leu Thr Gly Leu Arg Ala Pro Asn Asn Asn
Asn Pro Val Gln Asp 35 40 45Val Asn Ser Gln Asp Met Ile Cys Gly Gln
Ser Gly Ser Thr Ser Asn 50 55 60Thr Ile Ile Glu Val Lys Ala Gly Asp
Arg Ile Gly Ala Trp Tyr Gln65 70 75 80His Val Ile Gly Gly Ala Gln
Phe Pro Asn Asp Pro Asp Asn Pro Ile 85 90 95Ala Lys Ser His Lys Gly
Pro Val Met Ala Tyr Leu Ala Lys Val Asp 100 105 110Asn Ala Ala Thr
Ala Ser Lys Thr Gly Leu Lys Trp Phe Lys Ile Trp 115 120 125Glu Asp
Thr Phe Asn Pro Ser Thr Lys Thr Trp Gly Val Asp Asn Leu 130 135
140Ile Asn Asn Asn Gly Trp Val Tyr Phe Asn Leu Pro Gln Cys Ile
Ala145 150 155 160Asp Gly Asn Tyr Leu Leu Arg Val Glu Val Leu Ala
Leu His Ser Ala 165 170 175Tyr Ser Gln Gly Gln Ala Gln Phe Tyr Gln
Ser Cys Ala Gln Ile Asn 180 185 190Val Ser Gly Gly Gly Ser Phe Thr
Pro Pro Ser Thr Val Ser Phe Pro 195 200 205Gly Ala Tyr Ser Ala Ser
Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gly 210 215 220Ala Thr Gly Gln
Pro Asp Asn Asn Gly Gln Pro Tyr Thr Ala Pro Gly225 230 235 240Pro
Ala Pro Ile Ser Cys 245271115DNAThermoascus aurantiacus
27atgtcgttct cgaagattgc tgcgatcacc ggggccatta cctatgcgtc tctggccgcc
60gctcacggtt atgttacagg aatcgtagcc gatggcacct agtatgtaac gctcatgcca
120agatccgcat tgctgtacta acaattagca gctacggggg ctatatcgtg
acccaatacc 180cctacatgtc gacaccgccg gatgtcatcg cctggtctac
caaagcaact gatcttggtt 240tcgtggatcc cagtagctat gcttcgtctg
atattatctg ccacaagggt gctgagcctg 300gtgccctgag cgccaaggtg
gctgctggag ggaccgtcga gctgcagtgg acggattggc 360ctgagagtca
caagggcccg gtcattgact acctcgccgc ctgtaacggg gactgctcga
420ctgtcgacaa gaccaaacta gagttcttca agattgatga gagtggccta
attgacggca 480gcagcgcccc aggcacatgg gcctctgaca acttgattgc
caataacaac agctggaccg 540tcaccatccc gagcacgatt gctcccggca
actatgtcct gagacatgaa atcattgccc 600tccactccgc cggaaataca
aatggtgctc agaactaccc ccagtgtatc aaccttgagg 660tcacaggcag
tggcaccgac acccctgccg gcaccctcgg aacggagctt tataaggcaa
720cggaccctgg cattctggtc aacatctacc agaccctgac cagctacgat
attcccggcc 780ctgctctgta caccggtggt agctctggta gctctggttc
ctccaacacc gccaaggcca 840ccacttcgac ggcttctagc tctatcgtga
ccccgacgcc tgttaacaac ccaaccgtta 900ctcagactgc cgttgttgat
gtcacccaga ctgtttccca gaatgctgcc gtcgccacca 960cgactccggc
ctccactgca gttgctacag ctgtcccaac gggaaccacc tttagctttg
1020attcgatgac ctcggatgaa ttcgtcagcc tgatgcgtgc gaccgtgaat
tggctgcttt 1080ctaacaagaa gcatgcccgg gatctttctt actaa
111528354PRTThermoascus aurantiacus 28Met Ser Phe Ser Lys Ile Ala
Ala Ile Thr Gly Ala Ile Thr Tyr Ala1 5 10 15Ser Leu Ala Ala Ala His
Gly Tyr Val Thr Gly Ile Val Ala Asp Gly 20 25 30Thr Tyr Tyr Gly Gly
Tyr Ile Val Thr Gln Tyr Pro Tyr Met Ser Thr 35 40 45Pro Pro Asp Val
Ile Ala Trp Ser Thr Lys Ala Thr Asp Leu Gly Phe 50 55 60Val Asp Pro
Ser Ser Tyr Ala Ser Ser Asp Ile Ile Cys His Lys Gly65 70 75 80Ala
Glu Pro Gly Ala Leu Ser Ala Lys Val Ala Ala Gly Gly Thr Val 85 90
95Glu Leu Gln Trp Thr Asp Trp Pro Glu Ser His Lys Gly Pro Val Ile
100 105 110Asp Tyr Leu Ala Ala Cys Asn Gly Asp Cys Ser Thr Val Asp
Lys Thr 115 120 125Lys Leu Glu Phe Phe Lys Ile Asp Glu Ser Gly Leu
Ile Asp Gly Ser 130 135 140Ser Ala Pro Gly Thr Trp Ala Ser Asp Asn
Leu Ile Ala Asn Asn Asn145 150 155 160Ser Trp Thr Val Thr Ile Pro
Ser Thr Ile Ala Pro Gly Asn Tyr Val 165 170 175Leu Arg His Glu Ile
Ile Ala Leu His Ser Ala Gly Asn Thr Asn Gly 180 185 190Ala Gln Asn
Tyr Pro Gln Cys Ile Asn Leu Glu Val Thr Gly Ser Gly 195 200 205Thr
Asp Thr Pro Ala Gly Thr Leu Gly Thr Glu Leu Tyr Lys Ala Thr 210 215
220Asp Pro Gly Ile Leu Val Asn Ile Tyr Gln Thr Leu Thr Ser Tyr
Asp225 230 235 240Ile Pro Gly Pro Ala Leu Tyr Thr Gly Gly Ser Ser
Gly Ser Ser Gly 245 250 255Ser Ser Asn Thr Ala Lys Ala Thr Thr Ser
Thr Ala Ser Ser Ser Ile 260 265 270Val Thr Pro Thr Pro Val Asn Asn
Pro Thr Val Thr Gln Thr Ala Val 275 280 285Val Asp Val Thr Gln Thr
Val Ser Gln Asn Ala Ala Val Ala Thr Thr 290 295 300Thr Pro Ala Ser
Thr Ala Val Ala Thr Ala Val Pro Thr Gly Thr Thr305 310 315 320Phe
Ser Phe Asp Ser Met Thr Ser Asp Glu Phe Val Ser Leu Met Arg 325 330
335Ala Thr Val Asn Trp Leu Leu Ser Asn Lys Lys His Ala Arg Asp Leu
340 345 350Ser Tyr29862DNAAspergillus fumigatus 29atgactttgt
ccaagatcac ttccattgct ggccttctgg cctcagcgtc tctcgtggct 60ggccacggct
ttgtttctgg cattgttgct gatgggaaat agtatgtgct tgaaccacac
120aaatgacagc tgcaacagct aacttctatt ccagttacgg agggtacctt
gttaaccaat 180acccctacat gagcaaccct cccgacacca ttgcctggtc
caccaccgcc accgacctcg 240gctttgtgga cggcaccggc taccagtctc
cggatattat ctgccacaga gacgcaaaga 300atggcaagtt gaccgcaacc
gttgcagccg gttcacagat cgaattccag tggacgacgt 360ggccagagtc
tcaccatgga ccggtacgac gccgaagaga agagaacata ttgtgaccag
420ataggctaac atagcatagt tgattactta cctcgctcca tgcaacggcg
actgtgccac 480cgtggacaag accaccctga agtttgtcaa gatcgccgct
caaggcttga tcgacggctc 540caacccacct ggtgtttggg ctgatgatga
aatgatcgcc aacaacaaca cggccacagt 600gaccattcct gcctcctatg
cccccggaaa ctacgtcctt cgccacgaga tcatcgccct 660tcactctgcg
ggtaacctga acggcgcgca gaactacccc cagtgtttca acatccaaat
720caccggtggc ggcagtgctc agggatctgg caccgctggc acgtccctgt
acaagaatac 780tgatcctggc atcaagtttg acatctactc ggatctgagc
ggtggatacc ctattcctgg 840tcctgcactg ttcaacgctt aa
86230250PRTAspergillus fumigatus 30Met Thr Leu Ser Lys Ile Thr Ser
Ile Ala Gly Leu Leu Ala Ser Ala1 5 10 15Ser Leu Val Ala Gly His Gly
Phe Val Ser Gly Ile Val Ala Asp Gly 20 25 30Lys Tyr Tyr Gly Gly Tyr
Leu Val Asn Gln Tyr Pro Tyr Met Ser Asn 35 40 45Pro Pro Asp Thr Ile
Ala Trp Ser Thr Thr Ala Thr Asp Leu Gly Phe 50 55 60Val Asp Gly Thr
Gly Tyr Gln Ser Pro Asp Ile Ile Cys His Arg Asp65 70 75 80Ala Lys
Asn Gly Lys Leu Thr Ala Thr Val Ala Ala Gly Ser Gln Ile 85 90 95Glu
Phe Gln Trp Thr Thr Trp Pro Glu Ser His His Gly Pro Leu Ile 100 105
110Thr Tyr Leu Ala Pro Cys Asn Gly Asp Cys Ala Thr Val Asp Lys Thr
115 120 125Thr Leu Lys Phe Val Lys Ile Ala Ala Gln Gly Leu Ile Asp
Gly Ser 130 135 140Asn Pro Pro Gly Val Trp Ala Asp Asp Glu Met Ile
Ala Asn Asn Asn145 150 155 160Thr Ala Thr Val Thr Ile Pro Ala Ser
Tyr Ala Pro Gly Asn Tyr Val 165 170 175Leu Arg His Glu Ile Ile Ala
Leu His Ser Ala Gly Asn Leu Asn Gly 180 185 190Ala Gln Asn Tyr Pro
Gln Cys Phe Asn Ile Gln Ile Thr Gly Gly Gly 195 200 205Ser Ala Gln
Gly Ser Gly Thr Ala Gly Thr Ser Leu Tyr Lys Asn Thr 210 215 220Asp
Pro Gly Ile Lys Phe Asp Ile Tyr Ser Asp Leu Ser Gly Gly Tyr225 230
235 240Pro Ile Pro Gly Pro Ala Leu Phe Asn Ala 245
250311021DNAPenicillium pinophilum 31atgccttcta ctaaagtcgc
tgccctttct gctgttctag ctttggcctc cacggttgct 60ggccatggtt ttgtgcaaaa
catcgttatc gacggtaaat cgtaagcagt gatgcatcca 120ttattaaact
agacatgctt acaaaaaaat cagttactct ggataccttg tgaatcagtt
180cccctacgag tccaacccac cagctgttat tgggtgggca acaactgcaa
ccgacctggg 240attcgtcgct cccagtgagt acaccaatgc agacattatc
tgccacaaga acgccacacc 300tggcgcgctt tctgctccag ttgctgcagg
gggcactgtc gagctccagt ggactacatg 360gcccgatagt catcacggtc
ctgtcatcag ctacctcgcc aactgcaatg gcaattgttc 420taccgtggat
aagactaagc tagactttgt caagattgac caaggtggtt tgatcgacga
480tactaccccc ccgggtacat gggcttccga caaacttatc gctgccaaca
acagctggac 540tgtaactatc ccctccacca tcgcgcctgg aaactacgtt
ttgcgccacg aaatcattgc 600tcttcactcc gctggaaacg cagacggtgc
ccaaaactac cctcaatgca tcaacttgga 660gatcaccggc agcggaaccg
ccgctccctc tggtaccgct ggcgaaaagc tctacacctc 720tactgacccc
ggtatcttgg tcaatatcta ccaatccttg tcgacctacg ttattcccgg
780accaactctg tggagcggtg ctgccaatgg cgctgttgcc actggttctg
ctactgcggt 840tgctacgact gccactgctt ctgcgaccgc tactcctacc
acacttgtta cctctgtcgc 900tccagcttca tctacctttg ccactgctgt
tgtgaccact gtcgctcctg cagtaactga 960tgtcgtgact gtcaccgatg
tagttaccgt gaccaccgtc atcaccacta ctgtcctttg 1020a
102132322PRTPenicillium pinophilum 32Met Pro Ser Thr Lys Val Ala
Ala Leu Ser Ala Val Leu Ala Leu Ala1 5 10 15Ser Thr Val Ala Gly His
Gly Phe Val Gln Asn Ile Val Ile Asp Gly 20 25 30Lys Ser Tyr Ser Gly
Tyr Leu Val Asn Gln Phe Pro Tyr Glu Ser Asn 35 40 45Pro Pro Ala Val
Ile Gly Trp Ala Thr Thr Ala Thr Asp Leu Gly Phe 50 55 60Val Ala Pro
Ser Glu Tyr Thr Asn Ala Asp Ile Ile Cys His Lys Asn65 70 75 80Ala
Thr Pro Gly Ala Leu Ser Ala Pro Val Ala Ala Gly Gly Thr Val 85 90
95Glu Leu Gln Trp Thr Thr Trp Pro Asp Ser His His Gly Pro Val Ile
100 105 110Ser Tyr Leu Ala Asn Cys Asn Gly Asn Cys Ser Thr Val Asp
Lys Thr 115 120 125Lys Leu Asp Phe Val Lys Ile Asp Gln Gly Gly Leu
Ile Asp Asp Thr 130 135 140Thr Pro Pro Gly Thr Trp Ala Ser Asp Lys
Leu Ile Ala Ala Asn Asn145 150 155 160Ser Trp Thr Val Thr Ile Pro
Ser Thr Ile Ala Pro Gly Asn Tyr Val 165 170 175Leu Arg His Glu Ile
Ile Ala Leu His Ser Ala Gly Asn Ala Asp Gly 180 185 190Ala Gln Asn
Tyr Pro Gln Cys Ile Asn Leu Glu Ile Thr Gly Ser Gly 195 200 205Thr
Ala Ala Pro Ser Gly Thr Ala Gly Glu Lys Leu Tyr Thr Ser Thr 210 215
220Asp Pro Gly Ile Leu Val Asn Ile Tyr Gln Ser Leu Ser Thr Tyr
Val225 230 235 240Ile Pro Gly Pro Thr Leu Trp Ser Gly Ala Ala Asn
Gly Ala Val Ala 245 250 255Thr Gly Ser Ala Thr Ala Val Ala Thr Thr
Ala Thr Ala Ser Ala Thr 260 265 270Ala Thr Pro Thr Thr Leu Val Thr
Ser Val Ala Pro Ala Ser Ser Thr 275 280 285Phe Ala Thr Ala Val Val
Thr Thr Val Ala Pro Ala Val Thr Asp Val 290 295 300Val Thr Val Thr
Asp Val Val Thr Val Thr Thr Val Ile Thr Thr Thr305 310 315 320Val
Leu331486DNAThermoascus sp. 33atgttgtcgt tcgcttctgc caagtcagct
gtgctgacga cccttctact tcttggatcc 60gctcaggctc acactttgat gaccaccctg
tttgtggatg gcgtcaatca gggagatggt 120gtctgtattc gcatgaacaa
caacggtagt actgccaaca cctatatcca gcctgtcacg 180agcaaggata
ttgcctgcgg taagtacagt accggtccag atatcatact ctatttcaat
240ccgacaacag tcagagctgg agagcaatgc taaacatccc caggcattca
aggcgaaatt 300ggcgccgctc gagtctgtcc agccaaggct tcatccaccc
tcacgttcca attccgagag 360cagccatcca acccgaattc cgctcctctc
gatccctcgc acaaaggccc cgctgcggtg 420tacctgaaaa aggtagactc
cgccatcgcg agcaacaacg ccgctggaga cggctggttc 480aagatctggg
agtccgtcta cgacgagtcc acgggcaaat ggggtacgac caagatgatc
540gagaacaacg ggcacatctc tgtcaaggtc cccgacgata tcgagggtgg
gtattatctc 600gcgcgtacgg agcttctggc gctgcacgcg gcgaacgaag
gggatccgca gttctacgtt 660ggctgcgcgc agctgttcat cgattcagcg
gggacagcga aaccgcctac tgtctctatt 720ggagagggga cctacgatct
gagcatgcct gccatgacgt acaatatcta ccagactccg 780ttggctctac
catacccgat gtatgggcct cctgtctaca cacctggctc tggctcgggt
840tctggctctg gttccgggtc agcttctgca acgagatctt ctgctattcc
tactgccacc 900gctgttacgg actgttcttc cgaagaggac agggaagact
cagtcatggc aaccggtgtt 960cccgttgcaa gaagcacact cagaacctgg
gttgacagac tgtcatggca tggtaaggcc 1020cgtgagaacg tgaaaccagc
cgccaggaga agcgcccttg tccagaccga gggtctgaag 1080ccggaaggct
gcatcttcgt caacggcaac tggtgcggtt tcgaggtccc cgattacaac
1140gatgcggaaa gctgctgggc tgtacgttcc cgtctaatta cttaaaacga
aataaaagct 1200aacagtactt ttctttttct aatcccaggc ctccgacaac
tgctggaaac agtccgactc 1260gtgctggaac cagacccagc ccaccggcta
caacaactgc cagatctggc aagaccagaa 1320atgcaagccc atccaggact
cgtgtagcca atccaacccg actggaccgc cgaacaaggg 1380caaggatata
actccaacgt ggccgcccct ggagggctcg atgaagacct tcaccaagcg
1440cactgtcagt taccgtgatt ggattatgaa aaggaaagga gcataa
148634444PRTThermoascus sp. 34Met Leu Ser Phe Ala Ser Ala Lys Ser
Ala Val Leu Thr Thr Leu Leu1 5 10 15Leu Leu Gly Ser Ala Gln Ala His
Thr Leu Met Thr Thr Leu Phe Val 20 25 30Asp Gly Val Asn Gln Gly Asp
Gly Val Cys Ile Arg Met Asn Asn Asn 35 40 45Gly Ser Thr Ala Asn Thr
Tyr Ile Gln Pro Val Thr Ser Lys Asp Ile 50 55 60Ala Cys Gly Ile Gln
Gly Glu Ile Gly Ala Ala Arg Val Cys Pro Ala65 70 75 80Lys Ala Ser
Ser Thr Leu Thr Phe Gln Phe Arg Glu Gln Pro Ser Asn 85 90 95Pro Asn
Ser Ala Pro Leu Asp Pro Ser His Lys Gly Pro Ala Ala Val 100 105
110Tyr Leu Lys Lys Val Asp Ser Ala Ile Ala Ser Asn Asn Ala Ala Gly
115 120 125Asp Gly Trp Phe Lys Ile Trp Glu Ser Val Tyr Asp Glu Ser
Thr Gly 130 135 140Lys Trp Gly Thr Thr Lys Met Ile Glu Asn Asn Gly
His Ile Ser Val145 150 155 160Lys Val Pro Asp Asp Ile Glu Gly Gly
Tyr Tyr Leu Ala Arg Thr Glu 165 170 175Leu Leu Ala Leu His Ala Ala
Asn Glu Gly Asp Pro Gln Phe Tyr Val 180 185 190Gly Cys Ala Gln Leu
Phe Ile Asp Ser Ala Gly Thr Ala Lys Pro Pro 195 200 205Thr Val Ser
Ile Gly Glu Gly Thr Tyr Asp Leu Ser Met Pro Ala Met 210 215 220Thr
Tyr Asn Ile Tyr Gln Thr Pro Leu Ala Leu Pro Tyr Pro Met Tyr225 230
235 240Gly Pro Pro Val Tyr Thr Pro Gly Ser Gly Ser Gly Ser Gly Ser
Gly 245 250 255Ser Gly Ser Ala Ser Ala Thr Arg Ser Ser Ala Ile Pro
Thr Ala Thr 260 265 270Ala Val Thr Asp Cys Ser Ser Glu Glu Asp Arg
Glu Asp Ser Val Met 275 280 285Ala Thr Gly Val Pro Val Ala Arg Ser
Thr Leu Arg Thr Trp Val Asp 290 295 300Arg Leu Ser Trp His Gly Lys
Ala Arg Glu Asn Val Lys Pro Ala Ala305 310 315 320Arg Arg Ser Ala
Leu Val Gln Thr Glu Gly Leu Lys Pro Glu Gly Cys 325 330 335Ile Phe
Val Asn Gly Asn Trp Cys Gly Phe Glu Val Pro Asp Tyr Asn 340 345
350Asp Ala Glu Ser Cys Trp Ala Ala Ser Asp Asn Cys Trp Lys Gln Ser
355 360 365Asp Ser Cys Trp Asn Gln Thr Gln Pro Thr Gly Tyr Asn Asn
Cys Gln 370 375 380Ile Trp Gln Asp Gln Lys Cys Lys Pro Ile Gln Asp
Ser Cys Ser Gln385 390 395 400Ser Asn Pro Thr Gly Pro Pro Asn Lys
Gly Lys Asp Ile Thr Pro Thr 405 410 415Trp Pro Pro Leu Glu Gly Ser
Met Lys Thr Phe Thr Lys Arg Thr Val 420 425 430Ser Tyr Arg Asp Trp
Ile Met Lys Arg Lys Gly Ala 435 44035835DNAPenicillium sp.
35atgctgtctt cgacgactcg caccctcgcc tttacaggcc ttgcgggcct tctgtccgct
60cccctggtca aggcccatgg ctttgtccag ggcattgtca tcggtgacca attgtaagtc
120cctctcttgc agttctgtcg attaactgct ggactgcttg cttgactccc
tgctgactcc 180caacagctac agcgggtaca tcgtcaactc gttcccctac
gaatccaacc caccccccgt 240catcggctgg gccacgaccg ccaccgacct
gggcttcgtc gacggcacag gataccaagg 300cccggacatc atctgccacc
ggaatgcgac gcccgcgccg ctgacagccc ccgtggccgc 360cggcggcacc
gtcgagctgc agtggacgcc gtggccggac agccaccacg gacccgtcat
420cacctacctg gcgccgtgca acggcaactg ctcgaccgtc gacaagacga
cgctggagtt 480cttcaagatc gaccagcagg gcctgatcga cgacacgagc
ccgccgggca cctgggcgtc 540ggacaacctc atcgccaaca acaatagctg
gaccgtcacc attcccaaca gcgtcgcccc 600cggcaactac gtcctgcgcc
acgagatcat cgccctgcac tcggccaaca acaaggacgg 660cgcccagaac
tacccccagt gcatcaacat cgaggtcacg ggcggcggct ccgacgcgcc
720tgagggtact ctgggcgagg atctctacca tgacaccgac ccgggcattc
tggtcgacat 780ttacgagccc attgcgacgt ataccattcc ggggccgcct
gagccgacgt tctag 83536253PRTPenicillium sp. 36Met Leu Ser Ser Thr
Thr Arg Thr Leu Ala Phe Thr Gly Leu Ala Gly1 5 10 15Leu Leu Ser Ala
Pro Leu Val Lys Ala His Gly Phe Val Gln Gly Ile 20 25 30Val Ile Gly
Asp Gln Phe Tyr Ser Gly Tyr Ile Val Asn Ser Phe Pro 35 40 45Tyr Glu
Ser Asn Pro Pro Pro Val Ile Gly Trp Ala Thr Thr Ala Thr 50 55 60Asp
Leu Gly Phe Val Asp Gly Thr Gly Tyr Gln Gly Pro Asp Ile Ile65 70 75
80Cys His Arg Asn Ala Thr Pro Ala Pro Leu Thr Ala Pro Val Ala Ala
85 90 95Gly Gly Thr Val Glu Leu Gln Trp Thr Pro Trp Pro Asp Ser His
His 100 105 110Gly Pro Val Ile Thr Tyr Leu Ala Pro Cys Asn Gly Asn
Cys Ser Thr 115 120 125Val Asp Lys Thr Thr Leu Glu Phe Phe Lys Ile
Asp Gln Gln Gly Leu 130 135 140Ile Asp Asp Thr Ser Pro Pro Gly Thr
Trp Ala Ser Asp Asn Leu Ile145 150 155 160Ala Asn Asn Asn Ser Trp
Thr Val Thr Ile Pro Asn Ser Val Ala Pro 165 170 175Gly Asn Tyr Val
Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Asn 180 185 190Asn Lys
Asp Gly Ala Gln Asn Tyr Pro Gln Cys Ile Asn Ile Glu Val 195 200
205Thr Gly Gly Gly Ser Asp Ala Pro Glu Gly Thr Leu Gly Glu Asp Leu
210 215 220Tyr His Asp Thr Asp Pro Gly Ile Leu Val Asp Ile Tyr Glu
Pro Ile225 230 235 240Ala Thr Tyr Thr Ile Pro Gly Pro Pro Glu Pro
Thr Phe 245 25037878DNAThielavia terrestris 37atgaagttct cactggtgtc
tctgctggct tacggcctct cggtcgaggc gcactccatc 60ttccaggttc gtctcgcaca
tcacgctcaa ctcggctcgt ggcgtaaggg caaggattaa 120cacggccggc
agagagtctc ggtcaacggc caagaccaag gcctgctcac cggcctccgc
180gctccaagca acaacaaccc agtgcaagat gtcaacagcc agaacatgat
ttgcggccag 240tcgggctcca agtcgcagac cgttatcaac gtcaaggccg
gcgacaggat cggctcgctc 300tggcagcatg tcatcggcgg cgcccagttt
tcgggtgacc cggacaaccc gatcgcccac 360tcgcacaagg gccccgtgat
ggcgtacctt gctaaggtcg acaatgccgc gtccgcgagc 420caaacgggtc
tgaagtggta agtagcgggc gacgctcagg ggacggggat cgggggcctg
480ctccatccga gactaacacc gtggacaggt tcaagatctg gcaggacggg
ttcgatacca 540gcagcaagac atggggcgtc gacaacctga tcaagaacaa
cggctgggtg tacttccacc 600tgccgcagtg cctcgctccg ggccagtatc
tcctgcgcgt cgaggttctg gcgctgcact 660cggcgtacca gcagggccag
gcccagttct accagtcctg cgcccagatc aacgtctccg 720gctccgggtc
cttcagcccg tcccagacgg tcagcatccc gggcgtctac agcgccaccg
780acccgagcat cctcatcaac atctacggca gcacggggca gcccgacaac
ggcggcaagg 840cttacaaccc ccctggaccc gccccgatct cctgctga
87838246PRTThielavia terrestris 38Met Lys Phe Ser Leu Val Ser Leu
Leu Ala Tyr Gly Leu Ser Val Glu1 5 10 15Ala His Ser Ile Phe Gln Arg
Val Ser Val Asn Gly Gln Asp Gln Gly 20 25 30Leu Leu Thr Gly Leu Arg
Ala Pro Ser Asn Asn Asn Pro Val Gln Asp 35 40 45Val Asn Ser Gln Asn
Met Ile Cys Gly Gln Ser Gly Ser Lys Ser Gln 50 55 60Thr Val Ile Asn
Val Lys Ala Gly Asp Arg Ile Gly Ser Leu Trp Gln65 70 75 80His Val
Ile Gly Gly Ala Gln Phe Ser Gly Asp Pro Asp Asn Pro Ile 85 90 95Ala
His Ser His Lys Gly Pro Val Met Ala Tyr Leu Ala Lys Val Asp 100 105
110Asn Ala Ala Ser Ala Ser Gln Thr Gly Leu Lys Trp Phe Lys Ile Trp
115 120 125Gln Asp Gly Phe Asp Thr Ser Ser Lys Thr Trp Gly Val Asp
Asn Leu 130 135 140Ile Lys Asn Asn Gly Trp Val Tyr Phe His Leu Pro
Gln Cys Leu Ala145 150 155 160Pro Gly Gln Tyr Leu Leu Arg Val Glu
Val Leu Ala Leu His Ser Ala 165 170 175Tyr Gln Gln Gly Gln Ala Gln
Phe Tyr Gln Ser Cys Ala Gln Ile Asn 180 185 190Val Ser Gly Ser Gly
Ser Phe Ser Pro Ser Gln Thr Val Ser Ile Pro 195 200 205Gly Val Tyr
Ser Ala Thr Asp Pro Ser Ile Leu Ile Asn Ile Tyr Gly 210 215 220Ser
Thr Gly Gln Pro Asp Asn Gly Gly Lys Ala Tyr Asn Pro Pro Gly225 230
235 240Pro Ala Pro Ile Ser Cys 245391253DNAThielavia terrestris
39atgaggacga cattcgccgc cgcgttggca gccttcgctg cgcaggaagt ggcaggccat
60gccatcttcc aacagctctg ggtggacggc accgactata tacgtgctcc ccttttcctt
120ttgtgtttgc ccatcctcga ttgataaccc gaggccatcc aatgctgact
cttacagcac 180ggctcctcct gcgtccgcat gccgctgtcg aactcgcccg
tcacgaacgt cggcagcagg 240gacatgatct gcaacgccgg cacgcgcccc
gtcagcggga agtgccccgt caaggccggc 300ggcaccgtga cggttgagat
gcaccaggtg ggctgatttc ctgagcgtcc tattcctccc 360ggaagcccct
ttcccatcct ttgccctggc taacccctcc gcccctccca gcaacccggg
420gatcggtcgt gtaacaacga agccatcggc ggcgcccact ggggaccggt
gcaggtgtac 480ctcagcaagg tggaggacgc gagcacggcg gacgggtcga
cgggctggtt caagatcttc 540gcggacacgt ggtccaagaa ggcgggcagc
tcggtggggg acgacgacaa ctggggcacg 600cgcgacctca acgcgtgctg
cggcaagatg caggtcaaga tcccggcgga catcccgtcg 660ggcgactacc
tgctgcgggc ggaggcgctg gcgctgcaca cggcgggcca ggtgggcggc
720gcgcagttct acatgagctg ctaccagatc accgtgtcgg gcggcggcag
cgccagcccg 780gccaccgtca agttccccgg cgcctacagc gccaacgacc
cgggcatcca catcaacatc 840cacgcggccg tgtccaacta cgtcgcgccc
ggcccggccg tctattccgg cggcacgacc 900aaggtggccg ggtccgggtg
ccaaggctgc gagaacacgt gcaaggtcgg ctcgtcgccc 960acggcgacgg
cgccgtcggg caagagcggc gcgggttccg acggcggcgc tgggaccgac
1020ggcgggtctt cgtcttcgag ccccgacacg ggcagcgcgt gcagcgtgca
ggcctacggg 1080cagtgcggcg ggaacgggta ctcgggttgc acccagtgcg
cggtaagttc ggggtcgtct 1140gtcttttgta ggaacatccg agaggcttgg
ctgacgaggc gttgttgtag cccggctata 1200cttgcaaggc ggtctctccg
ccgtactatt cgcagtgcgc cccttcttct tag 125340334PRTThielavia
terrestris 40Met Arg Thr Thr Phe Ala Ala Ala Leu Ala Ala Phe Ala
Ala Gln Glu1 5 10 15Val Ala Gly His Ala Ile Phe Gln Gln Leu Trp His
Gly Ser Ser Cys 20 25 30Val Arg Met Pro Leu Ser Asn Ser Pro Val Thr
Asn Val Gly Ser Arg 35 40 45Asp Met Ile Cys Asn Ala Gly Thr Arg Pro
Val Ser Gly Lys Cys Pro 50 55 60Val Lys Ala Gly Gly Thr Val Thr Val
Glu Met His Gln Gln Pro Gly65 70 75 80Asp Arg Ser Cys Asn Asn Glu
Ala Ile Gly Gly Ala His Trp Gly Pro 85 90 95Val Gln Val Tyr Leu Ser
Lys Val Glu Asp Ala Ser Thr Ala Asp Gly 100 105 110Ser Thr Gly Trp
Phe Lys Ile Phe Ala Asp Thr Trp Ser Lys Lys Ala 115 120 125Gly Ser
Ser Val Gly Asp Asp Asp Asn Trp Gly Thr Arg Asp Leu Asn 130 135
140Ala Cys Cys Gly Lys Met Gln Val Lys Ile Pro Ala Asp Ile Pro
Ser145 150 155 160Gly Asp Tyr Leu Leu Arg Ala Glu Ala Leu Ala Leu
His Thr Ala Gly 165 170 175Gln Val Gly Gly Ala Gln Phe Tyr Met Ser
Cys Tyr Gln Ile Thr Val 180 185 190Ser Gly Gly Gly Ser Ala Ser Pro
Ala Thr Val Lys Phe Pro Gly Ala 195 200 205Tyr Ser Ala Asn Asp Pro
Gly Ile His Ile Asn Ile His Ala Ala Val 210 215 220Ser Asn Tyr Val
Ala Pro Gly Pro Ala Val Tyr Ser Gly Gly Thr Thr225 230 235 240Lys
Val Ala Gly Ser Gly Cys Gln Gly Cys Glu Asn Thr Cys Lys Val 245 250
255Gly Ser Ser Pro Thr Ala Thr Ala Pro Ser Gly Lys Ser Gly Ala Gly
260 265 270Ser Asp Gly Gly Ala Gly Thr Asp Gly Gly Ser Ser Ser Ser
Ser Pro 275 280 285Asp Thr Gly Ser Ala Cys Ser Val Gln Ala Tyr Gly
Gln Cys Gly Gly 290 295 300Asn Gly Tyr Ser Gly Cys Thr Gln Cys Ala
Pro Gly Tyr Thr Cys Lys305 310 315 320Ala Val Ser Pro Pro Tyr Tyr
Ser Gln Cys Ala Pro Ser Ser 325 33041798DNAThielavia terrestris
41atgaagctga gcgttgccat cgccgtgctg gcgtcggctc ttgccgaggc tcactgtgag
60tgcatcgtct cactccagct actgcgaagc ttgctgacga tggtccctag acaccttccc
120cagcatcgga aacaccgctg actggcagta tgtgcggatt acaacgaact
accagagcaa 180cgggccggtg acggacgtca cctcggatca aattcggtgc
tacgaacgga acccaggcac 240gggagcgcag ggcatataca acgtcaccgc
cggccagacc atcaactaca acgcgaaggc 300gtccatctcc cacccggggc
ccatgtcctt ctacattgct aaggttcccg ccggccaaac 360cgctgcgacc
tgggacggta agggggctgt gtggaccaag atctaccagg acatgcccaa
420gttcggcagc agcctgacct ggcccaccat gggtaagaat tctcaccctg
gaaatgaacg 480cacatttgca cagatctaac atggcctaca ggcgccaagt
ctgtccccgt caccatccct 540cgttgcctcc agaacggcga ttaccttctg
cgagccgagc acatcgctct acacagcgcg 600agcagcgtcg gtggcgccca
gttctacctc tcgtgcgccc agcttactgt cagcggcggc 660agtggcacct
ggaaccccaa gaaccgggtc tccttccccg gcgcttacaa ggcaacagac
720ccgggcatct tgatcaacat ctactacccc gtgccgacca gctactcgcc
gcccggcccg 780ccggctgaga cgtgctaa 79842227PRTThielavia terrestris
42Met Lys Leu Ser Val Ala Ile Ala Val Leu Ala Ser Ala Leu Ala Glu1
5 10 15Ala His Tyr Thr Phe Pro Ser Ile Gly Asn Thr Ala Asp Trp Gln
Tyr 20 25 30Val Arg Ile Thr Thr Asn Tyr Gln Ser Asn Gly Pro Val Thr
Asp Val 35 40 45Thr Ser Asp Gln Ile Arg Cys Tyr Glu Arg Asn Pro Gly
Thr Gly Ala 50 55 60Gln Gly Ile Tyr Asn Val Thr Ala Gly Gln Thr Ile
Asn Tyr Asn Ala65 70 75 80Lys Ala Ser Ile Ser His Pro Gly Pro Met
Ser Phe Tyr Ile Ala Lys 85 90 95Val Pro Ala Gly Gln Thr Ala Ala Thr
Trp Asp Gly Lys Gly Ala Val 100 105 110Trp Thr Lys Ile Tyr Gln Asp
Met Pro Lys Phe Gly Ser Ser Leu Thr 115 120 125Trp Pro Thr Met Gly
Ala Lys Ser Val Pro Val Thr Ile Pro Arg Cys 130 135 140Leu Gln Asn
Gly Asp Tyr Leu Leu Arg Ala Glu His Ile Ala Leu His145 150 155
160Ser Ala Ser Ser Val Gly Gly Ala Gln Phe Tyr Leu Ser Cys Ala Gln
165 170 175Leu Thr Val Ser Gly Gly Ser Gly Thr Trp Asn Pro Lys Asn
Arg Val 180 185 190Ser Phe Pro Gly Ala Tyr Lys Ala Thr Asp Pro Gly
Ile Leu Ile Asn 195 200 205Ile Tyr Tyr Pro Val Pro Thr Ser Tyr Ser
Pro Pro Gly Pro Pro Ala 210 215 220Glu Thr Cys22543977DNAThielavia
terrestris 43atgaagctgt catcccagct cgccgccctc acgctggccg cggcctccgt
gtcaggccac 60tacatcttcg agcagattgc ccatggcggc accaagttcc caccttacga
gtacatccga 120agaaacacga actataacag ccctgtcacc agtctctcgt
cgaacgacct gcgatgcaac 180gtaggcggcg agacggctgg caacacgacc
gtcctcgacg tgaaggcggg cgactccttc 240accttctact cggacgtggc
cgtgtaccac caggggccca tctcactgtg cgtgccccgg 300gccaactttg
atcagtccca agcggactgt ccgctcgcct ggataaccac aattgactga
360cagcccgcac agctacatgt ccaaggctcc cggctccgtc gtggactacg
acggctccgg 420cgactggttc aagatccacg actggggccc gaccttcagc
aacggccagg cctcgtggcc 480gctgcggggt gcgtcccttc cctttccctc
ccccttcctc ccccttcctc cccccctttc 540cccccttttc tgtctggtcg
cacgccctgc tgacgtcccc gtagacaact accagtacaa 600catcccgacg
tgcatcccga acggcgagta cctgctgcgc atccagtcgc tggcgatcca
660caacccgggc gccacgccgc agttctacat cagctgcgcg caggtccggg
tctcgggcgg 720cggcagcgcc tccccctccc caacggccaa gatccccggc
gcgttcaagg cgaccgatcc 780cgggtatacc gcgaatgtga gtgccctatg
ttccttgcgc tccttgttcc ttgctccttg 840ctcggcgtgc ttgaacgcta
cgggctgtgg
agggagggat ggatggatga ataggatgct 900gactgatggt gggacaccag
atttacaata acttccactc gtatacggtg ccgggtccgg 960cggtctttca gtgctag
97744223PRTThielavia terrestris 44Met Lys Leu Ser Ser Gln Leu Ala
Ala Leu Thr Leu Ala Ala Ala Ser1 5 10 15Val Ser Gly His Tyr Ile Phe
Glu Gln Ile Ala His Gly Gly Thr Lys 20 25 30Phe Pro Pro Tyr Glu Tyr
Ile Arg Arg Asn Thr Asn Tyr Asn Ser Pro 35 40 45Val Thr Ser Leu Ser
Ser Asn Asp Leu Arg Cys Asn Val Gly Gly Glu 50 55 60Thr Ala Gly Asn
Thr Thr Val Leu Asp Val Lys Ala Gly Asp Ser Phe65 70 75 80Thr Phe
Tyr Ser Asp Val Ala Val Tyr His Gln Gly Pro Ile Ser Leu 85 90 95Tyr
Met Ser Lys Ala Pro Gly Ser Val Val Asp Tyr Asp Gly Ser Gly 100 105
110Asp Trp Phe Lys Ile His Asp Trp Gly Pro Thr Phe Ser Asn Gly Gln
115 120 125Ala Ser Trp Pro Leu Arg Asp Asn Tyr Gln Tyr Asn Ile Pro
Thr Cys 130 135 140Ile Pro Asn Gly Glu Tyr Leu Leu Arg Ile Gln Ser
Leu Ala Ile His145 150 155 160Asn Pro Gly Ala Thr Pro Gln Phe Tyr
Ile Ser Cys Ala Gln Val Arg 165 170 175Val Ser Gly Gly Gly Ser Ala
Ser Pro Ser Pro Thr Ala Lys Ile Pro 180 185 190Gly Ala Phe Lys Ala
Thr Asp Pro Gly Tyr Thr Ala Asn Ile Tyr Asn 195 200 205Asn Phe His
Ser Tyr Thr Val Pro Gly Pro Ala Val Phe Gln Cys 210 215
220451107DNAThielavia terrestris 45atgccttctt tcgcctccaa gactctcctt
tccaccctgg cgggtgccgc atccgtggcc 60gcccacgggc acgtgtcgaa catcgtcatc
aacggggtct cgtaccaggg ttacgatccg 120acctccttcc cttacatgca
gaacccgccc atcgtggtcg gctggactgc cgccgacacg 180gacaacggct
ttgttgcccc ggatgccttc gccagtggcg atatcatctg ccacaagaac
240gccaccaacg ccaagggcca cgccgtggtc gccgcgggag acaagatctt
catccagtgg 300aacacatggc ccgagtccca ccacggcccc gtcatcgact
acctcgcgag ctgcggcagc 360gcgtcctgcg agaccgtcga caagaccaag
ctcgagttct tcaagatcga cgaggtcggc 420ctggtcgacg gcagctcggc
gcccggtgtg tggggctccg accagctcat cgccaacaac 480aactcgtggc
tcgtcgagat cccgcccacc atcgcgccgg gcaactacgt cctgcgccac
540gagatcatcg cgctgcacag cgccgaaaac gccgacggcg cccagaacta
cccgcagtgc 600ttcaacctgc agatcaccgg caccggcacc gccaccccct
ccggcgtccc cggcacctcg 660ctctacaccc cgaccgaccc gggcatcctc
gtcaacatct acagcgcccc gatcacctac 720accgtcccgg ggccggccct
catctccggc gccgtcagca tcgcccagtc ctcctccgcc 780atcaccgcct
ccggcaccgc cctgaccggc tctgccaccg cacccgccgc cgccgctgct
840accacaactt ccaccaccaa cgccgcggct gctgctacct ctgctgctgc
tgctgctggt 900acttccacaa ccaccaccag cgccgcggcc gtggtccaga
cctcctcctc ctcctcctcc 960gccccgtcct ctgccgccgc cgccgccacc
accaccgcgg ctgccagcgc ccgcccgacc 1020ggctgctcct ctggccgctc
caggaagcag ccgcgccgcc acgcgcggga tatggtggtt 1080gcgcgagggg
ctgaggaggc aaactga 110746368PRTThielavia terrestris 46Met Pro Ser
Phe Ala Ser Lys Thr Leu Leu Ser Thr Leu Ala Gly Ala1 5 10 15Ala Ser
Val Ala Ala His Gly His Val Ser Asn Ile Val Ile Asn Gly 20 25 30Val
Ser Tyr Gln Gly Tyr Asp Pro Thr Ser Phe Pro Tyr Met Gln Asn 35 40
45Pro Pro Ile Val Val Gly Trp Thr Ala Ala Asp Thr Asp Asn Gly Phe
50 55 60Val Ala Pro Asp Ala Phe Ala Ser Gly Asp Ile Ile Cys His Lys
Asn65 70 75 80Ala Thr Asn Ala Lys Gly His Ala Val Val Ala Ala Gly
Asp Lys Ile 85 90 95Phe Ile Gln Trp Asn Thr Trp Pro Glu Ser His His
Gly Pro Val Ile 100 105 110Asp Tyr Leu Ala Ser Cys Gly Ser Ala Ser
Cys Glu Thr Val Asp Lys 115 120 125Thr Lys Leu Glu Phe Phe Lys Ile
Asp Glu Val Gly Leu Val Asp Gly 130 135 140Ser Ser Ala Pro Gly Val
Trp Gly Ser Asp Gln Leu Ile Ala Asn Asn145 150 155 160Asn Ser Trp
Leu Val Glu Ile Pro Pro Thr Ile Ala Pro Gly Asn Tyr 165 170 175Val
Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Glu Asn Ala Asp 180 185
190Gly Ala Gln Asn Tyr Pro Gln Cys Phe Asn Leu Gln Ile Thr Gly Thr
195 200 205Gly Thr Ala Thr Pro Ser Gly Val Pro Gly Thr Ser Leu Tyr
Thr Pro 210 215 220Thr Asp Pro Gly Ile Leu Val Asn Ile Tyr Ser Ala
Pro Ile Thr Tyr225 230 235 240Thr Val Pro Gly Pro Ala Leu Ile Ser
Gly Ala Val Ser Ile Ala Gln 245 250 255Ser Ser Ser Ala Ile Thr Ala
Ser Gly Thr Ala Leu Thr Gly Ser Ala 260 265 270Thr Ala Pro Ala Ala
Ala Ala Ala Thr Thr Thr Ser Thr Thr Asn Ala 275 280 285Ala Ala Ala
Ala Thr Ser Ala Ala Ala Ala Ala Gly Thr Ser Thr Thr 290 295 300Thr
Thr Ser Ala Ala Ala Val Val Gln Thr Ser Ser Ser Ser Ser Ser305 310
315 320Ala Pro Ser Ser Ala Ala Ala Ala Ala Thr Thr Thr Ala Ala Ala
Ser 325 330 335Ala Arg Pro Thr Gly Cys Ser Ser Gly Arg Ser Arg Lys
Gln Pro Arg 340 345 350Arg His Ala Arg Asp Met Val Val Ala Arg Gly
Ala Glu Glu Ala Asn 355 360 36547993DNAThielavia terrestris
47atgccgcccg cactccctca actcctaacc acggtcctga ccgccctcac cctcggttcc
60accgccctcg cccactcaca cctcgcgtac attatcgtta acggcaagct ctaccagggc
120ttcgacccgc gcccgcacca ggccaactac ccttcccggg tcgggtggtc
caccggcgcc 180gtcgacgacg gcttcgtcac gccggccaac tactccaccc
cggacatcat ttgccacatc 240gccggcacca gcccggccgg ccacgcgccc
gtgcgcccgg gcgaccgcat ccacgtccag 300tggaacggct ggccggtcgg
ccacatcggt cccgtgctgt cgtacctcgc ccgctgcgag 360tcggacacgg
gctgcacggg ccagaacaag accgcgctgc ggtggaccaa gatcgacgac
420tccagcccga ccatgcagaa cgtcgccggc gcgggcaccc agggcgaggg
cacccccggc 480aagcgctggg ccaccgacgt gctgatcgcc gccaacaaca
gctggcaggt cgccgtgccg 540gcggggctgc cgaccggcgc gtacgtgctg
cgcaacgaga tcatcgcgct gcactacgcg 600gcgaggaaga acggggcgca
gaactatccg ctctgcatga acctgtgggt ggacgccagt 660ggtgataata
gtagtgtggc tgcaacgacg gcggcggtga cggcgggggg tctgcagatg
720gatgcgtatg acgcgcgcgg gttctacaag gagaacgatc cgggcgtgct
ggtcaatgtc 780acggccgcgc tgtcgtcgta tgtcgtgccc gggccgacgg
tggcggcggg cgccacgccg 840gtgccgtacg cgcagcagag cccgagcgtg
tcgacggcgg cgggcacgcc cgtcgtcgtt 900acaaggacta gcgagacggc
gccgtacacg ggcgccatga cgccgacggt tgcggcgagg 960atgaagggga
gggggtatga tcggcggggt tag 99348330PRTThielavia terrestris 48Met Pro
Pro Ala Leu Pro Gln Leu Leu Thr Thr Val Leu Thr Ala Leu1 5 10 15Thr
Leu Gly Ser Thr Ala Leu Ala His Ser His Leu Ala Tyr Ile Ile 20 25
30Val Asn Gly Lys Leu Tyr Gln Gly Phe Asp Pro Arg Pro His Gln Ala
35 40 45Asn Tyr Pro Ser Arg Val Gly Trp Ser Thr Gly Ala Val Asp Asp
Gly 50 55 60Phe Val Thr Pro Ala Asn Tyr Ser Thr Pro Asp Ile Ile Cys
His Ile65 70 75 80Ala Gly Thr Ser Pro Ala Gly His Ala Pro Val Arg
Pro Gly Asp Arg 85 90 95Ile His Val Gln Trp Asn Gly Trp Pro Val Gly
His Ile Gly Pro Val 100 105 110Leu Ser Tyr Leu Ala Arg Cys Glu Ser
Asp Thr Gly Cys Thr Gly Gln 115 120 125Asn Lys Thr Ala Leu Arg Trp
Thr Lys Ile Asp Asp Ser Ser Pro Thr 130 135 140Met Gln Asn Val Ala
Gly Ala Gly Thr Gln Gly Glu Gly Thr Pro Gly145 150 155 160Lys Arg
Trp Ala Thr Asp Val Leu Ile Ala Ala Asn Asn Ser Trp Gln 165 170
175Val Ala Val Pro Ala Gly Leu Pro Thr Gly Ala Tyr Val Leu Arg Asn
180 185 190Glu Ile Ile Ala Leu His Tyr Ala Ala Arg Lys Asn Gly Ala
Gln Asn 195 200 205Tyr Pro Leu Cys Met Asn Leu Trp Val Asp Ala Ser
Gly Asp Asn Ser 210 215 220Ser Val Ala Ala Thr Thr Ala Ala Val Thr
Ala Gly Gly Leu Gln Met225 230 235 240Asp Ala Tyr Asp Ala Arg Gly
Phe Tyr Lys Glu Asn Asp Pro Gly Val 245 250 255Leu Val Asn Val Thr
Ala Ala Leu Ser Ser Tyr Val Val Pro Gly Pro 260 265 270Thr Val Ala
Ala Gly Ala Thr Pro Val Pro Tyr Ala Gln Gln Ser Pro 275 280 285Ser
Val Ser Thr Ala Ala Gly Thr Pro Val Val Val Thr Arg Thr Ser 290 295
300Glu Thr Ala Pro Tyr Thr Gly Ala Met Thr Pro Thr Val Ala Ala
Arg305 310 315 320Met Lys Gly Arg Gly Tyr Asp Arg Arg Gly 325
330491221DNAThielavia terrestris 49atgaagacat tcaccgccct cctggccgca
gccggcctcg tcgccggcca tggatatgtc 60gacaacgcca ccattggcgg ccagttttat
caggtactct accgcttcac ccaaggtccg 120ctggccacaa ctctataggt
gtcataaatt aacaagccac cgtcccgcag ttctatcagg 180tgtgctcgct
accgaccatg tggtcccgtc tcagcaagcc actcacacgc ccatgatccc
240ctagccttac gtcgacccgt atttagcaac cttggcacgt agtatttatt
gtcccaaata 300ttgagctgaa ctgcacctcc ctagaatccc gcggtgctaa
cattctttca gcccgacagg 360gtctctcgat ccatcccggg caacggcccg
gtcacggacg tcactctcat cgacctgcag 420tgcaacgcca attccacccc
ggccaagctc cacgccactg ccgctgccgg ctcggacgtg 480attctccgct
ggacgctctg gcctgagtcg cacgttggcc ccgtcatcac ctacatggcc
540cgctgccccg acacgggctg ccaggactgg atgccgggca cttcgtagga
gcccatcttg 600caccatatcc atttcaaccg gccacacgca ctgacccata
tgtctgtcta cccctgcagt 660gcggtctggt tcaagatcaa ggagggcggc
cgcgacggca cttccaacac ctgggccgac 720gtacgtgtac cccgtcccag
agagccaaag cccccccttc aacaaagcaa acatctcaat 780agcccgagcc
tacgcactaa cccctctcct tccccctcga aaacacagac cccgctgatg
840acggcgccca cctcgtacac gtacacgatc ccctcctgcc tgaagaaggg
ctactacctg 900gtccgccacg agatcatcgc gctgcacgcc gcctacacct
accccggcgc gcagttctac 960ccgggctgcc accagctcaa cgtcacgggc
ggcgggtcca ccgtaccgtc gagcggcctg 1020gtggcctttc ccggggcgta
caagggcagt gaccccggga ttacgtacga tgcgtataaa 1080ggtgggttgg
ctggttggcc caggtcttgg tgatggggga atgtggtgat gaggtttatt
1140atttgggatc ccgtggctaa cgtaaccctg ggtgtagcgc aaacgtacca
gattcctggg 1200ccggcggtct ttacttgctg a 122150236PRTThielavia
terrestris 50Met Lys Thr Phe Thr Ala Leu Leu Ala Ala Ala Gly Leu
Val Ala Gly1 5 10 15His Gly Tyr Val Asp Asn Ala Thr Ile Gly Gly Gln
Phe Tyr Gln Asn 20 25 30Pro Ala Val Leu Thr Phe Phe Gln Pro Asp Arg
Val Ser Arg Ser Ile 35 40 45Pro Gly Asn Gly Pro Val Thr Asp Val Thr
Leu Ile Asp Leu Gln Cys 50 55 60Asn Ala Asn Ser Thr Pro Ala Lys Leu
His Ala Thr Ala Ala Ala Gly65 70 75 80Ser Asp Val Ile Leu Arg Trp
Thr Leu Trp Pro Glu Ser His Val Gly 85 90 95Pro Val Ile Thr Tyr Met
Ala Arg Cys Pro Asp Thr Gly Cys Gln Asp 100 105 110Trp Met Pro Gly
Thr Ser Ala Val Trp Phe Lys Ile Lys Glu Gly Gly 115 120 125Arg Asp
Gly Thr Ser Asn Thr Trp Ala Asp Thr Pro Leu Met Thr Ala 130 135
140Pro Thr Ser Tyr Thr Tyr Thr Ile Pro Ser Cys Leu Lys Lys Gly
Tyr145 150 155 160Tyr Leu Val Arg His Glu Ile Ile Ala Leu His Ala
Ala Tyr Thr Tyr 165 170 175Pro Gly Ala Gln Phe Tyr Pro Gly Cys His
Gln Leu Asn Val Thr Gly 180 185 190Gly Gly Ser Thr Val Pro Ser Ser
Gly Leu Val Ala Phe Pro Gly Ala 195 200 205Tyr Lys Gly Ser Asp Pro
Gly Ile Thr Tyr Asp Ala Tyr Lys Ala Gln 210 215 220Thr Tyr Gln Ile
Pro Gly Pro Ala Val Phe Thr Cys225 230 23551933DNAThielavia
terrestris 51atggccttgc tgctcttggc aggcttggcc attctggccg ggccggctca
tgcccacggc 60ggcctcgcca actacacagt gggcaacacc tggtataggg ggtgcgtaag
gggggcaccg 120acaacgcctg cttagtaact ccaccatttc gagcgggcta
acaccgggcg cagctacgac 180cccttcacgc cggcggccga ccagatcggc
cagccgtgga tgatccaacg cgcgtgggac 240tcgatcgacc cgatcttcag
cgtcaacgac aaggcgctcg cctgcaacac cccggccacg 300gcgccgacct
cttacattcc catccgcgcg ggcgagaaca tcacggccgt gtactggtac
360tggctgcacc cggtgggccc catgacggcg tggctggcgc ggtgcgacgg
cgactgccgc 420gacgccgacg tcaacgaggc gcgctggttc aagatctggg
aggccggcct gctcagcggg 480ccgaacctgg ccgagggcat gtggtaccag
aaggcgttcc agaactggga cggcagcccg 540gacctgtggc ccgtcacgat
cccggccggg ctgaagagcg gcctgtacat gatccggcac 600gagatcttgt
cgatccacgt cgaggataaa ccgcagtttt atcccgagtg tgcgcatctg
660aatgtgaccg ggggtgggga cctgctgccg cctgatgagt ttttggtgaa
gttcccgggc 720gcttacaaag aagatagtga gtgaaacgcg aagcttcggt
agccattggg ttgcgctgat 780ggaggttaga cccgtcgatc aagatcaata
tctactcgga ccagtacgcc aatacaacgg 840tgagtgtaac aggtcgagca
aaaccaaaca gatgccgatg actgatgatc tcagaattac 900acaattcccg
gagggccgat atgggatggg tga 93352250PRTThielavia terrestris 52Met Ala
Leu Leu Leu Leu Ala Gly Leu Ala Ile Leu Ala Gly Pro Ala1 5 10 15His
Ala His Gly Gly Leu Ala Asn Tyr Thr Val Gly Asn Thr Trp Tyr 20 25
30Arg Gly Tyr Asp Pro Phe Thr Pro Ala Ala Asp Gln Ile Gly Gln Pro
35 40 45Trp Met Ile Gln Arg Ala Trp Asp Ser Ile Asp Pro Ile Phe Ser
Val 50 55 60Asn Asp Lys Ala Leu Ala Cys Asn Thr Pro Ala Thr Ala Pro
Thr Ser65 70 75 80Tyr Ile Pro Ile Arg Ala Gly Glu Asn Ile Thr Ala
Val Tyr Trp Tyr 85 90 95Trp Leu His Pro Val Gly Pro Met Thr Ala Trp
Leu Ala Arg Cys Asp 100 105 110Gly Asp Cys Arg Asp Ala Asp Val Asn
Glu Ala Arg Trp Phe Lys Ile 115 120 125Trp Glu Ala Gly Leu Leu Ser
Gly Pro Asn Leu Ala Glu Gly Met Trp 130 135 140Tyr Gln Lys Ala Phe
Gln Asn Trp Asp Gly Ser Pro Asp Leu Trp Pro145 150 155 160Val Thr
Ile Pro Ala Gly Leu Lys Ser Gly Leu Tyr Met Ile Arg His 165 170
175Glu Ile Leu Ser Ile His Val Glu Asp Lys Pro Gln Phe Tyr Pro Glu
180 185 190Cys Ala His Leu Asn Val Thr Gly Gly Gly Asp Leu Leu Pro
Pro Asp 195 200 205Glu Phe Leu Val Lys Phe Pro Gly Ala Tyr Lys Glu
Asp Asn Pro Ser 210 215 220Ile Lys Ile Asn Ile Tyr Ser Asp Gln Tyr
Ala Asn Thr Thr Asn Tyr225 230 235 240Thr Ile Pro Gly Gly Pro Ile
Trp Asp Gly 245 250531584DNAThielavia terrestris 53atgatgccgt
cccttgttcg cttctcaatg ggtctggcga ccgccttcgc ctcgctgtcc 60acagcacata
ccgtcttcac cacgcttttc atcaacggcg tcgaccaagg ggacgggacc
120tgcatccgca tggccaagaa gggcagcgtt tgcacccatc ccattgctgg
tggcctcgac 180agcccagaca tggcttgtgg tatgccctct gcgtttcccc
tgcgagagct ttcctcgagc 240taacccaatg ccgcgttgcc caggccgaga
cggacaacaa gccgtggcat tcacctgccc 300agccccggcg ggctccaagt
tgagcttcga gttccgcatg tgggccgacg cctctcagcc 360cggctctatc
gacccatccc acctcggctc gacggcaatc tacctcaaac aagtctccaa
420catcagctcc gactcggctg ccggccctgg ctggttcaag atctacgccg
agggctacga 480cacagccgcc aagaagtggg ccacagagaa gctcatcgac
aacggcggcc tgctgagcat 540cgagcttccg cccactctgc cggcgggata
ctacctcgcc cgcagcgaga tcgtcaccat 600ccagaacgtc accaacgacc
acgtcgaccc gcagttctac gttggctgcg cacagctctt 660cgtccagggg
cctccgacca cccccaccgt cccgccagac agactcgtct ccatcccggg
720ccacgtccat gcctccgacc cggggctgac cttcaacatc tggcgcgacg
acccctccaa 780gacggcctac accgtcgtcg gcccggcccc cttctccccc
accgccgccc ccacccccac 840ctccaccaac accaacgggc agcaacaaca
acaacagcaa caggcgataa agcagacgga 900cggcgtgatc cccgccgact
gccagctcaa gaacgccaac tggtgcggcg ccgaggtgcc 960cgcgtacgcc
gacgaggccg gctgctgggc gtcgtcggcc gactgcttcg cccagctgga
1020cgcctgctac acgtcggcgc cgcccacggg cagccgcggc tgccggctgt
gggaggactg 1080gtgcaccggc attcagcagg gctgccgcgc ggggcggtgg
cgggggccgc cgccctttca 1140tggggagggg gcagcagcgg aggtgtgaac
ggttcgggga cgggtggcgg tggtggtggt 1200ggtggtggtg gcactggctc
