U.S. patent application number 11/872355 was filed with the patent office on 2008-04-17 for polypeptides having glucoamylase activity and polynucleotides encoding same.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Eric Allain, Shiro Fukuyama, Michiko Ihara, Sara Landvik, Jiyin Liu, Chee-Leong Soong, Hiroaki Udagawa.
Application Number | 20080090271 11/872355 |
Document ID | / |
Family ID | 36602341 |
Filed Date | 2008-04-17 |
United States Patent
Application |
20080090271 |
Kind Code |
A1 |
Udagawa; Hiroaki ; et
al. |
April 17, 2008 |
POLYPEPTIDES HAVING GLUCOAMYLASE ACTIVITY AND POLYNUCLEOTIDES
ENCODING SAME
Abstract
The present invention relates to polypeptides having
glucoamylase activity and isolated polynucleotides encoding said
polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods for producing and using the polypeptides. The
invention also relates to the composition comprising a glucoamylase
of the invention as well as the use such compositions for starch
conversion processes, brewing, including processes for producing
fermentation products or syrups.
Inventors: |
Udagawa; Hiroaki;
(Yokohama-shi, JP) ; Landvik; Sara; (Vedbaek,
DK) ; Ihara; Michiko; (Chiba, JP) ; Liu;
Jiyin; (Raleigh, NC) ; Soong; Chee-Leong;
(Raleigh, NC) ; Allain; Eric; (Boone, NC) ;
Fukuyama; Shiro; (Chiba, JP) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
NC
Novozymes North America, Inc.
Franklinton
|
Family ID: |
36602341 |
Appl. No.: |
11/872355 |
Filed: |
October 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11315730 |
Dec 22, 2005 |
7326548 |
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11872355 |
Oct 15, 2007 |
|
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60638614 |
Dec 22, 2004 |
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60650612 |
Feb 7, 2005 |
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Current U.S.
Class: |
435/96 ; 435/203;
435/205; 530/350 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12P 7/06 20130101; Y02E 50/17 20130101; C12Y 302/01001 20130101;
C12N 9/2428 20130101; C12N 9/242 20130101; Y02E 50/10 20130101;
C12P 19/02 20130101; C12Y 302/01003 20130101; C12P 19/14
20130101 |
Class at
Publication: |
435/096 ;
435/203; 435/205; 530/350 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C12N 9/30 20060101 C12N009/30; C12N 9/34 20060101
C12N009/34; C12P 19/20 20060101 C12P019/20 |
Claims
1-91. (canceled)
92. An isolated polypeptide having glucoamylase activity, selected
from the group consisting of: (a) a polypeptide having an amino
acid sequence which has at least 90% sequence identity with amino
acids 1 to 556 of SEQ ID NO: 2; and (b) a polypeptide having an
amino acid sequence which has at least 90% sequence identity with
amino acids 1 to 561 of SEQ ID NO: 37.
93. A fusion polypeptide comprising the polypeptide of claim 92 and
a second polypeptide.
94. A composition comprising the polypeptide of claim 92 and an
alpha-amylase.
95. The composition of claim 94, wherein the alpha-amylase is a
fungal alpha-amylase.
96. The composition of claim 94, wherein the alpha-amylase is
obtained from Aspergillus, Meriplus, or Rhizomucor.
97. The composition of claim 94, wherein the alpha-amylase is
obtained from Aspergillus awamori, Aspergillus kawachii,
Aspergillus niger, Aspergillus oryzae, Meripilus giganteus, or
Rhizomucor pusillus.
98. The composition of claim 94, wherein the alpha-amylase
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.
99. A process for producing a fermentation product from
starch-containing material comprising the steps of: (a) liquefying
the starch-containing material in the presence of an alpha-amylase;
(b) saccharifying the liquefied material obtained in step (a) using
a polypeptide of claim 92; and (c) fermenting the saccharified
material using a fermenting organism.
100. A process for producing a fermentation product from
starch-containing material comprising: (a) saccharifying
starch-containing material with a polypeptide of claim 92 at a
temperature below the initial gelatinization temperature of said
starch-containing material, and (b) fermenting using a fermenting
organism.
101. The process of claim 100, wherein the polypeptide has an amino
acid sequence which has at least 95% sequence identity with amino
acids 1 to 556 of SEQ ID NO: 2 or amino acids 1 to 561 of SEQ ID
NO: 37 or is a fragment of the sequence of amino acids 1 to 556 of
SEQ ID NO: 2 or of amino acids 1 to 561 of SEQ ID NO: 37 which has
glucoamylase activity.
102. The process of claim 100, which comprises the sequence of
amino acids 1 to 556 of SEQ ID NO: 2 or amino acids 1 to 561 of SEQ
ID NO: 37.
103. The process of claim 100, which is a fragment of the sequence
of amino acids 1 to 556 of SEQ ID NO: 2 or a fragment of the
sequence of amino acids 1 to 561 of SEQ ID NO: 37 which has
glucoamylase activity.
104. An isolated polypeptide having glucoamylase activity, selected
from the group consisting of: (a) a polypeptide comprising a
catalytic domain having an amino acid sequence which has at least
90% sequence identity with amino acids 1 to 455 of SEQ ID NO: 2;
and (b) a polypeptide comprising a catalytic domain having an amino
acid sequence which has at least 90% sequence identity with amino
acids 1 to 460 of SEQ ID NO: 37.
105. The isolated polypeptide of claim 104, which comprises a
foreign binding domain.
106. A fusion polypeptide comprising the polypeptide of claim 104
and a second polypeptide.
107. A composition comprising a polypeptide of claim 104 and an
alpha-amylase.
108. A process for producing a fermentation product from
starch-containing material comprising the steps of: (a) liquefying
the starch-containing material in the presence of an alpha-amylase;
(b) saccharifying the liquefied material obtained in step (a) using
a polypeptide of claim 104; and (c) fermenting the saccharified
material using a fermenting organism.
109. A process for producing a fermentation product from
starch-containing material comprising: (a) saccharifying
starch-containing material with a polypeptide of claim 104 at a
temperature below the initial gelatinization temperature of said
starch-containing material, and (b) fermenting using a fermenting
organism.
110. An isolated polypeptide having carbohydrate binding activity,
selected from the group consisting of: (a) a polypeptide comprising
a binding domain having an amino acid sequence which has at least
90% sequence identity with amino acids 466 to 556 of SEQ ID NO: 2;
and (b) a polypeptide comprising a binding domain having an amino
acid sequence which has at least 90% sequence identity with amino
acids 471 to 561 of SEQ ID NO: 37.
111. The polypeptide of claim 110, which further comprises a
catalytic domain obtained from a glucoamylase polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/315,730 filed Dec. 22, 2005, which claims the benefit under
35 U.S.C. 119 of U.S. provisional application Nos. 60/638,614 and
60/650,612 filed Dec. 22, 2004 and Feb. 7, 2005, respectively, the
contents of which are incorporated herein by reference.
CROSS-REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to polypeptides having
glucoamylase activity and polynucleotides encoding the
polypeptides. The invention also relates to nucleic acid
constructs, vectors, and host cells comprising the polynucleotides
as well as methods for producing and using the polypeptides, and to
the use of glucoamylases of the invention for starch conversion to
producing fermentation products, such as ethanol, and syrups, such
as glucose. The invention also relates to a composition comprising
a glucoamylase of the invention.
[0005] 2. Description of the Related Art
[0006] Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3)
is an enzyme, which catalyzes the release of D-glucose from the
non-reducing ends of starch or related oligo and polysaccharide
molecules. Glucoamylases are produced by several filamentous fungi
and yeast, with those from Aspergillus being commercially most
important.
[0007] Commercially, glucoamylases are used to convert starchy
material, which is already partially hydrolyzed by an
alpha-amylase, to glucose. The glucose may then be converted
directly or indirectly into a fermentation product using a
fermenting organism. Examples of commercial fermentation products
include alcohols (e.g., ethanol, methanol, butanol,
1,3-propanediol); organic acids (e.g., citric acid, acetic acid,
itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid,
succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g.,
acetone); amino acids (e.g., glutamic acid); gases (e.g., H.sub.2
and CO.sub.2), and more complex compounds, including, for example,
antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins
(e.g., riboflavin, B.sub.12, beta-carotene); hormones, and other
compounds which are difficult to produce synthetically.
Fermentation processes are also commonly used in the consumable
alcohol (e.g., beer and wine), dairy (e.g., in the production of
yoghurt and cheese), leather, and tobacco industries.
[0008] The end product may also be syrup. For instance, the end
product may be glucose, but may also be converted, e.g., by glucose
isomerase to fructose or a mixture composed almost equally of
glucose and fructose. This mixture, or a mixture further enriched
with fructose, is the most commonly used high fructose corn syrup
(HFCS) commercialized throughout the world.
[0009] Boel et al., 1984, EMBO J. 3(5): 1097-1102 disclose
Aspergillus niger G1 or G2 glucoamylase.
[0010] U.S. Pat. No. 4,727,046 discloses a glucoamylase derived
from Corticium rolfsii which is also referred to as Athelia
rolfsii.
[0011] WO 84/02921 discloses a glucoamylase derived from
Aspergillus awamori.
[0012] WO 99/28248 discloses a glucoamylase derived from
Talaromyces emersonii.
[0013] WO 00/75296 discloses a glucoamylase derived from
Thermoascus crustaceus.
[0014] It is an object of the present invention to provide
polypeptides having glucoamylase activity and polynucleotides
encoding the polypeptides and which provide a high yield in
fermentation product production processes, such as ethanol
production processes, including one-step ethanol fermentation
processes from un-gelatinized raw (or uncooked) starch.
SUMMARY OF THE INVENTION
[0015] The present invention relates to polypeptides having
glucoamylase activity selected from the group consisting of:
[0016] (a) a polypeptide having an amino acid sequence which has at
least 75% identity with amino acids for mature polypeptide amino
acids 1 to 556 of SEQ ID NO: 2; or
[0017] (a1) a polypeptide having an amino acid sequence which has
at least 75% identity with amino acids for mature polypeptide amino
acids 1 to 561 of SEQ ID NO: 37;
[0018] (b) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 55 to 2166 of SEQ ID NO: 1, or (ii) which hybridizes
under at least medium stringency conditions with the cDNA sequence
contained in nucleotides 55 to 1725 of SEQ ID NO: 3, or (iii) a
complementary strand of (i) or (ii); or
[0019] (b1) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 55 to 2166 of SEQ ID NO: 36, or (ii) which hybridizes
under at least medium stringency conditions with the cDNA sequence
contained in nucleotides 55 to 1737 of SEQ ID NO: 38, or (iii) a
complementary strand of (i) or (ii); and
[0020] (c) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of amino
acids 1 to 556 of SEQ ID NO: 2, or
[0021] (c1) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of amino
acids 1 to 561 of SEQ ID NO: 37, The present invention also relates
to polynucleotides encoding polypeptides having glucoamylase
activity, selected from the group consisting of:
[0022] (a) a polynucleotide encoding a polypeptide having an amino
acid sequence which has at least 75% identity with the mature
polypeptide amino acids 1 to 556 of SEQ ID NO: 2;
[0023] (a1) a polynucleotide encoding a polypeptide having an amino
acid sequence which has at least 75% identity with the mature
polypeptide amino acids 1 to 561 of SEQ ID NO: 37;
[0024] (b) a polynucleotide having at least 60% identity with
nucleotides 55 to 2166 of SEQ ID NO: 1; or
[0025] (b1) a polynucleotide having at least 60% identity with
nucleotides 55 to 2166 of SEQ ID NO: 36;
[0026] (c) a polynucleotide having at least 60% identity with
nucleotides 55 to 1725 of SEQ ID NO: 3; or
[0027] (c1) a polynucleotide having at least 60% identity with
nucleotides 55 to 1737 of SEQ ID NO: 38;
[0028] (d) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 55 to 2166 of SEQ ID NO: 1, or (ii) which hybridizes
under at least medium stringency conditions with the cDNA sequence
contained in nucleotides 55 to 1725 of SEQ ID NO: 3, or (iii) a
complementary strand of (i) or (ii), or
[0029] (d1) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 55 to 2166 of SEQ ID NO: 36, or (ii) which hybridizes
under at least medium stringency conditions with the cDNA sequence
contained in nucleotides 55 to 1737 of SEQ ID NO: 38, or (iii) a
complementary strand of (i) or (ii).
[0030] In a preferred embodiment the polypeptide is derivable from
a strain of the genus Trametes, preferably Trametes cingulata or
the E. coli strain deposited at DSMZ and given the no. DSM 17106.
Deposited strain DSM 17106 harbors plasmid HUda595 comprising a
sequence identical to SEQ ID NO: 1. A specific polypeptide of the
invention is the mature polypeptide obtained when expressing
plasmid pHUda440 in a suitable fungal host cell such as Aspergillus
oryzae as described in Example 7.
[0031] In a second aspect the present invention relates to
polypeptides having glucoamylase activity selected from the group
consisting of:
[0032] (a) a polypeptide having an amino acid sequence which has at
least 70% identity with amino acids for mature polypeptide amino
acids 1 to 575 of SEQ ID NO: 5; or
[0033] (a1) a polypeptide having an amino acid sequence which has
at least 70% identity with amino acids for mature polypeptide amino
acids 1 to 565 of SEQ ID NO: 40;
[0034] (b) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 55 to 2189 of SEQ ID NO: 4, or (ii) which hybridizes
under at least medium stringency conditions with the cDNA sequence
contained in nucleotides 55 to 1725 of SEQ ID NO: 6, or (iii) a
complementary strand of (i) or (ii); or
[0035] (b1) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 55 to 2182 of SEQ ID NO: 39, or (ii) which hybridizes
under at least medium stringency conditions with the cDNA sequence
contained in nucleotides 55 to 1749 of SEQ ID NO: 41, or (iii) a
complementary strand of (i) or (ii); and
[0036] (c) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of amino
acids 1 to 575 of SEQ ID NO: 5, or
[0037] (c1) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of amino
acids 1 to 565 of SEQ ID NO: 40.
[0038] The present invention also relates to polynucleotides
encoding polypeptides having glucoamylase activity, selected from
the group consisting of:
[0039] (a) a polynucleotide encoding a polypeptide having an amino
acid sequence which has at least 75% identity with the mature
polypeptide amino acids 1 to 575 of SEQ ID NO: 5; or
[0040] (a1) a polynucleotide encoding a polypeptide having an amino
acid sequence which has at least 75% identity with the mature
polypeptide amino acids 1 to 565 of SEQ ID NO: 40;
[0041] (b) a polynucleotide having at least 60% identity with
nucleotides 55 to 2189 of SEQ ID NO: 4; or
[0042] (b1) a polynucleotide having at least 60% identity with
nucleotides 55 to 2182 of SEQ ID NO: 39;
[0043] (c) a polynucleotide having at least 60% identity with
nucleotides 55 to 1725 of SEQ ID NO: 6; or
[0044] (c1) a polynucleotide having at least 60% identity with
nucleotides 55 to 1749 of SEQ ID NO: 41;
[0045] (d) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 55 to 2189 of SEQ ID NO: 4, or (ii) which hybridizes
under at least medium stringency conditions with the cDNA sequence
contained in nucleotides 55 to 1725 of SEQ ID NO: 6, or (iii) a
complementary strand of (i) or (ii); or
[0046] (d1) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 55 to 2182 of SEQ ID NO: 39, or (ii) which hybridizes
under at least medium stringency conditions with the cDNA sequence
contained in nucleotides 55 to 1749 of SEQ ID NO: 41, or (iii) a
complementary strand of (i) or (ii).
[0047] In a preferred embodiment the polypeptide is derivable from
a strain of the genus Pachykytospora, preferably Pachykytospora
papyracea or the E. coli strain deposited at DSMZ and given the no.
DSM 17105. Deposited strain DSM 17105 harbors plasmid HUda594
comprising a sequence identical to SEQ ID NO: 4. A specific
polypeptide of the invention is the mature polypeptide obtained
when expressing plasmid pHUda450 in a suitable fungal host cell
such as Aspergillus oryzae as described in Example 7.
[0048] In a third aspect the invention relates to polypeptides
having glucoamylase activity selected from the group consisting
of:
[0049] (a) a polypeptide having an amino acid sequence which has at
least 60% identity with amino acids for mature polypeptide amino
acids 1 to 556 of SEQ ID NO: 26; or
[0050] (a1) a polypeptide having an amino acid sequence which has
at least 60% identity with amino acids for mature polypeptide amino
acids 1 to 548 of SEQ ID NO: 24; or
[0051] (a2) a polypeptide having an amino acid sequence which has
at least 60% identity with amino acids for mature polypeptide amino
acids 1 to 523 of SEQ ID NO: 43;
[0052] (b) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 117 to 2249 of SEQ ID NO: 23, or (ii) which hybridizes
under at least low stringency conditions with the cDNA sequence
contained in nucleotides 52 to 1719 of SEQ ID NO: 25, or (iii) a
complementary strand of (i) or (ii);
[0053] (b1) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
the cDNA sequence contained in nucleotides 52 to 1620 of SEQ ID NO:
42 or (iii) a complementary strand of (i) or (ii); and
[0054] (c) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of amino
acids 1 to 556 of SEQ ID NO: 26, or
[0055] (c1) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of amino
acids 1 to 548 of SEQ ID NO: 24;
[0056] (c2) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of amino
acids 1 to 523 of SEQ ID NO: 43.
[0057] The present invention also relates to polynucleotides
encoding polypeptides having glucoamylase activity, selected from
the group consisting of:
[0058] (a) a polynucleotide encoding a polypeptide having an amino
acid sequence which has at least 60% identity with the mature
polypeptide amino acids 1 to 556 of SEQ ID NO: 26; or
[0059] (a1) a polynucleotide encoding a polypeptide having an amino
acid sequence which has at least 60% identity with the mature
polypeptide amino acids 1 to 548 of SEQ ID NO: 24; or
[0060] (a2) a polynucleotide encoding a polypeptide having an amino
acid sequence which has at least 60% identity with the mature
polypeptide amino acids 1 to 523 of SEQ ID NO: 43;
[0061] (b) a polynucleotide having at least 60% identity with
nucleotides 117 to 2249 of SEQ ID NO: 23; or
[0062] (c) a polynucleotide having at least 60% identity with
nucleotides 52 to 1719 of SEQ ID NO: 25; or
[0063] (c1) a polynucleotide having at least 60% identity with
nucleotides 52 to 1620 of SEQ ID NO: 42;
[0064] (d) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
nucleotides 117 to 2249 of SEQ ID NO: 23, or (ii) which hybridizes
under at least low stringency conditions with the cDNA sequence
contained in nucleotides 52 to 1620 of SEQ ID NO: 42, or (iii) a
complementary strand of (i) or (ii), or
[0065] (d1) a polypeptide which is encoded by a nucleotide sequence
(i) which hybridizes under at least low stringency conditions with
the cDNA sequence contained in nucleotides 52 to 1719 of SEQ ID NO:
25, or (iii) a complementary strand of (i) or (ii).
[0066] In a preferred embodiment the polypeptide is derivable from
a strain of the genus Leucopaxillus, preferably Leucopaxillus
giganteus or the sequence shown in SEQ ID NO: 26. A specific
polypeptide of the invention is the mature polypeptide obtained
when expressing plasmid pENI3372 in a suitable fungal host cell
such as Aspergillus niger as described in Example 12.
[0067] The present invention also relates to nucleic acid
constructs, recombinant expression vectors, and recombinant host
cells comprising the polynucleotides in SEQ ID NO: 1 or 3 (cDNA) or
36 or 38 (cDNA); or SEQ ID NO: 4 or 6 (cDNA) or 39 or 41 (cDNA), or
SEQ ID NO: 23 or 25 (cDNA) or 42 (cDNA), respectively.
[0068] Clones that, to the best of the inventors belief, are
identical to SEQ ID NO: 1 and 4 was deposited on 2 Feb. 2005 under
the terms of the Budapest Treaty on the International Recognition
of the Deposit of Microorganisms for the Purposes of Patent
Procedure at Deutshe Sammmlung von Microorganismen und Zellkulturen
GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig DE. The
clones were giving the deposit nos. DSM 17106 and DSM 17105,
respectively.
[0069] The present invention also relates to methods for producing
such polypeptides having glucoamylase activity comprising (a)
cultivating a recombinant host cell comprising a nucleic acid
construct comprising a polynucleotide encoding the polypeptide
under conditions conducive for production of the polypeptide; and
(b) recovering the polypeptide.
[0070] The present invention also relates to processes of producing
a fermentation product or syrup.
DEFINITIONS
[0071] Glucoamylase activity: The term glucoamylase
(1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is defined as an
enzyme, which catalyzes the release of D-glucose from the
non-reducing ends of starch or related oligo- and polysaccharide
molecules. For purposes of the present invention, glucoamylase
activity is determined according to the procedure described in the
`Materials & Methods`-section below.
[0072] The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 50%, more
preferably at least 60%, more preferably at least 70%, more
preferably at least 80%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 100% of
the glucoamylase activity of the polypeptide consisting of the
amino acid sequence shown as amino acids 1 to 556 of SEQ ID NO: 2
or amino acids 1 to 561 of SEQ ID NO: 37; or amino acids 1 to 575
of SEQ ID NO: 5 or amino acids 1 to 565 of SEQ ID NO: 40; or amino
acids 1 to 548 of SEQ ID NO: 24 or amino acids 1 to 556 of SEQ ID
NO: 26 or amino acids 1 to 523 of SEQ ID NO: 43, respectively.
[0073] Polypeptide: The term "polypeptide" as used herein refers to
a isolated polypeptide which is at least 20% pure, preferably at
least 40% pure, more preferably at least 60% pure, even more
preferably at least 80% pure, most preferably at least 90% pure,
and even most preferably at least 95% pure, as determined by
SDS-PAGE.
[0074] Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation which
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%, at
most 3%, even more preferably at most 2%, most preferably at most
1%, and even most preferably at most 0.5% by weight of other
polypeptide material with which it is natively associated. It is,
therefore, preferred that the substantially pure polypeptide is at
least 92% pure, preferably at least 94% pure, more preferably at
least 95% pure, more preferably at least 96% pure, more preferably
at least 96% pure, more preferably at least 97% pure, more
preferably at least 98% pure, even more preferably at least 99%,
most preferably at least 99.5% pure, and even most preferably 100%
pure by weight of the total polypeptide material present in the
preparation.
[0075] The polypeptides of the present invention are preferably in
a substantially pure form. In particular, it is preferred that the
polypeptides are in "essentially pure form", i.e., that the
polypeptide preparation is essentially free of other polypeptide
material with which it is natively associated. This can be
accomplished, for example, by preparing the polypeptide by means of
well-known recombinant methods or by classical purification
methods.
[0076] Herein, the term "substantially pure polypeptide" is
synonymous with the terms "isolated polypeptide" and "polypeptide
in isolated form".
[0077] Identity: The relatedness between two amino acid sequences
or between two nucleotide sequences is described by the parameter
"identity".
[0078] For purposes of the present invention, the degree of
identity between two amino acid sequences is determined by the
Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the
LASERGENE.TM. MEGALIGN.TM. software (DNASTAR, Inc., Madison, Wis.)
with an identity table and the following multiple alignment
parameters: Gap penalty of 10 and gap length penalty of 10.
Pairwise alignment parameters are Ktuple=1, gap penalty=3,
windows=5, and diagonals=5.
[0079] For purposes of the present invention, the degree of
identity between two nucleotide sequences is determined by the
Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the
National Academy of Science USA 80: 726-730) using the
LASERGENE.TM. MEGALIGN.TM. software (DNASTAR, Inc., Madison, Wis.)
with an identity table and the following multiple alignment
parameters: Gap penalty of 10 and gap length penalty of 10.
Pairwise alignment parameters are Ktuple=3, gap penalty=3, and
windows=20.
[0080] Polypeptide Fragment: The term "polypeptide fragment" is
defined herein as a polypeptide having one or more amino acids
deleted from the amino and/or carboxyl terminus of SEQ ID NO: 2 or
37; or SEQ ID NO: 5 or 40; or SEQ ID NO: 24, 26, or 43,
respectively, or homologous sequences thereof, wherein the fragment
has glucoamylase activity.
[0081] Subsequence: The term "subsequence" is defined herein as a
nucleotide sequence having one or more nucleotides deleted from the
5' and/or 3' end of SEQ ID NO: 1, 36, or 38, respectively; or SEQ
ID NO: 4, 39, or 41, or SEQ ID NO: 23, 25, or 42, respectively, or
homologous sequences thereof, wherein the subsequence encodes a
polypeptide fragment having glucoamylase activity.
[0082] Allelic variant: The term "allelic variant" denotes herein
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.
[0083] Substantially pure polynucleotide: The term "substantially
pure polynucleotide" as used herein refers to a polynucleotide
preparation free of other extraneous or unwanted nucleotides and in
a form suitable for use within genetically engineered protein
production systems. Thus, a substantially pure polynucleotide
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%,
more preferably at most 3%, even more preferably at most 2%, most
preferably at most 1%, and even most preferably at most 0.5% by
weight of other polynucleotide material with which it is natively
associated. A substantially pure polynucleotide may, however,
include naturally occurring 5' and 3' untranslated regions, such as
promoters and terminators. It is preferred that the substantially
pure polynucleotide is at least 90% pure, preferably at least 92%
pure, more preferably at least 94% pure, more preferably at least
95% pure, more preferably at least 96% pure, more preferably at
least 97% pure, even more preferably at least 98% pure, most
preferably at least 99%, and even most preferably at least 99.5%
pure by weight. The polynucleofides of the present invention are
preferably in a substantially pure form. In particular, it is
preferred that the polynucleotides disclosed herein are in
"essentially pure form", i.e., that the polynucleotide preparation
is essentially free of other polynucleotide material with which it
is natively associated. Herein, the term "substantially pure
polynucleotide" is synonymous with the terms "isolated
polynucleotide" and "polynucleotide in isolated form." The
polynucleotides may be of genomic, cDNA, RNA, semi-synthetic,
synthetic origin, or any combinations thereof.
[0084] cDNA: The term "cDNA" is defined herein as a DNA molecule
which can be prepared by reverse transcription from a mature,
spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks
intron sequences that are usually present in the corresponding
genomic DNA. The initial, primary RNA transcript is a precursor to
mRNA which is processed through a series of steps before appearing
as mature spliced mRNA. These steps include the removal of intron
sequences by a process called splicing. cDNA derived from mRNA
lacks, therefore, any intron sequences.
[0085] Nucleic acid construct: The term "nucleic acid construct" as
used herein refers to a nucleic acid molecule, either single- or
double-stranded, which is isolated from a naturally occurring gene
or which is modified to contain segments of nucleic acids in a
manner that would not otherwise exist in nature. The term nucleic
acid construct is synonymous with the term "expression cassette"
when the nucleic acid construct contains the control sequences
required for expression of a coding sequence of the present
invention.
[0086] Control sequence: The term "control sequences" is defined
herein to include all components, which are necessary or
advantageous for the expression of a polynucleotide encoding a
polypeptide of the present invention. Each control sequence may be
native or foreign to the nucleotide sequence encoding the
polypeptide. Such control sequences include, but are not limited
to, a leader, polyadenylation sequence, pro-peptide 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 nucleotide sequence
encoding a polypeptide.
[0087] Operably linked: The term "operably linked" denotes herein a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the
polynucleotide sequence such that the control sequence directs the
expression of the coding sequence of a polypeptide.
[0088] Coding sequence: When used herein the term "coding sequence"
means a nucleotide sequence, which directly specifies the amino
acid sequence of its protein product. The boundaries of the coding
sequence are generally determined by an open reading frame, which
usually begins with the ATG start codon or alternative start codons
such as GTG and TTG. The coding sequence may a DNA, cDNA, or
recombinant nucleotide sequence.
[0089] Expression: The term "expression" includes any step involved
in the production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0090] Expression vector: The term "expression vector" is defined
herein as a linear or circular DNA molecule that comprises a
polynucleotide encoding a polypeptide of the invention, and which
is operably linked to additional nucleotides that provide for its
expression.
[0091] Host cell: The term "host cell", as used herein, includes
any cell type which is susceptible to transformation, transfection,
transduction, and the like with a nucleic acid construct comprising
a polynucleotide of the present invention.
[0092] Modification: The term "modification" means herein any
chemical modification of the polypeptide consisting of the amino
acids 1 to 556 of SEQ ID NO: 2 or amino acids 1 to 561 of SEQ ID
NO: 37; or amino acids 1 to 675 of SEQ ID NO: 5 or amino acids 1 to
565 of SEQ ID NO: 40; or amino acids 1 to 556 of SEQ ID NO: 26 or
SEQ ID NO: 1 to 548 of SEQ ID NO: 24 or SEQ ID NO: 1 to 523 of SEQ
ID NO: 43, respectively, as well as genetic manipulation of the DNA
encoding the polypeptides. The modification(s) can be
substitution(s), deletion(s) and/or insertions(s) of the amino
acid(s) as well as replacement(s) of amino acid side chain(s).
[0093] Artificial variant: When used herein, the term "artificial
variant" means a polypeptide having glucoamylase activity produced
by an organism expressing a modified nucleotide sequence of SEQ ID
NO: 1 or 3 (cDNA) or SEQ ID NO: 36 or 38 (cDNA); or SEQ ID NO: 4 or
6 (cDNA), or SEQ ID NO: 39 or 41 (cDNA); or SEQ ID NO: 23 or 25
(cDNA) or 42 (cDNA). The modified nucleotide sequence is obtained
through human intervention by modification of the nucleotide
sequence disclosed in SEQ ID NO: 1 or 3, or SEQ ID NO: 36 or 38; or
SEQ ID NO: 4 or 6, or SEQ ID NO: 39 or 41; or SEQ ID NO: 23 or 25
or 42, respectively.
BRIEF DESCRIPTION OF THE DRAWING
[0094] FIG. 1 shows the debranching activity toward pullulan of
Trametes cingulata glucoamylase compared to glucoamylases from
Athelia rolfsii, Aspergillus niger, and Talaromyces emersonii.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Glucoamylase Activity
[0095] In a first aspect, the present invention relates to
polypeptides having an amino acid sequence which has a degree of
identity to amino acids 1 to 556 of SEQ ID NO: 2, or amino acids
1-561 of SEQ ID NO: 37; or amino acids 1-575 of SEQ ID NO: 5 or
amino acids 1-565 of SEQ ID NO: 40; or amino acids 1-556 of SEQ ID
NO: 26 or amino acids 1-548 of SEQ ID NO: 24 or amino acids 1-523
of SEQ ID NO: 43 (i.e., mature polypeptide), respectively.
[0096] In an embodiment the amino acid sequence has glucoamylase
activity and is at least 75%, preferably at least 80%, more
preferably at least 85%, even more preferably at least 90%, most
preferably at least 95%, more preferred at least 96%, even more
preferred at least 97%, even more preferred at least 98%, even more
preferably at least 99% identical to the mature part of SEQ ID NO:
2 or SEQ ID NO: 37 (hereinafter "homologous polypeptides").
[0097] In another embodiment the amino acid sequence has
glucoamylase activity and has at least 70%, more preferably at
least 75%, more preferably at least 80%, more preferably at least
85%, even more preferably at least 90%, most preferably at least
95%, more preferred at least 96%, even more preferred at least 97%,
even more preferred at least 98%, even more preferably at least 99%
identity to the mature part of SEQ ID NO: 5 or SEQ ID NO: 40
(hereinafter "homologous polypeptides").
[0098] In an embodiment the amino acid sequence has glucoamylase
activity and is at least 60%, at least 65%, at least 70%, at least
75%, preferably at least 80%, more preferably at least 85%, even
more preferably at least 90%, most preferably at least 95%, more
preferred at least 96%, even more preferred at least 97%, even more
preferred at least 98%, even more preferably at least 99% identical
to the mature part of SEQ ID NO: 26, 24 or 43, respectively
(hereinafter "homologous polypeptides").
[0099] In a preferred aspect, the homologous polypeptides have an
amino acid sequence which differs by ten amino acids, preferably by
five amino acids, more preferably by four amino acids, even more
preferably by three amino acids, most preferably by two amino
acids, and even most preferably by one amino acid from amino acids
1-556 of SEQ ID NO: 2, or amino acids 1-561 of SEQ ID NO: 37; or
amino acids 1-575 of SEQ ID NO: 5, or amino acids 1-565 of SEQ ID
NO: 40; or amino acids 1-556 of SEQ ID NO: 26 or amino acids 1-548
of SEQ ID NO: 24 or amino acids 1-523 of SEQ ID NO: 43,
respectively.