ttcttcggct tctgccccga cggagacggc ctctgctggc 1260cgggggggcg
caagaatagc tgccgtggcc ggctgcggag gcgggacagg agacatggtt
1320gaagaggttt tcctctttta ttgggacgct tgcagcggct ggcgacggag
ccgtggtggt 1380ggttcgattc ttgcgaggct tatccttcat gtccttcttc
cacttttgag accgaggcga 1440gcccctcgag tccatttact tctcttccac
ctgtacctca acttctgtta tccaggaacc 1500agtggtttct ataatcgcct
gagcattaaa ctaggcatat ggccaagcaa aatgtcgcct 1560gatgtagcgc
attacgtgaa ataa 158454478PRTThielavia terrestris 54Met Met Pro Ser
Leu Val Arg Phe Ser
Met Gly Leu Ala Thr Ala Phe1 5 10 15Ala Ser Leu Ser Thr Ala His Thr
Val Phe Thr Thr Leu Phe Ile Asn 20 25 30Gly Val Asp Gln Gly Asp Gly
Thr Cys Ile Arg Met Ala Lys Lys Gly 35 40 45Ser Val Cys Thr His Pro
Ile Ala Gly Gly Leu Asp Ser Pro Asp Met 50 55 60Ala Cys Gly Arg Asp
Gly Gln Gln Ala Val Ala Phe Thr Cys Pro Ala65 70 75 80Pro Ala Gly
Ser Lys Leu Ser Phe Glu Phe Arg Met Trp Ala Asp Ala 85 90 95Ser Gln
Pro Gly Ser Ile Asp Pro Ser His Leu Gly Ser Thr Ala Ile 100 105
110Tyr Leu Lys Gln Val Ser Asn Ile Ser Ser Asp Ser Ala Ala Gly Pro
115 120 125Gly Trp Phe Lys Ile Tyr Ala Glu Gly Tyr Asp Thr Ala Ala
Lys Lys 130 135 140Trp Ala Thr Glu Lys Leu Ile Asp Asn Gly Gly Leu
Leu Ser Ile Glu145 150 155 160Leu Pro Pro Thr Leu Pro Ala Gly Tyr
Tyr Leu Ala Arg Ser Glu Ile 165 170 175Val Thr Ile Gln Asn Val Thr
Asn Asp His Val Asp Pro Gln Phe Tyr 180 185 190Val Gly Cys Ala Gln
Leu Phe Val Gln Gly Pro Pro Thr Thr Pro Thr 195 200 205Val Pro Pro
Asp Arg Leu Val Ser Ile Pro Gly His Val His Ala Ser 210 215 220Asp
Pro Gly Leu Thr Phe Asn Ile Trp Arg Asp Asp Pro Ser Lys Thr225 230
235 240Ala Tyr Thr Val Val Gly Pro Ala Pro Phe Ser Pro Thr Ala Ala
Pro 245 250 255Thr Pro Thr Ser Thr Asn Thr Asn Gly Gln Gln Gln Gln
Gln Gln Gln 260 265 270Gln Ala Ile Lys Gln Thr Asp Gly Val Ile Pro
Ala Asp Cys Gln Leu 275 280 285Lys Asn Ala Asn Trp Cys Gly Ala Glu
Val Pro Ala Tyr Ala Asp Glu 290 295 300Ala Gly Cys Trp Ala Ser Ser
Ala Asp Cys Phe Ala Gln Leu Asp Ala305 310 315 320Cys Tyr Thr Ser
Ala Pro Pro Thr Gly Ser Arg Gly Cys Arg Leu Trp 325 330 335Glu Asp
Trp Cys Thr Gly Ile Gln Gln Gly Cys Arg Ala Gly Arg Trp 340 345
350Arg Gly Pro Pro Pro Phe His Gly Glu Gly Ala Ala Ala Glu Thr Ala
355 360 365Ser Ala Gly Arg Gly Gly Ala Arg Ile Ala Ala Val Ala Gly
Cys Gly 370 375 380Gly Gly Thr Gly Asp Met Val Glu Glu Val Phe Leu
Phe Tyr Trp Asp385 390 395 400Ala Cys Ser Gly Trp Arg Arg Ser Arg
Gly Gly Gly Ser Ile Leu Ala 405 410 415Arg Leu Ile Leu His Val Leu
Leu Pro Leu Leu Arg Pro Arg Arg Ala 420 425 430Pro Arg Val His Leu
Leu Leu Phe His Leu Tyr Leu Asn Phe Cys Tyr 435 440 445Pro Gly Thr
Ser Gly Phe Tyr Asn Arg Leu Ser Ile Lys Leu Gly Ile 450 455 460Trp
Pro Ser Lys Met Ser Pro Asp Val Ala His Tyr Val Lys465 470
47555868DNAThielavia terrestris 55atgcagctcc tcgtgggctt gctgcttgca
gccgtggctg ctcgagcaca ttgtatttct 60acccctttcc gcgtgcctcc cagcctcaag
gcaagaagac gcacgcagca gctaacggac 120cctatcagac acatttccca
gactcgtggt aaatgggcag cccgaggaca aggactggtc 180ggttacgcgc
atgaccaaga acgcgcagag caagcaggga gtccaggacc cgaccagtcc
240cgacattcgc tgctacacgt cgcagacggc gcctaacgtg gctacggtcc
ctgccggagc 300caccgtccat tacatatcga ctcagcagat caaccacccg
ggcccgacgc agtactacct 360cgccaaggta ccggcggggt cgtcggccaa
gacgtgggac gggtcagggg ccgtctggtt 420caagatctcg accaccatgc
cttacttgga caacaacaag cagcttgtct ggccgaatca 480gagtaggaac
aattcccgct ccaatcttcg atttggcctt gagctacggc cgattgcatg
540ggagagaccg ttgactgacg gggcaaccca accttcatca gacacgtaca
cgacggtcaa 600cacgaccatc cccgccgata cgcccagtgg ggaatacctc
ctccgggtcg agcagatcgc 660gctgcacctg gcctcgcagc ccaacggggc
tcagttctac ctggcctgct cgcagatcca 720gattacgggc ggcggcaacg
gcacgcccgg cccgctagtc gcgttgccgg gggcgtacaa 780gagcaacgac
ccgggcattt tggtcaacat ctactctatg cagcccggcg attacaagcc
840gcccgggccg ccggtgtgga gtggctga 86856230PRTThielavia terrestris
56Met Gln Leu Leu Val Gly Leu Leu Leu Ala Ala Val Ala Ala Arg Ala1
5 10 15His Tyr Thr Phe Pro Arg Leu Val Val Asn Gly Gln Pro Glu Asp
Lys 20 25 30Asp Trp Ser Val Thr Arg Met Thr Lys Asn Ala Gln Ser Lys
Gln Gly 35 40 45Val Gln Asp Pro Thr Ser Pro Asp Ile Arg Cys Tyr Thr
Ser Gln Thr 50 55 60Ala Pro Asn Val Ala Thr Val Pro Ala Gly Ala Thr
Val His Tyr Ile65 70 75 80Ser Thr Gln Gln Ile Asn His Pro Gly Pro
Thr Gln Tyr Tyr Leu Ala 85 90 95Lys Val Pro Ala Gly Ser Ser Ala Lys
Thr Trp Asp Gly Ser Gly Ala 100 105 110Val Trp Phe Lys Ile Ser Thr
Thr Met Pro Tyr Leu Asp Asn Asn Lys 115 120 125Gln Leu Val Trp Pro
Asn Gln Asn Thr Tyr Thr Thr Val Asn Thr Thr 130 135 140Ile Pro Ala
Asp Thr Pro Ser Gly Glu Tyr Leu Leu Arg Val Glu Gln145 150 155
160Ile Ala Leu His Leu Ala Ser Gln Pro Asn Gly Ala Gln Phe Tyr Leu
165 170 175Ala Cys Ser Gln Ile Gln Ile Thr Gly Gly Gly Asn Gly Thr
Pro Gly 180 185 190Pro Leu Val Ala Leu Pro Gly Ala Tyr Lys Ser Asn
Asp Pro Gly Ile 195 200 205Leu Val Asn Ile Tyr Ser Met Gln Pro Gly
Asp Tyr Lys Pro Pro Gly 210 215 220Pro Pro Val Trp Ser Gly225
230571068DNAThielavia terrestris 57atgaagctgt acctggcggc ctttctaggc
gccgtcgcca ccccgggagc gttcgctcat 60cgtaggttcc ccgtctatct ccctaggggt
agcaccacga ctaatttctc gtcgtccccc 120tgtagaaatc cacgggattc
tacttgtcaa cggcaccgaa acgccggaat ggaaatacgt 180ccggtaatat
ctaccttgct ctccttcttc cacaaccagc ctaacacatc atcagtgacg
240tggcctggga gggcgcctac gaaccggaaa aataccccaa caccgagttc
tttaagacgc 300ccccgcagac ggacatcaac aacccgaaca tcacctgcgg
caggaacgcg ttcgactcgg 360ccagcaagac tgagacggcc gacatactgg
ccggctcaga ggtcggcttc cgcgtctcgt 420gggacggcaa cggcaagtac
ggcgtgttct ggcatcccgg gccggggcag atctacctct 480ctcgtgctcc
gaacgacgac ctggaggact accgcggcga cggagactgg ttcaagatcg
540caaccggcgc cgccgtctcc aataccgagt ggctgctgtg gaacaagcat
gacgtgagcc 600ccaacattcc tcgcccaatc gatccccaac ctggtcacca
tggcggcgtc cgggatgcaa 660agagactaac tccagaggaa cctacctagt
tcaacttcac catccccaag acgacgccgc 720cgggcaagta cctgatgcgc
atcgagcagt tcatgccctc cacggtcgaa tacagccagt 780ggtacgtcaa
ctgcgcccac gtcaacatca tcggccccgg cggaggcacg ccgacgggct
840ttgccaggtt tcccggcacc tacactgttg acgatcccgg taagccggac
ctaccggaca 900cagaggcctc gggatagctt gctaaccttg tttgctctct
ctctttttct ctcccgacta 960ggcatcaagg tgccgttgaa ccagatcgtc
aacagcggag agttgccgca ggaccaactg 1020aggctgctcg agtacaagcc
cccgggccca gcgctgtgga ctggttga 106858257PRTThielavia terrestris
58Met Lys Leu Tyr Leu Ala Ala Phe Leu Gly Ala Val Ala Thr Pro Gly1
5 10 15Ala Phe Ala His Gln Ile His Gly Ile Leu Leu Val Asn Gly Thr
Glu 20 25 30Thr Pro Glu Trp Lys Tyr Val Arg Asp Val Ala Trp Glu Gly
Ala Tyr 35 40 45Glu Pro Glu Lys Tyr Pro Asn Thr Glu Phe Phe Lys Thr
Pro Pro Gln 50 55 60Thr Asp Ile Asn Asn Pro Asn Ile Thr Cys Gly Arg
Asn Ala Phe Asp65 70 75 80Ser Ala Ser Lys Thr Glu Thr Ala Asp Ile
Leu Ala Gly Ser Glu Val 85 90 95Gly Phe Arg Val Ser Trp Asp Gly Asn
Gly Lys Tyr Gly Val Phe Trp 100 105 110His Pro Gly Pro Gly Gln Ile
Tyr Leu Ser Arg Ala Pro Asn Asp Asp 115 120 125Leu Glu Asp Tyr Arg
Gly Asp Gly Asp Trp Phe Lys Ile Ala Thr Gly 130 135 140Ala Ala Val
Ser Asn Thr Glu Trp Leu Leu Trp Asn Lys His Asp Phe145 150 155
160Asn Phe Thr Ile Pro Lys Thr Thr Pro Pro Gly Lys Tyr Leu Met Arg
165 170 175Ile Glu Gln Phe Met Pro Ser Thr Val Glu Tyr Ser Gln Trp
Tyr Val 180 185 190Asn Cys Ala His Val Asn Ile Ile Gly Pro Gly Gly
Gly Thr Pro Thr 195 200 205Gly Phe Ala Arg Phe Pro Gly Thr Tyr Thr
Val Asp Asp Pro Gly Ile 210 215 220Lys Val Pro Leu Asn Gln Ile Val
Asn Ser Gly Glu Leu Pro Gln Asp225 230 235 240Gln Leu Arg Leu Leu
Glu Tyr Lys Pro Pro Gly Pro Ala Leu Trp Thr 245 250
255Gly59871DNAThermoascus crustaceus 59atggcctttt cccagataat
ggctattacc ggcgtttttc ttgcctctgc ttccctggtg 60gctggccatg gctttgttca
gaatatcgtg attgatggta aaaggtacct aactacctac 120cttactatct
gatgtcattt acaagaaagg gcacagacac aagcggcaaa aaaaagaaag
180aaagaaagaa agaaagaaag ctgacaaaaa ttcaacaagt tatggcgggt
acatcgtgaa 240ccaatatcca tacatgtcag atcctccgga ggtcgtcggc
tggtctacca ccgcaaccga 300cctcggattc gtggacggta ccggatacca
aggacctgat atcatctgcc acaggggcgc 360caagcctgca gccctgactg
cccaagtggc cgccggagga accgtcaagc tggaatggac 420tccatggcct
gattctcacc acggcccggt gatcaactac cttgctcctt gcaacggtga
480ctgttccacc gtggacaaga cccaattgaa attcttcaag atcgcccagg
ccggtctcat 540cgatgacaac agtcctcctg gtatctgggc ctcagacaat
ctgatagcgg ccaacaacag 600ctggactgtc accatcccaa ccacaactgc
acctggaaac tatgttctaa ggcatgagat 660cattgctctc cactcagctg
ggaacaagga tggtgcgcag aactatcccc agtgcatcaa 720cctgaaggtc
actggaaatg gttctggcaa tcctcctgct ggtgctcttg gaacggcact
780ctacaaggat acagatccgg gaattctgat caatatctac cagaaacttt
ccagctatgt 840tattcctggt cctgctttgt acactggtta g
87160251PRTThermoascus crustaceus 60Met Ala Phe Ser Gln Ile Met Ala
Ile Thr Gly Val Phe Leu Ala Ser1 5 10 15Ala Ser Leu Val Ala Gly His
Gly Phe Val Gln Asn Ile Val Ile Asp 20 25 30Gly Lys Ser Tyr Gly Gly
Tyr Ile Val Asn Gln Tyr Pro Tyr Met Ser 35 40 45Asp Pro Pro Glu Val
Val Gly Trp Ser Thr Thr Ala Thr Asp Leu Gly 50 55 60Phe Val Asp Gly
Thr Gly Tyr Gln Gly Pro Asp Ile Ile Cys His Arg65 70 75 80Gly Ala
Lys Pro Ala Ala Leu Thr Ala Gln Val Ala Ala Gly Gly Thr 85 90 95Val
Lys Leu Glu Trp Thr Pro Trp Pro Asp Ser His His Gly Pro Val 100 105
110Ile Asn Tyr Leu Ala Pro Cys Asn Gly Asp Cys Ser Thr Val Asp Lys
115 120 125Thr Gln Leu Lys Phe Phe Lys Ile Ala Gln Ala Gly Leu Ile
Asp Asp 130 135 140Asn Ser Pro Pro Gly Ile Trp Ala Ser Asp Asn Leu
Ile Ala Ala Asn145 150 155 160Asn Ser Trp Thr Val Thr Ile Pro Thr
Thr Thr Ala Pro Gly Asn Tyr 165 170 175Val Leu Arg His Glu Ile Ile
Ala Leu His Ser Ala Gly Asn Lys Asp 180 185 190Gly Ala Gln Asn Tyr
Pro Gln Cys Ile Asn Leu Lys Val Thr Gly Asn 195 200 205Gly Ser Gly
Asn Pro Pro Ala Gly Ala Leu Gly Thr Ala Leu Tyr Lys 210 215 220Asp
Thr Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gln Lys Leu Ser Ser225 230
235 240Tyr Val Ile Pro Gly Pro Ala Leu Tyr Thr Gly 245
250611102DNAThermoascus crustaceus 61atgtcattct cgaagatact
tgctatcgct ggggccatta cctacgcatc ttcagctgcc 60gctcatggtt atgtccaggg
aattgttgtc gatggcagct agtatgtcac tctggatgga 120accttcagca
cgtactgtac taacaatcag cagctacggg ggatatatgg tgacccaata
180tccctacacc gctcaacctc cggaactcat cgcctggtcc actaaagcaa
ccgatcttgg 240gtttgtggac ggcagtggct atacttctcc tgatatcatc
tgccataagg gtgctgagcc 300tggtgcccag agcgccaaag tggcagctgg
agggaccgtt gagctgcagt ggacggcatg 360gcccgagtct cacaagggcc
cagttattga ctacctcgcc gcctgcgacg gggactgctc 420atctgttgat
aagactgcac taaagttctt taagattgac gagagtggtc tgattgacgg
480caacggtgct ggaacatggg cctctgatac gttgatcaaa aataacaaca
gctggactgt 540caccatccca agcacaattg cttccggaaa ctacgtacta
agacacgaaa taattgcgct 600ccattctgcc ggaaacaaag atggtgctca
gaactatccc cagtgtatca acctcgaggt 660cactggtagt ggcaccgaaa
accctgctgg cactctcgga acagcgcttt acacagacac 720tgatcctggc
cttctggtca acatctacca gggtctgtcc aactattcaa tccctggtcc
780tgctctgtat agcggcaaca gtgataacgc tggttccctc aaccctacca
ccacgccgtc 840aattcagaat gctgctgctg ctccctccac ttccacagca
tctgttgtca ctgattcttc 900gtcagccacc cagactgcta gtgtcgccgc
cacgactcca gcctccactt cggctgttac 960agcctcacca gctcccgata
ctggaagcga cgtaaccaaa tatctggatt cgatgagctc 1020ggatgaggtc
ctcaccctgg tgcgcgggac cctgtcttgg ctggtttcta acaagaaaca
1080tgcgcgggat ctttctcact ga 110262349PRTThermoascus crustaceus
62Met Ser Phe Ser Lys Ile Leu Ala Ile Ala Gly Ala Ile Thr Tyr Ala1
5 10 15Ser Ser Ala Ala Ala His Gly Tyr Val Gln Gly Ile Val Val Asp
Gly 20 25 30Ser Tyr Tyr Gly Gly Tyr Met Val Thr Gln Tyr Pro Tyr Thr
Ala Gln 35 40 45Pro Pro Glu Leu Ile Ala Trp Ser Thr Lys Ala Thr Asp
Leu Gly Phe 50 55 60Val Asp Gly Ser Gly Tyr Thr Ser Pro Asp Ile Ile
Cys His Lys Gly65 70 75 80Ala Glu Pro Gly Ala Gln Ser Ala Lys Val
Ala Ala Gly Gly Thr Val 85 90 95Glu Leu Gln Trp Thr Ala Trp Pro Glu
Ser His Lys Gly Pro Val Ile 100 105 110Asp Tyr Leu Ala Ala Cys Asp
Gly Asp Cys Ser Ser Val Asp Lys Thr 115 120 125Ala Leu Lys Phe Phe
Lys Ile Asp Glu Ser Gly Leu Ile Asp Gly Asn 130 135 140Gly Ala Gly
Thr Trp Ala Ser Asp Thr Leu Ile Lys Asn Asn Asn Ser145 150 155
160Trp Thr Val Thr Ile Pro Ser Thr Ile Ala Ser Gly Asn Tyr Val Leu
165 170 175Arg His Glu Ile Ile Ala Leu His Ser Ala Gly Asn Lys Asp
Gly Ala 180 185 190Gln Asn Tyr Pro Gln Cys Ile Asn Leu Glu Val Thr
Gly Ser Gly Thr 195 200 205Glu Asn Pro Ala Gly Thr Leu Gly Thr Ala
Leu Tyr Thr Asp Thr Asp 210 215 220Pro Gly Leu Leu Val Asn Ile Tyr
Gln Gly Leu Ser Asn Tyr Ser Ile225 230 235 240Pro Gly Pro Ala Leu
Tyr Ser Gly Asn Ser Asp Asn Ala Gly Ser Leu 245 250 255Asn Pro Thr
Thr Thr Pro Ser Ile Gln Asn Ala Ala Ala Ala Pro Ser 260 265 270Thr
Ser Thr Ala Ser Val Val Thr Asp Ser Ser Ser Ala Thr Gln Thr 275 280
285Ala Ser Val Ala Ala Thr Thr Pro Ala Ser Thr Ser Ala Val Thr Ala
290 295 300Ser Pro Ala Pro Asp Thr Gly Ser Asp Val Thr Lys Tyr Leu
Asp Ser305 310 315 320Met Ser Ser Asp Glu Val Leu Thr Leu Val Arg
Gly Thr Leu Ser Trp 325 330 335Leu Val Ser Asn Lys Lys His Ala Arg
Asp Leu Ser His 340 345631493DNAThermoascus crustaceus 63atgttgtcat
tcattcccac caagtcagct gcgctgacga ctcttctact tcttggaaca 60gctcatgctc
acactttgat gaccaccatg tttgtggacg gcgtcaacca gggagatggt
120gtctgcattc gcatgaacaa tgacggcgga actgccaata cctatatcca
gcctatcacg 180agcaaggata tcgcctgcgg taagtaccca gatgtcatca
tactctgcca taacatccgt 240catatctact agaatcggag caatgttaag
tatttccagg catccaaggc gaaatcggcg 300cctcccgagt ctgcccagtc
aaggcatctt ccaccctaac cttccaattc cgcgagcaac 360ccaacaaccc
aaactcctcc cctctcgatc catcgcacaa aggccccgcc gcggtgtacc
420tgaaaaaggt cgactccgcc atcgcgagca acaacgccgc cggagacagc
tggttcaaga 480tctgggagtc cgtctacgac gagtccacgg gcaaatgggg
cacgaccaag atgatcgaga 540acaacgggca catctccgtc aaggtgcccg
atgatatcga gggtggttac tatcttgccc 600ggacggagct gctggcgcta
cattctgcgg atcaggggga tccgcagttc tatgttggct 660gtgcgcagct
gtttatcgat tcggatggga cggcgaaacc gcccactgtt tctattggag
720aggggacgta cgatctgagc atgcctgcca tgacgtataa tatctgggag
acaccgttgg 780ctctgccgta tccgatgtat gggcctcctg tctatacgcc
tggctctggt tctggatcag 840tccgtgcgac gagctcttct gctgtcccta
ctgcaaccga atcctctttt gtagaggaaa 900gagcaaaccc cgtcacggca
aacagtgttt attctgcaag gggcaaattc aaaacctgga 960ttgataaact
gtcatggcgc gggaaggtcc gtgagaacgt cagacaagcc gcgggaagaa
1020gaagcactct cgtccagact gtgggtctaa agccaaaagg ctgcatcttc
gtcaatggaa 1080actggtgcgg cttcgaggtt cccgactaca acgatgcgga
gagctgctgg gctgtatgtt 1140cccctcctta gcctcttaca tccctaagta
ctacatttga aaacaacaaa aagaaatgta 1200tatactaact acgtacgctc
tactctaggc ctccgacaac tgctggaaac agtccgacgc 1260ctgctggaac
aagacccaac ccacgggcta caataactgc cagatctggc aggacaagaa
1320atgcaaggtc atccaggatt cctgtagcgg acccaacccg catggaccac
cgaataaggg 1380caaggatttg actccggagt ggccgccact gaagggctcg
atggatacgt tctccaagcg 1440tactatcggt taccgcgatt ggattgttag
aaggagaggt gcatgagggt gta 149364436PRTThermoascus crustaceus 64Met
Leu Ser Phe Ile Pro Thr Lys Ser Ala Ala Leu Thr Thr Leu Leu1 5 10
15Leu Leu Gly Thr Ala His Ala His Thr Leu Met Thr Thr Met Phe Val
20 25 30Asp Gly Val Asn Gln Gly Asp Gly Val Cys Ile Arg Met Asn Asn
Asp 35 40 45Gly Gly Thr Ala Asn Thr Tyr Ile Gln Pro Ile Thr Ser Lys
Asp Ile 50 55 60Ala Cys Gly Ile Gln Gly Glu Ile Gly Ala Ser Arg Val
Cys Pro Val65 70 75 80Lys Ala Ser Ser Thr Leu Thr Phe Gln Phe Arg
Glu Gln Pro Asn Asn 85 90 95Pro Asn Ser Ser Pro Leu Asp Pro Ser His
Lys Gly Pro Ala Ala Val 100 105 110Tyr Leu Lys Lys Val Asp Ser Ala
Ile Ala Ser Asn Asn Ala Ala Gly 115 120 125Asp Ser Trp Phe Lys Ile
Trp Glu Ser Val Tyr Asp Glu Ser Thr Gly 130 135 140Lys Trp Gly Thr
Thr Lys Met Ile Glu Asn Asn Gly His Ile Ser Val145 150 155 160Lys
Val Pro Asp Asp Ile Glu Gly Gly Tyr Tyr Leu Ala Arg Thr Glu 165 170
175Leu Leu Ala Leu His Ser Ala Asp Gln Gly Asp Pro Gln Phe Tyr Val
180 185 190Gly Cys Ala Gln Leu Phe Ile Asp Ser Asp Gly Thr Ala Lys
Pro Pro 195 200 205Thr Val Ser Ile Gly Glu Gly Thr Tyr Asp Leu Ser
Met Pro Ala Met 210 215 220Thr Tyr Asn Ile Trp Glu Thr Pro Leu Ala
Leu Pro Tyr Pro Met Tyr225 230 235 240Gly Pro Pro Val Tyr Thr Pro
Gly Ser Gly Ser Gly Ser Val Arg Ala 245 250 255Thr Ser Ser Ser Ala
Val Pro Thr Ala Thr Glu Ser Ser Phe Val Glu 260 265 270Glu Arg Ala
Asn Pro Val Thr Ala Asn Ser Val Tyr Ser Ala Arg Gly 275 280 285Lys
Phe Lys Thr Trp Ile Asp Lys Leu Ser Trp Arg Gly Lys Val Arg 290 295
300Glu Asn Val Arg Gln Ala Ala Gly Arg Arg Ser Thr Leu Val Gln
Thr305 310 315 320Val Gly Leu Lys Pro Lys Gly Cys Ile Phe Val Asn
Gly Asn Trp Cys 325 330 335Gly Phe Glu Val Pro Asp Tyr Asn Asp Ala
Glu Ser Cys Trp Ala Ala 340 345 350Ser Asp Asn Cys Trp Lys Gln Ser
Asp Ala Cys Trp Asn Lys Thr Gln 355 360 365Pro Thr Gly Tyr Asn Asn
Cys Gln Ile Trp Gln Asp Lys Lys Cys Lys 370 375 380Val Ile Gln Asp
Ser Cys Ser Gly Pro Asn Pro His Gly Pro Pro Asn385 390 395 400Lys
Gly Lys Asp Leu Thr Pro Glu Trp Pro Pro Leu Lys Gly Ser Met 405 410
415Asp Thr Phe Ser Lys Arg Thr Ile Gly Tyr Arg Asp Trp Ile Val Arg
420 425 430Arg Arg Gly Ala 435651035DNAAspergillus aculeatus
65atgaagtata ttcctctcgt tattgcagtt gctgccggcc tggcacgtcc ggctactgcc
60cactacatct tcagcaagct cgtgctgaac ggagaggcat ctgcggactg gcaatacatc
120cgcgagacta ctcgcagcat agtctatgag ccgaccaagt acacctctac
cttcgataac 180ctaacaccca gcgatagcga cttccgctgt aatctcggtt
ccttcagcaa tgctgcgaag 240accgaggtcg ctgaggttgc ggcaggcgat
accatcgcaa tgaagctatt ctacgacacc 300agtattgcgc atcctggccc
gggacaagtt tatatgtcca aggcaccgac cggcaatgtt 360caggaatacc
aaggagacgg ggattggttc aaaatctggg aaaagaccct ttgcaacacg
420gatggtgatc tgactacaga ggcctggtgc acctggggca tgtcacagtt
tgaatttcaa 480atcccagctg cgaccccggc aggagagtac ctagtgcgcg
ccgagcatat aggcctgcat 540ggcgctcaag cgaacgaggc cgaattcttc
tacagctgtg cgcagatcaa ggttacaggc 600tcgggaactg gatctcccag
tctcacgtat caaattcctg gtctctataa cgacactatg 660accctgttca
atggcctcaa tctttggact gattcagccg agaaggtgca gctggatttc
720ctggagacgc caattgggga cgacgtgtgg agcggagcag gctcggggag
cccatctgct 780gccacctctt cgaccagcgg tgcaactctt gcagctcagg
gtacaactac ctctgccgcg 840catgctcagg cccagaccac cattaccacc
agcaccagca ccatcacgtc tctcgaatca 900gccagctcaa ccgatctcgt
tgcgcagtat ggtcagtgcg gaggccttaa ctggtccggt 960ccaaccgagt
gtgagacacc ttatacctgt gtgcagcaga acccttacta ccatcaatgc
1020gtgaattcgt gctga 103566344PRTAspergillus aculeatus 66Met Lys
Tyr Ile Pro Leu Val Ile Ala Val Ala Ala Gly Leu Ala Arg1 5 10 15Pro
Ala Thr Ala His Tyr Ile Phe Ser Lys Leu Val Leu Asn Gly Glu 20 25
30Ala Ser Ala Asp Trp Gln Tyr Ile Arg Glu Thr Thr Arg Ser Ile Val
35 40 45Tyr Glu Pro Thr Lys Tyr Thr Ser Thr Phe Asp Asn Leu Thr Pro
Ser 50 55 60Asp Ser Asp Phe Arg Cys Asn Leu Gly Ser Phe Ser Asn Ala
Ala Lys65 70 75 80Thr Glu Val Ala Glu Val Ala Ala Gly Asp Thr Ile
Ala Met Lys Leu 85 90 95Phe Tyr Asp Thr Ser Ile Ala His Pro Gly Pro
Gly Gln Val Tyr Met 100 105 110Ser Lys Ala Pro Thr Gly Asn Val Gln
Glu Tyr Gln Gly Asp Gly Asp 115 120 125Trp Phe Lys Ile Trp Glu Lys
Thr Leu Cys Asn Thr Asp Gly Asp Leu 130 135 140Thr Thr Glu Ala Trp
Cys Thr Trp Gly Met Ser Gln Phe Glu Phe Gln145 150 155 160Ile Pro
Ala Ala Thr Pro Ala Gly Glu Tyr Leu Val Arg Ala Glu His 165 170
175Ile Gly Leu His Gly Ala Gln Ala Asn Glu Ala Glu Phe Phe Tyr Ser
180 185 190Cys Ala Gln Ile Lys Val Thr Gly Ser Gly Thr Gly Ser Pro
Ser Leu 195 200 205Thr Tyr Gln Ile Pro Gly Leu Tyr Asn Asp Thr Met
Thr Leu Phe Asn 210 215 220Gly Leu Asn Leu Trp Thr Asp Ser Ala Glu
Lys Val Gln Leu Asp Phe225 230 235 240Leu Glu Thr Pro Ile Gly Asp
Asp Val Trp Ser Gly Ala Gly Ser Gly 245 250 255Ser Pro Ser Ala Ala
Thr Ser Ser Thr Ser Gly Ala Thr Leu Ala Ala 260 265 270Gln Gly Thr
Thr Thr Ser Ala Ala His Ala Gln Ala Gln Thr Thr Ile 275 280 285Thr
Thr Ser Thr Ser Thr Ile Thr Ser Leu Glu Ser Ala Ser Ser Thr 290 295
300Asp Leu Val Ala Gln Tyr Gly Gln Cys Gly Gly Leu Asn Trp Ser
Gly305 310 315 320Pro Thr Glu Cys Glu Thr Pro Tyr Thr Cys Val Gln
Gln Asn Pro Tyr 325 330 335Tyr His Gln Cys Val Asn Ser Cys
340671203DNAAspergillus aculeatus 67atgtctgttg ctaagtttgc
tggtgttatc ctcggttcgg ccgctctcgt cgctggccac 60ggttacgtgt cgggtgctgt
tgtcgacgga acctactatg gcggctacat tgtcacttcc 120tacccctatt
ccagcgatcc cccggagacc attggatggt ctaccgaggc gaccgacttg
180ggtttcgtcg atggtagcga gtatgctgat gccgacatca tttgccacaa
gagtgccaag 240cccggtgcca tctctgctga ggtcaaggcc ggtggtactg
ttgagctcca gtggactacc 300tggcccgaca gccaccacgg ccctgtcctg
acctaccttg ccaactgcaa tggtgactgc 360agcagcgtca ccaagaccga
cctcgagttt ttcaagattg acgagagcgg tctcatcaac 420gacgacgacg
tccccggtac ctgggccagt gataacttga tcgccaacaa caacagctgg
480actgtgacca tcccctctga cattgcggct ggcaactacg tcctccgtca
cgaaatcatt 540gcccttcact ctgctggtaa caaggatggt gctcagaact
accctcagtg cctcaacttg 600aaggtcactg gcggcggtga tctcgctcct
tctggcactg ctggtgagag cctgtacaag 660gacaccgatg ctggtatcct
cgtcaacatc taccagtctc tttcctccta cgatattccc 720ggacctgcta
tgtacaacgc tacctccagc tcctccagct cctccagctc cagctccagc
780tccagctcca gctccagctc cggctcttcc agctccgccg ccgcctccag
cagctccagc 840agctccagca ctactgccgc cgccgccgcc gctaccagcg
ctgcttcttc cgtcacctct 900gctgctggct ccgtcgttac tcagactgct
accgctgttg agactgatac tgccactgcc 960taccagacct ccactgaggt
tgcgcaagtc accgtcaccg gtagcgctcc ccagcagacc 1020tacgttgcca
ctcccagcag ctccagctct gcctccagca gctccagtgc ttccgtatcc
1080accagcacca gcctcaccag ctacttcgag tccctgagcg ctgatcagtt
cctcagcgtt 1140ctcaagcaga ctttcacctg gttggtcagc gagaagaagc
acgcccgtga cctctccgcc 1200taa 120368400PRTAspergillus aculeatus
68Met Ser Val Ala Lys Phe Ala Gly Val Ile Leu Gly Ser Ala Ala Leu1
5 10 15Val Ala Gly His Gly Tyr Val Ser Gly Ala Val Val Asp Gly Thr
Tyr 20 25 30Tyr Gly Gly Tyr Ile Val Thr Ser Tyr Pro Tyr Ser Ser Asp
Pro Pro 35 40 45Glu Thr Ile Gly Trp Ser Thr Glu Ala Thr Asp Leu Gly
Phe Val Asp 50 55 60Gly Ser Glu Tyr Ala Asp Ala Asp Ile Ile Cys His
Lys Ser Ala Lys65 70 75 80Pro Gly Ala Ile Ser Ala Glu Val Lys Ala
Gly Gly Thr Val Glu Leu 85 90 95Gln Trp Thr Thr Trp Pro Asp Ser His
His Gly Pro Val Leu Thr Tyr 100 105 110Leu Ala Asn Cys Asn Gly Asp
Cys Ser Ser Val Thr Lys Thr Asp Leu 115 120 125Glu Phe Phe Lys Ile
Asp Glu Ser Gly Leu Ile Asn Asp Asp Asp Val 130 135 140Pro Gly Thr
Trp Ala Ser Asp Asn Leu Ile Ala Asn Asn Asn Ser Trp145 150 155
160Thr Val Thr Ile Pro Ser Asp Ile Ala Ala Gly Asn Tyr Val Leu Arg
165 170 175His Glu Ile Ile Ala Leu His Ser Ala Gly Asn Lys Asp Gly
Ala Gln 180 185 190Asn Tyr Pro Gln Cys Leu Asn Leu Lys Val Thr Gly
Gly Gly Asp Leu 195 200 205Ala Pro Ser Gly Thr Ala Gly Glu Ser Leu
Tyr Lys Asp Thr Asp Ala 210 215 220Gly Ile Leu Val Asn Ile Tyr Gln
Ser Leu Ser Ser Tyr Asp Ile Pro225 230 235 240Gly Pro Ala Met Tyr
Asn Ala Thr Ser Ser Ser Ser Ser Ser Ser Ser 245 250 255Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser Gly Ser Ser Ser Ser 260 265 270Ala
Ala Ala Ser Ser Ser Ser Ser Ser Ser Ser Thr Thr Ala Ala Ala 275 280
285Ala Ala Ala Thr Ser Ala Ala Ser Ser Val Thr Ser Ala Ala Gly Ser
290 295 300Val Val Thr Gln Thr Ala Thr Ala Val Glu Thr Asp Thr Ala
Thr Ala305 310 315 320Tyr Gln Thr Ser Thr Glu Val Ala Gln Val Thr
Val Thr Gly Ser Ala 325 330 335Pro Gln Gln Thr Tyr Val Ala Thr Pro
Ser Ser Ser Ser Ser Ala Ser 340 345 350Ser Ser Ser Ser Ala Ser Val
Ser Thr Ser Thr Ser Leu Thr Ser Tyr 355 360 365Phe Glu Ser Leu Ser
Ala Asp Gln Phe Leu Ser Val Leu Lys Gln Thr 370 375 380Phe Thr Trp
Leu Val Ser Glu Lys Lys His Ala Arg Asp Leu Ser Ala385 390 395
400691170DNAAspergillus aculeatus 69atgaagtcct ctactttcgg
tatgctcgct ctggcagcag cagccaagat ggtcgatgcc 60cacaccaccg tcttcgccgt
ctggatcaac ggcgaggacc agggtctggg caacagtgcc 120agtggctaca
tccggtctcc ccccagcaac agccccgtca aggacgtgac ctcgaccgac
180atcacctgca acgtcaacgg cgaccaggcg gcggctaaga ccctctccgt
caagggcggc 240gacgtcgtca ccttcgagtg gcaccacgac agccgggacg
cctccgacga catcatcgcc 300tcctcccaca agggccccgt catggtctac
atggccccga ccaccgccgg cagcagcggc 360aagaactggg tcaagatcgc
cgaggacgga tactccgacg gcacctgggc cgtcgacacc 420ctgatcgcca
acagcggcaa gcacaacatc accgtccccg acgtccccgc cggcgactac
480ctcttccgcc cggagatcat cgccctccac gaggccgaga acgagggcgg
cgcccagttc 540tacatggagt gtgtccagtt caaggtcacc tccgacggtg
ccaacactct gcccgacggt 600gtcagcctgc ccggcgccta ctccgccact
gaccccggta tcctcttcaa catgtacggc 660tccttcgaca gctatcccat
ccccggtccc tccgtctggg atggcactag ctctggctct 720tcctcttctt
cctcttcttc ctcttccagc tcttccgccg ccgctgccgt tgttgccacc
780tcctcttcct cttcctctgc ttccatcgag gccgtgacca ccaagggtgc
cgtcgccgcc 840gtctccaccg ccgccgccgt ggctcctacc accaccaccg
ctgcccccac caccttcgcc 900acggccgtcg cctccaccaa gaaggccact
gcctgccgca acaagaccaa gtcctcctcc 960gctgccacca ccgccgccgc
cgtcgccgag accacctctt ccaccgctgc cgccaccgct 1020gctgcttcct
ctgcctcttc cgcctccggc accgccggca agtacgagcg ctgcggtggc
1080cagggctgga ccggtgccac cacctgcgtt gatggctgga cctgcaagca
gtggaaccct 1140tactactacc agtgcgttga gtctgcctag
117070389PRTAspergillus aculeatus 70Met Lys Ser Ser Thr Phe Gly Met
Leu Ala Leu Ala Ala Ala Ala Lys1 5 10 15Met Val Asp Ala His Thr Thr
Val Phe Ala Val Trp Ile Asn Gly Glu 20 25 30Asp Gln Gly Leu Gly Asn
Ser Ala Ser Gly Tyr Ile Arg Ser Pro Pro 35 40 45Ser Asn Ser Pro Val
Lys Asp Val Thr Ser Thr Asp Ile Thr Cys Asn 50 55 60Val Asn Gly Asp
Gln Ala Ala Ala Lys Thr Leu Ser Val Lys Gly Gly65 70 75 80Asp Val
Val Thr Phe Glu Trp His His Asp Ser Arg Asp Ala Ser Asp 85 90 95Asp
Ile Ile Ala Ser Ser His Lys Gly Pro Val Met Val Tyr Met Ala 100 105
110Pro Thr Thr Ala Gly Ser Ser Gly Lys Asn Trp Val Lys Ile Ala Glu
115 120 125Asp Gly Tyr Ser Asp Gly Thr Trp Ala Val Asp Thr Leu Ile
Ala Asn 130 135 140Ser Gly Lys His Asn Ile Thr Val Pro Asp Val Pro
Ala Gly Asp Tyr145 150 155 160Leu Phe Arg Pro Glu Ile Ile Ala Leu
His Glu Ala Glu Asn Glu Gly 165 170 175Gly Ala Gln Phe Tyr Met Glu
Cys Val Gln Phe Lys Val Thr Ser Asp 180 185 190Gly Ala Asn Thr Leu
Pro Asp Gly Val Ser Leu Pro Gly Ala Tyr Ser 195 200 205Ala Thr Asp
Pro Gly Ile Leu Phe Asn Met Tyr Gly Ser Phe Asp Ser 210 215 220Tyr
Pro Ile Pro Gly Pro Ser Val Trp Asp Gly Thr Ser Ser Gly Ser225 230
235 240Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ala Ala Ala
Ala 245 250 255Val Val Ala Thr Ser Ser Ser Ser Ser Ser Ala Ser Ile
Glu Ala Val 260 265 270Thr Thr Lys Gly Ala Val Ala Ala Val Ser Thr
Ala Ala Ala Val Ala 275 280 285Pro Thr Thr Thr Thr Ala Ala Pro Thr
Thr Phe Ala Thr Ala Val Ala 290 295 300Ser Thr Lys Lys Ala Thr Ala
Cys Arg Asn Lys Thr Lys Ser Ser Ser305 310 315 320Ala Ala Thr Thr
Ala Ala Ala Val Ala Glu Thr Thr Ser Ser Thr Ala 325 330 335Ala Ala
Thr Ala Ala Ala Ser Ser Ala Ser Ser Ala Ser Gly Thr Ala 340 345
350Gly Lys Tyr Glu Arg Cys Gly Gly Gln Gly Trp Thr Gly Ala Thr Thr
355 360 365Cys Val Asp Gly Trp Thr Cys Lys Gln Trp Asn Pro Tyr Tyr
Tyr Gln 370 375 380Cys Val Glu Ser Ala385711221DNAAspergillus
aculeatus 71atgcgtcagg ctcagtcttt gtccctcttg acagctcttc tgtctgccac
gcgtgtggct 60ggacacggtc acgtcactaa cgttgtcgtc aacggtgttt actacgaggg
cttcgatatc 120aacagcttcc cctacgagtc cgatccccct aaggtggcgg
cttggaccac tcctaacact 180ggcaacggtt tcatttcccc cagcgactac
ggtaccgatg acattatttg ccaccagaat 240gccaccaacg cccaggccca
cattgttgtt gcggctggtg acaagatcaa catccagtgg 300accgcgtggc
ccgattccca ccacggtcct gtccttgact acctcgctcg ctgcgacggt
360gagtgtgaga cggttgataa gaccactctt gagtttttca agatcgacgg
cgtcggtctc 420atcagtgaca ccgaagtgcc cggtacctgg ggagatgacc
agctgatcgc caacaacaac 480agctggttgg tcgagatccc cccgaccatt
gctcctggca actatgttct tcgccacgag 540cttatcgctc tccacagcgc
cggcactgaa gatggtgctc agaactaccc ccagtgtttc 600aacctccagg
tcactggctc cggtactgac gagcccgctg gtaccctcgg caccaagctc
660tacactgagg atgaggctgg tatcgttgtg aacatctaca cctctctgtc
ttcctatgcc 720gtccccggcc ccacccagta cagcggcgcc gtctctgtca
gccaatccac ttcggccatt 780acctccaccg gaactgctgt tgtcggtagc
ggcagcgctg ttgccacctc tgccgccgcg 840gctaccacca gcgctgctgc
ttcttctgcc gctgctgcta ccaccgctgc tgccgttacc 900agcgccaatg
ccaacactca gattgcccag cccagcagca gctcttctta ctcccagatc
960gccgtgcagg tgccctcctc ctggaccacc cttgtgaccg tcactcctcc
cgccgccgcc 1020gccaccaccc ctgctgccgt ccctgagcct cagaccccct
ctgccagctc tggagccacc 1080actaccagca gcagcagcgg cgccgcccag
tctctctacg gccagtgcgg tggtatcaac 1140tggaccggag ctacctcttg
cgttgagggc gctacttgct accagtacaa cccttactac 1200taccagtgca
tctctgccta a 122172406PRTAspergillus aculeatus 72Met Arg Gln Ala
Gln Ser Leu Ser Leu Leu Thr Ala Leu Leu Ser Ala1 5 10 15Thr Arg Val
Ala Gly His Gly His Val Thr Asn Val Val Val Asn Gly 20 25 30Val Tyr
Tyr Glu Gly Phe Asp Ile Asn Ser Phe Pro Tyr Glu Ser Asp 35 40 45Pro
Pro Lys Val
Ala Ala Trp Thr Thr Pro Asn Thr Gly Asn Gly Phe 50 55 60Ile Ser Pro
Ser Asp Tyr Gly Thr Asp Asp Ile Ile Cys His Gln Asn65 70 75 80Ala
Thr Asn Ala Gln Ala His Ile Val Val Ala Ala Gly Asp Lys Ile 85 90
95Asn Ile Gln Trp Thr Ala Trp Pro Asp Ser His His Gly Pro Val Leu
100 105 110Asp Tyr Leu Ala Arg Cys Asp Gly Glu Cys Glu Thr Val Asp
Lys Thr 115 120 125Thr Leu Glu Phe Phe Lys Ile Asp Gly Val Gly Leu
Ile Ser Asp Thr 130 135 140Glu Val Pro Gly Thr Trp Gly Asp Asp Gln
Leu Ile Ala Asn Asn Asn145 150 155 160Ser Trp Leu Val Glu Ile Pro
Pro Thr Ile Ala Pro Gly Asn Tyr Val 165 170 175Leu Arg His Glu Leu
Ile Ala Leu His Ser Ala Gly Thr Glu Asp Gly 180 185 190Ala Gln Asn
Tyr Pro Gln Cys Phe Asn Leu Gln Val Thr Gly Ser Gly 195 200 205Thr
Asp Glu Pro Ala Gly Thr Leu Gly Thr Lys Leu Tyr Thr Glu Asp 210 215
220Glu Ala Gly Ile Val Val Asn Ile Tyr Thr Ser Leu Ser Ser Tyr
Ala225 230 235 240Val Pro Gly Pro Thr Gln Tyr Ser Gly Ala Val Ser
Val Ser Gln Ser 245 250 255Thr Ser Ala Ile Thr Ser Thr Gly Thr Ala
Val Val Gly Ser Gly Ser 260 265 270Ala Val Ala Thr Ser Ala Ala Ala
Ala Thr Thr Ser Ala Ala Ala Ser 275 280 285Ser Ala Ala Ala Ala Thr
Thr Ala Ala Ala Val Thr Ser Ala Asn Ala 290 295 300Asn Thr Gln Ile
Ala Gln Pro Ser Ser Ser Ser Ser Tyr Ser Gln Ile305 310 315 320Ala
Val Gln Val Pro Ser Ser Trp Thr Thr Leu Val Thr Val Thr Pro 325 330
335Pro Ala Ala Ala Ala Thr Thr Pro Ala Ala Val Pro Glu Pro Gln Thr
340 345 350Pro Ser Ala Ser Ser Gly Ala Thr Thr Thr Ser Ser Ser Ser
Gly Ala 355 360 365Ala Gln Ser Leu Tyr Gly Gln Cys Gly Gly Ile Asn
Trp Thr Gly Ala 370 375 380Thr Ser Cys Val Glu Gly Ala Thr Cys Tyr
Gln Tyr Asn Pro Tyr Tyr385 390 395 400Tyr Gln Cys Ile Ser Ala
405731284DNAAspergillus aculeatus 73atgtctcttt ccaagattgc
cactcttctg ctgggctcgg tctcgctggt cgctggtcat 60gggtatgtct cgagcatcga
ggtggacggt accacctatg gagggtactt ggtcgacact 120tattactacg
aatccgaccc gcccgagtta atcgcctggt ccacaaatgc cacggatgat
180ggctatgtat cgccctccga ctacgagagc gtgaacatca tctgccacaa
ggggtctgcg 240cccggcgcgt tgtcggcccc tgtcgcgccc ggaggctggg
tgcagatgac ctggaacacc 300tggcccaccg accatcacgg ccctgtcatc
acgtatatgg ccaattgcca cggttcttgc 360gcagatgtgg acaagaccac
cctcgagttc ttcaagatcg atgctggcgg cttgatcgat 420gacacggacg
tgcctggaac ttgggcgacc gatgagctca ttgaagatag ctatagtcgc
480aacatcacta tccccagcga tattgccccc gggtactatg ttttgcgaca
cgagatcatt 540gctctgcaca gcgccgagaa cctggacgga gcccagaact
acccccagtg catcaatctg 600gaagtcaccg gcagcgagac agcaaccccg
agtggcacct tgggcactgc tctgtacaag 660gagaccgacc ccggcatcta
tgttgacatc tggaacacgt tgagcacgta tactattccc 720ggccccgcgc
tgtacactgc tggtagcact gcgaccgcag ccgctgctgc cgataccacc
780actacttctg ctggcaccac cgctgaggcc accaccgctg ccgccgccgt
gagtaccacc 840gcggacgctg ttccgaccga gtcttcagct ccttccgaga
ccagcgcgac taccgcgaac 900cctgctcggc ccactgccgg cagcgacatc
cgcttccagc ccggtcaggt caaggctggt 960gcttcagtca acaactcggc
tactgagact tcctctggtg agtctgccac gacgaccaca 1020acatcagtgg
ccactgcggc ttcgagcgcg gattcgtcga cgacttctgg ggttttgagt
1080ggcgcctgca gccaggaggg ctactggtac tgcaacgggg gcactgcgtt
ccagcgctgt 1140gtcaacgggg aatgggatgc gtcccagagt gtggctgcgg
gcacggtctg caccgccggt 1200atctcggaga ccatcaccat ttcagccgcc
gccacgcgcc gggatgccat gcgtcgtcat 1260ctggcgcgtc ccaagcgtca ctga
128474427PRTAspergillus aculeatus 74Met Ser Leu Ser Lys Ile Ala Thr
Leu Leu Leu Gly Ser Val Ser Leu1 5 10 15Val Ala Gly His Gly Tyr Val
Ser Ser Ile Glu Val Asp Gly Thr Thr 20 25 30Tyr Gly Gly Tyr Leu Val
Asp Thr Tyr Tyr Tyr Glu Ser Asp Pro Pro 35 40 45Glu Leu Ile Ala Trp
Ser Thr Asn Ala Thr Asp Asp Gly Tyr Val Ser 50 55 60Pro Ser Asp Tyr
Glu Ser Val Asn Ile Ile Cys His Lys Gly Ser Ala65 70 75 80Pro Gly
Ala Leu Ser Ala Pro Val Ala Pro Gly Gly Trp Val Gln Met 85 90 95Thr
Trp Asn Thr Trp Pro Thr Asp His His Gly Pro Val Ile Thr Tyr 100 105
110Met Ala Asn Cys His Gly Ser Cys Ala Asp Val Asp Lys Thr Thr Leu
115 120 125Glu Phe Phe Lys Ile Asp Ala Gly Gly Leu Ile Asp Asp Thr
Asp Val 130 135 140Pro Gly Thr Trp Ala Thr Asp Glu Leu Ile Glu Asp
Ser Tyr Ser Arg145 150 155 160Asn Ile Thr Ile Pro Ser Asp Ile Ala
Pro Gly Tyr Tyr Val Leu Arg 165 170 175His Glu Ile Ile Ala Leu His
Ser Ala Glu Asn Leu Asp Gly Ala Gln 180 185 190Asn Tyr Pro Gln Cys
Ile Asn Leu Glu Val Thr Gly Ser Glu Thr Ala 195 200 205Thr Pro Ser
Gly Thr Leu Gly Thr Ala Leu Tyr Lys Glu Thr Asp Pro 210 215 220Gly
Ile Tyr Val Asp Ile Trp Asn Thr Leu Ser Thr Tyr Thr Ile Pro225 230
235 240Gly Pro Ala Leu Tyr Thr Ala Gly Ser Thr Ala Thr Ala Ala Ala
Ala 245 250 255Ala Asp Thr Thr Thr Thr Ser Ala Gly Thr Thr Ala Glu
Ala Thr Thr 260 265 270Ala Ala Ala Ala Val Ser Thr Thr Ala Asp Ala
Val Pro Thr Glu Ser 275 280 285Ser Ala Pro Ser Glu Thr Ser Ala Thr
Thr Ala Asn Pro Ala Arg Pro 290 295 300Thr Ala Gly Ser Asp Ile Arg
Phe Gln Pro Gly Gln Val Lys Ala Gly305 310 315 320Ala Ser Val Asn
Asn Ser Ala Thr Glu Thr Ser Ser Gly Glu Ser Ala 325 330 335Thr Thr
Thr Thr Thr Ser Val Ala Thr Ala Ala Ser Ser Ala Asp Ser 340 345
350Ser Thr Thr Ser Gly Val Leu Ser Gly Ala Cys Ser Gln Glu Gly Tyr
355 360 365Trp Tyr Cys Asn Gly Gly Thr Ala Phe Gln Arg Cys Val Asn
Gly Glu 370 375 380Trp Asp Ala Ser Gln Ser Val Ala Ala Gly Thr Val
Cys Thr Ala Gly385 390 395 400Ile Ser Glu Thr Ile Thr Ile Ser Ala
Ala Ala Thr Arg Arg Asp Ala 405 410 415Met Arg Arg His Leu Ala Arg
Pro Lys Arg His 420 42575804DNAAspergillus aculeatus 75atgcttgtca
aactcatctc ttttctttca gctgctacca gcgtagctgc tcatggtcat 60gtgtcaaaca
ttgtgatcaa cggggtgtcc taccgcggat gggacatcaa ttcggaccct
120tacaattcca accctccggt ggtggttgca tggcaaacac ccaacacagc
taatggcttc 180atctcccctg atgcatacga cacagatgat gttatttgcc
atctgagcgc tacgaatgcc 240agaggccacg cagtcgtcgc tgctggcgac
aagatcagcc tccagtggac gacctggcct 300gacagtcacc atggccctgt
catcagctac ctagccaact gcggctccag ctgcgagaca 360gtcgataaga
ccaccctcga gttcttcaag atcgatggtg ttggcttggt ggatgagagc
420aatccccctg gtatctgggg agacgatgag ctcattgcca acaacaactc
ttggctggta 480gagattccag ctagtatcgc gccaggatac tatgtgctgc
gtcacgagtt gatcgctctg 540catggagcag ggagtgagaa tggagcccag
aattacatgc aatgtttcaa ccttcaggtt 600actgggactg gcacggtcca
gccttccggg gtcctgggca cggagctgta caaacccaca 660gacgctggaa
ttcttgtcaa tatctaccag tcgctctcca cctatgttgt tcctggcccg
720accctgatcc cccaggccgt ttccctcgtt cagtcgagct ccaccattac
cgcctcgggc 780acggcagtga caaccacggc ttga 80476267PRTAspergillus
aculeatus 76Met Leu Val Lys Leu Ile Ser Phe Leu Ser Ala Ala Thr Ser
Val Ala1 5 10 15Ala His Gly His Val Ser Asn Ile Val Ile Asn Gly Val
Ser Tyr Arg 20 25 30Gly Trp Asp Ile Asn Ser Asp Pro Tyr Asn Ser Asn
Pro Pro Val Val 35 40 45Val Ala Trp Gln Thr Pro Asn Thr Ala Asn Gly
Phe Ile Ser Pro Asp 50 55 60Ala Tyr Asp Thr Asp Asp Val Ile Cys His
Leu Ser Ala Thr Asn Ala65 70 75 80Arg Gly His Ala Val Val Ala Ala
Gly Asp Lys Ile Ser Leu Gln Trp 85 90 95Thr Thr Trp Pro Asp Ser His
His Gly Pro Val Ile Ser Tyr Leu Ala 100 105 110Asn Cys Gly Ser Ser
Cys Glu Thr Val Asp Lys Thr Thr Leu Glu Phe 115 120 125Phe Lys Ile
Asp Gly Val Gly Leu Val Asp Glu Ser Asn Pro Pro Gly 130 135 140Ile
Trp Gly Asp Asp Glu Leu Ile Ala Asn Asn Asn Ser Trp Leu Val145 150
155 160Glu Ile Pro Ala Ser Ile Ala Pro Gly Tyr Tyr Val Leu Arg His
Glu 165 170 175Leu Ile Ala Leu His Gly Ala Gly Ser Glu Asn Gly Ala
Gln Asn Tyr 180 185 190Met Gln Cys Phe Asn Leu Gln Val Thr Gly Thr
Gly Thr Val Gln Pro 195 200 205Ser Gly Val Leu Gly Thr Glu Leu Tyr
Lys Pro Thr Asp Ala Gly Ile 210 215 220Leu Val Asn Ile Tyr Gln Ser
Leu Ser Thr Tyr Val Val Pro Gly Pro225 230 235 240Thr Leu Ile Pro
Gln Ala Val Ser Leu Val Gln Ser Ser Ser Thr Ile 245 250 255Thr Ala
Ser Gly Thr Ala Val Thr Thr Thr Ala 260 26577822DNAAspergillus
aculeatus 77atgaagtatc ttgcgatctt cgcggcagca gcagctggac tggcccgccc
gacagcagcg 60cactacatct tcagcaagct gattctggac ggcgaagtct ctgaggactg
gcagtatatt 120cgtaaaacca cccgggagac atgctatttg ccgaccaagt
tcaccgacac cttcgacaac 180ttgactccga acgaccagga tttccggtgc
aatctcggct cgttcagcaa cgccgccaag 240accgaagtgg ccgaggtgga
agcgggctcc acgattggca tgcagctttt cgctggtagc 300cacatgcgtc
acccgggacc tgcgcaagtc ttcatgtcta aggccccgtc cggcaacgta
360cagagctacg agggtgacgg ctcctggttc aagatctggg agcgtacact
ctgcgacaaa 420agtggcgatc tgactggaga tgcgtggtgt acatacggcc
agaccgagat cgagtttcaa 480atccccgagg cgaccccgac gggcgaatac
ctggtccgag cggagcacat cggtcttcac 540cgcgcacaga gtaatcaagc
cgagttctac tacagctgcg cccaggtcaa ggtcacgggc 600aatggtaccg
gggtgccgag ccagacatat cagatccctg gcatgtacaa tgaccgctcg
660gagcttttca acgggctgaa cttgtggtcc tactcggtgg agaacgtcga
ggcagccatg 720aagaattcta tcgtgggtga tgaaatttgg aatggaagtt
ctgttccctc tgagtcccat 780gtcccgaagt ataagaagag tcatgcttgt
cgtgtttatt ga 82278273PRTAspergillus aculeatus 78Met Lys Tyr Leu
Ala Ile Phe Ala Ala Ala Ala Ala Gly Leu Ala Arg1 5 10 15Pro Thr Ala
Ala His Tyr Ile Phe Ser Lys Leu Ile Leu Asp Gly Glu 20 25 30Val Ser
Glu Asp Trp Gln Tyr Ile Arg Lys Thr Thr Arg Glu Thr Cys 35 40 45Tyr
Leu Pro Thr Lys Phe Thr Asp Thr Phe Asp Asn Leu Thr Pro Asn 50 55
60Asp Gln Asp Phe Arg Cys Asn Leu Gly Ser Phe Ser Asn Ala Ala Lys65
70 75 80Thr Glu Val Ala Glu Val Glu Ala Gly Ser Thr Ile Gly Met Gln
Leu 85 90 95Phe Ala Gly Ser His Met Arg His Pro Gly Pro Ala Gln Val
Phe Met 100 105 110Ser Lys Ala Pro Ser Gly Asn Val Gln Ser Tyr Glu
Gly Asp Gly Ser 115 120 125Trp Phe Lys Ile Trp Glu Arg Thr Leu Cys
Asp Lys Ser Gly Asp Leu 130 135 140Thr Gly Asp Ala Trp Cys Thr Tyr
Gly Gln Thr Glu Ile Glu Phe Gln145 150 155 160Ile Pro Glu Ala Thr