[0100] A polypeptide of the present invention preferably comprises
the mature amino acid sequences of SEQ ID NO: 2 or 37; or SEQ ID
NO: 5 or 40; or SEQ ID NO: 26 24 or 43, respectively, or allelic
variants thereof; or fragments thereof that have glucoamylase
activity, e.g., the catalytic domain.
Catalytic Domain
[0101] In an aspect, the invention relates to polypeptides that
comprise the catalytic region/domain of the amino acid sequences of
SEQ ID NO: 2 or 37; or SEQ ID NO: 5 or 40 or SEQ ID NO: 26, 24, or
43, respectively.
[0102] The catalytic region/domain of the Trametes cingulata
glucoamylase is located at amino acids 1-455 in SEQ ID NO: 2 or
amino acids 1-460 of SEQ ID NO: 37. In one embodiment the region
may be considered to include the linker region at amino acids
456-465 of SEQ ID NO: 2 or amino acids 461-470 of SEQ ID NO: 37,
respectively, or part thereof. The binding domain is encoded by
polynucleotides 1423-1725 in SEQ ID NO: 3 or polynucleotides
1774-2163 of SEQ ID NO: 36 or polynucleotides 1465-1737 of SEQ ID
NO: 38, respectively.
[0103] The catalytic region/domain of the Pachykytospora papyracea
glucoamylase is located at amino acids 1-475 in SEQ ID NO: 5 or
amino acids 1-465 of SEQ ID NO: 40. In one embodiment the region
may be considered to include the linker region at amino acids
476-484 of SEQ ID NO: 5 or amino acids 466-474 of SEQ ID NO: 40,
respectively, or part thereof. The binding domain is encoded by
polynucleotides 1420-1725 in SEQ ID NO: 6 or polynucleotides
1763-2182 of SEQ ID NO: 39 or polynucleotides 1477-1749 of SEQ ID
NO: 41, respectively.
[0104] The catalytic region/domain of the Leucopaxillus giganteus
glucoamylase is located at amino acids 1-451 of SEQ ID NO: 26 or
amino acids 1-455 of SEQ ID NO: 24 or amino acids 1-418 of SEQ ID
NO: 43, respectively. In one embodiment the region may be
considered to include the linker region at amino acids 452-461 of
SEQ ID NO: 26 or amino acids 456-466 of SEQ ID NO: 24 or amino
acids 419-429 of SEQ ID NO: 43, respectively, or part thereof. The
binding domain (CBM) is encoded by polynucleotides 1438-1719 in SEQ
ID NO: 25 or polynucleotides 1854-2249 of SEQ ID NO: 23 or
polynucleotides 1339-1620 of SEQ ID NO: 42, respectively.
[0105] In a preferred embodiment the invention relates to a
catalytic region which has at least 60% identity, preferably at
least 65% identity, more preferably at least 70% identity, more
preferably at least 75% identity, more preferably at least 80%
identity, more preferably at least 85% identity, even more
preferably at least 90% identity, most preferably at least 95%
identity, more preferred at least 96% identity, even more preferred
at least 97% identity, even more preferred at least 98% identity,
even more preferably at least 99% identity, especially 100%
identity to amino acids 1-455 in SEQ ID NO: 2 or amino acids 1-460
of SEQ ID NO: 37 (Trametes); or amino acids 1-475 in SEQ ID NO: 5
or amino acids 1-465 of SEQ ID NO: 40 (Pachykytospora); or amino
acids 1-451 in SEQ ID NO: 26 or amino acids 1-455 of SEQ ID NO: 24
or amino acids 1-418 in SEQ ID NO: 43 (Leucopaxillus),
respectively, and which have glucoamylase activity (hereinafter
"homologous polypeptides"). In a preferred aspect, the homologous
catalytic regions have amino acid sequences which differs by ten
amino acids, preferably by five amino acids, more preferably by
four amino acids, even more preferably by three amino acids, most
preferably by two amino acids, and even most preferably by one
amino acid from amino acids 1-455 of SEQ ID NO: 2 or amino acids
1460 of SEQ ID NO: 37 (Trametes cingulata); or amino acids 1-475 of
SEQ ID NO: 5 or amino acids 1-465 of SEQ ID NO: 40 (Pachykytospora)
or amino acids 1-451 in SEQ ID NO: 26 or amino acids 1-455 of SEQ
ID NO: 2424 or amino acids 1-418 in SEQ ID NO: 43 (Leucopaxillus
giganteus), respectively.
Binding Domain
[0106] In another aspect, the invention relates to polypeptides
having carbohydrate-binding affinity, preferably starch-binding
affinity.
[0107] The binding domain in Trametes glucoamylase is located at
amino acid 466-556 of SEQ ID NO: 2 and is encoded by
polynucleotides 1420-1725 in SEQ ID NO: 3 or is located at amino
acids 471-561 of SEQ ID NO: 37 and is encoded by polynucleotides
1465-1737 in SEQ ID NO: 38.
[0108] The binding domain in Pachykytospora glucoamylase is located
at amino acids 485-575 is SEQ ID NO: 5 (Pachykytspora) and is
encoded by polynucleotides 1423-1725 in SEQ ID NO: 6 or is located
at amino acids 475-565 of SEQ ID NO: 40 and is encoded by
polynucleotides 1477-1749 in SEQ ID NO: 41.
[0109] The binding domain in Leucopaxillus glucoamylase is located
at amino acids 463-556 of SEQ ID NO: 26 or amino acids 467-548 of
SEQ ID NO: 24 or amino acids 430-523 of SEQ ID NO: 43,
respectively, and is encoded by polynucleotides 1854-2249 in SEQ ID
NO: 23 or polynucleotides 1438-1719 in SEQ ID NO: 25 or
polynucleotides 1339-1620 in SEQ ID NO: 42, respectively.
[0110] Consequently, in this aspect the invention relates to a
polypeptide having carbohydrate binding affinity, selected from the
group consisting of:
(a) i) a polypeptide comprising an amino acid sequence which has at
least 60% identity with amino acids 466 to 556 of SEQ ID NO: 2 or
amino acids 471 to 561 of SEQ ID NO: 37, respectively; or
[0111] ii) a polypeptide comprising an amino acid sequence which
has at least 60% identity with amino acids 485 to 575 of SEQ ID NO:
5 or amino acids 475 to 565 of SEQ ID NO: 40, respectively; or
[0112] iii) a polypeptide comprising an amino acid sequence which
has at least 60% identity with amino acids 463 to 556 of SEQ ID NO:
26 or amino acids 467 to 548 of SEQ ID NO: 24, or amino acids 430
to 523 of SEQ ID NO: 43, respectively;
(b) a polypeptide which is encoded by a nucleotide sequence which
hybridizes under low stringency conditions with a polynucleotide
probe selected from the group consisting of
[0113] (i) the complementary strand of nucleotides 1420 to 1725 of
SEQ ID NO: 3 or nucleotides 1465 to 1737 of SEQ ID NO: 38,
respectively;
[0114] (ii) the complementary strand of nucleotides 1423 to 1725 of
SEQ ID NO: 6 or nucleotides 1477 to 1749 of SEQ ID NO: 41,
respectively;
[0115] (iii) the complementary strand of nucleotides 1438 to 1719
of SEQ ID NO: 25 or nucleotides 1854 to 2249 of SEQ ID NO: 23 or
nucleotides 1339 to 1620 of SEQ ID NO: 42, respectively;
(c) a fragment of (a) or (b) that has carbohydrate binding
affinity.
[0116] In a preferred embodiment the carbohydrate binding affinity
is starch-binding affinity.
[0117] In a preferred embodiment the invention relates to a
polypeptide having carbohydrate binding affinity which has at least
60% identity, preferably at least 70% identity, more preferably at
least 75% identity, more preferably at least 80% identity, more
preferably at least 85% identity, even more preferably at least 90%
identity, most preferably at least 95% identity, more preferred at
least 96% identity, even more preferred at least 97% identity, even
more preferred at least 98% identity, even more preferably at least
99% identity, especially 100% identity to amino acids 466 to 556 in
SEQ ID NO: 2 or amino acids 471 to 561 of SEQ ID NO: 37,
respectively, (Trametes), or amino acids 485 to 575 in SEQ ID NO: 5
or amino acids 475 to 565 of SEQ ID NO: 40, respectively,
(Pachykytospora), or amino acids 463 to 556 of SEQ ID NO: 26 or
amino acids 467 to 548 of SEQ ID NO: 24 or amino acids 430 to 523
of SEQ ID NO: 43, respectively (Leucopaxillus), respectively.
[0118] In a preferred aspect, homologous binding domains have amino
acid sequences which differs by ten amino acids, preferably by five
amino acids, more preferably by four amino acids, even more
preferably by three amino acids, most preferably by two amino
acids, and even most preferably by one amino acid from amino acids
466 to 556 of SEQ ID NO: 2 or amino acids 471 to 561 of SEQ ID NO:
37, respectively, (Trametes cingulata) or amino acids 485 to 575 of
SEQ ID NO: 5 or amino acids 475 to 565 of SEQ ID NO: 40,
respectively, (Pachykytospora) or amino acids 463 to 556 of SEQ ID
NO: 26 or amino acids 467 to 548 of SEQ ID NO: 24 or amino acids
430 to 523 of SEQ ID NO: 43, respectively (Leucopaxillus),
respectively.
[0119] In another embodiment the invention relates to a polypeptide
having carbohydrate-binding affinity, selected from the group
consisting of:
(a) a polypeptide which is encoded by a nucleotide sequence which
hybridizes under low stringency conditions, preferably under
medium, more preferably under high stringency conditions with a
polynucleotide probe selected from the group consisting of
[0120] (i) the complementary strand of nucleotides 1420 to 1725 of
SEQ ID NO: 3 or nucleotides 1465 to 1737 in SEQ ID NO: 38,
respectively;
[0121] (ii) the complementary strand of nucleotides 1423 to 1725 of
SEQ ID NO: 6 or nucleotides 1477 to 1749 in SEQ ID NO: 41,
respectively;
[0122] (iii) the complementary strand of nucleotides 1438 to 1719
of SEQ ID NO: 25 or nucleotides 1854 to 2249 in SEQ ID NO: 23 or
nucleotides 1339 to 1620 in SEQ ID NO: 42, respectively;
(b) a fragment of (a) that has carbohydrate-binding affinity.
[0123] The invention also relates to a polypeptide having
carbohydrate-binding affinity, where the polypeptide is an
artificial variant which comprises an amino acid sequence that has
at least one substitution, deletion and/or insertion of an amino
acid as compared to amino acids 466 to 556 of SEQ ID NO: 2 or amino
acids 471 to 561 of SEQ ID NO: 37 (Trametes); or amino acids 485 to
575 of SEQ ID NO: 5 or amino acids 475 to 565 of SEQ ID NO: 40
(Pachykytospora); or amino acids 463 to 556 of SEQ ID NO: 26 or
amino acids 467 to 548 of SEQ ID NO: 24 or amino acids 430 to 523
of SEQ ID NO: 43 (Leucopaxillus), respectively.
[0124] The invention also relates to a polypeptide having
carbohydrate-binding affinity, where the polypeptide is an
artificial variant which comprises an amino acid sequence that has
at least one substitution, deletion and/or insertion of an amino
acid as compared to the amino acid sequence encoded by the
carbohydrate-binding domain encoding part of the polynucleotide
sequences shown in position 1420 to 1725 in SEQ ID NO: 3 or
position 1465 to 1737 in SEQ ID NO: 38; or position 1423 to 1725 of
SEQ ID NO: 6 or position 1477 to 1749 in SEQ ID NO: 41; or position
1438 to 1719 of SEQ ID NO: 25 or position 1854 to 2249 in SEQ ID
NO: 23 or nucleotides 1339 to 1620 in SEQ ID NO: 42,
respectively.
Hybrids
[0125] The glucoamylases or catalytic regions of the invention may
be linked, via a linker sequence or directly, to one or more
foreign binding domains (also referred to as binding modules
(CBM)). A "foreign" binding domain is a binding-domain that is not
derived from the wild-type glucoamylases of the invention in
question. The binding-domain is preferably a carbohydrate-binding
domain (i.e., having affinity for binding to a carbohydrate),
especially a starch-binding domain or a cellulose-binding domain.
Preferred binding domains are of fungal or bacterial origin.
Examples of specifically contemplated starch-binding domains are
disclosed in WO 2005/003311 which is hereby incorporated by
reference.
[0126] In a preferred embodiment the linker in a glucoamylase of
the invention is replaced with a more stable linker, i.e., a linker
that is more difficult to cut than the parent linker. This is done
to avoid that the binding-domain is cleaved off. Specifically
contemplated stable linkers include the Aspergillus kawachli
linker: TABLE-US-00001 TTTTTTAAAT STSKATTSSSSSSAAATTSSS (SEQ ID NO:
22)
[0127] Thus, in a preferred embodiment the invention relates to a
hybrid glucoamylase having the amino acid sequence shown in SEQ ID
NO: 2 or 37, respectively, wherein the native linker located from
amino acids 456 to 465 of SEQ ID NO: 2 or from amino acids 461 to
470 in SEQ ID NO: 37, respectively, or part thereof, is replaced
with the Aspergillus kawachii linker shown in SEQ ID NO: 22.
[0128] Thus, in another preferred embodiment the invention relates
to a hybrid glucoamylase having the amino acid sequence shown in
SEQ ID NO: 5 or 40, respectively, wherein the native linker located
from 476 to 484 in SEQ ID NO: 5 or from amino acids 466 to 474 in
SEQ ID NO: 40, respectively, or part thereof is replaced with the
Aspergillus kawachii linker shown in SEQ ID NO: 22.
[0129] Thus, in another preferred embodiment the invention relates
to a hybrid glucoamylase having the amino acid sequence shown in
SEQ ID NO: 26 or 24, respectively, wherein the native linker
located from 452 to 462 in SEQ ID NO: 26 or from amino acids 456
466 in SEQ ID NO: 24 or from amino acids 419 to 429 in SEQ ID NO:
24, respectively, or part thereof is replaced with the Aspergillus
kawachii linker shown in SEQ ID NO: 22.
[0130] Thus, the invention also relates to hybrids consisting of a
glucoamylase of the invention or catalytic domain of the invention
having glucoamylase activity fused to a stable linker (e.g.,
Aspergillus kawachii linker) and one or more carbohydrate-binding
domains, e.g., a carbohydrate-binding module (CBM) disclosed in WO
2005/003311 on page 5, line 30 to page 8, line 12, hereby
incorporated by reference.
Hybridization
[0131] In another aspect, the present invention relates to
polypeptides having glucoamylase activity which are encoded by
polynucleotides (i) which hybridizes under at least low stringency
conditions, preferably medium stringency conditions, more
preferably medium-high stringency conditions, even more preferably
high stringency conditions, and most preferably very high
stringency conditions with a nucleotide sequence with nucleotides
55 to 2166 of SEQ ID NO: 1 or nucleotides 55 to 2166 of SEQ ID NO:
36, respectively (Trametes genomic DNA), or (ii) which hybridizes
under at least medium stringency conditions, preferably medium-high
stringency conditions, more preferably high stringency conditions,
and more preferably very high stringency conditions with a
nucleotide sequence with the cDNA sequence contained in nucleotides
55 to 1725 of SEQ ID NO: 3 or nucleotides 55 to 1737 of SEQ ID NO:
38, respectively (Trametes cDNA), or (iii) a subsequence of (i) or
(ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning,
A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A
subsequence of SEQ ID NOS: 1 or 3, or SEQ ID NOS: 36 or 38
(Trametes) contains at least 100 contiguous nucleotides or
preferably at least 200 continguous nucleotides. Moreover, the
subsequence may encode a polypeptide fragment which has
glucoamylase activity.
[0132] The invention also relates to isolated polypeptides having
glucoamylase activity which are encoded by polynucleotides (i)
which hybridizes under at least low stringency conditions,
preferably medium stringency conditions, more preferably
medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with a nucleotide sequence with nucleotides 55 to 2189
of SEQ ID NO: 4 or nucleotides 55 to 2182 of SEQ ID NO: 39,
respectively (Pachykytospora genomic DNA), or (ii) which hybridizes
under at least medium stringency conditions, preferably medium-high
stringency conditions, more preferably high stringency conditions,
and even more preferably very high stringency conditions with a
nucleotide sequence with the cDNA sequence contained in nucleotides
55 to 1725 of SEQ ID NO: 6 or nucleotides 55 to 1749 of SEQ ID NO:
41, respectively (Pachykytospora cDNA), or (iii) a subsequence of
(i) or (ii), or (iv) a complementary strand of (i), (ii), or
(iii).
[0133] The invention also relates to isolated polypeptides having
glucoamylase activity which are encoded by polynucleotides (i)
which hybridizes under at least low stringency conditions,
preferably medium stringency conditions, more preferably
medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with a nucleotide sequence with nucleotides 117 to 2249
of SEQ ID NO: 23 (Leucopaxillus genomic DNA), or (ii) which
hybridizes under at least low stringency conditions, preferably
medium, more preferably medium-high stringency conditions, more
preferably high stringency conditions, and even more preferably
very high stringency conditions with a nucleotide sequence with the
cDNA sequence contained in nucleotides 52 to 1719 of SEQ ID NO: 25
or nucleotides 52 to 1620 of SEQ ID NO: 42 (Leucopaxillus cDNA), or
(iii) a subsequence of (i) or (ii), or (iv) a complementary strand
of (i), (ii), or (iii)
[0134] The nucleotide sequence of SEQ ID NO: 1, 3, 36, or 38,
respectively, or a subsequence thereof, or the nucleotide sequence
of SEQ ID NO: 4, 6, 39, or 41, respectively, or a subsequence
thereof, or the nucleotide sequence of SEQ ID NO: 23, 25 or 42,
respectively, or a subsequence thereof, as well as the amino acid
sequence of SEQ ID NO: 2 or 37, respectively, or a fragment
thereof, or the amino acid sequence of SEQ ID NO: 5 or 40,
respectively, or a fragment thereof, or the amino acid sequence of
SEQ ID NO: 26, 24, or 43, respectively, or a fragment thereof, may
be used to design a nucleic acid probe to identify and clone DNA
encoding polypeptides having glucoamylase activity 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 or cDNA of the genus or species 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 14, preferably at least 25, more preferably at least 35, and
most preferably at least 70 nucleotides in length. It is however,
preferred that the nucleic acid probe is at least 100 nucleotides
in length. For example, the nucleic acid probe may be at least 200
nucleotides, preferably at least 300 nucleotides, more preferably
at least 400 nucleotides, or most preferably at least 500
nucleotides in length. Even longer probes may be used, e.g.,
nucleic acid probes which are at least 600 nucleotides, at least
preferably at least 700 nucleotides, more preferably at least 800
nucleotides, or most preferably 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.
[0135] A genomic DNA or cDNA library prepared from such other
organisms may, therefore, be screened for DNA which hybridizes with
the probes described above and which encodes a polypeptide having
glucoamylase activity. Genomic or other DNA from such other
organisms 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 which is homologous with SEQ ID
NO: 1, 3, 36, or 38, respectively, or a subsequence thereof, or SEQ
ID NO: 4, 6, 39 or 41, respectively, or a subsequence thereof, or
SEQ ID NO: 23, 25, or 42, respectively, or a subsequence thereof,
the carrier material is used in a Southern blot.
[0136] For purposes of the present invention, hybridization
indicates that the nucleotide sequences hybridize to labeled
nucleic acid probes corresponding to the nucleotide sequence shown
in SEQ ID NO: 1, 3, 36 or 38, respectively, or SEQ ID NO: 4, 6, 39,
or 41, respectively, or SEQ ID NO: 23, 25, or 42, respectively, its
complementary strands, or subsequences thereof, under low or medium
to very high stringency conditions. Molecules to which the nucleic
acid probe hybridizes under these conditions can be detected using
X-ray film.
[0137] In a preferred embodiment, the nucleic acid probe is
nucleotides 55 to 2166 of SEQ ID NO: 1 or nucleotides 55 to 2166 of
SEQ ID NO: 36, or nucleotides 1 to 1725 of SEQ ID NO: 3 or
nucleotides 55 to 1737 of SEQ ID NO: 38 (Trametes cDNA). In a
preferred embodiment, the nucleic acid probe is nucleotides 55 to
2186 of SEQ ID NO: 4 or nucleotides 55 to 2182 of SEQ ID NO: 39 or
nucleotides 1 to 1725 of SEQ ID NO: 6 or nucleotides 55 to 1749 of
SEQ ID NO: 41 (Pachykytospora cDNA). In a preferred embodiment, the
nucleic acid probe is nucleotides 117 to 2249 of SEQ ID NO: 23 or
nucleotides 52 to 1719 of SEQ ID NO: 25 (Leucopaxillus cDNA) or
nucleotides 52 to 1620 of SEQ ID NO: 42 (Leucopaxillus cDNA). In
other preferred aspect, the nucleic acid probe is a polynucleotide
sequence which encodes the catalytic region between amino acids 1
and 455 of SEQ ID NO: 2 or amino acids 1 to 460 of SEQ ID NO: 37
(Trametes) or between amino acids 1 and 475 of SEQ ID NO: 5 or
amino acids 1 to 465 of SEQ ID NO: 40 (Pachykytospora) or between
amino acids 1 and 455 of SEQ ID NO: 24 or amino acids 1 to 451 of
SEQ ID NO: 26 or amino acids 1 to 418 of SEQ ID NO: 43
(Leucopaxillus).
[0138] In another aspect the invention relates to nucleic acid
probes that encode the binding domain in amino acids 466 to 456 of
SEQ ID NO: 2 or amino acids 471 to 561 of SEQ ID NO: 37,
respectively, or amino acids 485 to 575 of SEQ ID NO: 5 or amino
acids 475 to 565 of SEQ ID NO: 40, respectively, or amino acids 463
to 556 of SEQ ID NO: 26 or amino acids 467 to 548 of SEQ ID NO: 24
or amino acids 430 to 523 of SEQ ID NO: 43, respectively.
[0139] In another preferred aspect, the nucleic acid probe is the
mature polypeptide coding region of SEQ ID NO: 1, 3, 36 or 38,
respectively (Trametes). In another preferred embodiment, the
nucleic acid probe is the mature polypeptide coding region of SEQ
ID NO: 4, 6, 39 or 41 (Pachykytospora). In another preferred
embodiment, the nucleic acid probe is the mature polypeptide coding
region of SEQ ID NOS: 23, 25, or 42 (Leucopaxillus). In another
preferred aspect, the nucleic acid probe is the part of the
sequences in plasmids pHUda595 and pHUda594, respectively, coding
for the mature polypeptides of the invention Plasmids pHUda595 and
pHUda594, which are contained in Escherichia coli DSM 17106 and
Escherichia coli DSM 17105, respectively, encode polypeptides
having glucoamylase activity.
[0140] For long probes of at least 100 nucleotides in length, low
to very high stringency conditions are defined as prehybridization
and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200
micro g/ml sheared and denatured salmon sperm DNA, and either 25%
formamide for low stringencies, 35% formamide for medium and
medium-high stringencies, or 50% formamide for high and very high
stringencies, following standard Southern blotting procedures for
12 to 24 hours optimally.
[0141] For long probes of at least 100 nucleotides in length, the
carrier material is finally washed three times each for 15 minutes
using 2.times.SSC, 0.2% SDS preferably at least at 50.degree. C.
(low stringency), more preferably at least at 55.degree. C. (medium
stringency), more preferably at least at 60.degree. C. (medium-high
stringency), even more preferably at least at 65.degree. C. (high
stringency), and most preferably at least at 70.degree. C. (very
high stringency).
[0142] For short probes which are about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization, hybridization, and washing post-hybridization at
about 5.degree. C. to about 10.degree. C. below the calculated
T.sub.m using the calculation according to Bolton and McCarthy
(1962, Proceedings of the National Academy of Sciences USA 48:1390)
in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,
1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium
monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml
following standard Southern blotting procedures.
[0143] For short probes which are about 15 nucleotides to about 70
nucleotides in length, the carrier material is washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0144] Under salt-containing hybridization conditions, the
effective T.sub.m is what controls the degree of identity required
between the probe and the filter bound DNA for successful
hybridization. The effective T.sub.m may be determined using the
formula below to determine the degree of identity required for two
DNAs to hybridize under various stringency conditions. Effective
T.sub.m=81.5+16.6(log M[Na.sup.+])+0.41(% G+C)-0.72(%
formamide)
[0145] (See
www.ndsu.nodak.edu/instruct/mccleanlplsc731/dna/dna6.htm)
[0146] The G+C content of SEQ ID NO: 1 or nucleotides 55 to 2166 of
SEQ ID NO: 1 is 60.5%.
[0147] The G+C content of SEQ ID NO: 3 (cDNA) or nucleotides 55 to
1725 of SEQ ID NO: 3 is 62.3%.
[0148] The G+C content of SEQ ID NO: 4 or nucleotides 55 to 2189 of
SEQ ID NO: 4 is 60.7%.
[0149] The G+C content of SEQ ID NO: 6 (cDNA) or nucleotides 55 to
1725 of SEQ ID NO: 6 is 63.7%.
[0150] For medium stringency, the formamide is 35% and the Na.sup.+
concentration for 5.times.SSPE is 0.75 M. Applying this formula to
these values, the Effective T.sub.m is 79.0.degree. C.
[0151] Another relevant relationship is that a 1% mismatch of two
DNAs lowers the T.sub.m by 1.4.degree. C. To determine the degree
of identity required for two DNAs to hybridize under medium
stringency conditions at 42.degree. C., the following formula is
used: % Homology=100-[(Effective T.sub.m-Hybridization
Temperature)/1.4]
[0152] (See
ndsu.nodak.edu/instruct/mcclean/plsc731/dna/dna6.htm)
[0153] Applying this formula to the values, the degree of identity
required for two DNAs to hybridize under medium stringency
conditions at 42.degree. C. is 100-[(79.0-42)/1.4]=51%.
Variants
[0154] In a further aspect, the present invention relates to
artificial variants comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids in SEQ ID
NOS: 2, 5, 24, 26, 37, 40, and 43, respectively, or the mature
polypeptide thereof. Preferably, amino acid changes are 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 one to about 30 amino acids;
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue; a small linker peptide of up to
about 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.
[0155] Examples of conservative substitutions are within the group
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 which
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. The most commonly occurring
exchanges 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.
[0156] In addition to the 20 standard amino acids, non-standard
amino acids (such as 4-hydroxyproline, 6-N-methyl lysine,
2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be
substituted for amino acid residues of a wild-type polypeptide. A
limited number of non-conservative amino acids, amino acids that
are not encoded by the genetic code, and unnatural amino acids may
be substituted for amino acid residues. "Unnatural amino acids"
have been modified after protein synthesis, and/or have a chemical
structure in their side chain(s) different from that of the
standard amino acids. Unnatural amino acids can be chemically
synthesized, and preferably, are commercially available, and
include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and
3,3-dimethylproline.
[0157] 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.
[0158] Essential amino acids in the parent polypeptides 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
biological activity (i.e., glucoamylase 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 enzymes 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.
309:59-64. The identities of essential amino acids can also be
inferred from analysis of identities with polypeptides which are
related to a polypeptide according to the invention.
[0159] Single or multiple amino acid substitutions 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, Biochem. 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).
[0160] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells.
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
of interest, and can be applied to polypeptides of unknown
structure.
[0161] The total number of amino acid substitutions, deletions
and/or insertions of amino acids in position 1 to 556 of SEQ ID NO:
2 or position 1 to 561 of SEQ ID NO: 37 (Trametes glucoamylase); or
in position 1 to 575 in SEQ ID NO: 5 or position 1 to 565 in SEQ ID
NO: 40 (Pachykytospora glucoamylase) or position 1 to 556 of SEQ ID
NO: 26 or position 1 to 548 of SEQ ID NO: 24 or position 1 to 523
of SEQ ID NO: 43 (Leucopaxilus glucoamylase), respectively, is 10,
preferably 9, more preferably 8, more preferably 7, more preferably
at most 6, more preferably at most 5, more preferably 4, even more
preferably 3, most preferably 2, and even most preferably 1.
Sources of Polypeptides Having Glucoamylase Activity
[0162] A polypeptide of the present invention 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 polypeptide encoded by a nucleotide
sequence is produced by the source or by a strain in which the
nucleotide sequence from the source has been inserted. In a
preferred aspect, the polypeptide obtained from a given source is
secreted extracellularly.
[0163] In a preferred embodiment, the glucoamylase of the invention
derived from the class Basidiomycetes. In a more preferred
embodiment a glucoamylase of the invention is derived from a strain
of the genus Trametes, more preferably from a strain of the species
Trametes cingulata, or deposited clone DSM 17106, or a strain of
the genus Pachykytospora more preferably a strain of the species
Pachykytospora papyracea, or the deposited clone DSM 17105, or a
strain of the genus Leucopaxililus, more preferably a strain of the
species Leucopaxillus giganteus.
[0164] 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.
[0165] The Trametes cingulata strain was collected in Zimbabwe in
the period from 1995 to 1997.
[0166] The Pachykytospora papyracea strain was collected in
Zimbabwe in the period from 1995 to 1997.
[0167] The Leucopaxillus giganteus strain was collected in Denmark
in 2003.
[0168] Furthermore, such polypeptides may be identified and
obtained from other sources including microorganisms isolated from
nature (e.g., soil, composts, water, etc.) using the
above-mentioned probes. Techniques for isolating microorganisms
from natural habitats are well known in the art. The polynucleotide
may then be obtained by similarly screening a genomic or cDNA
library of another microorganism. Once a polynucleotide sequence
encoding a polypeptide has been detected with the probe(s), the
polynucleotide can be isolated or cloned by utilizing techniques
which are well known to those of ordinary skill in the art (see,
e.g., Sambrook et al., 1989, supra).
[0169] Polypeptides of the present invention also include fused
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A fused polypeptide is produced by
fusing a nucleotide sequence (or a portion thereof) encoding
another polypeptide to a nucleotide sequence (or a portion thereof)
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 fused polypeptide is under control of the
same promoter(s) and terminator.
Polynucleotides
[0170] The present invention also relates to isolated
polynucleotides having a nucleotide sequence which encode a
polypeptide of the present invention. In a preferred aspect, the
nucleotide sequence is set forth in any of SEQ ID NO: 1, 3, 4, 6,
23, 25, 36, 38, 39, 41, or 42, respectively. In another more
preferred aspect, the nucleotide sequence is the sequence contained
in plasmid pHuda595 or pHuda594 that is contained in Escherichia
coli DSM 17106 and Escherichia coli DSM 17105, respectively. In
another preferred aspect, the nucleotide sequence is the mature
polypeptide coding region of any of SEQ ID NO: 1, 3, 4, 6, 23, 25,
36, 38, 39, 41, or 42, respectively. The present invention also
encompasses nucleotide sequences which encode a polypeptide having
the amino acid sequence of any of SEQ ID NO: 2, 5, 24, 26, 37, 40,
or 43, respectively, or the mature polypeptide thereof, which
differs from SEQ ID NO: 1, 3, 4, 6, 23, 25, 36, 38, 39, 41, or 42
respectively, by virtue of the degeneracy of the genetic code. The
present invention also relates to subsequences of any of SEQ ID NO:
1, 3, 4, 6, 23, 26, 36, 38, 39, 41, or 42, respectively, which
encode fragments of SEQ ID NO: 2, 5, 24, 26, 37, 39, 40, or 43
respectively, that have glucoamylase activity.
[0171] The present invention also relates to mutant polynucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of any of SEQ ID NO: 1, 3, 4, 6, 23, 25, 36, 38, 39, 41,
or 42, respectively, in which the mutant nucleotide sequence
encodes a polypeptide which consists of amino acids 1 to 556 of SEQ
ID NO: 2, amino acids 1 to 575 of SEQ ID NO: 5, amino acids 1 to
548 of SEQ ID NO: 24, amino acid 1 to 556 of SEQ ID NO: 26, amino
acids 1 to 561 of SEQ ID NO: 37, amino acids 1 to 565 of SEQ ID NO:
40, or amino acids 1 to 523 of SEQ ID NO: 43, respectively.
[0172] The techniques used to isolate or clone a polynucleotide
encoding a polypeptide are known in the art and include isolation
from genomic DNA, preparation from cDNA, or a combination thereof.
The cloning of the polynucleotides of the present invention from
such genomic DNA can be effected, e.g., by using the well known
polymerase chain reaction (PCR) or antibody screening of expression
libraries to detect cloned DNA fragments with shared structural
features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods
and Application, Academic Press, New York. Other nucleic acid
amplification procedures such as ligase chain reaction (LCR),
ligated activated transcription (LAT) and nucleotide sequence-based
amplification (NASBA) may be used. The polynucleotides may be
cloned from a strain of the genera Trametes, Pachykytospora,
Leucopaxillus or other or related organisms and thus, for example,
may be an allelic or species variant of the polypeptide encoding
region of the nucleotide sequences.
[0173] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 1 (i.e., nucleotides 55
to 2166), or SEQ ID NO: 3 (i.e., nucleotides 55 to 1725), or SEQ ID
NO: 4 (i.e., nucleotides 55 to 2182), or SEQ ID NO: 6 (i.e.,
nucleotides 55 to 1725), or SEQ ID NO: 25 (i.e., nucleotides 52 to
1719), or SEQ ID NO: 38 (i.e., nucleotide 55 to 1737), or SEQ ID
NO: 41 (i.e., nucleotide 55 to 1749), or SEQ ID NO: 42 (i.e.,
nucleotide 55 to 1620), respectively, of at least 60%, preferably
at least 65%, more preferably at least 70%, more preferably at
least 75%, more preferably at least 80%, more preferably at least
85%, more preferably at least 90%, even more preferably at least
95%, even more prefer ably 96%, even more 97%, even more 98%, and
most preferably at least 99% identity, which encode an active
polypeptide.
[0174] Modification of a nucleotide sequence encoding a polypeptide
of the present invention may be necessary for the synthesis of
polypeptides substantially similar to the polypeptide. The term
"substantially similar" to the polypeptide refers to non-naturally
occurring forms of the polypeptide. These polypeptides may differ
in some engineered way from the polypeptide isolated from its
native source, e.g., artificial variants that differ in specific
activity, thermostability, pH optimum, or the like. The variant
sequence may be constructed on the basis of the nucleotide sequence
presented as the mature polypeptide encoding region of any of SEQ
ID NO: 1, 3, 4, 6, 23, 25, 36, 38, 39, 41, or 42, respectively,
e.g., subsequences thereof, and/or by introduction of nucleotide
substitutions, which do not give rise to another amino acid
sequence of the polypeptide encoded by the nucleotide sequence, but
which correspond to the codon usage of the host organism intended
for production of the enzyme, or by introduction of nucleotide
substitutions which may give rise to a different amino acid
sequence. For a general description of nucleotide substitution,
see, e.g., Ford et al., 1991, Protein Expression and Purification
2: 95-107.
[0175] It will be apparent to those skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acid residues essential to the activity of the polypeptide
encoded by an isolated polynucleotide of the invention, and
therefore preferably not subject to substitution, may be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham
and Wells, 1989, Science 244: 1081-1085). In the latter technique,
mutations are introduced at every positively charged residue in the
molecule, and the resultant mutant molecules are tested for
glucoamylase activity to identify amino acid residues that are
critical to the activity of the molecule. Sites of substrate-enzyme
interaction can also be determined by analysis of the
three-dimensional structure as determined by such techniques as
nuclear magnetic resonance analysis, crystallography or
photoaffinity labelling (see, e.g., de Vos et al., 1992, Science
255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224:
899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
[0176] The present invention also relates to isolated
polynucleotides encoding a polypeptide of the present invention,
(i) which hybridize under low stringency conditions, more
preferably medium stringency conditions, more preferably
medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with nucleotides 55 to 2166 of SEQ ID NO: 1 or
nucleotides 55 to 2166 of SEQ ID NO: 36, respectively, or (ii)
which hybridize under medium stringency conditions, more preferably
medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with nucleotides the cDNA sequence contained in
nucleotides 55 to 1725 of SEQ ID NO: 3 or nucleotides 55 to 1737 of
SEQ ID NO: 38, respectively, or (iii) a complementary strand of (i)
or (ii); or allelic variants and subsequences thereof (Sambrook et
al., 1989, supra), as defined herein.
[0177] The present invention also relates to isolated
polynucleotides encoding a polypeptide of the present invention,
(i) which hybridize under low stringency conditions, more
preferably medium stringency conditions, more preferably
medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with nucleotides 55 to 2189 of SEQ ID NO: 4 or
nucleotides 55 to 2182 of SEQ ID NO: 39, respectively, or (ii)
which hybridize under medium stringency conditions, more preferably
medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with nucleotides the cDNA sequence contained in
nucleotides 55 to 1725 of SEQ ID NO: 6 or nucleotides 55 to 1749 of
SEQ ID NO: 41, or (iii) a complementary strand of (i) or (ii); or
allelic variants and subsequences thereof (Sambrook et al., 1989,
supra), as defined herein.
[0178] The present invention also relates to isolated
polynucleotides encoding a polypeptide of the present invention,
(i) which hybridize under low stringency conditions, more
preferably medium stringency conditions, more preferably
medium-high stringency conditions, even more preferably high
stringency conditions, and most preferably very high stringency
conditions with nucleotides 117 to 2249 of SEQ ID NO: 23, or (ii)
which hybridize under low stringency conditions, preferably medium
stringency conditions, more preferably medium-high stringency
conditions, even more preferably high stringency conditions, and
most preferably very high stringency conditions with nucleotides
the cDNA sequence contained in nucleotides 52 to 1719 of SEQ ID NO:
25 or nucleotides 52 to 1620 of SEQ ID NO: 42, respectively, or
(iii) a complementary strand of (i) or (ii); or allelic variants
and subsequences thereof (Sambrook et al., 1989, supra), as defined
herein.
[0179] The present invention also relates to isolated
polynucleotides obtained by (a) hybridizing a population of DNA
under low, medium, medium-high, high, or very high stringency
conditions with (i) nucleotides 55 to 2166 of SEQ ID NO: 1 or
nucleotides 55 to 2166 of SEQ ID NO: 36, respectively, or (ii)
hybridizing a population of DNA under medium, medium-high, high, or
very high stringency conditions with the cDNA sequence contained in
nucleotides 55 to 1725 of SEQ ID NO: 3 or nucleotides 55 to 1737 of
SEQ ID NO: 38, respectively, or (iii) a complementary strand of (i)
or (ii); and (b) isolating the hybridizing polynucleotide, which
encodes a polypeptide having glucoamylase activity.
[0180] The present invention also relates to isolated
polynucleotides obtained by (a) hybridizing a population of DNA
under low, medium, medium-high, high, or very high stringency
conditions with (i) nucleotides 55 to 2189 of SEQ ID NO: 4 or
nucleotides 55 to 2182 of SEQ ID NO: 39, respectively, or (ii)
hybridizing a population of DNA under medium, medium-high, high, or
very high stringency conditions with the cDNA sequence contained in
nucleotides 55 to 1725 of SEQ ID NO: 6 or nucleotides 55 to 1749 of
SEQ ID NO: 41, respectively, or (iii) a complementary strand of (i)
or (ii); and (b) isolating the hybridizing polynucleotide, which
encodes a polypeptide having glucoamylase activity.
[0181] The present invention also relates to isolated
polynucleotides obtained by (a) hybridizing a population of DNA
under low, medium, medium-high, high, or very high stringency
conditions with (i) nucleotides 117 to 2249 of SEQ ID NO: 23, or
(ii) hybridizing a population of DNA under medium, medium-high,
high, or very high stringency conditions with the cDNA sequence
contained in nucleotides 52 to 1719 of SEQ ID NO: 25 or nucleotides
52 to 1620 of SEQ ID NO: 42, respectively, or (iii) a complementary
strand of (i) or (ii); and (b) isolating the hybridizing
polynucleotide, which encodes a polypeptide having glucoamylase
activity.
Nucleic Acid Constructs
[0182] The present invention also relates to nucleic acid
constructs comprising an isolated polynucleotide of the present
invention operably linked to one or more control sequences which
direct the expression of the coding sequence in a suitable host
cell under conditions compatible with the control sequences.
[0183] An isolated polynucleotide encoding a polypeptide of the
present invention may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
polynucleotide's sequence prior to its insertion into a vector may
be desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotide sequences utilizing
recombinant DNA methods are well known in the art.
[0184] The control sequence may be an appropriate promoter
sequence, a nucleotide sequence which is recognized by a host cell
for expression of a polynucleotide encoding a polypeptide of the
present invention. The promoter sequence contains transcriptional
control sequences which mediate the expression of the polypeptide.
The promoter may be any nucleotide sequence which shows
transcriptional activity in the host cell of choice 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.
[0185] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium venenatum glucoamylase (WO 00/56900),
Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
(WO 00/56900), Fusarium oxysporum trypsin-like protease (WO
96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei endoglucanase I,
Trichoderma reesei endoglucanase II, Trichoderma reesei
endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma
reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma
reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as
the NA2-tpi promoter (a hybrid of the promoters from the genes for
Aspergillus niger neutral alpha-amylase and Aspergillus oryzae
triose phosphate isomerase); and mutant, truncated, and hybrid
promoters thereof.
[0186] 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 metallothionine (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.
[0187] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any terminator which is functional in the host cell of
choice may be used in the present invention.
[0188] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0189] 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.
[0190] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA which is important for
translation by the host cell. The leader sequence is operably
linked to the 5' terminus of the nucleotide sequence encoding the
polypeptide. Any leader sequence that is functional in the host
cell of choice may be used in the present invention.
[0191] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0192] 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).
[0193] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleotide
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence which is functional in the host cell
of choice may be used in the present invention.
[0194] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0195] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0196] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleotide sequence may inherently contain a signal peptide
coding region naturally linked in translation reading frame with
the segment of the coding region which encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region which is foreign to the
coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a
signal peptide coding region. Alternatively, the foreign signal
peptide coding region may simply replace the natural signal peptide
coding region in order to enhance secretion of the polypeptide.
However, any signal peptide coding region which directs the
expressed polypeptide into the secretory pathway of a host cell of
choice may be used in the present invention.
[0197] Effective signal peptide coding regions for filamentous
fungal host cells are the signal peptide coding regions obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, and
Humicola lanuginosa lipase.
[0198] 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 regions are described by Romanos et al., 1992, supra.
[0199] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a polypeptide. 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 a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Bacillus subtilis
alkaline protease (aprE), Bacillus subtilis neutral protease
(nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei
aspartic proteinase, and Myceliophthora thermophila laccase (WO
95/33836).
[0200] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0201] It may also be desirable to add regulatory sequences which
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those which cause the 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. In yeast, the ADH2 system or
GAL1 system may be used. In filamentous fungi, the TAKA
alpha-amylase promoter, Aspergillus niger glucoamylase promoter,
and Aspergillus oryzae glucoamylase promoter may be used as
regulatory sequences. Other examples of regulatory sequences are
those which allow for gene amplification. In eukaryotic systems,
these include the dihydrofolate reductase gene which is amplified
in the presence of methotrexate, and the metallothionein genes
which are amplified with heavy metals. In these cases, the
nucleotide sequence encoding the polypeptide would be operably
linked with the regulatory sequence.
Expression Vectors
[0202] The present invention also relates to recombinant expression
vectors comprising a polynucleotide of the present invention, a
promoter, and transcriptional and translational stop signals. The
various nucleic acids and control sequences described above may be
joined together to produce a recombinant expression vector which
may include one or more convenient restriction sites to allow for
insertion or substitution of the nucleotide sequence encoding the
polypeptide at such sites. Alternatively, a nucleotide sequence of
the present invention may be expressed by inserting the nucleotide
sequence or a nucleic acid construct comprising the sequence 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.
[0203] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to
recombinant DNA procedures and can bring about expression of the
nucleotide sequence. 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 vectors may be linear or closed
circular plasmids.
[0204] The vector may be an autonomously replicating vector, i.e.,
a vector which 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
which, 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 which together contain the total DNA to
be introduced into the genome of the host cell, or a transposon may
be used.
[0205] The vectors of the present invention preferably contain one
or more selectable markers which permit easy selection of
transformed 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.
[0206] Examples of suitable markers for yeast host cells are ADE2,
HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use
in a filamentous fungal host cell include, but are not limited to,
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
the amdS and pyrG genes of Aspergillus nidulans or Aspergillus
oryzae and the bar gene of Streptomyces hygroscopicus.
[0207] The vectors of the present invention preferably contain 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.
[0208] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the polypeptide or
any other element of the vector for integration into the genome by
homologous or non-homologous recombination. Alternatively, the
vector may contain additional nucleotide sequences 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 preferably contain a sufficient number of nucleic
acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000
base pairs, and most preferably 800 to 10,000 base pairs, which
have a high degree of identity with 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 nucleotide sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0209] 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 which functions in a cell. The term "origin of
replication" or "plasmid replicator" is defined herein as a
nucleotide sequence that enables a plasmid or vector to replicate
in vivo.
[0210] 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.
[0211] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67;
Cullen et al., 1987, Nucleic Acids Research 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.
[0212] More than one copy of a polynucleotide of the present
invention may be inserted into the host cell to increase production
of the gene product. 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.
[0213] 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
[0214] The present invention also relates to recombinant host
cells, comprising a polynucleotide of the present invention, which
are advantageously used in the recombinant production of the
polypeptides. A vector comprising a polynucleotide of the present
invention is introduced into a host cell so that the 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 polypeptide and its source.
[0215] The host cell may be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0216] In a preferred aspect, the host cell is a fungal cell.
"Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et
al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et
al., 1995, supra).
[0217] In a more preferred aspect, the fungal host cell is 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, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0218] In an even more preferred aspect, the yeast host cell is a
Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell.
[0219] In a most preferred aspect, the yeast host cell is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
In another most preferred aspect, the yeast host cell is a
Kluyveromyces lactis cell. In another most preferred aspect, the
yeast host cell is a Yarrowia lipolytica cell.
[0220] In another more preferred aspect, the fungal host cell is 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.
[0221] In an even more preferred aspect, the filamentous fungal
host cell is an Acremonium, Aspergillus, Aureobasidium,
Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus,
Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
Trametes, or Trichoderma cell.
[0222] In a most preferred aspect, the filamentous fungal host cell
is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus
niger or Aspergillus oryzae cell. In another most preferred aspect,
the filamentous fungal host cell is a 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, or Fusarium venenatum cell. In another
most preferred aspect, the filamentous fungal host cell is a
Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceeiporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or
Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,
Humicola insolens, Humicola lanuginosa, Mucor miehei,
Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichodermna harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride strain cell.
[0223] 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 238 023 and Yelton et al., 1984,
Proceedings of the National Academy of Sciences USA 81: 1470-1474.
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,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 1920.
Methods of Production
[0224] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising (a) cultivating
a cell, which in its wild-type form is capable of producing the
polypeptide, under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide. Preferably, the
cell is of the genus Trametes, Pachykytospora, or Leucopaxillus,
and more preferably Trametes cingulata, Pachykytospora papyracea,
or Leucopaxillus giganteus.
[0225] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising (a) cultivating
a host cell under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0226] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising (a) cultivating
a host cell under conditions conducive for production of the
polypeptide, wherein the host cell comprises a nucleotide sequence
having the mature polypeptide coding region of SEQ ID NO: 1, 3, 4,
6, 23, 25, 36, 38, 39, 41, or 42, respectively, wherein the
nucleotide sequence encodes a polypeptide which consists of amino
acids 1 to 556 of SEQ ID NO: 2 or amino acids 1 to 561 of SEQ ID
NO: 37, respectively; or amino acids 1 to 575 of SEQ ID NO: 5 or
amino acids 1 to 565 of SEQ ID NO: 40, respectively; or amino acids
1 to 556 of SEQ ID NO: 26 or amino acids 1 to 548 of SEQ ID NO: 24
or amino acids 1 to 523 of SEQ ID NO: 43, respectively, and (b)
recovering the polypeptide.
[0227] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the polypeptide using methods well known in the art. For
example, the cell may be cultivated by shake flask cultivation, and
small-scale 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 polypeptide 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 polypeptide is secreted into the nutrient
medium, the polypeptide can be recovered directly from the medium.
If the polypeptide is not secreted, it can be recovered from cell
lysates.
[0228] The polypeptides may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include 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
polypeptide as described herein.
[0229] The resulting polypeptide may be recovered using methods
known in the art. For example, the polypeptide may be recovered
from the nutrient medium by conventional procedures including, but
not limited to, centrifugation, filtration, extraction,
spray-drying, evaporation, or precipitation.
[0230] The polypeptides of the present invention 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, J.-C. Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989).
Plants
[0231] The present invention also relates to a transgenic plant,
plant part, or plant cell which has been transformed with a
nucleotide sequence encoding a polypeptide having glucoamylase
activity of the present invention so as to express and produce the
polypeptide in recoverable quantities. The polypeptide may be
recovered from the plant or plant part. Alternatively, the plant or
plant part containing the recombinant polypeptide 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.
[0232] 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).
[0233] 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.
[0234] 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 utilisation of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seeds coats.
[0235] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0236] The transgenic plant or plant cell expressing a polypeptide
of the present invention 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 polypeptide of the present invention into the plant host
genome and propagating the resulting modified plant or plant cell
into a transgenic plant or plant cell.
[0237] The expression construct is conveniently a nucleic acid
construct which comprises a polynucleotide encoding a polypeptide
of the present invention operably linked with appropriate
regulatory sequences required for expression of the nucleotide
sequence in the plant or plant part of choice. Furthermore, the
expression construct may comprise a selectable marker useful for
identifying host 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).
[0238] 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
polypeptide is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide of the present
invention 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.
[0239] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, and the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294, Christensen et al., 1992, Plant Mo.
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 & 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 at
al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba
promoter from the legumin B4 and the unknown seed protein gene from
Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152:
708-711), a promoter from a seed oil body protein (Chen et al.,
1998, Plant and Cell Physiology 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
Physiology 102: 991-1000, the chlorella virus adenine
methyltransferase gene promoter (Mitra and Higgins, 1994, Plant
Molecular Biology 26: 85-93), or the aldP gene promoter from rice
(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674),
or a wound inducible promoter such as the potato pin2 promoter (Xu
et al., 1993, Plant Molecular Biology 22: 573-588). Likewise, the
promoter may inducible 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.
[0240] A promoter enhancer element may also be used to achieve
higher expression of a polypeptide of the present invention in the
plant. For instance, the promoter enhancer element may be an intron
which is placed between the promoter and the nucleotide sequence
encoding a polypeptide of the present invention. For instance, Xu
et al., 1993, supra, disclose the use of the first intron of the
rice actin 1 gene to enhance expression.
[0241] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0242] 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).
[0243] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology
19: 1538) and can also be used for transforming monocots, although
other transformation methods are often used for these plants.
Presently, the method of choice 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 Journal 2: 275-281; Shimamoto, 1994,
Current Opinion Biotechnology 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 Molecular Biology 21:
415-428.
[0244] 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.
[0245] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding a polypeptide having glucoamylase activity of the present
invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
Compositions
[0246] The present invention also relates to compositions
comprising a polypeptide of the present invention. Preferably, the
compositions are enriched in such a polypeptide. The term
"enriched" indicates that the glucoamylase activity of the
composition has been increased, e.g., by an enrichment factor of
1.1.
[0247] The composition may comprise a polypeptide of the present
invention as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the composition may comprise multiple
enzymatic activities, such as an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,
cutinase, cycliodextrin glycosyltransferase, deoxyribonuclease,
esterase, alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase,
laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,
peptidoglutaminase, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The additional enzyme(s) may be produced, for example, by a
microorganism belonging to the genus Aspergillus, preferably
Aspergillus aculeatus, Aspertgillus awamori, Aspergillus fumigatus,
Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, or Aspergillus oryzae; Fusarium, preferably
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 sulphureum, Fusarium toruiloseum,
Fusarium trichothecioides, or Fusarium venenatum; Humicola,
preferably Humicola insolens or Humicola lanuginosa; or
Trichoderma, preferably Trichoderma harzianum, Trichoderma
koningli, Trichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma viride.
[0248] The polypeptide 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. For instance, the polypeptide composition may be
in the form of a granulate or a microgranulate. The polypeptide to
be included in the composition may be stabilized in accordance with
methods known in the art.
Combination of Glucoamylase and Acid Alpha-Amylase
[0249] According to this aspect of the invention a glucoamylase of
the invention may be combined with an acid alpha-amylase in a ratio
of between 0.3 and 5.0 AFAU/AGU. More preferably the ratio between
acid alpha-amylase activity and glucoamylase activity is at least
0.35, at least 0.40, at least 0.50, at least 0.60, at least 0.7, at
least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2,
at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least
1.7, at least 1.8, at least 1.85, or even at least 1.9 AFAU/AGU.
However, the ratio between acid alpha-amylase activity and
glucoamylase activity should preferably be less than 4.5, less than
4.0, less than 3.5, less than 3.0, less than 2.5, or even less than
2.25 AFAU/AGU. In AUU/AGI the activities of acid alpha-amylase and
glucoamylase are preferably present in a ratio of between 0.4 and
6.5 AUU/AGI. More preferably the ratio between acid alpha-amylase
activity and glucoamylase activity is at least 0.45, at least 0.50,
at least 0.60, at least 0.7, at least 0.8, at least 0.9, at least
1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at
least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9,
at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least
2.4, or even at least 2.5 AUU/AGI. However, the ratio between acid
alpha-amylase activity and glucoamylase activity is preferably less
than 6.0, less than 5.5, less than 4.5, less than 4.0, less than
3.5, or even less than 3.0 AUU/AGI.
[0250] Above composition is suitable for use in a starch conversion
process mentioned below for producing syrup and fermentation
products such as ethanol.
[0251] Examples are given below of preferred uses of the
polypeptide compositions of the invention. The dosage of the
polypeptide composition of the invention and other conditions under
which the composition is used may be determined on the basis of
methods known in the art.
[0252] Combination of Trametes cingulata Glucoamylase with Another
Glucoamylase and an Acid Alpha-Amylase
[0253] The Trametes cingulata glucoamylase of the invention have
been found to have a 4-7 fold higher alpha-1,6-debranching activity
than other glucoamylases, such as Athelia rolfsii, Aspergillus
niger and Talaromyces emersonii (see Example 13).
[0254] Therefore, according to the invention the Trametes cingulata
glucoamylase may be combined with acid alpha-amylase and further
another glucoamylase. Such combination of enzymes would be suitable
in processes comprises starch conversion, include ethanol
production, including one step fermentation processes.
[0255] The alpha-amylase may be any alpha-amylase. In a preferred
embodiment the alpha-amylase is any of those listed in the
"Alpha-Amylase"-section below. In a preferred embodiment the
alpha-amylase is a fungal alpha-amylase, especially those disclosed
below in the "Fungal Alpha-Amylases"-section, especially the
Aspergillus kawachii alpha-amylase. Preferred are also hybrid
alpha-amylases disclosed below in the "Fungal hybrid
alpha-amylase"-section below, including hybrids disclosed in U.S.
Patent Publication no. 2005/0054071 (hybrids listed in Table 3 is
especially contemplated), and further the hybrids disclosed in
co-pending U.S. application No. 60/638,614, including especially
the Fungamyl variant with catalytic domain JA118 and Athelia
rolfsii SBD (SEQ ID NO: 28 herein and SEQ ID NO: 100 in U.S.
60/638,614); Rhizomucor pusillus alpha-amylase with Athelia rolfsii
AMG linker and SBD (SEQ ID NO: 29 herein and SEQ ID NO: 101 in U.S.
application No. 60/638,614); and Meripilus giganteus alpha-amylase
with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 30
herein and SEQ ID NO: 102 in U.S. application No. 60/638,614).
[0256] The glucoamylase may be any glucoamylase, including
glucoamylases of fungal or bacterial origin selected from the group
consisting of Aspergillus glucoamylases, in particular A. niger G1
or G2 glucoamylase (Boel et al., 1984, EMBO J. 3 (5): 1097-1102),
or variants thereof, such as disclosed in WO 92/00381, WO 00/04136
add WO 01/04273 (from Novozymes, Denmark); the A. awamori
glucoamylase (WO 84/02921), A. oryzae (Agric. Biol. Chem., 1991, 55
(4): 941-949), or variants or fragments thereof. Other Aspergillus
glucoamylase variants include variants to enhance the thermal
stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9:
499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Engng. 8:
575-582); N182 (Chen et al., 1994, Biochem. J. 301: 275-281);
disulphide bonds, A246C (Fierobe et al., 1996, Biochemistry, 35:
8698-8704; and introduction of Pro residues in position A435 and
S436 (Li et al., 1997, Protein Engng. 10: 1199-1204. Other
glucoamylases include Corticium rolfsii glucoamylase (U.S. Pat. No.
4,727,046) also referred to as Athelia rolfsii, Talaromyces
glucoamylases, in particular, derived from Talaromyces emersonii
(WO 99/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32,153),
Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No.
4,587,215), Rhizopus nivius (e.g., the enzyme available from Shin
Nihon Chemicals, Japan, under the tradename "CU CONC"), Humicola
grisea var. thermoidea (e.g., ATCC 16453, NRRL 15222, NRRL 15223,
NRRL 15224, NRRL 15225).
[0257] Bacterial glucoamylases contemplated include glucoamylases
from the genus Clostridium, in particular C. thermoamylolyticum (EP
135,138), and C. thermohydrosulfuricum (WO 86/01831).
[0258] Examples of commercially available compositions comprising
other glucoamylase include AMG 200L; AMG 300 L; SAN.TM. SUPER,
SAN.TM. EXTRA L, SPIRIZYME.TM. PLUS, SPIRIZYME.TM. FUEL,
SPIRIZYME.TM. B4U and AMG.TM. E (from Novozymes A/S); OPTIDEX.TM.
300 (from Genencor Int.); AMIGASE.TM. and AMIGASE.TM. PLUS (from
DSM); G-ZYME.TM. G900, G-ZYME.TM. and G990 ZR (from Genencor
Int.).
[0259] In a specific embodiment the Trametes cingulata glucoamylase
of the invention is combined with glucoamylase derived from one of
Aspergillus niger, Athea rolfsii, or Talaromyces emersonii and the
Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker
and SBD (SEQ ID NO: 29 herein and SEQ ID NO: 101 in U.S.
application No. 60/638,614).
Uses
[0260] The present invention is also directed to process/methods
for using the polypeptides having glucoamylase activity of the
invention.
[0261] Uses according to the invention include starch conversion of
starch to e.g., syrup and fermentation products, including ethanol
and beverages. Examples of processes where a glucoamylase of the
invention may be used include the ones described in: WO
2004/081193, WO 2004/080923, WO 2003/66816, WO 2003166826, and WO
92/20777 which are hereby all incorporated by reference.
Production of Fermentation Products
Processes for Producing Fermentation Products from Gelatinized
Starch-Containing Material
[0262] In this aspect the present invention relates to a process
for producing a fermentation product, especially ethanol, from
starch-containing material, which process includes a liquefaction
step and separately or simultaneously performed saccharification
and fermentation step(s).
[0263] The invention relates to a process for producing a
fermentation product from starch-containing material comprising the
steps of:
[0264] (a) liquefying starch-containing material in the presence of
an alpha-amylase;
[0265] (b) saccharifying the liquefied material obtained in step
(a) using a glucoamylase of the invention;
[0266] (c) fermenting the saccharified material using a fermenting
organism.
[0267] The fermentation product, such as especially ethanol, may
optionally be recovered after fermentation, e.g., by distillation.
Suitable starch-containing starting materials are listed in the
section "Starch-containing materials"-section below. Contemplated
enzymes are listed in the "Enzymes"-section below. The fermentation
is preferably carried out in the presence of yeast, preferably a
strain of Saccharomyces. Suitable fermenting organisms are listed
in the "Fermenting Organisms"-section below. In a preferred
embodiment step (b) and (c) are carried out simultaneously (SSF
process).
[0268] In a particular embodiment, the process of the invention
further comprises, prior to the step (a), the steps of:
[0269] x) reducing the particle size of the starch-containing
material, preferably by milling;
[0270] y) forming a slurry comprising the starch-containing
material and water.
[0271] The aqueous slurry may contain from 10-40 wt-%, preferably
25-35 wt-% starch-containing material. The slurry is heated to
above the gelatinization temperature and alpha-amylase, preferably
bacterial and/or acid fungal alpha-amylase, may be added to
initiate liquefaction (thinning). The slurry may in an embodiment
be jet-cooked to further gelatinize the slurry before being
subjected to an alpha-amylase in step (a) of the invention.
[0272] More specifically liquefaction may be carried out as a
three-step hot slurry process. The slurry is heated to between
60-95.degree. C., preferably 80-85.degree. C., and alpha-amylase is
added to initiate liquefaction (thinning). Then the slurry may be
jet-cooked at a temperature between 95-140.degree. C., preferably
105-125.degree. C., for 1-15 minutes, preferably for 3-10 minutes,
especially around 5 minutes. The slurry is cooled to 60-95.degree.
C. and more alpha-amylase is added to finalize hydrolysis
(secondary liquefaction). The liquefaction process is usually
carried out at pH 4.5-6.5, in particular at a pH between 5 and 6.
Milled and liquefied whole grains are known as mash.
[0273] The saccharification in step (b) may be carried out using
conditions well know in the art. For instance, a full
saccharification process may lasts up to from about 24 to about 72
hours, however, it is common only to do a pre-saccharification of
typically 40-90 minutes at a temperature between 30-65.degree. C.,
typically about 60.degree. C., followed by complete
saccharification during fermentation in a simultaneous
saccharification and fermentation process (SSF).
[0274] Saccharification is typically carried out at temperatures
from 30-65.degree. C., typically around 60.degree. C., and at a pH
between 4 and 5, normally at about pH 4.5.
[0275] The most widely used process in ethanol production is the
simultaneous saccharification and fermentation (SSF) process, in
which there is no holding stage for the saccharification, meaning
that fermenting organism, such as yeast, and enzyme(s) may be added
together.
[0276] When doing SSF it is common to introduce a
pre-saccharification step at a temperature above 50.degree. C.,
just prior to the fermentation.
[0277] In accordance with the present invention the fermentation
step (c) includes, without limitation, fermentation processes used
to produce alcohols (e.g., ethanol, methanol, butanol); organic
acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid,
gluconic acid); ketones (e.g., acetone); amino acids (e.g.,
glutamic acid); gases (e.g., H.sub.2 and CO.sub.2); antibiotics
(e.g., penicillin and tetracycline); enzymes; vitamins (e.g.,
riboflavin, B12, beta-carotene); and hormones. Preferred
fermentation processes include alcohol fermentation processes, as
are well known in the art. Preferred fermentation processes are
anaerobic fermentation processes, as are well known in the art.
Processes for Producing Fermentation Products from Un-Gelatinized
Starch-Containing
[0278] In this aspect the invention relates to processes for
producing a fermentation product from starch-containing material
without gelatinization of the starch-containing material. In one
embodiment only a glucoamylase of the invention is used during
saccharification and fermentation. According to the invention the
desired fermentation product, such as ethanol, can be produced
without liquefying the aqueous slurry containing the
starch-containing material. In one embodiment a process of the
invention includes saccharifying milled starch-containing material
below the gelatinization temperature in the presence of a
glucoamylase of the invention to produce sugars that can be
fermented into the desired fermentation product by a suitable
fermenting organism.
[0279] Examples 8 and 9 below disclose production of ethanol from
un-gelatinized (uncooked) milled corn using glucoamylases of the
invention derived from Trametes cingulata and Pachykytospora
papyracea. Both glucoamylases show significantly higher ethanol
yields compared to corresponding processes carried out using
glucoamylases derived from Aspergillus niger or Talaromyces
emersonii, respectively.