Pro Thr Gly Glu Tyr Leu Val Arg Ala Glu His 165 170 175Ile Gly Leu
His Arg Ala Gln Ser Asn Gln Ala Glu Phe Tyr Tyr Ser 180 185 190Cys
Ala Gln Val Lys Val Thr Gly Asn Gly Thr Gly Val Pro Ser Gln 195 200
205Thr Tyr Gln Ile Pro Gly Met Tyr Asn Asp Arg Ser Glu Leu Phe Asn
210 215 220Gly Leu Asn Leu Trp Ser Tyr Ser Val Glu Asn Val Glu Ala
Ala Met225 230 235 240Lys Asn Ser Ile Val Gly Asp Glu Ile Trp Asn
Gly Ser Ser Val Pro 245 250 255Ser Glu Ser His Val Pro Lys Tyr Lys
Lys Ser His Ala Cys Arg Val 260 265 270Tyr79872DNAThermomyces
lanuginosus 79atgaagggct ccagcgctgc gtcggtgctt cttaccttcc
tcgcgggcat ctcccgtacc 60tctgcgcacg ggtatgtctc caacctcgtt atcaacggcg
tctactatcg gggctggctc 120cccggcgaag acccctacaa ccctgacccc
ccgattggcg ttggctggga gacgcccaac 180ctgggcaacg gcttcgtgac
gccgtcggaa gcgtcgaccg acgccgtcat ctgccacaag 240gaagccacac
cagcccgcgg tcatgtctcc gtgaaggccg gtgacaagat ctacatccaa
300tggcagccga atccatggcc ggattcccac cacggtgcgt caaacttctg
cccgaaagct 360gttcacactc actaacaaca cttttaggcc ccgtcctgga
ctatctggcc ccttgcaacg 420ggccctgtga gtccgtcgac aagaccagcc
tgcgcttctt caagatcgac ggagtgggtc 480ttatcgacgg ctcttctcct
ccgggctact gggccgacga cgaactcatt gcgaacggca 540acgggtggct
ggttcagatc cccgaggaca tcaagccggg taactacgtc ctgcgacacg
600agatcatcgc cttgcacagc gccgggaacc cggacggcgc ccagctgtac
ccgcagtgct 660tcaaccttga gattacggga tccggcaccg tcgagccgga
gggcgtgcca gccaccgagt 720tctactcgcc cgatgacccg ggcatcctgg
tcaacatcta cgagcccctg tccacgtatg 780aggtgccggg tccctcgctc
atcccgcagg cggttcagat cgagcagtct tcgtctgcga 840ttacggcgac
gggcacgccg acgccggcat ga 87280272PRTThermomyces lanuginosus 80Met
Lys Gly Ser Ser Ala Ala Ser Val Leu Leu Thr Phe Leu Ala Gly1 5 10
15Ile Ser Arg Thr Ser Ala His Gly Tyr Val Ser Asn Leu Val Ile Asn
20 25 30Gly Val Tyr Tyr Arg Gly Trp Leu Pro Gly Glu Asp Pro Tyr Asn
Pro 35 40 45Asp Pro Pro Ile Gly Val Gly Trp Glu Thr Pro Asn Leu Gly
Asn Gly 50 55 60Phe Val Thr Pro Ser Glu Ala Ser Thr Asp Ala Val Ile
Cys His Lys65 70 75 80Glu Ala Thr Pro Ala Arg Gly His Val Ser Val
Lys Ala Gly Asp Lys 85 90 95Ile Tyr Ile Gln Trp Gln Pro Asn Pro Trp
Pro Asp Ser His His Gly 100 105 110Pro Val Leu Asp Tyr Leu Ala Pro
Cys Asn Gly Pro Cys Glu Ser Val 115 120 125Asp Lys Thr Ser Leu Arg
Phe Phe Lys Ile Asp Gly Val Gly Leu Ile 130 135 140Asp Gly Ser Ser
Pro Pro Gly Tyr Trp Ala Asp Asp Glu Leu Ile Ala145 150 155 160Asn
Gly Asn Gly Trp Leu Val Gln Ile Pro Glu Asp Ile Lys Pro Gly 165 170
175Asn Tyr Val Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Gly Asn
180 185 190Pro Asp Gly Ala Gln Leu Tyr Pro Gln Cys Phe Asn Leu Glu
Ile Thr 195 200 205Gly Ser Gly Thr Val Glu Pro Glu Gly Val Pro Ala
Thr Glu Phe Tyr 210 215 220Ser Pro Asp Asp Pro Gly Ile Leu Val Asn
Ile Tyr Glu Pro Leu Ser225 230 235 240Thr Tyr Glu Val Pro Gly Pro
Ser Leu Ile Pro Gln Ala Val Gln Ile 245 250 255Glu Gln Ser Ser Ser
Ala Ile Thr Ala Thr Gly Thr Pro Thr Pro Ala 260 265
270811039DNAThermomyces lanuginosus 81atggcattct ctacggttac
agtttttgtt acgttcctgg ccttcatctc catagcttct 60gctcatggct tcgtgacaaa
aatcaccgta ctcggagata ataataagga gtacgtctca 120gtctcgctag
gttgctaaca caggagagat cgctgaccat tgcagctacc ccggctttga
180cccgagcact cccaaggagg ttcctccggg tctcgatgtc gcttggtcta
ctagtgccag 240tgatcaggga tacatgagca gttcaaatgc ctcgtatcac
agtaaggact ttatctgcca 300cagaaacgcc aaacctgctc cagacgcagc
tcaagttcat gcgggcgaca aggtgcagct 360tcactggact caatggcctg
gacctgagga tcaccagggt cctatccttg attacctcgc 420gagctgcaac
ggaccctgct caaacgtgga gaaggcgagc cttaagtgga cgaagattga
480cgaggcaggg cgctttccca acggaacgtg ggcaacggac ctgctcagga
atgggggaaa 540cacgtggaat gtgacgattc catcggatct tgctcctgga
gaatatgtcc tccgcaacga 600gatcattgca cttcactcgg cgagaaatat
gggtggagct cagcactaca tgcaatgtgt 660caatctgaac gtcactggca
ccggccatag agagctacag ggcgtctccg ccgcagaatt 720ttacaatcct
acggatcctg gaattttgat taacgtctgg caaactcaaa gcctttcctc
780ctaccatatt cccggaccta cactgttagc cgccgatacc ggcaacgacg
gtggccattc 840tgcatcatct accttggcga ctgtgacaag cagacgtctt
tccactccga gcgacgccat 900gcccgggaat ggttcatacg gtgcaatttc
gccgcccctc aaacctgcta aaggattcca 960tcctgtttgt aacgcccgat
tcagacatgg cagcactttc actttgacta ccctggtcgc 1020accaccagcc
aggacctaa 103982327PRTThermomyces lanuginosus 82Met Ala Phe Ser Thr
Val Thr Val Phe Val Thr Phe Leu Ala Phe Ile1 5 10
15Ser Ile Ala Ser Ala His Gly Phe Val Thr Lys Ile Thr Val Leu Gly
20 25 30Asp Asn Asn Lys Asp Tyr Pro Gly Phe Asp Pro Ser Thr Pro Lys
Glu 35 40 45Val Pro Pro Gly Leu Asp Val Ala Trp Ser Thr Ser Ala Ser
Asp Gln 50 55 60Gly Tyr Met Ser Ser Ser Asn Ala Ser Tyr His Ser Lys
Asp Phe Ile65 70 75 80Cys His Arg Asn Ala Lys Pro Ala Pro Asp Ala
Ala Gln Val His Ala 85 90 95Gly Asp Lys Val Gln Leu His Trp Thr Gln
Trp Pro Gly Pro Glu Asp 100 105 110His Gln Gly Pro Ile Leu Asp Tyr
Leu Ala Ser Cys Asn Gly Pro Cys 115 120 125Ser Asn Val Glu Lys Ala
Ser Leu Lys Trp Thr Lys Ile Asp Glu Ala 130 135 140Gly Arg Phe Pro
Asn Gly Thr Trp Ala Thr Asp Leu Leu Arg Asn Gly145 150 155 160Gly
Asn Thr Trp Asn Val Thr Ile Pro Ser Asp Leu Ala Pro Gly Glu 165 170
175Tyr Val Leu Arg Asn Glu Ile Ile Ala Leu His Ser Ala Arg Asn Met
180 185 190Gly Gly Ala Gln His Tyr Met Gln Cys Val Asn Leu Asn Val
Thr Gly 195 200 205Thr Gly His Arg Glu Leu Gln Gly Val Ser Ala Ala
Glu Phe Tyr Asn 210 215 220Pro Thr Asp Pro Gly Ile Leu Ile Asn Val
Trp Gln Thr Gln Ser Leu225 230 235 240Ser Ser Tyr His Ile Pro Gly
Pro Thr Leu Leu Ala Ala Asp Thr Gly 245 250 255Asn Asp Gly Gly His
Ser Ala Ser Ser Thr Leu Ala Thr Val Thr Ser 260 265 270Arg Arg Leu
Ser Thr Pro Ser Asp Ala Met Pro Gly Asn Gly Ser Tyr 275 280 285Gly
Ala Ile Ser Pro Pro Leu Lys Pro Ala Lys Gly Phe His Pro Val 290 295
300Cys Asn Ala Arg Phe Arg His Gly Ser Thr Phe Thr Leu Thr Thr
Leu305 310 315 320Val Ala Pro Pro Ala Arg Thr
32583881DNAThermomyces lanuginosus 83atgaaaggct ccaccactgc
gtctttgctt cttccgctcc tggcgagcgt tactcgcacc 60tctgcgcacg ggtttgtctc
caacctcgtc atcaatggcg tcttctatcg gggctggctc 120ccgaccgagg
acccctacaa ggctgacccc ccgattggcg tcggctggga gacgcctaac
180ctgggcaacg gcttcgtgct gcccgaagaa gcgtcgaccg atgccatcgt
ctgccacaaa 240gaggccgagc cggcccgcgg ctatgccagc gtcgctgccg
gtgacaagat ctacattcag 300tggcagccga acccatggcc ggagtctcat
cacggtacgt caaactgccc attgttgcaa 360ttcagaatca tctactaaca
actcttcaag gccccgtcat tgactacctg gccccttgca 420acggtgactg
ctcgactgtc aacaagacca gtttggagtt cttcaagatc gacggcgtgg
480gcctcatcga cggctcctcc ccgccgggta agtgggctga cgacgagctc
attgccaacg 540gcaacggctg gctggtccag atccccgagg acatcaagcc
gggcaactac gtcctgcgcc 600atgagatcat cgccttgcac gaggcgttca
accagaacgg cgctcagatc tacccgcagt 660gcttcaacct ccagattacc
ggctccggca ctgtcgagcc cgagggcacg ccggctaccg 720agctgtattc
gcccaccgat ccgggcattc tggttgacat ctacaacccc ttgagcacgt
780acgtcgtgcc cggcccgacg ctcatcccgc aggcggttga gattgagcag
tcttcgtcgg 840ctgtcacggc gactggtacg ccgacgccgg cggcggcgta a
88184274PRTThermomyces lanuginosus 84Met Lys Gly Ser Thr Thr Ala
Ser Leu Leu Leu Pro Leu Leu Ala Ser1 5 10 15Val Thr Arg Thr Ser Ala
His Gly Phe Val Ser Asn Leu Val Ile Asn 20 25 30Gly Val Phe Tyr Arg
Gly Trp Leu Pro Thr Glu Asp Pro Tyr Lys Ala 35 40 45Asp Pro Pro Ile
Gly Val Gly Trp Glu Thr Pro Asn Leu Gly Asn Gly 50 55 60Phe Val Leu
Pro Glu Glu Ala Ser Thr Asp Ala Ile Val Cys His Lys65 70 75 80Glu
Ala Glu Pro Ala Arg Gly Tyr Ala Ser Val Ala Ala Gly Asp Lys 85 90
95Ile Tyr Ile Gln Trp Gln Pro Asn Pro Trp Pro Glu Ser His His Gly
100 105 110Pro Val Ile Asp Tyr Leu Ala Pro Cys Asn Gly Asp Cys Ser
Thr Val 115 120 125Asn Lys Thr Ser Leu Glu Phe Phe Lys Ile Asp Gly
Val Gly Leu Ile 130 135 140Asp Gly Ser Ser Pro Pro Gly Lys Trp Ala
Asp Asp Glu Leu Ile Ala145 150 155 160Asn Gly Asn Gly Trp Leu Val
Gln Ile Pro Glu Asp Ile Lys Pro Gly 165 170 175Asn Tyr Val Leu Arg
His Glu Ile Ile Ala Leu His Glu Ala Phe Asn 180 185 190Gln Asn Gly
Ala Gln Ile Tyr Pro Gln Cys Phe Asn Leu Gln Ile Thr 195 200 205Gly
Ser Gly Thr Val Glu Pro Glu Gly Thr Pro Ala Thr Glu Leu Tyr 210 215
220Ser Pro Thr Asp Pro Gly Ile Leu Val Asp Ile Tyr Asn Pro Leu
Ser225 230 235 240Thr Tyr Val Val Pro Gly Pro Thr Leu Ile Pro Gln
Ala Val Glu Ile 245 250 255Glu Gln Ser Ser Ser Ala Val Thr Ala Thr
Gly Thr Pro Thr Pro Ala 260 265 270Ala Ala85969DNAAurantiporus
alborubescens 85atgcgaacca tcgccacgtt tgttacgctt gtagcctcag
ttctccctgc ggtcctcgca 60cacggaggtg tcctctccta ttcsaacggg gggaattggt
actggggatg gaagccttac 120aattcacctg acgggcagac caccatccaa
cgcccgtggg caacatacaa tccgatcact 180gatgcgacgg atcctaccat
tgcttgcaac aacgacggga catctggagc tctgcagttg 240actgcgacag
tcgcggcggg atctgccatc acggcgtatt ggaaccaggt gtggccgcat
300gataaagggc cgatgacgac atacctcgca caatgccccg gcagtacctg
cacaggagtc 360aacgcgaaga ctctgaaatg gttcaagatc gatcacgccg
ggttgctttc tggtactgtc 420tacagtggct cgtgggcatc aggcaagatg
attgcacaga actcgacctg gacaactacc 480attccagcga cggtgccttc
agggaactat ctgatacgtt tcgagactat tgccctgcac 540tctttgccag
cgcaatttta ccctgagtgc gcacaaattc aaatcacggg cggaggttcc
600cgtgctccaa ccgctgcaga gcttgttagc ttccctggcg cgtacagcaa
caatgatcct 660ggtgtcaaca ttgacatcta ctccaatgcc gcgcagagtg
caaccacata cgtaatacca 720ggacctccat tgtacggcgg tgcttccgga
tctggtccat cttccgcgcc tccatcaagt 780accccaggta gttcgtccac
ttcccacggt cccacgtccg tcagcacgtc cagcagtgct 840gcaccatcga
cgacaggaac cgtgacgcag tacggtcagt gcggtggcat tggttgggct
900ggagctaccg gctgtatctc accattcaag tgcacggtca tcaacgatta
ttactaccag 960tgcctctga 96986322PRTAurantiporus alborubescens 86Met
Arg Thr Ile Ala Thr Phe Val Thr Leu Val Ala Ser Val Leu Pro1 5 10
15Ala Val Leu Ala His Gly Gly Val Leu Ser Tyr Ser Asn Gly Gly Asn
20 25 30Trp Tyr Trp Gly Trp Lys Pro Tyr Asn Ser Pro Asp Gly Gln Thr
Thr 35 40 45Ile Gln Arg Pro Trp Ala Thr Tyr Asn Pro Ile Thr Asp Ala
Thr Asp 50 55 60Pro Thr Ile Ala Cys Asn Asn Asp Gly Thr Ser Gly Ala
Leu Gln Leu65 70 75 80Thr Ala Thr Val Ala Ala Gly Ser Ala Ile Thr
Ala Tyr Trp Asn Gln 85 90 95Val Trp Pro His Asp Lys Gly Pro Met Thr
Thr Tyr Leu Ala Gln Cys 100 105 110Pro Gly Ser Thr Cys Thr Gly Val
Asn Ala Lys Thr Leu Lys Trp Phe 115 120 125Lys Ile Asp His Ala Gly
Leu Leu Ser Gly Thr Val Tyr Ser Gly Ser 130 135 140Trp Ala Ser Gly
Lys Met Ile Ala Gln Asn Ser Thr Trp Thr Thr Thr145 150 155 160Ile
Pro Ala Thr Val Pro Ser Gly Asn Tyr Leu Ile Arg Phe Glu Thr 165 170
175Ile Ala Leu His Ser Leu Pro Ala Gln Phe Tyr Pro Glu Cys Ala Gln
180 185 190Ile Gln Ile Thr Gly Gly Gly Ser Arg Ala Pro Thr Ala Ala
Glu Leu 195 200 205Val Ser Phe Pro Gly Ala Tyr Ser Asn Asn Asp Pro
Gly Val Asn Ile 210 215 220Asp Ile Tyr Ser Asn Ala Ala Gln Ser Ala
Thr Thr Tyr Val Ile Pro225 230 235 240Gly Pro Pro Leu Tyr Gly Gly
Ala Ser Gly Ser Gly Pro Ser Ser Ala 245 250 255Pro Pro Ser Ser Thr
Pro Gly Ser Ser Ser Thr Ser His Gly Pro Thr 260 265 270Ser Val Ser
Thr Ser Ser Ser Ala Ala Pro Ser Thr Thr Gly Thr Val 275 280 285Thr
Gln Tyr Gly Gln Cys Gly Gly Ile Gly Trp Ala Gly Ala Thr Gly 290 295
300Cys Ile Ser Pro Phe Lys Cys Thr Val Ile Asn Asp Tyr Tyr Tyr
Gln305 310 315 320Cys Leu87705DNAAurantiporus alborubescens
87atgaaggcta tcttggctat tttctcggcc cttgctccac ttgccgctgc gcattatacc
60ttccctgatt ttattgtcaa cggaacaaca actgccgatt gggtctacat ccgagagacc
120gcgaaccact actcgaatgg tcctgtaacc aacgtgaacg atccagaatt
ccgatgctac 180gagctggacc tgcaaaacac ggcagcgagt accctcaccg
ccacggtctc tgcaggctcc 240agcgtcggct ttaaagctaa cagcgccctt
taccatcctg gttatctcga tgtgtatatg 300tccaaagcga ccccagctgc
taattcaccc agtgctggaa cggaccaaag ctggttcaag 360gtctatgaat
ccgctccggt cttcgcgaat ggggccctaa gcttcccttc ggagaacatc
420caatctttca cgttcacaat cccgaagtcc cttcccagtg gccaatatct
catccgtgtg 480gaacacatcg ctctccactc cgccagtagc tacggaggtg
cacaattcta catcagctgc 540gctcaagtca atgtcgtcaa cggcgggaac
ggaaacccag gaccgttagt caagattccc 600ggcgtttaca ctgggaacga
gcctggcatc ctcatcaaca tctacagctt cccaccgggt 660ttcagtggct
accaatcccc gggacctgct gtgtggcgtg gttga 70588234PRTAurantiporus
alborubescens 88Met Lys Ala Ile Leu Ala Ile Phe Ser Ala Leu Ala Pro
Leu Ala Ala1 5 10 15Ala His Tyr Thr Phe Pro Asp Phe Ile Val Asn Gly
Thr Thr Thr Ala 20 25 30Asp Trp Val Tyr Ile Arg Glu Thr Ala Asn His
Tyr Ser Asn Gly Pro 35 40 45Val Thr Asn Val Asn Asp Pro Glu Phe Arg
Cys Tyr Glu Leu Asp Leu 50 55 60Gln Asn Thr Ala Ala Ser Thr Leu Thr
Ala Thr Val Ser Ala Gly Ser65 70 75 80Ser Val Gly Phe Lys Ala Asn
Ser Ala Leu Tyr His Pro Gly Tyr Leu 85 90 95Asp Val Tyr Met Ser Lys
Ala Thr Pro Ala Ala Asn Ser Pro Ser Ala 100 105 110Gly Thr Asp Gln
Ser Trp Phe Lys Val Tyr Glu Ser Ala Pro Val Phe 115 120 125Ala Asn
Gly Ala Leu Ser Phe Pro Ser Glu Asn Ile Gln Ser Phe Thr 130 135
140Phe Thr Ile Pro Lys Ser Leu Pro Ser Gly Gln Tyr Leu Ile Arg
Val145 150 155 160Glu His Ile Ala Leu His Ser Ala Ser Ser Tyr Gly
Gly Ala Gln Phe 165 170 175Tyr Ile Ser Cys Ala Gln Val Asn Val Val
Asn Gly Gly Asn Gly Asn 180 185 190Pro Gly Pro Leu Val Lys Ile Pro
Gly Val Tyr Thr Gly Asn Glu Pro 195 200 205Gly Ile Leu Ile Asn Ile
Tyr Ser Phe Pro Pro Gly Phe Ser Gly Tyr 210 215 220Gln Ser Pro Gly
Pro Ala Val Trp Arg Gly225 23089702DNATrichophaea saccata
89atgacgcccc tgaaactccg cccccttctc ctcctggtgc tttccacgac cctcagcctc
60gtgcacgcgc actatcgctt ctacgaactg atcgccaacg gggccaccca cgcttccttc
120gaatacatcc gccaatgggt gcccatctac agcaactctc ccgtaaccga
cgtcaccagc 180gtcaacctcc gctgcaacgt caacgccact cccgccgccg
aggtgatcac cgttgctgcc 240ggtagcaccg tcggcttcgt agcagacaca
acagtaacgc accccggtgc gttcaccgcg 300tacatggcga aagcgcccga
agacatcacg gaatgggatg gcaacgggga ctggttcaag 360atctgggaga
agggtccaac gagtataacc agtagcggga taacctggga cgtcacggat
420acccaatgga ccttcaccat cccttccgcg acaccaaacg gtcaatacct
actccgcttc 480gagcacatag cgctccacgc cgccagcacc gtggggggtg
ctcaattcta catgtcgtgc 540gcgcagatac aagtaacgaa cggcggcaac
gggagtcccg ggcccaccat caagttcccg 600ggcggataca gcgccacaga
ccccggtatc ctgatcaata tctattatcc catccccact 660agttacacta
ttcctggtcc accggtttgg accggtaagt aa 70290233PRTTrichophaea saccata
90Met Thr Pro Leu Lys Leu Arg Pro Leu Leu Leu Leu Val Leu Ser Thr1
5 10 15Thr Leu Ser Leu Val His Ala His Tyr Arg Phe Tyr Glu Leu Ile
Ala 20 25 30Asn Gly Ala Thr His Ala Ser Phe Glu Tyr Ile Arg Gln Trp
Val Pro 35 40 45Ile Tyr Ser Asn Ser Pro Val Thr Asp Val Thr Ser Val
Asn Leu Arg 50 55 60Cys Asn Val Asn Ala Thr Pro Ala Ala Glu Val Ile
Thr Val Ala Ala65 70 75 80Gly Ser Thr Val Gly Phe Val Ala Asp Thr
Thr Val Thr His Pro Gly 85 90 95Ala Phe Thr Ala Tyr Met Ala Lys Ala
Pro Glu Asp Ile Thr Glu Trp 100 105 110Asp Gly Asn Gly Asp Trp Phe
Lys Ile Trp Glu Lys Gly Pro Thr Ser 115 120 125Ile Thr Ser Ser Gly
Ile Thr Trp Asp Val Thr Asp Thr Gln Trp Thr 130 135 140Phe Thr Ile
Pro Ser Ala Thr Pro Asn Gly Gln Tyr Leu Leu Arg Phe145 150 155
160Glu His Ile Ala Leu His Ala Ala Ser Thr Val Gly Gly Ala Gln Phe
165 170 175Tyr Met Ser Cys Ala Gln Ile Gln Val Thr Asn Gly Gly Asn
Gly Ser 180 185 190Pro Gly Pro Thr Ile Lys Phe Pro Gly Gly Tyr Ser
Ala Thr Asp Pro 195 200 205Gly Ile Leu Ile Asn Ile Tyr Tyr Pro Ile
Pro Thr Ser Tyr Thr Ile 210 215 220Pro Gly Pro Pro Val Trp Thr Gly
Lys225 23091714DNATrichophaea saccata 91atgaaatgcc ttctctccct
ccttctcgcc gcgacagcgg tctccgctca cacgatcttc 60caagaaatcg gcataaacgg
ggtgatgcaa gctcgctacg actacatgcg gctgccgtcc 120tacgacggtc
ccattacgga cgtaacgagc acctacatgg cgtgcaacgg tggtcccaat
180ccattggtcc aaatctcgaa cgacgtcgct ttcgtaaaag ccggcgacag
catcacgctg 240caatgggcgc aaacgttgac gacagatttc aacacggggc
tgatcatcga tccatcgcac 300ttgggtcctg tgatggtcta catggccaaa
gtaccctccg ccaccggtcc gatccccaac 360agcggctggt tcaaaatcta
cgaagacggc tacgacccga caacaaagac atgggcggta 420accaagctca
tcaacaacaa gggaaaagtg accgtcacca tcccatcgtg tctaccggca
480ggggactact tgctgcgcgg tgaaatcatt gccttgcacg cggctagtac
ctatccaggc 540gcacagtttt acatggagtg tgcgcagttg cggcttacca
gtggcggcac taagatgcct 600accacgtata acattccggg gatctattcg
cccactgatc cgggtgttac gttcaatctt 660tacaatggat tcacgagtta
taccattcct ggcccaaggc cgtttacatg ctag 71492237PRTTrichophaea
saccata 92Met Lys Cys Leu Leu Ser Leu Leu Leu Ala Ala Thr Ala Val
Ser Ala1 5 10 15His Thr Ile Phe Gln Glu Ile Gly Ile Asn Gly Val Met
Gln Ala Arg 20 25 30Tyr Asp Tyr Met Arg Leu Pro Ser Tyr Asp Gly Pro
Ile Thr Asp Val 35 40 45Thr Ser Thr Tyr Met Ala Cys Asn Gly Gly Pro
Asn Pro Leu Val Gln 50 55 60Ile Ser Asn Asp Val Ala Phe Val Lys Ala
Gly Asp Ser Ile Thr Leu65 70 75 80Gln Trp Ala Gln Thr Leu Thr Thr
Asp Phe Asn Thr Gly Leu Ile Ile 85 90 95Asp Pro Ser His Leu Gly Pro
Val Met Val Tyr Met Ala Lys Val Pro 100 105 110Ser Ala Thr Gly Pro
Ile Pro Asn Ser Gly Trp Phe Lys Ile Tyr Glu 115 120 125Asp Gly Tyr
Asp Pro Thr Thr Lys Thr Trp Ala Val Thr Lys Leu Ile 130 135 140Asn
Asn Lys Gly Lys Val Thr Val Thr Ile Pro Ser Cys Leu Pro Ala145 150
155 160Gly Asp Tyr Leu Leu Arg Gly Glu Ile Ile Ala Leu His Ala Ala
Ser 165 170 175Thr Tyr Pro Gly Ala Gln Phe Tyr Met Glu Cys Ala Gln
Leu Arg Leu 180 185 190Thr Ser Gly Gly Thr Lys Met Pro Thr Thr Tyr
Asn Ile Pro Gly Ile 195 200 205Tyr Ser Pro Thr Asp Pro Gly Val Thr
Phe Asn Leu Tyr Asn Gly Phe 210 215 220Thr Ser Tyr Thr Ile Pro Gly
Pro Arg Pro Phe Thr Cys225 230 235931455DNAPenicillium thomii
93atgtctctgt ctaagatttc tggattgatc ctcggatctg ctgccttggt ggctggccac
60ggttacgtga gcggaatcgt cgttgacgat acctactatg gtggatacct tgtcacccag
120tacccttatg agagtgacgc cccagagctc attgcctggt cggagcaaga
gaccgatctg 180ggttacatcg atggctctga gtatgccaac tccaacatca
tctgtcacaa ggaggccaaa 240cctggtgctt tggaagcacc cgttaaggct
ggtggctccg tcgagctcca gtggaccact 300tggcctacca gccaccacgg
tcctgtcatt acctacatgg ccaactgtaa cggcgactgt 360gacgacgttg
acaagactac tttgcagttc ttcaagattg accagggtgg tttgatcagc
420gataccaccg agcccggtac ctgggcaact gacaacctca tcgccaacaa
caatagccgt 480actgtcaccg tccccagcga cattgccgat ggaaactacg
tcctccgtca cgagatcatt 540gccctccact ccgccgggga gaccaacggt
gcccagaact acccccaatg tatcaacttg 600aaggtcactg gcggcggtag
cgctactcct tctggtaccc tgggtaccgc cctgtacaag 660aacaccgacc
ccggtatcct gatcaacatc tacacttccc tcagcaccta cgatatcccc
720ggcccaaccc tgtacactgc cggcgccgcc gctgctaccg ctgcctccac
ggctgcctct 780tccaccgccg ctgccgttac tactgccgac gccgtcacta
ccgccgctgc cgtcaccagc 840agctctgcat ccgtggaagt tgtgcccaca
actactccca gctcatcaat cgtcagtgcc 900ttcccaacct ggagcccctc
ttctacccca
cccttctcca actcttccaa cggatggcgt 960ccgtcattca gccgcggacc
tggtggcccc cgcttcacat ctgctcctgc tcctcagttc 1020tccgctccta
gcggcgctca gcagaagcag tctgccactg ctacccccat cgtggctacc
1080cctgtcgtga tcaccatgac cgagaccagc acctcctggg tcaccgaaat
ggttactctt 1140actgacaagt ctgttgtgca gaccaccagc gctgtcccag
tcgtcgtcgc cgccaccact 1200acccttaccg agggaagcga gcctgctcag
acagcctccc ccagcgttgt ctccggctcc 1260tctagctccg gctctagctc
ctcatctacc accaccacct caaagacctc aactggatcc 1320gactacgtct
ccagcgactg gatgtcttac ctcagctcct tgagcgctgc tgaggtcctc
1380cagatgctgc gccagacctt ccgttggatg gtcagcaacg acaaggtgca
cgctcgtgat 1440attaccatca actag 145594484PRTPenicillium thomii
94Met Ser Leu Ser Lys Ile Ser Gly Leu Ile Leu Gly Ser Ala Ala Leu1
5 10 15Val Ala Gly His Gly Tyr Val Ser Gly Ile Val Val Asp Asp Thr
Tyr 20 25 30Tyr Gly Gly Tyr Leu Val Thr Gln Tyr Pro Tyr Glu Ser Asp
Ala Pro 35 40 45Glu Leu Ile Ala Trp Ser Glu Gln Glu Thr Asp Leu Gly
Tyr Ile Asp 50 55 60Gly Ser Glu Tyr Ala Asn Ser Asn Ile Ile Cys His
Lys Glu Ala Lys65 70 75 80Pro Gly Ala Leu Glu Ala Pro Val Lys Ala
Gly Gly Ser Val Glu Leu 85 90 95Gln Trp Thr Thr Trp Pro Thr Ser His
His Gly Pro Val Ile Thr Tyr 100 105 110Met Ala Asn Cys Asn Gly Asp
Cys Asp Asp Val Asp Lys Thr Thr Leu 115 120 125Gln Phe Phe Lys Ile
Asp Gln Gly Gly Leu Ile Ser Asp Thr Thr Glu 130 135 140Pro Gly Thr
Trp Ala Thr Asp Asn Leu Ile Ala Asn Asn Asn Ser Arg145 150 155
160Thr Val Thr Val Pro Ser Asp Ile Ala Asp Gly Asn Tyr Val Leu Arg
165 170 175His Glu Ile Ile Ala Leu His Ser Ala Gly Glu Thr Asn Gly
Ala Gln 180 185 190Asn Tyr Pro Gln Cys Ile Asn Leu Lys Val Thr Gly
Gly Gly Ser Ala 195 200 205Thr Pro Ser Gly Thr Leu Gly Thr Ala Leu
Tyr Lys Asn Thr Asp Pro 210 215 220Gly Ile Leu Ile Asn Ile Tyr Thr
Ser Leu Ser Thr Tyr Asp Ile Pro225 230 235 240Gly Pro Thr Leu Tyr
Thr Ala Gly Ala Ala Ala Ala Thr Ala Ala Ser 245 250 255Thr Ala Ala
Ser Ser Thr Ala Ala Ala Val Thr Thr Ala Asp Ala Val 260 265 270Thr
Thr Ala Ala Ala Val Thr Ser Ser Ser Ala Ser Val Glu Val Val 275 280
285Pro Thr Thr Thr Pro Ser Ser Ser Ile Val Ser Ala Phe Pro Thr Trp
290 295 300Ser Pro Ser Ser Thr Pro Pro Phe Ser Asn Ser Ser Asn Gly
Trp Arg305 310 315 320Pro Ser Phe Ser Arg Gly Pro Gly Gly Pro Arg
Phe Thr Ser Ala Pro 325 330 335Ala Pro Gln Phe Ser Ala Pro Ser Gly
Ala Gln Gln Lys Gln Ser Ala 340 345 350Thr Ala Thr Pro Ile Val Ala
Thr Pro Val Val Ile Thr Met Thr Glu 355 360 365Thr Ser Thr Ser Trp
Val Thr Glu Met Val Thr Leu Thr Asp Lys Ser 370 375 380Val Val Gln
Thr Thr Ser Ala Val Pro Val Val Val Ala Ala Thr Thr385 390 395
400Thr Leu Thr Glu Gly Ser Glu Pro Ala Gln Thr Ala Ser Pro Ser Val
405 410 415Val Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Ser Thr
Thr Thr 420 425 430Thr Ser Lys Thr Ser Thr Gly Ser Asp Tyr Val Ser
Ser Asp Trp Met 435 440 445Ser Tyr Leu Ser Ser Leu Ser Ala Ala Glu
Val Leu Gln Met Leu Arg 450 455 460Gln Thr Phe Arg Trp Met Val Ser
Asn Asp Lys Val His Ala Arg Asp465 470 475 480Ile Thr Ile
Asn951021DNATalaromyces stipitatus 95atgccttcca ctaaagttgc
tgctctatct gccgtcctgg ctttggcctc cacggttgct 60ggccatggct ttgtgcaaaa
tattgtcatt gacggtaaat cgtaagtgac ttgcttttgt 120actatagagc
tagataaata cttatactaa ataattcagc tacactggct acctcgtgaa
180ccagtatcct taccagtcca acccaccagc tgttattggg tggtcaacca
ctgcaaccga 240cttgggattt gtcgatggat ctggatacac caacccggat
atcatctgcc acaaaaacgc 300caaacccggt cagctttctg ctccggttgc
cgcaggaggc aaggttgagc tcgaatggac 360aacatggccc gagagccatc
acggccctgt catcagctat ctcgccaatt gcaatggcga 420ttgtactacc
gtggataaga cgaagctcga atttgtcaaa atcgatcagc ggggtctgat
480cgacgacagc aatcctcccg gtacatgggc cgccgaccag ctcatcgccg
ccaacaacag 540ctggactgta actattcccg agagcatcgc gcctggaaac
tacgtccttc gccacgaaat 600catcgctctt cactccgcca acaacgcaac
cggagctcaa aactaccctc aatgcatcaa 660cttgcaaatc actggcagcg
ggacggccaa cccatctggt acccctggcg agaaactcta 720taccccaact
gacccaggta tcttggtcaa catctaccag tcattgtcgt cttatgttat
780tcccggtccg actttgtgga gtggtgctgc agcgcacgtt gttgccactg
cagccggttc 840tgctactggg gttgcttctg ccaccgctac tccgaccact
cttgtgactg ccgtttcatc 900gcctaccggt gctccttcag tggtgactcc
tgaggctcct tcagtaacct cgttcgcccc 960agtggtgact gttactgatg
tcgttactgt gactaccgtc atcactacta ctatctctta 1020g
102196320PRTTalaromyces stipitatus 96Met Pro Ser Thr Lys Val Ala
Ala Leu Ser Ala Val Leu Ala Leu Ala1 5 10 15Ser Thr Val Ala Gly His
Gly Phe Val Gln Asn Ile Val Ile Asp Gly 20 25 30Lys Ser Tyr Thr Gly
Tyr Leu Val Asn Gln Tyr Pro Tyr Gln Ser Asn 35 40 45Pro Pro Ala Val
Ile Gly Trp Ser Thr Thr Ala Thr Asp Leu Gly Phe 50 55 60Val Asp Gly
Ser Gly Tyr Thr Asn Pro Asp Ile Ile Cys His Lys Asn65 70 75 80Ala
Lys Pro Gly Gln Leu Ser Ala Pro Val Ala Ala Gly Gly Lys Val 85 90
95Glu Leu Glu Trp Thr Thr Trp Pro Glu Ser His His Gly Pro Val Ile
100 105 110Ser Tyr Leu Ala Asn Cys Asn Gly Asp Cys Thr Thr Val Asp
Lys Thr 115 120 125Lys Leu Glu Phe Val Lys Ile Asp Gln Arg Gly Leu
Ile Asp Asp Ser 130 135 140Asn Pro Pro Gly Thr Trp Ala Ala Asp Gln
Leu Ile Ala Ala Asn Asn145 150 155 160Ser Trp Thr Val Thr Ile Pro
Glu Ser Ile Ala Pro Gly Asn Tyr Val 165 170 175Leu Arg His Glu Ile
Ile Ala Leu His Ser Ala Asn Asn Ala Thr Gly 180 185 190Ala Gln Asn
Tyr Pro Gln Cys Ile Asn Leu Gln Ile Thr Gly Ser Gly 195 200 205Thr
Ala Asn Pro Ser Gly Thr Pro Gly Glu Lys Leu Tyr Thr Pro Thr 210 215
220Asp Pro Gly Ile Leu Val Asn Ile Tyr Gln Ser Leu Ser Ser Tyr
Val225 230 235 240Ile Pro Gly Pro Thr Leu Trp Ser Gly Ala Ala Ala
His Val Val Ala 245 250 255Thr Ala Ala Gly Ser Ala Thr Gly Val Ala
Ser Ala Thr Ala Thr Pro 260 265 270Thr Thr Leu Val Thr Ala Val Ser
Ser Pro Thr Gly Ala Pro Ser Val 275 280 285Val Thr Pro Glu Ala Pro
Ser Val Thr Ser Phe Ala Pro Val Val Thr 290 295 300Val Thr Asp Val
Val Thr Val Thr Thr Val Ile Thr Thr Thr Ile Ser305 310 315
32097821DNAHumicola insolens 97atgaagctca gcgttgtcct cacaggcctg
gcggcagccc tcgccgaggc tcattgtcag 60tccatacgac agcgaaaccc ctggatgatc
acgagactaa ccagtcctac cagacacctt 120ccccagcgtc ggcaacaccg
ccgactggca ggtcgtgcgc cagacgacca acttccagag 180caacggcccc
gtgacggacg tcaactcgga ccagatccgg tgctacgagc gcttccccgg
240ccagggggcg cccggcatct acaacgtcac cgccggccag accatctcgt
acaacgccaa 300ggcctctatc tcccacccgg gccccatggc cttctacatc
gccaaggtcc ctgccggcta 360caccgccgcc aactgggatg gcaggggcgc
cgtgtggtcc aagatctacc aggacatgcc 420gcgcattgcg gggagtctga
cctggcctac caatggtacg aaatcctctt ctatccttca 480tacttgctat
tcctccaact gcctggcagc tcacactaac ttccacacac ccaggcgccc
540gttccgtctc ggtaaccatc ccccgctgcc tgcaagacgg ccactacctg
ttgcgcgccg 600agcacatcgg cctgcacagc gcgagcggcg tgggcggcgc
gcagttctac atctcgtgtg 660cccagctcta cgtcagcggc ggcaccggca
cttggaaccc gcgcaacaag gtcgcgttcc 720ccggcgccta cagcccgacg
cacccgggca tcatgatcaa catctactgg ccggtgccga 780cgagctacac
gccgccgggg ccgccggttg agacgtgctg a 82198227PRTHumicola insolens
98Met Lys Leu Ser Val Val Leu Thr Gly Leu Ala Ala Ala Leu Ala Glu1
5 10 15Ala His Tyr Thr Phe Pro Ser Val Gly Asn Thr Ala Asp Trp Gln
Val 20 25 30Val Arg Gln Thr Thr Asn Phe Gln Ser Asn Gly Pro Val Thr
Asp Val 35 40 45Asn Ser Asp Gln Ile Arg Cys Tyr Glu Arg Phe Pro Gly
Gln Gly Ala 50 55 60Pro Gly Ile Tyr Asn Val Thr Ala Gly Gln Thr Ile
Ser Tyr Asn Ala65 70 75 80Lys Ala Ser Ile Ser His Pro Gly Pro Met
Ala Phe Tyr Ile Ala Lys 85 90 95Val Pro Ala Gly Tyr Thr Ala Ala Asn
Trp Asp Gly Arg Gly Ala Val 100 105 110Trp Ser Lys Ile Tyr Gln Asp
Met Pro Arg Ile Ala Gly Ser Leu Thr 115 120 125Trp Pro Thr Asn Gly
Ala Arg Ser Val Ser Val Thr Ile Pro Arg Cys 130 135 140Leu Gln Asp
Gly His Tyr Leu Leu Arg Ala Glu His Ile Gly Leu His145 150 155
160Ser Ala Ser Gly Val Gly Gly Ala Gln Phe Tyr Ile Ser Cys Ala Gln
165 170 175Leu Tyr Val Ser Gly Gly Thr Gly Thr Trp Asn Pro Arg Asn
Lys Val 180 185 190Ala Phe Pro Gly Ala Tyr Ser Pro Thr His Pro Gly
Ile Met Ile Asn 195 200 205Ile Tyr Trp Pro Val Pro Thr Ser Tyr Thr
Pro Pro Gly Pro Pro Val 210 215 220Glu Thr Cys225991120DNAHumicola
insolens 99atgcgcccct tcctcgccgc cctcgccgcg gccaccacgg tccacgccca
cggctgggtc 60gacaacgcca ccatcgacgg cgtcttctac cagctctacc acccgtacat
ggacccgtac 120atgggcgagt tcgccccgcc tcgcatctcg cgcaagctgg
tgtggaacgg ctacgtgaac 180gacgtgacgt ccatcgacct gcaatgcggc
ggacacacgg ccgaagggca aatcggcacg 240gaacccgcgc cgctgcacgc
ccccgccacg gccgggtcga cggtcaacct ccgctggacg 300ctgtggccgg
actcgcacat ggggcccatc atgacgtaca tggcgcggtg tccggacgag
360ggttgtgata agtggttgcc gggggaggag taagtgtttc ctggcgggaa
tggctgtgta 420tttgagaagg agatattatg agtgaaactg ggagaggcga
gaagaagaga tgctgacgcg 480ggttttgctc tcctcagacc agtctggttc
aaaatccacg aagccggccg gtacaccacc 540gacaagtctt accccgacga
catctgggaa gttgtaagtg ccctgcctac ctatccatcc 600ctaattccct
ccctcccctc tccacctcct ccttccgcgc ccccctcccc ccccttattt
660gctaaccaac cccctccctt acagacccgc ctcatgtacc ccgccaacga
aggctacaac 720tacaccatcc ccgcctgcct cgcatccggc cactacctgg
tccggcacga gatcatcgcc 780ttacactcgg cctgggccaa aggcgaagcg
cagttctatc cctcgtgcca ccagctgacc 840gtcacctcca tcggcggtaa
cgtgcgcgaa gcgccggccg agtaccgcgt cagtttcccc 900ggcgcgtaca
aggacgatga tccgggtatt ttcatcaacg tttggaaccg taagttcttt
960ttttgttccc cttcctccca acctacctag gtgtcgtaat gtggtccgta
agggtttgtt 1020tgttgttgag ggatatagct gacaatggat gtgtgataac
acagctggcc cctacaccat 1080tcccggaccg ccggtctgga cgtgccccga
gtctgagtaa 1120100257PRTHumicola insolens 100Met Arg Pro Phe Leu
Ala Ala Leu Ala Ala Ala Thr Thr Val His Ala1 5 10 15His Gly Trp Val
Asp Asn Ala Thr Ile Asp Gly Val Phe Tyr Gln Leu 20 25 30Tyr His Pro
Tyr Met Asp Pro Tyr Met Gly Glu Phe Ala Pro Pro Arg 35 40 45Ile Ser
Arg Lys Leu Val Trp Asn Gly Tyr Val Asn Asp Val Thr Ser 50 55 60Ile
Asp Leu Gln Cys Gly Gly His Thr Ala Glu Gly Gln Ile Gly Thr65 70 75
80Glu Pro Ala Pro Leu His Ala Pro Ala Thr Ala Gly Ser Thr Val Asn
85 90 95Leu Arg Trp Thr Leu Trp Pro Asp Ser His Met Gly Pro Ile Met
Thr 100 105 110Tyr Met Ala Arg Cys Pro Asp Glu Gly Cys Asp Lys Trp
Leu Pro Val 115 120 125Trp Phe Lys Ile His Glu Ala Gly Arg Tyr Thr
Thr Asp Lys Ser Tyr 130 135 140Pro Asp Asp Ile Trp Glu Val Thr Arg
Leu Met Tyr Pro Ala Asn Glu145 150 155 160Gly Tyr Asn Tyr Thr Ile
Pro Ala Cys Leu Ala Ser Gly His Tyr Leu 165 170 175Val Arg His Glu
Ile Ile Ala Leu His Ser Ala Trp Ala Lys Gly Glu 180 185 190Ala Gln
Phe Tyr Pro Ser Cys His Gln Leu Thr Val Thr Ser Ile Gly 195 200
205Gly Asn Val Arg Glu Ala Pro Ala Glu Tyr Arg Val Ser Phe Pro Gly
210 215 220Ala Tyr Lys Asp Asp Asp Pro Gly Ile Phe Ile Asn Val Trp
Asn Pro225 230 235 240Gly Pro Tyr Thr Ile Pro Gly Pro Pro Val Trp
Thr Cys Pro Glu Ser 245 250 255Glu101878DNAHumicola insolens
101atgagactct ccctgacaac cctcctggcc tctgccctgt ccgtccaggg
tcacgccatc 60ttccaggtgc gttcctttca ccacccacat catcatgatg aacctcaaag
ttgctaaccc 120ccgctgggca gagagttacc gtcaacggcc aggaccaagg
ctcgttgact ggtctccggg 180ccccgaataa caacaacccc gtgcagaacg
tcaacagcca ggacatcatc tgtggcgctc 240ccgggtcgcg gtcacagtcc
gtcatcaacg tcaatgccgg cgaccgcatc ggtgcctggt 300accagcatgt
catcggcggc gcccagttcc ccggcgaccc ggacaacccg atcgccaggt
360cccacaaggg ccccatctcc gtctatctgg ccaaggtgga caacgctgcc
acggcgaacc 420accagggtct gcaatggtaa acatacctcg ggtcaagtca
gaacctctgt gatcgccgag 480acgactaacc cctctttccc ataaacaggt
tcaagatctg gcacgacggc ttcaacccct 540ccacccggca atgggccgtc
gacaccatga tcaacaacaa cggctgggtc tatttcaacc 600tcccgcagtg
catcgctccc ggccactatc tcatgcgcgt cgagctgctc gctctccact
660cggccaccta ccaaggccag gcgcagttct acatctcgtg cgcccagatc
aacgtccagt 720cgggcggcaa ctttactccc tggcagacgg ttagcttccc
cggcgcctac caggccaacc 780accccggcat tcaggtcaac atttacggcg
ccatgggcca gccggataac ggcggcaggc 840cctaccagat tccgggcccg
gagccgattc agtgctga 878102246PRTHumicola insolens 102Met Arg Leu
Ser Leu Thr Thr Leu Leu Ala Ser Ala Leu Ser Val Gln1 5 10 15Gly His
Ala Ile Phe Gln Arg Val Thr Val Asn Gly Gln Asp Gln Gly 20 25 30Ser
Leu Thr Gly Leu Arg Ala Pro Asn Asn Asn Asn Pro Val Gln Asn 35 40
45Val Asn Ser Gln Asp Ile Ile Cys Gly Ala Pro Gly Ser Arg Ser Gln
50 55 60Ser Val Ile Asn Val Asn Ala Gly Asp Arg Ile Gly Ala Trp Tyr
Gln65 70 75 80His Val Ile Gly Gly Ala Gln Phe Pro Gly Asp Pro Asp
Asn Pro Ile 85 90 95Ala Arg Ser His Lys Gly Pro Ile Ser Val Tyr Leu
Ala Lys Val Asp 100 105 110Asn Ala Ala Thr Ala Asn His Gln Gly Leu
Gln Trp Phe Lys Ile Trp 115 120 125His Asp Gly Phe Asn Pro Ser Thr
Arg Gln Trp Ala Val Asp Thr Met 130 135 140Ile Asn Asn Asn Gly Trp
Val Tyr Phe Asn Leu Pro Gln Cys Ile Ala145 150 155 160Pro Gly His
Tyr Leu Met Arg Val Glu Leu Leu Ala Leu His Ser Ala 165 170 175Thr
Tyr Gln Gly Gln Ala Gln Phe Tyr Ile Ser Cys Ala Gln Ile Asn 180 185
190Val Gln Ser Gly Gly Asn Phe Thr Pro Trp Gln Thr Val Ser Phe Pro
195 200 205Gly Ala Tyr Gln Ala Asn His Pro Gly Ile Gln Val Asn Ile
Tyr Gly 210 215 220Ala Met Gly Gln Pro Asp Asn Gly Gly Arg Pro Tyr
Gln Ile Pro Gly225 230 235 240Pro Glu Pro Ile Gln Cys
2451031067DNAHumicola insolens 103atgggaccga cctgggcagt gattctgggg
ctgattgctc cttctgtgct cagtcacagt 60tgcgtctccc aacagacctc tcgactttta
tcaagctggt actgactcat aacccaactc 120acctagatat ccatgggatc
ctcctggtca atggcacaga gacaccagag tggaaatacg 180tcctgtatgt
ttcctcatat cctagcccca ttgtacgagt tgttgacgtg atacagcgat
240gttgcgccgg cggttccaat ttcaaaccca gactctctcc cccctggata
ccaaggctat 300aaggttgatc ccatcatcgg atccgggaac cccaacatca
cttgtggccg gctagcattt 360gactcggcac ccaagacgca aatcgccgat
gtgctagccg gctccgaggt aggattccga 420gtctcggctg atggcttggg
aaatcgggat ctggagaagg gctacatccc gacgttctgg 480cacccaggtc
cggcccaggc atacttgtca cgtgccccga acgacgacct gtacagctac
540aaaggcgacg gggactggtt caagattgcc tacgctggcc cggtggacga
cctgacgtgg 600tccctttggc cgggagtttc agatgtatgt tcatcctcca
tagtcctgtt tttgccctct 660ccaggaccaa attattaata tcgagtcgca
gttcaacttc accattccgt tgtcgacacc 720ccctggcaag tatttgctcc
gaatcgagaa cttcatgcca acggcctcga caggatatct 780tcagttctac
gtcaattgtg catttgtcaa catcattgga ccaggaggtg ggaccccgac
840cgagttcatt cgaattcccg gggattacac cgacgaggat ccaggtgagt
ttgtgttatg 900agacatgttc aactcgcacc gacgaatgct tgtttcctga
cagagatttg taaaaactag 960gctttctcgt
tcccccggag caaagctcct tggatggcag agtcccaagg gaccagttga
1020aactgatgag ctacacgcca ccaggtcctg cggtgtggac ggggtga
1067104265PRTHumicola insolens 104Met Gly Pro Thr Trp Ala Val Ile
Leu Gly Leu Ile Ala Pro Ser Val1 5 10 15Leu Asn Ile His Gly Ile Leu
Leu Val Asn Gly Thr Glu Thr Pro Glu 20 25 30Trp Lys Tyr Val Leu Asp
Val Ala Pro Ala Val Pro Ile Ser Asn Pro 35 40 45Asp Ser Leu Pro Pro
Gly Tyr Gln Gly Tyr Lys Val Asp Pro Ile Ile 50 55 60Gly Ser Gly Asn
Pro Asn Ile Thr Cys Gly Arg Leu Ala Phe Asp Ser65 70 75 80Ala Pro
Lys Thr Gln Ile Ala Asp Val Leu Ala Gly Ser Glu Val Gly 85 90 95Phe
Arg Val Ser Ala Asp Gly Leu Gly Asn Arg Asp Leu Glu Lys Gly 100 105
110Tyr Ile Pro Thr Phe Trp His Pro Gly Pro Ala Gln Ala Tyr Leu Ser
115 120 125Arg Ala Pro Asn Asp Asp Leu Tyr Ser Tyr Lys Gly Asp Gly
Asp Trp 130 135 140Phe Lys Ile Ala Tyr Ala Gly Pro Val Asp Asp Leu
Thr Trp Ser Leu145 150 155 160Trp Pro Gly Val Ser Asp Phe Asn Phe
Thr Ile Pro Leu Ser Thr Pro 165 170 175Pro Gly Lys Tyr Leu Leu Arg
Ile Glu Asn Phe Met Pro Thr Ala Ser 180 185 190Thr Gly Tyr Leu Gln
Phe Tyr Val Asn Cys Ala Phe Val Asn Ile Ile 195 200 205Gly Pro Gly
Gly Gly Thr Pro Thr Glu Phe Ile Arg Ile Pro Gly Asp 210 215 220Tyr
Thr Asp Glu Asp Pro Gly Phe Leu Val Pro Pro Glu Gln Ser Ser225 230
235 240Leu Asp Gly Arg Val Pro Arg Asp Gln Leu Lys Leu Met Ser Tyr
Thr 245 250 255Pro Pro Gly Pro Ala Val Trp Thr Gly 260
2651051035DNAHumicola insolens 105atgaaggccc tcaccctcct cgccgccgcg
accgcggcct cggcgcacac catcttcgtg 60cagctcgagg ccgacggcac gcgctacccc
gtctcgcacg gcgtgcgcac cccgcagtac 120gacggcccca tcaccgacgt
ctcgtccaac gacctggcct gcaacggcgg gcccaacccg 180accatgaaga
cggacaagat catcaccgtg acggcgggca gcaccgtcaa ggccatctgg
240cggcacacgc tgcagtcggg ccccaacgac gtcatggacc ccagccacaa
gggcccgacg 300ctggcgtacc tgaagaaggt ggacaacgcg ctgacggatt
cgggcgtggg cggcggctgg 360ttcaagatcc aggaggacgg gcacagcaat
gggaattggg gcacgctcaa ggtaatcaac 420aaccagggca ttcactatat
cgatatcccc gactgcatcg acagcgggca gtatttgttg 480cgggccgaga
tgatcgctct gcacgctgcc gggtcgccgg gcggtgcgca gctttatgtg
540agtttcttcc ttcttttctt cttctctccc tttgtgataa gaataaagat
ccacaccaca 600gtcaaaccaa agcatcctaa cctcggcatc tactcacaga
tggaatgcgc ccaaatcgaa 660atcgtcggcg gcaagggcac cgtcaagccc
cagacctact ccatcccggg catctacaag 720tccaacgacc cgggcatcct
catcaacatc tactccatgt cgccctcgag ccagtacatc 780atccccggcc
cgcccctctt cacctgcaac ggcggcggcg gcagcaacaa cggcggcggc
840aacaacggcg gcagcaaccc ccccgtccag cagccccccg ccaccaccct
caccaccgcc 900atcgcccagc ccacgcccat ctgctccgtc cagcagtggg
gtcagtgcgg cggccagggc 960tatagcggct gcaccacctg cgcgtcgccg
tataggtgta acgagatcaa cgcgtggtat 1020tcgcagtgct tgtaa
1035106310PRTHumicola insolens 106Met Lys Ala Leu Thr Leu Leu Ala
Ala Ala Thr Ala Ala Ser Ala His1 5 10 15Thr Ile Phe Val Gln Leu Glu
Ala Asp Gly Thr Arg Tyr Pro Val Ser 20 25 30His Gly Val Arg Thr Pro
Gln Tyr Asp Gly Pro Ile Thr Asp Val Ser 35 40 45Ser Asn Asp Leu Ala
Cys Asn Gly Gly Pro Asn Pro Thr Met Lys Thr 50 55 60Asp Lys Ile Ile
Thr Val Thr Ala Gly Ser Thr Val Lys Ala Ile Trp65 70 75 80Arg His
Thr Leu Gln Ser Gly Pro Asn Asp Val Met Asp Pro Ser His 85 90 95Lys
Gly Pro Thr Leu Ala Tyr Leu Lys Lys Val Asp Asn Ala Leu Thr 100 105
110Asp Ser Gly Val Gly Gly Gly Trp Phe Lys Ile Gln Glu Asp Gly His
115 120 125Ser Asn Gly Asn Trp Gly Thr Leu Lys Val Ile Asn Asn Gln
Gly Ile 130 135 140His Tyr Ile Asp Ile Pro Asp Cys Ile Asp Ser Gly
Gln Tyr Leu Leu145 150 155 160Arg Ala Glu Met Ile Ala Leu His Ala
Ala Gly Ser Pro Gly Gly Ala 165 170 175Gln Leu Tyr Met Glu Cys Ala
Gln Ile Glu Ile Val Gly Gly Lys Gly 180 185 190Thr Val Lys Pro Gln
Thr Tyr Ser Ile Pro Gly Ile Tyr Lys Ser Asn 195 200 205Asp Pro Gly
Ile Leu Ile Asn Ile Tyr Ser Met Ser Pro Ser Ser Gln 210 215 220Tyr
Ile Ile Pro Gly Pro Pro Leu Phe Thr Cys Asn Gly Gly Gly Gly225 230
235 240Ser Asn Asn Gly Gly Gly Asn Asn Gly Gly Ser Asn Pro Pro Val
Gln 245 250 255Gln Pro Pro Ala Thr Thr Leu Thr Thr Ala Ile Ala Gln
Pro Thr Pro 260 265 270Ile Cys Ser Val Gln Gln Trp Gly Gln Cys Gly
Gly Gln Gly Tyr Ser 275 280 285Gly Cys Thr Thr Cys Ala Ser Pro Tyr
Arg Cys Asn Glu Ile Asn Ala 290 295 300Trp Tyr Ser Gln Cys Leu305
3101071065DNAHumicola insolens 107atggctccca agacctcgac gttccttgcc
tccctcacgg gcgccgccct cgtggctgcc 60cacggccatg tcagccacat cattgtcaat
ggcgtccagt accggaacta cgaccccacc 120accgacttct acagcggcaa
ccctccgacc gtgatcggct ggtcggccct caaccaggac 180aacggcttca
tcgagcccaa caacttcggc acccccgaca tcatctgcca taagtcggcc
240aagcccggcg gcggccacgt cacggtgagg gccggtgaca agatcagcat
cgtctggacc 300cccgagtggc ccgagtcgca cgtcggcccc gtcatcgact
accttgccgc gtgcaacggc 360gactgcgaga cggtcgacaa gacctccctc
cgcttcttca agatcgacgg cgccggctac 420gacgccgcgg ccggccgctg
ggccgccgac gctctgcgcg ccaacggcaa ctcgtggctt 480gtgcagatcc
ccgccgacct caaggccggc aactacgtgc ttcggcacga gatcatcgcc
540ctgcacggcg ccgccaaccc caacggcgcc caggcctacc cgcagtgcat
caacatccgc 600gtcaccggcg gcggcaacaa ccagccctcg ggcgtccccg
gcacccagct ctacaaggcc 660tcggacccgg gcatcctctt caacccctgg
gtcgccaacc ctcagtaccc cgtcccgggc 720ccggccctca tccccggcgc
cgtgagctcc atccctcaga gccgctcgac cgccaccgcc 780acgggcaccg
ccacccgccc cggcgccgac acggacccga cgggcgtccc tcccgtcgtc
840accaccactt ctgccccggc tcaggtgacc accaccacca gcagccgcac
cacctccctc 900cctcagatca ccaccacctt cgcgaccagc accaccccgc
cgcccccggc cgctacccag 960agcaagtggg gccagtgcgg cggcaacggc
tggaccggcc cgaccgtctg cgcgccgggc 1020tcgagctgca acaagctcaa
cgactggtac tcgcagtgca tctaa 1065108354PRTHumicola insolens 108Met
Ala Pro Lys Thr Ser Thr Phe Leu Ala Ser Leu Thr Gly Ala Ala1 5 10
15Leu Val Ala Ala His Gly His Val Ser His Ile Ile Val Asn Gly Val
20 25 30Gln Tyr Arg Asn Tyr Asp Pro Thr Thr Asp Phe Tyr Ser Gly Asn
Pro 35 40 45Pro Thr Val Ile Gly Trp Ser Ala Leu Asn Gln Asp Asn Gly
Phe Ile 50 55 60Glu Pro Asn Asn Phe Gly Thr Pro Asp Ile Ile Cys His
Lys Ser Ala65 70 75 80Lys Pro Gly Gly Gly His Val Thr Val Arg Ala
Gly Asp Lys Ile Ser 85 90 95Ile Val Trp Thr Pro Glu Trp Pro Glu Ser
His Val Gly Pro Val Ile 100 105 110Asp Tyr Leu Ala Ala Cys Asn Gly
Asp Cys Glu Thr Val Asp Lys Thr 115 120 125Ser Leu Arg Phe Phe Lys
Ile Asp Gly Ala Gly Tyr Asp Ala Ala Ala 130 135 140Gly Arg Trp Ala
Ala Asp Ala Leu Arg Ala Asn Gly Asn Ser Trp Leu145 150 155 160Val
Gln Ile Pro Ala Asp Leu Lys Ala Gly Asn Tyr Val Leu Arg His 165 170
175Glu Ile Ile Ala Leu His Gly Ala Ala Asn Pro Asn Gly Ala Gln Ala
180 185 190Tyr Pro Gln Cys Ile Asn Ile Arg Val Thr Gly Gly Gly Asn
Asn Gln 195 200 205Pro Ser Gly Val Pro Gly Thr Gln Leu Tyr Lys Ala
Ser Asp Pro Gly 210 215 220Ile Leu Phe Asn Pro Trp Val Ala Asn Pro
Gln Tyr Pro Val Pro Gly225 230 235 240Pro Ala Leu Ile Pro Gly Ala
Val Ser Ser Ile Pro Gln Ser Arg Ser 245 250 255Thr Ala Thr Ala Thr
Gly Thr Ala Thr Arg Pro Gly Ala Asp Thr Asp 260 265 270Pro Thr Gly
Val Pro Pro Val Val Thr Thr Thr Ser Ala Pro Ala Gln 275 280 285Val
Thr Thr Thr Thr Ser Ser Arg Thr Thr Ser Leu Pro Gln Ile Thr 290 295
300Thr Thr Phe Ala Thr Ser Thr Thr Pro Pro Pro Pro Ala Ala Thr
Gln305 310 315 320Ser Lys Trp Gly Gln Cys Gly Gly Asn Gly Trp Thr
Gly Pro Thr Val 325 330 335Cys Ala Pro Gly Ser Ser Cys Asn Lys Leu
Asn Asp Trp Tyr Ser Gln 340 345 350Cys Ile109804DNAHumicola
insolens 109atgtatcttt tacctatcgc cgcggccgcc ctagcgttca ccaccaccgc
atacgcccac 60gcccaagtct acggcttgcg tgtcaacgac caacaccaag gcgatgggcg
caacaaatac 120atccgctcgc ccagcagcaa ttcccccatc cggtgggacc
acgtaaccca cccattcctc 180atctgcaaca tccgcgacga caaccaaccc
ccgggtcccg cgcctgactt tgtccgcgcc 240ttcgccggcg accgcgtggc
gttccaatgg taccacgccc gccccaacga cccgacggat 300tacgtcctcg
acagctccca cctcggcgtc ctcgttacct ggatcgcgcc gtacacggac
360gggcccggga ccggccccat ttggaccaag atccaccagg acgggtggaa
cggcacgcac 420tgggccacga gccggctcat cagcaacggc gggttcgtcg
agttccggct gcccggctcg 480ctaaagcccg ggaagtacct ggtgcggcag
gagattatcg ctctgcacca ggccgacatg 540cccggtccga accgcgggcc
tgagttctac cccagctgcg cgcaattgga ggtttttggg 600tctggtgagg
cggcgccgcc gcaggggtat gatatcaaca aggggtatgc ggagagcggg
660gataagttgt ggttcaacat ttacatcaac aagaatgatg agttcaaaat
gcctggaccg 720gaggtttggg atggtgggtg tcggtttgga gagcgatggg
caaccgagga accaggcaag 780cccaaggtga accaacacgg ataa
804110267PRTHumicola insolens 110Met Tyr Leu Leu Pro Ile Ala Ala
Ala Ala Leu Ala Phe Thr Thr Thr1 5 10 15Ala Tyr Ala His Ala Gln Val
Tyr Gly Leu Arg Val Asn Asp Gln His 20 25 30Gln Gly Asp Gly Arg Asn
Lys Tyr Ile Arg Ser Pro Ser Ser Asn Ser 35 40 45Pro Ile Arg Trp Asp
His Val Thr His Pro Phe Leu Ile Cys Asn Ile 50 55 60Arg Asp Asp Asn
Gln Pro Pro Gly Pro Ala Pro Asp Phe Val Arg Ala65 70 75 80Phe Ala
Gly Asp Arg Val Ala Phe Gln Trp Tyr His Ala Arg Pro Asn 85 90 95Asp
Pro Thr Asp Tyr Val Leu Asp Ser Ser His Leu Gly Val Leu Val 100 105
110Thr Trp Ile Ala Pro Tyr Thr Asp Gly Pro Gly Thr Gly Pro Ile Trp
115 120 125Thr Lys Ile His Gln Asp Gly Trp Asn Gly Thr His Trp Ala
Thr Ser 130 135 140Arg Leu Ile Ser Asn Gly Gly Phe Val Glu Phe Arg
Leu Pro Gly Ser145 150 155 160Leu Lys Pro Gly Lys Tyr Leu Val Arg
Gln Glu Ile Ile Ala Leu His 165 170 175Gln Ala Asp Met Pro Gly Pro
Asn Arg Gly Pro Glu Phe Tyr Pro Ser 180 185 190Cys Ala Gln Leu Glu
Val Phe Gly Ser Gly Glu Ala Ala Pro Pro Gln 195 200 205Gly Tyr Asp
Ile Asn Lys Gly Tyr Ala Glu Ser Gly Asp Lys Leu Trp 210 215 220Phe
Asn Ile Tyr Ile Asn Lys Asn Asp Glu Phe Lys Met Pro Gly Pro225 230
235 240Glu Val Trp Asp Gly Gly Cys Arg Phe Gly Glu Arg Trp Ala Thr
Glu 245 250 255Glu Pro Gly Lys Pro Lys Val Asn Gln His Gly 260
265111843DNAHumicola insolens 111atgaagctcc tcgctcctct gatgctggct
ggcgccgcca gcgcccgtga gtaacccctg 60gctggatctc atgctggtgc cagtgttcca
tgactgacaa ccaccctcag acaccatctt 120cacctccctc gaggttgatg
gccgcaacta cggcacgggc aacggcgtcc gcgtcccctc 180ctacaacggc
cccgtcgagg atgtcacgtc caactcgatc gcctgcaacg gcccgccgaa
240cccgaccagc ccgaccgaca cggtcatcac cgtccaggct ggccagaacg
tgactgccat 300ctggcggtac atgctcaaca cccagggcac ctcgcccaac
gacatcatgg acagcagcca 360caagggtcct actctcgcct acctcaagaa
ggtcaacgat gcccggactg actcgggcgt 420cggcgatggc tggttcaaga
tccagcacga cggcttcgac ggcaccacct ggggcaccga 480gcgcgtcatc
ttcggccagg gccgtcacac catcaagatc cccgagtgca tcgagcccgg
540ccagtacctg ctgcgtgctg agatgatcgc cctccacggc gcccagaact
acccgggtgc 600tcagttctac atggagtgcg cccagctcaa cattgtcggt
ggcaccggca ccaagaaacc 660cagcaccgtc agcttccctg gcgcttacaa
ggtatgtccg agtttggtac cgagataact 720ggagatgaga aaagtgatgc
taacaaacca tgacagggca ccgaccccgg cgtcaagctc 780agcatctggt
ggccgcccgt caccaactac gtcattcccg gccccgatgt cttcaagtgc 840taa
843112237PRTHumicola insolens 112Met Lys Leu Leu Ala Pro Leu Met
Leu Ala Gly Ala Ala Ser Ala His1 5 10 15Thr Ile Phe Thr Ser Leu Glu
Val Asp Gly Arg Asn Tyr Gly Thr Gly 20 25 30Asn Gly Val Arg Val Pro
Ser Tyr Asn Gly Pro Val Glu Asp Val Thr 35 40 45Ser Asn Ser Ile Ala
Cys Asn Gly Pro Pro Asn Pro Thr Ser Pro Thr 50 55 60Asp Thr Val Ile
Thr Val Gln Ala Gly Gln Asn Val Thr Ala Ile Trp65 70 75 80Arg Tyr
Met Leu Asn Thr Gln Gly Thr Ser Pro Asn Asp Ile Met Asp 85 90 95Ser
Ser His Lys Gly Pro Thr Leu Ala Tyr Leu Lys Lys Val Asn Asp 100 105
110Ala Arg Thr Asp Ser Gly Val Gly Asp Gly Trp Phe Lys Ile Gln His
115 120 125Asp Gly Phe Asp Gly Thr Thr Trp Gly Thr Glu Arg Val Ile
Phe Gly 130 135 140Gln Gly Arg His Thr Ile Lys Ile Pro Glu Cys Ile
Glu Pro Gly Gln145 150 155 160Tyr Leu Leu Arg Ala Glu Met Ile Ala
Leu His Gly Ala Gln Asn Tyr 165 170 175Pro Gly Ala Gln Phe Tyr Met
Glu Cys Ala Gln Leu Asn Ile Val Gly 180 185 190Gly Thr Gly Thr Lys
Lys Pro Ser Thr Val Ser Phe Pro Gly Ala Tyr 195 200 205Lys Gly Thr
Asp Pro Gly Val Lys Leu Ser Ile Trp Trp Pro Pro Val 210 215 220Thr
Asn Tyr Val Ile Pro Gly Pro Asp Val Phe Lys Cys225 230
235113705DNAHumicola insolens 113atgaagctcc tctcaaccct cgccgccatt
gcggccacct tggccacggc ggatgcgcac 60tacatcttca acatcctgta cgtcaacggc
cagcgcatgg gcggcgagta cacctacgtg 120cggcgcaact ccaactcgta
cttccccgtg ttccccgaca tcctcaactc caacgacatg 180cgttgcaacg
tgggtgccag accgggcaac acccaaaccg ccaccgtcag ggccggcgac
240aggatcggct tcaaggtctt caacaacgag gtcatcgagc accctggtcc
cggcttcatc 300tacatgtcca aagccccggg cagcgtcaac aactatgacg
gcagcgggga ctggttcaag 360gtttacgaga ccggtctctg ccgcggtggt
ggcaacgtcg acaccaactg gtgctcgtac 420tacaaggacc ggctcgagtt
taccatcccg cccaagactc ctcccggcga gtatctggtg 480cgtatcgagc
atatcggtct gcacgagggc cacgtcaaca gggcgcagtt ctacatcacc
540tgcgcgcagc tcaagattga ggggcccggc ggcggcaacc cgaacccact
cgtgaagatc 600ccgggcatct acagggccaa cgaccccggc atcgcctaca
acaagtggac caacaacccg 660gcgccgtaca tcatgccggg tcccaaggtg
tgggatggca actaa 705114234PRTHumicola insolens 114Met Lys Leu Leu
Ser Thr Leu Ala Ala Ile Ala Ala Thr Leu Ala Thr1 5 10 15Ala Asp Ala
His Tyr Ile Phe Asn Ile Leu Tyr Val Asn Gly Gln Arg 20 25 30Met Gly
Gly Glu Tyr Thr Tyr Val Arg Arg Asn Ser Asn Ser Tyr Phe 35 40 45Pro
Val Phe Pro Asp Ile Leu Asn Ser Asn Asp Met Arg Cys Asn Val 50 55
60Gly Ala Arg Pro Gly Asn Thr Gln Thr Ala Thr Val Arg Ala Gly Asp65
70 75 80Arg Ile Gly Phe Lys Val Phe Asn Asn Glu Val Ile Glu His Pro
Gly 85 90 95Pro Gly Phe Ile Tyr Met Ser Lys Ala Pro Gly Ser Val Asn
Asn Tyr 100 105 110Asp Gly Ser Gly Asp Trp Phe Lys Val Tyr Glu Thr
Gly Leu Cys Arg 115 120 125Gly Gly Gly Asn Val Asp Thr Asn Trp Cys
Ser Tyr Tyr Lys Asp Arg 130 135 140Leu Glu Phe Thr Ile Pro Pro Lys
Thr Pro Pro Gly Glu Tyr Leu Val145 150 155 160Arg Ile Glu His Ile
Gly Leu His Glu Gly His Val Asn Arg Ala Gln 165 170 175Phe Tyr Ile
Thr Cys Ala Gln Leu Lys Ile Glu Gly Pro Gly Gly Gly 180 185 190Asn
Pro Asn Pro Leu Val Lys Ile Pro Gly Ile Tyr Arg Ala Asn Asp 195 200
205Pro Gly Ile Ala Tyr Asn Lys Trp Thr
Asn Asn Pro Ala Pro Tyr Ile 210 215 220Met Pro Gly Pro Lys Val Trp
Asp Gly Asn225 230115753DNAHumicola insolens 115atgctgggaa
gcgctcttct gctcctgggc actgccctgg gcgccaccgc ccactacacg 60ttccctagga
tcaacagcgg cggcgactgg cagtatgtcc gccgggccga caactggcag
120gacaacggct tcgttggcaa cgtcaactcg cctcagatcc ggtgcttcca
gagcaggcac 180caggccgccc cggccaccct caacgtcacc gccggctcca
cggtgaccta ctacgccaat 240cccaacgtct atcaccccgg cccgatggcc
ttctacatgg cccgcgtccc cgatggccag 300gatatcaact cgtggaccgg
cgagggtgcc gtgtggttca agatctacca cgagcagcct 360accggcctgg
gccagcagct gaggtggtct agcgatggta cgtgaatggt gatcctgtgg
420catctcaacc tcttccagac ttctgacccg agcccccgcg gccctacagg
caagaactcg 480ttccaggttc agatcccccg ctgcatccgc tctggctact
acctgctccg tgctgagcac 540atcggcttgc acagcgccgg cagccctggt
ggcgctcagt tctacatctc ttgcgcccag 600ctcgccgtca acggcggtgg
cagcaccgag ccccccaaca aggtgtcctt ccctggtgcc 660tacagcccgt
ccgaccccgg cattcagatc aacatctact ggcctgttcc gacctcgtac
720aagaaccccg gccccccggt cttccagtgc taa 753116226PRTHumicola
insolens 116Met Leu Gly Ser Ala Leu Leu Leu Leu Gly Thr Ala Leu Gly
Ala Thr1 5 10 15Ala His Tyr Thr Phe Pro Arg Ile Asn Ser Gly Gly Asp
Trp Gln Tyr 20 25 30Val Arg Arg Ala Asp Asn Trp Gln Asp Asn Gly Phe
Val Gly Asn Val 35 40 45Asn Ser Pro Gln Ile Arg Cys Phe Gln Ser Arg
His Gln Ala Ala Pro 50 55 60Ala Thr Leu Asn Val Thr Ala Gly Ser Thr
Val Thr Tyr Tyr Ala Asn65 70 75 80Pro Asn Val Tyr His Pro Gly Pro
Met Ala Phe Tyr Met Ala Arg Val 85 90 95Pro Asp Gly Gln Asp Ile Asn
Ser Trp Thr Gly Glu Gly Ala Val Trp 100 105 110Phe Lys Ile Tyr His
Glu Gln Pro Thr Gly Leu Gly Gln Gln Leu Arg 115 120 125Trp Ser Ser
Asp Gly Lys Asn Ser Phe Gln Val Gln Ile Pro Arg Cys 130 135 140Ile
Arg Ser Gly Tyr Tyr Leu Leu Arg Ala Glu His Ile Gly Leu His145 150
155 160Ser Ala Gly Ser Pro Gly Gly Ala Gln Phe Tyr Ile Ser Cys Ala
Gln 165 170 175Leu Ala Val Asn Gly Gly Gly Ser Thr Glu Pro Pro Asn
Lys Val Ser 180 185 190Phe Pro Gly Ala Tyr Ser Pro Ser Asp Pro Gly
Ile Gln Ile Asn Ile 195 200 205Tyr Trp Pro Val Pro Thr Ser Tyr Lys
Asn Pro Gly Pro Pro Val Phe 210 215 220Gln Cys225117854DNAHumicola
insolens 117atgaagctgc ttcctgggtt gcttctggca gccacggctg cccaagccca
ttgtacgttt 60ccgatcccca agaccatctt cgagaatttt cgagccagat ctttctgaga
gagttgctga 120caattcctgc tagacacatt ccccaggctc gttgtcaacg
ggcagcctga ggagagggac 180tggtcggtca ctcggatgac aaagaaccac
cagagcaagt cgggaattga aaacccaact 240agccccgaca tccgttgcta
cagctcgcag actgccccta acgtggcgat tgtgccggcc 300gggtctacca
tccactacat ctcgacccaa caaatcaacc atcctggccc gactcagtac
360tatctcgcca aggtcccagc tggtcagtca gccaagacct gggatggctc
tggcaacgtg 420tggttcaaga tcgccacgag catgccggag tacgatcaaa
acaggcagct ggtttggccc 480ggtcatagta aggactcact ctcgtccgat
catctctttt gagtgagtct tgggcatacc 540cactgactac gtctgctatg
acagatacct atcagaccat caacgccacc atcccggcca 600acacgccgag
cggagagtac ctcctgcgtg tcgagcaaat tgccctccac atggccagcc
660agccgaacaa ggcccagttc tacatctcgt gctctcagat tcagattacc
aatggcggaa 720acggcactcc gggccctcta gttgcattcc cgggggcata
caggagcaac gaccctggca 780tcctggtcaa tctctacagc ggcatgcagc
cttcgcagta ccagccccct ggaccggccg 840tgtggcgtgg ctga
854118231PRTHumicola insolens 118Met Lys Leu Leu Pro Gly Leu Leu
Leu Ala Ala Thr Ala Ala Gln Ala1 5 10 15His Tyr Thr Phe Pro Arg Leu
Val Val Asn Gly Gln Pro Glu Glu Arg 20 25 30Asp Trp Ser Val Thr Arg
Met Thr Lys Asn His Gln Ser Lys Ser Gly 35 40 45Ile Glu Asn Pro Thr
Ser Pro Asp Ile Arg Cys Tyr Ser Ser Gln Thr 50 55 60Ala Pro Asn Val
Ala Ile Val Pro Ala Gly Ser Thr Ile His Tyr Ile65 70 75 80Ser Thr
Gln Gln Ile Asn His Pro Gly Pro Thr Gln Tyr Tyr Leu Ala 85 90 95Lys
Val Pro Ala Gly Gln Ser Ala Lys Thr Trp Asp Gly Ser Gly Asn 100 105
110Val Trp Phe Lys Ile Ala Thr Ser Met Pro Glu Tyr Asp Gln Asn Arg
115 120 125Gln Leu Val Trp Pro Gly His Asn Thr Tyr Gln Thr Ile Asn
Ala Thr 130 135 140Ile Pro Ala Asn Thr Pro Ser Gly Glu Tyr Leu Leu
Arg Val Glu Gln145 150 155 160Ile Ala Leu His Met Ala Ser Gln Pro
Asn Lys Ala Gln Phe Tyr Ile 165 170 175Ser Cys Ser Gln Ile Gln Ile
Thr Asn Gly Gly Asn Gly Thr Pro Gly 180 185 190Pro Leu Val Ala Phe
Pro Gly Ala Tyr Arg Ser Asn Asp Pro Gly Ile 195 200 205Leu Val Asn
Leu Tyr Ser Gly Met Gln Pro Ser Gln Tyr Gln Pro Pro 210 215 220Gly
Pro Ala Val Trp Arg Gly225 230119863DNAHumicola insolens
119atgctcctga actcggtcat cggctcggcc gtcctcctgg ccaccggcgc
cgccgcccac 60ggtgccgtga ccagctacgt cattgccggg aagaactacc ctgggtaggt
aacctcgtgg 120aagcgaatgc aggcagttca ttcactaaca catacctccg
ttagctacaa cggctacgcc 180ccgtccacca cccccaacac gatccagtgg
caatggtcga cctacgaccc catctactcc 240gccaccgacc ccaagctccg
ctgcaacggc ggccgctcgg ccacgcagtc cgccccggct 300gctccgggcg
acaacatcac cgccatctgg cagcagtgga cgcatagcca gggccccatc
360ctcgtctgga tgtacaagtg tcccggcgcc ttcagctcgt gcgacggctc
gggccagggc 420tggttcaaga ttgacgaggc cggcttcaat ggcgacggca
agaccgtgtt cctcgacacc 480gagcgcccct ccggctggga gatcgccaag
ctggttggcg gcaacaaggg ctggaccagc 540accatcccca agaacctggc
cccgggcaac tacctggtcc gccacgagtt gattgccctt 600caccaggcca
acgccccgca gtggtaccct gagtgcgcgc aggtcgtgat caccggctcg
660ggcactaagg agccgcctgc gtcgtacaag gctgccattc ccggctactg
caaccagaac 720gatcccaaca ttcgggtatg tgaggcctat ttggagttcg
gctaaggcat gatactaact 780ctacccccca ggttcctatc aacgaccact
ccatccccca gacctacaag atccctggcc 840ctccggtctg gcgcggcgag taa
863120248PRTHumicola insolens 120Met Leu Leu Asn Ser Val Ile Gly
Ser Ala Val Leu Leu Ala Thr Gly1 5 10 15Ala Ala Ala His Gly Ala Val
Thr Ser Tyr Val Ile Ala Gly Lys Asn 20 25 30Tyr Pro Gly Tyr Asn Gly
Tyr Ala Pro Ser Thr Thr Pro Asn Thr Ile 35 40 45Gln Trp Gln Trp Ser
Thr Tyr Asp Pro Ile Tyr Ser Ala Thr Asp Pro 50 55 60Lys Leu Arg Cys
Asn Gly Gly Arg Ser Ala Thr Gln Ser Ala Pro Ala65 70 75 80Ala Pro
Gly Asp Asn Ile Thr Ala Ile Trp Gln Gln Trp Thr His Ser 85 90 95Gln
Gly Pro Ile Leu Val Trp Met Tyr Lys Cys Pro Gly Ala Phe Ser 100 105
110Ser Cys Asp Gly Ser Gly Gln Gly Trp Phe Lys Ile Asp Glu Ala Gly
115 120 125Phe Asn Gly Asp Gly Lys Thr Val Phe Leu Asp Thr Glu Arg
Pro Ser 130 135 140Gly Trp Glu Ile Ala Lys Leu Val Gly Gly Asn Lys
Gly Trp Thr Ser145 150 155 160Thr Ile Pro Lys Asn Leu Ala Pro Gly
Asn Tyr Leu Val Arg His Glu 165 170 175Leu Ile Ala Leu His Gln Ala
Asn Ala Pro Gln Trp Tyr Pro Glu Cys 180 185 190Ala Gln Val Val Ile
Thr Gly Ser Gly Thr Lys Glu Pro Pro Ala Ser 195 200 205Tyr Lys Ala
Ala Ile Pro Gly Tyr Cys Asn Gln Asn Asp Pro Asn Ile 210 215 220Arg
Val Pro Ile Asn Asp His Ser Ile Pro Gln Thr Tyr Lys Ile Pro225 230
235 240Gly Pro Pro Val Trp Arg Gly Glu 245121883DNAHumicola
insolens 121atgaagctca ccacctccat cgccctgctg gctgcggccg gcgcgcaggc
tcactgtacg 60tgctccctca tctcatccat ctcctcagac catgttttac ctattggtta
ctaacaagct 120ctcacgcaga caccttcccc cgcaccaagg tcgacggcgt
cacctcgggc gagtgggaga 180cgatccgcat caccgagaac cactggtcgc
acggccccgt gacggacgtg acctcgcagg 240ccatgacgtg ctacgagaag
acgcccggcc agggcgcgcc caagacggtt aacgtgaagg 300ccggcggcac
cgtcaccttc accgtcgaca cggacgtggg ccacccgggc ccgctgcact
360tctacttggc caaggtgccc gcgggcaaga cggccgcgac gtttgacggc
aagggcgccg 420tgtggttcaa gatttaccag gacggccccg gcgggttggg
gaccagctcg ttgacttggc 480ctagctttgg tgagctttct tttctttatt
ttcttcaatc ctcccataat tacctcccga 540cgaggaaata aatatacctt
acctgatatt aacccatccc cccccacctc ctccaggcaa 600gaaggaagtc
tctgtccaaa tccccccctg cgtgcaggac ggcgagtacc tgctgcgcgt
660cgagcacatt gcgctgcaca gcgccgcgag cgtcggcggc gcgcagctct
acatttcgtg 720cgcgcaaatc aacgtcaccg gcggcaccgg cacgctcaac
ccgggccagc tcgtctcgtt 780cccgggcgcc tacaagccca ccgacccggg
catcctgttc cagctctact ggccgccgcc 840gacccagtac atcaaccccg
gtccggcgcc ggtgaagtgc tga 883122233PRTHumicola insolens 122Met Lys
Leu Thr Thr Ser Ile Ala Leu Leu Ala Ala Ala Gly Ala Gln1 5 10 15Ala
His Tyr Thr Phe Pro Arg Thr Lys Val Asp Gly Val Thr Ser Gly 20 25
30Glu Trp Glu Thr Ile Arg Ile Thr Glu Asn His Trp Ser His Gly Pro
35 40 45Val Thr Asp Val Thr Ser Gln Ala Met Thr Cys Tyr Glu Lys Thr
Pro 50 55 60Gly Gln Gly Ala Pro Lys Thr Val Asn Val Lys Ala Gly Gly
Thr Val65 70 75 80Thr Phe Thr Val Asp Thr Asp Val Gly His Pro Gly
Pro Leu His Phe 85 90 95Tyr Leu Ala Lys Val Pro Ala Gly Lys Thr Ala
Ala Thr Phe Asp Gly 100 105 110Lys Gly Ala Val Trp Phe Lys Ile Tyr
Gln Asp Gly Pro Gly Gly Leu 115 120 125Gly Thr Ser Ser Leu Thr Trp
Pro Ser Phe Gly Lys Lys Glu Val Ser 130 135 140Val Gln Ile Pro Pro
Cys Val Gln Asp Gly Glu Tyr Leu Leu Arg Val145 150 155 160Glu His
Ile Ala Leu His Ser Ala Ala Ser Val Gly Gly Ala Gln Leu 165 170
175Tyr Ile Ser Cys Ala Gln Ile Asn Val Thr Gly Gly Thr Gly Thr Leu
180 185 190Asn Pro Gly Gln Leu Val Ser Phe Pro Gly Ala Tyr Lys Pro
Thr Asp 195 200 205Pro Gly Ile Leu Phe Gln Leu Tyr Trp Pro Pro Pro
Thr Gln Tyr Ile 210 215 220Asn Pro Gly Pro Ala Pro Val Lys Cys225
230123928DNAHumicola insolens 123atgaagactc tcgcatccgc cctcattgcc
gcgggccttc tggcccagta cgccgctgcc 60catgccattt tccagtttgc cagcagcggt
ggcactgact ttgggacgtc ctgtgttagg 120atgccggtga gtgaacgggt
gcccctgaac atgtgttgct cacgaaacaa ggttatgttg 180actctataca
gcccaacaac tctcccgtca cgagcgtcac cagcagtgac atggcttgca
240atgttggcgg atctcgcggt gtatctggca tttgcgaggt gaacggtaag
agttctcctc 300agccttttct ctgtcaagca ctaaacagca ctcgctaacc
atttcaatct cagccggctc 360cgacttcacc gtcgagatgc acgcgcagcc
caacgaccgg tcgtgcgcca gcgaagccat 420tggcggcaac cacttcgggc
ccgtcatggt gtacatggcc aaggtggacg acgcgacgcg 480ggcggacggt
gcgtcggcgt cttggttcaa ggtggacgag ttcggctacg acgccggctc
540caagacatgg ggaaccgaca tgctcaacaa gaactgcggc aagcggacgt
tccgcatccc 600gagcaaaatc ccgtctgggg actatctggt gcgtgcggag
gctattgctt tgcacaccgc 660gggccagccg tcgggtgcgc agttttatat
gagctgctat gtgagttctt ccatgcttcc 720ccttgtggtg tcactgtata
gaagatgcta atatctccca cagcaagttc gcatcaaggg 780cagcaacaac
ggtcagcttc cggctggtgt tcggattcct ggcgcctaca gcgcgacgga
840cccgggcatc ctcgtcgata tctggggcaa tggtttcagc cagtacacta
ttcctggccc 900tcgtgtcatt gatgggagct ttttctga 928124243PRTHumicola
insolens 124Met Lys Thr Leu Ala Ser Ala Leu Ile Ala Ala Gly Leu Leu
Ala Gln1 5 10 15Tyr Ala Ala Ala His Ala Ile Phe Gln Phe Ala Ser Ser
Gly Gly Thr 20 25 30Asp Phe Gly Thr Ser Cys Val Arg Met Pro Pro Asn
Asn Ser Pro Val 35 40 45Thr Ser Val Thr Ser Ser Asp Met Ala Cys Asn
Val Gly Gly Ser Arg 50 55 60Gly Val Ser Gly Ile Cys Glu Val Asn Ala
Gly Ser Asp Phe Thr Val65 70 75 80Glu Met His Ala Gln Pro Asn Asp
Arg Ser Cys Ala Ser Glu Ala Ile 85 90 95Gly Gly Asn His Phe Gly Pro
Val Met Val Tyr Met Ala Lys Val Asp 100 105 110Asp Ala Thr Arg Ala
Asp Gly Ala Ser Ala Ser Trp Phe Lys Val Asp 115 120 125Glu Phe Gly
Tyr Asp Ala Gly Ser Lys Thr Trp Gly Thr Asp Met Leu 130 135 140Asn
Lys Asn Cys Gly Lys Arg Thr Phe Arg Ile Pro Ser Lys Ile Pro145 150
155 160Ser Gly Asp Tyr Leu Val Arg Ala Glu Ala Ile Ala Leu His Thr
Ala 165 170 175Gly Gln Pro Ser Gly Ala Gln Phe Tyr Met Ser Cys Tyr
Gln Val Arg 180 185 190Ile Lys Gly Ser Asn Asn Gly Gln Leu Pro Ala
Gly Val Arg Ile Pro 195 200 205Gly Ala Tyr Ser Ala Thr Asp Pro Gly
Ile Leu Val Asp Ile Trp Gly 210 215 220Asn Gly Phe Ser Gln Tyr Thr
Ile Pro Gly Pro Arg Val Ile Asp Gly225 230 235 240Ser Phe
Phe1251092DNAHumicola insolens 125atgcctcgct tcaccaagtc cattgtctcg
gccctggccg gcgcttccct ggtcgcagcc 60cacggccatg tcacccacat cgtcatcaac
ggcgtgctgt acccgaactt cgaccctaca 120tcccaccctt acctgcagaa
cccgccgacc gttgtgggct ggaccgccgc caacaccgac 180aacggcttcg
ttgctcccga ccagttcgcc tcgggcgata tcatctgcca caaccaggcc
240accaacgcgg gcggccacgc cgtggtcgcg gccggcgaca agatttggat
ccagtgggac 300cagtggcctg agagccacca cggccccgtc ctcgactacc
tcgcctcctg cggcagctcg 360ggctgcgagt cggtcaacaa gctcgacctc
gagttcttca agatcggcga aaagggcctg 420atcgacggct cctccgcgcc
gggccggtgg gcgtcggacg agctgatcgc caacaacgcc 480ggctggctgg
tccagatccc cgccgacatt gcgcccggcc actacgtcct gcgccacgaa
540atcatcgccc tccacgccgc cggccagccc aacggcgccc agaactaccc
gcagtgcttc 600aacctcctcg tcacgggctc cggcaccgcg cggccgcagg
gcgtcaaggg aacagcgctg 660tacaccccca acgacaaggg catcttggcg
ggcatctaca atgcccccgt ctcgtacgag 720attcccggcc ccgcgctcta
ctccggcgcc gccaggaact tgcagcagag ctcgtcccag 780gccacgtcga
ctgccacggc tttgactggg gacgcggtgc ctgttccgac ccaagccccc
840gtcactacca cttcctcttc ttcggccgat gccgccaccg ccacctccac
caccgtccag 900ccgccccagc aaaccaccct cacgaccgcc atcgccacgt
cgaccgctgc tgctgccccg 960acgaccaccg ccggcagcgg aaacggtggc
aaccggccct tcccaacccg ctgccctggc 1020ctggctgggc tcgggtttga
caagcgccgt cgccagctcc gcgctgagga gggtgtgcag 1080gtggttgctt ga
1092126363PRTHumicola insolens 126Met Pro Arg Phe Thr Lys Ser Ile
Val Ser Ala Leu Ala Gly Ala Ser1 5 10 15Leu Val Ala Ala His Gly His
Val Thr His Ile Val Ile Asn Gly Val 20 25 30Leu Tyr Pro Asn Phe Asp
Pro Thr Ser His Pro Tyr Leu Gln Asn Pro 35 40 45Pro Thr Val Val Gly
Trp Thr Ala Ala Asn Thr Asp Asn Gly Phe Val 50 55 60Ala Pro Asp Gln
Phe Ala Ser Gly Asp Ile Ile Cys His Asn Gln Ala65 70 75 80Thr Asn
Ala Gly Gly His Ala Val Val Ala Ala Gly Asp Lys Ile Trp 85 90 95Ile
Gln Trp Asp Gln Trp Pro Glu Ser His His Gly Pro Val Leu Asp 100 105
110Tyr Leu Ala Ser Cys Gly Ser Ser Gly Cys Glu Ser Val Asn Lys Leu
115 120 125Asp Leu Glu Phe Phe Lys Ile Gly Glu Lys Gly Leu Ile Asp
Gly Ser 130 135 140Ser Ala Pro Gly Arg Trp Ala Ser Asp Glu Leu Ile
Ala Asn Asn Ala145 150 155 160Gly Trp Leu Val Gln Ile Pro Ala Asp
Ile Ala Pro Gly His Tyr Val 165 170 175Leu Arg His Glu Ile Ile Ala
Leu His Ala Ala Gly Gln Pro Asn Gly 180 185 190Ala Gln Asn Tyr Pro
Gln Cys Phe Asn Leu Leu Val Thr Gly Ser Gly 195 200 205Thr Ala Arg
Pro Gln Gly Val Lys Gly Thr Ala Leu Tyr Thr Pro Asn 210 215 220Asp
Lys Gly Ile Leu Ala Gly Ile Tyr Asn Ala Pro Val Ser Tyr Glu225 230
235 240Ile Pro Gly Pro Ala Leu Tyr Ser Gly Ala Ala Arg Asn Leu Gln
Gln 245 250 255Ser Ser Ser Gln Ala Thr Ser Thr Ala Thr Ala Leu Thr
Gly Asp Ala 260 265 270Val Pro Val Pro Thr Gln Ala Pro Val Thr Thr
Thr Ser Ser Ser Ser 275 280 285Ala Asp Ala Ala Thr Ala Thr Ser Thr
Thr Val Gln Pro Pro Gln Gln 290 295 300Thr Thr Leu Thr Thr Ala Ile
Ala Thr Ser Thr Ala Ala Ala Ala
Pro305 310 315 320Thr Thr Thr Ala Gly Ser Gly Asn Gly Gly Asn Arg
Pro Phe Pro Thr 325 330 335Arg Cys Pro Gly Leu Ala Gly Leu Gly Phe
Asp Lys Arg Arg Arg Gln 340 345 350Leu Arg Ala Glu Glu Gly Val Gln
Val Val Ala 355 3601271086DNAHumicola insolens 127atgaagggac
ttctcagcat cgccgccctt tccctggcgg ttggtgaggc ttcggcccac 60tacatcttcc
agcagctctc gacgggtggc accaagcacc ccatgtggaa gtacatccgc
120cagcacacca actacaactc tcccgtcatc gacctcgact ccaacgacct
ccgctgcaat 180gtcggtgccc ggggtgctgg aactgagacc gttacggtcg
ctgctggctc gagcctgacc 240ttccacctcg acacccccgt ctaccaccag
ggccctgtgt cggtgtaagt agaagttctc 300agacgaacca ccaatgtcgg
cagataattt ctaactccga tgtccagcta tatgtccaag 360gctcccggct
ccgtgtcgga ctatgacggc agcggcggct ggttcaagat tcaagactgg
420ggcccgacct tcaccggcag cggcgccacc tggaagctgg atgactccta
caccttcaac 480atcccctcgt gcattcccga cggcgagtac ctcgtccgca
tccagtccct gggtatccac 540aacccctggc cggcgggtat tccgcagttc
tatatctcgt gcgctcaggt gcgcgtcacc 600ggcggtggca acgcgaaccc
gagcccgcag gtgtcgatcc caggtgcctt caaggagacc 660gacccgggct
acactgccaa cgtgagtttc catccatgct acatatccct tttacgctct
720cgatcccatg actaaccccc ccctgaaaag atctacaaca acttccgcag
ctacaccgtc 780cccggcccgt ccgtcttcac ctgcagcggc aacagcggcg
gcggctccaa ccccagcaac 840cctaaccccc cgaccccgac gaccttcacc
acccaggtga ccaccccgac cccggcgtct 900ccgccctctt gcaccgtcgc
gaagtggtac gtctgaaaaa aaatctcctc caggccggac 960atgagaaaac
taacatgaac gaaaaacagg ggccagtgcg gtggccaggg ctacagcggc
1020tgcaccaact gcgaggccgg ctcgacctgc aggcagcaga acgcttacta
ttctcagtgc 1080atctaa 1086128296PRTHumicola insolens 128Met Lys Gly
Leu Leu Ser Ile Ala Ala Leu Ser Leu Ala Val Gly Glu1 5 10 15Ala Ser
Ala His Tyr Ile Phe Gln Gln Leu Ser Thr Gly Gly Thr Lys 20 25 30His
Pro Met Trp Lys Tyr Ile Arg Gln His Thr Asn Tyr Asn Ser Pro 35 40
45Val Ile Asp Leu Asp Ser Asn Asp Leu Arg Cys Asn Val Gly Ala Arg
50 55 60Gly Ala Gly Thr Glu Thr Val Thr Val Ala Ala Gly Ser Ser Leu
Thr65 70 75 80Phe His Leu Asp Thr Pro Val Tyr His Gln Gly Pro Val
Ser Val Tyr 85 90 95Met Ser Lys Ala Pro Gly Ser Val Ser Asp Tyr Asp
Gly Ser Gly Gly 100 105 110Trp Phe Lys Ile Gln Asp Trp Gly Pro Thr
Phe Thr Gly Ser Gly Ala 115 120 125Thr Trp Lys Leu Asp Asp Ser Tyr
Thr Phe Asn Ile Pro Ser Cys Ile 130 135 140Pro Asp Gly Glu Tyr Leu
Val Arg Ile Gln Ser Leu Gly Ile His Asn145 150 155 160Pro Trp Pro
Ala Gly Ile Pro Gln Phe Tyr Ile Ser Cys Ala Gln Val 165 170 175Arg
Val Thr Gly Gly Gly Asn Ala Asn Pro Ser Pro Gln Val Ser Ile 180 185
190Pro Gly Ala Phe Lys Glu Thr Asp Pro Gly Tyr Thr Ala Asn Ile Tyr
195 200 205Asn Asn Phe Arg Ser Tyr Thr Val Pro Gly Pro Ser Val Phe
Thr Cys 210 215 220Ser Gly Asn Ser Gly Gly Gly Ser Asn Pro Ser Asn
Pro Asn Pro Pro225 230 235 240Thr Pro Thr Thr Phe Thr Thr Gln Val
Thr Thr Pro Thr Pro Ala Ser 245 250 255Pro Pro Ser Cys Thr Val Ala
Lys Trp Gly Gln Cys Gly Gly Gln Gly 260 265 270Tyr Ser Gly Cys Thr
Asn Cys Glu Ala Gly Ser Thr Cys Arg Gln Gln 275 280 285Asn Ala Tyr
Tyr Ser Gln Cys Ile 290 2951291032DNAHumicola insolens
129atgaggccct tctcactcgt cgctctggcg acggccgtca gcggccacgc
catcttccag 60cgcgtgtcgg ttaacggcgt cgaccaaggc cagctcaagg gcgtccgcgc
tccctcgagc 120aactacccca tcgagaacgt caaccacccc gactttgcct
gcaacaccaa catccagcac 180cgcgacggca ccgtcatcaa gatccccgcc
ggcgccaccg tcggcgcctg gtggcagcac 240gagatcggcg ggccctcgtt
cccgggtgac ccggataacc cgatcgctgc ttcgcacaag 300ggtgagttcc
catagataga tctcttctct cccgacccct tgtatcctct cataactaac
360cacctcaacc ccccaggccc tatccaagtc tacctcgcca aggtcgacaa
cgccgcgacc 420gcctccccca acggcctgcg gtggttcaag attgccgaga
agggcctgtc gggcggcgtc 480tgggccgtcg acgagatgat ccgcaacaac
ggctggcact acttcaccat gccgcagtgc 540atcgcgcccg gccactacct
gatgcgcgtc gagctgttgg cgctgcactc ggccagcttc 600cccggcggcg
cccagttcta catggagtgc gcccagatcg aggtcaccgg ctcgggcaac
660ttctcgccct ccgagacggt cagcttcccc ggcgcctacc cggccaacca
cccgggtatc 720gtcgtcagca tctacgacgc ccagggtaac gccaacaacg
gcgggcgcga gtaccagatc 780cccgggccgc ggccgatcac ctgctccggc
ggtggaagca acaatggtgg cgggaacaac 840aatggtggtg gaaacaacaa
cggcggcggc aacaacaacg gcggtgggaa caacaacggt 900ggtggtaaca
ccggtggcgg ctcggcgccg ctctggggcc agtgcggcgg caatgggtat
960accggcccga cgacttgtgc cgagggtact tgcaagaagc agaatgactg
gtactcgcag 1020tgtacgcctt ag 1032130318PRTHumicola insolens 130Met
Arg Pro Phe Ser Leu Val Ala Leu Ala Thr Ala Val Ser Gly His1 5 10
15Ala Ile Phe Gln Arg Val Ser Val Asn Gly Val Asp Gln Gly Gln Leu
20 25 30Lys Gly Val Arg Ala Pro Ser Ser Asn Tyr Pro Ile Glu Asn Val
Asn 35 40 45His Pro Asp Phe Ala Cys Asn Thr Asn Ile Gln His Arg Asp
Gly Thr 50 55 60Val Ile Lys Ile Pro Ala Gly Ala Thr Val Gly Ala Trp
Trp Gln His65 70 75 80Glu Ile Gly Gly Pro Ser Phe Pro Gly Asp Pro
Asp Asn Pro Ile Ala 85 90 95Ala Ser His Lys Gly Pro Ile Gln Val Tyr
Leu Ala Lys Val Asp Asn 100 105 110Ala Ala Thr Ala Ser Pro Asn Gly
Leu Arg Trp Phe Lys Ile Ala Glu 115 120 125Lys Gly Leu Ser Gly Gly
Val Trp Ala Val Asp Glu Met Ile Arg Asn 130 135 140Asn Gly Trp His
Tyr Phe Thr Met Pro Gln Cys Ile Ala Pro Gly His145 150 155 160Tyr
Leu Met Arg Val Glu Leu Leu Ala Leu His Ser Ala Ser Phe Pro 165 170
175Gly Gly Ala Gln Phe Tyr Met Glu Cys Ala Gln Ile Glu Val Thr Gly
180 185 190Ser Gly Asn Phe Ser Pro Ser Glu Thr Val Ser Phe Pro Gly
Ala Tyr 195 200 205Pro Ala Asn His Pro Gly Ile Val Val Ser Ile Tyr
Asp Ala Gln Gly 210 215 220Asn Ala Asn Asn Gly Gly Arg Glu Tyr Gln
Ile Pro Gly Pro Arg Pro225 230 235 240Ile Thr Cys Ser Gly Gly Gly
Ser Asn Asn Gly Gly Gly Asn Asn Asn 245 250 255Gly Gly Gly Asn Asn
Asn Gly Gly Gly Asn Asn Asn Gly Gly Gly Asn 260 265 270Asn Asn Gly
Gly Gly Asn Thr Gly Gly Gly Ser Ala Pro Leu Trp Gly 275 280 285Gln
Cys Gly Gly Asn Gly Tyr Thr Gly Pro Thr Thr Cys Ala Glu Gly 290 295
300Thr Cys Lys Lys Gln Asn Asp Trp Tyr Ser Gln Cys Thr Pro305 310
3151311113DNAHumicola insolens 131atggtgttgc ggtctctctc tatcctggcc
ttcgtagcca gaggcgtctt cgcccacggt 60ggcctctcca actacacggt cggcgacacg
tggtatagcg ggtgcgtcca tgaacaactc 120ctatatcttc cccccctcca
cattgcgacc gctgcacatc tcactcgtcc ataaacaaca 180acatcaatcg
gtagacactg tccaaaagct aaccaccgta cctcctgaac acagctacga
240ccccttcacc cccgccgccg cccaactctc ccaaccctgg ctgatccaac
gccaatggac 300cagcatcgac ccgctcttct ccccgacctc tccctacctc
gcctgcaact tccccggcac 360cgcgccacca tcttacatcc ctctccgcgc
cggcgacatc ctcaccgcgg tttactggtt 420ctggctgcac cccgtggggc
cgatgagcgt ttggctggcg cggtgcgcag gggactgccg 480cgacgaggac
gtgacgcggg cgcgctggtt caagatctgg catgcggggt ttctggaggg
540gccgaatttg gagctcggga tgtggtatca gaagaagttc cagcggtggg
atggcgggcc 600ggcgctctgg cgggtgagga taccgagggg gttgaagaag
gggttgtaca tggtcaggca 660tgagattttg tcgattcatg tgggtggacg
gccccagttt tatcccgagt gtgcgcactt 720gaatgtgacg gagggtggtg
aggtggtagt gccgggggag tggacgagaa ggttccctgg 780ggcgtatgac
gatgatggtg agtgccttgc tagacgggaa ggctctatgg atggggcgga
840tgagacgaaa ggctggtgtg agactgtcag cactgacggc ctgcagacaa
gtcagtcttc 900atcgatatct accggccgga acatgaaaac aggacggtac
gtgggacaag caagcctcgg 960atttttcaga ttttcgactc tgacaacgaa
caggactatg agatccctgg aggcccgatt 1020tgggaaaggt acgtacaatc
gcatcatctt gactctgtat tcaggggcta acataaacac 1080agcttggggg
agatggagtt atggcctgaa tga 1113132259PRTHumicola insolens 132Met Val
Leu Arg Ser Leu Ser Ile Leu Ala Phe Val Ala Arg Gly Val1 5 10 15Phe
Ala His Gly Gly Leu Ser Asn Tyr Thr Val Gly Asp Thr Trp Tyr 20 25
30Ser Gly Tyr Asp Pro Phe Thr Pro Ala Ala Ala Gln Leu Ser Gln Pro
35 40 45Trp Leu Ile Gln Arg Gln Trp Thr Ser Ile Asp Pro Leu Phe Ser
Pro 50 55 60Thr Ser Pro Tyr Leu Ala Cys Asn Phe Pro Gly Thr Ala Pro
Pro Ser65 70 75 80Tyr Ile Pro Leu Arg Ala Gly Asp Ile Leu Thr Ala
Val Tyr Trp Phe 85 90 95Trp Leu His Pro Val Gly Pro Met Ser Val Trp
Leu Ala Arg Cys Ala 100 105 110Gly Asp Cys Arg Asp Glu Asp Val Thr
Arg Ala Arg Trp Phe Lys Ile 115 120 125Trp His Ala Gly Phe Leu Glu
Gly Pro Asn Leu Glu Leu Gly Met Trp 130 135 140Tyr Gln Lys Lys Phe
Gln Arg Trp Asp Gly Gly Pro Ala Leu Trp Arg145 150 155 160Val Arg
Ile Pro Arg Gly Leu Lys Lys Gly Leu Tyr Met Val Arg His 165 170
175Glu Ile Leu Ser Ile His Val Gly Gly Arg Pro Gln Phe Tyr Pro Glu
180 185 190Cys Ala His Leu Asn Val Thr Glu Gly Gly Glu Val Val Val
Pro Gly 195 200 205Glu Trp Thr Arg Arg Phe Pro Gly Ala Tyr Asp Asp
Asp Asp Lys Ser 210 215 220Val Phe Ile Asp Ile Tyr Arg Pro Glu His
Glu Asn Arg Thr Asp Tyr225 230 235 240Glu Ile Pro Gly Gly Pro Ile
Trp Glu Ser Leu Gly Glu Met Glu Leu 245 250 255Trp Pro
Glu1331103DNAHumicola insolens 133atgaggaccg tcttcgccgc cgcactggca
gcactcgctg cccgggaagt cgccggccat 60gccacgttcc agcaactctg ggttgacgga
accgattata taagtgcccc ccttttctcg 120gttccatttg atatcatgat
gctgacaccc ccagcacggc agcacctgcg tccgcctccc 180cgccagcaac
agccccctga ccgacgtcac cagcagcgac ttcgcctgca acatcggcgg
240ccggcgcggc gtgggcggca aatgccccgt caaagccggc ggcgtggtca
cgatcgagat 300gcatcagcag cccaacgacc ggaactgccg cagcgaggcc
atcggcggca tgcactgggg 360tccggtgcag gtctacctca gcaaggtccc
cgacgcgtcg accgccgagc cgacgcaggt 420gggctggttc aagatcttct
ccaacgcgtg ggccaagaag cccggcggca actcgggcga 480cgacgactac
tggggcacgc gcgagctcaa cggctgctgc gggcgcatgg acgtgccgat
540ccccaccgac ctggaagacg gcgactacct gctgcgcgcc gaggcgctgg
cgctgcacgc 600catgccgggc cagttctaca tgtcgtgcta ccagatcacc
atcacgggcg gcacgggcac 660cgcgaagccg gcgactgtcc gcttccccgg
agcgtacacc aacaacgacg ccggcatccg 720cgccaacatc cacgccccgc
tgagcaccta catcgcgccc ggcccggagg tgtactccgg 780cggtaccacc
cgggcgcccg gtgagggctg cccgggatgt gctacgacct gccaggttgg
840ctcgtcgccc agcgcgcagg ctccaggcca tggcacggcc gtgggcggcg
gagctggtgg 900cccgtctgct tgcaccgtcc aggcgtatgg ccagtgcggt
ggccagggat acacgggttg 960caccgagtgc gcggtaagtt gggacttcct
tgtcattaaa atcgcaaatg gaacggatgg 1020gctaacattt gcgggtgcag
gatggtttcg tttgccgcga cgtctcggct ccgtggtact 1080ctcagtgcca
gcctgctttc taa 1103134325PRTHumicola insolens 134Met Arg Thr Val
Phe Ala Ala Ala Leu Ala Ala Leu Ala Ala Arg Glu1 5 10 15Val Ala Gly
His Ala Thr Phe Gln Gln Leu Trp Val Asp Gly Thr Asp 20 25 30Tyr Gly
Ser Thr Cys Val Arg Leu Pro Ala Ser Asn Ser Pro Leu Thr 35 40 45Asp
Val Thr Ser Ser Asp Phe Ala Cys Asn Ile Gly Gly Arg Arg Gly 50 55
60Val Gly Gly Lys Cys Pro Val Lys Ala Gly Gly Val Val Thr Ile Glu65
70 75 80Met His Gln Gln Pro Asn Asp Arg Asn Cys Arg Ser Glu Ala Ile
Gly 85 90 95Gly Met His Trp Gly Pro Val Gln Val Tyr Leu Ser Lys Val
Pro Asp 100 105 110Ala Ser Thr Ala Glu Pro Thr Gln Val Gly Trp Phe
Lys Ile Phe Ser 115 120 125Asn Ala Trp Ala Lys Lys Pro Gly Gly Asn
Ser Gly Asp Asp Asp Tyr 130 135 140Trp Gly Thr Arg Glu Leu Asn Gly
Cys Cys Gly Arg Met Asp Val Pro145 150 155 160Ile Pro Thr Asp Leu
Glu Asp Gly Asp Tyr Leu Leu Arg Ala Glu Ala 165 170 175Leu Ala Leu
His Ala Met Pro Gly Gln Phe Tyr Met Ser Cys Tyr Gln 180 185 190Ile
Thr Ile Thr Gly Gly Thr Gly Thr Ala Lys Pro Ala Thr Val Arg 195 200
205Phe Pro Gly Ala Tyr Thr Asn Asn Asp Ala Gly Ile Arg Ala Asn Ile
210 215 220His Ala Pro Leu Ser Thr Tyr Ile Ala Pro Gly Pro Glu Val
Tyr Ser225 230 235 240Gly Gly Thr Thr Arg Ala Pro Gly Glu Gly Cys
Pro Gly Cys Ala Thr 245 250 255Thr Cys Gln Val Gly Ser Ser Pro Ser
Ala Gln Ala Pro Gly His Gly 260 265 270Thr Ala Val Gly Gly Gly Ala
Gly Gly Pro Ser Ala Cys Thr Val Gln 275 280 285Ala Tyr Gly Gln Cys
Gly Gly Gln Gly Tyr Thr Gly Cys Thr Glu Cys 290 295 300Ala Asp Gly
Phe Val Cys Arg Asp Val Ser Ala Pro Trp Tyr Ser Gln305 310 315
320Cys Gln Pro Ala Phe 3251351039DNAHumicola insolens 135atgaggctcc
cccaagtggc ttccgttctg gccctcgcgg cccaggtcca cggtcacggc 60tacatctacc
gtgtcaccgc cgacaacatt gtgtaagcgc cctcagattc cggacctctt
120cctacctggt ggctaacctt ctctcaactc ttcagctacc cgggatacga
catctatgtc 180gatcccctcc tccaaccgcc cccgtaccgc attgcctacg
gtggtggcca gacgggtccc 240gtctatgata tcaacagcaa ggatatcgcc
tgccagcgcg tccacagccc cgctccgggt 300ctgattgccc aggctcgcgc
gggcagcaac atcaccttct ggtggtcgcg gtggctgtac 360agccacaagg
gtcccatctc ggcatggatg gctccgtatg agggcgacat tgccaatgtg
420gacgtcaacc agctcgagtt cttcaagatt ggcgaggagt tccacgatga
gaccggcaag 480tgggcgacgg agaagctggt ggacgacccc gagggcaagt
ggaccgtcaa gatccccgcc 540gatatcaagc ccggtctcta tgtcgtgcgg
aacgaggtaa gtttcatccg tcccaaaaaa 600ggggtcccat cccatgcatg
gtgcatgccc agtctaatca tcatctcccg gatagatcat 660cgccctccac
ttcgccgtcc gcatgcctcc cttctttgcc gccttcaccc ccctcggacc
720gcagttctac atgacctgct tcgccttcaa catcaccggc gacggcacgg
ccactcccca 780gggctacaag ttccctggcg cctacagcaa ggacgatccg
gccctgtggt gggatctgga 840ggagaacaag aacccgtacc ccggcgccgg
ccccaagccc cacgtctcgg cctacgatgt 900cgacctcgtc cccaacgagt
tgtacatcgt cagcccgacg aacaacgcga cggctgatga 960gctctactgg
gaggcccaga ggcaggcgct tgctgcccag gcggcgacga cggagtactt
1020tgactcgatt ggtggctaa 1039136298PRTHumicola insolens 136Met Arg
Leu Pro Gln Val Ala Ser Val Leu Ala Leu Ala Ala Gln Val1 5 10 15His
Gly His Gly Tyr Ile Tyr Arg Val Thr Ala Asp Asn Ile Val Tyr 20 25
30Pro Gly Tyr Asp Ile Tyr Val Asp Pro Leu Leu Gln Pro Pro Pro Tyr
35 40 45Arg Ile Ala Tyr Gly Gly Gly Gln Thr Gly Pro Val Tyr Asp Ile
Asn 50 55 60Ser Lys Asp Ile Ala Cys Gln Arg Val His Ser Pro Ala Pro
Gly Leu65 70 75 80Ile Ala Gln Ala Arg Ala Gly Ser Asn Ile Thr Phe
Trp Trp Ser Arg 85 90 95Trp Leu Tyr Ser His Lys Gly Pro Ile Ser Ala
Trp Met Ala Pro Tyr 100 105 110Glu Gly Asp Ile Ala Asn Val Asp Val
Asn Gln Leu Glu Phe Phe Lys 115 120 125Ile Gly Glu Glu Phe His Asp
Glu Thr Gly Lys Trp Ala Thr Glu Lys 130 135 140Leu Val Asp Asp Pro
Glu Gly Lys Trp Thr Val Lys Ile Pro Ala Asp145 150 155 160Ile Lys
Pro Gly Leu Tyr Val Val Arg Asn Glu Ile Ile Ala Leu His 165 170
175Phe Ala Val Arg Met Pro Pro Phe Phe Ala Ala Phe Thr Pro Leu Gly
180 185 190Pro Gln Phe Tyr Met Thr Cys Phe Ala Phe Asn Ile Thr Gly
Asp Gly 195 200 205Thr Ala Thr Pro Gln Gly Tyr Lys Phe Pro Gly Ala
Tyr Ser Lys Asp 210 215 220Asp Pro Ala Leu Trp Trp Asp Leu Glu Glu
Asn Lys Asn Pro Tyr Pro225 230 235 240Gly Ala Gly Pro Lys Pro His
Val Ser Ala Tyr Asp Val Asp Leu Val 245 250 255Pro Asn Glu Leu Tyr
Ile Val Ser Pro Thr Asn Asn Ala Thr Ala Asp 260
265 270Glu Leu Tyr Trp Glu Ala Gln Arg Gln Ala Leu Ala Ala Gln Ala
Ala 275 280 285Thr Thr Glu Tyr Phe Asp Ser Ile Gly Gly 290
2951371025DNAHumicola insolens 137atgcacgtcc agtctctcct tgccggagcg
ctcgctctgg ctccgtcggc gtctgctcac 60ttcctcttcc cgcacctgat gctgaacggt
gtccgcacgg gagcctacga gtatgtccgg 120gagcacgact tcggcttcat
gccgcacaac aacgactgga tcaactcgcc cgatttccgt 180tgcaacgagg
ggtcctggcg tcatcgccgc gagcccaaga ccgccgtagt cactgccggc
240gttgacgtcg tgggcttcaa cctgcacctg gactttgacc tgtaccatcc
gggccccgtg 300acggtaagca catctgagtc agaacatacc tccctgtgac
gtagactaat gagtctctta 360ccgcagatct atctctcccg cgcccccggc
gacgtgcgtg actacgacgg atctggtgac 420tggttcaagg tgtaccagct
gggcacccgc caacccttca acggcactga cgagggctgg 480gccacttgga
agatgaagaa ctggcagttc cgcctgcccg ctgagatccc ggcgggcgag
540tacctgatgc gcatcgagca gatgagcgtg caccctcctt accgccagaa
ggagtggtac 600gtgcagtgcg cccacctaaa gatcaacagc aactacaacg
gccccgcgcc cggcccgacc 660atcaagattc ccggagggta caagatcagc
gatcctgcga ttcaatatga ccagtgggcg 720cagccgccgc cgacgtacgc
gcccatgccg ggaccgccgc tgtggcccaa caacaatcct 780cagcagggca
acccgaatca gggcggaaat aacggcggtg gcaaccaggg cggcggcaat
840ggtggctgca ccgttccgaa gtggtatgta gagttcttca ctattatcat
gagatgcagc 900gttggacttg tgcttacacc tagaacaggg gccaatgcgg
tggtcagggt tacagcgggt 960gcaggaactg cgagtctggc tcgacatgcc
gtgcccagaa cgactggtac tcgcagtgcc 1020tgtaa 1025138298PRTHumicola
insolens 138Met His Val Gln Ser Leu Leu Ala Gly Ala Leu Ala Leu Ala
Pro Ser1 5 10 15Ala Ser Ala His Phe Leu Phe Pro His Leu Met Leu Asn
Gly Val Arg 20 25 30Thr Gly Ala Tyr Glu Tyr Val Arg Glu His Asp Phe
Gly Phe Met Pro 35 40 45His Asn Asn Asp Trp Ile Asn Ser Pro Asp Phe
Arg Cys Asn Glu Gly 50 55 60Ser Trp Arg His Arg Arg Glu Pro Lys Thr