[0280] Accordingly, in this aspect the invention relates to a
process for producing a fermentation product from starch-containing
material comprising:
[0281] (a) saccharifying starch-containing material with a
glucoamylase having [0282] i) the sequence shown as amino acids 1
to 556 in SEQ ID NO: 2 or amino acids 1 to 561 in SEQ ID NO: 37, or
a glucoamylase having at least 75% identity thereto, and/or [0283]
ii) the sequence shown as amino acids 1 to 575 in SEQ ID NO: 5 or
amino acids 1 to 565 in SEQ ID NO: 40, or a glucoamylase having at
least 70% identity thereto, and/or [0284] iii) the sequence shown
as amino acids 1 to 548 in SEQ ID NO: 24 or amino acids 1 to 556 in
SEQ ID NO: 26 or amino acids 1 to 523 in SEQ ID NO: 43, or a
glucoamylase having at least 60% identity thereto, at a temperature
below the initial gelatinization temperature of said
starch-containing material,
[0285] (b) fermenting using a fermenting organism.
[0286] Steps (a) and (b) of the process of the invention may be
carried out sequentially or simultaneously.
[0287] The term "initial gelatinization temperature" means the
lowest temperature at which gelatinization of the starch commences.
Starch heated in water begins to gelatinize between 50.degree. C.
and 75.degree. C.; the exact temperature of gelatinization depends
on the specific starch, and can readily be determined by the
skilled artisan. Thus, the initial gelatinization temperature may
vary according to the plant species, to the particular variety of
the plant species as well as with the growth conditions. In the
context of this invention the initial gelatinization temperature of
a given starch-containing material is the temperature at which
birefringence is lost in 5% of the starch granules using the method
described by Gorinstein and Lii, 1992, Starch/Starke 44 (12):
461-466.
[0288] Before step (a) a slurry of starch-containing material, such
as granular starch, having 20-55 wt.-% dry solids, preferably 25-40
wt.-% dry solids, more preferably 30-35% dry solids of
starch-containing material may be prepared. The slurry may include
water and/or process waters, such as stillage (backset), scrubber
water, evaporator condensate or distillate, side stripper water
from distillation, or other fermentation product plant process
water. Because the process of the invention is carried out below
the gelatinization temperature and thus no significant viscosity
increase takes place, high levels of stillage may be used if
desired. In an embodiment the aqueous slurry contains from about 1
to about 70 vol.-% stillage, preferably 15-60% vol.-% stillage,
especially from about 30 to 50 vol.-% stillage.
[0289] The starch-containing material may be prepared by reducing
the particle size, preferably by milling, to 0.05 to 3.0 mm,
preferably 0.1-0.5 mm. After being subjected to a process of the
invention 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 preferably at least 99% of the dry solids of the
starch-containing material is converted into a soluble starch
hydrolysate.
[0290] The process of the invention is conducted at a temperature
below the initial gelatinization temperature. Preferably the
temperature at which step (a) is carried out is between
30-75.degree. C., preferably between 45-60.degree. C.
[0291] In a preferred embodiment step (a) and step (b) are carried
out as a simultaneous saccharification and fermentation process. In
such preferred embodiment the process is typically carried at a
temperature between 28.degree. C. and 36.degree. C., such as
between 29.degree. C. and 35.degree. C., such as between 30.degree.
C. and 34.degree. C., such as around 32.degree. C. According to the
invention the temperature may be adjusted up or down during
fermentation.
[0292] In an embodiment simultaneous saccharification and
fermentation is carried out so that the sugar level, such as
glucose level, is kept at a low level such as below 6 wt.-%,
preferably below about 3 wt.-%, preferably below about 2 wt.-%,
more preferred below about 1 wt.-%, even more preferred below about
0.5%, or even more preferred 0.25% wt.-%, such as below about 0.1
wt.-%. Such low levels of sugar can be accomplished by simply
employing adjusted quantities of enzyme and fermenting organism. A
skilled person in the art can easily determine which quantities of
enzyme and fermenting organism to use. The employed quantities of
enzyme and fermenting organism may also be selected to maintain low
concentrations of maltose in the fermentation broth. For instance,
the maltose level may be kept below about 0.5 wt.-% or below about
0.2 wt.-%.
[0293] The process of the invention may be carried out at a pH in
the range between 3 and 7, preferably from pH 3.5 to 6, or more
preferably from pH 4 to 5.
Starch-Containing Materials
[0294] Any suitable starch-containing starting material, including
granular starch, may be used according to the present invention.
The starting material is generally selected based on the desired
fermentation product. Examples of starch-containing starting
materials, suitable for use in a process of present invention,
include tubers, roots, stems, whole grains, corms, cobs, wheat,
barley, rye, milo, sago, cassaya, tapioca, sorghum, rice peas,
beans, or sweet potatoes, or mixtures thereof, or cereals,
sugar-containing raw materials, such as molasses, fruit materials,
sugar cane or sugar beet, potatoes, and cellulose-containing
materials, such as wood or plant residues, or mixtures thereof.
Contemplated are both waxy and non-waxy types of corn and
barley.
[0295] The term "granular starch" means raw uncooked starch, i.e.,
starch in its natural form found in cereal, tubers or grains.
Starch is formed within plant cells as tiny granules insoluble in
water. When put in cold water, the starch granules may absorb a
small amount of the liquid and swell. At temperatures up to
50.degree. C. to 75.degree. C. the swelling may be reversible.
However, with higher temperatures an irreversible swelling called
"gelatinization" begins. Granular starch to be processed may be a
highly refined starch quality, preferably at least 90%, at least
95%, at least 97% or at least 99.5% pure or it may be a more crude
starch containing material comprising milled whole grain including
non-starch fractions such as germ residues and fibers. The raw
material, such as whole grain, is milled in order to open up the
structure and allowing for further processing. Two milling
processes are preferred according to the invention: wet and dry
milling. In dry milling whole kernels are milled and used. Wet
milling gives a good separation of germ and meal (starch granules
and protein) and is often applied at locations where the starch
hydrolysate is used in production of syrups. Both dry and wet
milling is well known in the art of starch processing and is
equally contemplated for the process of the invention.
[0296] The starch-containing material is reduced in size,
preferably by milling, in order to expose more surface area. In an
embodiment the particle size is between 0.05 to 3.0 mm, preferably
0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more
preferably at least 70%, even more preferably at least 90% of the
milled starch-containing material fit through a sieve with a 0.05
to 3.0 mm screen, preferably 0.1-0.5 mm screen.
Fermentation Products
[0297] The term "fermentation product" means a product produced by
a process including a fermentation step using a fermenting
organism. Fermentation products contemplated according to the
invention include alcohols (e.g., ethanol, methanol, butanol);
organic acids (e.g., citric acid, acetic acid, itaconic acid,
lactic acid, gluconic acid); ketones (e.g., acetone); amino acids
(e.g., glutamic acid); gases (e.g., H.sub.2 and CO.sub.2);
antibiotics (e.g., penicillin and tetracydine); enzymes; vitamins
(e.g., riboflavin, B.sub.12, beta-carotene); and hormones. In a
preferred embodiment the fermentation product is ethanol, e.g.,
fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or
industrial ethanol or products used in the consumable alcohol
industry (e.g., beer and wine), dairy industry (e.g., fermented
dairy products), leather industry and tobacco industry. Preferred
beer types comprise ales, stouts, porters, lagers, bitters, malt
liquors, happoushu, high-alcohol beer, low-alcohol beer,
low-calorie beer or light beer. Preferred fermentation processes
used include alcohol fermentation processes, as are well known in
the art. Preferred fermentation processes are anaerobic
fermentation processes, as are well known in the art.
Fermenting Organisms
[0298] "Fermenting organism" refers to any organism, including
bacterial and fungal organisms, suitable for use in a fermentation
process and capable of producing desired a fermentation product.
Especially suitable fermenting organisms are able to ferment, i.e.,
convert, sugars, such as glucose or maltose, directly or indirectly
into the desired fermentation product, Examples of fermenting
organisms include fungal organisms, such as yeast. Preferred yeast
includes strains of Saccharomyces spp., in particular,
Saccharomyces cerevisiae. Commercially available yeast include,
e.g., Red Star.TM./Lesaffre Ethanol Red (available from Red
Star/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a
division of Burns Philp Food Inc., USA), SUPERSTART (available from
Alitech), GERT STRAND (available from Gert Strand AB, Sweden) and
FERMIOL (available from DSM Specialties).
Enzymes
Glucoamylase
[0299] The glucoamylase is preferably a glucoamylase of the
invention. However, as mentioned above a glucoamylase of the
invention may also be combined with other glucoamylases.
[0300] The glucoamylase may added in an amount of 0.001 to 10 AGU/g
DS, preferably from 0.01 to 5 AGU/g DS, such as around 0.1, 0.3,
0.5, 1 or 2 AGU/g DS, especially 0.1 to 0.5 AGU/g DS or 0.02-20
AGU/g DS, preferably 0.1-10 AGU/g DS.
[0301] Alpha-Amylase
[0302] The alpha-amylase may according to the invention be of any
origin. Preferred are alpha-amylases of fungal or bacterial
origin.
[0303] In a preferred embodiment the alpha-amylase is an acid
alpha-amylase, e.g., fungal acid alpha-amylase or bacterial acid
alpha-amylase. The term "acid alpha-amylase" means an alpha-amylase
(E.C. 3.2.1.1) which added in an effective amount has activity
optimum at a pH in the range of 3 to 7, preferably from 3.5 to 6,
or more preferably from 4-5.
Bacterial Alpha-Amylases
[0304] According to the invention a bacterial alpha-amylase may
preferably be derived from the genus Bacillus.
[0305] In a preferred embodiment the Bacillus alpha-amylase is
derived from a strain of B. licheniformis, B. amyloliquefaciens, B.
subtilis or B. stearothermophilus, but may also be derived from
other Bacillus sp. Specific examples of contemplated alpha-amylases
include the Bacillus licheniformis alpha-amylase (BLA) shown in SEQ
ID NO: 4 in WO 99/19467, the Bacillus amyloliquefaciens
alpha-amylase (BAN) shown in SEQ ID NO: 5 in WO 99/19467, and the
Bacillus stearothermophilus alpha-amylase (BSG) shown in SEQ ID NO:
3 in WO 99/19467. In an embodiment of the invention the
alpha-amylase is an enzyme having a degree of identity of at least
60%, preferably at least 70%, more preferred at least 80%, even
more preferred at least 90%, such as at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% identity to any of the
sequences shown as SEQ ID NOS: 1, 2, 3, 4, or 5, respectively, in
WO 99/19467.
[0306] The Bacillus alpha-amylase may also be a variant and/or
hybrid, especially one described in any of WO 96/23873, WO
96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355
(all documents hereby incorporated by reference). Specifically
contemplated alpha-amylase variants are disclosed in U.S. Pat. Nos.
6,093,562, 6,297,038 or U.S. Pat. No. 6,187,576 (hereby
incorporated by reference) and include Bacillus stearothermophilus
alpha-amylase (BSG alpha-amylase) variants having a deletion of one
or two amino acids in position 179 to 182, preferably a double
deletion disclosed in WO 1996/023873--see e.g., page 20, lines 1-10
(hereby incorporated by reference), preferably corresponding to
delta (181-182) compared to the wild-type BSG alpha-amylase amino
acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or
deletion of amino acids 179 and 180 using SEQ ID NO: 3 in WO
99/19467 for numbering (which reference is hereby incorporated by
reference). Even more preferred are Bacillus alpha-amylases,
especially Bacillus stearothermophilus alpha-amylase, which have a
double deletion corresponding to delta (181-182) and further
comprise a N193F substitution (also denoted I181*+G182*+N193F)
compared to the wild-type BSG alpha-amylase amino acid sequence set
forth in SEQ ID NO: 3 disclosed in WO 99/19467.
[0307] The alpha-amylase may also be a maltogenic alpha-amylase. A
"maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, E.C.
3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose
in the alpha-configuration. A maltogenic alpha-amylase from
Bacillus stearothermophilus strain NCIB 11837 is commercially
available from Novozymes A/S, Denmark. The maltogenic alpha-amylase
is described in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628,
which are hereby incorporated by reference.
Bacterial Hybrid Alpha-Amylases
[0308] A hybrid alpha-amylase specifically contemplated comprises
445 C-terminal amino acid residues of the Bacillus licheniformis
alpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37
N-terminal amino acid residues of the alpha-amylase derived from
Bacillus amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/194676),
with one or more, especially all, of the following
substitutions:
[0309] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using
the Bacillus licheniformis numbering). Also preferred are variants
having one or more of the following mutations (or corresponding
mutations in other Bacillus alpha-amylase backbones): H154Y, A181T,
N190F, A209V and Q264S and/or deletion of two residues between
positions 176 and 179, preferably deletion of E178 and G179 (using
the SEQ ID NO: 5 numbering of WO 99/19467).
[0310] The bacterial alpha-amylase may be added in amounts as are
well-known in the art. When measured in KNU units (described below
in the "Materials & Methods"-section) the alpha-amylase
activity is preferably present in an amount of 0.5-5,000 NU/g of
DS, in an amount of 1-500 NU/g of DS, or more preferably in an
amount of 51,000 NU/g of DS, such as 10-100 NU/g DS.
Fungal Alpha-Amylases
[0311] Fungal acid alpha-amylases include acid alpha-amylases
derived from a strain of the genus Aspergillus, such as Aspergillus
oryzae, Aspergillus niger, Aspergillus kawachii alpha-amylases.
[0312] A preferred acid fungal alpha-amylase is a Fungamyl-like
alpha-amylase which is preferably derived from a strain of
Aspergillus oryzae. In the present disclosure, the term
"Fungamyl-like alpha-amylase" indicates an alpha-amylase which
exhibits a high identity, i.e., more than 70%, more than 75%, more
than 80%, more than 85% more than 90%, more than 95%, more than
96%, more than 97%, more than 98%, more than 99% or even 100%
identity to the mature part of the amino acid sequence shown in SEQ
ID NO: 10 in WO 96/23874.
[0313] Another preferred acid alpha-amylase is derived from a
strain Aspergillus niger. In a preferred embodiment the acid fungal
alpha-amylase is the one from A. niger disclosed as "AMYA_ASPNG" in
the Swiss-prot/TeEMBL database under the primary accession no.
P56271 and described in more detail in WO 89/01969 (Example 3). The
acid Aspergillus niger acid alpha-amylase is also shown as SEQ ID
NO: 1 in WO 2004/080923 (Novozymes) which is hereby incorporated by
reference. Also variants of said acid fungal amylase having at
least 70% identity, such as at least 80% or even at least 90%
identity, such as at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99% identity to SEQ ID NO: 1 in WO
2004/080923 are contemplated. A suitable commercially available
acid fungal alpha-amylase derived from Aspergillus niger is SP288
(available from Novozymes A/S, Denmark).
[0314] In a preferred embodiment the alpha-amylase is derived from
Aspergillus kawachii and disclosed by Kaneko et al., 1996, J.
Ferment. Bioeng. 81: 292-298, "Molecular-cloning and determination
of the nucleotide-sequence of a gene encoding an acid-stable
alpha-amylase from Aspergillus kawachii"; and further as
EMBL:#AB008370.
[0315] The fungal acid alpha-amylase may also be a wild-type enzyme
comprising a carbohydrate-binding module (CBM) and an alpha-amylase
catalytic domain (i.e., a none-hybrid), or a variant thereof. In an
embodiment the wild-type acid alpha-amylase is derived from a
strain of Aspergillus kawachii.
Fungal Hybrid Alpha-Amylases
[0316] In a preferred embodiment the fungal acid alpha-amylase is a
hybrid alpha-amylase. Preferred examples of fungal hybrid
alpha-amylases include the ones disclosed in WO 2005/003311 or U.S.
Patent Publication no. 2005/0054071 (Novozymes) or U.S. patent
application No. 60/638,614 (Novozymes) which is hereby incorporated
by reference. A hybrid alpha-amylase may comprise an alpha-amylase
catalytic domain (CD) and a carbohydrate-binding domain/module
(CBM) and optional a linker.
[0317] Specific examples of contemplated hybrid alpha-amylases
include those disclosed in U.S. patent application No. 60/638,614
including Fungamyl variant with catalytic domain JA118 and Athelia
rolfsii SBD (SEQ ID NO: 28 herein and SEQ ID NO: 100 in U.S.
application No. 60/638,614), Rhizomucor pusillus alpha-amylase with
Athelia rolfsii AMG linker and SBD (SEQ ID NO: 29 herein and SEQ ID
NO: 101 in U.S. application No. 60/638,614) and Meripilus giganteus
alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ
ID NO: 30 herein and SEQ ID NO: 102 in U.S. application No.
60/638,614).
[0318] Other specific examples of contemplated hybrid
alpha-amylases include those disclosed in U.S. Patent Application
Publication no. 2005/0054071, including those disclosed in Table 3
on page 15, such as Aspergillus niger alpha-amylase with
Aspergillus kawachii linker and starch binding domain.
Commercial Alpha-Amylase Products
[0319] Preferred commercial compositions comprising alpha-amylase
include MYCOLASE from DSM (Gist Brocades), BAN.TM., TERMAMYL.TM.
SC, FUNGAMYL.TM., LIQUOZYME.TM. X and SAN.TM. SUPER, SAN.TM. EXTRA
L (Novozymes A/S) and CLARASE.TM. L-40,000, DEX-LO.TM., SPEZYME.TM.
FRED, SPEZYME.TM. AA, and SPEZYME.TM. DELTA M (Genencor Int.), and
the acid fungal alpha-amylase sold under the trade name SP288
(available from Novozymes A/S, Denmark).
[0320] An acid alpha-amylases may according to the invention be
added in an amount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5
AFAU/g DS, especially 0.3 to 2 AFAU/g DS.
Production of Syrup
[0321] The present invention also provides a process of using a
glucoamylase of the invention for producing syrup, such as glucose
and the like, from starch-containing material. Suitable starting
materials are exemplified in the "Starch-containing
materials"-section above. Generally, the process comprises the
steps of partially hydrolyzing starch-containing material
(liquefaction) in the presence of alpha-amylase and then further
saccharifying the release of glucose from the non-reducing ends of
the starch or related oligo- and polysaccharide molecules in the
presence of glucoamylase of the invention.
[0322] Liquefaction and saccharification may be carried our as
described above for fermentation product production.
[0323] The glucoamylase of the invention may also be used in
immobilized form. This is suitable and often used for producing
speciality syrups, such as maltose syrups, and further for the
raffinate stream of oligosaccharides in connection with the
production of fructose syrups, e.g., high fructose syrup (HFS).
[0324] Consequently, this aspect of the invention relates to a
process of producing syrup from starch-containing material,
comprising
[0325] (a) liquefying starch-containing material in the presence of
an alpha-amylase,
[0326] (b) saccharifying the material obtained in step (a) using a
glucoamylase of the invention.
[0327] A syrup may be recovered from the saccharified material
obtained in step (b).
[0328] Details on suitable conditions can be found above.
Brewing
[0329] A glucoamylase of the invention can also be used in a
brewing process. The glucoamylases of the invention is added in
effective amounts which can be easily determined by the skilled
person in the art. For instance, in the production of "low carb" or
super attenuated beers, a higher proportion of alcohol and a lower
amount of residual dextrin are desired. These beers are formulated
using exogenous enzymes compositions comprising enzyme activities
capable of debranching the limit dextrins. A glucoamylase of the
invention, preferably Trametes cingulata, may be applied to reduce
the content of limit dextrins as well as hydrolyzing the alpha-1,4
bonds.
[0330] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
de-scribed 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.
[0331] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties. The
present invention is further described by the following examples
which should not be construed as limiting the scope of the
invention.
Materials & Methods
Glucoamylases:
[0332] Glucoamylase derived from Trametes cingulata disclosed in
SEQ ID NO: 2 and available from Novozymes A/S.
[0333] Glucoamylase derived from Pachykytospora papyraceae
disclosed in SEQ ID NO: 5 and available from Novozymes A/S.
[0334] Glucoamylase derived from Leucopaxillus giganteus disclosed
in SEQ ID NO: 24 and available from Novozymes A/S.
[0335] Glucoamylase derived from Aspergillus niger disclosed in
Boel et al., 1984, EMBO J. 3 (5): 1097-1102 and available from
Novozymes A/S.
[0336] Glucoamylase derived from Talaromyces emersonii disclosed in
WO 99/28448 and available from Novozymes A/S.
[0337] Enzymes for DNA manipulations (e.g., restriction
endonucleases, ligases etc.) are obtainable from New England
Biolabs, Inc. and were used according to the manufacturer's
instructions.
Alpha-Amylase:
[0338] Hybrid Alpha-Amylase A: Rhizomucor pusillus alpha-amylase
with Athelia rolfsii glucoamylase linker and SBD disclosed in U.S.
patent application No. 60/638,614 and SEQ ID NO: 29.
Yeast: Red Star.TM. available from Red Star/Lesaffre, USA
Microbial Strains
[0339] E. coli DH12alpha (GIBCO BRL, Life Technologies, USA)
[0340] Aspergillus oryzae IFO 4177 is available from Institute for
Fermentation, Osaka (IFO) Culture Collection of Microorganisms,
17-85, Juso-honmachi, 2-chome, Yodogawa-ku, Osaka 532-8686,
Japan.
[0341] Aspergillus oryzae BECh-2 is described in WO 2000/39322
(Novozymes). It is a mutant of JaL228 (described in WO 98/12300)
which is a mutant of IFO 4177.
[0342] Aspergillus niger strain Mbin119 is described in WO
2004/090155 (see Example 12).
Other Materials
[0343] Pullulan available from Wako Pure Chemical (Japan).
Deposit of Biological Material
[0344] The following biological material has been deposited under
the terms of the Budapest Treaty at Deutshe Sammmlung von
Microorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b,
D-38124 Braunschweig DE, and given the following accession number:
TABLE-US-00002 Deposit Accession Number Date of Deposit Escherichia
coli NN049798 DSM 17106 2 Feb. 2005 Escherichia coli NN049797 DSM
17105 2 Feb. 2005
[0345] The strain has been deposited under conditions that assure
that access to the culture will be available during the pendency of
this patent application to one determined by the Commissioner of
Patents and Trademarks to be entitled thereto under 37 C.F.R.
.sctn.1.14 and 35 U.S.C. .sctn.122. The deposit represents a
substantially pure culture of the deposited strain. The deposit is
available as required by foreign patent laws in countries wherein
counterparts of the subject application, or its progeny are filed.
However, it should be understood that the availability of a deposit
does not constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
Media and Reagents:
[0346] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
PDA2: 39 g/L Potato Dextrose Agar, 20 g/L agar, 50 mL glycerol
Cove: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10 mM
Acetamide, 30 g/L noble agar.
Cove salt solution: per liter 26 g KCl, 26 g MgSO.sub.4-7 aq, 76 g
KH.sub.2PO.sub.4, 50 ml Cove trace metals.
Cove trace metals: per liter 0.04 g NaB407-10 aq, 0.4 g CuSO4-5 aq,
1.2 g FeSO.sub.4-7 aq, 0.7 g MnSO.sub.4-aq, 0.7 g
Na.sub.2MoO.sub.2-2 aq, 0.7 g ZnSO.sub.4-7 aq.
YPG: 4 g/L Yeast extract, 1 g/L KH2PO4, 0.5 g/L MgSO.sub.4-7 aq, 5
g/L Glucose, pH 6.0.
STC: 0.8 M Sorbitol, 25 mM Tris pH 8, 25 mM CaCl.sub.2.
STPC: 40% PEG4000 in STC buffer.
Cove top agarose: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10
mM Acetamide, 10 g/L low melt agarose.
MS-9: per liter 30 g soybean powder, 20 g glycerol, pH 6.0.
MDU-pH5: per liter 45 g maltose-1 aq, 7 g yeast extract, 12 g
KH.sub.2PO.sub.4, 1 g MgSO.sub.4-7 aq, 2 g K.sub.2SO.sub.4, 0.5 ml
AMG trace metal solution and 25 g 2-morpholinoethanesulfonic acid,
pH 5.0.
Methods
[0347] Unless otherwise stated, DNA manipulations and
transformations were performed using standard methods of molecular
biology as described in Sambrook et al., 1989, Molecular cloning: A
laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor,
N.Y.; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular
Biology", John Wiley and Sons, 1995; Harwood, C. R., and Cutting,
S. M. (eds.) "Molecular Biological Methods for Bacillus". John
Wiley and Sons, 1990.
Glucoamylase Activity
[0348] Glucoamylase activity may be measured in AGI units or in
Glucoamylase Units (AGU).
Glucoamylase Activity (AGI)
[0349] Glucoamylase (equivalent to amyloglucosidase) converts
starch into glucose. The amount of glucose is determined here by
the glucose oxidase method for the activity determination. The
method described in the section 76-11 Starch--Glucoamylase Method
with Subsequent Measurement of Glucose with Glucose Oxidase in
"Approved methods of the American Association of Cereal Chemists".
Vol. 1-2 AACC, from American Association of Cereal Chemists,
(2000); ISBN: 1-891127-12-8.
[0350] One glucoamylase unit (AGI) is the quantity of enzyme which
will form 1 micro mole of glucose per minute under the standard
conditions of the method.
[0351] Standard Conditions/Reaction Conditions: TABLE-US-00003
Substrate: Soluble starch, concentration approx. 16 g dry matter/L.
Buffer: Acetate, approx. 0.04 M, pH = 4.3 pH: 4.3 Incubation
temperature: 60.degree. C. Reaction time: 15 minutes Termination of
the reaction: NaOH to a concentration of approximately 0.2 g/L
(pH.about.9) Enzyme concentration: 0.15-0.55 AAU/mL.
[0352] The starch should be Lintner starch, which is a thin-boiling
starch used in the laboratory as calorimetric indicator. Lintner
starch is obtained by dilute hydrochloric acid treatment of native
starch so that it retains the ability to color blue with
iodine.
Glucoamylase Activity (AGU)
[0353] The Novo Glucoamylase Unit (AGU) is defined as the amount of
enzyme, which hydrolyzes 1 micromole maltose per minute under the
standard conditions 37.degree. C., pH 4.3, substrate: maltose 23.2
mM, buffer: acetate 0.1 M, reaction time 5 minutes.
[0354] An autoanalyzer system may be used. Mutarotase is added to
the glucose dehydrogenase reagent so that any alpha-D-glucose
present is turned into beta-D-glucose. Glucose dehydrogenase reacts
specifically with beta-D-glucose in the reaction mentioned above,
forming NADH which is determined using a photometer at 340 nm as a
measure of the original glucose concentration. TABLE-US-00004 AMG
incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1 M pH:
4.30 .+-. 0.05 Incubation temperature: 37.degree. C. .+-. 1
Reaction time: 5 minutes Enzyme working range: 0.5-4.0 AGU/mL Color
reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:
phosphate 0.12 M; 0.15 M NaCl pH: 7.60 .+-. 0.05 Incubation
temperature: 37.degree. C. .+-. 1 Reaction time: 5 minutes
Wavelength: 340 nm
[0355] A folder (EB-SM-0131.02/01) describing this analytical
method in more detail is available on request from Novozymes A/S,
Denmark, which folder is hereby included by reference.
Alpha-Amylase Activity (KNU)
[0356] The alpha-amylase activity may be determined using potato
starch as substrate. This method is based on the break-down of
modified potato starch by the enzyme, and the reaction is followed
by mixing samples of the starch/enzyme solution with an iodine
solution. Initially, a blackish-blue color is formed, but during
the break-down of the starch the blue color gets weaker and
gradually turns into a reddish-brown, which is compared to a
colored glass standard.
[0357] One Kilo Novo alpha amylase Unit (KNU) is defined as the
amount of enzyme which, under standard conditions (i.e., at
37.degree. C.+/-0.05; 0.0003 M Ca.sup.2+; and pH 5.6) dextrinizes
5260 mg starch dry substance Merck Amylum solubile.
[0358] A folder EB-SM-0009.02/01 describing this analytical method
in more detail is available upon request to Novozymes A/S, Denmark,
which folder is hereby included by reference.
Acid Alpha-Amylase Activity
[0359] When used according to the present invention the activity of
any acid alpha-amylase may be measured in AFAU (Acid Fungal
Alpha-amylase Units). Alternatively activity of acid alpha-amylase
may be measured in MU (Acid Alpha-amylase Units).
Acid Alpha-Amylase Units (AAU)
[0360] The acid alpha-amylase activity can be measured in AAU (Acid
Alpha-amylase Units), which is an absolute method. One Acid Amylase
Unit (AAU) is the quantity of enzyme converting 1 g of starch (100%
of dry matter) per hour under standardized conditions into a
product having a transmission at 620 nm after reaction with an
iodine solution of known strength equal to the one of a color
reference.
[0361] Standard Conditions/Reaction Conditions: TABLE-US-00005
Substrate: Soluble starch. Concentration approx. 20 g DS/L. Buffer:
Citrate, approx. 0.13 M, pH = 4.2 Iodine solution: 40.176 g
potassium iodide + 0.088 g iodine/L City water
15.degree.-20.degree.dH (German degree hardness) pH: 4.2 Incubation
temperature: 30.degree. C. Reaction time: 11 minutes Wavelength:
620 nm Enzyme concentration: 0.13-0.19 AAU/mL Enzyme working range:
0.13-0.19 AAU/mL
[0362] The starch should be Lintner starch, which is a thin-boiling
starch used in the laboratory as colorimetric indicator. Lintner
starch is obtained by dilute hydrochloric acid treatment of native
starch so that it retains the ability to color blue with iodine.
Further details can be found in EP 0140410 B2, which disclosure is
hereby included by reference.
Acid Alpha-Amylase Activity (AFAU)
[0363] Acid alpha-amylase activity may be measured in AFAU (Acid
Fungal Alpha-amylase Units), which are determined relative to an
enzyme standard. 1 AFAU is defined as the amount of enzyme which
degrades 5.260 mg starch dry matter per hour under the below
mentioned standard conditions.
[0364] Acid alpha-amylase, an endo-alpha-amylase
(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzes
alpha-1,4-glucosidic bonds in the inner regions of the starch
molecule to form dextrins and oligosaccharides with different chain
lengths. The intensity of color formed with iodine is directly
proportional to the concentration of starch. Amylase activity is
determined using reverse colorimetry as a reduction in the
concentration of starch under the specified analytical conditions.
##STR1##
[0365] Standard Conditions/Reaction Conditions: TABLE-US-00006
Substrate: Soluble starch, approx. 0.17 g/L Buffer: Citrate,
approx. 0.03 M Iodine (I2): 0.03 g/L CaCl.sub.2: 1.85 mM pH: 2.50
.+-. 0.05 Incubation temperature: 40.degree. C. Reaction time: 23
seconds Wavelength: 590 nm Enzyme concentration: 0.025 AFAU/mL
Enzyme working range: 0.01-0.04 AFAU/mL
[0366] A folder EB-SM-0259.02/01 describing this analytical method
in more detail is available upon request to Novozymes A/S, Denmark,
which folder is hereby included by reference.
EXAMPLES
Example 1
Molecular Screening of Glucoamylase Genes
[0367] Trametes cingulata was grown on PDA2 medium and genome DNA
was isolated from 0.2 g mycelium using FastDNA SPIN Kit for Soil
(Qbiogene, USA) according to the manufacturer's instructions.
[0368] PCR reaction was done on genome DNA with the degenerated
primers ArAF1 and ArAR3 TABLE-US-00007 ArAF1
5'-CRTRCTYDVCAACATYGG-3' (SEQ ID NO: 7) ArAR3 5'
GTCAGARCADGGYTGRRASGTG-3' (SEQ ID NO: 8)
wherein D=A or G or T; R=A or G; S.dbd.C or G; V=A or C or G;
Y.dbd.C or T
[0369] The amplification reaction (13 microL) was composed of 1
microL genome DNA solution, 1 micro M primer ArAF1, 1 micro M
primer ArAR3, 11 microL Extensor Hi-Fidelity PCR Master Mix
(ABgene, UK). The reaction was incubated in a DNA Engine Dyad
PTC-0220 (MJ Research, USA) programmed as follows: 1 cycle at
94.degree. C. for 2 minutes; 20 cycles each at 94.degree. C. for 30
seconds, 65.degree. C. for 45 seconds, with an annealing
temperature decline of 1.degree. C. per cycle, and 72.degree. C.
for 1 minute 30 seconds; followed by 20 cycles each at 94.degree.
C. for 30 seconds, 45.degree. C. for 45 seconds and 72.degree. C.
for 1 minute 30 seconds; 1 cycle at 72.degree. C. for 7 minutes;
and a hold at 4.degree. C. The PCR product was purified using
ExoSAP-IT (USB, USA) according to the manufacturer's instructions
and sequenced. The sequence was subsequently compared to the
Aspergillus niger glucoamylase gene, showing that the PCR product
encoded a part of a glucoamylase.
Example 2
Molecular Screening of Glucoamylase Genes
[0370] Pachykytospora papyracea was grown on PDA2 medium and genome
DNA was isolated from 0.2 g mycelium using FastDNA SPIN Kit for
Soil (Qbiogene, USA) according to the manufacturer's
instructions.
[0371] PCR reaction (PCR 1) was done on genome DNA with the
degenerated primers AM2F and AM4R2: TABLE-US-00008 AM2F
5'-TGGGGIMGNCCNCARMGNGAYGG-3' (SEQ ID NO: 9) AM4R2
5'-RTCYTCNGGRTANCKNCC-3' (SEQ ID NO: 10)
wherein I=inosine; K=G or T; M=A or C; N=A or C or G or T; R=A or
G; Y.dbd.C or T
[0372] The amplification reaction (25 microL) was composed of 1
microL genome DNA solution, 2 micro M primer AM2F, 2 micro M primer
AM4R2, 22 microL Reddy PCR Master Mix (ABgene, UK). The reaction
was incubated in a DNA Engine Dyad PTC-0220 (MJ Research, USA)
programmed as follows: 1 cycle at 94.degree. C. for 2 minutes; 20
cycles each at 94.degree. C. for 1 minute, 55.degree. C. for 1
minute, with an annealing temperature decline of 1.degree. C. per
cycle, and 72.degree. C. for 1 minute; followed by 20 cycles each
at 94.degree. C. for 1 minute, 40.degree. C. for 1 minute and
72.degree. C. for 1 minute; 1 cycle at 72.degree. C. for 7 minutes;
and a hold at 4.degree. C.
[0373] Subsequently a PCR reaction was done on an aliquot of the
first PCR reaction (PCR 1) with the degenerated primers AM3F and
AM4R2: TABLE-US-00009 AM3F 5'-TAYGAYYTNYGGGARGA-3' (SEQ ID NO: 11)
AM4R2 5'-RTCYTCNGGRTANCKNCC-3' (SEQ ID NO: 10)
wherein K=G or T; N=A or C or G or T; R=A or G; Y=C or T
[0374] The amplification reaction (13 microLI) was composed of 1
microL of the first PCR reaction (PCR 1), 1 microM primer AM3F, 1
micro M primer AM4R2, 11 microL Reddy PCR Master Mix (ABgene, UK).
The reaction was incubated in a DNA Engine Dyad PTC-0220 (MJ
Research, USA) programmed as follows: 1 cycle at 94.degree. C. for
2 minutes; 5 cycles each at 94.degree. C. for 45 seconds,
45.degree. C. for 45 seconds and 72.degree. C. for 1 minute;
followed by 30 cycles each at 94.degree. C. for 45 seconds,
40.degree. C. for 45 seconds and 72.degree. C. for 1 minute; 1
cycle at 72.degree. C. for 7 minutes; and a hold at 4.degree. C. A
0.5 kb amplified PCR band was obtained. The reaction product was
isolated on a 1.0% agarose gel using TBE buffer and it was excised
from the gel and purified using GFX PCR DNA and Gel band
Purification Kit (Amersham Biosciences, UK). The excised band was
sequenced and subsequently compared to the Aspergillus niger
glucoamylase gene, showing that the PCR product encoded a part of a
glucoamylase.
Example 3
Cloning of Glucoamylase Gene from Trametes cingulata
[0375] From the partial sequence of the Trametes cingulata
glucoamylase more gene sequence was obtained with PCR based gene
walking using the Vectorette Kit from SIGMA-Genosys. The gene
walking was basically done as described in the manufacturer's
protocol. 0.15 micro g genomic DNA of Trametes cingulata was
digested with EcoRI, BamHI and HindIII, independently. The digested
DNA was ligated with the corresponding Vectorette units supplied by
the manufacturer using a DNA Engine Dyad PTC-0220 (MJ Research,
USA) programmed as follows: 1 cycle at 16.degree. C. for 60
minutes; 4 cycles each at 37.degree. C. for 20 minutes, 16.degree.
C. for 60 minutes, 37.degree. C. for 10 minutes; followed by 1
cycle at 16.degree. C. for 60 minutes and a hold at 4.degree. C.
The ligation reactions were subsequent diluted 5 times with sterile
water.
[0376] PCR reactions with linker-ligated genome DNA of the Trametes
cingulata as template was performed with a DNA Engine Dyad PTC-0220
(MJ Research, USA) programmed as follows. 1 cycle at 94.degree. C.
for 2 minutes; 40 cycles each at 94.degree. C. for 15 seconds,
72.degree. C. for 1 minute, 72.degree. C. for 1 minute, 1 cycle at
72.degree. C. for 7 minutes; and a hold at 40 using the supplied
Vectorette primer and primer TraF1 as shown below. TABLE-US-00010
TraF1: 5'- TAGTCGTACTGGAACCCCACC -3' (SEQ ID NO: 12)
[0377] The amplification reactions (12.5 microL) were composed of
0.5 microL of linker-ligated genome DNAs, 400 nM Vectorette primer,
400 nM TraF1 primer, 11 microL Extensor Hi-Fidelity PCR Master Mix
(ABgene, UK).
[0378] A 0.5 kb amplified band was obtained by the PCR reaction
from HindIII digested genome DNA. The reaction product was isolated
on a 1.0% agarose gel using TBE buffer and was excised from the
gel. 100 microL sterile water was added to the excised agarose gel
fragment and it was melted by incubation at 95.degree. C. for 5
minutes to release the DNA. The DNA band was reamplified by
repeating the PCR reaction described above using 0.5 microL of the
isolated DNA fragment instead of linker-ligated genome DNA.
[0379] After the PCR reaction the DNA was purified using ExoSAP-IT
(USB, USA) according to the manufacturers instructions and
sequenced and subsequently compared to the Aspergillus niger
glucoamylase gene, showing that it encoded a further 250 bp part of
the glucoamylase gene.
[0380] In order to clone the missing parts of the glucoamylase gene
from Trametes cingulata, PCR based gene walking was carried out
using LA PCR.TM. in vitro Cloning Kit (TAKARA, Japan) according to
the manufacturer's instructions.
[0381] Five micro g of genome DNA of Trametes cingulata was
digested with BamHI, EcoRI, HindIII, PstI, SalI and XbaI,
independently. 200 ml of ice-cold ethanol was added to the reaction
mixture (50 microL) and then digested DNA was recovered by
centrifugation at 15,000.times.g for 30 minutes at 4.degree. C. The
recovered DNA was ligated with a corresponding artificial linkers
supplied by manufactures. The linker ligated DNA was recovered by
adding 200 ml of ice-cold ethanol to the reaction mixture (50
microL) followed by centrifugation at 15,000.times.g for 30 minutes
at 4.degree. C.
[0382] PCR reactions with linker-ligated genome DNA of the Trametes
cingulata as template was performed with a LA PCR system (TAKARA,
Japan) using primer C1 and TC5' for cloning of missing
5'-glucoamylase gene and primer C1 and TC3' for cloning of missing
3'-glucoamylase gene, as shown below. TABLE-US-00011 (SEQ ID NO:
13) C1: 5'-gtacatattgtcgttagaacgcgtaatacgactca-3' (SEQ ID NO: 14)
TC5': 5'-cgtatatgtcagcgctaccatgt-3' (SEQ ID NO: 15) TC3':
5'-aaacgtgagcgaccattttctgt-3'
[0383] The amplification reactions (50 microL) were composed of 1
ng of template DNA per microL, 250 mM dNTP each, 250 nM primer, 250
nM primer, 0.1 U of LA Taq polymerase per microL in 1.times. buffer
(TAKARA, Japan). The reactions were incubated in a DNA Engine
PTC-200 (MJ-Research, Japan) programmed as follows: 1 cycle at
94.degree. C. for 2 minutes; 30 cycles each at 94.degree. C. for
0.5 minute, 55.degree. C. for 2 minutes, and 72.degree. C. for 2
minutes; 1 cycle at 72.degree. C. for 10 minutes; and a hold at
4.degree. C.
[0384] 0.4 kb and 1.0 kb amplified bands were obtained from SalI
digested genome DNA with primer C1 and TC5' and XbaI digested
genome DNA with primer C1 and TC3', respectively. These reaction
products were isolated on a 1.0% agarose gel using TAE buffer and
was excised from the gel and purified using a QIAquick.TM. Gel
Extraction Kit (QIAGEN Inc., Valencia, Calif.) according to the
manufacturer's instructions.
[0385] The amplified DNA fragments were ligated into pT7BIue
(Invitrogen, Netherlands), independently. The ligation mixture was
then transformed into E. coli DH12alpha (GIBCO BRL, Life
Technologies, USA) to create pHUda438 and pHUda439 for a 0.4 kb
amplified band and a 1.0 kb amplified band, respectively. The
resultant plasmids were sequenced and compared to the Aspergillus
niger glucoamylase gene, showing that clones encode the missing
parts of the glucoamylase.
Example 4
Construction of pHUda440 Expression Vector
[0386] Expression vector pHUda440 was constructed for transcription
of the glucoamylase gene from Trametes cingulata. A PCR reaction
with the genome DNA of the Trametes cingulata as template was
performed with an Expand.TM. PCR system (Roche Diagnostics, Japan)
using primers TFF to introduce a BamH I site and primer TFR to
introduce an Xho I site, as shown below. TABLE-US-00012 (SEQ ID NO:
16) TFF: 5'-tttggatccaccatgcgtttcacgctcctcacctcc-3' (SEQ ID NO: 17)
TFR: 5'-tttctcgagctaccgccaggtgtcattctg-3'
[0387] The amplification reactions (50 microL) were composed of 1
ng of template DNA per microL, 250 mM dNTP each, 250 nM primer TFF,
250 nM primer TFR, 0.1 U of Taq polymerase per microL in 1.times.
buffer (Roche Diagnostics, Japan). The reactions were incubated in
a DNA Engine PTC-200 (MJ-Research, Japan) programmed as follows: 1
cycle at 94.degree. C. for 2 minutes; 30 cycles each at 92.degree.
C. for 1 minute, 55.degree. C. for 1 minute, and 72.degree. C. for
2 minutes; 1 cycle at 72.degree. C. for 10 minutes; and a hold at
4.degree. C.
[0388] The reaction products were isolated on a 1.0% agarose gel
using TAE buffer where a 2.2 kb product band was excised from the
gel and purified using a QIAquick.TM. Gel Extraction Kit (QIAGEN
Inc., Valencia, Calif.) according to the manufacturer's
instructions.
[0389] The 2.2 kb amplified DNA fragment was digested with BamHI
and XhoI, and ligated into the Aspergillus expression cassette
pCaHj483 digested with BamH I and XhoI. The ligation mixture was
transformed into E. coli DH12alpha (GIBCO BRL, Life Technologies,
USA) to create the expression plasmid pHUda440. The amplified
plasmid was recovered using a QIAprep.RTM. Spin Miniprep kit
(QIAGEN Inc., Valencia, Calif.) according to the manufacturer's
instructions.
[0390] Plasmid pCaHj483 comprised an expression cassette based on
the Aspergillus niger neutral amylase II promoter fused to the
Aspergillus nidulans triose phosphate isomerase non translated
leader sequence (Na2/tpi promoter) and the Aspergillus niger
glucoamylase terminator (AMG terminator), the selective marker amdS
from Aspergillus nidulans enabling growth on acetamide as sole
nitrogen source.
Example 5
Cloning of the glucoamylase gene from Pachykytospora Papyraceae
[0391] In order to clone the missing parts of the glucoamylase gene
from Pachykytospora papyraceae, PCR based gene walking was carried
out using LA PCR.TM. in vitro Cloning Kit (TAKARA, Japan) according
to the manufacturers instructions.
[0392] Five micro g of genome DNA of Pachykytospora papyraceae was
digested with BamHI, EcoRI, HindIII, PstI, SalI and XbaI,
independently. 200 mL of ice-cold ethanol was added to the reaction
mixture (50 microL) and then digested DNA was recovered by
centrifugation at 15,000.times.g for 30 minutes at 4.degree. C. The
recovered DNA was ligated with a corresponding artificial linkers
supplied by manufactures. The linker ligated DNA was recovered by
adding 200 mL of ice-cold ethanol to the reaction mixture (50
microL followed by centrifugation at 15,000.times.g for 30 minutes
at 4.degree. C.
[0393] PCR reactions with linker-ligated genome DNA of the
Pachykytospora papyraceae as template was performed with a LA PCR
system (TAKARA, Japan) using primer C1 and PP5' for cloning of
missing 5'-glucoamylase gene and primer C1 and PP3' for cloning of
missing 3'-glucoamylase gene, as shown below. TABLE-US-00013 (SEQ
ID NO: 13) C1: 5'-gtacatattgtcgttagaacgcgtaatacgactca-3' (SEQ ID
NO: 18) PP5': 5'-cctccctgagtgagcgatgctgc-3' (SEQ ID NO: 19) PP3':
5'-caactccggcctctcctccagcg-3'
[0394] The amplification reactions (50 microL) were composed of 1
ng of template DNA per microL, 250 mM dNTP each, 250 nM primer, 250
nM primer, 0.1 U of LA Taq polymerase per microL in 1.times. buffer
(TAKARA, Japan). The reactions were incubated in a DNA Engine
PTC-200 (MJ-Research, Japan) programmed as follows: 1 cycle at
94.degree. C. for 2 minutes; 30 cydes each at 94.degree. C. for 0.5
minute, 55.degree. C. for 2 minutes, and 72.degree. C. for 2
minutes; 1 cycle at 72.degree. C. for 10 minutes; and a hold at
4.degree. C.
[0395] 0.5 kb and 0.9 kb amplified bands were obtained from XbaI
digested genome DNA with primer C1 and PP5' and EcoRI digested
genome DNA with primer C1 and PP3', respectively. These reaction
products were isolated on a 1.0% agarose gel using TAE buffer and
was excised from the gel and purified using a QIAquick.TM. Gel
Extraction Kit (QIAGEN Inc., Valencia, Calif.) according to the
manufacturer's instructions.
[0396] The amplified DNA fragments were ligated into pT7Blue
(Invitrogen, Netherlands), independently. The ligation mixture was
then transformed into E. coli DH12alpha (GIBCO BRL, Life
Technologies, USA) to create pHUda448 and pHUda449 for a 0.5 kb
amplified band and a 0.9 kb amplified band, respectively. The
resultant plasmids were sequenced and compared to the Aspergillus
niger glucoamylase gene, showing that clones encode the missing
parts of the glucoamylase.
Example 6
Construction of pHUda450 Expression Vector
[0397] Expression vector pHUda450 was constructed for transcription
of the glucoamylase gene from Pachykytospora papyraceae. A PCR
reaction with the genome DNA of the Pachykytospora papyraceae as
template was performed with an Expand.TM. PCR system (Roche
Diagnostics, Japan) using primers PPF to introduce a BamH I site
and primer PPR to to introduce an Xho I site, as shown below.
TABLE-US-00014 (SEQ ID NO: 20) PPF:
5'-tttggatccaccatgcgcttcaccctcctctcctcc-3' (SEQ ID NO: 21) PPR:
5'-tttctcgagtcaccgccaggtgtcgttctg-3'
[0398] The amplification reactions (50 microL) were composed of 1
ng of template DNA per microL, 250 mM dNTP each, 250 nM primer PPF,
250 nM primer PPR, 0.1 U of Taq polymerase per microL in 1.times.
buffer (Roche Diagnostics, Japan). The reactions were incubated in
a DNA Engine PTC-200 (MJ-Research, Japan) programmed as follows: 1
cycle at 94.degree. C. for 2 minutes; 30 cycles each at 92.degree.
C. for 1 minute, 55.degree. C. for 1 minute, and 72.degree. C. for
2 minutes; 1 cycle at 72.degree. C. for 10 minutes; and a hold at
4.degree. C.
[0399] The reaction products were isolated on a 1.0% agarose gel
using TAE buffer where a 2.2 kb product band was excised from the
gel and purified using a QIAquick.TM. Gel Extraction Kit (QIAGEN
Inc., Valencia, Calif.) according to the manufacturer's
instructions.
[0400] The 2.2 kb amplified DNA fragment was digested with BamHI
and XhoI, and ligated into the Aspergillus expression cassette
pCaHj483 digested with BamH I and XhoI. The ligation mixture was
transformed into E. coli DH12alpha (GIBCO BRL, Life Technologies,
USA) to create the expression plasmid pHUda450. The amplified
plasmid was recovered using a QIAprep.RTM. Spin Miniprep kit
(QIAGEN Inc., Valencia, Calif.) according to the manufacturer's
instructions.
Example 7
Expression of Glucoamylase Genes Derived from Trametes cingulata
and Pachykytospora papyraceae in Aspergillus oryzae
[0401] Aspergillus oryzae strain BECh-2 was inoculated to 100 mL of
YPG medium and incubated for 16 hours at 32.degree. C. at 80 rpm.
Pellets were collected and washed with 0.6 M KCl, and resuspended
20 ml 0.6 M KCl containing a commercial beta-glucanase product
(GLUCANEX.TM., Novozymes A/S, Bagsv.ae butted.rd, Denmark) at a
final concentration of 600 microL per mL. The suspension was
incubated at 32.degree. C. and 80 rpm until protoplasts were
formed, and then washed twice with STC buffer. The protoplasts were
counted with a hematometer and resuspended and adjusted in an
8:2:0.1 solution of STC:STPC:DMSO to a final concentration of
2.5.times.10.sup.7 protoplasts/ml. Approximately 3 micro g of
pHUda440 or pHUda450 was added to 100 microL of the protoplast
suspension, mixed gently, and incubated on ice for 20 minutes. One
mL of SPTC was added and the protoplast suspension was incubated
for 30 minutes at 37.degree. C. After the addition of 10 mL of
50.degree. C. COVE top agarose, the reaction was poured onto COVE
agar plates and the plates were incubated at 32.degree. C. After 5
days transformants were selected from the COVE medium.
[0402] Four randomly selected transformants were inoculated into
100 mL of MS-9 medium and cultivated at 32.degree. C. for 1 day.
Three ml of MS-9 medium was inoculated into 100 mL of MDU-pH5
medium and cultivated at 30.degree. C. for 3 days. Supernatants
were obtained by centrifugation at 3,000.times.g for 10
minutes.
[0403] Glucoamylase activity in the supernatant samples was
determined as an increase in NADH production by glucose
dehydrogenase and mutarotase reaction with generating glucose and
measured the absorbance at 340 nm. Six microL of enzyme samples
dissolved in 100 mM sodium acetate pH 4.3 buffer was mixed with 31
microL of 23.2 mM of maltose in 100 mM sodium acetate pH 4.3 buffer
and incubated at 37.degree. C. for 5 minutes. Then, 313 microL of
color reagent (430 U of glucose dehydrogenase per liter, 9 U
mutarotase per liter, 0.21 mM NAD, and 0.15 M NaCl in 0.12 M
phosphate pH 7.6 buffer) was added to the reaction mixture and
incubated at 37.degree. C. for 5 minutes. Activity was measured at
340 nm on a spectrophotometer. Six microL of distilled water was
used in place of the enzyme samples as controls.
[0404] Tables 1 and 2 show the glucoamylase activities of the
selected transformants, relative to the activity of the host
strain, Aspergillus oryzae BECh-2, which was normalized to 1.0.
TABLE-US-00015 TABLE 1 Shake flask results of the selected
transformants expressing Trametes cingulata glucoamylase T.
cingulata glucoamylase (AGU/ml) Strains Relative activities #13-1
180 #13-2 199 #19-1 148 #19-2 169 BECh-2 1.0
[0405] TABLE-US-00016 TABLE 2 Shake flask results of the selected
transformants expressing Pachykytospora papyraceae glucaoamylase P.
papyraceae glucoamylase (AGU/ml) Strains Relative activities #B11-1
42 #B11-2 48 #B11-3 36 #B11-4 50 BECh-2 1.0
Example 8
Evaluation of Trametes cingulata Glucoamylase in One-Step Fuel
Ethanol Fermentations
[0406] The relative performance of Trametes cingulata glucoamylase
to Aspergillus niger glucoamylase and Talaromyces emersonii
glucoamylase was evaluated via mini-scale fermentations. About 380
g of milled corn (ground in a pilot scale hammer mill through a
1.65 mm screen) was added to about 620 g tap water. This mixture
was supplemented with 3 mL 1 g/L penicillin. The pH of this slurry
was adjusted to 5.0 with 40% H.sub.2SO.sub.4. The dry solid (DS)
level was determined in triplicate to be about 32%. Approximately 5
g of this slurry was added to 15 mL tubes.
[0407] A two dose dose-response was conducted with each enzyme.
Dosages used were 0.3 and 0.6 nmol/g DS. Six replicates of each
treatment were run.
[0408] After dosing the tubes were inoculated with 0.04 mL/g mash
of yeast propagate (RED STAR.TM. yeast) that had been grown for
22.5 hours on corn mash. Tubes were capped with a screw on top
which had been punctured with a small needle to allow gas release
and vortexed briefly before weighing and incubation at 32.degree.
C. 70 hour fermentations were carried out and ethanol yields were
determined by weighing the tubes. Tubes were vortexed briefly
before weighing. The result of the experiment is shown in Table
1.
[0409] It can be seen from Table 1 the ethanol yield per gram DS is
significantly higher when using the Trametes cingulata glucoamylase
compared to yields for the wild-type Aspergillus niger and
Talaromyces emersonii glucoamylases. TABLE-US-00017 TABLE 1
Glucoamylase nmol/g DS Ethanol yields Trametes cingulata 0.3 56.2
Aspergillus niger 47.2 Talaromyces emersonii 30.5 Trametes
cingulata 0.6 100.8 Aspergillus niger 87.2 Talaromyces emersonii
43.4
Example 9
Evaluation of Pachykytospora papyracea Glucoamylase in One Step
Fuel Ethanol Fermentations
[0410] The relative performance of Pachykytospora papyracea
glucoamylase to Aspergillus niger glucoamylase and Talaromyces
emersonii glucoamylase was evaluated via mini-scale fermentations.
About 380 g of milled corn (ground in a pilot scale hammer mill
through a 1.65 mm screen) was added to about 620 g tap water. This
mixture was supplemented with 3 mL 1 g/L penicillin. The pH of this
slurry was adjusted to 5.0 with 40% H.sub.2SO.sub.4. The dry solid
(DS) level was determined in triplicate to be about 32%.
Approximately 5 g of this slurry was added to 15 mL tubes.
[0411] A two dose dose-response was conducted with each enzyme.
Dosages used were 0.3 and 0.6 nmol/g DS. Six replicates of each
treatment were run.
[0412] After dosing the tubes were inoculated with 0.04 mL/g mash
of yeast propagate (RED STAR.TM. yeast) that had been grown for
22.5 hours on corn mash. Tubes were capped with a screw on top
which had been punctured with a small needle to allow gas release
and vortexed briefly before weighing and incubation at 32.degree.
C. 70 hour fermentations were carried out and ethanol yields were
determined by weighing the tubes. Tubes were vortexed briefly
before weighing. The result of the experiment is shown in Table
2.
[0413] It can be seen from Table 2 the ethanol yield per gram DS is
significantly higher when using the Pachykytospora papyracea
glucoamylase compared to yields for the wild-type Aspergillus niger
and Talaromyces emersonii glucoamylases. TABLE-US-00018 TABLE 2
Glucoamylase Nmol/g DS Ethanol yields Pachykytospora papyracea 0.3
76.3 Aspergillus niger 47.2 Talaromyces emersonii 30.5
Pachykytospora papyracea 0.6 102.0 Aspergillus niger 87.2
Talaromyces emersonii 43.4
Example 10
Trametes cingulata Glucoamylase in Combination with Hybrid
Alpha-Amylase A from Rhizomucor pusillus for One Step
Fermentation
[0414] All treatments were evaluated via mini-scale fermentations.
410 g of ground corn was added to 590 g tap water. This mixture was
supplemented with 3.0 ml 1 g/L penicillin and 1 g of urea. The pH
of this slurry was adjusted to 4.5 with 5 N NaOH (initial pH,
before adjustment was about 3.8). Dry Solid (DS) level was
determined to be 35%. Approximately 5 g of this slurry was added to
20 ml vials. Each vial was dosed with the appropriate amount of
enzyme followed by addition of 200 micro liter yeast propagate/5 g
fermentation. Actual dosages were based on the exact weight of corn
slurry in each vial. Vials were incubated at 32.degree. C. 9
replicate fermentations of each treatment were run. Three
replicates were selected for 24 hour, 48 hour and 70 hour time
point analysis. Vials were vortexed at 24, 48 and 70 hours. The
time point analysis consisted of weighing the vials and prepping
the sample for HPLC. The HPLC preparation consisted of stopping the
reaction by addition of 50 micro liters of 40% H.sub.2SO.sub.4,
centrifuging, and filtering through a 0.45 micro m filter. Samples
awaiting HPLC analysis were stored at 4.degree. C.
[0415] Enzymes used in this study: TABLE-US-00019 % enzyme dose
AFAU/g DS Alpha-Amylase A AGU/g DS Alpha-Amylase A T. cingulata
from Rhizomucor T. cingulata from Trial # glucoamylase pusillus
glucoamylase Rhizomucor pusillus 1 100% 0% 0.43 0 2 90% 10% 0.387
0.01 3 80% 20% 0.344 0.02 4 70% 30% 0.301 0.03 5 60% 40% 0.258 0.04
6 45% 55% 0.1935 0.055 7 30% 70% 0.129 0.07 8 15% 85% 0.0645 0.085
9 0% 100% 0 0.1 Note: T. cingulata glucoamylase, 49 AGU/ml) and
hybrid Alpha-Amylase A from Rhizomucor pusillus (17 AFAU/ml) are
purified enzymes from Novozymes Japan. DS = dry solid.
Results
[0416] The synergistic effect of alpha-amylase and glucoamylase is
presented in a table below. When T. cingulata glucoamylase was used
alone in one step fermentation, it produced 54.1, 81.2 and 99.0 g/l
ethanol after 24, 48, and 70 hours fermentation, respectively. When
the hybrid alpha-amylase A from Rhizomucor pusillus is used alone
in fermentation, it produced 90.5, 124.6, and 138.1 g/l ethanol
after 24, 48, and 70 hour fermentation, respectively.
TABLE-US-00020 T. cingulata Hybrid Alpha- glucoamylase Amylase A
Ethanol (g/l) Ratio Trial # AGU/g DS AFAU/g DS 24 hrs 48 hrs 70 hrs
AGU/AFAU 1 0.430 0.000 54.1 81.2 99.0 N/A 2 0.387 0.010 88.5 130.7
145.0 38.70 3 0.344 0.020 92.9 132.1 145.9 17.20 4 0.301 0.030 96.7
135.3 146.6 10.03 5 0.258 0.040 96.1 136.6 147.1 6.45 6 0.194 0.055
97.1 135.5 145.6 3.52 7 0.129 0.070 95.4 132.9 144.6 1.84 8 0.065
0.085 93.3 130.4 142.9 0.76 9 0.000 0.100 90.5 124.6 138.1 0.00
[0417] The optimal ratio of T. cingulata glucoamylase to hybrid
Alpha-Amylase A from Rhizomucor pusillus alpha-amylase is about 6.5
AGU/AFAU (Table above). Essentially similar performance in term of
ethanol yield after 70 hours fermentation was observed in the range
of 0.76-38.7 AGU/AFAU ratio, indicating robust performance for a
broad activity ration range of the mixtures of T. cingulata
glucoamylase to hybrid Alpha-Amylase A.
Example 11
DNA Extraction and PCR Amplification of Leucopaxillus giganteus
[0418] 0.2-2 g of the spore forming layer (lamellas) of the fresh
fruit-bodies of Leucopaxillus giganteus were used for genomic DNA
extraction using FastDNA SPIN Kit for Soil (Qbiogene, USA)
according to the manufacturer's instructions.
[0419] PCR reaction was done on genome DNA with the degenerated
primers ArAF1 and ArAR3 TABLE-US-00021 ArAF1
5'-CRTRCTYDVCAACATYGG-3' (SEQ ID NO: 7) ArAR3 5'
GTCAGARCADGGYTGRRASGTG-3' (SEQ ID NO: 8)
wherein D=A or G or T; R=A or G; S=C or G; V=A or C or G; Y=C or
T
[0420] The amplification reaction (13 microL) was composed of 1
microL genome DNA solution, 1 micro M primer ArAF1 (25
.mu.mol/microL), 1 micro M primer ArAR3 (25 .mu.mol/microL), 11
microL Extensor Hi-Fidelity PCR Master Mix (ABgene, UK). The
reaction was incubated in a DNA Engine Dyad PTC-0220 (MJ Research,
USA) programmed as follows: 1 cycle at 94.degree. C. for 2 minutes;
20 cycles each at 94.degree. C. for 30 seconds, 65.degree. C. for
45 seconds, with an annealing temperature decline of 1.degree. C.
per cycle, and 72.degree. C. for 1 minute 30 seconds; followed by
20 cycles each at 94.degree. C. for 30 seconds, 45.degree. C. for
45 seconds and 72.degree. C. for 1 minute 30 seconds; 1 cycle at
72.degree. C. for 7 minutes; and a hold at 4.degree. C. The PCR
product was purified using ExoSAP-IT (USB, USA) according to the
manufacturer's instructions and sequenced using the primers as used
in the amplification reaction. The sequence was subsequently
compared to the Aspergillus niger glucoamylase gene, showing that
the PCR product encoded a part of a glucoamylase.
[0421] From the partial sequence of the Leucopaxillus giganteus
glucoamylase more gene sequence was obtained with PCR based gene
walking using the Vectorette Kit from SIGMA-Genosys. The gene
walking was performed as described in the manufacturer's protocol.
0.15 micro 9 genomic DNA of Leucopaxillus giganteus was digested
with EcoRI, BamHI and HindIII, independently. The digested DNA was
ligated with the corresponding Vectorette units supplied by the
manufacture using a DNA Engine Dyad PTC-0220 (MJ Research, USA)
programmed as follows: 1 cycle at 16.degree. C. for 60 minutes; 4
cycles each at 37.degree. C. for 20 minutes, 16.degree. C. for 60
minutes, 37.degree. C. for 10 minutes; followed by 1 cycle at
16.degree. C. for 60 minutes and a hold at 4.degree. C. The
ligation reactions were subsequent diluted 5 times with sterile
water.
[0422] PCR reactions with linker-ligated genome DNA of the
Leucopaxillus giganteus as template was performed with a DNA Engine
Dyad PTC-0220 (MJ Research, USA) programmed as follows: 1 cycle at
94.degree. C. for 2 minutes; 40 cycles each at 94.degree. C. for 15
seconds, 72.degree. C. for 1 minute, 72.degree. C. for 1 minute, 1
cycle at 72.degree. C. for 7 minutes; and a hold at 4.degree. C.
using the supplied Vectorette primer and the specific Leucopaxillus
giganteus AMG primers Nc1R2 and NC1F0, respectively, as shown
below. TABLE-US-00022 Nc1R2: (SEQ ID NO:31)
5'-GGTAGACTAGTTACCTCGTTGG-3' Nc1F0: (SEQ ID NO:32)
5'-GCTTCCCTAGCCACTGCCATTGG-3'
[0423] The amplification reactions (12.5 microL) were composed of
0.5 microL of linker-ligated genome DNAs, 400 nM Vectorette primer,
400 nM Leucopaxillus giganteus specific primer, 11 microL Extensor
Hi-Fidelity PCR Master Mix (ABgene, UK).
[0424] After the PCR reaction the PCR products were purified using
ExoSAP-IT (USB, USA) according to the manufacturer's instructions
and sequenced and subsequently compared to the Aspergillus niger
glucoamylase gene.
[0425] A 1.7 kb amplified band was obtained by the PCR reaction
from HindIII digested genome DNA amplified with the primer Nc1R2.
Sequencing of the PCR product using this primer showed that it
encoded the remaining 600 base pairs of the glucoamylase gene in
the 5' direction.
[0426] A 1.8 amplified band was obtained by the PCR reaction from
HindIII digested genome DNA amplified with the primer Nc1F0.