Ala Val Val Thr Ala Gly65 70 75 80Val Asp Val Val Gly Phe Asn Leu
His Leu Asp Phe Asp Leu Tyr His 85 90 95Pro Gly Pro Val Thr Ile Tyr
Leu Ser Arg Ala Pro Gly Asp Val Arg 100 105 110Asp Tyr Asp Gly Ser
Gly Asp Trp Phe Lys Val Tyr Gln Leu Gly Thr 115 120 125Arg Gln Pro
Phe Asn Gly Thr Asp Glu Gly Trp Ala Thr Trp Lys Met 130 135 140Lys
Asn Trp Gln Phe Arg Leu Pro Ala Glu Ile Pro Ala Gly Glu Tyr145 150
155 160Leu Met Arg Ile Glu Gln Met Ser Val His Pro Pro Tyr Arg Gln
Lys 165 170 175Glu Trp Tyr Val Gln Cys Ala His Leu Lys Ile Asn Ser
Asn Tyr Asn 180 185 190Gly Pro Ala Pro Gly Pro Thr Ile Lys Ile Pro
Gly Gly Tyr Lys Ile 195 200 205Ser Asp Pro Ala Ile Gln Tyr Asp Gln
Trp Ala Gln Pro Pro Pro Thr 210 215 220Tyr Ala Pro Met Pro Gly Pro
Pro Leu Trp Pro Asn Asn Asn Pro Gln225 230 235 240Gln Gly Asn Pro
Asn Gln Gly Gly Asn Asn Gly Gly Gly Asn Gln Gly 245 250 255Gly Gly
Asn Gly Gly Cys Thr Val Pro Lys Trp Gly Gln Cys Gly Gly 260 265
270Gln Gly Tyr Ser Gly Cys Arg Asn Cys Glu Ser Gly Ser Thr Cys Arg
275 280 285Ala Gln Asn Asp Trp Tyr Ser Gln Cys Leu 290
2951391035DNAHumicola insolens 139atgccaccac cactactggc caccgtcctc
tccttgctag ccctcacccg cggcgccctt 60tcccattccc acctagccca cgtcatcatc
aacggccagc tctaccacgg cttcgaccca 120cgtccaaacc aaaacaacca
tccagcccgt gtcggctggt ccacgaccgc cacagatgac 180ggcttcgtca
ccccgggcaa ttactcccat cccgacatca tctgccaccg cggcggcgtc
240agcccgcgcg cccacgctcc cgtcaccgcc ggcggcaagg tccaggtcca
atggaacggc 300tggccgatcg gacacgtcgg gccgatcctg acctacatcg
cgccgtgcgg cggactgccg 360ggcgccgaag aagggtgtac gggcgtggac
aaaaccgacc tgcggtggac caagatcgac 420gactcgatgc cgccgttccg
gtttaccgac gccaccaagc cagtctctgg cagagcgcag 480ttcccgatag
gccaggtctg ggcgacggat gcgctggtcg aggcgaataa tagctggtcg
540gtggtcattc ccaggaatat cccgccgggg ccgtacgttt tgaggcagga
gattgtggcc 600ctgcattacg cggcgaagtt gaacggggcg cagaactatc
cgttgtgtct gaacctctgg 660gtggaaaagg ggcagcagga tcagggagag
cccttcaaat tcgatgctta cgacgcgagg 720gagttttaca gcgaggacca
tccgggtgtg ttgattgatg ttatgacgat ggttgggccg 780agagccgtgt
accggatacc tggaccgacc gtggccagtg gtgccacgag aattccgcac
840tcattgcaga cgagcgccga gacgtgggtg gaagggacgc cggtggccgt
gacgagggcg 900acggaaacgg ttcagatgga gataactacg acacctgcag
gtcagggagc tggtgtgagg 960acagctaccc ctgccatgcc aacaccaaca
gtgacgaaga ggtggaaggg aagatttgag 1020atgggtaggc catga
1035140344PRTHumicola insolens 140Met Pro Pro Pro Leu Leu Ala Thr
Val Leu Ser Leu Leu Ala Leu Thr1 5 10 15Arg Gly Ala Leu Ser His Ser
His Leu Ala His Val Ile Ile Asn Gly 20 25 30Gln Leu Tyr His Gly Phe
Asp Pro Arg Pro Asn Gln Asn Asn His Pro 35 40 45Ala Arg Val Gly Trp
Ser Thr Thr Ala Thr Asp Asp Gly Phe Val Thr 50 55 60Pro Gly Asn Tyr
Ser His Pro Asp Ile Ile Cys His Arg Gly Gly Val65 70 75 80Ser Pro
Arg Ala His Ala Pro Val Thr Ala Gly Gly Lys Val Gln Val 85 90 95Gln
Trp Asn Gly Trp Pro Ile Gly His Val Gly Pro Ile Leu Thr Tyr 100 105
110Ile Ala Pro Cys Gly Gly Leu Pro Gly Ala Glu Glu Gly Cys Thr Gly
115 120 125Val Asp Lys Thr Asp Leu Arg Trp Thr Lys Ile Asp Asp Ser
Met Pro 130 135 140Pro Phe Arg Phe Thr Asp Ala Thr Lys Pro Val Ser
Gly Arg Ala Gln145 150 155 160Phe Pro Ile Gly Gln Val Trp Ala Thr
Asp Ala Leu Val Glu Ala Asn 165 170 175Asn Ser Trp Ser Val Val Ile
Pro Arg Asn Ile Pro Pro Gly Pro Tyr 180 185 190Val Leu Arg Gln Glu
Ile Val Ala Leu His Tyr Ala Ala Lys Leu Asn 195 200 205Gly Ala Gln
Asn Tyr Pro Leu Cys Leu Asn Leu Trp Val Glu Lys Gly 210 215 220Gln
Gln Asp Gln Gly Glu Pro Phe Lys Phe Asp Ala Tyr Asp Ala Arg225 230
235 240Glu Phe Tyr Ser Glu Asp His Pro Gly Val Leu Ile Asp Val Met
Thr 245 250 255Met Val Gly Pro Arg Ala Val Tyr Arg Ile Pro Gly Pro
Thr Val Ala 260 265 270Ser Gly Ala Thr Arg Ile Pro His Ser Leu Gln
Thr Ser Ala Glu Thr 275 280 285Trp Val Glu Gly Thr Pro Val Ala Val
Thr Arg Ala Thr Glu Thr Val 290 295 300Gln Met Glu Ile Thr Thr Thr
Pro Ala Gly Gln Gly Ala Gly Val Arg305 310 315 320Thr Ala Thr Pro
Ala Met Pro Thr Pro Thr Val Thr Lys Arg Trp Lys 325 330 335Gly Arg
Phe Glu Met Gly Arg Pro 3401411057DNAHumicola insolens
141atgaagtccc tgacctacgc cgcgctggcc gccctctggg cccagcagac
cgctgctcat 60gccaccttcc agcaactctg ggtcgacggc gtcgactacg gcagtcagtg
cgcccgcctg 120ccgccgtcca actcccccat cgccagcgtc acctcgaccg
ccatgcgctg caacaacggt 180ccccgcgctg ccgccaagtg ccccgtcaag
gctggcggca ccgtcaccat cgagatgcac 240caggttggtt tccttgaagt
gttcccctac cacatataca gaccgtagct aacacaccca 300tccttagcaa
cccggtgacc ggtcctgcaa ccaggacgcc attggcggtg cccaccacgg
360ccccgtgatg gtgtacatgt ccaaggtctc tgatgccttc accgccgacg
gctcgtcagg 420ctggttcaag atcttccagg acggctgggc caagaacccc
aacggccgcg ttggcgacga 480cgacttctgg ggcaccaagg acctcaacac
ctgctgcggc aagatgaacg tcaagatccc 540cgccgacatc gcccccggcg
actacctgct ccgcgccgag gccatcgcgc tgcacgccgc 600cggccccagc
ggtggcgccc agccctacgt cacctgctac cagctcaccg tcacgggcgg
660cggcaacgcc aacccgccca ccgtcaactt ccccggcgcc tacagcgagc
gtgaccctgg 720catcgccgtc agcatccacg gcgctctgtc caactacgtc
gtccccggtc ctccggtcta 780ctcgggcggc agcgagaagc gcgctggcag
cccctgcgag ggctgcgagg ccacctgcaa 840ggtcggctcg agccccagcc
agactcttgc tccttccaac ccggccccga cctctcccgc 900caacggcggc
ggcaacaacg gtggtggcaa cactggcggc ggctgcaccg tgcccaagtg
960gcagcagtgc ggcggccagg gctactcggg ctgcaccgtc tgcgagtctg
gctcgacttg 1020ccgcgctcag aaccagtggt actctcagtg cgtgtaa
1057142330PRTHumicola insolens 142Met Lys Ser Leu Thr Tyr Ala Ala
Leu Ala Ala Leu Trp Ala Gln Gln1 5 10 15Thr Ala Ala His Ala Thr Phe
Gln Gln Leu Trp Val Asp Gly Val Asp 20 25 30Tyr Gly Ser Gln Cys Ala
Arg Leu Pro Pro Ser Asn Ser Pro Ile Ala 35 40 45Ser Val Thr Ser Thr
Ala Met Arg Cys Asn Asn Gly Pro Arg Ala Ala 50 55 60Ala Lys Cys Pro
Val Lys Ala Gly Gly Thr Val Thr Ile Glu Met His65 70 75 80Gln Gln
Pro Gly Asp Arg Ser Cys Asn Gln Asp Ala Ile Gly Gly Ala 85 90 95His
His Gly Pro Val Met Val Tyr Met Ser Lys Val Ser Asp Ala Phe 100 105
110Thr Ala Asp Gly Ser Ser Gly Trp Phe Lys Ile Phe Gln Asp Gly Trp
115 120 125Ala Lys Asn Pro Asn Gly Arg Val Gly Asp Asp Asp Phe Trp
Gly Thr 130 135 140Lys Asp Leu Asn Thr Cys Cys Gly Lys Met Asn Val
Lys Ile Pro Ala145 150 155 160Asp Ile Ala Pro Gly Asp Tyr Leu Leu
Arg Ala Glu Ala Ile Ala Leu 165 170 175His Ala Ala Gly Pro Ser Gly
Gly Ala Gln Pro Tyr Val Thr Cys Tyr 180 185 190Gln Leu Thr Val Thr
Gly Gly Gly Asn Ala Asn Pro Pro Thr Val Asn 195 200 205Phe Pro Gly
Ala Tyr Ser Glu Arg Asp Pro Gly Ile Ala Val Ser Ile 210 215 220His
Gly Ala Leu Ser Asn Tyr Val Val Pro Gly Pro Pro Val Tyr Ser225 230
235 240Gly Gly Ser Glu Lys Arg Ala Gly Ser Pro Cys Glu Gly Cys Glu
Ala 245 250 255Thr Cys Lys Val Gly Ser Ser Pro Ser Gln Thr Leu Ala
Pro Ser Asn 260 265 270Pro Ala Pro Thr Ser Pro Ala Asn Gly Gly Gly
Asn Asn Gly Gly Gly 275 280 285Asn Thr Gly Gly Gly Cys Thr Val Pro
Lys Trp Gln Gln Cys Gly Gly 290 295 300Gln Gly Tyr Ser Gly Cys Thr
Val Cys Glu Ser Gly Ser Thr Cys Arg305 310 315 320Ala Gln Asn Gln
Trp Tyr Ser Gln Cys Val 325 330143772DNAHumicola insolens
143atgaagctcc tcctccccgc cctcctggct ctggccgccg agtccgtctc
ggcgcactac 60atcttccaac aactcaccgt cgccggcacc aagtaccccg tgtggaagta
catccggcgc 120aacagcaatc cggcgtggct tcaaaacggc cctgtgaccg
acctcgcctc gaccgacctg 180cgctgcaacg tgggcgggca ggtcagcaac
ggcaccgaga ctctcacggt ccgcgcgggc 240gaccagttca cgttccacct
cgacacggcg gtgtaccacc agggcccgac ctcgctgtac 300atgtcgcgcg
ctccgggcaa ggtggaggac tatgatggca gcgggccgtg gtttaagatt
360tatgattggg ggccgacagg gaataattgg gtcatgaggg gtatggtttc
ccctattaat 420tattattatt gtttacttgg ggcatcatct ggtggtggtg
ctggtgacga tgataagagt 480gatggagaag gacctggctg acgacctaaa
aacccgatca gattcgtaca cgtacaacat 540cccccgctgc atccccgacg
gcgagtatct cctgcgcatc cagcagctgg gtctgcacaa 600tccgggcgcc
gcgccgcagt tctacatcag ctgcgcccag atcaaggtca ccggcggcgg
660cactaccaac ccgaccccca cggctctgat tccgggagcg ttcagggcta
cggatccggg 720atacactgtc aacgtaagtc aaactttgag caactccata
tcaacctcgt ga 772144216PRTHumicola insolens 144Met Lys Leu Leu Leu
Pro Ala Leu Leu Ala Leu Ala Ala Glu Ser Val1 5 10 15Ser Ala His Tyr
Ile Phe Gln Gln Leu Thr Val Ala Gly Thr Lys Tyr 20 25 30Pro Val Trp
Lys Tyr Ile Arg Arg Asn Ser Asn Pro Ala Trp Leu Gln 35 40 45Asn Gly
Pro Val Thr Asp Leu Ala Ser Thr Asp Leu Arg Cys Asn Val 50 55 60Gly
Gly Gln Val Ser Asn Gly Thr Glu Thr Leu Thr Val Arg Ala Gly65 70 75
80Asp Gln Phe Thr Phe His Leu Asp Thr Ala Val Tyr His Gln Gly Pro
85 90 95Thr Ser Leu Tyr Met Ser Arg Ala Pro Gly Lys Val Glu Asp Tyr
Asp 100 105 110Gly Ser Gly Pro Trp Phe Lys Ile Tyr Asp Trp Gly Pro
Thr Gly Asn 115 120 125Asn Trp Val Met Arg Asp Ser Tyr Thr Tyr Asn
Ile Pro Arg Cys Ile 130 135 140Pro Asp Gly Glu Tyr Leu Leu Arg Ile
Gln Gln Leu Gly Leu His Asn145 150 155 160Pro Gly Ala Ala Pro Gln
Phe Tyr Ile Ser Cys Ala Gln Ile Lys Val 165 170 175Thr Gly Gly Gly
Thr Thr Asn Pro Thr Pro Thr Ala Leu Ile Pro Gly 180 185 190Ala Phe
Arg Ala Thr Asp Pro Gly Tyr Thr Val Asn Val Ser Gln Thr 195 200
205Leu Ser Asn Ser Ile Ser Thr Ser 210 2151451536DNAHumicola
insolens 145atgcgttctg tttcccttct tgcggccgct ttcgcgccgc tggctacggc
acacacggtc 60tttacagctc ttttcatcaa caatgtccac cagggcgacg gcacttgcgt
ccgtatggct 120aagcagggca acctcgccac ccatcccgtc agtctgaaca
gcaatgagat ggcctgcggt 180gggtaggccc cgttcctcga gcagctgatc
tcgaactaac atgttgattc ttgaactcca 240ggtcgcgatg gccaacaacc
agtggcattt acttgcccag cacctgcggg agccaagctg 300accttattgt
ttcgtatgtg ggcagatggc tctcagccag gttccatcga caagtctcac
360gttggtccca tgtccatcta cctcaagaaa gtctcagata tgaacaccga
ctcggccgca 420gggcccgggt ggttcaagat ctggagtgag ggctacgacg
ctgcgacgaa gaaatgggcc 480acggagaaac tcatcgccaa caacggtttg
ctcagcgtca acctacctcc cggcctccct 540gcaggctact acctcgcccg
ccacgaaatc gtcactctcc aaaacgtcac caacaacaag 600gccgatccgc
agttctacgt cggctgtgcg cagctgttcg tccaagggtt gggcaccgcc
660gcctccgtgc ctgctgacaa aaccgtttcc atccccggcc atctgaaccc
caacgacccg 720gcgctggtat tcaaccccta tacccaaaac gctgcgacat
acccaagctt cggcccaccg 780ctcttcttcc caaatgctgc ttcggcggga
tcaaacaagg cccagtcaac actcaagcaa 840acctccggcg tcatcccctc
cgactgcctc atcaaaaacg ccaactggtg cggccgtgaa 900gttccagact
ataccaacga ggcgggatgc tggacggcgg cggggaactg ttgggagcag
960gctgatcaat gctacaagac agccccgcca tcgggccata agggatgcaa
gacctgggag 1020gagcagaagt gcaacgtcat ccagaactcc tgtgaagcga
agaggttttc gggcccgcca 1080aacagggggg tcaagtttgc tgatatggat
gtgaatcagc ttgttccggg ggcgatccct 1140gaagcagtga acgccggtca
gaatggggag gcggttgttg ttgacggcac aacgagctct 1200gcagatgaga
aggcgagtgt ggatttgaca acatcgtctc taccgacgcc gacgcctgcg
1260gctgaagaaa acgggaagga ggatgaaaga ctggctcttg atccgaccct
gacggaggac 1320gagtcgtttt tctcagttga gccaacgtct gagcccactg
gtgttcaggt tgaggtgcct 1380ttgacaactg tggtcctcct tccaacgctc
acctcatctt tgaatccatt gccaaccccg 1440acctcaattt cccagccggc
tcacccggga agaccatgca caggtcgccg tcgtaggccg 1500aggccagggt
ttccgaaaca cccgcgcgat ttttaa 1536146490PRTHumicola insolens 146Met
Arg Ser Val Ser Leu Leu Ala Ala Ala Phe Ala Pro Leu Ala Thr1 5 10
15Ala His Thr Val Phe Thr Ala Leu Phe Ile Asn Asn Val His Gln Gly
20 25 30Asp Gly Thr Cys Val Arg Met Ala Lys Gln Gly Asn Leu Ala Thr
His 35 40 45Pro Val Ser Leu Asn Ser Asn Glu Met Ala Cys Gly Arg Asp
Gly Gln 50 55 60Gln Pro Val Ala Phe Thr Cys Pro Ala Pro Ala Gly Ala
Lys Leu Thr65 70 75 80Leu Leu Phe Arg Met Trp Ala Asp Gly Ser Gln
Pro Gly Ser Ile Asp 85 90 95Lys Ser His Val Gly Pro Met Ser Ile Tyr
Leu Lys Lys Val Ser Asp 100 105 110Met Asn Thr Asp Ser Ala Ala Gly
Pro Gly Trp Phe Lys Ile Trp Ser 115 120 125Glu Gly Tyr Asp Ala Ala
Thr Lys Lys Trp Ala Thr Glu Lys Leu Ile 130 135 140Ala Asn Asn Gly
Leu Leu Ser Val Asn Leu Pro Pro Gly Leu Pro Ala145 150 155 160Gly
Tyr Tyr Leu Ala Arg His Glu Ile Val Thr Leu Gln Asn Val Thr 165 170
175Asn Asn Lys Ala Asp Pro Gln Phe Tyr Val Gly Cys Ala Gln Leu Phe
180 185 190Val Gln Gly Leu Gly Thr Ala Ala Ser Val Pro Ala Asp Lys
Thr Val 195 200 205Ser Ile Pro Gly His Leu Asn Pro Asn Asp Pro Ala
Leu Val Phe Asn 210 215 220Pro Tyr Thr Gln Asn Ala Ala Thr Tyr Pro
Ser Phe Gly Pro Pro Leu225 230 235 240Phe Phe Pro Asn Ala Ala Ser
Ala Gly Ser Asn Lys Ala Gln Ser Thr 245 250 255Leu Lys Gln Thr Ser
Gly Val Ile Pro Ser Asp Cys Leu Ile Lys Asn 260 265 270Ala Asn Trp
Cys Gly Arg Glu Val Pro Asp Tyr Thr Asn Glu Ala Gly 275 280 285Cys
Trp Thr Ala Ala Gly Asn Cys Trp Glu Gln Ala Asp Gln Cys Tyr 290
295 300Lys Thr Ala Pro Pro Ser Gly His Lys Gly Cys Lys Thr Trp Glu
Glu305 310 315 320Gln Lys Cys Asn Val Ile Gln Asn Ser Cys Glu Ala
Lys Arg Phe Ser 325 330 335Gly Pro Pro Asn Arg Gly Val Lys Phe Ala
Asp Met Asp Val Asn Gln 340 345 350Leu Val Pro Gly Ala Ile Pro Glu
Ala Val Asn Ala Gly Gln Asn Gly 355 360 365Glu Ala Val Val Val Asp
Gly Thr Thr Ser Ser Ala Asp Glu Lys Ala 370 375 380Ser Val Asp Leu
Thr Thr Ser Ser Leu Pro Thr Pro Thr Pro Ala Ala385 390 395 400Glu
Glu Asn Gly Lys Glu Asp Glu Arg Leu Ala Leu Asp Pro Thr Leu 405 410
415Thr Glu Asp Glu Ser Phe Phe Ser Val Glu Pro Thr Ser Glu Pro Thr
420 425 430Gly Val Gln Val Glu Val Pro Leu Thr Thr Val Val Leu Leu
Pro Thr 435 440 445Leu Thr Ser Ser Leu Asn Pro Leu Pro Thr Pro Thr
Ser Ile Ser Gln 450 455 460Pro Ala His Pro Gly Arg Pro Cys Thr Gly
Arg Arg Arg Arg Pro Arg465 470 475 480Pro Gly Phe Pro Lys His Pro
Arg Asp Phe 485 490147921DNAHumicola insolens 147atgttcttcc
gcaacgccgc cactcttgct ctggcctacg ccaccaccgg cgtctcggcc 60cacgcgctca
tgtacggcgt ctgggtcaac ggcgtcgacc aaggcgacgg ccgcaacgtc
120tacatccgca cgccccccaa caacagcccg gtcaaagacc tcgccagccc
ggacatcgtc 180tgcaacgtca acggcgggcg cgccgttccg gacttcgtcc
aggcctcggc gggggacacc 240ctcaccttcg agtggctgca caacacccgc
ggcgacgaca tcatcgaccg ctcccacctc 300ggccccatca tcacctacat
cgcccctttt accacgggca acccgacggg gcccgtctgg 360accaaaatcg
ccgaacaggg cttcaaccct tccacccgcc gctgggccgt cgacgatctg
420atcgacaacg gcggcaagac cgacttcgtc ctgcccgcgt ccctcgcgcc
gggcaggtac 480atcatccggc aggagatcat cgcgcaccac gagtccgaaa
ccacgttcga atccaacccg 540gcgcggggtg cccagttcta cccgtcgtgc
gtgcagatcc aagtctcttc tggctcgggc 600accgccgtgc cggatcagaa
ctttgacttc aacacgggct acacgtacgc cgaccccggc 660atccacttca
acatctacac ctcgttcaac agctactcca tccccggccc ggaggtttgg
720acgggcgcta gcaccggcgg cggcaacggc aacggcaacg gcaacggcaa
tgccacgcct 780acgcagccta ctcccactcc cactgtcact cccactccca
tcgagaccgc ccagccggtt 840accacgacga ccacctcgac ccggccgttc
cctacccgct gccctggccg ccgcctcaag 900cgtgaggagc ccaaggcttg a
921148306PRTHumicola insolens 148Met Phe Phe Arg Asn Ala Ala Thr
Leu Ala Leu Ala Tyr Ala Thr Thr1 5 10 15Gly Val Ser Ala His Ala Leu
Met Tyr Gly Val Trp Val Asn Gly Val 20 25 30Asp Gln Gly Asp Gly Arg
Asn Val Tyr Ile Arg Thr Pro Pro Asn Asn 35 40 45Ser Pro Val Lys Asp
Leu Ala Ser Pro Asp Ile Val Cys Asn Val Asn 50 55 60Gly Gly Arg Ala
Val Pro Asp Phe Val Gln Ala Ser Ala Gly Asp Thr65 70 75 80Leu Thr
Phe Glu Trp Leu His Asn Thr Arg Gly Asp Asp Ile Ile Asp 85 90 95Arg
Ser His Leu Gly Pro Ile Ile Thr Tyr Ile Ala Pro Phe Thr Thr 100 105
110Gly Asn Pro Thr Gly Pro Val Trp Thr Lys Ile Ala Glu Gln Gly Phe
115 120 125Asn Pro Ser Thr Arg Arg Trp Ala Val Asp Asp Leu Ile Asp
Asn Gly 130 135 140Gly Lys Thr Asp Phe Val Leu Pro Ala Ser Leu Ala
Pro Gly Arg Tyr145 150 155 160Ile Ile Arg Gln Glu Ile Ile Ala His
His Glu Ser Glu Thr Thr Phe 165 170 175Glu Ser Asn Pro Ala Arg Gly
Ala Gln Phe Tyr Pro Ser Cys Val Gln 180 185 190Ile Gln Val Ser Ser
Gly Ser Gly Thr Ala Val Pro Asp Gln Asn Phe 195 200 205Asp Phe Asn
Thr Gly Tyr Thr Tyr Ala Asp Pro Gly Ile His Phe Asn 210 215 220Ile
Tyr Thr Ser Phe Asn Ser Tyr Ser Ile Pro Gly Pro Glu Val Trp225 230
235 240Thr Gly Ala Ser Thr Gly Gly Gly Asn Gly Asn Gly Asn Gly Asn
Gly 245 250 255Asn Ala Thr Pro Thr Gln Pro Thr Pro Thr Pro Thr Val
Thr Pro Thr 260 265 270Pro Ile Glu Thr Ala Gln Pro Val Thr Thr Thr
Thr Thr Ser Thr Arg 275 280 285Pro Phe Pro Thr Arg Cys Pro Gly Arg
Arg Leu Lys Arg Glu Glu Pro 290 295 300Lys Ala3051491092DNAHumicola
insolens 149atggctcatc catgggcacg ttgcgtctat acagccatct ggctcgctgc
ctccgcttct 60ggacgtaggt acaagactcc ggcagtgcca tttatgaacc cacaacgtgg
actggtcccg 120tgctaacaca tcacagactc gcgcgtttgg agtgtctcgg
tcaatggacg ctaccaggga 180ccgggtgttg atgactacct gcgcgcaccg
ccaagtgact ctccggtggt ggacctggac 240tcaccaaccc tcaactgcaa
tgtcaatgga aacaagcctg ttccagggtt tgttgaggtg 300tctgcgggag
attctctgga atggaagtgg tactacatca acccgtacaa cccaagcgac
360atgatcatcg cggcagaaca ccgcggaccg atcatcacct acatcacgaa
ttacaccgat 420ggccagcctc aaggagctgt ctggaccaag attgatcacg
aaggctacga tcctgtgaca 480gaccggttcg ccgtcgacaa cttgatcgcc
aacaggggat ggaaagcaat caagcttccc 540atgctcgccg acgggaagta
catcctgcga caggagatca tcgcactcca cagcgcacac 600aaccaaggcg
gggcccagct gtatccgaac tgcattcaga tcaaggtcgt tggtggcaag
660ggaagcgcgg tgcccaacca gaactttgat ctcaacaagg ggtacacatc
cgatcacccg 720ggacttcggt tcaacctgtg gcaaccattc aacaattaca
ccattcccgg tcctgaggtc 780tggaagggag ttgtggttgc gagcaatggt
acaacgaaca gcaccacaaa tctcaccaac 840aacaccggca ccggttttgc
gaacagcact atggccactg gtgaaacaag gaccgagagg 900agttttatga
cacttaccgc atcacattca gacactggcg tccccgccaa atctcatact
960gtggctgtaa gctggacaac atccgccgcc gttgttgggt ctccgattag
cgttaccaca 1020actttcagtt cctttaccac aacaccggtt ccgacgaact
ctaccggtgc ttatctctac 1080cggtacaagt ga 1092150339PRTHumicola
insolens 150Met Ala His Pro Trp Ala Arg Cys Val Tyr Thr Ala Ile Trp
Leu Ala1 5 10 15Ala Ser Ala Ser Gly His Ser Arg Val Trp Ser Val Ser
Val Asn Gly 20 25 30Arg Tyr Gln Gly Pro Gly Val Asp Asp Tyr Leu Arg
Ala Pro Pro Ser 35 40 45Asp Ser Pro Val Val Asp Leu Asp Ser Pro Thr
Leu Asn Cys Asn Val 50 55 60Asn Gly Asn Lys Pro Val Pro Gly Phe Val
Glu Val Ser Ala Gly Asp65 70 75 80Ser Leu Glu Trp Lys Trp Tyr Tyr
Ile Asn Pro Tyr Asn Pro Ser Asp 85 90 95Met Ile Ile Ala Ala Glu His
Arg Gly Pro Ile Ile Thr Tyr Ile Thr 100 105 110Asn Tyr Thr Asp Gly
Gln Pro Gln Gly Ala Val Trp Thr Lys Ile Asp 115 120 125His Glu Gly
Tyr Asp Pro Val Thr Asp Arg Phe Ala Val Asp Asn Leu 130 135 140Ile
Ala Asn Arg Gly Trp Lys Ala Ile Lys Leu Pro Met Leu Ala Asp145 150
155 160Gly Lys Tyr Ile Leu Arg Gln Glu Ile Ile Ala Leu His Ser Ala
His 165 170 175Asn Gln Gly Gly Ala Gln Leu Tyr Pro Asn Cys Ile Gln
Ile Lys Val 180 185 190Val Gly Gly Lys Gly Ser Ala Val Pro Asn Gln
Asn Phe Asp Leu Asn 195 200 205Lys Gly Tyr Thr Ser Asp His Pro Gly
Leu Arg Phe Asn Leu Trp Gln 210 215 220Pro Phe Asn Asn Tyr Thr Ile
Pro Gly Pro Glu Val Trp Lys Gly Val225 230 235 240Val Val Ala Ser
Asn Gly Thr Thr Asn Ser Thr Thr Asn Leu Thr Asn 245 250 255Asn Thr
Gly Thr Gly Phe Ala Asn Ser Thr Met Ala Thr Gly Glu Thr 260 265
270Arg Thr Glu Arg Ser Phe Met Thr Leu Thr Ala Ser His Ser Asp Thr
275 280 285Gly Val Pro Ala Lys Ser His Thr Val Ala Val Ser Trp Thr
Thr Ser 290 295 300Ala Ala Val Val Gly Ser Pro Ile Ser Val Thr Thr
Thr Phe Ser Ser305 310 315 320Phe Thr Thr Thr Pro Val Pro Thr Asn
Ser Thr Gly Ala Tyr Leu Tyr 325 330 335Arg Tyr
Lys1511089DNATrichoderma reesei 151atgatccaga agctttccaa cctccttgtc
accgcactgg cggtggctac tggcgttgtc 60ggacatggac atattaatga cattgtcatc
aacggggtgt ggtatcaggc ctatgatcct 120acaacgtttc catacgagtc
aaaccccccc atagtagtgg gctggacggc tgccgacctt 180gacaacggta
cgtgatcctc atctctatct gtacaacgct catgctaatc caactcaata
240ggcttcgttt cacccgacgc ataccaaaac cctgacatca tctgccacaa
gaatgctacg 300aatgccaagg ggcacgcgtc tgtcaaggcc ggagacacta
ttctcttcca gtgggtgcca 360gttccatggc cgcaccctgg tcccattgtc
gactacctgg ccaactgcaa tggtgactgc 420gagaccgttg acaagacgac
gcttgagttc ttcaagatcg atggcgttgg tctcctcagc 480ggcggggatc
cgggcacctg ggcctcagac gtgctgatct ccaacaacaa cacctgggtc
540gtcaagatcc ccgacaatct tgcgccaggc aattacgtgc tccgccacga
gatcatcgcg 600ttacacagcg ccgggcaggc aaacggcgct cagaactacc
cccagtgctt caacattgcc 660gtctcaggct cgggttctct gcagcccagc
ggcgttctag ggaccgacct ctatcacgcg 720acggaccctg gtgttctcat
caacatctac accagcccgc tcaactacat catccctgga 780cctaccgtgg
tatcaggcct gccaacgagt gttgcccagg ggagctccgc cgcgacggcc
840accgccagcg ccactgttcc tggaggcggt agcggcccga ccagcagaac
cacgacaacg 900gcgaggacga cgcaggcctc aagcaggccc agctctacgc
ctcccgcaac cacgtcggca 960cctgctggcg gcccaaccca gactctgtac
ggccagtgtg gtggcagcgg ttacagcggg 1020cctactcgat gcgcgccgcc
agccacttgc tctaccttga acccctacta cgcccagtgc 1080cttaactag
1089152344PRTTrichoderma reesei 152Met Ile Gln Lys Leu Ser Asn Leu
Leu Val Thr Ala Leu Ala Val Ala1 5 10 15Thr Gly Val Val Gly His Gly
His Ile Asn Asp Ile Val Ile Asn Gly 20 25 30Val Trp Tyr Gln Ala Tyr
Asp Pro Thr Thr Phe Pro Tyr Glu Ser Asn 35 40 45Pro Pro Ile Val Val
Gly Trp Thr Ala Ala Asp Leu Asp Asn Gly Phe 50 55 60Val Ser Pro Asp
Ala Tyr Gln Asn Pro Asp Ile Ile Cys His Lys Asn65 70 75 80Ala Thr
Asn Ala Lys Gly His Ala Ser Val Lys Ala Gly Asp Thr Ile 85 90 95Leu
Phe Gln Trp Val Pro Val Pro Trp Pro His Pro Gly Pro Ile Val 100 105
110Asp Tyr Leu Ala Asn Cys Asn Gly Asp Cys Glu Thr Val Asp Lys Thr
115 120 125Thr Leu Glu Phe Phe Lys Ile Asp Gly Val Gly Leu Leu Ser
Gly Gly 130 135 140Asp Pro Gly Thr Trp Ala Ser Asp Val Leu Ile Ser
Asn Asn Asn Thr145 150 155 160Trp Val Val Lys Ile Pro Asp Asn Leu
Ala Pro Gly Asn Tyr Val Leu 165 170 175Arg His Glu Ile Ile Ala Leu
His Ser Ala Gly Gln Ala Asn Gly Ala 180 185 190Gln Asn Tyr Pro Gln
Cys Phe Asn Ile Ala Val Ser Gly Ser Gly Ser 195 200 205Leu Gln Pro
Ser Gly Val Leu Gly Thr Asp Leu Tyr His Ala Thr Asp 210 215 220Pro
Gly Val Leu Ile Asn Ile Tyr Thr Ser Pro Leu Asn Tyr Ile Ile225 230
235 240Pro Gly Pro Thr Val Val Ser Gly Leu Pro Thr Ser Val Ala Gln
Gly 245 250 255Ser Ser Ala Ala Thr Ala Thr Ala Ser Ala Thr Val Pro
Gly Gly Gly 260 265 270Ser Gly Pro Thr Ser Arg Thr Thr Thr Thr Ala
Arg Thr Thr Gln Ala 275 280 285Ser Ser Arg Pro Ser Ser Thr Pro Pro
Ala Thr Thr Ser Ala Pro Ala 290 295 300Gly Gly Pro Thr Gln Thr Leu
Tyr Gly Gln Cys Gly Gly Ser Gly Tyr305 310 315 320Ser Gly Pro Thr
Arg Cys Ala Pro Pro Ala Thr Cys Ser Thr Leu Asn 325 330 335Pro Tyr
Tyr Ala Gln Cys Leu Asn 3401531398DNAPenicillium emersonii
153atgtttggat tcaagtctac taccttggct gtttggatgc tcagtcttcc
ggcaacatcg 60catgcccaca ctgtcatgac cacactgttt gtcgatggcg tcaaccaggg
agacggtgtc 120tgtatccgta tgaacaagaa cggctctacg tccaatttct
tcgtcagtcc tgtcagtagc 180agggacgttg cttgtggtag ggaaaaagag
caccaatccc tacatgtact ctacttttgt 240tttcaaaaca taagaactga
caagcaaaaa ggaatcgatg gagaaatcgg tgtcgcaaga 300gtctgtccgg
ccaaggcctc gtcgatcctg accttcgagt tccgcgaaca tcccgacaac
360gtgagctctg cacctctcga tccctcacac aagggtcccg cgtcggtgta
cttgaagaag 420gtcgattccg ccatcgccag caacaacgcc gcaggagacg
ggtggttcaa gatctgggaa 480tccgtctacg acgagacgtc agacaaatgg
ggcacgacga agatgatcga gaacgatgga 540cacatctccg ttcagatccc
ggaggagatt gagggagggt actatctcgc gcgaacggag 600cttctggcgc
ttcacgcggc gagctcgaat ccgcccaatc cgcagttctt tgtcggctgc
660gcgcagctct tcatcgagtc gaatgggacc gcaaagccgt cgactgttcg
catcggtgag 720ggtacctata acctgtccat gccgggactg acctacaata
tctgggaaaa gccgctgtcc 780ctgccgtatg cgatggttgg tccgacggtt
tacagagctg gctcgggggc tagctcatca 840gcagtcgctc ccacggcagc
gagtgctact gctgctgcta ccgttacgca ggcggtagct 900ccattaccaa
ctaccagtgc tccaagttca caacaaaatg tcggctcatg cggagttgtt
960gttgcggacg agatagaaaa gcgagacact ctcgttcaga cggaaggact
caagccagaa 1020ggctgcatct ttgtcaatgg taactggtgc ggtttcgaag
tgccttcgta cacggaccaa 1080gacagctgct gggccgtaag tctcttgctt
tcaccttact actttttttt ttctttcgaa 1140agaaaaatga aaaatgtgac
caagctgaca acctgtctcc atataatata gtcatcgaac 1200aactgctggg
cccaatccga cgactgctgg aacaagacac aaccgacggg atacaacctc
1260tgtccgatct ggcaggccaa atgccgagag atctccaacg ggtgcgagaa
agggacttgg 1320acggggcctc ctcatcaggg agaggacatg actccgtcgt
ggccgtcgtt gaggggggaa 1380ttgaagatct ttacctga
1398154408PRTPenicillium emersonii 154Met Phe Gly Phe Lys Ser Thr
Thr Leu Ala Val Trp Met Leu Ser Leu1 5 10 15Pro Ala Thr Ser His Ala
His Thr Val Met Thr Thr Leu Phe Val Asp 20 25 30Gly Val Asn Gln Gly
Asp Gly Val Cys Ile Arg Met Asn Lys Asn Gly 35 40 45Ser Thr Ser Asn
Phe Phe Val Ser Pro Val Ser Ser Arg Asp Val Ala 50 55 60Cys Gly Ile
Asp Gly Glu Ile Gly Val Ala Arg Val Cys Pro Ala Lys65 70 75 80Ala
Ser Ser Ile Leu Thr Phe Glu Phe Arg Glu His Pro Asp Asn Val 85 90
95Ser Ser Ala Pro Leu Asp Pro Ser His Lys Gly Pro Ala Ser Val Tyr
100 105 110Leu Lys Lys Val Asp Ser Ala Ile Ala Ser Asn Asn Ala Ala
Gly Asp 115 120 125Gly Trp Phe Lys Ile Trp Glu Ser Val Tyr Asp Glu
Thr Ser Asp Lys 130 135 140Trp Gly Thr Thr Lys Met Ile Glu Asn Asp
Gly His Ile Ser Val Gln145 150 155 160Ile Pro Glu Glu Ile Glu Gly
Gly Tyr Tyr Leu Ala Arg Thr Glu Leu 165 170 175Leu Ala Leu His Ala
Ala Ser Ser Asn Pro Pro Asn Pro Gln Phe Phe 180 185 190Val Gly Cys
Ala Gln Leu Phe Ile Glu Ser Asn Gly Thr Ala Lys Pro 195 200 205Ser
Thr Val Arg Ile Gly Glu Gly Thr Tyr Asn Leu Ser Met Pro Gly 210 215
220Leu Thr Tyr Asn Ile Trp Glu Lys Pro Leu Ser Leu Pro Tyr Ala
Met225 230 235 240Val Gly Pro Thr Val Tyr Arg Ala Gly Ser Gly Ala
Ser Ser Ser Ala 245 250 255Val Ala Pro Thr Ala Ala Ser Ala Thr Ala
Ala Ala Thr Val Thr Gln 260 265 270Ala Val Ala Pro Leu Pro Thr Thr
Ser Ala Pro Ser Ser Gln Gln Asn 275 280 285Val Gly Ser Cys Gly Val
Val Val Ala Asp Glu Ile Glu Lys Arg Asp 290 295 300Thr Leu Val Gln
Thr Glu Gly Leu Lys Pro Glu Gly Cys Ile Phe Val305 310 315 320Asn
Gly Asn Trp Cys Gly Phe Glu Val Pro Ser Tyr Thr Asp Gln Asp 325 330
335Ser Cys Trp Ala Ser Ser Asn Asn Cys Trp Ala Gln Ser Asp Asp Cys
340 345 350Trp Asn Lys Thr Gln Pro Thr Gly Tyr Asn Leu Cys Pro Ile
Trp Gln 355 360 365Ala Lys Cys Arg Glu Ile Ser Asn Gly Cys Glu Lys
Gly Thr Trp Thr 370 375 380Gly Pro Pro His Gln Gly Glu Asp Met Thr
Pro Ser Trp Pro Ser Leu385 390 395 400Arg Gly Glu Leu Lys Ile Phe
Thr 405155902DNAMalbranchea cinnamomea 155atgagaattg aaaagcttct
aaacgctgcg ctcctcgctg gagcagtatc tgctcacacc 60atcatgacca gcttggttgt
tgatggaacc gaatatcgta tgttcaatct caccttgagc 120tttcttttca
cactagtttg ttgagaatct ggccattttc agctcctgga catgctgtcc
180gcatcccaag ttacaacggg gtatgcatgt ggatgggctc tgtttcctat
taagagcccc 240atgagctgat cgccttgaca tagcccatta ccgatgtcac
ctccaacagc gtggcttgca 300atggtccccc taatcccact acccctagct
ccgagatcat catggtccgg gctggatcga 360ctatccaggg gaaatggaga
cataccgaga ccgacgtgat tgacccaagc cacaaggtat 420gactataccg
ccacgagaga tatccttgac tcacttacac cctctttcta acctctctat
480attagggccc cgtaatggcg
tacctgaaga aagtcgacga cgccatcaac gaccctggaa 540cgggtgacgg
ctggttcaag atttgggaag acggcctgca cgacgatggc acctgggccg
600tcgacgacct catcgccgcc aacggttacc aggacattcc catcccccca
tgtctcgcgg 660acggccagta cttgctgcgg gctgagatca ttgctctgca
tggtgctagc cagccgggtg 720gtgcgcaact gtatatggaa tgcgcccaga
tcggagttgt gggtggctct ggaacagcta 780acccgtccac agttgcgttc
cccggcgcct acaaggccga tgatcctggc atcactgtca 840acatttattg
gccgcctctt gaggaataca tcattcctgg tcctgaccca ttcacctgct 900aa
902156234PRTMalbranchea cinnamomea 156Met Arg Ile Glu Lys Leu Leu
Asn Ala Ala Leu Leu Ala Gly Ala Val1 5 10 15Ser Ala His Thr Ile Met
Thr Ser Leu Val Val Asp Gly Thr Glu Tyr 20 25 30Pro Pro Gly His Ala
Val Arg Ile Pro Ser Tyr Asn Gly Pro Ile Thr 35 40 45Asp Val Thr Ser
Asn Ser Val Ala Cys Asn Gly Pro Pro Asn Pro Thr 50 55 60Thr Pro Ser
Ser Glu Ile Ile Met Val Arg Ala Gly Ser Thr Ile Gln65 70 75 80Gly
Lys Trp Arg His Thr Glu Thr Asp Val Ile Asp Pro Ser His Lys 85 90
95Gly Pro Val Met Ala Tyr Leu Lys Lys Val Asp Asp Ala Ile Asn Asp
100 105 110Pro Gly Thr Gly Asp Gly Trp Phe Lys Ile Trp Glu Asp Gly
Leu His 115 120 125Asp Asp Gly Thr Trp Ala Val Asp Asp Leu Ile Ala
Ala Asn Gly Tyr 130 135 140Gln Asp Ile Pro Ile Pro Pro Cys Leu Ala
Asp Gly Gln Tyr Leu Leu145 150 155 160Arg Ala Glu Ile Ile Ala Leu
His Gly Ala Ser Gln Pro Gly Gly Ala 165 170 175Gln Leu Tyr Met Glu
Cys Ala Gln Ile Gly Val Val Gly Gly Ser Gly 180 185 190Thr Ala Asn
Pro Ser Thr Val Ala Phe Pro Gly Ala Tyr Lys Ala Asp 195 200 205Asp
Pro Gly Ile Thr Val Asn Ile Tyr Trp Pro Pro Leu Glu Glu Tyr 210 215
220Ile Ile Pro Gly Pro Asp Pro Phe Thr Cys225
230157810DNAMalbranchea cinnamomea 157atgcttccga acgcagctgg
tctgcttgta gcaggtgtcg tctctctttc tggagtagcc 60gcacatggac atgtctcgaa
aattttcctg gacggccagg agtaagccaa ttgtgcgtca 120tttttttagt
atccgttggt taatcattga tttgtattcc tcagatacgg tggttggatc
180gccgatgtct atccgtacat gcccgaaccc ccagagacaa ttggctggcc
tacaaatgtg 240accgacaatg gcttcgtgtc ccccgacagg ttcagctctc
cagaaatcat ctgtcaccgc 300gggggcgttc caagcgccat ctcggcgccc
gtttccgcgg gcgggaccgt cgagttggaa 360tggagcacgt ggcccgagag
ccatcatggt cctgtgctca actaccttgc caaggtcgac 420ggcgacttca
gcgacatcgg ccccagcacg ctccagttct tcaagtttga cgagaccggc
480cttgtatccg gctcaaaccc agggtactgg ggtacggatg tgatgctcgc
gaatggccgg 540cggtactcca tgatgatccc aagcaccatc gctcccggga
agtacgtctt gcgccatgag 600ctcgtcgccc tgcagaacgt cggcgccgcc
cagctgtacc cgcagtgtat caacattgaa 660gtgacaagta ataccactgg
aggagacgtc aaccccggcg gtacgcctgc taccgagctc 720tactctcctg
cggaccccgg tttcttgttc aacatctacg aacagtatga ttcgtaccca
780attcccggtc cggatgtttt ccgcgattaa 810158248PRTMalbranchea
cinnamomea 158Met Leu Pro Asn Ala Ala Gly Leu Leu Val Ala Gly Val
Val Ser Leu1 5 10 15Ser Gly Val Ala Ala His Gly His Val Ser Lys Ile
Phe Leu Asp Gly 20 25 30Gln Glu Tyr Gly Gly Trp Ile Ala Asp Val Tyr
Pro Tyr Met Pro Glu 35 40 45Pro Pro Glu Thr Ile Gly Trp Pro Thr Asn
Val Thr Asp Asn Gly Phe 50 55 60Val Ser Pro Asp Arg Phe Ser Ser Pro
Glu Ile Ile Cys His Arg Gly65 70 75 80Gly Val Pro Ser Ala Ile Ser
Ala Pro Val Ser Ala Gly Gly Thr Val 85 90 95Glu Leu Glu Trp Ser Thr
Trp Pro Glu Ser His His Gly Pro Val Leu 100 105 110Asn Tyr Leu Ala
Lys Val Asp Gly Asp Phe Ser Asp Ile Gly Pro Ser 115 120 125Thr Leu
Gln Phe Phe Lys Phe Asp Glu Thr Gly Leu Val Ser Gly Ser 130 135
140Asn Pro Gly Tyr Trp Gly Thr Asp Val Met Leu Ala Asn Gly Arg
Arg145 150 155 160Tyr Ser Met Met Ile Pro Ser Thr Ile Ala Pro Gly
Lys Tyr Val Leu 165 170 175Arg His Glu Leu Val Ala Leu Gln Asn Val
Gly Ala Ala Gln Leu Tyr 180 185 190Pro Gln Cys Ile Asn Ile Glu Val
Thr Ser Asn Thr Thr Gly Gly Asp 195 200 205Val Asn Pro Gly Gly Thr
Pro Ala Thr Glu Leu Tyr Ser Pro Ala Asp 210 215 220Pro Gly Phe Leu
Phe Asn Ile Tyr Glu Gln Tyr Asp Ser Tyr Pro Ile225 230 235 240Pro
Gly Pro Asp Val Phe Arg Asp 245159729DNAMalbranchea cinnamomea
159atggttcagt ttaagctgag cacggcctct cttctggctc ttgcctccta
cgccgccgcc 60cacggctacg tgagctcgat ccaagccgac ggacagacct atcccggcgc
tgatccacac 120aaccccaacc cagagtcccc cggctggcag gcggagaaca
ccgatctggg cttcgtcgag 180ccctccgcct tctcgacccc cgccatcgcc
tgccacaaga acgcgcgggc tccgcccgcc 240cacgcgaccg tccaggccgg
gagcaccatc aagctgacgt ggaacacctg gccggagtcg 300caccacggac
ccgtgctcga ctacatcgcg ccgtgcaacg gcgactgctc tagcgcgtcg
360gcgggctcgc tgaacttcgt caagatcgcc gagaagggcc tgatctccgg
ctccaaccca 420ggcttctggg ccgccgacga gctgatccag aacggcaact
cgtgggaggt caccatcccc 480gcgaacctgg cgccgggcaa gtacgtgctg
cgccacgaga tcatcgcgct gcactcggcc 540ggcaacccca acggcgccca
ggcctacccg cagtgcatca acctcgaggt cactggcggc 600ggctccgcga
ctccctctgg ccagccggcg acttcgttct actcccccaa cgaccccggc
660atcctgttca acctgtacca gtcgttcgac tcctacccga tccccggccc
cgccgtgtgg 720agcggttaa 729160242PRTMalbranchea cinnamomea 160Met
Val Gln Phe Lys Leu Ser Thr Ala Ser Leu Leu Ala Leu Ala Ser1 5 10
15Tyr Ala Ala Ala His Gly Tyr Val Ser Ser Ile Gln Ala Asp Gly Gln
20 25 30Thr Tyr Pro Gly Ala Asp Pro His Asn Pro Asn Pro Glu Ser Pro
Gly 35 40 45Trp Gln Ala Glu Asn Thr Asp Leu Gly Phe Val Glu Pro Ser
Ala Phe 50 55 60Ser Thr Pro Ala Ile Ala Cys His Lys Asn Ala Arg Ala
Pro Pro Ala65 70 75 80His Ala Thr Val Gln Ala Gly Ser Thr Ile Lys
Leu Thr Trp Asn Thr 85 90 95Trp Pro Glu Ser His His Gly Pro Val Leu
Asp Tyr Ile Ala Pro Cys 100 105 110Asn Gly Asp Cys Ser Ser Ala Ser
Ala Gly Ser Leu Asn Phe Val Lys 115 120 125Ile Ala Glu Lys Gly Leu
Ile Ser Gly Ser Asn Pro Gly Phe Trp Ala 130 135 140Ala Asp Glu Leu
Ile Gln Asn Gly Asn Ser Trp Glu Val Thr Ile Pro145 150 155 160Ala
Asn Leu Ala Pro Gly Lys Tyr Val Leu Arg His Glu Ile Ile Ala 165 170
175Leu His Ser Ala Gly Asn Pro Asn Gly Ala Gln Ala Tyr Pro Gln Cys
180 185 190Ile Asn Leu Glu Val Thr Gly Gly Gly Ser Ala Thr Pro Ser
Gly Gln 195 200 205Pro Ala Thr Ser Phe Tyr Ser Pro Asn Asp Pro Gly
Ile Leu Phe Asn 210 215 220Leu Tyr Gln Ser Phe Asp Ser Tyr Pro Ile
Pro Gly Pro Ala Val Trp225 230 235 240Ser Gly1611081DNAMalbranchea
cinnamomea 161atgtcaccct ccttcaagtc cactgccatc ctcggagccg
ttgctctggc cgcccgcgtg 60cgcgcccacg gctacgtgtc tggaatcgtc gttgacggtg
cttagtacgt ctgcatgttc 120ctctcttcgt tacaggtaac atctttcttg
gtattgctcg tgctgaccct tttctttcag 180ccatggcggt tacatcgtcg
acaagtaccc ctacatgccc aacccgcccg atgtggtcgg 240ctggtcgact
acggccacgg acctgggctt cgtcgcccct gacgcctttg gcgacccgga
300catcatctgc caccgggacg gtgcccccgg tgccatccac gccaaagtca
acgccggtgc 360caccatcgag ctgcagtgga acacctggcc cgagagccac
cacggtcccg tcatcgacta 420cctggctaac tgcaacggtg actgctcgtc
cgtcgacaag acctcgctca agttcttcaa 480gatcagcgag gccggcctaa
acgacggctc caacgccccc ggccagtggg cgtccgacga 540tctcattgcc
aacaacaaca gctggactgt gaccatcccc aagtcgatcg ccccgggcaa
600ctacgtgctg cgccacgaga tcatcgccct gcacagcgcc ggcaaccaga
atggcgcgca 660gaactacccc cagtgcttca acctcgagat caccagcaac
ggcagcgaca acccggaggg 720cgtgctggga accgagctgt acaaggccga
cgacccgggc attctgttca acatctacca 780gcccatggac tcgtacccga
ttcccggccc tgctctctac accggcggct cttctccctc 840ccctaatccg
cccacctcta cccagtcgcc tgtgccccag cccacccagt ctcccccatc
900gggcagcaac cccggcaacg gcaacggcga cgacgacaac gacaacggca
acgagacccc 960atccccgtct ctccccgtcg agatccctga cgacctgacc
tcgcgcgagc tactcctcgt 1020ggcccaggag atcattgccc gtctgcttga
gctgcagaat cagctggtcg tctcgaacta 1080a 1081162334PRTMalbranchea
cinnamomea 162Met Ser Pro Ser Phe Lys Ser Thr Ala Ile Leu Gly Ala
Val Ala Leu1 5 10 15Ala Ala Arg Val Arg Ala His Gly Tyr Val Ser Gly
Ile Val Val Asp 20 25 30Gly Ala Tyr His Gly Gly Tyr Ile Val Asp Lys
Tyr Pro Tyr Met Pro 35 40 45Asn Pro Pro Asp Val Val Gly Trp Ser Thr
Thr Ala Thr Asp Leu Gly 50 55 60Phe Val Ala Pro Asp Ala Phe Gly Asp
Pro Asp Ile Ile Cys His Arg65 70 75 80Asp Gly Ala Pro Gly Ala Ile
His Ala Lys Val Asn Ala Gly Ala Thr 85 90 95Ile Glu Leu Gln Trp Asn
Thr Trp Pro Glu Ser His His Gly Pro Val 100 105 110Ile Asp Tyr Leu
Ala Asn Cys Asn Gly Asp Cys Ser Ser Val Asp Lys 115 120 125Thr Ser
Leu Lys Phe Phe Lys Ile Ser Glu Ala Gly Leu Asn Asp Gly 130 135
140Ser Asn Ala Pro Gly Gln Trp Ala Ser Asp Asp Leu Ile Ala Asn
Asn145 150 155 160Asn Ser Trp Thr Val Thr Ile Pro Lys Ser Ile Ala
Pro Gly Asn Tyr 165 170 175Val Leu Arg His Glu Ile Ile Ala Leu His
Ser Ala Gly Asn Gln Asn 180 185 190Gly Ala Gln Asn Tyr Pro Gln Cys
Phe Asn Leu Glu Ile Thr Ser Asn 195 200 205Gly Ser Asp Asn Pro Glu
Gly Val Leu Gly Thr Glu Leu Tyr Lys Ala 210 215 220Asp Asp Pro Gly
Ile Leu Phe Asn Ile Tyr Gln Pro Met Asp Ser Tyr225 230 235 240Pro
Ile Pro Gly Pro Ala Leu Tyr Thr Gly Gly Ser Ser Pro Ser Pro 245 250
255Asn Pro Pro Thr Ser Thr Gln Ser Pro Val Pro Gln Pro Thr Gln Ser
260 265 270Pro Pro Ser Gly Ser Asn Pro Gly Asn Gly Asn Gly Asp Asp
Asp Asn 275 280 285Asp Asn Gly Asn Glu Thr Pro Ser Pro Ser Leu Pro
Val Glu Ile Pro 290 295 300Asp Asp Leu Thr Ser Arg Glu Leu Leu Leu
Val Ala Gln Glu Ile Ile305 310 315 320Ala Arg Leu Leu Glu Leu Gln
Asn Gln Leu Val Val Ser Asn 325 330163875DNAMalbranchea cinnamomea
163atgaagacgc tctctgcggg tctccttgcg cttgccagcg ctgccagcgc
tcactgtccg 60tccagccttc ccttgctctc tcctgacatt cgcgccagca tttccatcag
gtccaataaa 120actaacacac gagccatggg acgacggtca tagacacctt
cccctccctg atcgccaacg 180gcgtcgtcac cggcgaatgg gagtatgtcc
ggcagacgga gaaccattac tcaaacgccc 240ccgtcaccga cgtctccagc
gaggccatcc gctgctacga gaatcccggg cggcccgccg 300caaagaccct
gagcgttgcc gccggctcga ccgtgggctt caccgtctcc cccagcatct
360accacccggg cccactgcag ttctacatgg ccagggtgcc cgacggccag
accgcggact 420cgtgggacgg cagcgggcag gtgtggttca agatcttcga
gcagggaccg cagatcgatc 480cgtcgggatt gacgtggccg agtgacggtg
cgacgagaaa accccctttt tttttttttt 540tttttttttt tccctctcgc
catctgctaa ctgcgagtga actggctcta ggactctccc 600aggtccaagt
caccatcccc agctccctcc cgtcgggcga ctacctgctg cgcgtcgagc
660agattggcct gcactccgcg tcgtccgtca acggcgccca gttctacctc
tcctgcgcac 720agctcaccgt caccggcggc ggaaacggga acccaggccc
gctcgtctcg ttcccgggcg 780cgtacagccc cacggacccg ggtctgctga
tcaacatcta ctggccgatt ccgaccagct 840acgagctgcc cgggccaccg
gtgtggcgcg gttag 875164230PRTMalbranchea cinnamomea 164Met Lys Thr
Leu Ser Ala Gly Leu Leu Ala Leu Ala Ser Ala Ala Ser1 5 10 15Ala His
Tyr Thr Phe Pro Ser Leu Ile Ala Asn Gly Val Val Thr Gly 20 25 30Glu
Trp Glu Tyr Val Arg Gln Thr Glu Asn His Tyr Ser Asn Ala Pro 35 40
45Val Thr Asp Val Ser Ser Glu Ala Ile Arg Cys Tyr Glu Asn Pro Gly
50 55 60Arg Pro Ala Ala Lys Thr Leu Ser Val Ala Ala Gly Ser Thr Val
Gly65 70 75 80Phe Thr Val Ser Pro Ser Ile Tyr His Pro Gly Pro Leu
Gln Phe Tyr 85 90 95Met Ala Arg Val Pro Asp Gly Gln Thr Ala Asp Ser
Trp Asp Gly Ser 100 105 110Gly Gln Val Trp Phe Lys Ile Phe Glu Gln
Gly Pro Gln Ile Asp Pro 115 120 125Ser Gly Leu Thr Trp Pro Ser Asp
Gly Leu Ser Gln Val Gln Val Thr 130 135 140Ile Pro Ser Ser Leu Pro
Ser Gly Asp Tyr Leu Leu Arg Val Glu Gln145 150 155 160Ile Gly Leu
His Ser Ala Ser Ser Val Asn Gly Ala Gln Phe Tyr Leu 165 170 175Ser
Cys Ala Gln Leu Thr Val Thr Gly Gly Gly Asn Gly Asn Pro Gly 180 185
190Pro Leu Val Ser Phe Pro Gly Ala Tyr Ser Pro Thr Asp Pro Gly Leu
195 200 205Leu Ile Asn Ile Tyr Trp Pro Ile Pro Thr Ser Tyr Glu Leu
Pro Gly 210 215 220Pro Pro Val Trp Arg Gly225
2301651194DNAMalbranchea cinnamomea 165atgcggttct cagccgtggc
cgcggttggc tttctgatgg gcaccgccag cgcacacatg 60aagatgaaga ctccatatcc
atttggcccc gacacgctca acaccagccc cctccaggca 120aacctgaccg
acttcccctg caagcacaga cccggcgtct acgacccacc gctactccca
180caccccgacg cgaatacctt caccgtcggc gtccccgtca ccctcagctt
catcggcagc 240gccgtccacg gcggcggctc gtgtcagatc agcctgacca
cggaccgaca gccgaccagg 300gactcggtct ggaaggtcat ccactccatc
gagggcggct gtcccgccaa caccgacggc 360aacctgggag gcggcgcgga
cgccgaggtc gccagcacct tcgagttcca gattcccccc 420agcattccgc