Sequencing of the PCR product using this primer showed that it
encoded further approximately 530 base pairs of the glucoamylase
gene, however not reaching the end of the gene. Therefore, an
additional sequencing primer Nc1F2, were designed based on the
newly obtained additional sequence of the glucoamylase gene. Using
Nc1F2 as a downstream primer of Nc1F0 on the same PCR product
showed that it encoded the remaining approximately 520 base pairs
of the glucoamylase gene in the 3' direction. TABLE-US-00023 Nc1F2
(SEQ ID NO:33) 5' GTTGATTTAACTTGGAGCTATGC
Example 12
Cloning and Expression of Leucopaxillus giganteus Glucoamylase
[0427] From the partial sequence of Leucopaxillus giganteus
glucoamylase more gene sequence was obtained.
[0428] The following PCR cloning primers were used: TABLE-US-00024
Forward primer: (SEQ ID NO:34) 5'
TCCCTTGGATCCAGGATGCATTTCTCTGTCCTCTC 3' BamHI Reverse primer: (SEQ
ID NO:35) 5' CTTATCCTCGAGCTACTTCCACGAGTCATTCTGG 3' Xhol
[0429] PCR was made with gDNA from Leucopaxillus giganteus as
template using Phusion as polymerase and the above primers
introducing respectively BamHI and XhoI. 5 micro L of the PCR
product was tested in a 1% agarose gel, and showed a band at about
2.2 kb. The PCR product was purified on a QIAquick column.
[0430] The purified product and Aspergillus vector pEN12516
Leucopaxillus giganteus (see WO 2004/069872) were digested with
BamHI and XhoI. The vector and insert fragments were purified from
a 1% preparative agarose gel using the QIAquick method. The 2.2 kb
fragment was ligated into the vector pEN12516 and transformed into
TOP10 E. coli competent cells. The resulting plasmid was termed as
pEN13372.
Transformation in Aspergillus niger
[0431] Protoplasts of the Aspergillus niger strain Mbin119 (see WO
2004/090155) were made. About 5 micro g of pEN13372 was transformed
into the protoplasts. The resulting Aspergillus niger transformants
were tested for glucoamylase activity.
Example 13
Debranching Activity Toward Pullulan of Trametes cingulata
Glucoamylase
[0432] The alpha-1,6-debranching activity of glucoamylases derived
from Trametes cingulata, Athelia rolfsii, Aspergillus niger and
Talaromyces emersonii was investigated.
[0433] Pullulan (MW 50,000.about.100,000) was dissolved in MilliQ
water and added into a reaction mixture to a 3% final concentration
containing 50 mM NaAc buffer, pH 4.0, with enzyme dosage of 0.42
micro 9 enzyme/mg pullulan at 37.degree. C. Oligosaccharide profile
was analyzed periodically by HPLC.
[0434] The result of the test is displayed in FIG. 1.
[0435] 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.
[0436] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
43 1 2166 DNA Trametes cingulata sig_peptide (1)..(54) CDS
(1)..(162) mat_peptide (55)..(2166) Intron (163)..(247) CDS
(248)..(521) Intron (522)..(577) CDS (578)..(722) Intron
(723)..(772) CDS (773)..(932) Intron (933)..(1001) CDS
(1002)..(1277) Intron (1278)..(1341) CDS (1342)..(1807)
misc_feature (1744)..(1773) Linker region misc_feature
(1774)..(2166) binding domain Intron (1808)..(1864) CDS
(1865)..(1963) Intron (1964)..(2023) CDS (2024)..(2163) 1 atg cgt
ttc acg ctc ctc acc tcc ctc ctg ggc ctc gcc ctc ggc gcg 48 Met Arg
Phe Thr Leu Leu Thr Ser Leu Leu Gly Leu Ala Leu Gly Ala -15 -10 -5
ttc gcg cag tcg agt gcg gcc gac gcg tac gtc gcg tcc gaa tcg ccc 96
Phe Ala Gln Ser Ser Ala Ala Asp Ala Tyr Val Ala Ser Glu Ser Pro -1
1 5 10 atc gcc aag gcg ggt gtg ctc gcc aac atc ggg ccc agc ggc tcc
aag 144 Ile Ala Lys Ala Gly Val Leu Ala Asn Ile Gly Pro Ser Gly Ser
Lys 15 20 25 30 tcc aac gga gca aag gca agtgacacag tgacactccg
gggcgcccat 192 Ser Asn Gly Ala Lys Ala 35 gcttcattct tctgtgcaca
tggtagcgct gacatatcgt tgtttttgac agccc ggc 250 Gly atc gtg att gca
agt ccg agc aca tcc aac ccg aac tac ctg tac aca 298 Ile Val Ile Ala
Ser Pro Ser Thr Ser Asn Pro Asn Tyr Leu Tyr Thr 40 45 50 tgg acg
cgc gac tcg tcc ctc gtg ttc aag gcg ctc atc gac cag ttc 346 Trp Thr
Arg Asp Ser Ser Leu Val Phe Lys Ala Leu Ile Asp Gln Phe 55 60 65
acc act ggc gaa gat acc tcg ctc cga act ctg att gac gag ttc acc 394
Thr Thr Gly Glu Asp Thr Ser Leu Arg Thr Leu Ile Asp Glu Phe Thr 70
75 80 85 tcg gcg gag gcc ata ctc cag cag gtg ccg aac ccg agc ggg
aca gtc 442 Ser Ala Glu Ala Ile Leu Gln Gln Val Pro Asn Pro Ser Gly
Thr Val 90 95 100 agc act gga ggc ctc ggc gag ccc aag ttc aac atc
gac gag acc gcg 490 Ser Thr Gly Gly Leu Gly Glu Pro Lys Phe Asn Ile
Asp Glu Thr Ala 105 110 115 ttc acg gat gcc tgg ggt cgt cct cag cgc
g gtaagtcgga ggttgcctcg 541 Phe Thr Asp Ala Trp Gly Arg Pro Gln Arg
120 125 acggagatac gcccagactg acttcaagac tctcag at ggt ccc gct ctc
cgg 594 Asp Gly Pro Ala Leu Arg 130 gcg act gcc atc atc acc tac gcc
aac tgg ctc ctc gac aac aag aac 642 Ala Thr Ala Ile Ile Thr Tyr Ala
Asn Trp Leu Leu Asp Asn Lys Asn 135 140 145 acg acc tac gtg acc aac
act ctc tgg cct atc atc aag ctc gac ctc 690 Thr Thr Tyr Val Thr Asn
Thr Leu Trp Pro Ile Ile Lys Leu Asp Leu 150 155 160 165 gac tac gtc
gcc agc aac tgg aac cag tcc ac gtatgttctc taaattctct 742 Asp Tyr
Val Ala Ser Asn Trp Asn Gln Ser Thr 170 175 cccgtgggta accagtctga
acgttcatag g ttt gat ctc tgg gag gag att 794 Phe Asp Leu Trp Glu
Glu Ile 180 aac tcc tcg tcg ttc ttc act acc gcc gtc cag cac cgt gct
ctg cgc 842 Asn Ser Ser Ser Phe Phe Thr Thr Ala Val Gln His Arg Ala
Leu Arg 185 190 195 gag ggc gcg act ttc gct aat cgc atc gga caa acc
tcg gtg gtc agc 890 Glu Gly Ala Thr Phe Ala Asn Arg Ile Gly Gln Thr
Ser Val Val Ser 200 205 210 215 ggg tac acc acc caa gca aac aac ctt
ctc tgc ttc ctg cag 932 Gly Tyr Thr Thr Gln Ala Asn Asn Leu Leu Cys
Phe Leu Gln 220 225 gcagtctatc ccgtcacacg tctgtctgtt tccgttttcc
cacagctcac ctcgtcccgg 992 gccctgtag tcg tac tgg aac ccc acc ggc ggc
tat atc acc gca aac acg 1043 Ser Tyr Trp Asn Pro Thr Gly Gly Tyr
Ile Thr Ala Asn Thr 230 235 240 ggc ggc ggc cgc tct ggc aag gac gcg
aac acc gtt ctc acg tcg atc 1091 Gly Gly Gly Arg Ser Gly Lys Asp
Ala Asn Thr Val Leu Thr Ser Ile 245 250 255 cac acc ttc gac ccg gcc
gct gga tgc gac gct gtt acg ttc cag ccg 1139 His Thr Phe Asp Pro
Ala Ala Gly Cys Asp Ala Val Thr Phe Gln Pro 260 265 270 275 tgc tcg
gac aag gcg ctg tcg aac ttg aag gtg tac gtc gat gcg ttc 1187 Cys
Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Tyr Val Asp Ala Phe 280 285
290 cgc tcg atc tac tcc atc aac agc ggg atc gcc tcg aat gcg gcc gtt
1235 Arg Ser Ile Tyr Ser Ile Asn Ser Gly Ile Ala Ser Asn Ala Ala
Val 295 300 305 gct acc ggc cgc tac ccc gag gac agc tac atg ggc gga
aac 1277 Ala Thr Gly Arg Tyr Pro Glu Asp Ser Tyr Met Gly Gly Asn
310 315 320 gtgagcgacc atttctgtgc gtacaccgcg gtcgcgttaa ctgagatgtt
ctcctctcct 1337 gtag cca tgg tac ctc acc acc tcc gcc gtc gct gag
cag ctc tac gat 1386 Pro Trp Tyr Leu Thr Thr Ser Ala Val Ala Glu
Gln Leu Tyr Asp 325 330 335 gcg ctc att gtg tgg aac aag ctt ggc gcc
ctg aac gtc acg agc acc 1434 Ala Leu Ile Val Trp Asn Lys Leu Gly
Ala Leu Asn Val Thr Ser Thr 340 345 350 tcc ctc ccc ttc ttc cag cag
ttc tcg tca ggc gtc acc gtc ggc acc 1482 Ser Leu Pro Phe Phe Gln
Gln Phe Ser Ser Gly Val Thr Val Gly Thr 355 360 365 tat gcc tca tcc
tcg tcc acc ttc aag acg ctc act tcc gcc atc aag 1530 Tyr Ala Ser
Ser Ser Ser Thr Phe Lys Thr Leu Thr Ser Ala Ile Lys 370 375 380 acc
ttc gcc gac ggc ttc ctc gcg gtc aac gcc aag tac acg ccc tcg 1578
Thr Phe Ala Asp Gly Phe Leu Ala Val Asn Ala Lys Tyr Thr Pro Ser 385
390 395 400 aac ggc ggc ctt gct gaa cag tac agc cgg agc aac ggc tcg
ccc gtc 1626 Asn Gly Gly Leu Ala Glu Gln Tyr Ser Arg Ser Asn Gly
Ser Pro Val 405 410 415 agc gct gtg gac ctg acg tgg agc tat gct gct
gcc ctc acg tcg ttt 1674 Ser Ala Val Asp Leu Thr Trp Ser Tyr Ala
Ala Ala Leu Thr Ser Phe 420 425 430 gct gcg cgc tca ggc aag acg tat
gcg agc tgg ggc gcg gcg ggt ttg 1722 Ala Ala Arg Ser Gly Lys Thr
Tyr Ala Ser Trp Gly Ala Ala Gly Leu 435 440 445 act gtc ccg acg act
tgc tcg ggg agt ggc ggt gct ggg act gtg gcc 1770 Thr Val Pro Thr
Thr Cys Ser Gly Ser Gly Gly Ala Gly Thr Val Ala 450 455 460 gtc acc
ttc aac gtg cag gcg acc acc gtg ttc ggc g gtgagtacgc 1817 Val Thr
Phe Asn Val Gln Ala Thr Thr Val Phe Gly 465 470 475 catcgtatgc
tactagggca gttactcata gcttgtcgga cttgtag ag aac att 1872 Glu Asn
Ile tac atc aca ggc tcg gtc ccc gct ctc cag aac tgg tcg ccc gac aac
1920 Tyr Ile Thr Gly Ser Val Pro Ala Leu Gln Asn Trp Ser Pro Asp
Asn 480 485 490 495 gcg ctc atc ctc tca gcg gcc aac tac ccc act tgg
agc agt a 1963 Ala Leu Ile Leu Ser Ala Ala Asn Tyr Pro Thr Trp Ser
Ser 500 505 cgtctgaacc gccttcagcc tgcttcatac gttcgctgac atcgggcatc
catctagtca 2023 cc gtg aac ctg ccg gcg agc acg acg atc gag tac aag
tac att cgc 2070 Thr Val Asn Leu Pro Ala Ser Thr Thr Ile Glu Tyr
Lys Tyr Ile Arg 515 520 525 aag ttc aac ggc gcg gtc acc tgg gag tcc
gac ccg aac aac tcg atc 2118 Lys Phe Asn Gly Ala Val Thr Trp Glu
Ser Asp Pro Asn Asn Ser Ile 530 535 540 acg acg ccc gcg agc ggc acg
ttc acc cag aac gac acc tgg cgg tag 2166 Thr Thr Pro Ala Ser Gly
Thr Phe Thr Gln Asn Asp Thr Trp Arg 545 550 555 2 574 PRT Trametes
cingulata 2 Met Arg Phe Thr Leu Leu Thr Ser Leu Leu Gly Leu Ala Leu
Gly Ala -15 -10 -5 Phe Ala Gln Ser Ser Ala Ala Asp Ala Tyr Val Ala
Ser Glu Ser Pro -1 1 5 10 Ile Ala Lys Ala Gly Val Leu Ala Asn Ile
Gly Pro Ser Gly Ser Lys 15 20 25 30 Ser Asn Gly Ala Lys Ala Gly Ile
Val Ile Ala Ser Pro Ser Thr Ser 35 40 45 Asn Pro Asn Tyr Leu Tyr
Thr Trp Thr Arg Asp Ser Ser Leu Val Phe 50 55 60 Lys Ala Leu Ile
Asp Gln Phe Thr Thr Gly Glu Asp Thr Ser Leu Arg 65 70 75 Thr Leu
Ile Asp Glu Phe Thr Ser Ala Glu Ala Ile Leu Gln Gln Val 80 85 90
Pro Asn Pro Ser Gly Thr Val Ser Thr Gly Gly Leu Gly Glu Pro Lys 95
100 105 110 Phe Asn Ile Asp Glu Thr Ala Phe Thr Asp Ala Trp Gly Arg
Pro Gln 115 120 125 Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Ile Ile
Thr Tyr Ala Asn 130 135 140 Trp Leu Leu Asp Asn Lys Asn Thr Thr Tyr
Val Thr Asn Thr Leu Trp 145 150 155 Pro Ile Ile Lys Leu Asp Leu Asp
Tyr Val Ala Ser Asn Trp Asn Gln 160 165 170 Ser Thr Phe Asp Leu Trp
Glu Glu Ile Asn Ser Ser Ser Phe Phe Thr 175 180 185 190 Thr Ala Val
Gln His Arg Ala Leu Arg Glu Gly Ala Thr Phe Ala Asn 195 200 205 Arg
Ile Gly Gln Thr Ser Val Val Ser Gly Tyr Thr Thr Gln Ala Asn 210 215
220 Asn Leu Leu Cys Phe Leu Gln Ser Tyr Trp Asn Pro Thr Gly Gly Tyr
225 230 235 Ile Thr Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala
Asn Thr 240 245 250 Val Leu Thr Ser Ile His Thr Phe Asp Pro Ala Ala
Gly Cys Asp Ala 255 260 265 270 Val Thr Phe Gln Pro Cys Ser Asp Lys
Ala Leu Ser Asn Leu Lys Val 275 280 285 Tyr Val Asp Ala Phe Arg Ser
Ile Tyr Ser Ile Asn Ser Gly Ile Ala 290 295 300 Ser Asn Ala Ala Val
Ala Thr Gly Arg Tyr Pro Glu Asp Ser Tyr Met 305 310 315 Gly Gly Asn
Pro Trp Tyr Leu Thr Thr Ser Ala Val Ala Glu Gln Leu 320 325 330 Tyr
Asp Ala Leu Ile Val Trp Asn Lys Leu Gly Ala Leu Asn Val Thr 335 340
345 350 Ser Thr Ser Leu Pro Phe Phe Gln Gln Phe Ser Ser Gly Val Thr
Val 355 360 365 Gly Thr Tyr Ala Ser Ser Ser Ser Thr Phe Lys Thr Leu
Thr Ser Ala 370 375 380 Ile Lys Thr Phe Ala Asp Gly Phe Leu Ala Val
Asn Ala Lys Tyr Thr 385 390 395 Pro Ser Asn Gly Gly Leu Ala Glu Gln
Tyr Ser Arg Ser Asn Gly Ser 400 405 410 Pro Val Ser Ala Val Asp Leu
Thr Trp Ser Tyr Ala Ala Ala Leu Thr 415 420 425 430 Ser Phe Ala Ala
Arg Ser Gly Lys Thr Tyr Ala Ser Trp Gly Ala Ala 435 440 445 Gly Leu
Thr Val Pro Thr Thr Cys Ser Gly Ser Gly Gly Ala Gly Thr 450 455 460
Val Ala Val Thr Phe Asn Val Gln Ala Thr Thr Val Phe Gly Glu Asn 465
470 475 Ile Tyr Ile Thr Gly Ser Val Pro Ala Leu Gln Asn Trp Ser Pro
Asp 480 485 490 Asn Ala Leu Ile Leu Ser Ala Ala Asn Tyr Pro Thr Trp
Ser Ser Thr 495 500 505 510 Val Asn Leu Pro Ala Ser Thr Thr Ile Glu
Tyr Lys Tyr Ile Arg Lys 515 520 525 Phe Asn Gly Ala Val Thr Trp Glu
Ser Asp Pro Asn Asn Ser Ile Thr 530 535 540 Thr Pro Ala Ser Gly Thr
Phe Thr Gln Asn Asp Thr Trp Arg 545 550 555 3 1725 DNA Trametes
cingulata misc_feature (1)..(1725) cDNA misc_feature (55)..(1725)
coding region of cDNA misc_feature (1420)..(1725) binding domain 3
atgcgtttca cgctcctcac ctccctcctg ggcctcgccc tcggcgcgtt cgcgcagtcg
60 agtgcggccg acgcgtacgt cgcgtccgaa tcgcccatcg ccaaggcggg
tgtgctcgcc 120 aacatcgggc ccagcggctc caagtccaac ggagcaaagg
caggcatcgt gattgcaagt 180 ccgagcacat ccaacccgaa ctacctgtac
acatggacgc gcgactcgtc cctcgtgttc 240 aaggcgctca tcgaccagtt
caccactggc gaagatacct cgctccgaac tctgattgac 300 gagttcacct
cggcggaggc catactccag caggtgccga acccgagcgg gacagtcagc 360
actggaggcc tcggcgagcc caagttcaac atcgacgaga ccgcgttcac ggatgcctgg
420 ggtcgtcctc agcgcgatgg tcccgctctc cgggcgactg ccatcatcac
ctacgccaac 480 tggctcctcg acaacaagaa cacgacctac gtgaccaaca
ctctctggcc tatcatcaag 540 ctcgacctcg actacgtcgc cagcaactgg
aaccagtcca cgtttgatct ctgggaggag 600 attaactcct cgtcgttctt
cactaccgcc gtccagcacc gtgctctgcg cgagggcgcg 660 actttcgcta
atcgcatcgg acaaacctcg gtggtcagcg ggtacaccac ccaagcaaac 720
aaccttctct gcttcctgca gtcgtactgg aaccccaccg gcggctatat caccgcaaac
780 acgggcggcg gccgctctgg caaggacgcg aacaccgttc tcacgtcgat
ccacaccttc 840 gacccggccg ctggatgcga cgctgttacg ttccagccgt
gctcggacaa ggcgctgtcg 900 aacttgaagg tgtacgtcga tgcgttccgc
tcgatctact ccatcaacag cgggatcgcc 960 tcgaatgcgg ccgttgctac
cggccgctac cccgaggaca gctacatggg cggaaaccca 1020 tggtacctca
ccacctccgc cgtcgctgag cagctctacg atgcgctcat tgtgtggaac 1080
aagcttggcg ccctgaacgt cacgagcacc tccctcccct tcttccagca gttctcgtca
1140 ggcgtcaccg tcggcaccta tgcctcatcc tcgtccacct tcaagacgct
cacttccgcc 1200 atcaagacct tcgccgacgg cttcctcgcg gtcaacgcca
agtacacgcc ctcgaacggc 1260 ggccttgctg aacagtacag ccggagcaac
ggctcgcccg tcagcgctgt ggacctgacg 1320 tggagctatg ctgctgccct
cacgtcgttt gctgcgcgct caggcaagac gtatgcgagc 1380 tggggcgcgg
cgggtttgac tgtcccgacg acttgctcgg ggagtggcgg tgctgggact 1440
gtggccgtca ccttcaacgt gcaggcgacc accgtgttcg gcgagaacat ttacatcaca
1500 ggctcggtcc ccgctctcca gaactggtcg cccgacaacg cgctcatcct
ctcagcggcc 1560 aactacccca cttggagcag taccgtgaac ctgccggcga
gcacgacgat cgagtacaag 1620 tacattcgca agttcaacgg cgcggtcacc
tgggagtccg acccgaacaa ctcgatcacg 1680 acgcccgcga gcggcacgtt
cacccagaac gacacctggc ggtag 1725 4 2189 DNA Pachykytospora
papyracea CDS (1)..(159) sig_peptide (1)..(54) mat_peptide
(55)..(2186) Intron (160)..(238) CDS (239)..(720) Intron
(516)..(572) Intron (721)..(782) CDS (783)..(942) Intron
(943)..(1005) CDS (1006)..(1281) Intron (1282)..(1340) CDS
(1341)..(1803) misc_feature (1743)..(1769) linker misc_feature
(1770)..(2189) binding region Intron (1804)..(1882) CDS
(1883)..(1978) Intron (1979)..(2043) CDS (2044)..(2186) 4 atg cgc
ttc acc ctc ctc tcc tcc ctc gtc gcc ctc gcc acc ggc gcg 48 Met Arg
Phe Thr Leu Leu Ser Ser Leu Val Ala Leu Ala Thr Gly Ala -15 -10 -5
ttc gcc cag acc agc cag gcc gac gcg tac gtc aag tcc gag ggc ccc 96
Phe Ala Gln Thr Ser Gln Ala Asp Ala Tyr Val Lys Ser Glu Gly Pro -1
1 5 10 atc gcg aag gcg ggc ctc ctc gcc aac atc ggg ccc agc ggc tcc
aag 144 Ile Ala Lys Ala Gly Leu Leu Ala Asn Ile Gly Pro Ser Gly Ser
Lys 15 20 25 30 tcg cac ggg gcg aag gtgcgcttct ctttttccca
ttctacgtcg cttaaagcgc 199 Ser His Gly Ala Lys 35 gctcatacat
gtgcatgacc gcgttccgcg tgcgcgcag gcc ggt ctc gtc gtc 253 Ala Gly Leu
Val Val 40 gcc tcc ccc agc acg tcg gac ccc gac tac gtc tac acc tgg
acg cgt 301 Ala Ser Pro Ser Thr Ser Asp Pro Asp Tyr Val Tyr Thr Trp
Thr Arg 45 50 55 gat tcg tca ctc gtc ttc aag act atc atc gac cag
ttc acc tcc ggg 349 Asp Ser Ser Leu Val Phe Lys Thr Ile Ile Asp Gln
Phe Thr Ser Gly 60 65 70 gaa gac acc tcc ctc cgc aca ctc att gac
cag ttc act agc gcg gag 397 Glu Asp Thr Ser Leu Arg Thr Leu Ile Asp
Gln Phe Thr Ser Ala Glu 75 80 85 aag gac ctc cag cag acg tcc aac
cct agt ggc act gtt tcc acc ggc 445 Lys Asp Leu Gln Gln Thr Ser Asn
Pro Ser Gly Thr Val Ser Thr Gly 90 95 100 ggt ctc ggc gag ccc aag
ttc aac atc gat ggg tcc gcg ttc acc ggt 493 Gly Leu Gly Glu Pro Lys
Phe Asn Ile Asp Gly Ser Ala Phe Thr Gly 105 110 115 120 gcc tgg ggt
cgc cct cag cgc ggt atg cac act cta cca cag ttg aag 541 Ala Trp Gly
Arg Pro Gln Arg Gly Met His Thr Leu Pro Gln Leu Lys 125 130 135 ctt
gtt aag cgc tta cat gtt ttg tgc aca gac ggt cct gct ctc cgc 589 Leu
Val Lys Arg Leu His Val Leu Cys Thr Asp Gly Pro Ala Leu Arg 140 145
150 gcg act gct atc ata gcc tac gct aac tgg ctg ctc gac aac aac aac
637 Ala Thr Ala Ile Ile Ala Tyr Ala Asn Trp Leu Leu Asp Asn Asn Asn
155 160 165 ggc acg tcc tac gtc acc aac acc ctc tgg ccc atc atc aag
ctt gac 685 Gly Thr Ser Tyr Val Thr Asn Thr Leu Trp Pro Ile Ile Lys
Leu Asp 170 175 180 ttg gac tac acc cag aac aac tgg aac cag tcg ac
gtaagttcat 730 Leu Asp Tyr Thr Gln Asn Asn Trp Asn Gln Ser Thr 185
190 195 tattccagct ttggctgcta gaactgcatt gatcctcatg tcttatgccc ag g
ttc 786 Phe gac ctt tgg gag gag gtc aac tcc tcc tct ttc ttc acg act
gcc gtc 834 Asp Leu
Trp Glu Glu Val Asn Ser Ser Ser Phe Phe Thr Thr Ala Val 200 205 210
cag cac cgt gct ctc cgc gag ggt atc gcc ttc gcg aag aag atc ggc 882
Gln His Arg Ala Leu Arg Glu Gly Ile Ala Phe Ala Lys Lys Ile Gly 215
220 225 caa acg tcg gtc gtg agc ggc tac acc acg cag gcg acc aac ctt
ctc 930 Gln Thr Ser Val Val Ser Gly Tyr Thr Thr Gln Ala Thr Asn Leu
Leu 230 235 240 245 tgc ttc ctg cag gtcagtgtgc atgtgcagca
cgccttatgg ctatagctta 982 Cys Phe Leu Gln acccgtgttc cgcatcttcg cag
tcg tac tgg aac ccc tcg ggc ggc tat gtc 1035 Ser Tyr Trp Asn Pro
Ser Gly Gly Tyr Val 250 255 act gcg aac aca ggc ggc ggc cgg tcc ggc
aag gac tcg aac acc gtc 1083 Thr Ala Asn Thr Gly Gly Gly Arg Ser
Gly Lys Asp Ser Asn Thr Val 260 265 270 275 ctg acc tcg atc cac acc
ttc gac ccc gcc gct ggc tgc gac gcc gcg 1131 Leu Thr Ser Ile His
Thr Phe Asp Pro Ala Ala Gly Cys Asp Ala Ala 280 285 290 acg ttc cag
ccg tgc tct gac aag gcc ctg tcc aac ctc aag gtc tac 1179 Thr Phe
Gln Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Tyr 295 300 305
gtc gac tcg ttc cgt tcc atc tac tcc atc aac agt ggc atc gcc tcc
1227 Val Asp Ser Phe Arg Ser Ile Tyr Ser Ile Asn Ser Gly Ile Ala
Ser 310 315 320 aac gcc gct gtc gct gtt ggc cgc tac ccc gag gat gtg
tac tac aac 1275 Asn Ala Ala Val Ala Val Gly Arg Tyr Pro Glu Asp
Val Tyr Tyr Asn 325 330 335 ggc aac gtgagttccg tgtcccctgc
atcattgtca acagcagaaa ctgaatccca 1331 Gly Asn 340 tccgcgtag ccc tgg
tac ctc tcc acg tcc gcc gtc gct gag cag ctc tac 1382 Pro Trp Tyr
Leu Ser Thr Ser Ala Val Ala Glu Gln Leu Tyr 345 350 355 gac gcg atc
atc gtc tgg aac aag ctc ggc tcg ctc gaa gtg acg agc 1430 Asp Ala
Ile Ile Val Trp Asn Lys Leu Gly Ser Leu Glu Val Thr Ser 360 365 370
acc tcg ctc gcg ttc ttc aag cag ctc tcc tcg gac gcc gcc gtc ggc
1478 Thr Ser Leu Ala Phe Phe Lys Gln Leu Ser Ser Asp Ala Ala Val
Gly 375 380 385 acc tac tcg tcc tcg tcc gcg acg ttc aag acg ctc act
gca gcc gcg 1526 Thr Tyr Ser Ser Ser Ser Ala Thr Phe Lys Thr Leu
Thr Ala Ala Ala 390 395 400 aag aca ctc gcg gat ggc ttc ctc gct gtg
aac gcg aag tac acg ccc 1574 Lys Thr Leu Ala Asp Gly Phe Leu Ala
Val Asn Ala Lys Tyr Thr Pro 405 410 415 tcg aac ggc ggc ctc gcg gag
cag ttc agc aag agc aac ggc tcg ccg 1622 Ser Asn Gly Gly Leu Ala
Glu Gln Phe Ser Lys Ser Asn Gly Ser Pro 420 425 430 435 ctc agc gcc
gtc gac ctc acg tgg agc tac gcc gcc gcg ctc acg tcc 1670 Leu Ser
Ala Val Asp Leu Thr Trp Ser Tyr Ala Ala Ala Leu Thr Ser 440 445 450
ttt gcc gcg cgt gag ggc aag acc ccc gcg agc tgg ggc gct gcg ggc
1718 Phe Ala Ala Arg Glu Gly Lys Thr Pro Ala Ser Trp Gly Ala Ala
Gly 455 460 465 ctc acc gtg ccg tcg acg tgc tcg ggt aac gcg ggc ccc
agc gtg aag 1766 Leu Thr Val Pro Ser Thr Cys Ser Gly Asn Ala Gly
Pro Ser Val Lys 470 475 480 gtg acg ttc aac gtc cag gct acg act acc
ttc ggc g gtcagtcctc 1813 Val Thr Phe Asn Val Gln Ala Thr Thr Thr
Phe Gly 485 490 495 ttctccaact cgtttcggtc ggtgatgttg agcattcgtc
tgacgtgtgt gtgttactgc 1873 tgcttgcag ag aac atc tac atc acc ggt aac
acc gct gcg ctc cag aac 1923 Glu Asn Ile Tyr Ile Thr Gly Asn Thr
Ala Ala Leu Gln Asn 500 505 tgg tcg ccc gat aac gcg ctc ctc ctc tct
gct gac aag tac ccc acc 1971 Trp Ser Pro Asp Asn Ala Leu Leu Leu
Ser Ala Asp Lys Tyr Pro Thr 510 515 520 525 tgg agc a gtacgtgtca
tctcatctcc agcctctcat attacgttgt ttgctcatct 2028 Trp Ser gcatgtgctt
cgcag tc acg ctc gac ctc ccc gcg aac acc gtc gtc gag 2078 Ile Thr
Leu Asp Leu Pro Ala Asn Thr Val Val Glu 530 535 tac aaa tac atc cgc
aag ttc aac ggc cag gtc acc tgg gaa tcg gac 2126 Tyr Lys Tyr Ile
Arg Lys Phe Asn Gly Gln Val Thr Trp Glu Ser Asp 540 545 550 555 ccc
aac aac tcg atc acg acg ccc gcc gac ggt acc ttc acc cag aac 2174
Pro Asn Asn Ser Ile Thr Thr Pro Ala Asp Gly Thr Phe Thr Gln Asn 560
565 570 gac acc tgg cgg tga 2189 Asp Thr Trp Arg 575 5 593 PRT
Pachykytospora papyracea 5 Met Arg Phe Thr Leu Leu Ser Ser Leu Val
Ala Leu Ala Thr Gly Ala -15 -10 -5 Phe Ala Gln Thr Ser Gln Ala Asp
Ala Tyr Val Lys Ser Glu Gly Pro -1 1 5 10 Ile Ala Lys Ala Gly Leu
Leu Ala Asn Ile Gly Pro Ser Gly Ser Lys 15 20 25 30 Ser His Gly Ala
Lys Ala Gly Leu Val Val Ala Ser Pro Ser Thr Ser 35 40 45 Asp Pro
Asp Tyr Val Tyr Thr Trp Thr Arg Asp Ser Ser Leu Val Phe 50 55 60
Lys Thr Ile Ile Asp Gln Phe Thr Ser Gly Glu Asp Thr Ser Leu Arg 65
70 75 Thr Leu Ile Asp Gln Phe Thr Ser Ala Glu Lys Asp Leu Gln Gln
Thr 80 85 90 Ser Asn Pro Ser Gly Thr Val Ser Thr Gly Gly Leu Gly
Glu Pro Lys 95 100 105 110 Phe Asn Ile Asp Gly Ser Ala Phe Thr Gly
Ala Trp Gly Arg Pro Gln 115 120 125 Arg Gly Met His Thr Leu Pro Gln
Leu Lys Leu Val Lys Arg Leu His 130 135 140 Val Leu Cys Thr Asp Gly
Pro Ala Leu Arg Ala Thr Ala Ile Ile Ala 145 150 155 Tyr Ala Asn Trp
Leu Leu Asp Asn Asn Asn Gly Thr Ser Tyr Val Thr 160 165 170 Asn Thr
Leu Trp Pro Ile Ile Lys Leu Asp Leu Asp Tyr Thr Gln Asn 175 180 185
190 Asn Trp Asn Gln Ser Thr Phe Asp Leu Trp Glu Glu Val Asn Ser Ser
195 200 205 Ser Phe Phe Thr Thr Ala Val Gln His Arg Ala Leu Arg Glu
Gly Ile 210 215 220 Ala Phe Ala Lys Lys Ile Gly Gln Thr Ser Val Val
Ser Gly Tyr Thr 225 230 235 Thr Gln Ala Thr Asn Leu Leu Cys Phe Leu
Gln Ser Tyr Trp Asn Pro 240 245 250 Ser Gly Gly Tyr Val Thr Ala Asn
Thr Gly Gly Gly Arg Ser Gly Lys 255 260 265 270 Asp Ser Asn Thr Val
Leu Thr Ser Ile His Thr Phe Asp Pro Ala Ala 275 280 285 Gly Cys Asp
Ala Ala Thr Phe Gln Pro Cys Ser Asp Lys Ala Leu Ser 290 295 300 Asn
Leu Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr Ser Ile Asn 305 310
315 Ser Gly Ile Ala Ser Asn Ala Ala Val Ala Val Gly Arg Tyr Pro Glu
320 325 330 Asp Val Tyr Tyr Asn Gly Asn Pro Trp Tyr Leu Ser Thr Ser
Ala Val 335 340 345 350 Ala Glu Gln Leu Tyr Asp Ala Ile Ile Val Trp
Asn Lys Leu Gly Ser 355 360 365 Leu Glu Val Thr Ser Thr Ser Leu Ala
Phe Phe Lys Gln Leu Ser Ser 370 375 380 Asp Ala Ala Val Gly Thr Tyr
Ser Ser Ser Ser Ala Thr Phe Lys Thr 385 390 395 Leu Thr Ala Ala Ala
Lys Thr Leu Ala Asp Gly Phe Leu Ala Val Asn 400 405 410 Ala Lys Tyr
Thr Pro Ser Asn Gly Gly Leu Ala Glu Gln Phe Ser Lys 415 420 425 430
Ser Asn Gly Ser Pro Leu Ser Ala Val Asp Leu Thr Trp Ser Tyr Ala 435
440 445 Ala Ala Leu Thr Ser Phe Ala Ala Arg Glu Gly Lys Thr Pro Ala
Ser 450 455 460 Trp Gly Ala Ala Gly Leu Thr Val Pro Ser Thr Cys Ser
Gly Asn Ala 465 470 475 Gly Pro Ser Val Lys Val Thr Phe Asn Val Gln
Ala Thr Thr Thr Phe 480 485 490 Gly Glu Asn Ile Tyr Ile Thr Gly Asn
Thr Ala Ala Leu Gln Asn Trp 495 500 505 510 Ser Pro Asp Asn Ala Leu
Leu Leu Ser Ala Asp Lys Tyr Pro Thr Trp 515 520 525 Ser Ile Thr Leu
Asp Leu Pro Ala Asn Thr Val Val Glu Tyr Lys Tyr 530 535 540 Ile Arg
Lys Phe Asn Gly Gln Val Thr Trp Glu Ser Asp Pro Asn Asn 545 550 555
Ser Ile Thr Thr Pro Ala Asp Gly Thr Phe Thr Gln Asn Asp Thr Trp 560
565 570 Arg 575 6 1725 DNA Pachykytospora papyracea misc_feature
(1)..(1725) cDNA misc_feature (55)..(1725) coding region of cDNA
misc_feature (1423)..(1725) binding domain 6 atgcgcttca ccctcctctc
ctccctcgtc gccctcgcca ccggcgcgtt cgcccagacc 60 agccaggccg
acgcgtacgt caagtccgag ggccccatcg cgaaggcggg cctcctcgcc 120
aacatcgggc ccagcggctc caagtcgcac ggggcgaagg ccggtctcgt cgtcgcctcc
180 cccagcacgt cggaccccga ctacgtctac acctggacgc gtgattcgtc
actcgtcttc 240 aagactatca tcgaccagtt cacctccggg gaagacacct
ccctccgcac actcattgac 300 cagttcacta gcgcggagaa ggacctccag
cagacgtcca accctagtgg cactgtttcc 360 accggcggtc tcggcgagcc
caagttcaac atcgatgggt ccgcgttcac cggtgcctgg 420 ggtcgccctc
agcgcgacgg tcctgctctc cgcgcgactg ctatcatagc ctacgctaac 480
tggctgctcg acaacaacaa cggcacgtcc tacgtcacca acaccctctg gcccatcatc
540 aagcttgact tggactacac ccagaacaac tggaaccagt cgacgttcga
cctttgggag 600 gaggtcaact cctcctcttt cttcacgact gccgtccagc
accgtgctct ccgcgagggt 660 atcgccttcg cgaagaagat cggccaaacg
tcggtcgtga gcggctacac cacgcaggcg 720 accaaccttc tctgcttcct
gcagtcgtac tggaacccct cgggcggcta tgtcactgcg 780 aacacaggcg
gcggccggtc cggcaaggac tcgaacaccg tcctgacctc gatccacacc 840
ttcgaccccg ccgctggctg cgacgccgcg acgttccagc cgtgctctga caaggccctg
900 tccaacctca aggtctacgt cgactcgttc cgttccatct actccatcaa
cagtggcatc 960 gcctccaacg ccgctgtcgc tgttggccgc taccccgagg
atgtgtacta caacggcaac 1020 ccctggtacc tctccacgtc cgccgtcgct
gagcagctct acgacgcgat catcgtctgg 1080 aacaagctcg gctcgctcga
agtgacgagc acctcgctcg cgttcttcaa gcagctctcc 1140 tcggacgccg
ccgtcggcac ctactcgtcc tcgtccgcga cgttcaagac gctcactgca 1200
gccgcgaaga cactcgcgga tggcttcctc gctgtgaacg cgaagtacac gccctcgaac
1260 ggcggcctcg cggagcagtt cagcaagagc aacggctcgc cgctcagcgc
cgtcgacctc 1320 acgtggagct acgccgccgc gctcacgtcc tttgccgcgc
gtgagggcaa gacccccgcg 1380 agctggggcg ctgcgggcct caccgtgccg
tcgacgtgct cgggtaacgc gggccccagc 1440 gtgaaggtga cgttcaacgt
ccaggctacg actaccttcg gcgagaacat ctacatcacc 1500 ggtaacaccg
ctgcgctcca gaactggtcg cccgataacg cgctcctcct ctctgctgac 1560
aagtacccca cctggagcat cacgctcgac ctccccgcga acaccgtcgt cgagtacaaa
1620 tacatccgca agttcaacgg ccaggtcacc tgggaatcgg accccaacaa
ctcgatcacg 1680 acgcccgccg acggtacctt cacccagaac gacacctggc ggtga
1725 7 18 DNA Artificial Degenerated Primer ArAF1 misc_feature
(2)..(2) R = A or G misc_feature (4)..(4) R = A or G misc_feature
(7)..(7) Y = C or T misc_feature (8)..(8) D = A or G or T
misc_feature (9)..(9) V = A or C or G misc_feature (16)..(16) Y = C
or T 7 crtrctydvc aacatygg 18 8 22 DNA Artificial Degenerated
Primer ArAF3 misc_feature (7)..(7) R = A or G misc_feature
(10)..(10) D = A or G or T misc_feature (13)..(13) Y = C or T
misc_feature (16)..(16) R = A or G misc_feature (17)..(17) R = A or
G misc_feature (19)..(19) S = C or G 8 gtcagarcad ggytgrrasg tg 22
9 23 DNA Artificial AM2F degenerated primer misc_feature (6)..(6) n
is inosine misc_feature (7)..(7) M = A or C misc_feature (9)..(9) N
= A or C or G or T misc_feature (12)..(12) N = A or C or G or T
misc_feature (15)..(15) R = A or G misc_feature (16)..(16) M = A or
C misc_feature (18)..(18) N = A or C or G or T misc_feature
(21)..(21) Y = C or T 9 tggggnmgnc cncarmgnga ygg 23 10 18 DNA
Artificial AM4R2 degenerated primer misc_feature (1)..(1) R = A or
G misc_feature (4)..(4) Y = C or T misc_feature (7)..(7) N = A or C
or G or T misc_feature (10)..(10) R = A or G misc_feature
(13)..(13) N = A or C or G or T misc_feature (15)..(15) K = G or T
misc_feature (16)..(16) n is a, c, g, or t misc_feature (136)..(16)
N = A or C or G or T 10 rtcytcnggr tanckncc 18 11 17 DNA Artificial
AMF3 degenerated primer misc_feature (3)..(3) Y = C or T
misc_feature (6)..(6) Y = C or T misc_feature (7)..(7) Y = C or T
misc_feature (9)..(9) N = A or C or G or T misc_feature (10)..(10)
Y = C or T misc_feature (15)..(15) R = A or G 11 taygayytny gggarga
17 12 21 DNA Artificial TraF1 primer misc_feature (1)..(21) TraF1
primer 12 tagtcgtact ggaaccccac c 21 13 35 DNA Artificial C1 primer
13 gtacatattg tcgttagaac gcgtaatacg actca 35 14 23 DNA Artificial
TC5' primer misc_feature (1)..(23) TC5' primer 14 cgtatatgtc
agcgctacca tgt 23 15 23 DNA Artificial TC3' primer misc_feature
(1)..(23) TC3' primer 15 aaacgtgagc gaccattttc tgt 23 16 36 DNA
Artificial TFF primer 16 tttggatcca ccatgcgttt cacgctcctc acctcc 36
17 30 DNA Artificial TFR primer 17 tttctcgagc taccgccagg tgtcattctg
30 18 23 DNA Artificial PP5' primer 18 cctccctgag tgagcgatgc tgc 23
19 23 DNA Artificial PP3' primer 19 caactccggc ctctcctcca gcg 23 20
36 DNA Artificial PPF primer 20 tttggatcca ccatgcgctt caccctcctc
tcctcc 36 21 30 DNA Artificial PPR primer 21 tttctcgagt caccgccagg
tgtcgttctg 30 22 31 PRT Aspergillus kawachii PEPTIDE (1)..(31)
Linker 22 Thr Thr Thr Thr Thr Thr Ala Ala Ala Thr Ser Thr Ser Lys
Ala Thr 1 5 10 15 Thr Ser Ser Ser Ser Ser Ser Ala Ala Ala Thr Thr
Ser Ser Ser 20 25 30 23 2494 DNA Leucopaxillus giganteus
misc_feature (29)..(29) n is a, c, g, or t misc_feature (38)..(38)
n is a, c, g, or t sig_peptide (66)..(128) CDS (66)..(319)
mat_peptide (117)..(2249) Intron (320)..(375) CDS (376)..(540)
Intron (541)..(591) CDS (592)..(605) Intron (606)..(664) CDS
(665)..(809) Intron (810)..(863) CDS (864)..(1023) Intron
(1024)..(1088) CDS (1089)..(1361) Intron (1362)..(1415) CDS
(1416)..(1896) misc_feature (1821)..(1853) Linker misc_feature
(1854)..(2249) binding domain Intron (1897)..(1954) CDS
(1955)..(2014) Intron (2015)..(2106) CDS (2107)..(2249) 23
tataaagagc gtcgcttcag cgatacctnt tcttcagngc atttcgcctc tcccttctaa
60 gcagg atg cat ttc tct gtc ctc tcc gta ttt ctc gcg att agt tct
gct 110 Met His Phe Ser Val Leu Ser Val Phe Leu Ala Ile Ser Ser Ala
-15 -10 -5 tgg gct cag tct agc gca gtc gat gcc tat ctc gct ctc gaa
tcc tcc 158 Trp Ala Gln Ser Ser Ala Val Asp Ala Tyr Leu Ala Leu Glu
Ser Ser -1 1 5 10 gtc gcc aag gcc ggg ttg ctc gcc aac att ggc cca
tct ggt tca aag 206 Val Ala Lys Ala Gly Leu Leu Ala Asn Ile Gly Pro
Ser Gly Ser Lys 15 20 25 30 tct tcg ggt gcc aag tct ggg att gtc att
gcg tcg cct tcg cat agc 254 Ser Ser Gly Ala Lys Ser Gly Ile Val Ile
Ala Ser Pro Ser His Ser 35 40 45 aac cct gac tac ctg ttc acc tgg
acc cgc gat tct tcg ctt gtg ttc 302 Asn Pro Asp Tyr Leu Phe Thr Trp
Thr Arg Asp Ser Ser Leu Val Phe 50 55 60 cag act atc atc aac ca
gtaggtgtct tcctcttcta ggtcgctgct 349 Gln Thr Ile Ile Asn Gln 65
tgtcgttgac acgaggacac gcccag g ttc acg ttg gga cac gac aat agt 400
Phe Thr Leu Gly His Asp Asn Ser 70 75 ttg agg cct gag att gac aat
ttt gtt gat tcc caa agg aag atc caa 448 Leu Arg Pro Glu Ile Asp Asn
Phe Val Asp Ser Gln Arg Lys Ile Gln 80 85 90 caa gtc tca aac cct
tcg gga act gtt agt tct ggc ggc ctt ggc gag 496 Gln Val Ser Asn Pro
Ser Gly Thr Val Ser Ser Gly Gly Leu Gly Glu 95 100 105 ccc aag ttc
aat atc gac gaa acc gcc ttt aca ggg gca tgg gg 540 Pro Lys Phe Asn
Ile Asp Glu Thr Ala Phe Thr Gly Ala Trp Gly 110 115 120 gtgagtcctt
cctggactgc gtcatataca taattcacag atattgtcta g g cgg 595 Arg ccc caa
cga g gtaactagtc taccatgatt accgggatgc aacatcaaca 645 Pro Gln Arg
125 gttttcgcat tatttgtag at gga cct gct ctc cga tcc acc gcg ctc att
696 Asp Gly Pro Ala Leu Arg Ser Thr Ala Leu Ile 130 135 acc tgg gcc
aat tac ctg atc gct aac agc aac aca tcc tac gtc acc 744 Thr Trp Ala
Asn Tyr Leu Ile Ala Asn Ser Asn Thr Ser Tyr Val Thr 140 145 150 aac
acc cta tgg ccc atc atc aaa ttg gac ctc gac tac gtc gcg
tcc 792 Asn Thr Leu Trp Pro Ile Ile Lys Leu Asp Leu Asp Tyr Val Ala
Ser 155 160 165 170 aac tgg aac cag act gg gtgagtcact tgactatttt
cgcaactttc 839 Asn Trp Asn Gln Thr Gly 175 ttggttcatg aaagctactc
ccag t ttc gat ttg tgg gaa gaa gta tcc tct 891 Phe Asp Leu Trp Glu
Glu Val Ser Ser 180 185 tct tcc ttc ttc act act gcg gtt caa cac cgc
tcc ctt cgc caa ggt 939 Ser Ser Phe Phe Thr Thr Ala Val Gln His Arg
Ser Leu Arg Gln Gly 190 195 200 gct tcc cta gcc act gcc att gga caa
acc tct gtc gtt cct ggc tac 987 Ala Ser Leu Ala Thr Ala Ile Gly Gln
Thr Ser Val Val Pro Gly Tyr 205 210 215 acc acc cag gcc aac aat ata
ctc tgc ttt caa cag gtggctcctt 1033 Thr Thr Gln Ala Asn Asn Ile Leu
Cys Phe Gln Gln 220 225 tctttctttt cttacaacta gcatacacga agaacctgac
actcaaattt gctag tcc 1091 Ser 230 tac tgg aac tca gct ggg tat atg
act gcc aat acc gga ggc ggg cgt 1139 Tyr Trp Asn Ser Ala Gly Tyr
Met Thr Ala Asn Thr Gly Gly Gly Arg 235 240 245 tct ggg aaa gac gcc
aac acc gtc ctc aca agt att cac aca ttc gat 1187 Ser Gly Lys Asp
Ala Asn Thr Val Leu Thr Ser Ile His Thr Phe Asp 250 255 260 ccc gat
gcc ggc tgc gat tcc atc act ttc caa cct tgt tca gac cgt 1235 Pro
Asp Ala Gly Cys Asp Ser Ile Thr Phe Gln Pro Cys Ser Asp Arg 265 270
275 gcg ctc atc aac ctt gtc aca tac gtc aat gca ttc cga agc atc tac
1283 Ala Leu Ile Asn Leu Val Thr Tyr Val Asn Ala Phe Arg Ser Ile
Tyr 280 285 290 gct atc aac gcg ggc atc gct aat aac caa ggc gtt gcc
act ggt agg 1331 Ala Ile Asn Ala Gly Ile Ala Asn Asn Gln Gly Val
Ala Thr Gly Arg 295 300 305 310 tat cct gaa gat ggc tac atg ggc gga
aac gtatgccttg tccactcgcc 1381 Tyr Pro Glu Asp Gly Tyr Met Gly Gly
Asn 315 320 gtccacagtc ctcgaagcct gatcgctgcc ttag cct tgg tat ctg
act act tta 1436 Pro Trp Tyr Leu Thr Thr Leu 325 gcc gtt tct gaa
cag ctc tac tac gct ctc tcc act tgg aag aaa cat 1484 Ala Val Ser
Glu Gln Leu Tyr Tyr Ala Leu Ser Thr Trp Lys Lys His 330 335 340 agc
tcc ctc acc att acg gcg aca tca caa cct ttt ttc gcg ctc ttc 1532
Ser Ser Leu Thr Ile Thr Ala Thr Ser Gln Pro Phe Phe Ala Leu Phe 345
350 355 tcg ccg ggt gtt gct act ggc aca tat gcg tcc tct acg act acc
tat 1580 Ser Pro Gly Val Ala Thr Gly Thr Tyr Ala Ser Ser Thr Thr
Thr Tyr 360 365 370 375 gct aca ctt act act gct att cag aat tac gcg
gat agc ttc atc gct 1628 Ala Thr Leu Thr Thr Ala Ile Gln Asn Tyr
Ala Asp Ser Phe Ile Ala 380 385 390 gtc gtg gct aag tat acg cct gcc
aat ggc gga ctg gcg gaa cag tac 1676 Val Val Ala Lys Tyr Thr Pro
Ala Asn Gly Gly Leu Ala Glu Gln Tyr 395 400 405 agc agg agt aac ggt
ttg ccc gtt agt gcc gtt gat tta act tgg agc 1724 Ser Arg Ser Asn
Gly Leu Pro Val Ser Ala Val Asp Leu Thr Trp Ser 410 415 420 tat gcc
gct ctc ttg acg gcg gct gat gcg cga gcg ggg cta aca ccc 1772 Tyr
Ala Ala Leu Leu Thr Ala Ala Asp Ala Arg Ala Gly Leu Thr Pro 425 430
435 gct gca tgg gga gca gcg ggg ttg acc gtg cca agc act tgc tct act
1820 Ala Ala Trp Gly Ala Ala Gly Leu Thr Val Pro Ser Thr Cys Ser
Thr 440 445 450 455 ggg ggt ggt tca aac cca ggt ggt gga ggg tcg gtc
tct gtt acg ttc 1868 Gly Gly Gly Ser Asn Pro Gly Gly Gly Gly Ser
Val Ser Val Thr Phe 460 465 470 aat gtt caa gct aca acc acc ttt ggt
g gtaggtccca ttcaacacgc 1916 Asn Val Gln Ala Thr Thr Thr Phe Gly
475 480 gcagattttg ctgggaaatc tcatgattgg tttgacag aa aac att ttt
ttg acc 1971 Glu Asn Ile Phe Leu Thr 485 ggc tcg atc aac gag tta
gct aac tgg tct cct gat aat gct c 2014 Gly Ser Ile Asn Glu Leu Ala
Asn Trp Ser Pro Asp Asn Ala 490 495 500 tcgccctctc tgcggccaat
tatcccacct ggagcagtca gtcccagtcc atcgctccac 2074 tacaagccat
caaccgctga ccatatctct ag ta acc gtc aac gtt ccc gca 2126 Leu Thr
Val Asn Val Pro Ala 505 agc act acg atc caa tac aag ttt atc cgt aaa
ttc aac gga gcc atc 2174 Ser Thr Thr Ile Gln Tyr Lys Phe Ile Arg
Lys Phe Asn Gly Ala Ile 510 515 520 acc tgg gag tcc gac ccg aat agg
cag atc aca acg ccg tct tcg gga 2222 Thr Trp Glu Ser Asp Pro Asn
Arg Gln Ile Thr Thr Pro Ser Ser Gly 525 530 535 agt ttt gtc cag aat
gac tcg tgg aag tagtcggtag ataagatgtg 2269 Ser Phe Val Gln Asn Asp
Ser Trp Lys 540 545 caagatgagg tccatggctc acccaaacgt tactcatagt
aaatttgata ctgaaatttg 2329 ttcagcacat gaaatcgtta ttcctcctct
gacgtttagt gaagaataaa gcgagatccc 2389 gcccaggaag gtgctatagt
gtagtggtta tcactcggga ttttgatgtg gtactaagta 2449 tcatacaaca
ttcccgagac ccaggttcga accctggtag cacct 2494 24 565 PRT
Leucopaxillus giganteus 24 Met His Phe Ser Val Leu Ser Val Phe Leu
Ala Ile Ser Ser Ala Trp -15 -10 -5 Ala Gln Ser Ser Ala Val Asp Ala
Tyr Leu Ala Leu Glu Ser Ser Val -1 1 5 10 15 Ala Lys Ala Gly Leu
Leu Ala Asn Ile Gly Pro Ser Gly Ser Lys Ser 20 25 30 Ser Gly Ala
Lys Ser Gly Ile Val Ile Ala Ser Pro Ser His Ser Asn 35 40 45 Pro
Asp Tyr Leu Phe Thr Trp Thr Arg Asp Ser Ser Leu Val Phe Gln 50 55
60 Thr Ile Ile Asn Gln Phe Thr Leu Gly His Asp Asn Ser Leu Arg Pro
65 70 75 Glu Ile Asp Asn Phe Val Asp Ser Gln Arg Lys Ile Gln Gln
Val Ser 80 85 90 95 Asn Pro Ser Gly Thr Val Ser Ser Gly Gly Leu Gly
Glu Pro Lys Phe 100 105 110 Asn Ile Asp Glu Thr Ala Phe Thr Gly Ala
Trp Gly Arg Pro Gln Arg 115 120 125 Asp Gly Pro Ala Leu Arg Ser Thr
Ala Leu Ile Thr Trp Ala Asn Tyr 130 135 140 Leu Ile Ala Asn Ser Asn
Thr Ser Tyr Val Thr Asn Thr Leu Trp Pro 145 150 155 Ile Ile Lys Leu
Asp Leu Asp Tyr Val Ala Ser Asn Trp Asn Gln Thr 160 165 170 175 Gly
Phe Asp Leu Trp Glu Glu Val Ser Ser Ser Ser Phe Phe Thr Thr 180 185
190 Ala Val Gln His Arg Ser Leu Arg Gln Gly Ala Ser Leu Ala Thr Ala
195 200 205 Ile Gly Gln Thr Ser Val Val Pro Gly Tyr Thr Thr Gln Ala
Asn Asn 210 215 220 Ile Leu Cys Phe Gln Gln Ser Tyr Trp Asn Ser Ala
Gly Tyr Met Thr 225 230 235 Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys
Asp Ala Asn Thr Val Leu 240 245 250 255 Thr Ser Ile His Thr Phe Asp
Pro Asp Ala Gly Cys Asp Ser Ile Thr 260 265 270 Phe Gln Pro Cys Ser
Asp Arg Ala Leu Ile Asn Leu Val Thr Tyr Val 275 280 285 Asn Ala Phe
Arg Ser Ile Tyr Ala Ile Asn Ala Gly Ile Ala Asn Asn 290 295 300 Gln
Gly Val Ala Thr Gly Arg Tyr Pro Glu Asp Gly Tyr Met Gly Gly 305 310
315 Asn Pro Trp Tyr Leu Thr Thr Leu Ala Val Ser Glu Gln Leu Tyr Tyr
320 325 330 335 Ala Leu Ser Thr Trp Lys Lys His Ser Ser Leu Thr Ile
Thr Ala Thr 340 345 350 Ser Gln Pro Phe Phe Ala Leu Phe Ser Pro Gly
Val Ala Thr Gly Thr 355 360 365 Tyr Ala Ser Ser Thr Thr Thr Tyr Ala
Thr Leu Thr Thr Ala Ile Gln 370 375 380 Asn Tyr Ala Asp Ser Phe Ile
Ala Val Val Ala Lys Tyr Thr Pro Ala 385 390 395 Asn Gly Gly Leu Ala
Glu Gln Tyr Ser Arg Ser Asn Gly Leu Pro Val 400 405 410 415 Ser Ala
Val Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr Ala Ala 420 425 430
Asp Ala Arg Ala Gly Leu Thr Pro Ala Ala Trp Gly Ala Ala Gly Leu 435
440 445 Thr Val Pro Ser Thr Cys Ser Thr Gly Gly Gly Ser Asn Pro Gly
Gly 450 455 460 Gly Gly Ser Val Ser Val Thr Phe Asn Val Gln Ala Thr
Thr Thr Phe 465 470 475 Gly Glu Asn Ile Phe Leu Thr Gly Ser Ile Asn
Glu Leu Ala Asn Trp 480 485 490 495 Ser Pro Asp Asn Ala Leu Thr Val
Asn Val Pro Ala Ser Thr Thr Ile 500 505 510 Gln Tyr Lys Phe Ile Arg
Lys Phe Asn Gly Ala Ile Thr Trp Glu Ser 515 520 525 Asp Pro Asn Arg
Gln Ile Thr Thr Pro Ser Ser Gly Ser Phe Val Gln 530 535 540 Asn Asp
Ser Trp Lys 545 25 1722 DNA Leucopaxillus giganteus CDS (1)..(1719)
cDNA sig_peptide (1)..(51) misc_feature (1)..(1404) Catalytic
Domain mat_peptide (52)..(1719) misc_feature (1405)..(1437) Linker
misc_feature (1438)..(1719) binding domain 25 atg cat ttc tct gtc
ctc tcc gta ttt ctc gcg att agt tct gct tgg 48 Met His Phe Ser Val
Leu Ser Val Phe Leu Ala Ile Ser Ser Ala Trp -15 -10 -5 gct cag tct
agc gca gtc gat gcc tat ctc gct ctc gaa tcc tcc gtc 96 Ala Gln Ser
Ser Ala Val Asp Ala Tyr Leu Ala Leu Glu Ser Ser Val -1 1 5 10 15
gcc aag gcc ggg ttg ctc gcc aac att ggc cca tct ggt tca aag tct 144
Ala Lys Ala Gly Leu Leu Ala Asn Ile Gly Pro Ser Gly Ser Lys Ser 20
25 30 tcg ggt gcc aag tct ggg att gtc att gcg tcg cct tcg cat agc
aac 192 Ser Gly Ala Lys Ser Gly Ile Val Ile Ala Ser Pro Ser His Ser
Asn 35 40 45 cct gac tac ctg ttc acc tgg acc cgc gat tct tcg ctt
gtg ttc cag 240 Pro Asp Tyr Leu Phe Thr Trp Thr Arg Asp Ser Ser Leu
Val Phe Gln 50 55 60 act atc atc aac cag ttc acg ttg gga cac gac
aat agt ttg agg cct 288 Thr Ile Ile Asn Gln Phe Thr Leu Gly His Asp
Asn Ser Leu Arg Pro 65 70 75 gag att gac aat ttt gtt gat tcc caa
agg aag atc caa caa gtc tca 336 Glu Ile Asp Asn Phe Val Asp Ser Gln
Arg Lys Ile Gln Gln Val Ser 80 85 90 95 aac cct tcg gga act gtt agt
tct ggc ggc ctt ggc gag ccc aag ttc 384 Asn Pro Ser Gly Thr Val Ser
Ser Gly Gly Leu Gly Glu Pro Lys Phe 100 105 110 aat atc gac gaa acc
gcc ttt aca ggg gca tgg ggg gat gga cct gct 432 Asn Ile Asp Glu Thr
Ala Phe Thr Gly Ala Trp Gly Asp Gly Pro Ala 115 120 125 ctc cga tcc
acc gcg ctc att acc tgg gcc aat tac ctg atc gct aac 480 Leu Arg Ser
Thr Ala Leu Ile Thr Trp Ala Asn Tyr Leu Ile Ala Asn 130 135 140 agc
aac aca tcc tac gtc acc aac acc cta tgg ccc atc atc aaa ttg 528 Ser
Asn Thr Ser Tyr Val Thr Asn Thr Leu Trp Pro Ile Ile Lys Leu 145 150
155 gac ctc gac tac gtc gcg tcc aac tgg aac cag act agt ttc gat ttg
576 Asp Leu Asp Tyr Val Ala Ser Asn Trp Asn Gln Thr Ser Phe Asp Leu
160 165 170 175 tgg gaa gaa gta tcc tct tct tcc ttc ttc act act gcg
gtt caa cac 624 Trp Glu Glu Val Ser Ser Ser Ser Phe Phe Thr Thr Ala
Val Gln His 180 185 190 cgc tcc ctt cgc caa ggt gct tcc cta gcc act
gcc att gga caa acc 672 Arg Ser Leu Arg Gln Gly Ala Ser Leu Ala Thr
Ala Ile Gly Gln Thr 195 200 205 tct gtc gtt ccw ggc tac acc acc cag
gcc aac aat ata ctc tgc ttt 720 Ser Val Val Xaa Gly Tyr Thr Thr Gln
Ala Asn Asn Ile Leu Cys Phe 210 215 220 caa cag tcc tac tgg aac tca
gct ggg tat atg act gcc aat acc gga 768 Gln Gln Ser Tyr Trp Asn Ser
Ala Gly Tyr Met Thr Ala Asn Thr Gly 225 230 235 ggc ggg cgt tct ggg
aaa gac gcc aac acc gtc ctc aca agt att cac 816 Gly Gly Arg Ser Gly
Lys Asp Ala Asn Thr Val Leu Thr Ser Ile His 240 245 250 255 aca ttc
gat ccc gat gcc ggc tgc gat tcc atc act ttc caa cct tgt 864 Thr Phe
Asp Pro Asp Ala Gly Cys Asp Ser Ile Thr Phe Gln Pro Cys 260 265 270
tca gac cgt gcg ctc atc aac ctt gtc aca tac gtc aat gca ttc cga 912
Ser Asp Arg Ala Leu Ile Asn Leu Val Thr Tyr Val Asn Ala Phe Arg 275
280 285 agc atc tac gct atc aac gcg ggc atc gct aat aac caa ggc gtt
gcc 960 Ser Ile Tyr Ala Ile Asn Ala Gly Ile Ala Asn Asn Gln Gly Val
Ala 290 295 300 act ggt agg tat cct gaa gat ggc tac atg ggc gga aac
cct tgg tat 1008 Thr Gly Arg Tyr Pro Glu Asp Gly Tyr Met Gly Gly
Asn Pro Trp Tyr 305 310 315 ctg act act tta gcc gtt tct gaa cag ctc
tac tac gct ctc tcc act 1056 Leu Thr Thr Leu Ala Val Ser Glu Gln
Leu Tyr Tyr Ala Leu Ser Thr 320 325 330 335 tgg aag aaa cat agc tcc
ctc acc att acg gcg aca tca caa cct ttt 1104 Trp Lys Lys His Ser
Ser Leu Thr Ile Thr Ala Thr Ser Gln Pro Phe 340 345 350 ttc gcg ctc
ttc tcg ccg ggt gtt gct act ggc aca tat gcg tcc tct 1152 Phe Ala
Leu Phe Ser Pro Gly Val Ala Thr Gly Thr Tyr Ala Ser Ser 355 360 365
acg act acc tat gct aca ctt act act gct att cag aat tac gcg gat
1200 Thr Thr Thr Tyr Ala Thr Leu Thr Thr Ala Ile Gln Asn Tyr Ala
Asp 370 375 380 agc ttc atc gct gtc gtg gct aag tat acg cct gcc aat
ggc gga ctg 1248 Ser Phe Ile Ala Val Val Ala Lys Tyr Thr Pro Ala
Asn Gly Gly Leu 385 390 395 gcg gaa cag tac agc agg agt aac ggt ttg
ccc gtt agt gcc gtt gat 1296 Ala Glu Gln Tyr Ser Arg Ser Asn Gly
Leu Pro Val Ser Ala Val Asp 400 405 410 415 tta act tgg agc tat gcc
gct ctc ttg acg gcg gct gat gcg cga gcg 1344 Leu Thr Trp Ser Tyr
Ala Ala Leu Leu Thr Ala Ala Asp Ala Arg Ala 420 425 430 ggg cta aca
ccc gct gca tgg gga gca gcg ggg ttg acc gtg cca agc 1392 Gly Leu
Thr Pro Ala Ala Trp Gly Ala Ala Gly Leu Thr Val Pro Ser 435 440 445
act tgc tct act ggg ggt ggt tca aac cca ggt ggt gga ggg tcg gtc
1440 Thr Cys Ser Thr Gly Gly Gly Ser Asn Pro Gly Gly Gly Gly Ser
Val 450 455 460 tct gtt acg ttc aat gtt caa gct aca acc acc ttt ggt
gaa aac att 1488 Ser Val Thr Phe Asn Val Gln Ala Thr Thr Thr Phe
Gly Glu Asn Ile 465 470 475 ttt ttg acc ggc tcg atc aac gag tta gct
aac tgg tct cct gat aat 1536 Phe Leu Thr Gly Ser Ile Asn Glu Leu
Ala Asn Trp Ser Pro Asp Asn 480 485 490 495 gct ctc gcc ctc tct gcg
gcc aat tat ccc acc tgg agc agt acc gtc 1584 Ala Leu Ala Leu Ser
Ala Ala Asn Tyr Pro Thr Trp Ser Ser Thr Val 500 505 510 aac gtt ccc