ccggcgagta cacgctggcg tggacctggc tcaaccgcct cggcaaccgc
480gagttctaca tgaactgcgc cccgatcacc gtcgtcgcgc ccaagaagcg
atacgccccg 540tcgtcgtcgt cgtcggaccc gcggcccctt gcgtccctcg
cccagcgcca ggacctcccg 600gacatgttca tcgcgaacat caacggctgc
acgaccgagg agggcatcga cgtgcgcttc 660cccgaccccg gcccctccgt
cgaacgcgca gggaacccca gcaggctcct ggcgagcggc 720gaggtgatct
gcaagatgcg cggcagcgac acgggaccca tcgcgggggg tggcagcggc
780ggcggagacg gaggcaacga ctcggaggcc agcccagctc ccagtcaaga
tcctcacccc 840agttcaaacc caagccccga cccgaatcca gacccggacc
cgcagtgcct gccgccagcg 900ccatcaacat caaccgcgtc gaccccgacg
tctccgtcgg cgacaccacc cgcctccccg 960tcctccaccg gcgacagcgg
cgacggtggc ccggggctca ccggctcctg caccgagggc 1020tggttcaact
gcatcggcgg cacgcacttc cagcagtgca cggccagcgg gcagtggagc
1080gtcgcgcggc ccgtggcccc gggcacggtc tgccgcgaag gagtggggcc
tgatctgcag 1140atcggttatg ccaaggggga cgacgttaga ggcgtccgtc
gggcgcaccg ctga 1194166397PRTMalbranchea cinnamomea 166Met Arg Phe
Ser Ala Val Ala Ala Val Gly Phe Leu Met Gly Thr Ala1 5 10 15Ser Ala
His Met Lys Met Lys Thr Pro Tyr Pro Phe Gly Pro Asp Thr 20 25 30Leu
Asn Thr Ser Pro Leu Gln Ala Asn Leu Thr Asp Phe Pro Cys Lys 35 40
45His Arg Pro Gly Val Tyr Asp Pro Pro Leu Leu Pro His Pro Asp Ala
50 55 60Asn Thr Phe Thr Val Gly Val Pro Val Thr Leu Ser Phe Ile Gly
Ser65 70 75 80Ala Val His Gly Gly Gly Ser Cys Gln Ile Ser Leu Thr
Thr Asp Arg 85 90 95Gln Pro Thr Arg Asp Ser Val Trp Lys Val Ile His
Ser Ile Glu Gly 100 105 110Gly Cys Pro Ala Asn Thr Asp Gly Asn Leu
Gly Gly Gly Ala Asp Ala 115 120 125Glu Val Ala Ser Thr Phe Glu Phe
Gln Ile Pro Pro Ser Ile Pro Pro 130 135 140Gly Glu Tyr Thr Leu Ala
Trp Thr Trp Leu Asn Arg Leu Gly Asn Arg145 150 155 160Glu Phe Tyr
Met Asn Cys Ala Pro Ile Thr Val Val Ala Pro Lys Lys 165 170 175Arg
Tyr Ala Pro Ser Ser Ser Ser Ser Asp Pro Arg Pro Leu Ala Ser 180 185
190Leu Ala Gln Arg Gln Asp Leu Pro Asp Met Phe Ile Ala Asn Ile Asn
195 200 205Gly Cys Thr Thr Glu Glu Gly Ile Asp Val Arg Phe Pro Asp
Pro Gly 210 215 220Pro Ser Val Glu Arg Ala Gly Asn Pro Ser Arg Leu
Leu Ala Ser Gly225 230 235 240Glu Val Ile Cys Lys Met Arg Gly Ser
Asp Thr Gly Pro Ile Ala Gly 245 250 255Gly Gly Ser Gly Gly Gly Asp
Gly Gly Asn Asp Ser Glu Ala Ser Pro 260
265 270Ala Pro Ser Gln Asp Pro His Pro Ser Ser Asn Pro Ser Pro Asp
Pro 275 280 285Asn Pro Asp Pro Asp Pro Gln Cys Leu Pro Pro Ala Pro
Ser Thr Ser 290 295 300Thr Ala Ser Thr Pro Thr Ser Pro Ser Ala Thr
Pro Pro Ala Ser Pro305 310 315 320Ser Ser Thr Gly Asp Ser Gly Asp
Gly Gly Pro Gly Leu Thr Gly Ser 325 330 335Cys Thr Glu Gly Trp Phe
Asn Cys Ile Gly Gly Thr His Phe Gln Gln 340 345 350Cys Thr Ala Ser
Gly Gln Trp Ser Val Ala Arg Pro Val Ala Pro Gly 355 360 365Thr Val
Cys Arg Glu Gly Val Gly Pro Asp Leu Gln Ile Gly Tyr Ala 370 375
380Lys Gly Asp Asp Val Arg Gly Val Arg Arg Ala His Arg385 390
3951671233DNAMalbranchea cinnamomea 167atgcgtttct cggcttccac
cgttgcagca gcggctgctt tcctgtccgc tggggttgtc 60aacgcccata tgaacatgaa
gtttccctat ccatatgggc ctgatactct gaacaacagc 120cctcttcaga
acaacctcgc cgactttccc tgcaagcaac ggcctggagt gtatgaccct
180ccagcgctcc ccagtcctga tgccaacacc ctcaccatcg gcgtccctgc
gaccctggag 240ttccttggcg atgctgtcca tggtggtggt tcttgccaaa
tcagcttgac aactgacaag 300gagcctacca aagactctgt ctggaaggtt
atccattcca ttgagggtgg atgccctgcc 360aacactgaag ggaaccttgg
aagtgatcca aagggagaac gtgccagcaa gttccagttc 420actatccctc
ccagcatcgc accaggcgag tatactctgg cttggacctg gatcaaccgc
480attggtaacc gcgagtatta tatggactgc gccccaatca gagttcaaga
agcgcccaag 540aagcggtata ccccaacccc gccaactgag ccacgaagcc
tgattccctt ggcgaaacgt 600gacgaccttc ctgatatgtt tatcgccaac
atcaacggct gcaatacccc tgaaggtatc 660gacatccgtt acccaaaccc
cggtccttct attgagtatg cgggcaatcc catgaacctt 720atgaaggtcg
gtgagccagt ttgcgtgtac gcagatggca ctcctggccc tcttgctgga
780ggtgaagagg gtggtgacgg tggtaacact gagcaaccgc aaccgtcgaa
caacgcgggc 840ccttcgcctg gtgtcttcgc gcctctccag tccgagactg
ttgttcctcc agtgcccaca 900ccacccactc cggctcccca gccgtcgacc
ccgactgtcc agactcaggc cccggccccc 960accgttccct ccaatccaac
tccaacggtg gtgccagctc cggctcctaa cagcggcaac 1020ggtggctctg
ctttgactgg tccttgtact gaagagggca tttacaactg cattggcggc
1080acttccttcc agcgatgtgc tagtggtgaa tggaccgcag ttctgccagt
tgctgagggc 1140actgtctgca aggaagggtt cagtactgat ttgggaatta
cccatgcgaa aaagcgtggc 1200attcatcgtc gccgtggcca ctttcgcgca taa
1233168410PRTMalbranchea cinnamomea 168Met Arg Phe Ser Ala Ser Thr
Val Ala Ala Ala Ala Ala Phe Leu Ser1 5 10 15Ala Gly Val Val Asn Ala
His Met Asn Met Lys Phe Pro Tyr Pro Tyr 20 25 30Gly Pro Asp Thr Leu
Asn Asn Ser Pro Leu Gln Asn Asn Leu Ala Asp 35 40 45Phe Pro Cys Lys
Gln Arg Pro Gly Val Tyr Asp Pro Pro Ala Leu Pro 50 55 60Ser Pro Asp
Ala Asn Thr Leu Thr Ile Gly Val Pro Ala Thr Leu Glu65 70 75 80Phe
Leu Gly Asp Ala Val His Gly Gly Gly Ser Cys Gln Ile Ser Leu 85 90
95Thr Thr Asp Lys Glu Pro Thr Lys Asp Ser Val Trp Lys Val Ile His
100 105 110Ser Ile Glu Gly Gly Cys Pro Ala Asn Thr Glu Gly Asn Leu
Gly Ser 115 120 125Asp Pro Lys Gly Glu Arg Ala Ser Lys Phe Gln Phe
Thr Ile Pro Pro 130 135 140Ser Ile Ala Pro Gly Glu Tyr Thr Leu Ala
Trp Thr Trp Ile Asn Arg145 150 155 160Ile Gly Asn Arg Glu Tyr Tyr
Met Asp Cys Ala Pro Ile Arg Val Gln 165 170 175Glu Ala Pro Lys Lys
Arg Tyr Thr Pro Thr Pro Pro Thr Glu Pro Arg 180 185 190Ser Leu Ile
Pro Leu Ala Lys Arg Asp Asp Leu Pro Asp Met Phe Ile 195 200 205Ala
Asn Ile Asn Gly Cys Asn Thr Pro Glu Gly Ile Asp Ile Arg Tyr 210 215
220Pro Asn Pro Gly Pro Ser Ile Glu Tyr Ala Gly Asn Pro Met Asn
Leu225 230 235 240Met Lys Val Gly Glu Pro Val Cys Val Tyr Ala Asp
Gly Thr Pro Gly 245 250 255Pro Leu Ala Gly Gly Glu Glu Gly Gly Asp
Gly Gly Asn Thr Glu Gln 260 265 270Pro Gln Pro Ser Asn Asn Ala Gly
Pro Ser Pro Gly Val Phe Ala Pro 275 280 285Leu Gln Ser Glu Thr Val
Val Pro Pro Val Pro Thr Pro Pro Thr Pro 290 295 300Ala Pro Gln Pro
Ser Thr Pro Thr Val Gln Thr Gln Ala Pro Ala Pro305 310 315 320Thr
Val Pro Ser Asn Pro Thr Pro Thr Val Val Pro Ala Pro Ala Pro 325 330
335Asn Ser Gly Asn Gly Gly Ser Ala Leu Thr Gly Pro Cys Thr Glu Glu
340 345 350Gly Ile Tyr Asn Cys Ile Gly Gly Thr Ser Phe Gln Arg Cys
Ala Ser 355 360 365Gly Glu Trp Thr Ala Val Leu Pro Val Ala Glu Gly
Thr Val Cys Lys 370 375 380Glu Gly Phe Ser Thr Asp Leu Gly Ile Thr
His Ala Lys Lys Arg Gly385 390 395 400Ile His Arg Arg Arg Gly His
Phe Arg Ala 405 410169699DNAMalbranchea cinnamomea 169atgaaggcag
cagtatcctt ggcccttctt gttgccgtag ctggagcagc ctctgctcac 60aatatcttcc
ccaacctcat cgtcgacggg acggttacgg gagactggga attcgtccgc
120accaccgcca acaaatggtc ccgcgagggc ttaaccaacg tcaacagcga
gagtatgaga 180tgctacgagg aggccggccg tccaccctcg gaggtgaaaa
ccgtcaaagc ggggtcgaga 240gtcggtttcg cttcccaggc gcctattcgc
catattgggc cagtgctctt ttacatggcc 300cgtgttcccg atgggcagga
tgtggactcg tggaccccat cgggggacgt ctggttcaag 360atccatcagc
agggaccgga gcggtctgag tcgggatgga cctggcctac gcaagaccaa
420accgaactct tcgtcgacat cccagcctcc gtcccagacg gcaactatct
cctgcgaatc 480gagcaaatcg cgctccacga cgcacagtac gttggtggtg
cgcagtttta cctcgcatgc 540ggccaaatca acgtcaccgg gggcggcagc
ggggacccag gtcccaaggt ctcattccct 600ggcgcgtata aacccaccga
tcctggtatt ttgctggacc tccatggaca tccgccggcg 660aattatcagt
tcccgggacc agccgtatgg caaggctga 699170232PRTMalbranchea cinnamomea
170Met Lys Ala Ala Val Ser Leu Ala Leu Leu Val Ala Val Ala Gly Ala1
5 10 15Ala Ser Ala His Asn Ile Phe Pro Asn Leu Ile Val Asp Gly Thr
Val 20 25 30Thr Gly Asp Trp Glu Phe Val Arg Thr Thr Ala Asn Lys Trp
Ser Arg 35 40 45Glu Gly Leu Thr Asn Val Asn Ser Glu Ser Met Arg Cys
Tyr Glu Glu 50 55 60Ala Gly Arg Pro Pro Ser Glu Val Lys Thr Val Lys
Ala Gly Ser Arg65 70 75 80Val Gly Phe Ala Ser Gln Ala Pro Ile Arg
His Ile Gly Pro Val Leu 85 90 95Phe Tyr Met Ala Arg Val Pro Asp Gly
Gln Asp Val Asp Ser Trp Thr 100 105 110Pro Ser Gly Asp Val Trp Phe
Lys Ile His Gln Gln Gly Pro Glu Arg 115 120 125Ser Glu Ser Gly Trp
Thr Trp Pro Thr Gln Asp Gln Thr Glu Leu Phe 130 135 140Val Asp Ile
Pro Ala Ser Val Pro Asp Gly Asn Tyr Leu Leu Arg Ile145 150 155
160Glu Gln Ile Ala Leu His Asp Ala Gln Tyr Val Gly Gly Ala Gln Phe
165 170 175Tyr Leu Ala Cys Gly Gln Ile Asn Val Thr Gly Gly Gly Ser
Gly Asp 180 185 190Pro Gly Pro Lys Val Ser Phe Pro Gly Ala Tyr Lys
Pro Thr Asp Pro 195 200 205Gly Ile Leu Leu Asp Leu His Gly His Pro
Pro Ala Asn Tyr Gln Phe 210 215 220Pro Gly Pro Ala Val Trp Gln
Gly225 230171801DNAMalbranchea cinnamomea 171atgaaggcca ccgttctagc
tggcctcgcg gccgtgattg ctgctcaagg tgtagctggg 60catgcgacat tccagcagct
atgggttgat ggagaggata agggaggtgc ttgcgcaaga 120ttgcctttga
gcaactcacc cgtgacagat gtcaatagcg cagagatcgc atgcaacgcg
180aacagcggtc ctgcggccga gaagtgtacc gtttccgcag gcggagtcgt
caccgtcgag 240atgcaccagc agcccggaga tcggtcgtgt gacaacgaag
ccattggagg caaccactgg 300ggtcccgtgc tcgtatacat gagtaaagtc
gacgactccg ccaccgcaga cgggtccggc 360gggtggttca agatcttcga
agacacctgg gctcccgcac ccgactccaa ttccggctcc 420gacgactact
ggggcgtcaa ggatctgaac gcccactgcg gccgcatgga cgtgccaatc
480cccgctgacc tggcgccagg agactacctg ctgagagcgg aggtgattgc
gctgcacacc 540gcgtcgtcgc ccggcggtgc gcagttctac atgacctgct
accagctcac ggttgatgga 600gaggggtcac agagcccgca aaccgtgtcg
ttccccggcg cttattcgcc aagtgaccct 660gggattcaga tcaatatcta
tcagaagttg acggaatatg tctctcctgg accagctgtg 720attgagggtg
gaaccaccgt tgaggccggc acaggcggta gcaccattcc cggcaaacta
780aatgacgtgt tcttgtcttg a 801172266PRTMalbranchea cinnamomea
172Met Lys Ala Thr Val Leu Ala Gly Leu Ala Ala Val Ile Ala Ala Gln1
5 10 15Gly Val Ala Gly His Ala Thr Phe Gln Gln Leu Trp Val Asp Gly
Glu 20 25 30Asp Lys Gly Gly Ala Cys Ala Arg Leu Pro Leu Ser Asn Ser
Pro Val 35 40 45Thr Asp Val Asn Ser Ala Glu Ile Ala Cys Asn Ala Asn
Ser Gly Pro 50 55 60Ala Ala Glu Lys Cys Thr Val Ser Ala Gly Gly Val
Val Thr Val Glu65 70 75 80Met His Gln Gln Pro Gly Asp Arg Ser Cys
Asp Asn Glu Ala Ile Gly 85 90 95Gly Asn His Trp Gly Pro Val Leu Val
Tyr Met Ser Lys Val Asp Asp 100 105 110Ser Ala Thr Ala Asp Gly Ser
Gly Gly Trp Phe Lys Ile Phe Glu Asp 115 120 125Thr Trp Ala Pro Ala
Pro Asp Ser Asn Ser Gly Ser Asp Asp Tyr Trp 130 135 140Gly Val Lys
Asp Leu Asn Ala His Cys Gly Arg Met Asp Val Pro Ile145 150 155
160Pro Ala Asp Leu Ala Pro Gly Asp Tyr Leu Leu Arg Ala Glu Val Ile
165 170 175Ala Leu His Thr Ala Ser Ser Pro Gly Gly Ala Gln Phe Tyr
Met Thr 180 185 190Cys Tyr Gln Leu Thr Val Asp Gly Glu Gly Ser Gln
Ser Pro Gln Thr 195 200 205Val Ser Phe Pro Gly Ala Tyr Ser Pro Ser
Asp Pro Gly Ile Gln Ile 210 215 220Asn Ile Tyr Gln Lys Leu Thr Glu
Tyr Val Ser Pro Gly Pro Ala Val225 230 235 240Ile Glu Gly Gly Thr
Thr Val Glu Ala Gly Thr Gly Gly Ser Thr Ile 245 250 255Pro Gly Lys
Leu Asn Asp Val Phe Leu Ser 260 265173975DNACorynascus thermophilus
173atgccccctc cacggctaca cacgttcctt gccctcttgg ccctggtatc
agcccccacc 60gcacgggggc attcccatct cgcatacatc atcatcaacg gcgaggtgta
ccacggattc 120gacccgcggc cgggggagga gaactcgccg gcgcgcgtgg
gctggtcgac gggggcggtc 180gacgacgggt tcgtggggcc ggccgactac
tcgtcgcccg acataatctg ccacgtcgag 240ggggccagcc cgccggcgca
cgcgcccgtc cgggccggcg accgggttca cgtgcagtgg 300aacggctggc
cgctcgggca tgtggggccg gtgctgtcgt acctggcccc ctgcggcggc
360ctggaggggg ccgagcgcgg gtgtgccgga gtggacaagc ggcagctgcg
gtggaccaag 420gtggacgact cgctgccggc gatggagaga ctgtccacca
cggtcggggc cgcggacggc 480ggcggcgtgc ccgggcagcg ctgggccacc
gacgtgctgg tcgcggccaa caacagctgg 540caggtcgaga tcccgcgcgg
gctccgggac gggccgtacg tgctgcggca cgagatcgtc 600gcgctgcact
tcgcggccga ccgcggcggc gcgcagaact acccggtctg cgtcaacctc
660tgggtcgagg gcggcgacgg caccatggag ctggacggct tcgacgccac
cgagctctac 720cggcccgacg acccgggcat cctgctcgac gtgacggccg
gcccgcgctc gtacgtcgtg 780cccggcccga cgctggtcgc gggggccacg
cgggtgccgt acgcgcagca gaacagcagc 840tcggcgaggg cggagggaac
ccccgtgatg gtcatcagga gcacagagac ggtgcccctg 900acggtagcac
ctaccccgac caatagtacg ggtcgggctt acgggaggag gtacggaagc
960aggtttcagg ggtag 975174324PRTCorynascus thermophilus 174Met Pro
Pro Pro Arg Leu His Thr Phe Leu Ala Leu Leu Ala Leu Val1 5 10 15Ser
Ala Pro Thr Ala Arg Gly His Ser His Leu Ala Tyr Ile Ile Ile 20 25
30Asn Gly Glu Val Tyr His Gly Phe Asp Pro Arg Pro Gly Glu Glu Asn
35 40 45Ser Pro Ala Arg Val Gly Trp Ser Thr Gly Ala Val Asp Asp Gly
Phe 50 55 60Val Gly Pro Ala Asp Tyr Ser Ser Pro Asp Ile Ile Cys His
Val Glu65 70 75 80Gly Ala Ser Pro Pro Ala His Ala Pro Val Arg Ala
Gly Asp Arg Val 85 90 95His Val Gln Trp Asn Gly Trp Pro Leu Gly His
Val Gly Pro Val Leu 100 105 110Ser Tyr Leu Ala Pro Cys Gly Gly Leu
Glu Gly Ala Glu Arg Gly Cys 115 120 125Ala Gly Val Asp Lys Arg Gln
Leu Arg Trp Thr Lys Val Asp Asp Ser 130 135 140Leu Pro Ala Met Glu
Arg Leu Ser Thr Thr Val Gly Ala Ala Asp Gly145 150 155 160Gly Gly
Val Pro Gly Gln Arg Trp Ala Thr Asp Val Leu Val Ala Ala 165 170
175Asn Asn Ser Trp Gln Val Glu Ile Pro Arg Gly Leu Arg Asp Gly Pro
180 185 190Tyr Val Leu Arg His Glu Ile Val Ala Leu His Phe Ala Ala
Asp Arg 195 200 205Gly Gly Ala Gln Asn Tyr Pro Val Cys Val Asn Leu
Trp Val Glu Gly 210 215 220Gly Asp Gly Thr Met Glu Leu Asp Gly Phe
Asp Ala Thr Glu Leu Tyr225 230 235 240Arg Pro Asp Asp Pro Gly Ile
Leu Leu Asp Val Thr Ala Gly Pro Arg 245 250 255Ser Tyr Val Val Pro
Gly Pro Thr Leu Val Ala Gly Ala Thr Arg Val 260 265 270Pro Tyr Ala
Gln Gln Asn Ser Ser Ser Ala Arg Ala Glu Gly Thr Pro 275 280 285Val
Met Val Ile Arg Ser Thr Glu Thr Val Pro Leu Thr Val Ala Pro 290 295
300Thr Pro Thr Asn Ser Thr Gly Arg Ala Tyr Gly Arg Arg Tyr Gly
Ser305 310 315 320Arg Phe Gln Gly1751115DNACorynascus thermophilus
175atggctccat taacgtccgc agccctgatc ctgggcaccc ttatcagctt
ggtctcgggc 60catggctatc tgaagagcat caccgtcaac ggcaaggagt acctcgcttg
gcaggttggc 120caggacgact atatcaaccc gactccggtc cgatatgccc
gcaggcttgc aaacaacggg 180ccagtcccgg atttcaccac caaggatatc
acgtacgttt ccgtggaggc cggcactggc 240tgtggcggaa gagggcaaga
ccgccggact gacgcgtgcc atgactttac agctgcggcg 300ccggtggtaa
tgagccggct gagggaatca tcgagctgaa ggctggcgac actgtgtacg
360cgccgtcccc tccccagcta acgttacccg atcgacctca tctggacggt
tagctgacag 420ggtcgtcttc tctcgcacac gcaaatagga ccctcaactg
ggaccagtgg ggtagcagcc 480actccggccc agtcatgaag tgagtcttgc
ggccttcccg gcgacggacc gtaccagagg 540ttattacggg agtagcagtc
gtaatcagcg aacccattcg aactaacccc tcccgcacca 600gctatctcgc
ccattgcacc aacgacgact gcaagtcgtt caagggcgac agcggcaacg
660tctgggtcaa gatcgagcag ctcgcgtaca acccgtcggc caaccccccc
tgggcgtccg 720acctcctccg cgagcagggc gccaagtgga aggtgacgat
cccgcccacc ctcgcccccg 780gcgagtacct gctgcggcac gagatcctgg
gcctgcacgt cgccggaacc gtgatgggcg 840cccagttcta ccccagctgc
acccagatca gggtcaccca gggcgggaac acgcagctgc 900cctccggcat
cgcgcttccc ggtgcttacg acccgcatga cgggggtgta agtctcggat
960gtatgatctg gaattgtctc gacgcttgct gacagtgttt attccagatc
ttggtcgagt 1020tgtggagggt taaccagggc caggtcaact acaccgcgcc
tggaggaccc gtctggagcg 1080cggcggcgcc ggatcccaac cgctctggcc cctga
1115176240PRTCorynascus thermophilus 176Met Ala Pro Leu Thr Ser Ala
Ala Leu Ile Leu Gly Thr Leu Ile Ser1 5 10 15Leu Val Ser Gly His Gly
Tyr Leu Lys Ser Ile Thr Val Asn Gly Lys 20 25 30Glu Tyr Leu Ala Trp
Gln Val Gly Gln Asp Asp Tyr Ile Asn Pro Thr 35 40 45Pro Val Arg Tyr
Ala Arg Arg Leu Ala Asn Asn Gly Pro Val Pro Asp 50 55 60Phe Thr Thr
Lys Asp Ile Thr Cys Gly Ala Gly Gly Asn Glu Pro Ala65 70 75 80Glu
Gly Ile Ile Glu Leu Lys Ala Gly Asp Thr Val Thr Leu Asn Trp 85 90
95Asp Gln Trp Gly Ser Ser His Ser Gly Pro Val Met Asn Tyr Leu Ala
100 105 110His Cys Thr Asn Asp Asp Cys Lys Ser Phe Lys Gly Asp Ser
Gly Asn 115 120 125Val Trp Val Lys Ile Glu Gln Leu Ala Tyr Asn Pro
Ser Ala Asn Pro 130 135 140Pro Trp Ala Ser Asp Leu Leu Arg Glu Gln
Gly Ala Lys Trp Lys Val145 150 155 160Thr Ile Pro Pro Thr Leu Ala
Pro Gly Glu Tyr Leu Leu Arg His Glu 165 170 175Ile Leu Gly Leu His
Val Ala Gly Thr Val Met Gly Ala Gln Phe Tyr 180 185 190Pro Ser Cys
Thr Gln Ile Arg Val Thr Gln Gly Gly Asn Thr Gln Leu 195 200 205Pro
Ser Gly Ile Ala Leu Pro Gly Ala Tyr Asp Pro His Asp Gly Gly 210 215
220Gly Pro Val Trp Ser Ala Ala Ala Pro Asp Pro Asn Arg Ser Gly
Pro225 230 235 240177988DNACorynascus thermophilus 177atgaaatacg
ccctccagct cgctgcggcc
gcggcttttg cggtgaacag cgcggccggc 60cactacatct tccagcagtt tgcgacaggc
gggacgacgt acccgccctg gaagtacatc 120cgccgcaaca ccaacccgga
ctggctgcag aacgggccgg tgacggacct gtcgtcgacc 180gacctgcgct
gtaacgtggg cgggcaggtc agcaacggga ccgagaccat caccgtcaac
240gccggcgacg aattcacctt catcctcgac acgcccgtct accacgccgg
ccccacctcg 300ctctacatgt ccaaggcgcc cggcgcggcg gccgactacg
acggcagcgg gtcctggttc 360aagatctatg actggggccc gcagggaacg
agctggacgc tgagcggtac gtgtgcctgt 420ttctcatcat caccacgacc
atcctcatga tgattaccgc tctcgttatg attatgctgc 480tgttgcggtt
ctgctggaag agtatctgac ccgtctaccg tatccaggct cgtacaccca
540gagaattccc aggtgcatcc ctgacggcga atacctcctc cgcatccagc
agatcggact 600tcacaacccc ggcgccgagc cacaggtacg gtcctggact
tccgggtctc ctcttgcgca 660ccgtcgctga cgcaggacga acaaaaacag
ttctacatca gctgcgccca agtcaaggtg 720gtcaatggcg gcagcaccaa
cccgagcccg accgcccaga ttccgggagc cttccacagc 780aacgatcccg
gcttgaccgt caacgtaagc ccggcctcgc atcatttccc cgggaaccga
840aatagcaatg agctgacaac cgatcgtaga tctacaccga ccctctcaac
aactacgtcg 900tccccggacc ccgggttgta agtctctccg gatgccctcc
tccgttgatg gtcacgcctt 960gctaatgtcg tccaagttct cctgctag
988178225PRTCorynascus thermophilus 178Met Lys Tyr Ala Leu Gln Leu
Ala Ala Ala Ala Ala Phe Ala Val Asn1 5 10 15Ser Ala Ala Gly His Tyr
Ile Phe Gln Gln Phe Ala Thr Gly Gly Thr 20 25 30Thr Tyr Pro Pro Trp
Lys Tyr Ile Arg Arg Asn Thr Asn Pro Asp Trp 35 40 45Leu Gln Asn Gly
Pro Val Thr Asp Leu Ser Ser Thr Asp Leu Arg Cys 50 55 60Asn Val Gly
Gly Gln Val Ser Asn Gly Thr Glu Thr Ile Thr Val Asn65 70 75 80Ala
Gly Asp Glu Phe Thr Phe Ile Leu Asp Thr Pro Val Tyr His Ala 85 90
95Gly Pro Thr Ser Leu Tyr Met Ser Lys Ala Pro Gly Ala Ala Ala Asp
100 105 110Tyr Asp Gly Ser Gly Ser Trp Phe Lys Ile Tyr Asp Trp Gly
Pro Gln 115 120 125Gly Thr Ser Trp Thr Leu Ser Gly Ser Tyr Thr Gln
Arg Ile Pro Arg 130 135 140Cys Ile Pro Asp Gly Glu Tyr Leu Leu Arg
Ile Gln Gln Ile Gly Leu145 150 155 160His Asn Pro Gly Ala Glu Pro
Gln Phe Tyr Ile Ser Cys Ala Gln Val 165 170 175Lys Val Val Asn Gly
Gly Ser Thr Asn Pro Ser Pro Thr Ala Gln Ile 180 185 190Pro Gly Ala
Phe His Ser Asn Asp Pro Gly Leu Thr Val Asn Ile Tyr 195 200 205Thr
Asp Pro Leu Asn Asn Tyr Val Val Pro Gly Pro Arg Val Phe Ser 210 215
220Cys225179859DNACorynascus thermophilus 179atgaaggccc tctctctcct
tgcggctgcc tcggcggtct ctgcccacac catcttcgtc 60cagctcgaag cggacggcac
gaggtacccg gtctcgtacg gcatccggac gccgacgtac 120gacggcccca
tcaccgacgt cacgtccaac gacgttgcct gcaacggcgg gccgaacccg
180acgaccccgt ccggcgacgt catcacggtc acggcgggca ccacggtcaa
ggccatctgg 240agacacacgc tccagtccgg cccggacgac gtcatggacg
ccagccacaa gggcccgacc 300ctggcctacc tcaagaaggt cgacgacgcc
accacggact cgggcatcgg cggcggctgg 360ttcaagattc aggaggacgg
ctacaacaac ggcgagtggg gcaccagcaa ggtgatctcc 420aacggcggcg
agcactacat gtgagtcctt tctccgacag agcgaggaga aacacagaga
480gggagagaga gagaggccga ccaatctcgc tgacccgctg caacagcgac
atcccggcct 540gcattccccc gggccagtac ctcctccgcg ccgagatgat
tgctctccac agcgccgggt 600ctcccggcgg tgctcagctc tacgtaagcc
tctctgccct tccttattac cacccccccc 660ccaaacctct gactgacacg
cttggcagat ggaatgcgcc cagatcaaca tcgtcggcag 720ctccggctcc
ctgcccagct cgaccgtcag cttccccggc gcgtacagcg ccaacgaccc
780gggcatcctc atcaacatct actccatgtc cccctcggac acgtacatca
ttccgggccc 840ggaggtcttc acttgctag 859180235PRTCorynascus
thermophilus 180Met Lys Ala Leu Ser Leu Leu Ala Ala Ala Ser Ala Val
Ser Ala His1 5 10 15Thr Ile Phe Val Gln Leu Glu Ala Asp Gly Thr Arg
Tyr Pro Val Ser 20 25 30Tyr Gly Ile Arg Thr Pro Thr Tyr Asp Gly Pro
Ile Thr Asp Val Thr 35 40 45Ser Asn Asp Val Ala Cys Asn Gly Gly Pro
Asn Pro Thr Thr Pro Ser 50 55 60Gly Asp Val Ile Thr Val Thr Ala Gly
Thr Thr Val Lys Ala Ile Trp65 70 75 80Arg His Thr Leu Gln Ser Gly
Pro Asp Asp Val Met Asp Ala Ser His 85 90 95Lys Gly Pro Thr Leu Ala
Tyr Leu Lys Lys Val Asp Asp Ala Thr Thr 100 105 110Asp Ser Gly Ile
Gly Gly Gly Trp Phe Lys Ile Gln Glu Asp Gly Tyr 115 120 125Asn Asn
Gly Glu Trp Gly Thr Ser Lys Val Ile Ser Asn Gly Gly Glu 130 135
140His Tyr Ile Asp Ile Pro Ala Cys Ile Pro Pro Gly Gln Tyr Leu
Leu145 150 155 160Arg Ala Glu Met Ile Ala Leu His Ser Ala Gly Ser
Pro Gly Gly Ala 165 170 175Gln Leu Tyr Met Glu Cys Ala Gln Ile Asn
Ile Val Gly Ser Ser Gly 180 185 190Ser Leu Pro Ser Ser Thr Val Ser
Phe Pro Gly Ala Tyr Ser Ala Asn 195 200 205Asp Pro Gly Ile Leu Ile
Asn Ile Tyr Ser Met Ser Pro Ser Asp Thr 210 215 220Tyr Ile Ile Pro
Gly Pro Glu Val Phe Thr Cys225 230 2351811011DNACorynascus
thermophilus 181atggccaaga cctctgctct cctcgccggc ctgacgggcg
cggccctcgt cgctgcccac 60ggccacgtca gccacatcat cgtcaacggc gtgtactaca
ggaactacga cccgacgacc 120gactcgtacc agaccaaccc gccgagggtc
atcggctggg cggccgccca gcaggacaat 180ggcttcgtcg agcccaacaa
ctttggctcg ccggatgtca tctgccacaa gagcgccact 240cccggcggcg
gccacgccac cgtcgctgcc ggagacaaga tcagcctcgt ctggacgccc
300gagtggcccg agtcccacat cggcccggtc atcgactatc tggcggcctg
caacggcgac 360tgcgagacgg tcgacaagac gtcgctgcgc tggttcaaga
tcgacggcgc cggctacgac 420aagtcgaccg gccgctgggc cgccgacgcc
ctgcgcgcca acggcaacag ctggctcgtc 480cagatcccgt cggacctcaa
ggcgggcaac tacgtgctcc gccacgagat catcgccctc 540cacggcgcca
acaacgccaa cggcgcccag tcgtacccgc agtgcatcaa cctccgcgtc
600acgggcggcg gcaacaacct gcccagcggc gtgcccggca cctcgctgta
cagggccaac 660gacccgggca tcctcttcaa cccctacgtc ccctcgcccg
actacccggt ccccggcccg 720tccctcattc ccggcgccgt cagctccatc
gcccagagca agtcggtcgc cacggccacg 780gccacggcca cccctcccgg
cggcggcaac aacaaccccc ccgccaccac caccgccggc 840ggccccacca
gcaccaccag cagcccctcc cagcagacca ccaccccgcc gtcgggcagc
900gtgcagacca agtacggcca gtgcggcggc aacggctgga ccggcccgac
cctgtgcgcc 960cccggctcga gctgcaccgt tctcaacgag tggtactccc
agtgcgtgta a 1011182336PRTCorynascus thermophilus 182Met Ala Lys
Thr Ser Ala Leu Leu Ala Gly Leu Thr Gly Ala Ala Leu1 5 10 15Val Ala
Ala His Gly His Val Ser His Ile Ile Val Asn Gly Val Tyr 20 25 30Tyr
Arg Asn Tyr Asp Pro Thr Thr Asp Ser Tyr Gln Thr Asn Pro Pro 35 40
45Arg Val Ile Gly Trp Ala Ala Ala Gln Gln Asp Asn Gly Phe Val Glu
50 55 60Pro Asn Asn Phe Gly Ser Pro Asp Val Ile Cys His Lys Ser Ala
Thr65 70 75 80Pro Gly Gly Gly His Ala Thr Val Ala Ala Gly Asp Lys
Ile Ser Leu 85 90 95Val Trp Thr Pro Glu Trp Pro Glu Ser His Ile Gly
Pro Val Ile Asp 100 105 110Tyr Leu Ala Ala Cys Asn Gly Asp Cys Glu
Thr Val Asp Lys Thr Ser 115 120 125Leu Arg Trp Phe Lys Ile Asp Gly
Ala Gly Tyr Asp Lys Ser Thr Gly 130 135 140Arg Trp Ala Ala Asp Ala
Leu Arg Ala Asn Gly Asn Ser Trp Leu Val145 150 155 160Gln Ile Pro
Ser Asp Leu Lys Ala Gly Asn Tyr Val Leu Arg His Glu 165 170 175Ile
Ile Ala Leu His Gly Ala Asn Asn Ala Asn Gly Ala Gln Ser Tyr 180 185
190Pro Gln Cys Ile Asn Leu Arg Val Thr Gly Gly Gly Asn Asn Leu Pro
195 200 205Ser Gly Val Pro Gly Thr Ser Leu Tyr Arg Ala Asn Asp Pro
Gly Ile 210 215 220Leu Phe Asn Pro Tyr Val Pro Ser Pro Asp Tyr Pro
Val Pro Gly Pro225 230 235 240Ser Leu Ile Pro Gly Ala Val Ser Ser
Ile Ala Gln Ser Lys Ser Val 245 250 255Ala Thr Ala Thr Ala Thr Ala
Thr Pro Pro Gly Gly Gly Asn Asn Asn 260 265 270Pro Pro Ala Thr Thr
Thr Ala Gly Gly Pro Thr Ser Thr Thr Ser Ser 275 280 285Pro Ser Gln
Gln Thr Thr Thr Pro Pro Ser Gly Ser Val Gln Thr Lys 290 295 300Tyr
Gly Gln Cys Gly Gly Asn Gly Trp Thr Gly Pro Thr Leu Cys Ala305 310
315 320Pro Gly Ser Ser Cys Thr Val Leu Asn Glu Trp Tyr Ser Gln Cys
Val 325 330 3351831315DNACorynascus thermophilus 183atgaaaacgc
ttgccgccct cctcgtctcc gccggcctcg tggctgcgca cggctatgtt 60gaccgtgcca
cgatcggcgg caaggagtac caggtaatga caacaaacac ggctactccc
120gtgtggatgc gtcgtcgaag agtagctaac aatacggtcc ctatagttct
accaggtggg 180ctcggtaccg gccagttggc tctccatgcc ggcagttcct
gacatgcatc tcgcatattt 240agccgtacgt tgatccgtac atgggcgaca
acaaggtaac aacaaacctt aatataacaa 300gaacaaccta tccatcctcc
ctcccccccc ctctccacac cccccccctc tctctctctt 360tctctccttt
ctcctctgat gcaccggtcg agcacgcact aaacaggggg taattacggg
420gggcatttca gcccgacagg gtctcccgct cgatcccggg caacggcccc
gtggaggacg 480tcaactcgct cgacatccag tgcaacgcgg gcgcgcagcc
ggccaagctc cacgcccccg 540ccgccgccgg ctcgaccgtg acgctcaact
ggaccctctg gcccgactcg cacgtcggcc 600ccgtcatcac ctacatggcg
cgctgccccg acagcggctg ccagaactgg tcgcccggaa 660cccagtatgg
cccattccaa tcctgtttgt tgatattgat ggggggtaaa gacggagggg
720atggttggcg gtgctaaatg gtttactttc ctgatgacag gcccgtctgg
ttcaagatca 780aggagggcgg ccgtgagggc acgtccaacg tctgggcggc
cgtacgtgat cacaccccgt 840tccgaaaaca acgaggcaca caccaaagcc
aactaacccc tcccttcttt cgctctctat 900ctctctcgac agaccccgct
catgaaggcg ccgtcggcgt acacgtacac gatcccggcc 960tgcctcaaga
gcggctacta cctggtgcgg cacgagatca tcgcgctgca ctcggcctgg
1020cagtaccccg gcgcgcagtt ctacccgggc tgccaccagc tccaggtcac
cggcggcggc 1080tcgaccgtgc cctcggccaa cctggtcgcc ttccccggcg
cctacaaggg cagcgacccc 1140ggcatcacct acgacgcgta caagggtgag
ccatctcttt ctctctttct ctctgtctcg 1200cttttctctt tccttgtgcc
tcttggttgt ccgtcttgga gcagggcagg gcgactgacg 1260cggagtggca
gcgcaacctt acacgatccc gggcccgccc gtgtttactt gctaa
1315184253PRTCorynascus thermophilus 184Met Lys Thr Leu Ala Ala Leu
Leu Val Ser Ala Gly Leu Val Ala Ala1 5 10 15His Gly Tyr Val Asp Arg
Ala Thr Ile Gly Gly Lys Glu Tyr Gln Phe 20 25 30Tyr Gln Val Gly Ser
Val Pro Ala Ser Trp Leu Ser Met Pro Ala Val 35 40 45Pro Asp Met His
Leu Ala Tyr Leu Ala Pro Asp Arg Val Ser Arg Ser 50 55 60Ile Pro Gly
Asn Gly Pro Val Glu Asp Val Asn Ser Leu Asp Ile Gln65 70 75 80Cys
Asn Ala Gly Ala Gln Pro Ala Lys Leu His Ala Pro Ala Ala Ala 85 90
95Gly Ser Thr Val Thr Leu Asn Trp Thr Leu Trp Pro Asp Ser His Val
100 105 110Gly Pro Val Ile Thr Tyr Met Ala Arg Cys Pro Asp Ser Gly
Cys Gln 115 120 125Asn Trp Ser Pro Gly Thr Gln Pro Val Trp Phe Lys
Ile Lys Glu Gly 130 135 140Gly Arg Glu Gly Thr Ser Asn Val Trp Ala
Ala Thr Pro Leu Met Lys145 150 155 160Ala Pro Ser Ala Tyr Thr Tyr
Thr Ile Pro Ala Cys Leu Lys Ser Gly 165 170 175Tyr Tyr Leu Val Arg
His Glu Ile Ile Ala Leu His Ser Ala Trp Gln 180 185 190Tyr Pro Gly
Ala Gln Phe Tyr Pro Gly Cys His Gln Leu Gln Val Thr 195 200 205Gly
Gly Gly Ser Thr Val Pro Ser Ala Asn Leu Val Ala Phe Pro Gly 210 215
220Ala Tyr Lys Gly Ser Asp Pro Gly Ile Thr Tyr Asp Ala Tyr Lys
Ala225 230 235 240Gln Pro Tyr Thr Ile Pro Gly Pro Pro Val Phe Thr
Cys 245 250185924DNACorynascus thermophilus 185atgtaccgca
cgctcggttc ccttgccctg ctcgctggag gcgctgctgc ccacggtgcc 60gtgaccagct
acaacatcgc gggcaaggac taccctgggt aaggaaggag atctctctct
120ctctctctct ctctctctct ctctctctct ctcgttctct tgctaacaca
aaggcacctc 180tgcagatact cgggctttgc cccgaccggc gaacccgtca
tccagtggca atggcccgac 240tacaaccccg tcatgtccgc tagcgacttc
aagctccgct gcaacggcgg caccaacgcg 300cagctgtatg ctgaggcggc
ccccggcgat accatcacgg ccacctgggc ccagtggacg 360cacgcccagg
gcccgatcct ggtgtggatg tacaagtgcc ccggcgactt cagctcctgc
420gacggctccg gcgagggctg gttcaagatc gacgaggccg gcttccacgg
cgacggccag 480actgtcttcc tcgacagcga gaacccctcg ggctgggaca
tcgccaagct ggtcggcggc 540aacaagtcgt ggagcagcaa gatccccgag
ggcctcgctc cgggcaacta cctggtccgc 600cacgagctca tcgccctgca
ccaggccaac gccccgcagt tctaccccga gtgcgcccag 660gtcaaggtta
ccggctccgg caccgccgag cccgactcct cgtacaaggc cgccatcccc
720ggctactgct cgcagagcga ccccaacatt tcggtaagga gggactcccg
gccgagagag 780agagaggact cattcctggt gctaacccgt tcacttccgc
agttcaacat caacgaccac 840tccctcccgc aggagtacaa gatccccggc
ccgccggtct tcaagggcac tgcctccgcc 900aaggctcgct ccttccaggc ctaa
924186255PRTCorynascus thermophilus 186Met Tyr Arg Thr Leu Gly Ser
Leu Ala Leu Leu Ala Gly Gly Ala Ala1 5 10 15Ala His Gly Ala Val Thr
Ser Tyr Asn Ile Ala Gly Lys Asp Tyr Pro 20 25 30Gly Tyr Ser Gly Phe
Ala Pro Thr Gly Glu Pro Val Ile Gln Trp Gln 35 40 45Trp Pro Asp Tyr
Asn Pro Val Met Ser Ala Ser Asp Phe Lys Leu Arg 50 55 60Cys Asn Gly
Gly Thr Asn Ala Gln Leu Tyr Ala Glu Ala Ala Pro Gly65 70 75 80Asp
Thr Ile Thr Ala Thr Trp Ala Gln Trp Thr His Ala Gln Gly Pro 85 90
95Ile Leu Val Trp Met Tyr Lys Cys Pro Gly Asp Phe Ser Ser Cys Asp
100 105 110Gly Ser Gly Glu Gly Trp Phe Lys Ile Asp Glu Ala Gly Phe
His Gly 115 120 125Asp Gly Gln Thr Val Phe Leu Asp Ser Glu Asn Pro
Ser Gly Trp Asp 130 135 140Ile Ala Lys Leu Val Gly Gly Asn Lys Ser
Trp Ser Ser Lys Ile Pro145 150 155 160Glu Gly Leu Ala Pro Gly Asn
Tyr Leu Val Arg His Glu Leu Ile Ala 165 170 175Leu His Gln Ala Asn
Ala Pro Gln Phe Tyr Pro Glu Cys Ala Gln Val 180 185 190Lys Val Thr
Gly Ser Gly Thr Ala Glu Pro Asp Ser Ser Tyr Lys Ala 195 200 205Ala
Ile Pro Gly Tyr Cys Ser Gln Ser Asp Pro Asn Ile Ser Phe Asn 210 215
220Ile Asn Asp His Ser Leu Pro Gln Glu Tyr Lys Ile Pro Gly Pro
Pro225 230 235 240Val Phe Lys Gly Thr Ala Ser Ala Lys Ala Arg Ser
Phe Gln Ala 245 250 255187742DNACorynascus thermophilus
187atgctggcga caaccttcgc tctcctgacg gccgctctcg gcgtcagcgc
ccattatacc 60ctcccccggg tcgggtccgg ctccgagtgg cagcacgtgc gccgggctga
caactggcaa 120aacaacggct tcgtcgacaa cgtctactcg cagcagatcc
gctgcttcca gtcgagcaat 180gccggcgccc cggatgtcta caccgtccag
gcgggctcga gcgtgaccta ctacgccaac 240cccagcatct accaccccgg
ccccatgcag ttctacctcg cccgcgttcc ggacggacag 300gacgtcaagt
cgtggaacgg cgacggcgct gtgtggttca aggtgtacga ggagcagcct
360cagttcggct cccagcttac ctggcctagc aacggtgcgt cgaccatgct
ctctcgtttg 420gcccgttgcc aggtgctaac tgtccttccc gtccgcaggc
aagaactcgt tccaggttcc 480catccccagc tgcatccgcc cgggcaagta
cctcctccgc gccgagcaca tcgccctgca 540cgttgcccag agccagggcg
gtgcccagtt ctacatctcg tgcgcccagc tcgacgtcac 600tggcggcggc
agcaccgagc cttcccagaa ggttgccttc ccgggtgcct actcgcccac
660cgaccccggc attctcatca acatcaactg gcccatcccg acctcgtaca
agaaccccgg 720cccgccggtc ttccgctgct aa 742188225PRTCorynascus
thermophilus 188Met Leu Ala Thr Thr Phe Ala Leu Leu Thr Ala Ala Leu
Gly Val Ser1 5 10 15Ala His Tyr Thr Leu Pro Arg Val Gly Ser Gly Ser
Glu Trp Gln His 20 25 30Val Arg Arg Ala Asp Asn Trp Gln Asn Asn Gly
Phe Val Asp Asn Val 35 40 45Tyr Ser Gln Gln Ile Arg Cys Phe Gln Ser
Ser Asn Ala Gly Ala Pro 50 55 60Asp Val Tyr Thr Val Gln Ala Gly Ser
Ser Val Thr Tyr Tyr Ala Asn65 70 75 80Pro Ser Ile Tyr His Pro Gly
Pro Met Gln Phe Tyr Leu Ala Arg Val 85 90 95Pro Asp Gly Gln Asp Val
Lys Ser Trp Asn Gly Asp Gly Ala Val Trp 100 105 110Phe Lys Val Tyr
Glu Glu Gln Pro Gln Phe Gly Ser Gln Leu Thr Trp 115 120 125Pro Ser
Asn Gly Lys Asn Ser Phe Gln Val Pro Ile Pro Ser Cys Ile 130
135 140Arg Pro Gly Lys Tyr Leu Leu Arg Ala Glu His Ile Ala Leu His
Val145 150 155 160Ala Gln Ser Gln Gly Gly Ala Gln Phe Tyr Ile Ser
Cys Ala Gln Leu 165 170 175Asp Val Thr Gly Gly Gly Ser Thr Glu Pro
Ser Gln Lys Val Ala Phe 180 185 190Pro Gly Ala Tyr Ser Pro Thr Asp
Pro Gly Ile Leu Ile Asn Ile Asn 195 200 205Trp Pro Ile Pro Thr Ser
Tyr Lys Asn Pro Gly Pro Pro Val Phe Arg 210 215
220Cys225189901DNACorynascus thermophilus 189atgaaggttc tcgcgcccct
ggttctggcc ggcgccgcca gcgcccacac catcttcacg 60tcgctcgagg tgggcggcgt
caaccatggc gtcggccagg gcgtccgcgt gccgtcgtac 120aacggcccga
tcgaggacgt gacgtccaac tcgatcgcct gcaacggccc ccccaacccg
180acgacgccga cggacaaggt gatcacggtc caggccggcc agacggtgac
ggccatctgg 240cggtacatgc tcagcaccac cggctcggcc cccaacgacg
tcatggacag cagccacaag 300ggcccgacca tggcctacct caagaaggtc
ggcaacgcca ccaccgactc gggcgtcggc 360ggcggctggt tcaagatcca
ggaggacggg ctgaacaacg gcgtctgggg cacggagcgc 420gtcatcaacg
gccagggccg ccacaacatc aagatccccg agtgcatcgc ccccggccag
480tacctcctcc gcgccgagat gctcgccctg cacggagcct ccaactaccc
cggcgcccag 540ttctacatgg agtgcgctca gctcaacagt acgtttgtcc
acgagagacg gaaaaacaaa 600acagaagcaa ggggaggcgg ggcagatgtg
atggctaaca ttgatgcttt cttcttcagt 660cgtcggcggc agcggcagca
agaccccgtc caccgtcagc ttcccgggtg cttacagcgt 720acgttgttcc
aaaaggcttt ttcttcgcgt ttttttttct ttgaactgat acagccccct
780ctgtgacgac tactaacacg gccacaatca acagggcaac gaccccggtg
tcaagatcaa 840catctactgg cctcccgtca ccgaatacaa ggttcccggc
cccagcgtct tcacttgcta 900a 901190237PRTCorynascus thermophilus
190Met Lys Val Leu Ala Pro Leu Val Leu Ala Gly Ala Ala Ser Ala His1
5 10 15Thr Ile Phe Thr Ser Leu Glu Val Gly Gly Val Asn His Gly Val
Gly 20 25 30Gln Gly Val Arg Val Pro Ser Tyr Asn Gly Pro Ile Glu Asp
Val Thr 35 40 45Ser Asn Ser Ile Ala Cys Asn Gly Pro Pro Asn Pro Thr
Thr Pro Thr 50 55 60Asp Lys Val Ile Thr Val Gln Ala Gly Gln Thr Val
Thr Ala Ile Trp65 70 75 80Arg Tyr Met Leu Ser Thr Thr Gly Ser Ala
Pro Asn Asp Val Met Asp 85 90 95Ser Ser His Lys Gly Pro Thr Met Ala
Tyr Leu Lys Lys Val Gly Asn 100 105 110Ala Thr Thr Asp Ser Gly Val
Gly Gly Gly Trp Phe Lys Ile Gln Glu 115 120 125Asp Gly Leu Asn Asn
Gly Val Trp Gly Thr Glu Arg Val Ile Asn Gly 130 135 140Gln Gly Arg
His Asn Ile Lys Ile Pro Glu Cys Ile Ala Pro Gly Gln145 150 155
160Tyr Leu Leu Arg Ala Glu Met Leu Ala Leu His Gly Ala Ser Asn Tyr
165 170 175Pro Gly Ala Gln Phe Tyr Met Glu Cys Ala Gln Leu Asn Ile
Val Gly 180 185 190Gly Ser Gly Ser Lys Thr Pro Ser Thr Val Ser Phe
Pro Gly Ala Tyr 195 200 205Ser Gly Asn Asp Pro Gly Val Lys Ile Asn
Ile Tyr Trp Pro Pro Val 210 215 220Thr Glu Tyr Lys Val Pro Gly Pro
Ser Val Phe Thr Cys225 230 235191944DNACorynascus thermophilus
191atgaagctga gcgctgccat cgccgtgctc gcggccgccc ttgccgaggc
gcactgtaag 60ctggcttgcc ggtcctcccc cttctcaacg acgccgagct cgagcgcgtg
ggactaatga 120cgatgtgacg acgacatcaa gatacctttc ccagcatcgc
caacacgccc gactggcagt 180atgtgcgcat cacgaccaac taccagagca
acggccccgt gacggacgtc aactcggacc 240agatccgctg ctacgagcgc
aacccgggca cgggcgcgcc cggcatctac aacgtcaccg 300ccggcaccac
catcaactac aacgccaagt cgtccatctc ccacccgggc cccatggcct
360tctacatcgc caaggtcccc gccggccagt cggccgccac ctgggacggc
aagggcgccg 420tctggtccaa gatctaccag gagatgccgc actttggctc
gagcctgacc tgggactcga 480acggtatgat gagttctctc tctccttctc
tctttgatgc tctccttgtg atgctaaacg 540acgacccccg ccaggccgcg
tctccatgcc cgtcaccatc ccccgctgtc tgcagaacgg 600cgagtacctg
ctgcgtgccg agcacattgc cctccacagc gccggcagcg tcggcggcgc
660ccagttctac atctcgtgcg ctcagatctc gggtatgcat tatatacttc
catattgtcc 720acccactcac cccccatccc ccacgcttaa tagctcgagc
agcggaacca tctgaagcta 780acacgtcccc cccagtcacc ggcggcaccg
gcacctggaa cccccgcaac aaggtgtcct 840tccccggcgc ctacaaggcc
accgacccgg gcatcctgat caacatctac tggcccatcc 900cgaccagcta
cacgcccgcc ggcccggccg tcgacacctg ctag 944192227PRTCorynascus
thermophilus 192Met Lys Leu Ser Ala Ala Ile Ala Val Leu Ala Ala Ala
Leu Ala Glu1 5 10 15Ala His Tyr Thr Phe Pro Ser Ile Ala Asn Thr Pro
Asp Trp Gln Tyr 20 25 30Val Arg Ile Thr Thr Asn Tyr Gln Ser Asn Gly
Pro Val Thr Asp Val 35 40 45Asn Ser Asp Gln Ile Arg Cys Tyr Glu Arg
Asn Pro Gly Thr Gly Ala 50 55 60Pro Gly Ile Tyr Asn Val Thr Ala Gly
Thr Thr Ile Asn Tyr Asn Ala65 70 75 80Lys Ser Ser Ile Ser His Pro
Gly Pro Met Ala Phe Tyr Ile Ala Lys 85 90 95Val Pro Ala Gly Gln Ser
Ala Ala Thr Trp Asp Gly Lys Gly Ala Val 100 105 110Trp Ser Lys Ile
Tyr Gln Glu Met Pro His Phe Gly Ser Ser Leu Thr 115 120 125Trp Asp
Ser Asn Gly Arg Val Ser Met Pro Val Thr Ile Pro Arg Cys 130 135
140Leu Gln Asn Gly Glu Tyr Leu Leu Arg Ala Glu His Ile Ala Leu
His145 150 155 160Ser Ala Gly Ser Val Gly Gly Ala Gln Phe Tyr Ile
Ser Cys Ala Gln 165 170 175Ile Ser Val Thr Gly Gly Thr Gly Thr Trp
Asn Pro Arg Asn Lys Val 180 185 190Ser Phe Pro Gly Ala Tyr Lys Ala
Thr Asp Pro Gly Ile Leu Ile Asn 195 200 205Ile Tyr Trp Pro Ile Pro
Thr Ser Tyr Thr Pro Ala Gly Pro Ala Val 210 215 220Asp Thr
Cys225193948DNACorynascus thermophilus 193atgtcttcct tcacctccaa
gggcctcctt tccgccctca tgggcgctgc cacggttgcc 60gcccacggcc acgtcaccaa
tatcgtcatc aacggcgtct cgtaccagaa ctacgatccg 120ttcagccacc
cttacatgcg gaaccccccg acggttgtcg gctggacggc gagcaacacg
180gacaacggct tcgtcggccc cgagtccttc tctagcccgg acatcatctg
ccacaagtcg 240gccaccaacg ccggcggtca tgccgttgtt gccgccggcg
acaagatttc catccagtgg 300gacacctggc ccgagtcgca ccacggtccg
gtcatcgact acctcgccga ctgcggcgac 360gcgggctgcg agaaggtcga
caagaccacg ctcgagttct tcaagatcag cgagaagggc 420ctgatcgacg
gcagcagcgc gcccggcagg tgggcgtccg acgagctgat cgccaacaac
480aactcgtggc tggtccagat cccgcccgac atcgcccccg gcaactacgt
cctgcgccac 540gagatcatcg ccctgcacag cgccggccag cagaacggcg
cgcagaacta cccccagtgc 600gtcaacctgc acatcaccgg ctccggcacc
cggaaaccct cgggcgtccc cggcaccgag 660ctctaccggc cgaccgaccc
cggcatcctg gccaacatct acacctcccc cgtcgcctac 720cagatccccg
gcccggccat catcccgggc gcctccgccg tcgagcagac cacctcggcc
780atcaccgcct ccgccagcgc ggttcttccc ggcttcgcta ccgccgcgcc
cccggctgcg 840accaccacaa ccaccaccgc ctccgctacc agtgctcccc
gcccgaccgg ctgtgccggt 900ctgaggaagc gccgtcgcca cgcccgtgat
gtcaaggttg ccctctag 948194315PRTCorynascus thermophilus 194Met Ser
Ser Phe Thr Ser Lys Gly Leu Leu Ser Ala Leu Met Gly Ala1 5 10 15Ala
Thr Val Ala Ala His Gly His Val Thr Asn Ile Val Ile Asn Gly 20 25
30Val Ser Tyr Gln Asn Tyr Asp Pro Phe Ser His Pro Tyr Met Arg Asn
35 40 45Pro Pro Thr Val Val Gly Trp Thr Ala Ser Asn Thr Asp Asn Gly
Phe 50 55 60Val Gly Pro Glu Ser Phe Ser Ser Pro Asp Ile Ile Cys His
Lys Ser65 70 75 80Ala Thr Asn Ala Gly Gly His Ala Val Val Ala Ala
Gly Asp Lys Ile 85 90 95Ser Ile Gln Trp Asp Thr Trp Pro Glu Ser His
His Gly Pro Val Ile 100 105 110Asp Tyr Leu Ala Asp Cys Gly Asp Ala
Gly Cys Glu Lys Val Asp Lys 115 120 125Thr Thr Leu Glu Phe Phe Lys
Ile Ser Glu Lys Gly Leu Ile Asp Gly 130 135 140Ser Ser Ala Pro Gly
Arg Trp Ala Ser Asp Glu Leu Ile Ala Asn Asn145 150 155 160Asn Ser
Trp Leu Val Gln Ile Pro Pro Asp Ile Ala Pro Gly Asn Tyr 165 170
175Val Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Gly Gln Gln Asn
180 185 190Gly Ala Gln Asn Tyr Pro Gln Cys Val Asn Leu His Ile Thr
Gly Ser 195 200 205Gly Thr Arg Lys Pro Ser Gly Val Pro Gly Thr Glu
Leu Tyr Arg Pro 210 215 220Thr Asp Pro Gly Ile Leu Ala Asn Ile Tyr
Thr Ser Pro Val Ala Tyr225 230 235 240Gln Ile Pro Gly Pro Ala Ile
Ile Pro Gly Ala Ser Ala Val Glu Gln 245 250 255Thr Thr Ser Ala Ile
Thr Ala Ser Ala Ser Ala Val Leu Pro Gly Phe 260 265 270Ala Thr Ala
Ala Pro Pro Ala Ala Thr Thr Thr Thr Thr Thr Ala Ser 275 280 285Ala
Thr Ser Ala Pro Arg Pro Thr Gly Cys Ala Gly Leu Arg Lys Arg 290 295
300Arg Arg His Ala Arg Asp Val Lys Val Ala Leu305 310
3151951380DNACorynascus thermophilus 195atgcatcctc ccatctttgt
tcttgggctt gcgagcctgc tttgccccct ctcgtctgca 60cacactactt tcaccaccct
cttcatcaat gatgtcaacc aaggtgacgg aacctgcatt 120cgcatggcga
aggagggcaa cgtcgctact catcctctcg cgggcggcct cgactctgaa
180gacatggcct gtggtacgtt gacacgtcct tgaccccgcc gagactgtcc
cgtgtatcta 240aacttctcat caggccggga tggccaagaa cccgttgcat
ttacctgccc ggccccagct 300ggtgccaagt tgaccttcga gtttcgcatg
tgggccgacg cttcgcagcc cggatcgatc 360gacccgtccc atcttggcgc
tatggccatc tacctcaaga aggtttctaa catgaaatct 420gacgcggccg
ctgggccggg ctggttcaag atttgggacc aaggctacga cacggaggcc
480aagaagtggg ccaccgagaa tctcattgag aacaacggcc tgctgagcgt
caaccttccc 540tcgggcttgt cgaccggcta ctacctcgtc cgtcaggaga
ccattacctt ccaaaacgtc 600accaatgaca tgccagatcc ccagttctac
gtcggttgcg cgcagctcta cgtcgaaggc 660acctcggact cacccatccc
cccagacaag accgtctcca ttcccggcca catcagcgac 720ccggccgacc
cgggcctgac ctttaacatc tacacggacg acgtgtccgc ctacaagccc
780cccggcccgg aggtttactt ccccaccgcc atcacctcct ccggaagcag
cgacgacagg 840ggggccgcgc gccagcagac tcccgccgac aagcaggccg
gagaaggcct cgttcccacc 900gactgcgtcg tcaagaacgc aaactggtgc
gccgccgccc tgccgcccta caccgacgag 960gccggctgct gggccgccgt
ggaggactgc aacaggcagc tggacgagtg ctacaccagc 1020gcgcccccct
cgggcagcag ggggtgcaag atctgggagg agcaggtatg catcgtcgtc
1080tcgcggaagt gcgaggcccg ggatttccag cccctcccgc ggctgtggaa
ggatctaaga 1140gagggaattg atgagccgat cccgggtggg aagttgcctc
cggcgctcaa cgcgggagag 1200agcggggatc atggcggaag aggctgcggc
caccatggtg gcgaggagga ggctggggct 1260ggggcggcct ccactcctgc
ttttgctgct ccccatgcgg ccaggattca caacccaaat 1320ttcaagaggg
gccggcgccg tgagtcgcgt tggcggcgac tggcatctgg tgagcaatag
1380196439PRTCorynascus thermophilus 196Met His Pro Pro Ile Phe Val
Leu Gly Leu Ala Ser Leu Leu Cys Pro1 5 10 15Leu Ser Ser Ala His Thr
Thr Phe Thr Thr Leu Phe Ile Asn Asp Val 20 25 30Asn Gln Gly Asp Gly
Thr Cys Ile Arg Met Ala Lys Glu Gly Asn Val 35 40 45Ala Thr His Pro
Leu Ala Gly Gly Leu Asp Ser Glu Asp Met Ala Cys 50 55 60Gly Arg Asp
Gly Gln Glu Pro Val Ala Phe Thr Cys Pro Ala Pro Ala65 70 75 80Gly
Ala Lys Leu Thr Phe Glu Phe Arg Met Trp Ala Asp Ala Ser Gln 85 90
95Pro Gly Ser Ile Asp Pro Ser His Leu Gly Ala Met Ala Ile Tyr Leu
100 105 110Lys Lys Val Ser Asn Met Lys Ser Asp Ala Ala Ala Gly Pro
Gly Trp 115 120 125Phe Lys Ile Trp Asp Gln Gly Tyr Asp Thr Glu Ala
Lys Lys Trp Ala 130 135 140Thr Glu Asn Leu Ile Glu Asn Asn Gly Leu
Leu Ser Val Asn Leu Pro145 150 155 160Ser Gly Leu Ser Thr Gly Tyr
Tyr Leu Val Arg Gln Glu Thr Ile Thr 165 170 175Phe Gln Asn Val Thr
Asn Asp Met Pro Asp Pro Gln Phe Tyr Val Gly 180 185 190Cys Ala Gln
Leu Tyr Val Glu Gly Thr Ser Asp Ser Pro Ile Pro Pro 195 200 205Asp
Lys Thr Val Ser Ile Pro Gly His Ile Ser Asp Pro Ala Asp Pro 210 215
220Gly Leu Thr Phe Asn Ile Tyr Thr Asp Asp Val Ser Ala Tyr Lys
Pro225 230 235 240Pro Gly Pro Glu Val Tyr Phe Pro Thr Ala Ile Thr
Ser Ser Gly Ser 245 250 255Ser Asp Asp Arg Gly Ala Ala Arg Gln Gln
Thr Pro Ala Asp Lys Gln 260 265 270Ala Gly Glu Gly Leu Val Pro Thr
Asp Cys Val Val Lys Asn Ala Asn 275 280 285Trp Cys Ala Ala Ala Leu
Pro Pro Tyr Thr Asp Glu Ala Gly Cys Trp 290 295 300Ala Ala Val Glu
Asp Cys Asn Arg Gln Leu Asp Glu Cys Tyr Thr Ser305 310 315 320Ala
Pro Pro Ser Gly Ser Arg Gly Cys Lys Ile Trp Glu Glu Gln Val 325 330
335Cys Ile Val Val Ser Arg Lys Cys Glu Ala Arg Asp Phe Gln Pro Leu
340 345 350Pro Arg Leu Trp Lys Asp Leu Arg Glu Gly Ile Asp Glu Pro
Ile Pro 355 360 365Gly Gly Lys Leu Pro Pro Ala Leu Asn Ala Gly Glu
Ser Gly Asp His 370 375 380Gly Gly Arg Gly Cys Gly His His Gly Gly
Glu Glu Glu Ala Gly Ala385 390 395 400Gly Ala Ala Ser Thr Pro Ala
Phe Ala Ala Pro His Ala Ala Arg Ile 405 410 415His Asn Pro Asn Phe
Lys Arg Gly Arg Arg Arg Glu Ser Arg Trp Arg 420 425 430Arg Leu Ala
Ser Gly Glu Gln 435197821DNACorynascus thermophilus 197atgaagctct
ctctcttttc cgtcctggcc gctgccctca ccgtcgaggg gcatgccatc 60ttccagaagg
tctccgtcaa cggggcggac cagggctccc tcaccggcct ccgcgctccc
120aacaacaaca acccggtgca ggatgtcagc agccaggaca tgatctgcgg
ccagccggga 180tcgacgtcga gcacggtcat cgaggtcaag gccggcgaca
ggatcggcgc ctggtaccag 240cacgtcatcg gcggtgccca gttccccggc
gaccctgaca acccgatcgc cgcgtcgcac 300aagggccccg tcatggccta
cctcgccaag gttgacaatg ccgcaaccgc cgacaagacg 360ggcctgcagt
ggtatgtgtt cccgccgccc gagggacgtc agcttggggc aagtcgcgtc
420tgaccgggct cgcttctttc tctctgtata ggttcaagat ctgggaggac
acctttgatc 480ccagcagcaa gacctggggt gtcgacaacc tcatcaacaa
caacggctgg gtgtacttca 540acatcccgca gtgcatcgcc gacggccact
acctcctccg ggttgaggtc ctcgccctgc 600actcggccta ccagaccggc
ggggctcagt tctaccagtc ctgcgcccag atcagcgtgt 660ccggcggcgg
ctccttcacg ccgtcgtcga ctgtgagctt cccgggcgcc tacaacgcca
720acgaccccgg catcacgatc aacatctacg gcgctaccgg tcagcccgac
aacaacggcc 780agccgtacac tgcccctggc cccgcgccca tctcctgctg a
821198246PRTCorynascus thermophilus 198Met Lys Leu Ser Leu Phe Ser
Val Leu Ala Ala Ala Leu Thr Val Glu1 5 10 15Gly His Ala Ile Phe Gln
Lys Val Ser Val Asn Gly Ala Asp Gln Gly 20 25 30Ser Leu Thr Gly Leu
Arg Ala Pro Asn Asn Asn Asn Pro Val Gln Asp 35 40 45Val Ser Ser Gln
Asp Met Ile Cys Gly Gln Pro Gly Ser Thr Ser Ser 50 55 60Thr Val Ile
Glu Val Lys Ala Gly Asp Arg Ile Gly Ala Trp Tyr Gln65 70 75 80His
Val Ile Gly Gly Ala Gln Phe Pro Gly Asp Pro Asp Asn Pro Ile 85 90
95Ala Ala Ser His Lys Gly Pro Val Met Ala Tyr Leu Ala Lys Val Asp
100 105 110Asn Ala Ala Thr Ala Asp Lys Thr Gly Leu Gln Trp Phe Lys
Ile Trp 115 120 125Glu Asp Thr Phe Asp Pro Ser Ser Lys Thr Trp Gly
Val Asp Asn Leu 130 135 140Ile Asn Asn Asn Gly Trp Val Tyr Phe Asn
Ile Pro Gln Cys Ile Ala145 150 155 160Asp Gly His Tyr Leu Leu Arg
Val Glu Val Leu Ala Leu His Ser Ala 165 170 175Tyr Gln Thr Gly Gly
Ala Gln Phe Tyr Gln Ser Cys Ala Gln Ile Ser 180 185 190Val Ser Gly
Gly Gly Ser Phe Thr Pro Ser Ser Thr Val Ser Phe Pro 195 200 205Gly
Ala Tyr Asn Ala Asn Asp Pro Gly Ile Thr Ile Asn Ile Tyr Gly 210 215
220Ala Thr Gly Gln Pro Asp Asn Asn Gly Gln Pro Tyr Thr Ala Pro
Gly225 230 235 240Pro Ala Pro Ile Ser Cys 2451991125DNACorynascus
thermophilus 199atgaagtcct tcaccctcac cgccctggcc gccctggccg
gcaacgccgc cgcccacgcg 60accttccagg ccctctgggt cgacggcgtc gactacggct
cgcagtgcgc ccgtcttccc 120ggatccaact ccccgatcac cgacgtgagc
tcgacggcca tccgctgcaa tgccaacgcc 180ggccgcgccc agggcaagtg
cccggtcaag
gccggctcga ccgtgacgat cgagatgcac 240caggtatgtt ccactaaaag
gaggaaaaga aaaaaaacag agtggaacgg tcaggctgac 300tgaggctctc
tcgctacgat cagcaacccg gtgaccggtc gtgcggcagc gacgccatcg
360gcggcgccca ccacggcccc gtcctcgtgt acatgtccaa ggtgtcggat
gcggcgtcgg 420ccgacggctc gtccggctgg ttcaaggtgt tcgaggacgg
ctgggccaag aacccgtcgg 480gcggctccgg cgacgacgac tactggggca
ccaaggacct caacgcctgc tgcggcaaga 540tgaacgtcaa gatcccgtcc
gacctgccgt cgggcgacta cctgctccgt gccgaggcca 600tcgccctgca
cacggccggc ggctcgggcg gcgcccagtt ctacatcacc tgctaccagc
660tcaccgtcga gggttccggc aacgccagcc cggccaccgt ctccttccct
ggcgcctaca 720aggcctccga cccgggcatc ctggtcaaca tccacgccgc
catgtccggc tacaccgtgc 780ccggcccgtc cgtctactcg ggcggcagca
ccaagaaggc cggcagcggc tgctccggct 840gcgaggccac ctgcgccgtc
ggctctagcc ccagcgccac cgtcacctcg tcgcccggca 900gccagcccac
ctcccccggc ggcggcgacg gcggcggctg caccgtcccc aagtaccagc
960agtgcggtgg ccagggctac agcggctgca ccaactgcga ggtgagttcc
cctgcttact 1020tgttgtcctc tgtacccctt ccatgttttc gatgctgact
ttctgcgtta gtctggctct 1080acttgcagcg ccgtctcgcc gccgtactac
taccagtgcg tgtaa 1125200324PRTCorynascus thermophilus 200Met Lys
Ser Phe Thr Leu Thr Ala Leu Ala Ala Leu Ala Gly Asn Ala1 5 10 15Ala
Ala His Ala Thr Phe Gln Ala Leu Trp Val Asp Gly Val Asp Tyr 20 25
30Gly Ser Gln Cys Ala Arg Leu Pro Gly Ser Asn Ser Pro Ile Thr Asp
35 40 45Val Ser Ser Thr Ala Ile Arg Cys Asn Ala Asn Ala Gly Arg Ala
Gln 50 55 60Gly Lys Cys Pro Val Lys Ala Gly Ser Thr Val Thr Ile Glu
Met His65 70 75 80Gln Gln Pro Gly Asp Arg Ser Cys Gly Ser Asp Ala
Ile Gly Gly Ala 85 90 95His His Gly Pro Val Leu Val Tyr Met Ser Lys
Val Ser Asp Ala Ala 100 105 110Ser Ala Asp Gly Ser Ser Gly Trp Phe
Lys Val Phe Glu Asp Gly Trp 115 120 125Ala Lys Asn Pro Ser Gly Gly
Ser Gly Asp Asp Asp Tyr Trp Gly Thr 130 135 140Lys Asp Leu Asn Ala
Cys Cys Gly Lys Met Asn Val Lys Ile Pro Ser145 150 155 160Asp Leu
Pro Ser Gly Asp Tyr Leu Leu Arg Ala Glu Ala Ile Ala Leu 165 170
175His Thr Ala Gly Gly Ser Gly Gly Ala Gln Phe Tyr Ile Thr Cys Tyr
180 185 190Gln Leu Thr Val Glu Gly Ser Gly Asn Ala Ser Pro Ala Thr
Val Ser 195 200 205Phe Pro Gly Ala Tyr Lys Ala Ser Asp Pro Gly Ile
Leu Val Asn Ile 210 215 220His Ala Ala Met Ser Gly Tyr Thr Val Pro
Gly Pro Ser Val Tyr Ser225 230 235 240Gly Gly Ser Thr Lys Lys Ala
Gly Ser Gly Cys Ser Gly Cys Glu Ala 245 250 255Thr Cys Ala Val Gly
Ser Ser Pro Ser Ala Thr Val Thr Ser Ser Pro 260 265 270Gly Ser Gln
Pro Thr Ser Pro Gly Gly Gly Asp Gly Gly Gly Cys Thr 275 280 285Val
Pro Lys Tyr Gln Gln Cys Gly Gly Gln Gly Tyr Ser Gly Cys Thr 290 295
300Asn Cys Glu Ser Gly Ser Thr Cys Ser Ala Val Ser Pro Pro Tyr
Tyr305 310 315 320Tyr Gln Cys Val2011037DNACorynascus thermophilus
201atgaagtcgt tcacctcagc cttgttcgcc gctgggctcc ttgctcagca
tgccgcagcc 60cactccatct tccagcaggc aagcagcggc tcgatcgact tcgacacgct
gtgcacccgg 120atgccggtca gtcccgaggg cccttgggtg atggatcatc
tgcccataga cttgttaccg 180acgagctgac gggttctcgt tattaatagc
ccaacaatag ccctgtcact agcgtgacca 240gcggcgacat gacctgcaac
gtcggcggca ccaacggagt gtcgggcttc tgcgaggtga 300acggtatggt
ttcccgagtt ttcgaccagt ccccccgttt gatttttacc gccgcctgac
360acgtgggctt cttgcttcgc tccttcggct agccggcgac gagtttacgg
ttgagatgca 420cgcgcagccc ggcgaccgct cgtgcgacaa cgaggccatc
ggcgggaacc acttcggccc 480ggtcctcatc tacatgagca aggtcgacga
cgcctcgact gccgacgggt ccggcgactg 540gttcaaggtg gacgagttcg
gctacgaccc gagcaccaag acctggggca ccgacaagct 600caacgagaac
tgcggcaagc gcactttcaa gatcccccgc aacatccctg cgggcgacta
660tctcgtccgg gccgaggcca tcgcgctgca cactgccagc cagccgggcg
gcgcgcagtt 720ctacatgagc tgctatgtaa gtttctagag tctctctctc
tctctcgctt tctctctctc 780gctcgccccg tctctccatt tgtcttcgtt
cttccttttc ccttccttca aatgatgtct 840ccccgctaac tttctctctc
cccacaactt agcaagtccg gatttccggc ggcaacggag 900gccagctgcc
tgccggagtc aagatcccgg gcgcgtacag tgccaacgac cccggtatcc
960tcatcgacat ctggggcaac gacttcaacg agtacatcat cccgggcccg
cccgttatcg 1020acagcagcta cttctaa 1037202242PRTCorynascus
thermophilus 202Met Lys Ser Phe Thr Ser Ala Leu Phe Ala Ala Gly Leu
Leu Ala Gln1 5 10 15His Ala Ala Ala His Ser Ile Phe Gln Gln Ala Ser
Ser Gly Ser Ile 20 25 30Asp Phe Asp Thr Leu Cys Thr Arg Met Pro Pro
Asn Asn Ser Pro Val 35 40 45Thr Ser Val Thr Ser Gly Asp Met Thr Cys
Asn Val Gly Gly Thr Asn 50 55 60Gly Val Ser Gly Phe Cys Glu Val Asn
Ala Gly Asp Glu Phe Thr Val65 70 75 80Glu Met His Ala Gln Pro Gly
Asp Arg Ser Cys Asp Asn Glu Ala Ile 85 90 95Gly Gly Asn His Phe Gly
Pro Val Leu Ile Tyr Met Ser Lys Val Asp 100 105 110Asp Ala Ser Thr
Ala Asp Gly Ser Gly Asp Trp Phe Lys Val Asp Glu 115 120 125Phe Gly
Tyr Asp Pro Ser Thr Lys Thr Trp Gly Thr Asp Lys Leu Asn 130 135
140Glu Asn Cys Gly Lys Arg Thr Phe Lys Ile Pro Arg Asn Ile Pro
Ala145 150 155 160Gly Asp Tyr Leu Val Arg Ala Glu Ala Ile Ala Leu
His Thr Ala Ser 165 170 175Gln Pro Gly Gly Ala Gln Phe Tyr Met Ser
Cys Tyr Gln Val Arg Ile 180 185 190Ser Gly Gly Asn Gly Gly Gln Leu
Pro Ala Gly Val Lys Ile Pro Gly 195 200 205Ala Tyr Ser Ala Asn Asp
Pro Gly Ile Leu Ile Asp Ile Trp Gly Asn 210 215 220Asp Phe Asn Glu
Tyr Ile Ile Pro Gly Pro Pro Val Ile Asp Ser Ser225 230 235 240Tyr
Phe2031200DNACorynascus thermophilus 203atgaaggcct ttagcctcgt
cgccctggcg acggccgtga gcggccatac catcttccag 60cgggtgtcgg tcaacgggca
agaccagggc cagctcaagg gcgtgcgggc gccgtcgagc 120aacttcccga
tccagaacgt caacgattcc aacttcgcct gcaacgcaaa catcgtgtac
180aaggacgaca ccatcatcaa gatccccgcg ggagcccgcg tgggttcgtg
gtggcagcac 240gtcatcggcg gcccgcaggg ctccaacgac ccggacaacc
cgatcgccgc ctcccacaag 300ggtatgctga gatggcgaac caacccgcgc
cccctttccc cccctcaacc tcccggaaca 360cgcgtagctg acgggcaaat
ccaggcccca tccaggtcta cctggccaag gttgacaacg 420cggcgacagc
gtcgcccacg ggcctcaggt ggttcaaggt tgccgagcgc gggctgaaca
480acggcgtgtg ggcggtcgac gagctcatcg ccaacaacgg ctggcactac
ttcgacctgc 540cgtcgtgcgt ggcccccggc cagtacctga tgcgcgtcga
gctgctcgcc ctgcacagcg 600cctcgagccc cggcggcgcc cagttctaca
tgggctgcgc ccagatcgaa ggtgggtgca 660attctcgttc tgcttccccg
tcccttccgg ccctttcttt ctctctctcc ccttgtgctt 720tcttcgctcc
ttgacgaacc cgaggaaaga gggaagagga aagaggaaag agggaggaaa
780cggggcggag agacagacgg gatcgaatga gagagacaag acaagatcgg
ctgacgagga 840caaccagtca ccggctcggg cacccacacg ggctccgact
tcgtctcgtt cccgggcgcc 900tactcggcca acgacccggg catcctgctg
agcatctacg actcctcggg caagcccacc 960aacggcgggc gggcgtacca
gatccccggc ccgcgcccca tctcgtgctc gggcggcagc 1020aacggcggcg
gtgacaacgg cggcggcgac aacggcggcg gcaacaacgg cggcggcaac
1080agcggcggca ccgtccccct ctacggccag tgcggcggca acggatacac
cggcccgacc 1140acctgcgccg agggaacctg caaggtgtcg aacgagtggt
acagccagtg cctcccctag 1200204306PRTCorynascus thermophilus 204Met
Lys Ala Phe Ser Leu Val Ala Leu Ala Thr Ala Val Ser Gly His1 5 10
15Thr Ile Phe Gln Arg Val Ser Val Asn Gly Gln Asp Gln Gly Gln Leu
20 25 30Lys Gly Val Arg Ala Pro Ser Ser Asn Phe Pro Ile Gln Asn Val
Asn 35 40 45Asp Ser Asn Phe Ala Cys Asn Ala Asn Ile Val Tyr Lys Asp
Asp Thr 50 55 60Ile Ile Lys Ile Pro Ala Gly Ala Arg Val Gly Ser Trp
Trp Gln His65 70 75 80Val Ile Gly Gly Pro Gln Gly Ser Asn Asp Pro
Asp Asn Pro Ile Ala 85 90 95Ala Ser His Lys Gly Pro Ile Gln Val Tyr
Leu Ala Lys Val Asp Asn 100 105 110Ala Ala Thr Ala Ser Pro Thr Gly
Leu Arg Trp Phe Lys Val Ala Glu 115 120 125Arg Gly Leu Asn Asn Gly
Val Trp Ala Val Asp Glu Leu Ile Ala Asn 130 135 140Asn Gly Trp His
Tyr Phe Asp Leu Pro Ser Cys Val Ala Pro Gly Gln145 150 155 160Tyr
Leu Met Arg Val Glu Leu Leu Ala Leu His Ser Ala Ser Ser Pro 165 170
175Gly Gly Ala Gln Phe Tyr Met Gly Cys Ala Gln Ile Glu Val Thr Gly
180 185 190Ser Gly Thr His Thr Gly Ser Asp Phe Val Ser Phe Pro Gly
Ala Tyr 195 200 205Ser Ala Asn Asp Pro Gly Ile Leu Leu Ser Ile Tyr
Asp Ser Ser Gly 210 215 220Lys Pro Thr Asn Gly Gly Arg Ala Tyr Gln
Ile Pro Gly Pro Arg Pro225 230 235 240Ile Ser Cys Ser Gly Gly Ser
Asn Gly Gly Gly Asp Asn Gly Gly Gly 245 250 255Asp Asn Gly Gly Gly
Asn Asn Gly Gly Gly Asn Ser Gly Gly Thr Val 260 265 270Pro Leu Tyr
Gly Gln Cys Gly Gly Asn Gly Tyr Thr Gly Pro Thr Thr 275 280 285Cys
Ala Glu Gly Thr Cys Lys Val Ser Asn Glu Trp Tyr Ser Gln Cys 290 295
300Leu Pro305205759DNAAcrophialophora fusispora 205atgcgcatag
aagctatcac aggcctcgtg ctggcctcgg ccggtgcagt gtctgcccat 60ggctgggtcg
atgtctgggc tattggcggc aagaactaca caggcttcaa ccccacggtg
120gcgccatggg tcccggatca gggcaccatt gcgtggccgg cctggaacac
cgacacagga 180ccggtgtaca gcaaggacgt caacaccaca gacatcatct
gctcaatcaa tgccaccaac 240gccaagatct actccgaccc catcgccgct
gggaacgtca tcaacctgca ctggacggtg 300tggccagact cacaccacgg
gcccatcctg tcgtacctgg ccgcgtgcaa cggcgactgc 360gccaaggccg
acaagaccaa gctcaagtgg ttcaagattg cccatgccgg tcaaatcagc
420ctgggcaccg gcggcggcca ggttggctac tgggccagcg acaagctgca
agacgacaac 480ggcacctggc ccgtcaccat tccggcctcc atcaagcccg
gcaattacgt gctgcggaac 540gagattattg ccctccattc ggcgtacgac
gtcggcgccg cccagctcta cccgcagtgc 600gttaatatca agatcacggg
caacggccgc gtcacccctg ccggcgtggt gggaaccaag 660ctctacaagg
agaccgatcc tggcctgcat tataacatct ataacgacga gtctaagcct
720gtctatcaga tccccggccc ggccttgtgt aagtgctaa
759206252PRTAcrophialophora fusispora 206Met Arg Ile Glu Ala Ile
Thr Gly Leu Val Leu Ala Ser Ala Gly Ala1 5 10 15Val Ser Ala His Gly
Trp Val Asp Val Trp Ala Ile Gly Gly Lys Asn 20 25 30Tyr Thr Gly Phe
Asn Pro Thr Val Ala Pro Trp Val Pro Asp Gln Gly 35 40 45Thr Ile Ala
Trp Pro Ala Trp Asn Thr Asp Thr Gly Pro Val Tyr Ser 50 55 60Lys Asp
Val Asn Thr Thr Asp Ile Ile Cys Ser Ile Asn Ala Thr Asn65 70 75
80Ala Lys Ile Tyr Ser Asp Pro Ile Ala Ala Gly Asn Val Ile Asn Leu
85 90 95His Trp Thr Val Trp Pro Asp Ser His His Gly Pro Ile Leu Ser
Tyr 100 105 110Leu Ala Ala Cys Asn Gly Asp Cys Ala Lys Ala Asp Lys
Thr Lys Leu 115 120 125Lys Trp Phe Lys Ile Ala His Ala Gly Gln Ile
Ser Leu Gly Thr Gly 130 135 140Gly Gly Gln Val Gly Tyr Trp Ala Ser
Asp Lys Leu Gln Asp Asp Asn145 150 155 160Gly Thr Trp Pro Val Thr
Ile Pro Ala Ser Ile Lys Pro Gly Asn Tyr 165 170 175Val Leu Arg Asn
Glu Ile Ile Ala Leu His Ser Ala Tyr Asp Val Gly 180 185 190Ala Ala
Gln Leu Tyr Pro Gln Cys Val Asn Ile Lys Ile Thr Gly Asn 195 200
205Gly Arg Val Thr Pro Ala Gly Val Val Gly Thr Lys Leu Tyr Lys Glu
210 215 220Thr Asp Pro Gly Leu His Tyr Asn Ile Tyr Asn Asp Glu Ser
Lys Pro225 230 235 240Val Tyr Gln Ile Pro Gly Pro Ala Leu Cys Lys
Cys 245 2502071035DNACorynascus sepedonium 207atgtctaaga cttctgctct
ccttgctggc ctaacgggcg cggccctcgt cgctgcccac 60gggcacgtca gccatatcat
tgtcaacggt gtctactatg agaactacga ccccacgaca 120cactggtacc
agcccaaccc accaacagtc atcggctgga cggcagccca gcaggacaac
180ggcttcatcg agcccaacaa ctttggcacg tcggacatca tctgccacaa
gagcggttct 240ccaggcggcg gtcacgctac cgtcgctgcg ggcgacaaga
tcaacatcgt ctggactccg 300gagtggcccg actcccatat cggcccggtc
attgactacc tggctgcctg caacggtgac 360tgcgagaccg taaacaagga
gtcgctgcgc ttctttaaga ttgacggggc cggctatgac 420aaggccgctg
gccgctgggc cgccgagact ctgcgccaga acggcaacag ctggctcgtc
480cagatcccgt ctgaccttaa ggctggcaac tacgtgctcc gccacgaaat
catcgccctc 540cacggcgctg gaagcgccaa cggtgctcaa gcctacccgc
agtgcatcaa ccttcgcgtg 600acgggcggcg gcagcagcgt gcccagcggc
gtggccggca cctcgctcta caaagcctcc 660gacgcaggca tcctcttcaa
cccctacgtc gcctctcccg attacccggt cccaggcccg 720gcgctcattg
ctggtgccgc cagctctatc gtacagagca cgtcggcagt gaccgctacc
780gcctcggcca ccgctcccgg tggcggcggc gccaacccca accctacgcc
caccaccacc 840tcctcgagca atcccgcccc aagcaccacc ctcaggacaa
ccacctcggc cgcgcaaacc 900acgcccccgc ctaccaatgg caacgtccag
acaaagtacg gtcagtgtgg tggtagggac 960tggagcggcc caacggcgtg
cgcggctggt tccagctgct cggtgctcaa cgactggtac 1020tcccagtgcg tgtaa
1035208344PRTCorynascus sepedonium 208Met Ser Lys Thr Ser Ala Leu
Leu Ala Gly Leu Thr Gly Ala Ala Leu1 5 10 15Val Ala Ala His Gly His
Val Ser His Ile Ile Val Asn Gly Val Tyr 20 25 30Tyr Glu Asn Tyr Asp
Pro Thr Thr His Trp Tyr Gln Pro Asn Pro Pro 35 40 45Thr Val Ile Gly
Trp Thr Ala Ala Gln Gln Asp Asn Gly Phe Ile Glu 50 55 60Pro Asn Asn
Phe Gly Thr Ser Asp Ile Ile Cys His Lys Ser Gly Ser65 70 75 80Pro
Gly Gly Gly His Ala Thr Val Ala Ala Gly Asp Lys Ile Asn Ile 85 90
95Val Trp Thr Pro Glu Trp Pro Asp Ser His Ile Gly Pro Val Ile Asp
100 105 110Tyr Leu Ala Ala Cys Asn Gly Asp Cys Glu Thr Val Asn Lys
Glu Ser 115 120 125Leu Arg Phe Phe Lys Ile Asp Gly Ala Gly Tyr Asp
Lys Ala Ala Gly 130 135 140Arg Trp Ala Ala Glu Thr Leu Arg Gln Asn
Gly Asn Ser Trp Leu Val145 150 155 160Gln Ile Pro Ser Asp Leu Lys
Ala Gly Asn Tyr Val Leu Arg His Glu 165 170 175Ile Ile Ala Leu His
Gly Ala Gly Ser Ala Asn Gly Ala Gln Ala Tyr 180 185 190Pro Gln Cys
Ile Asn Leu Arg Val Thr Gly Gly Gly Ser Ser Val Pro 195 200 205Ser
Gly Val Ala Gly Thr Ser Leu Tyr Lys Ala Ser Asp Ala Gly Ile 210 215
220Leu Phe Asn Pro Tyr Val Ala Ser Pro Asp Tyr Pro Val Pro Gly
Pro225 230 235 240Ala Leu Ile Ala Gly Ala Ala Ser Ser Ile Val Gln
Ser Thr Ser Ala 245 250 255Val Thr Ala Thr Ala Ser Ala Thr Ala Pro
Gly Gly Gly Gly Ala Asn 260 265 270Pro Asn Pro Thr Pro Thr Thr Thr
Ser Ser Ser Asn Pro Ala Pro Ser 275 280 285Thr Thr Leu Arg Thr Thr
Thr Ser Ala Ala Gln Thr Thr Pro Pro Pro 290 295 300Thr Asn Gly Asn
Val Gln Thr Lys Tyr Gly Gln Cys Gly Gly Arg Asp305 310 315 320Trp
Ser Gly Pro Thr Ala Cys Ala Ala Gly Ser Ser Cys Ser Val Leu 325 330
335Asn Asp Trp Tyr Ser Gln Cys Val 3402091044DNACorynascus
sepedonium 209atgccttctt ctacctccaa gggtcttttc tccgccctca
tgggcgcggc gtcggttgcc 60gcccatggtc atgtcaccaa cattgtcatc aacggtgtgt
cgtaccagaa ctacgacccg 120accagcttcc cttacatgca gaacccgccg
acggttgttg gctggacggc aagcaacact 180gataacggct tcgtcgctcc
tgatgcgttt gctagcggcg acatcatctg ccacagggac 240gccaccaatg
ctggtggtca tgccgtcgtt gctgctggtg acaaggtctt catccagtgg
300gatacctggc ctgagtcgca ccatggcccc gtccttgatt acctcgccag
ctgcggtgac 360gccggctgcg aaacggtcga caagaacact ctcgagttct
tcaagatcgg cgaggctggc 420ctgatcgacg gcagcagtgc tcccggcaag
tgggcgtcgg accagctgat tgagaacaat 480aactcgtgga tggttcagat
ccctgccaac cttgcgcccg gaaactatgt gctgcggcat 540gagattattg
ctttgcacag cgctgggcaa gctaacggtg cccaaaacta cccccagtgc
600ttcaacctgc aagttaccgg ctccggcacg gacaagcctg ccggtgtgct
cggcaccgag 660ctctacactc ccaccgacgc cggcatcttg gccaacatct
acacctcgcc tgttcagtac 720gagattcctg gcccggctct gatctcgggc
gcttcggccg ttgaacagtc ctcctcggct 780atcaccgcct ccgccagcgc
tgagaccggc
tccgccacag caccccctgc cggctctgcc 840acggccgccc ccaccactac
cactaccacg gctggctcgg atgctagcgc tacgccctcg 900tcctcgtcca
gctctggtgc gagcaccacc gccgagccca ccccttcggc tactactacc
960gccggcggca gcaccccgcg cccgacccgg tgccctggcc tgaagcgccg
ccgccacgcc 1020cgtgatgtca agctcgccct ctaa 1044210347PRTCorynascus
sepedonium 210Met Pro Ser Ser Thr Ser Lys Gly Leu Phe Ser Ala Leu
Met Gly Ala1 5 10 15Ala Ser Val Ala Ala His Gly His Val Thr Asn Ile
Val Ile Asn Gly 20 25 30Val Ser Tyr Gln Asn Tyr Asp Pro Thr Ser Phe
Pro Tyr Met Gln Asn 35 40 45Pro Pro Thr Val Val Gly Trp Thr Ala Ser
Asn Thr Asp Asn Gly Phe 50 55 60Val Ala Pro Asp Ala Phe Ala Ser Gly
Asp Ile Ile Cys His Arg Asp65 70 75 80Ala Thr Asn Ala Gly Gly His
Ala Val Val Ala Ala Gly Asp Lys Val 85 90 95Phe Ile Gln Trp Asp Thr
Trp Pro Glu Ser His His Gly Pro Val Leu 100 105 110Asp Tyr Leu Ala
Ser Cys Gly Asp Ala Gly Cys Glu Thr Val Asp Lys 115 120 125Asn Thr
Leu Glu Phe Phe Lys Ile Gly Glu Ala Gly Leu Ile Asp Gly 130 135
140Ser Ser Ala Pro Gly Lys Trp Ala Ser Asp Gln Leu Ile Glu Asn
Asn145 150 155 160Asn Ser Trp Met Val Gln Ile Pro Ala Asn Leu Ala
Pro Gly Asn Tyr 165 170 175Val Leu Arg His Glu Ile Ile Ala Leu His
Ser Ala Gly Gln Ala Asn 180 185 190Gly Ala Gln Asn Tyr Pro Gln Cys
Phe Asn Leu Gln Val Thr Gly Ser 195 200 205Gly Thr Asp Lys Pro Ala
Gly Val Leu Gly Thr Glu Leu Tyr Thr Pro 210 215 220Thr Asp Ala Gly
Ile Leu Ala Asn Ile Tyr Thr Ser Pro Val Gln Tyr225 230 235 240Glu
Ile Pro Gly Pro Ala Leu Ile Ser Gly Ala Ser Ala Val Glu Gln 245 250
255Ser Ser Ser Ala Ile Thr Ala Ser Ala Ser Ala Glu Thr Gly Ser Ala
260 265 270Thr Ala Pro Pro Ala Gly Ser Ala Thr Ala Ala Pro Thr Thr
Thr Thr 275 280 285Thr Thr Ala Gly Ser Asp Ala Ser Ala Thr Pro Ser
Ser Ser Ser Ser 290 295 300Ser Gly Ala Ser Thr Thr Ala Glu Pro Thr
Pro Ser Ala Thr Thr Thr305 310 315 320Ala Gly Gly Ser Thr Pro Arg
Pro Thr Arg Cys Pro Gly Leu Lys Arg 325 330 335Arg Arg His Ala Arg
Asp Val Lys Leu Ala Leu 340 3452111005DNAMalbranchea cinnamomea
211atgtcaccct ccttcaagtc cactgccatc ctcggagccg ttgctctggc
cgcccgcgtg 60cgcgcccacg gctacgtgtc tggaatcgtc gttgacggtg cttaccatgg
cggttacatc 120gtcgacaagt acccctacat gcccaaccca cccgatgtgg
tcggctggtc gactacggcc 180acggacctgg gcttcgtcgc ccctgacgcc
tttggcgacc cggacatcat ctgccaccgg 240gacggtgccc ccggtgccat
ccacgccaaa gtcaacgccg gtgccaccat cgagctgcag 300tggaacacct
ggcccgaaag ccaccacggg cccgtcatcg actacctggc taactgcaac
360ggtgactgct cgtccgtcga caagacctcg ctcaagttct tcaagatcag
cgaggccggc 420ctaaacgacg gctccaacgc ccccggccag tgggcgtccg
acgatctcat tgccaacaac 480aacagctgga ctgtgaccat ccccaagtcg
atcgccccgg gcaactacgt gctgcgccac 540gagatcatcg ccctgcacag
cgccggcaac cagaatggcg cgcagaacta cccccagtgc 600ttcaacctcg
agatcaccag caacggcagc gacaacccgg agggcgtgct gggaaccgag
660ctgtacaagg ccgacgaccc gggcattctg ttcaacatct accagcccat
ggactcgtac 720ccgattcccg gccctgctct ctacaccggc ggctcttctc
cctcccctaa tccgcccacc 780tctacccagt cgcctgtgcc ccagcccacc
cagtctcccc catcgggcag caaccccggc 840aacggcaacg gcgacgacga
caacgacaac ggcaacgaga ccccatcccc gtctctcccc 900gtcgagatcc
ctgacgacct gacctcgcgc gagctactcc ttgtggccca ggagatcatt
960gcccgtctgc ttgagctgca gaatcagctg gtcgtctcga actaa
1005212334PRTMalbranchea cinnamomea 212Met Ser Pro Ser Phe Lys Ser
Thr Ala Ile Leu Gly Ala Val Ala Leu1 5 10 15Ala Ala Arg Val Arg Ala
His Gly Tyr Val Ser Gly Ile Val Val Asp 20 25 30Gly Ala Tyr His Gly
Gly Tyr Ile Val Asp Lys Tyr Pro Tyr Met Pro 35 40 45Asn Pro Pro Asp
Val Val Gly Trp Ser Thr Thr Ala Thr Asp Leu Gly 50 55 60Phe Val Ala
Pro Asp Ala Phe Gly Asp Pro Asp Ile Ile Cys His Arg65 70 75 80Asp
Gly Ala Pro Gly Ala Ile His Ala Lys Val Asn Ala Gly Ala Thr 85 90
95Ile Glu Leu Gln Trp Asn Thr Trp Pro Glu Ser His His Gly Pro Val
100 105 110Ile Asp Tyr Leu Ala Asn Cys Asn Gly Asp Cys Ser Ser Val
Asp Lys 115 120 125Thr Ser Leu Lys Phe Phe Lys Ile Ser Glu Ala Gly
Leu Asn Asp Gly 130 135 140Ser Asn Ala Pro Gly Gln Trp Ala Ser Asp
Asp Leu Ile Ala Asn Asn145 150 155 160Asn Ser Trp Thr Val Thr Ile
Pro Lys Ser Ile Ala Pro Gly Asn Tyr 165 170 175Val Leu Arg His Glu
Ile Ile Ala Leu His Ser Ala Gly Asn Gln Asn 180 185 190Gly Ala Gln
Asn Tyr Pro Gln Cys Phe Asn Leu Glu Ile Thr Ser Asn 195 200 205Gly
Ser Asp Asn Pro Glu Gly Val Leu Gly Thr Glu Leu Tyr Lys Ala 210 215
220Asp Asp Pro Gly Ile Leu Phe Asn Ile Tyr Gln Pro Met Asp Ser
Tyr225 230 235 240Pro Ile Pro Gly Pro Ala Leu Tyr Thr Gly Gly Ser
Ser Pro Ser Pro 245 250 255Asn Pro Pro Thr Ser Thr Gln Ser Pro Val
Pro Gln Pro Thr Gln Ser 260 265 270Pro Pro Ser Gly Ser Asn Pro Gly
Asn Gly Asn Gly Asp Asp Asp Asn 275 280 285Asp Asn Gly Asn Glu Thr
Pro Ser Pro Ser Leu Pro Val Glu Ile Pro 290 295 300Asp Asp Leu Thr
Ser Arg Glu Leu Leu Leu Val Ala Gln Glu Ile Ile305 310 315 320Ala
Arg Leu Leu Glu Leu Gln Asn Gln Leu Val Val Ser Asn 325
3302131101DNATalaromyces leycettanus 213atgcatcaac acttccgata
cactgcgctc ctgacagcgt tgctgtcagc atcaacccga 60gtcgcatccc acggccatgt
cagcaacatt gtcattaatg gcgttcccta tcaaggatgg 120gatatcgatt
ccatgcccta cgagtcagac ccaccagtgg ttgtcgcctg ggagacacct
180aacacgtcaa acggtttcat taccccggat cagtacggta cgagtgatat
tatctgccat 240ctgaacgcaa ccaacgcaaa gggccatgcc gtcgttgctg
ccggagacaa gatcagcatt 300caatggactg cctggcccag ctcccaccac
ggccctgtca tcagctacct ggccaactgt 360ggcgccagct gtgagacagt
cgacaaaacg acgttgcaat tctttaagat cgacaacatc 420ggtttcatag
atgactcttc ccccccaggc atctgggcag ccgatcaatt ggaagcaaac
480aacaacacct ggctcgtgga gatccccccg accatcgctc caggatacta
cgtcctgcgc 540aacgagatca tcgccctaca cggtgcagag aatcaggatg
gcgcccagaa ctatccgcag 600tgcttcaatc tgcaggtcac cggctcgggt
accgataaac ccgccggcgt tcttggaact 660cagctctatt ctcccactga
cccgggcatt ctcgtgaaca tttacacgag cctttcgacc 720tacatcgtcc
ccggtccaac cccgtacagt ggttgggtgt ccgtcgtgca gtctagctct
780gctatcaccg cttctggaac cccggtgacg ggcactggcg gagttagccc
aaccacggct 840gctactacga cttcttcttc tcactccacg acttctacta
ctaccgggcc cactgtaacc 900tcgactagcc acactactac cactactact
cctactaccc tcagaaccac gactacaact 960gcagctggtg gtggtgcgac
acagaccgtc tacggccaat gcggcggtag tggttggact 1020ggcgcaactg
cctgcgcagc cggagctact tgcagcactc tgaatcccta ctatgcccaa
1080tgccttccta ctggtgcttg a 1101214366PRTTalaromyces leycettanus
214Met His Gln His Phe Arg Tyr Thr Ala Leu Leu Thr Ala Leu Leu Ser1
5 10 15Ala Ser Thr Arg Val Ala Ser His Gly His Val Ser Asn Ile Val
Ile 20 25 30Asn Gly Val Pro Tyr Gln Gly Trp Asp Ile Asp Ser Met Pro
Tyr Glu 35 40 45Ser Asp Pro Pro Val Val Val Ala Trp Glu Thr Pro Asn
Thr Ser Asn 50 55 60Gly Phe Ile Thr Pro Asp Gln Tyr Gly Thr Ser Asp
Ile Ile Cys His65 70 75 80Leu Asn Ala Thr Asn Ala Lys Gly His Ala
Val Val Ala Ala Gly Asp 85 90 95Lys Ile Ser Ile Gln Trp Thr Ala Trp
Pro Ser Ser His His Gly Pro 100 105 110Val Ile Ser Tyr Leu Ala Asn
Cys Gly Ala Ser Cys Glu Thr Val Asp 115 120 125Lys Thr Thr Leu Gln
Phe Phe Lys Ile Asp Asn Ile Gly Phe Ile Asp 130 135 140Asp Ser Ser
Pro Pro Gly Ile Trp Ala Ala Asp Gln Leu Glu Ala Asn145 150 155
160Asn Asn Thr Trp Leu Val Glu Ile Pro Pro Thr Ile Ala Pro Gly Tyr
165 170 175Tyr Val Leu Arg Asn Glu Ile Ile Ala Leu His Gly Ala Glu
Asn Gln 180 185 190Asp Gly Ala Gln Asn Tyr Pro Gln Cys Phe Asn Leu
Gln Val Thr Gly 195 200 205Ser Gly Thr Asp Lys Pro Ala Gly Val Leu
Gly Thr Gln Leu Tyr Ser 210 215 220Pro Thr Asp Pro Gly Ile Leu Val
Asn Ile Tyr Thr Ser Leu Ser Thr225 230 235 240Tyr Ile Val Pro Gly
Pro Thr Pro Tyr Ser Gly Trp Val Ser Val Val 245 250 255Gln Ser Ser
Ser Ala Ile Thr Ala Ser Gly Thr Pro Val Thr Gly Thr 260 265 270Gly
Gly Val Ser Pro Thr Thr Ala Ala Thr Thr Thr Ser Ser Ser His 275 280
285Ser Thr Thr Ser Thr Thr Thr Gly Pro Thr Val Thr Ser Thr Ser His
290 295 300Thr Thr Thr Thr Thr Thr Pro Thr Thr Leu Arg Thr Thr Thr
Thr Thr305 310 315 320Ala Ala Gly Gly Gly Ala Thr Gln Thr Val Tyr
Gly Gln Cys Gly Gly 325 330 335Ser Gly Trp Thr Gly Ala Thr Ala Cys
Ala Ala Gly Ala Thr Cys Ser 340 345 350Thr Leu Asn Pro Tyr Tyr Ala
Gln Cys Leu Pro Thr Gly Ala 355 360 3652151091DNAChaetomium
thermophilummisc_feature(522)..(524)n is a, c, g, or t
215atgccttcct tcgcttcgaa gactctcatt tctgccctcg ccggcgctgc
cagcgtcgcc 60gctcacggcc acgtcaagaa cttcgtcatc aacggtctgt cgtaccaggc
ctacgacccg 120accgtcttcc cgtacatgca gaaccctccc atcgtcgccg
gctggacggc ctccaacact 180gacaacggct tcgtgggccc cgagtcctac
tcgagccccg atatcatctg ccacaagtcg 240gccacgaacg ccaagggcca
tgccgtcatc aaggccggtg actctgtcta catccagtgg 300gacacctggc
ccgagtcgca ccacggcccg gtcatcgact acctcgccag ctgcggcagc
360gccggctgcg agacggtcga caagacccag ctcgagttct tcaagatcgc
cgaggccggt 420ctgattgacg gctcccaggc tcccggaaag tgggctgccg
atcagctcat cgcccagaac 480aactcgtggc tggtcaccat ccccgagaat
atcaagccgc tnnnggctcc tacgtcctcc 540gccacgagat catcgccctg
cacagcgctg gccagaccaa cggtgcccag aactaccccg 600tctgcatcaa
cctcgaggtc actggtggcg gcagcgacgt tccctcgggt gtcaagggta
660ctgagctcta caagcccacc gaccccggca tcctcatcaa catctaccag
tcgctctcga 720actacaccat ccctggccct gctctgatgc ccggcgccaa
gccagtcacc cagcacacct 780cagccatcat cggcagcacc accgccatca
ctggcaccgc caccgctgct ccggccgcgc 840cgacctcgac cgccgctgcc
atcaccacca gctctgctaa tgccaacccc gccccgacca 900ccacccgcgg
caacgccaac cccgtcccga ctaccaccct ccgcacgagc accatcgctc
960ctcagcccac tgctgccccc atccagaccc cgacctccag cgtcggccgg
cccccgcgcc 1020cgacccgctg ccctggtctg gacaacttca agcgcgctcg
tcgccacgct cgtgaccttg 1080ctgcccacta a 1091216364PRTChaetomium
thermophilummisc_feature(174)..(176)Xaa can be any naturally
occurring amino acid 216Met Pro Ser Phe Ala Ser Lys Thr Leu Ile Ser
Ala Leu Ala Gly Ala1 5 10 15Ala Ser Val Ala Ala His Gly His Val Lys
Asn Phe Val Ile Asn Gly 20 25 30Leu Ser Tyr Gln Ala Tyr Asp Pro Thr
Val Phe Pro Tyr Met Gln Asn 35 40 45Pro Pro Ile Val Ala Gly Trp Thr
Ala Ser Asn Thr Asp Asn Gly Phe 50 55 60Val Gly Pro Glu Ser Tyr Ser
Ser Pro Asp Ile Ile Cys His Lys Ser65 70 75 80Ala Thr Asn Ala Lys
Gly His Ala Val Ile Lys Ala Gly Asp Ser Val 85 90 95Tyr Ile Gln Trp
Asp Thr Trp Pro Glu Ser His His Gly Pro Val Ile 100 105 110Asp Tyr
Leu Ala Ser Cys Gly Ser Ala Gly Cys Glu Thr Val Asp Lys 115 120
125Thr Gln Leu Glu Phe Phe Lys Ile Ala Glu Ala Gly Leu Ile Asp Gly
130 135 140Ser Gln Ala Pro Gly Lys Trp Ala Ala Asp Gln Leu Ile Ala
Gln Asn145 150 155 160Asn Ser Trp Leu Val Thr Ile Pro Glu Asn Ile
Lys Pro Xaa Xaa Xaa 165 170 175Gly Ser Tyr Val Leu Arg His Glu Ile
Ile Ala Leu His Ser Ala Gly 180 185 190Gln Thr Asn Gly Ala Gln Asn
Tyr Pro Val Cys Ile Asn Leu Glu Val 195 200 205Thr Gly Gly Gly Ser
Asp Val Pro Ser Gly Val Lys Gly Thr Glu Leu 210 215 220Tyr Lys Pro
Thr Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gln Ser Leu225 230 235
240Ser Asn Tyr Thr Ile Pro Gly Pro Ala Leu Met Pro Gly Ala Lys Pro
245 250 255Val Thr Gln His Thr Ser Ala Ile Ile Gly Ser Thr Thr Ala
Ile Thr 260 265 270Gly Thr Ala Thr Ala Ala Pro Ala Ala Pro Thr Ser
Thr Ala Ala Ala 275 280 285Ile Thr Thr Ser Ser Ala Asn Ala Asn Pro
Ala Pro Thr Thr Thr Arg 290 295 300Gly Asn Ala Asn Pro Val Pro Thr
Thr Thr Leu Arg Thr Ser Thr Ile305 310 315 320Ala Pro Gln Pro Thr
Ala Ala Pro Ile Gln Thr Pro Thr Ser Ser Val 325 330 335Gly Arg Pro
Pro Arg Pro Thr Arg Cys Pro Gly Leu Asp Asn Phe Lys 340 345 350Arg
Ala Arg Arg His Ala Arg Asp Leu Ala Ala His 355
36021740DNAArtificial SequenceArtificial DNA Primer 217cacaactggg
gatccatgac tttgtccaag atcacttcca 4021840DNAArtificial
SequenceArtificial DNA Primer 218ggcctccgcg gccgcttaag cgttgaacag
tgcaggacca 4021940DNAArtificial SequenceArtificial DNA Primer
219cacaactggg gatccatgac tttgtccaag atcacttcca 4022040DNAArtificial
SequenceArtificial DNA Primer 220ggcctccgcg gccgcttaag cgttgaacag
tgcaggacca 4022140DNAArtificial SequenceArtificial DNA Primer
221cacaactggg gatccatgct gtcttcgacg actcgcaccc 4022240DNAArtificial
SequenceArtificial DNA Primer 222ggcctccgcg gccgcctaga acgtcggctc
aggcggcccc 4022327DNAArtificial SequenceArtificial DNA Primer
223ctggggatcc atgtcctttt ccaagat 2722429DNAArtificial
SequenceArtificial DNA Primer 224ctccgcggcc gcttaaccag tatacagag
2922525DNAArtificial SequenceArtificial DNA Primer 225actcaattta
cctctatcca cactt 2522625DNAArtificial SequenceArtificial DNA Primer
226gaattgtgag cggataacaa tttca 2522736DNAArtificial
SequenceArtificial DNA Primer 227cggactgcgc accatgctgt cttcgacgac
tcgcac 3622835DNAArtificial SequenceArtificial DNA Primer
228tcgccacgga gcttatcgac ttcttctaga acgtc 3522945DNAArtificial
SequenceArtificial DNA Primer 229atcatcgccc ttcactctgc ggccaacctg
aacggcgcgc agaac 4523045DNAArtificial SequenceArtificial DNA Primer
230gttctgcgcg ccgttcaggt tggccgcaga gtgaagggcg atgat
4523145DNAArtificial SequenceArtificial DNA Primer 231atcatcgccc
ttcactctgc gtggaacctg aacggcgcgc agaac 4523245DNAArtificial
SequenceArtificial DNA Primer 232gttctgcgcg ccgttcaggt tccacgcaga
gtgaagggcg atgat 4523349DNAArtificial SequenceArtificial DNA Primer
233acaagaatac tgatcctggc atctggtttg acatctactc ggatctgag
4923449DNAArtificial SequenceArtificial DNA Primer 234ctcagatccg
agtagatgtc aaaccagatg ccaggatcag tattcttgt 4923544DNAArtificial
SequenceArtificial DNA Primer 235tcgcccttca ctctgcgggt aagctgaacg
gcgcgcagaa ctac 4423644DNAArtificial SequenceArtificial DNA Primer
236gtagttctgc gcgccgttca gcttacccgc agagtgaagg gcga
4423745DNAArtificial SequenceArtificial DNA Primer 237atcatcgccc
ttcactctgc gtttaacctg aacggcgcgc agaac 4523845DNAArtificial
SequenceArtificial DNA Primer 238gttctgcgcg
ccgttcaggt taaacgcaga gtgaagggcg atgat 4523944DNAArtificial
SequenceArtificial DNA Primer 239tcgcccttca ctctgcgggt aagctgaacg
gcgcgcagaa ctac 4424044DNAArtificial SequenceArtificial DNA Primer
240gtagttctgc gcgccgttca gcttacccgc agagtgaagg gcga
4424145DNAArtificial SequenceArtificial DNA Primer 241gtgctcaggg
atctggcacc tacggcacgt ccctgtacaa gaata 4524245DNAArtificial
SequenceArtificial DNA Primer 242tattcttgta cagggacgtg ccgtaggtgc
cagatccctg agcac 452433060DNAAspergillus fumigatus 243atgagattcg
gttggctcga ggtggccgct ctgacggccg cttctgtagc caatgcccag 60gtttgtgatg
ctttcccgtc attgtttcgg atatagttga caatagtcat ggaaataatc
120aggaattggc tttctctcca ccattctacc cttcgccttg ggctgatggc
cagggagagt 180gggcagatgc ccatcgacgc gccgtcgaga tcgtttctca
gatgacactg gcggagaagg 240ttaaccttac aacgggtact gggtgggttg
cgactttttt gttgacagtg agctttcttc 300actgaccatc tacacagatg
ggaaatggac cgatgcgtcg gtcaaaccgg cagcgttccc 360aggtaagctt
gcaattctgc aacaacgtgc aagtgtagtt gctaaaacgc ggtggtgcag
420acttggtatc aactggggtc tttgtggcca ggattcccct ttgggtatcc
gtttctgtga 480gctatacccg cggagtcttt cagtccttgt attatgtgct
gatgattgtc tctgtatagc 540tgacctcaac tccgccttcc ctgctggtac
taatgtcgcc gcgacatggg acaagacact 600cgcctacctt cgtggcaagg
ccatgggtga ggaattcaac gacaagggcg tggacatttt 660gctggggcct
gctgctggtc ctctcggcaa atacccggac ggcggcagaa tctgggaagg
720cttctctcct gatccggttc tcactggtgt acttttcgcc gaaactatca
agggtatcca 780agacgcgggt gtgattgcta ctgccaagca ttacattctg
aatgaacagg agcatttccg 840acaggttggc gaggcccagg gatatggtta
caacatcacg gagacgatca gctccaacgt 900ggatgacaag accatgcacg
agttgtacct ttggtgagta gttgacactg caaatgagga 960ccttgattga
tttgactgac ctggaatgca ggccctttgc agatgctgtg cgcggtaaga
1020ttttccgtag acttgacctc gcgacgaaga aatcgctgac gaaccatcgt
agctggcgtt 1080ggcgctgtca tgtgttccta caatcaaatc aacaacagct
acggttgtca aaacagtcaa 1140actctcaaca agctcctcaa ggctgagctg
ggcttccaag gcttcgtcat gagtgactgg 1200agcgctcacc acagcggtgt
cggcgctgcc ctcgctgggt tggatatgtc gatgcctgga 1260gacatttcct
tcgacgacgg actctccttc tggggcacga acctaactgt cagtgttctt
1320aacggcaccg ttccagcctg gcgtgtcgat gacatggctg ttcgtatcat
gaccgcgtac 1380tacaaggttg gtcgtgaccg tcttcgtatt ccccctaact
tcagctcctg gacccgggat 1440gagtacggct gggagcattc tgctgtctcc
gagggagcct ggaccaaggt gaacgacttc 1500gtcaatgtgc agcgcagtca
ctctcagatc atccgtgaga ttggtgccgc tagtacagtg 1560ctcttgaaga
acacgggtgc tcttcctttg accggcaagg aggttaaagt gggtgttctc
1620ggtgaagacg ctggttccaa cccgtggggt gctaacggct gccccgaccg
cggctgtgat 1680aacggcactc ttgctatggc ctggggtagt ggtactgcca
acttccctta ccttgtcacc 1740cccgagcagg ctatccagcg agaggtcatc
agcaacggcg gcaatgtctt tgctgtgact 1800gataacgggg ctctcagcca
gatggcagat gttgcatctc aatccaggtg agtgcgggct 1860cttagaaaaa
gaacgttctc tgaatgaagt tttttaacca ttgcgaacag cgtgtctttg
1920gtgtttgtca acgccgactc tggagagggt ttcatcagtg tcgacggcaa
cgagggtgac 1980cgcaaaaatc tcactctgtg gaagaacggc gaggccgtca
ttgacactgt tgtcagccac 2040tgcaacaaca cgattgtggt tattcacagt
gttgggcccg tcttgatcga ccggtggtat 2100gataacccca acgtcactgc
catcatctgg gccggcttgc ccggtcagga gagtggcaac 2160tccctggtcg
acgtgctcta tggccgcgtc aaccccagcg ccaagacccc gttcacctgg
2220ggcaagactc gggagtctta cggggctccc ttgctcaccg agcctaacaa
tggcaatggt 2280gctccccagg atgatttcaa cgagggcgtc ttcattgact
accgtcactt tgacaagcgc 2340aatgagaccc ccatttatga gtttggccat
ggcttgagct acaccacctt tggttactct 2400caccttcggg ttcaggccct
caatagttcg agttcggcat atgtcccgac tagcggagag 2460accaagcctg
cgccaaccta tggtgagatc ggtagtgccg ccgactacct gtatcccgag
2520ggtctcaaaa gaattaccaa gtttatttac ccttggctca actcgaccga
cctcgaggat 2580tcttctgacg acccgaacta cggctgggag gactcggagt
acattcccga aggcgctagg 2640gatgggtctc ctcaacccct cctgaaggct
ggcggcgctc ctggtggtaa ccctaccctt 2700tatcaggatc ttgttagggt
gtcggccacc ataaccaaca ctggtaacgt cgccggttat 2760gaagtccctc
aattggtgag tgacccgcat gttccttgcg ttgcaatttg gctaactcgc
2820ttctagtatg tttcactggg cggaccgaac gagcctcggg tcgttctgcg
caagttcgac 2880cgaatcttcc tggctcctgg ggagcaaaag gtttggacca
cgactcttaa ccgtcgtgat 2940ctcgccaatt gggatgtgga ggctcaggac
tgggtcatca caaagtaccc caagaaagtg 3000cacgtcggca gctcctcgcg
taagctgcct ctgagagcgc ctctgccccg tgtctactag
3060244863PRTAspergillus fumigatus 244Met Arg Phe Gly Trp Leu Glu
Val Ala Ala Leu Thr Ala Ala Ser Val1 5 10 15Ala Asn Ala Gln Glu Leu
Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25 30Trp Ala Asp Gly Gln
Gly Glu Trp Ala Asp Ala His Arg Arg Ala Val 35 40 45Glu Ile Val Ser
Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr 50 55 60Gly Thr Gly
Trp Glu Met Asp Arg Cys Val Gly Gln Thr Gly Ser Val65 70 75 80Pro
Arg Leu Gly Ile Asn Trp Gly Leu Cys Gly Gln Asp Ser Pro Leu 85 90
95Gly Ile Arg Phe Ser Asp Leu Asn Ser Ala Phe Pro Ala Gly Thr Asn
100 105 110Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly
Lys Ala 115 120 125Met Gly Glu Glu Phe Asn Asp Lys Gly Val Asp Ile
Leu Leu Gly Pro 130 135 140Ala Ala Gly Pro Leu Gly Lys Tyr Pro Asp
Gly Gly Arg Ile Trp Glu145 150 155 160Gly Phe Ser Pro Asp Pro Val
Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175Ile Lys Gly Ile Gln
Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190Ile Leu Asn
Glu Gln Glu His Phe Arg Gln Val Gly Glu Ala Gln Gly 195 200 205Tyr
Gly Tyr Asn Ile Thr Glu Thr Ile Ser Ser Asn Val Asp Asp Lys 210 215
220Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg
Ala225 230 235 240Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile
Asn Asn Ser Tyr 245 250 255Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys
Leu Leu Lys Ala Glu Leu 260 265 270Gly Phe Gln Gly Phe Val Met Ser
Asp Trp Ser Ala His His Ser Gly 275 280 285Val Gly Ala Ala Leu Ala
Gly Leu Asp Met Ser Met Pro Gly Asp Ile 290 295 300Ser Phe Asp Asp
Gly Leu Ser Phe Trp Gly Thr Asn Leu Thr Val Ser305 310 315 320Val
Leu Asn Gly Thr Val Pro Ala Trp Arg Val Asp Asp Met Ala Val 325 330
335Arg Ile Met Thr Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Arg Ile
340 345 350Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Trp
Glu His 355 360 365Ser Ala Val Ser Glu Gly Ala Trp Thr Lys Val Asn
Asp Phe Val Asn 370 375 380Val Gln Arg Ser His Ser Gln Ile Ile Arg
Glu Ile Gly Ala Ala Ser385 390 395 400Thr Val Leu Leu Lys Asn Thr
Gly Ala Leu Pro Leu Thr Gly Lys Glu 405 410 415Val Lys Val Gly Val
Leu Gly Glu Asp Ala Gly Ser Asn Pro Trp Gly 420 425 430Ala Asn Gly
Cys Pro Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445Ala
Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455
460Gln Ala Ile Gln Arg Glu Val Ile Ser Asn Gly Gly Asn Val Phe
Ala465 470 475 480Val Thr Asp Asn Gly Ala Leu Ser Gln Met Ala Asp
Val Ala Ser Gln 485 490 495Ser Ser Val Ser Leu Val Phe Val Asn Ala
Asp Ser Gly Glu Gly Phe 500 505 510Ile Ser Val Asp Gly Asn Glu Gly
Asp Arg Lys Asn Leu Thr Leu Trp 515 520 525Lys Asn Gly Glu Ala Val
Ile Asp Thr Val Val Ser His Cys Asn Asn 530 535 540Thr Ile Val Val
Ile His Ser Val Gly Pro Val Leu Ile Asp Arg Trp545 550 555 560Tyr
Asp Asn Pro Asn Val Thr Ala Ile Ile Trp Ala Gly Leu Pro Gly 565 570
575Gln Glu Ser Gly Asn Ser Leu Val Asp Val Leu Tyr Gly Arg Val Asn
580 585 590Pro Ser Ala Lys Thr Pro Phe Thr Trp Gly Lys Thr Arg Glu
Ser Tyr 595 600 605Gly Ala Pro Leu Leu Thr Glu Pro Asn Asn Gly Asn
Gly Ala Pro Gln 610 615 620Asp Asp Phe Asn Glu Gly Val Phe Ile Asp
Tyr Arg His Phe Asp Lys625 630 635 640Arg Asn Glu Thr Pro Ile Tyr
Glu Phe Gly His Gly Leu Ser Tyr Thr 645 650 655Thr Phe Gly Tyr Ser
His Leu Arg Val Gln Ala Leu Asn Ser Ser Ser 660 665 670Ser Ala Tyr
Val Pro Thr Ser Gly Glu Thr Lys Pro Ala Pro Thr Tyr 675 680 685Gly
Glu Ile Gly Ser Ala Ala Asp Tyr Leu Tyr Pro Glu Gly Leu Lys 690 695
700Arg Ile Thr Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu
Glu705 710 715 720Asp Ser Ser Asp Asp Pro Asn Tyr Gly Trp Glu Asp
Ser Glu Tyr Ile 725 730 735Pro Glu Gly Ala Arg Asp Gly Ser Pro Gln
Pro Leu Leu Lys Ala Gly 740 745 750Gly Ala Pro Gly Gly Asn Pro Thr
Leu Tyr Gln Asp Leu Val Arg Val 755 760 765Ser Ala Thr Ile Thr Asn
Thr Gly Asn Val Ala Gly Tyr Glu Val Pro 770 775 780Gln Leu Tyr Val
Ser Leu Gly Gly Pro Asn Glu Pro Arg Val Val Leu785 790 795 800Arg
Lys Phe Asp Arg Ile Phe Leu Ala Pro Gly Glu Gln Lys Val Trp 805 810
815Thr Thr Thr Leu Asn Arg Arg Asp Leu Ala Asn Trp Asp Val Glu Ala
820 825 830Gln Asp Trp Val Ile Thr Lys Tyr Pro Lys Lys Val His Val
Gly Ser 835 840 845Ser Ser Arg Lys Leu Pro Leu Arg Ala Pro Leu Pro
Arg Val Tyr 850 855 8602451599DNAAspergillus fumigatus
245atgctggcct ccaccttctc ctaccgcatg tacaagaccg cgctcatcct
ggccgccctt 60ctgggctctg gccaggctca gcaggtcggt acttcccagg cggaagtgca
tccgtccatg 120acctggcaga gctgcacggc tggcggcagc tgcaccacca
acaacggcaa ggtggtcatc 180gacgcgaact ggcgttgggt gcacaaagtc
ggcgactaca ccaactgcta caccggcaac 240acctgggaca cgactatctg
ccctgacgat gcgacctgcg catccaactg cgcccttgag 300ggtgccaact
acgaatccac ctatggtgtg accgccagcg gcaattccct ccgcctcaac
360ttcgtcacca ccagccagca gaagaacatt ggctcgcgtc tgtacatgat
gaaggacgac 420tcgacctacg agatgtttaa gctgctgaac caggagttca
ccttcgatgt cgatgtctcc 480aacctcccct gcggtctcaa cggtgctctg
tactttgtcg ccatggacgc cgacggtggc 540atgtccaagt acccaaccaa
caaggccggt gccaagtacg gtactggata ctgtgactcg 600cagtgccctc
gcgacctcaa gttcatcaac ggtcaggcca acgtcgaagg gtggcagccc
660tcctccaacg atgccaatgc gggtaccggc aaccacgggt cctgctgcgc
ggagatggat 720atctgggagg ccaacagcat ctccacggcc ttcacccccc
atccgtgcga cacgcccggc 780caggtgatgt gcaccggtga tgcctgcggt
ggcacctaca gctccgaccg ctacggcggc 840acctgcgacc ccgacggatg
tgatttcaac tccttccgcc agggcaacaa gaccttctac 900ggccctggca
tgaccgtcga caccaagagc aagtttaccg tcgtcaccca gttcatcacc
960gacgacggca cctccagcgg caccctcaag gagatcaagc gcttctacgt
gcagaacggc 1020aaggtgatcc ccaactcgga gtcgacctgg accggcgtca
gcggcaactc catcaccacc 1080gagtactgca ccgcccagaa gagcctgttc
caggaccaga acgtcttcga aaagcacggc 1140ggcctcgagg gcatgggtgc
tgccctcgcc cagggtatgg ttctcgtcat gtccctgtgg 1200gatgatcact
cggccaacat gctctggctc gacagcaact acccgaccac tgcctcttcc
1260accactcccg gcgtcgcccg tggtacctgc gacatctcct ccggcgtccc
tgcggatgtc 1320gaggcgaacc accccgacgc ctacgtcgtc tactccaaca
tcaaggtcgg ccccatcggc 1380tcgaccttca acagcggtgg ctcgaacccc
ggtggcggaa ccaccacgac aactaccacc 1440cagcctacta ccaccacgac
cacggctgga aaccctggcg gcaccggagt cgcacagcac 1500tatggccagt
gtggtggaat cggatggacc ggacccacaa cctgtgccag cccttatacc
1560tgccagaagc tgaatgatta ttactctcag tgcctgtag
1599246532PRTAspergillus fumigatus 246Met Leu Ala Ser Thr Phe Ser
Tyr Arg Met Tyr Lys Thr Ala Leu Ile1 5 10 15Leu Ala Ala Leu Leu Gly
Ser Gly Gln Ala Gln Gln Val Gly Thr Ser 20 25 30Gln Ala Glu Val His
Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 35 40 45Gly Ser Cys Thr
Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 50 55 60Arg Trp Val
His Lys Val Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn65 70 75 80Thr
Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 85 90
95Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala
100 105 110Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln
Gln Lys 115 120 125Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp
Ser Thr Tyr Glu 130 135 140Met Phe Lys Leu Leu Asn Gln Glu Phe Thr
Phe Asp Val Asp Val Ser145 150 155 160Asn Leu Pro Cys Gly Leu Asn
Gly Ala Leu Tyr Phe Val Ala Met Asp 165 170 175Ala Asp Gly Gly Met
Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 180 185 190Tyr Gly Thr
Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 195 200 205Ile
Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 210 215
220Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met
Asp225 230 235 240Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr
Pro His Pro Cys 245 250 255Asp Thr Pro Gly Gln Val Met Cys Thr Gly
Asp Ala Cys Gly Gly Thr 260 265 270Tyr Ser Ser Asp Arg Tyr Gly Gly
Thr Cys Asp Pro Asp Gly Cys Asp 275 280 285Phe Asn Ser Phe Arg Gln
Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 290 295 300Thr Val Asp Thr
Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr305 310 315 320Asp
Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 325 330
335Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly
340 345 350Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln
Lys Ser 355 360 365Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly
Gly Leu Glu Gly 370 375 380Met Gly Ala Ala Leu Ala Gln Gly Met Val
Leu Val Met Ser Leu Trp385 390 395 400Asp Asp His Ser Ala Asn Met
Leu Trp Leu Asp Ser Asn Tyr Pro Thr 405 410 415Thr Ala Ser Ser Thr
Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 420 425 430Ser Ser Gly
Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 435 440 445Val
Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 450 455
460Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr
Thr465 470 475 480Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 485 490 495Val Ala Gln His Tyr Gly Gln Cys Gly Gly
Ile Gly Trp Thr Gly Pro 500 505 510Thr Thr Cys Ala Ser Pro Tyr Thr
Cys Gln Lys Leu Asn Asp Tyr Tyr 515 520 525Ser Gln Cys Leu
5302471713DNAAspergillus fumigatus 247atgaagcacc ttgcatcttc
catcgcattg actctactgt tgcctgccgt gcaggcccag 60cagaccgtat ggggccaatg
tatgttctgg ctgtcactgg aataagactg tatcaactgc 120tgatatgctt
ctaggtggcg gccaaggctg gtctggcccg acgagctgtg ttgccggcgc
180agcctgtagc acactgaatc cctgtatgtt agatatcgtc ctgagtggag
acttatactg 240acttccttag actacgctca gtgtatcccg ggagccaccg
cgacgtccac caccctcacg 300acgacgacgg cggcgacgac gacatcccag
accaccacca aacctaccac gactggtcca 360actacatccg cacccaccgt
gaccgcatcc ggtaaccctt tcagcggcta ccagctgtat 420gccaacccct
actactcctc cgaggtccat actctggcca tgccttctct gcccagctcg
480ctgcagccca aggctagtgc tgttgctgaa gtgccctcat ttgtttggct
gtaagtggcc 540ttatcccaat actgagacca actctctgac agtcgtagcg
acgttgccgc caaggtgccc 600actatgggaa cctacctggc cgacattcag
gccaagaaca aggccggcgc caaccctcct 660atcgctggta tcttcgtggt
ctacgacttg ccggaccgtg actgcgccgc tctggccagt 720aatggcgagt
actcaattgc caacaacggt gtggccaact acaaggcgta cattgacgcc
780atccgtgctc agctggtgaa gtactctgac gttcacacca tcctcgtcat
cggtaggccg 840tacacctccg ttgcgcgccg cctttctctg acatcttgca
gaacccgaca gcttggccaa 900cctggtgacc aacctcaacg tcgccaaatg
cgccaatgcg cagagcgcct acctggagtg 960tgtcgactat gctctgaagc
agctcaacct gcccaacgtc gccatgtacc tcgacgcagg 1020tatgcctcac
ttcccgcatt ctgtatccct tccagacact aactcatcag gccatgcggg
1080ctggctcgga tggcccgcca acttgggccc cgccgcaaca ctcttcgcca
aagtctacac 1140cgacgcgggt tcccccgcgg ctgttcgtgg cctggccacc
aacgtcgcca actacaacgc 1200ctggtcgctc
agtacctgcc cctcctacac ccagggagac cccaactgcg acgagaagaa
1260gtacatcaac gccatggcgc ctcttctcaa ggaagccggc ttcgatgccc
acttcatcat 1320ggatacctgt aagtgcttat tccaatcgcc gatgtgtgcc
gactaatcaa tgtttcagcc 1380cggaatggcg tccagcccac gaagcaaaac
gcctggggtg actggtgcaa cgtcatcggc 1440accggcttcg gtgttcgccc
ctcgactaac accggcgatc cgctccagga tgcctttgtg 1500tggatcaagc
ccggtggaga gagtgatggc acgtccaact cgacttcccc ccggtatgac
1560gcgcactgcg gatatagtga tgctctgcag cctgctcctg aggctggtac
ttggttccag 1620gtatgtcatc cattagccag atgagggata agtgactgac
ggacctaggc ctactttgag 1680cagcttctga ccaacgctaa cccgtccttt taa
1713248454PRTAspergillus fumigatus 248Met Lys His Leu Ala Ser Ser
Ile Ala Leu Thr Leu Leu Leu Pro Ala1 5 10 15Val Gln Ala Gln Gln Thr
Val Trp Gly Gln Cys Gly Gly Gln Gly Trp 20 25 30Ser Gly Pro Thr Ser
Cys Val Ala Gly Ala Ala Cys Ser Thr Leu Asn 35 40 45Pro Tyr Tyr Ala
Gln Cys Ile Pro Gly Ala Thr Ala Thr Ser Thr Thr 50 55 60Leu Thr Thr
Thr Thr Ala Ala Thr Thr Thr Ser Gln Thr Thr Thr Lys65 70 75 80Pro
Thr Thr Thr Gly Pro Thr Thr Ser Ala Pro Thr Val Thr Ala Ser 85 90
95Gly Asn Pro Phe Ser Gly Tyr Gln Leu Tyr Ala Asn Pro Tyr Tyr Ser
100 105 110Ser Glu Val His Thr Leu Ala Met Pro Ser Leu Pro Ser Ser
Leu Gln 115 120 125Pro Lys Ala Ser Ala Val Ala Glu Val Pro Ser Phe
Val Trp Leu Asp 130 135 140Val Ala Ala Lys Val Pro Thr Met Gly Thr
Tyr Leu Ala Asp Ile Gln145 150 155 160Ala Lys Asn Lys Ala Gly Ala
Asn Pro Pro Ile Ala Gly Ile Phe Val 165 170 175Val Tyr Asp Leu Pro
Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly 180 185 190Glu Tyr Ser
Ile Ala Asn Asn Gly Val Ala Asn Tyr Lys Ala Tyr Ile 195 200 205Asp
Ala Ile Arg Ala Gln Leu Val Lys Tyr Ser Asp Val His Thr Ile 210 215
220Leu Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu
Asn225 230 235 240Val Ala Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu
Glu Cys Val Asp 245 250 255Tyr Ala Leu Lys Gln Leu Asn Leu Pro Asn
Val Ala Met Tyr Leu Asp 260 265 270Ala Gly His Ala Gly Trp Leu Gly
Trp Pro Ala Asn Leu Gly Pro Ala 275 280 285Ala Thr Leu Phe Ala Lys
Val Tyr Thr Asp Ala Gly Ser Pro Ala Ala 290 295 300Val Arg Gly Leu
Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Leu305 310 315 320Ser
Thr Cys Pro Ser Tyr Thr Gln Gly Asp Pro Asn Cys Asp Glu Lys 325 330
335Lys Tyr Ile Asn Ala Met Ala Pro Leu Leu Lys Glu Ala Gly Phe Asp
340 345 350Ala His Phe Ile Met Asp Thr Ser Arg Asn Gly Val Gln Pro
Thr Lys 355 360 365Gln Asn Ala Trp Gly Asp Trp Cys Asn Val Ile Gly
Thr Gly Phe Gly 370 375 380Val Arg Pro Ser Thr Asn Thr Gly Asp Pro
Leu Gln Asp Ala Phe Val385 390 395 400Trp Ile Lys Pro Gly Gly Glu
Ser Asp Gly Thr Ser Asn Ser Thr Ser 405 410 415Pro Arg Tyr Asp Ala
His Cys Gly Tyr Ser Asp Ala Leu Gln Pro Ala 420 425 430Pro Glu Ala
Gly Thr Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr 435 440 445Asn
Ala Asn Pro Ser Phe 4502491849DNATrichoderma reesei 249tgccatttct
gacctggata ggttttccta tggtcattcc tataagagac acgctctttc 60gtcggcccgt
agatatcaga ttggtattca gtcgcacaga cgaaggtgag ttgatcctcc
120aacatgagtt ctatgagccc cccccttgcc cccccccgtt caccttgacc
tgcaatgaga 180atcccacctt ttacaagagc atcaagaagt attaatggcg
ctgaatagcc tctgctcgat 240aatatctccc cgtcatcgac aatgaacaag
tccgtggctc cattgctgct tgcagcgtcc 300atactatatg gcggcgccgt
cgcacagcag actgtctggg gccagtgtgg aggtattggt 360tggagcggac
ctacgaattg tgctcctggc tcagcttgtt cgaccctcaa tccttattat
420gcgcaatgta ttccgggagc cactactatc accacttcga cccggccacc
atccggtcca 480accaccacca ccagggctac ctcaacaagc tcatcaactc
cacccacgag ctctggggtc 540cgatttgccg gcgttaacat cgcgggtttt
gactttggct gtaccacaga gtgagtaccc 600ttgtttcctg gtgttgctgg
ctggttgggc gggtatacag cgaagcggac gcaagaacac 660cgccggtccg
ccaccatcaa gatgtgggtg gtaagcggcg gtgttttgta caactacctg
720acagctcact caggaaatga gaattaatgg aagtcttgtt acagtggcac
ttgcgttacc 780tcgaaggttt atcctccgtt gaagaacttc accggctcaa
acaactaccc cgatggcatc 840ggccagatgc agcacttcgt caacgaggac
gggatgacta ttttccgctt acctgtcgga 900tggcagtacc tcgtcaacaa
caatttgggc ggcaatcttg attccacgag catttccaag 960tatgatcagc
ttgttcaggg gtgcctgtct ctgggcgcat actgcatcgt cgacatccac
1020aattatgctc gatggaacgg tgggatcatt ggtcagggcg gccctactaa
tgctcaattc 1080acgagccttt ggtcgcagtt ggcatcaaag tacgcatctc
agtcgagggt gtggttcggc 1140atcatgaatg agccccacga cgtgaacatc
aacacctggg ctgccacggt ccaagaggtt 1200gtaaccgcaa tccgcaacgc
tggtgctacg tcgcaattca tctctttgcc tggaaatgat 1260tggcaatctg
ctggggcttt catatccgat ggcagtgcag ccgccctgtc tcaagtcacg
1320aacccggatg ggtcaacaac gaatctgatt tttgacgtgc acaaatactt
ggactcagac 1380aactccggta ctcacgccga atgtactaca aataacattg
acggcgcctt ttctccgctt 1440gccacttggc tccgacagaa caatcgccag
gctatcctga cagaaaccgg tggtggcaac 1500gttcagtcct gcatacaaga
catgtgccag caaatccaat atctcaacca gaactcagat 1560gtctatcttg
gctatgttgg ttggggtgcc ggatcatttg atagcacgta tgtcctgacg
1620gaaacaccga ctggcagtgg taactcatgg acggacacat ccttggtcag
ctcgtgtctc 1680gcaagaaagt agcactctga gctgaatgca gaagcctcgc
caacgtttgt atctcgctat 1740caaacatagt agctactcta tgaggctgtc
tgttctcgat ttcagcttta tatagtttca 1800tcaaacagta catattccct
ctgtggccac gcaaaaaaaa aaaaaaaaa 1849250418PRTTrichoderma reesei
250Met Asn Lys Ser Val Ala Pro Leu Leu Leu Ala Ala Ser Ile Leu Tyr1
5 10 15Gly Gly Ala Val Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Gly
Ile 20 25 30Gly Trp Ser Gly Pro Thr Asn Cys Ala Pro Gly Ser Ala Cys
Ser Thr 35 40 45Leu Asn Pro Tyr Tyr Ala Gln Cys Ile Pro Gly Ala Thr
Thr Ile Thr 50 55 60Thr Ser Thr Arg Pro Pro Ser Gly Pro Thr Thr Thr
Thr Arg Ala Thr65 70 75 80Ser Thr Ser Ser Ser Thr Pro Pro Thr Ser
Ser Gly Val Arg Phe Ala 85 90 95Gly Val Asn Ile Ala Gly Phe Asp Phe
Gly Cys Thr Thr Asp Gly Thr 100 105 110Cys Val Thr Ser Lys Val Tyr
Pro Pro Leu Lys Asn Phe Thr Gly Ser 115 120 125Asn Asn Tyr Pro Asp
Gly Ile Gly Gln Met Gln His Phe Val Asn Glu 130 135 140Asp Gly Met
Thr Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu Val145 150 155
160Asn Asn Asn Leu Gly Gly Asn Leu Asp Ser Thr Ser Ile Ser Lys Tyr
165 170 175Asp Gln Leu Val Gln Gly Cys Leu Ser Leu Gly Ala Tyr Cys
Ile Val 180 185 190Asp Ile His Asn Tyr Ala Arg Trp Asn Gly Gly Ile
Ile Gly Gln Gly 195 200 205Gly Pro Thr Asn Ala Gln Phe Thr Ser Leu
Trp Ser Gln Leu Ala Ser 210 215 220Lys Tyr Ala Ser Gln Ser Arg Val
Trp Phe Gly Ile Met Asn Glu Pro225 230 235 240His Asp Val Asn Ile
Asn Thr Trp Ala Ala Thr Val Gln Glu Val Val 245 250 255Thr Ala Ile
Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro 260 265 270Gly
Asn Asp Trp Gln Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala 275 280
285Ala Ala Leu Ser Gln Val Thr Asn Pro Asp Gly Ser Thr Thr Asn Leu
290 295 300Ile Phe Asp Val His Lys Tyr Leu Asp Ser Asp Asn Ser Gly
Thr His305 310 315 320Ala Glu Cys Thr Thr Asn Asn Ile Asp Gly Ala
Phe Ser Pro Leu Ala 325 330 335Thr Trp Leu Arg Gln Asn Asn Arg Gln
Ala Ile Leu Thr Glu Thr Gly 340 345 350Gly Gly Asn Val Gln Ser Cys
Ile Gln Asp Met Cys Gln Gln Ile Gln 355 360 365Tyr Leu Asn Gln Asn
Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp Gly 370 375 380Ala Gly Ser
Phe Asp Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr Gly385 390 395
400Ser Gly Asn Ser Trp Thr Asp Thr Ser Leu Val Ser Ser Cys Leu Ala
405 410 415Arg Lys2511415DNAAspergillus fumigatus 251atggtccatc
tatcttcatt ggcagcagcc ctggctgctc tgcctctgta tgtttaccca 60ctcacgagag
gaggaacagc tttgacattg ctatagtgta tatggagctg gcctgaacac
120agcagccaaa gccaaaggac taaagtactt tggttccgcc acggacaatc
cagagctcac 180ggactctgcg tatgtcgcgc aactgagcaa caccgatgat
tttggtcaaa tcacacccgg 240aaactccatg aaggtttgct tacgtctgcc
tccctggagc attgcctcaa aagctaattg 300gttgttttgt ttggatagtg
ggatgccacc gagccttctc agaattcttt ttcgttcgca 360aatggagacg
ccgtggtcaa tctggcgaac aagaatggcc agctgatgcg atgccatact
420ctggtctggc acagtcagct accgaactgg ggtatgtaaa cgtcttgtct
attctcaaat 480actctctaac agttgacagt ctctagcggg tcatggacca
atgcgaccct tttggcggcc 540atgaagaatc atatcaccaa tgtggttact
cactacaagg ggaagtgcta cgcctgggat 600gttgtcaatg aaggtttgtt
gctccatcta tcctcaatag ttcttttgaa actgacaagc 660ctgtcaatct
agccctgaac gaggacggta ctttccgtaa ctctgtcttc taccagatca
720tcggcccagc atacattcct attgcgttcg ccacggctgc tgccgcagat
cccgacgtga 780aactctacta caacgactac aacattgaat actcaggcgc
caaagcgact gctgcgcaga 840atatcgtcaa gatgatcaag gcctacggcg
cgaagatcga cggcgtcggc ctccaggcac 900actttatcgt cggcagcact
ccgagtcaat cggatctgac gaccgtcttg aagggctaca 960ctgctctcgg
cgttgaggtg gcctataccg aacttgacat ccgcatgcag ctgccctcga
1020ccgccgcaaa gctggcccag cagtccactg acttccaagg cgtggccgca
gcatgcgtta 1080gcaccactgg ctgcgtgggt gtcactatct gggactggac
cgacaagtac tcctgggtcc 1140ccagcgtgtt ccaaggctac ggcgccccat
tgccttggga tgagaactat gtgaagaagc 1200cagcgtacga tggcctgatg
gcgggtcttg gagcaagcgg ctccggcacc acaacgacca 1260ctactactac
ttctactacg acaggaggta cggaccctac tggagtcgct cagaaatggg
1320gacagtgtgg cggtattggc tggaccgggc caacaacttg tgtcagtggt
accacttgcc 1380aaaagctgaa tgactggtac tcacagtgcc tgtaa
1415252397PRTAspergillus fumigatus 252Met Val His Leu Ser Ser Leu
Ala Ala Ala Leu Ala Ala Leu Pro Leu1 5 10 15Val Tyr Gly Ala Gly Leu
Asn Thr Ala Ala Lys Ala Lys Gly Leu Lys 20 25 30Tyr Phe Gly Ser Ala
Thr Asp Asn Pro Glu Leu Thr Asp Ser Ala Tyr 35 40 45Val Ala Gln Leu
Ser Asn Thr Asp Asp Phe Gly Gln Ile Thr Pro Gly 50 55 60Asn Ser Met
Lys Trp Asp Ala Thr Glu Pro Ser Gln Asn Ser Phe Ser65 70 75 80Phe
Ala Asn Gly Asp Ala Val Val Asn Leu Ala Asn Lys Asn Gly Gln 85 90
95Leu Met Arg Cys His Thr Leu Val Trp His Ser Gln Leu Pro Asn Trp
100 105 110Val Ser Ser Gly Ser Trp Thr Asn Ala Thr Leu Leu Ala Ala
Met Lys 115 120 125Asn His Ile Thr Asn Val Val Thr His Tyr Lys Gly
Lys Cys Tyr Ala 130 135 140Trp Asp Val Val Asn Glu Ala Leu Asn Glu
Asp Gly Thr Phe Arg Asn145 150 155 160Ser Val Phe Tyr Gln Ile Ile
Gly Pro Ala Tyr Ile Pro Ile Ala Phe 165 170 175Ala Thr Ala Ala Ala
Ala Asp Pro Asp Val Lys Leu Tyr Tyr Asn Asp 180 185 190Tyr Asn Ile
Glu Tyr Ser Gly Ala Lys Ala Thr Ala Ala Gln Asn Ile 195 200 205Val
Lys Met Ile Lys Ala Tyr Gly Ala Lys Ile Asp Gly Val Gly Leu 210 215
220Gln Ala His Phe Ile Val Gly Ser Thr Pro Ser Gln Ser Asp Leu
Thr225 230 235 240Thr Val Leu Lys Gly Tyr Thr Ala Leu Gly Val Glu
Val Ala Tyr Thr 245 250 255Glu Leu Asp Ile Arg Met Gln Leu Pro Ser
Thr Ala Ala Lys Leu Ala 260 265 270Gln Gln Ser Thr Asp Phe Gln Gly
Val Ala Ala Ala Cys Val Ser Thr 275 280 285Thr Gly Cys Val Gly Val
Thr Ile Trp Asp Trp Thr Asp Lys Tyr Ser 290 295 300Trp Val Pro Ser
Val Phe Gln Gly Tyr Gly Ala Pro Leu Pro Trp Asp305 310 315 320Glu
Asn Tyr Val Lys Lys Pro Ala Tyr Asp Gly Leu Met Ala Gly Leu 325 330
335Gly Ala Ser Gly Ser Gly Thr Thr Thr Thr Thr Thr Thr Thr Ser Thr
340 345 350Thr Thr Gly Gly Thr Asp Pro Thr Gly Val Ala Gln Lys Trp
Gly Gln 355 360 365Cys Gly Gly Ile Gly Trp Thr Gly Pro Thr Thr Cys
Val Ser Gly Thr 370 375 380Thr Cys Gln Lys Leu Asn Asp Trp Tyr Ser
Gln Cys Leu385 390 3952532376DNAAspergillus fumigatus 253atggcggttg
ccaaatctat tgctgccgtg ctggtagcac tgttgcctgg tgcgcttgct 60caggcgaata
caagctatgt tgattacaat gtggaggcga atccggatct cacccctcag
120tcggtcgcta cgattgacct gtcctttccc gactgcgaga atggaccgct
cagcaagact 180ctcgtttgcg acacgtcggc tcggccgcat gaccgagctg
ctgccctggt ttccatgttc 240accttcgagg agctggtgaa caacacaggc
aacactagcc ctggtgttcc aagacttggt 300ctccctccgt accaagtatg
gagcgaggct ctccatggac ttgaccgcgc caacttcaca 360aacgagggag
agtacagctg ggccacctcg ttccccatgc ctatcctgac aatgtcggcc
420ttgaaccgaa ccctgatcaa ccagatcgcg accatcatcg caactcaagg
acgagctttc 480aataacgttg ggcggtatgg gctggacgtg tacgccccga
atataaatgc attcagatcg 540gctatgtggg gaagaggtca agagaccccc
ggagaagacg cttactgcct ggcatcggcg 600tatgcgtacg agtatatcac
tggcatccag ggtggtgttg atccggaaca cctcaagttg 660gtggccactg
ccaaacacta tgcgggctac gatcttgaga actgggacgg tcactcccgt
720ttgggcaacg atatgaacat tacacagcag gaactttccg aatactacac
ccctcagttc 780cttgttgcag ccagagacgc caaagtgcac agtgtcatgt
gctcctacaa cgcggtaaat 840ggggtgccca gctgcgcaaa ctcgttcttc
ctccagaccc tcctccgtga cacattcggc 900ttcgtcgagg atggttatgt
atccagcgac tgcgactcgg cgtacaatgt ctggaacccg 960cacgagtttg
cggccaacat cacgggggcc gctgcagact ctatccgggc ggggacggac
1020attgattgcg gcactactta tcaatactat ttcggcgaag cctttgacga
gcaagaggtc 1080acccgtgcag aaatcgaaag aggtgtgatc cgcctgtaca
gcaacttggt gcgtctcggc 1140tatttcgatg gcaatggaag cgtgtatcgg
gacctgacgt ggaatgatgt cgtgaccacg 1200gatgcctgga atatctcata
cgaagccgct gtagaaggca ttgtcctact gaagaacgat 1260ggaaccttgc
ctctcgccaa gtcggtccgc agtgttgcat tgattgggcc ctggatgaat
1320gtgacgactc agcttcaggg caactacttt ggaccggcgc cttatctgat
tagtccgttg 1380aatgccttcc agaattctga cttcgacgtg aactacgctt
tcggcacgaa catttcatcc 1440cactccacag atgggttttc cgaggcgttg
tctgctgcga agaaatccga cgtcatcata 1500ttcgcgggcg ggattgacaa
cactttggaa gcagaagcca tggatcgcat gaatatcaca 1560tggcccggca
atcagctaca gctcatcgac cagttgagcc aactcggcaa accgctgatc
1620gtcctccaga tgggcggcgg ccaagtcgac tcctcctcgc tcaagtccaa
caagaatgtc 1680aactccctga tctggggtgg ataccccgga caatccggcg
ggcaggctct cctagacatc 1740atcaccggca agcgcgcccc cgccggccga
ctcgtggtca cgcagtaccc ggccgaatac 1800gcaacccagt tccccgccac
cgacatgagc ctgcggcctc acggcaataa tcccggccag 1860acctacatgt
ggtacaccgg cacccccgtc tacgagtttg gccacgggct cttctacacg
1920accttccacg cctccctccc tggcaccggc aaggacaaga cctccttcaa
catccaagac 1980ctcctcacgc agccgcatcc gggcttcgca aacgtcgagc
aaatgccttt gctcaacttc 2040accgtgacga tcaccaatac cggcaaggtc
gcttccgact acactgctat gctcttcgcg 2100aacaccaccg cgggacctgc
tccatacccg aacaagtggc tcgtcggctt cgaccggctg 2160gcgagcctgg
aaccgcacag gtcgcagact atgaccatcc ccgtgactat cgacagcgtg
2220gctcgtacgg atgaggccgg caatcgggtt ctctacccgg gaaagtacga
gttggccctg 2280aacaatgagc ggtcggttgt ccttcagttt gtgctgacag
gccgagaggc tgtgattttc 2340aagtggcctg tagagcagca gcagatttcg tctgcg
2376254792PRTAspergillus fumigatus 254Met Ala Val Ala Lys Ser Ile
Ala Ala Val Leu Val Ala Leu Leu Pro1 5 10 15Gly Ala Leu Ala Gln Ala
Asn Thr Ser Tyr Val Asp Tyr Asn Val Glu 20 25 30Ala Asn Pro Asp Leu
Thr Pro Gln Ser Val Ala Thr Ile Asp Leu Ser 35 40 45Phe Pro Asp Cys
Glu Asn Gly Pro Leu Ser Lys Thr Leu Val Cys Asp 50 55 60Thr Ser Ala
Arg Pro His Asp Arg Ala Ala Ala Leu Val Ser Met Phe65 70 75 80Thr
Phe Glu Glu Leu Val Asn Asn Thr Gly Asn Thr Ser Pro Gly Val 85 90
95Pro Arg Leu Gly Leu Pro Pro Tyr Gln Val Trp Ser Glu Ala Leu His
100 105 110Gly Leu Asp Arg Ala Asn Phe Thr Asn Glu Gly Glu Tyr Ser
Trp Ala 115 120 125Thr Ser Phe Pro Met Pro Ile Leu Thr Met Ser Ala
Leu Asn Arg Thr 130 135 140Leu Ile Asn Gln Ile Ala Thr Ile Ile Ala
Thr
Gln Gly Arg Ala Phe145 150 155 160Asn Asn Val Gly Arg Tyr Gly Leu
Asp Val Tyr Ala Pro Asn Ile Asn 165 170 175Ala Phe Arg Ser Ala Met
Trp Gly Arg Gly Gln Glu Thr Pro Gly Glu 180 185 190Asp Ala Tyr Cys
Leu Ala Ser Ala Tyr Ala Tyr Glu Tyr Ile Thr Gly 195 200 205Ile Gln
Gly Gly Val Asp Pro Glu His Leu Lys Leu Val Ala Thr Ala 210 215
220Lys His Tyr Ala Gly Tyr Asp Leu Glu Asn Trp Asp Gly His Ser
Arg225 230 235 240Leu Gly Asn Asp Met Asn Ile Thr Gln Gln Glu Leu
Ser Glu Tyr Tyr 245 250 255Thr Pro Gln Phe Leu Val Ala Ala Arg Asp
Ala Lys Val His Ser Val 260 265 270Met Cys Ser Tyr Asn Ala Val Asn
Gly Val Pro Ser Cys Ala Asn Ser 275 280 285Phe Phe Leu Gln Thr Leu
Leu Arg Asp Thr Phe Gly Phe Val Glu Asp 290 295 300Gly Tyr Val Ser
Ser Asp Cys Asp Ser Ala Tyr Asn Val Trp Asn Pro305 310 315 320His
Glu Phe Ala Ala Asn Ile Thr Gly Ala Ala Ala Asp Ser Ile Arg 325 330
335Ala Gly Thr Asp Ile Asp Cys Gly Thr Thr Tyr Gln Tyr Tyr Phe Gly
340 345 350Glu Ala Phe Asp Glu Gln Glu Val Thr Arg Ala Glu Ile Glu
Arg Gly 355 360 365Val Ile Arg Leu Tyr Ser Asn Leu Val Arg Leu Gly
Tyr Phe Asp Gly 370 375 380Asn Gly Ser Val Tyr Arg Asp Leu Thr Trp
Asn Asp Val Val Thr Thr385 390 395 400Asp Ala Trp Asn Ile Ser Tyr
Glu Ala Ala Val Glu Gly Ile Val Leu 405 410 415Leu Lys Asn Asp Gly
Thr Leu Pro Leu Ala Lys Ser Val Arg Ser Val 420 425 430Ala Leu Ile
Gly Pro Trp Met Asn Val Thr Thr Gln Leu Gln Gly Asn 435 440 445Tyr
Phe Gly Pro Ala Pro Tyr Leu Ile Ser Pro Leu Asn Ala Phe Gln 450 455
460Asn Ser Asp Phe Asp Val Asn Tyr Ala Phe Gly Thr Asn Ile Ser
Ser465 470 475 480His Ser Thr Asp Gly Phe Ser Glu Ala Leu Ser Ala
Ala Lys Lys Ser 485 490 495Asp Val Ile Ile Phe Ala Gly Gly Ile Asp
Asn Thr Leu Glu Ala Glu 500 505 510Ala Met Asp Arg Met Asn Ile Thr
Trp Pro Gly Asn Gln Leu Gln Leu 515 520 525Ile Asp Gln Leu Ser Gln
Leu Gly Lys Pro Leu Ile Val Leu Gln Met 530 535 540Gly Gly Gly Gln
Val Asp Ser Ser Ser Leu Lys Ser Asn Lys Asn Val545 550 555 560Asn
Ser Leu Ile Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Gln Ala 565 570
575Leu Leu Asp Ile Ile Thr Gly Lys Arg Ala Pro Ala Gly Arg Leu Val
580 585 590Val Thr Gln Tyr Pro Ala Glu Tyr Ala Thr Gln Phe Pro Ala
Thr Asp 595 600 605Met Ser Leu Arg Pro His Gly Asn Asn Pro Gly Gln
Thr Tyr Met Trp 610 615 620Tyr Thr Gly Thr Pro Val Tyr Glu Phe Gly
His Gly Leu Phe Tyr Thr625 630 635 640Thr Phe His Ala Ser Leu Pro
Gly Thr Gly Lys Asp Lys Thr Ser Phe 645 650 655Asn Ile Gln Asp Leu
Leu Thr Gln Pro His Pro Gly Phe Ala Asn Val 660 665 670Glu Gln Met
Pro Leu Leu Asn Phe Thr Val Thr Ile Thr Asn Thr Gly 675 680 685Lys
Val Ala Ser Asp Tyr Thr Ala Met Leu Phe Ala Asn Thr Thr Ala 690 695
700Gly Pro Ala Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe Asp Arg
Leu705 710 715 720Ala Ser Leu Glu Pro His Arg Ser Gln Thr Met Thr
Ile Pro Val Thr 725 730 735Ile Asp Ser Val Ala Arg Thr Asp Glu Ala
Gly Asn Arg Val Leu Tyr 740 745 750Pro Gly Lys Tyr Glu Leu Ala Leu
Asn Asn Glu Arg Ser Val Val Leu 755 760 765Gln Phe Val Leu Thr Gly
Arg Glu Ala Val Ile Phe Lys Trp Pro Val 770 775 780Glu Gln Gln Gln
Ile Ser Ser Ala785 79025542DNAArtificial SequenceArtificial DNA
Primer 255gcagtggacg ccgtggccgc cgagccacca cggacccgtc at
4225642DNAArtificial SequenceArtificial DNA Primer 256atgacgggtc
cgtggtggct cggcggccac ggcgtccact gc 4225742DNAArtificial
SequenceArtificial DNA Primer 257gcagtggacg ccgtggccga agagccacca
cggacccgtc at 4225842DNAArtificial SequenceArtificial DNA Primer
258atgacgggtc cgtggtggct cttcggccac ggcgtccact gc
4225943DNAArtificial SequenceArtificial DNA Primer 259catcgccctg
cactcggccg ccaacaagga cggcgcccag aac 4326043DNAArtificial
SequenceArtificial DNA Primer 260gttctgggcg ccgtccttgt tggcggccga
gtgcagggcg atg 4326143DNAArtificial SequenceArtificial DNA Primer
261catcgccctg cactcggcct ggaacaagga cggcgcccag aac
4326243DNAArtificial SequenceArtificial DNA Primer 262gttctgggcg
ccgtccttgt tccaggccga gtgcagggcg atg 4326343DNAArtificial
SequenceArtificial DNA Primer 263cgccctgcac tcggccaaca agaaggacgg
cgcccagaac tac 4326443DNAArtificial SequenceArtificial DNA Primer
264gtagttctgg gcgccgtcct tcttgttggc cgagtgcagg gcg
4326547DNAArtificial SequenceArtificial DNA Primer 265gcttcaatgg
actccatggc ctaaatctca ccatggccca gttatca 4726647DNAArtificial
SequenceArtificial DNA Primer 266tgataactgg gccatggtga gatttaggcc
atggagtcca ttgaagc 4726747DNAArtificial SequenceArtificial DNA
Primer 267gcttcaatgg actccatggc ctccttctca ccatggccca gttatca
4726847DNAArtificial SequenceArtificial DNA Primer 268tgataactgg
gccatggtga gaaggaggcc atggagtcca ttgaagc 4726948DNAArtificial
SequenceArtificial DNA Primer 269gagattattg ctcttcactc agcttggaac
caggatggtg cccagaac 4827048DNAArtificial SequenceArtificial DNA
Primer 270gttctgggca ccatcctggt tccaagctga gtgaagagca ataatctc
4827127DNAArtificial SequenceArtificial DNA
Primermisc_feature(10)..(10)N=URACIL 271atgcagcgcn gccataacca
tgagtga 2727228DNAArtificial SequenceArtificial DNA
Primermisc_feature(10)..(10)N=URACIL 272agcgctgcan aattctctta
ctgtcatg 2827336DNAArtificial SequenceArtificial DNA
Primermisc_feature(8)..(8)N=URACIL 273aattaagncc tcagcgtgat
ttaaaacgcc attgct 3627441DNAArtificial SequenceArtificial DNA
Primermisc_feature(8)..(8)N=URACIL 274acttaatnaa accctcagcg
cagttaggtt ggtgttcttc t 4127529DNAArtificial SequenceArtificial DNA
Primermisc_feature(11)..(11)N=URACIL 275agctcaagga nacctacagt
tattcgaaa 2927635DNAArtificial SequenceArtificial DNA
Primermisc_feature(11)..(11)N=URACIL 276atccttgagc ngtttcctgt
gtgaaattgt tatcc 3527737DNAArtificial SequenceArtificial DNA
Primermisc_feature(9)..(9)N=URACIL 277atctcctcng ctggtctggt
taagccagcc ccgacac 3727827DNAArtificial SequenceArtificial DNA
Primermisc_feature(9)..(9)N=URACIL 278agaggagana atactctgcg ctccgcc
2727932DNAArtificial SequenceArtificial DNA
Primermisc_feature(9)..(9)N=URACIL 279gggtttaanc ctcacacagg
aaacagctat ga 3228046DNAArtificial SequenceArtificial DNA
Primermisc_feature(12)..(12)N=URACIL 280agtgtctgcg ancgctctca
ctgcccccag ttgtgtatat agagga 4628136DNAArtificial
SequenceArtificial DNA Primermisc_feature(12)..(12)N=URACIL
281atcgcagaca cngctggcgg tagacaatca atccat 3628232DNAArtificial
SequenceArtificial DNA Primermisc_feature(9)..(9)N=URACIL
282ggacttaang gatctaagat gagctcatgg ct 3228324DNAArtificial
SequenceArtificial DNA Primer 283acgccattgc tatgatgctt gaag
2428421DNAArtificial SequenceArtificial DNA Primer 284tggtgaggtg
ctatcgtcct t 2128521DNAArtificial SequenceArtificial DNA Primer
285cttcctgtag gtgcaccgaa g 2128621DNAArtificial SequenceArtificial
DNA Primer 286acagaacgat atcggacctc g 2128723DNAArtificial
SequenceArtificial DNA Primer 287tcgttatgtt aagtcttcta tca
2328821DNAArtificial SequenceArtificial DNA Primer 288agagctcgaa
gttcctccga g 2128922DNAArtificial SequenceArtificial DNA Primer
289tatcacgagg ccctttcgtc tc 2229022DNAArtificial SequenceArtificial
DNA Primer 290tccgtcggct cctctccttc gt 2229124DNAArtificial
SequenceArtificial DNA Primer 291tgcatatcct ctgacagtat atga
2429223DNAArtificial SequenceArtificial DNA Primer 292cagtgaagag
ggcagtcgat agt 2329322DNAArtificial SequenceArtificial DNA Primer
293acgaggaaca tggctatctg ga 2229423DNAArtificial SequenceArtificial
DNA Primer 294tcagctcatt ctgggaggtg gga 2329522DNAArtificial
SequenceArtificial DNA Primer 295actccaggat cctttaaatc ca
2229621DNAArtificial SequenceArtificial DNA Primer 296actggcaagg
gatgccatgc t 2129722DNAArtificial SequenceArtificial DNA Primer
297tgatcatata accaattgcc ct 2229822DNAArtificial SequenceArtificial
DNA Primer 298agttgtgtat atagaggatt ga 2229923DNAArtificial
SequenceArtificial DNA Primer 299tggtccttcg ctcgtgatgt gga
2330020DNAArtificial SequenceArtificial DNA Primer 300agtcctcagc
gttaccggca 2030120DNAArtificial SequenceArtificial DNA Primer
301accctcagct gtgtccggga 2030221DNAArtificial SequenceArtificial
DNA Primer 302tggtatgtga acgccagtct g 2130328DNAArtificial
SequenceArtificial DNA Primermisc_feature(8)..(8)N=URACIL
303agagcganat gtccttttcc aagataat 2830449DNAArtificial
SequenceArtificial DNA Primermisc_feature(8)..(8)N=URACIL
304tctgcgantt agtgatggtg gtgatgatga ccagtataca gaggaggac
4930528DNAArtificial SequenceArtificial DNA
Primermisc_feature(8)..(8)N=URACIL 305agagcganat gctgtcttcg
acgactcg 2830649DNAArtificial SequenceArtificial DNA
Primermisc_feature(8)..(8)N=URACIL 306tctgcganct agtgatggtg
gtgatgatgg aacgtcggct caggcggcc 4930733DNAArtificial
SequenceArtificial DNA Primermisc_feature(8)..(8)N=URACIL
307agagcganat gtctgttgct aagtttgctg gtg 3330849DNAArtificial
SequenceArtificial DNA Primermisc_feature(8)..(8)N=URACIL
308tctgcgantt agtgatggtg gtgatgatgg gcggagaggt cacgggcgt
4930920DNAArtificial SequenceArtificial DNA Primer 309cccagttatc
aactaccttg 2031020DNAArtificial SequenceArtificial DNA Primer
310ctcaatttac ctctatccac 2031120DNAArtificial SequenceArtificial
DNA Primer 311tataaccaat tgccctcatc 2031220DNAArtificial
SequenceArtificial DNA Primer 312gcaccgtcga gctgcagtgg
2031320DNAArtificial SequenceArtificial DNA Primer 313ccttgccaac
tgcaatggtg 2031420DNAArtificial SequenceArtificial DNA Primer
314gcaccgtcga gctgcagtgg 2031545DNAArtificial SequenceArtificial
DNA Primer 315attattgctc ttcactcagc tttcaaccag gatggtgccc agaac
4531645DNAArtificial SequenceArtificial DNA Primer 316gttctgggca
ccatcctggt tgaaagctga gtgaagagca ataat 4531745DNAArtificial
SequenceArtificial DNA Primer 317attattgctc ttcactcagc tatgaaccag
gatggtgccc agaac 4531845DNAArtificial SequenceArtificial DNA Primer
318gttctgggca ccatcctggt tcatagctga gtgaagagca ataat
4531931DNAArtificial SequenceArtificial DNA Primer 319ccctgcactc
ggccatgaac aaggacggcg c 3132031DNAArtificial SequenceArtificial DNA
Primer 320gcgccgtcct tgttcatggc cgagtgcagg g 3132129DNAArtificial
SequenceArtificial DNA Primer 321gcactcggcc aaccacaagg acggcgccc
2932229DNAArtificial SequenceArtificial DNA Primer 322gggcgccgtc
cttgtggttg gccgagtgc 2932325DNAArtificial SequenceArtificial DNA
Primer 323ccagaccagc agaggagata atact 2532423DNAArtificial
SequenceArtificial DNA Primer 324caaggatacc tacagttatt cga
2332530DNAArtificial SequenceArtificial DNA Primer 325ccagaccagc
agaggagata atactctgcg 3032632DNAArtificial SequenceArtificial DNA
Primer 326caaggatacc tacagttatt cgaaacctcc tg 3232742DNAArtificial
SequenceArtificial DNA Primer 327tccagtggac tacctggccc aagagccacc
acggccctgt cc 4232832DNAArtificial SequenceArtificial DNA Primer
328gggccaggta gtccactgga gctcaacagt ac 3232942DNAArtificial
SequenceArtificial DNA Primer 329tccagtggac tacctggccc cccagccacc
acggccctgt cc 4233032DNAArtificial SequenceArtificial DNA Primer
330gggccaggta gtccactgga gctcaacagt ac 3233146DNAArtificial
SequenceArtificial DNA Primer 331ccggtacctg ggccagtgat atcttgatcg
ccaacaacaa cagctg 4633230DNAArtificial SequenceArtificial DNA
Primer 332atcactggcc caggtaccgg ggacgtcgtc 3033346DNAArtificial
SequenceArtificial DNA Primer 333ccggtacctg ggccagtgat ctcttgatcg
ccaacaacaa cagctg 4633430DNAArtificial SequenceArtificial DNA
Primer 334atcactggcc caggtaccgg ggacgtcgtc 3033549DNAArtificial
SequenceArtificial DNA Primer 335aaatcattgc ccttcactct gctttcaaca
aggatggtgc tcagaacta 4933633DNAArtificial SequenceArtificial DNA
Primer 336agcagagtga agggcaatga tttcgtgacg gag 3333749DNAArtificial
SequenceArtificial DNA Primer 337aaatcattgc ccttcactct gctatgaaca
aggatggtgc tcagaacta 4933833DNAArtificial SequenceArtificial DNA
Primer 338agcagagtga agggcaatga tttcgtgacg gag 3333949DNAArtificial
SequenceArtificial DNA Primer 339aaatcattgc ccttcactct gctgccaaca
aggatggtgc tcagaacta 4934033DNAArtificial SequenceArtificial DNA
Primer 340agcagagtga agggcaatga tttcgtgacg gag 3334149DNAArtificial
SequenceArtificial DNA Primer 341aaatcattgc ccttcactct gcttggaaca
aggatggtgc tcagaacta 4934233DNAArtificial SequenceArtificial DNA
Primer 342agcagagtga agggcaatga tttcgtgacg gag 3334347DNAArtificial
SequenceArtificial DNA Primer 343cattgccctt cactctgctg gtcacaagga
tggtgctcag aactacc 4734434DNAArtificial SequenceArtificial DNA
Primer 344accagcagag
tgaagggcaa tgatttcgtg acgg 3434547DNAArtificial SequenceArtificial
DNA Primer 345cattgccctt cactctgctg gtaagaagga tggtgctcag aactacc
4734634DNAArtificial SequenceArtificial DNA Primer 346accagcagag
tgaagggcaa tgatttcgtg acgg 34
* * * * *