gca agc act acg atc caa tac aag ttt atc cgt aaa ttc 1632 Asn Val
Pro Ala Ser Thr Thr Ile Gln Tyr Lys Phe Ile Arg Lys Phe 515 520 525
aac gga gcc atc acc tgg gag tcc gac ccg aat agg cag atc aca acg
1680 Asn Gly Ala Ile Thr Trp Glu Ser Asp Pro Asn Arg Gln Ile Thr
Thr 530 535 540 ccg tct tcg gga agt ttt gtc cag aat gac tcg tgg aag
tag 1722 Pro Ser Ser Gly Ser Phe Val Gln Asn Asp Ser Trp Lys 545
550 555 26 573 PRT Leucopaxillus giganteus misc_feature
(211)..(211) The 'Xaa' at location 211 stands for Pro. 26 Met His
Phe Ser Val Leu Ser Val Phe Leu Ala Ile Ser Ser Ala Trp -15 -10 -5
Ala Gln Ser Ser Ala Val Asp Ala Tyr Leu Ala Leu Glu Ser Ser Val -1
1 5 10 15 Ala Lys Ala Gly Leu Leu Ala Asn Ile Gly Pro Ser Gly Ser
Lys Ser 20 25 30 Ser Gly Ala Lys Ser Gly Ile Val Ile Ala Ser Pro
Ser His Ser Asn 35 40 45 Pro Asp Tyr Leu Phe Thr Trp Thr Arg Asp
Ser Ser Leu Val Phe Gln 50 55 60 Thr Ile Ile Asn Gln Phe Thr Leu
Gly His Asp Asn Ser Leu Arg Pro 65 70 75 Glu Ile Asp Asn Phe Val
Asp Ser Gln Arg Lys Ile Gln Gln Val Ser 80 85 90 95 Asn Pro Ser Gly
Thr Val Ser Ser Gly Gly Leu Gly Glu Pro
Lys Phe 100 105 110 Asn Ile Asp Glu Thr Ala Phe Thr Gly Ala Trp Gly
Asp Gly Pro Ala 115 120 125 Leu Arg Ser Thr Ala Leu Ile Thr Trp Ala
Asn Tyr Leu Ile Ala Asn 130 135 140 Ser Asn Thr Ser Tyr Val Thr Asn
Thr Leu Trp Pro Ile Ile Lys Leu 145 150 155 Asp Leu Asp Tyr Val Ala
Ser Asn Trp Asn Gln Thr Ser Phe Asp Leu 160 165 170 175 Trp Glu Glu
Val Ser Ser Ser Ser Phe Phe Thr Thr Ala Val Gln His 180 185 190 Arg
Ser Leu Arg Gln Gly Ala Ser Leu Ala Thr Ala Ile Gly Gln Thr 195 200
205 Ser Val Val Xaa Gly Tyr Thr Thr Gln Ala Asn Asn Ile Leu Cys Phe
210 215 220 Gln Gln Ser Tyr Trp Asn Ser Ala Gly Tyr Met Thr Ala Asn
Thr Gly 225 230 235 Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr Val Leu
Thr Ser Ile His 240 245 250 255 Thr Phe Asp Pro Asp Ala Gly Cys Asp
Ser Ile Thr Phe Gln Pro Cys 260 265 270 Ser Asp Arg Ala Leu Ile Asn
Leu Val Thr Tyr Val Asn Ala Phe Arg 275 280 285 Ser Ile Tyr Ala Ile
Asn Ala Gly Ile Ala Asn Asn Gln Gly Val Ala 290 295 300 Thr Gly Arg
Tyr Pro Glu Asp Gly Tyr Met Gly Gly Asn Pro Trp Tyr 305 310 315 Leu
Thr Thr Leu Ala Val Ser Glu Gln Leu Tyr Tyr Ala Leu Ser Thr 320 325
330 335 Trp Lys Lys His Ser Ser Leu Thr Ile Thr Ala Thr Ser Gln Pro
Phe 340 345 350 Phe Ala Leu Phe Ser Pro Gly Val Ala Thr Gly Thr Tyr
Ala Ser Ser 355 360 365 Thr Thr Thr Tyr Ala Thr Leu Thr Thr Ala Ile
Gln Asn Tyr Ala Asp 370 375 380 Ser Phe Ile Ala Val Val Ala Lys Tyr
Thr Pro Ala Asn Gly Gly Leu 385 390 395 Ala Glu Gln Tyr Ser Arg Ser
Asn Gly Leu Pro Val Ser Ala Val Asp 400 405 410 415 Leu Thr Trp Ser
Tyr Ala Ala Leu Leu Thr Ala Ala Asp Ala Arg Ala 420 425 430 Gly Leu
Thr Pro Ala Ala Trp Gly Ala Ala Gly Leu Thr Val Pro Ser 435 440 445
Thr Cys Ser Thr Gly Gly Gly Ser Asn Pro Gly Gly Gly Gly Ser Val 450
455 460 Ser Val Thr Phe Asn Val Gln Ala Thr Thr Thr Phe Gly Glu Asn
Ile 465 470 475 Phe Leu Thr Gly Ser Ile Asn Glu Leu Ala Asn Trp Ser
Pro Asp Asn 480 485 490 495 Ala Leu Ala Leu Ser Ala Ala Asn Tyr Pro
Thr Trp Ser Ser Thr Val 500 505 510 Asn Val Pro Ala Ser Thr Thr Ile
Gln Tyr Lys Phe Ile Arg Lys Phe 515 520 525 Asn Gly Ala Ile Thr Trp
Glu Ser Asp Pro Asn Arg Gln Ile Thr Thr 530 535 540 Pro Ser Ser Gly
Ser Phe Val Gln Asn Asp Ser Trp Lys 545 550 555 27 1761 DNA
Artificial Hybrid Fungamyl variant JA118 with A. rolfsii SBD
mat_peptide (1)..(1758) CDS (1)..(1758) 27 gca acg cct gcg gac tgg
cga tcg caa tcc att tat ttc ctt ctc acg 48 Ala Thr Pro Ala Asp Trp
Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15 gat cga ttt gca
agg acg gat ggg tcg acg act gcg act tgt aat act 96 Asp Arg Phe Ala
Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30 gcg gat
cag aaa tac tgt ggt gga aca tgg cag ggc atc atc gac aag 144 Ala Asp
Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45
ttg gac tat atc cag gga atg ggc ttc aca gcc atc tgg atc acc ccc 192
Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50
55 60 gtt aca gcc cag ctg ccc cag acc acc gca tat gga gat gcc tac
cat 240 Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr
His 65 70 75 80 ggc tac tgg cag cag gat ata tac tct ctg aac gaa aac
tac ggc act 288 Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn
Tyr Gly Thr 85 90 95 gca gat gac ttg aag gcg ctc tct tcg gcc ctt
cat gag agg ggg atg 336 Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu
His Glu Arg Gly Met 100 105 110 tat ctt atg gtc gat gtg gtt gct aac
cat atg ggc tat gat gga ccg 384 Tyr Leu Met Val Asp Val Val Ala Asn
His Met Gly Tyr Asp Gly Pro 115 120 125 ggt agc tca gtc gat tac agt
gtg ttt gtt ccg ttc aat tcc gct agc 432 Gly Ser Ser Val Asp Tyr Ser
Val Phe Val Pro Phe Asn Ser Ala Ser 130 135 140 tac ttc cac ccg ttc
tgt ttc att caa aac tgg aat gat cag act cag 480 Tyr Phe His Pro Phe
Cys Phe Ile Gln Asn Trp Asn Asp Gln Thr Gln 145 150 155 160 gtt gag
gat tgc tgg cta gga gat aac act gtc tcc ttg cct gat ctc 528 Val Glu
Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175
gat acc acc aag gat gtg gtc aag aat gaa tgg tac gac tgg gtg gga 576
Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180
185 190 tca ttg gta tcg aac tac tcc att gac ggc ctc cgt atc gac aca
gta 624 Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr
Val 195 200 205 aaa cac gtc cag aag gac ttc tgg ccc ggg tac aac aaa
gcc gca ggc 672 Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys
Ala Ala Gly 210 215 220 gtg tac tgt atc ggc gag gtg ctc gac ggt gat
ccg gcc tac act tgt 720 Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp
Pro Ala Tyr Thr Cys 225 230 235 240 ccc tac cag gaa gtc ctg gac ggc
gta ctg aac tac ccc att tac tat 768 Pro Tyr Gln Glu Val Leu Asp Gly
Val Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255 cca ctc ctc aac gcc ttc
aag tca acc tcc ggc agc atg gac gac ctc 816 Pro Leu Leu Asn Ala Phe
Lys Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265 270 tac aac atg atc
aac acc gtc aaa tcc gac tgt cca gac tca aca ctc 864 Tyr Asn Met Ile
Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285 ctg ggc
aca ttc gtc gag aac cac gac aac cca cgg ttc gct tct tac 912 Leu Gly
Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300
acc aac gac ata gcc ctc gcc aag aac gtc gca gca ttc atc atc ctc 960
Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu 305
310 315 320 aac gac gga atc ccc atc atc tac gcc ggc caa gaa cag cac
tac gcc 1008 Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln
His Tyr Ala 325 330 335 ggc gga aac gac ccc gcg aac cgc gaa gca acc
tgg ctc tcg ggc tac 1056 Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala
Thr Trp Leu Ser Gly Tyr 340 345 350 ccg acc gac agc gag ctg tac aag
tta att gcc tcc gcg aac gca atc 1104 Pro Thr Asp Ser Glu Leu Tyr
Lys Leu Ile Ala Ser Ala Asn Ala Ile 355 360 365 cgg aac tat gcc att
agc aaa gat aca gga ttc gtg acc tac aag aac 1152 Arg Asn Tyr Ala
Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn 370 375 380 tgg ccc
atc tac aaa gac gac aca acg atc gcc atg cgc aag ggc aca 1200 Trp
Pro Ile Tyr Lys Asp Asp Thr Thr Ile Ala Met Arg Lys Gly Thr 385 390
395 400 gat ggg tcg cag atc gtg act atc ttg tcc aac aag ggt gct tcg
ggt 1248 Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala
Ser Gly 405 410 415 gat tcg tat acc ctc tcc ttg agt ggt gcg ggt tac
aca gcc ggc cag 1296 Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly
Tyr Thr Ala Gly Gln 420 425 430 caa ttg acg gag gtc att ggc tgc acg
acc gtg acg gtt gat tcg tcg 1344 Gln Leu Thr Glu Val Ile Gly Cys
Thr Thr Val Thr Val Asp Ser Ser 435 440 445 gga gat gtg cct gtt cct
atg gcg ggt ggg cta cct agg gta ttg tat 1392 Gly Asp Val Pro Val
Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr 450 455 460 ccg act gag
aag ttg gca ggt agc aag atc tgt agt agc tcg ggt gct 1440 Pro Thr
Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser Gly Ala 465 470 475
480 aca agc ccg ggt ggc tcc tcg ggt agt gtc gag gtc act ttc gac gtt
1488 Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu Val Thr Phe Asp
Val 485 490 495 tac gct acc aca gta tat ggc cag aac atc tat atc acc
ggt gat gtg 1536 Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile Tyr Ile
Thr Gly Asp Val 500 505 510 agt gag ctc ggc aac tgg aca ccc gcc aat
ggt gtt gca ctc tct tct 1584 Ser Glu Leu Gly Asn Trp Thr Pro Ala
Asn Gly Val Ala Leu Ser Ser 515 520 525 gct aac tac ccc acc tgg agt
gcc acg atc gct ctc ccc gct gac acg 1632 Ala Asn Tyr Pro Thr Trp
Ser Ala Thr Ile Ala Leu Pro Ala Asp Thr 530 535 540 aca atc cag tac
aag tat gtc aac att gac ggc agc acc gtc atc tgg 1680 Thr Ile Gln
Tyr Lys Tyr Val Asn Ile Asp Gly Ser Thr Val Ile Trp 545 550 555 560
gag gat gct atc agc aat cgc gag atc acg acg ccc gcc agc ggc aca
1728 Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro Ala Ser Gly
Thr 565 570 575 tac acc gaa aaa gac act tgg gat gaa tct tag 1761
Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 580 585 28 586 PRT
Artificial Synthetic Construct 28 Ala Thr Pro Ala Asp Trp Arg Ser
Gln Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15 Asp Arg Phe Ala Arg Thr
Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30 Ala Asp Gln Lys
Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45 Leu Asp
Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60
Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His 65
70 75 80 Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr
Gly Thr 85 90 95 Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His
Glu Arg Gly Met 100 105 110 Tyr Leu Met Val Asp Val Val Ala Asn His
Met Gly Tyr Asp Gly Pro 115 120 125 Gly Ser Ser Val Asp Tyr Ser Val
Phe Val Pro Phe Asn Ser Ala Ser 130 135 140 Tyr Phe His Pro Phe Cys
Phe Ile Gln Asn Trp Asn Asp Gln Thr Gln 145 150 155 160 Val Glu Asp
Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175 Asp
Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185
190 Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val
195 200 205 Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala
Ala Gly 210 215 220 Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro
Ala Tyr Thr Cys 225 230 235 240 Pro Tyr Gln Glu Val Leu Asp Gly Val
Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255 Pro Leu Leu Asn Ala Phe Lys
Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265 270 Tyr Asn Met Ile Asn
Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285 Leu Gly Thr
Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300 Thr
Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu 305 310
315 320 Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr
Ala 325 330 335 Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu
Ser Gly Tyr 340 345 350 Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala
Ser Ala Asn Ala Ile 355 360 365 Arg Asn Tyr Ala Ile Ser Lys Asp Thr
Gly Phe Val Thr Tyr Lys Asn 370 375 380 Trp Pro Ile Tyr Lys Asp Asp
Thr Thr Ile Ala Met Arg Lys Gly Thr 385 390 395 400 Asp Gly Ser Gln
Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415 Asp Ser
Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430
Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr Val Asp Ser Ser 435
440 445 Gly Asp Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu
Tyr 450 455 460 Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser
Ser Gly Ala 465 470 475 480 Thr Ser Pro Gly Gly Ser Ser Gly Ser Val
Glu Val Thr Phe Asp Val 485 490 495 Tyr Ala Thr Thr Val Tyr Gly Gln
Asn Ile Tyr Ile Thr Gly Asp Val 500 505 510 Ser Glu Leu Gly Asn Trp
Thr Pro Ala Asn Gly Val Ala Leu Ser Ser 515 520 525 Ala Asn Tyr Pro
Thr Trp Ser Ala Thr Ile Ala Leu Pro Ala Asp Thr 530 535 540 Thr Ile
Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser Thr Val Ile Trp 545 550 555
560 Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr Thr Pro Ala Ser Gly Thr
565 570 575 Tyr Thr Glu Lys Asp Thr Trp Asp Glu Ser 580 585 29 558
PRT Artificial Hybrid alpha-amylase with Rhizomucor pusillus
catalytic domain and A. rolfsii linker and SBD 29 Ser Pro Leu Pro
Gln Gln Gln Arg Tyr Gly Lys Arg Ala Thr Ser Asp 1 5 10 15 Asp Trp
Lys Ser Lys Ala Ile Tyr Gln Leu Leu Thr Asp Arg Phe Gly 20 25 30
Arg Ala Asp Asp Ser Thr Ser Asn Cys Ser Asn Leu Ser Asn Tyr Cys 35
40 45 Gly Gly Thr Tyr Glu Gly Ile Thr Lys His Leu Asp Tyr Ile Ser
Gly 50 55 60 Met Gly Phe Asp Ala Ile Trp Ile Ser Pro Ile Pro Lys
Asn Ser Asp 65 70 75 80 Gly Gly Tyr His Gly Tyr Trp Ala Thr Asp Phe
Tyr Gln Leu Asn Ser 85 90 95 Asn Phe Gly Asp Glu Ser Gln Leu Lys
Ala Leu Ile Gln Ala Ala His 100 105 110 Glu Arg Asp Met Tyr Val Met
Leu Asp Val Val Ala Asn His Ala Gly 115 120 125 Pro Thr Ser Asn Gly
Tyr Ser Gly Tyr Thr Phe Gly Asp Ala Ser Leu 130 135 140 Tyr His Pro
Lys Cys Thr Ile Asp Tyr Asn Asp Gln Thr Ser Ile Glu 145 150 155 160
Gln Cys Trp Val Ala Asp Glu Leu Pro Asp Ile Asp Thr Glu Asn Ser 165
170 175 Asp Asn Val Ala Ile Leu Asn Asp Ile Val Ser Gly Trp Val Gly
Asn 180 185 190 Tyr Ser Phe Asp Gly Ile Arg Ile Asp Thr Val Lys His
Ile Arg Lys 195 200 205 Asp Phe Trp Thr Gly Tyr Ala Glu Ala Ala Gly
Val Phe Ala Thr Gly 210 215 220 Glu Val Phe Asn Gly Asp Pro Ala Tyr
Val Gly Pro Tyr Gln Lys Tyr 225 230 235 240 Leu Pro Ser Leu Ile Asn
Tyr Pro Met Tyr Tyr Ala Leu Asn Asp Val 245 250 255 Phe Val Ser Lys
Ser Lys Gly Phe Ser Arg Ile Ser Glu Met Leu Gly 260 265 270 Ser Asn
Arg Asn Ala Phe Glu Asp Thr Ser Val Leu Thr Thr Phe Val 275 280 285
Asp Asn His Asp Asn Pro Arg Phe Leu Asn Ser Gln Ser Asp Lys Ala 290
295 300 Leu Phe Lys Asn Ala Leu Thr Tyr Val Leu Leu Gly Glu Gly Ile
Pro 305 310 315 320 Ile Val Tyr Tyr Gly Ser Glu Gln Gly Phe Ser Gly
Gly Ala Asp Pro 325 330 335 Ala Asn Arg Glu Val Leu Trp Thr Thr Asn
Tyr Asp Thr Ser Ser Asp 340 345 350 Leu Tyr Gln Phe Ile Lys Thr Val
Asn Ser Val Arg Met Lys Ser Asn 355 360 365 Lys Ala Val Tyr Met Asp
Ile Tyr Val Gly Asp Asn Ala Tyr Ala Phe 370 375 380 Lys His Gly Asp
Ala Leu Val Val Leu Asn Asn Tyr Gly Ser Gly Ser 385 390 395 400 Thr
Asn Gln Val Ser Phe Ser Val Ser Gly Lys Phe Asp Ser Gly Ala 405 410
415 Ser Leu Met Asp Ile Val Ser Asn Ile Thr Thr Thr
Val Ser Ser Asp 420 425 430 Gly Thr Val Thr Phe Asn Leu Lys Asp Gly
Leu Pro Ala Ile Phe Thr 435 440 445 Ser Ala Gly Ala Thr Ser Pro Gly
Gly Ser Ser Gly Ser Val Glu Val 450 455 460 Thr Phe Asp Val Tyr Ala
Thr Thr Val Tyr Gly Gln Asn Ile Tyr Ile 465 470 475 480 Thr Gly Asp
Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val 485 490 495 Ala
Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile Ala Leu 500 505
510 Pro Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn Ile Asp Gly Ser
515 520 525 Thr Val Ile Trp Glu Asp Ala Ile Ser Asn Arg Glu Ile Thr
Thr Pro 530 535 540 Ala Ser Gly Thr Tyr Thr Glu Lys Asp Thr Trp Asp
Glu Ser 545 550 555 30 574 PRT Artificial Hybrid alpha-amylase with
Meripilus giganteous catalytic domain with A. rolfsii linker and
SBD. 30 Arg Pro Thr Val Phe Asp Ala Gly Ala Asp Ala His Ser Leu His
Ala 1 5 10 15 Arg Ala Pro Ser Gly Ser Lys Asp Val Ile Ile Gln Met
Phe Glu Trp 20 25 30 Asn Trp Asp Ser Val Ala Ala Glu Cys Thr Asn
Phe Ile Gly Pro Ala 35 40 45 Gly Tyr Gly Phe Val Gln Val Ser Pro
Pro Gln Glu Thr Ile Gln Gly 50 55 60 Ala Gln Trp Trp Thr Asp Tyr
Gln Pro Val Ser Tyr Thr Leu Thr Gly 65 70 75 80 Lys Arg Gly Asp Arg
Ser Gln Phe Ala Asn Met Ile Thr Thr Cys His 85 90 95 Ala Ala Gly
Val Gly Val Ile Val Asp Thr Ile Trp Asn His Met Ala 100 105 110 Gly
Val Asp Ser Gly Thr Gly Thr Ala Gly Ser Ser Phe Thr His Tyr 115 120
125 Asn Tyr Pro Gly Ile Tyr Gln Asn Gln Asp Phe His His Cys Gly Leu
130 135 140 Glu Pro Gly Asp Asp Ile Val Asn Tyr Asp Asn Ala Val Glu
Val Gln 145 150 155 160 Thr Cys Glu Leu Val Asn Leu Ala Asp Leu Ala
Thr Asp Thr Glu Tyr 165 170 175 Val Arg Gly Arg Leu Ala Gln Tyr Gly
Asn Asp Leu Leu Ser Leu Gly 180 185 190 Ala Asp Gly Leu Arg Leu Asp
Ala Ser Lys His Ile Pro Val Gly Asp 195 200 205 Ile Ala Asn Ile Leu
Ser Arg Leu Ser Arg Ser Val Tyr Ile Thr Gln 210 215 220 Glu Val Ile
Phe Gly Ala Gly Glu Pro Ile Thr Pro Asn Gln Tyr Thr 225 230 235 240
Gly Asn Gly Asp Val Gln Glu Phe Arg Tyr Thr Ser Ala Leu Lys Asp 245
250 255 Ala Phe Leu Ser Ser Gly Ile Ser Asn Leu Gln Asp Phe Glu Asn
Arg 260 265 270 Gly Trp Val Pro Gly Ser Gly Ala Asn Val Phe Val Val
Asn His Asp 275 280 285 Thr Glu Arg Asn Gly Ala Ser Leu Asn Asn Asn
Ser Pro Ser Asn Thr 290 295 300 Tyr Val Thr Ala Thr Ile Phe Ser Leu
Ala His Pro Tyr Gly Thr Pro 305 310 315 320 Thr Ile Leu Ser Ser Tyr
Asp Gly Phe Thr Asn Thr Asp Ala Gly Ala 325 330 335 Pro Asn Asn Asn
Val Gly Thr Cys Ser Thr Ser Gly Gly Ala Asn Gly 340 345 350 Trp Leu
Cys Gln His Arg Trp Thr Ala Ile Ala Gly Met Val Gly Phe 355 360 365
Arg Asn Asn Val Gly Ser Ala Ala Leu Asn Asn Trp Gln Ala Pro Gln 370
375 380 Ser Gln Gln Ile Ala Phe Gly Arg Gly Ala Leu Gly Phe Val Ala
Ile 385 390 395 400 Asn Asn Ala Asp Ser Ala Trp Ser Thr Thr Phe Thr
Thr Ser Leu Pro 405 410 415 Asp Gly Ser Tyr Cys Asp Val Ile Ser Gly
Lys Ala Ser Gly Ser Ser 420 425 430 Cys Thr Gly Ser Ser Phe Thr Val
Ser Gly Gly Lys Leu Thr Ala Thr 435 440 445 Val Pro Ala Arg Ser Ala
Ile Ala Val His Thr Gly Gln Lys Gly Ser 450 455 460 Gly Gly Gly Ala
Thr Ser Pro Gly Gly Ser Ser Gly Ser Val Glu Val 465 470 475 480 Thr
Phe Asp Val Tyr Ala Thr Thr Val Tyr Gly Gln Asn Ile Tyr Ile 485 490
495 Thr Gly Asp Val Ser Glu Leu Gly Asn Trp Thr Pro Ala Asn Gly Val
500 505 510 Ala Leu Ser Ser Ala Asn Tyr Pro Thr Trp Ser Ala Thr Ile
Ala Leu 515 520 525 Pro Ala Asp Thr Thr Ile Gln Tyr Lys Tyr Val Asn
Ile Asp Gly Ser 530 535 540 Thr Val Ile Trp Glu Asp Ala Ile Ser Asn
Arg Glu Ile Thr Thr Pro 545 550 555 560 Ala Ser Gly Thr Tyr Thr Glu
Lys Asp Thr Trp Asp Glu Ser 565 570 31 22 DNA Artificial Primer 31
ggtagactag ttacctcgtt gg 22 32 23 DNA Artificial Primer 32
gcttccctag ccactgccat tgg 23 33 23 DNA Artificial Primer 33
gttgatttaa cttggagcta tgc 23 34 35 DNA Artificial Leucopaxillus
forward Primer 34 tcccttggat ccaggatgca tttctctgtc ctctc 35 35 34
DNA Artificial Leucopaxillus reverse Primer 35 cttatcctcg
agctacttcc acgagtcatt ctgg 34 36 2166 DNA Trametes cingulata CDS
(1)..(171) misc_signal (1)..(54) mat_peptide (55)..(2166) Intron
(172)..(244) CDS (245)..(521) Intron (522)..(577) CDS (578)..(722)
Intron (723)..(772) CDS (773)..(935) Intron (936)..(1001) CDS
(1002)..(1277) Intron (1278)..(1341) CDS (1342)..(1807)
misc_feature (1744)..(1773) Linker misc_feature (1774)..(2163)
binding domain Intron (1808)..(1864) CDS (1865)..(1960) Intron
(1961)..(2020) CDS (2021)..(2163) 36 atg cgt ttc acg ctc ctc acc
tcc ctc ctg ggc ctc gcc ctc ggc gcg 48 Met Arg Phe Thr Leu Leu Thr
Ser Leu Leu Gly Leu Ala Leu Gly Ala -15 -10 -5 ttc gcg cag tcg agt
gcg gcc gac gcg tac gtc gcg tcc gaa tcg ccc 96 Phe Ala Gln Ser Ser
Ala Ala Asp Ala Tyr Val Ala Ser Glu Ser Pro -1 1 5 10 atc gcc aag
gcg ggt gtg ctc gcc aac atc ggg ccc agc ggc tcc aag 144 Ile Ala Lys
Ala Gly Val Leu Ala Asn Ile Gly Pro Ser Gly Ser Lys 15 20 25 30 tcc
aac gga gca aag gca agt gac aca gtgacactcc ggggcgccca 191 Ser Asn
Gly Ala Lys Ala Ser Asp Thr 35 tgcttcattc ttctgtgcac atggtagcgc
tgacatatcg ttgtttttga cag ccc 247 Pro 40 ggc atc gtg att gca agt
ccg agc aca tcc aac ccg aac tac ctg tac 295 Gly Ile Val Ile Ala Ser
Pro Ser Thr Ser Asn Pro Asn Tyr Leu Tyr 45 50 55 aca tgg acg cgc
gac tcg tcc ctc gtg ttc aag gcg ctc atc gac cag 343 Thr Trp Thr Arg
Asp Ser Ser Leu Val Phe Lys Ala Leu Ile Asp Gln 60 65 70 ttc acc
act ggc gaa gat acc tcg ctc cga act ctg att gac gag ttc 391 Phe Thr
Thr Gly Glu Asp Thr Ser Leu Arg Thr Leu Ile Asp Glu Phe 75 80 85
acc tcg gcg gag gcc ata ctc cag cag gtg ccg aac ccg agc ggg aca 439
Thr Ser Ala Glu Ala Ile Leu Gln Gln Val Pro Asn Pro Ser Gly Thr 90
95 100 gtc agc act gga ggc ctc ggc gag ccc aag ttc aac atc gac gag
acc 487 Val Ser Thr Gly Gly Leu Gly Glu Pro Lys Phe Asn Ile Asp Glu
Thr 105 110 115 120 gcg ttc acg gat gcc tgg ggt cgt cct cag cgc g
gtaagtcgga 531 Ala Phe Thr Asp Ala Trp Gly Arg Pro Gln Arg 125 130
ggttgcctcg acggagatac gcccagactg acttcaagac tctcag at ggt ccc 585
Asp Gly Pro gct ctc cgg gcg act gcc atc atc acc tac gcc aac tgg ctc
ctc gac 633 Ala Leu Arg Ala Thr Ala Ile Ile Thr Tyr Ala Asn Trp Leu
Leu Asp 135 140 145 150 aac aag aac acg acc tac gtg acc aac act ctc
tgg cct atc atc aag 681 Asn Lys Asn Thr Thr Tyr Val Thr Asn Thr Leu
Trp Pro Ile Ile Lys 155 160 165 ctc gac ctc gac tac gtc gcc agc aac
tgg aac cag tcc ac 722 Leu Asp Leu Asp Tyr Val Ala Ser Asn Trp Asn
Gln Ser Thr 170 175 gtatgttctc taaattctct cccgtgggta accagtctga
acgttcatag g ttt gat 779 Phe Asp ctc tgg gag gag att aac tcc tcg
tcg ttc ttc act acc gcc gtc cag 827 Leu Trp Glu Glu Ile Asn Ser Ser
Ser Phe Phe Thr Thr Ala Val Gln 185 190 195 cac cgt gct ctg cgc gag
ggc gcg act ttc gct aat cgc atc gga caa 875 His Arg Ala Leu Arg Glu
Gly Ala Thr Phe Ala Asn Arg Ile Gly Gln 200 205 210 acc tcg gtg gtc
agc ggg tac acc acc caa gca aac aac ctt ctc tgc 923 Thr Ser Val Val
Ser Gly Tyr Thr Thr Gln Ala Asn Asn Leu Leu Cys 215 220 225 230 ttc
ctg cag gca gtctatcccg tcacacgtct gtctgtttcc gttttcccac 975 Phe Leu
Gln Ala agctcacctc gtcccgggcc ctgtag tcg tac tgg aac ccc acc ggc
ggc tat 1028 Ser Tyr Trp Asn Pro Thr Gly Gly Tyr 235 240 atc acc
gca aac acg ggc ggc ggc cgc tct ggc aag gac gcg aac acc 1076 Ile
Thr Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr 245 250
255 gtt ctc acg tcg atc cac acc ttc gac ccg gcc gct gga tgc gac gct
1124 Val Leu Thr Ser Ile His Thr Phe Asp Pro Ala Ala Gly Cys Asp
Ala 260 265 270 275 gtt acg ttc cag ccg tgc tcg gac aag gcg ctg tcg
aac ttg aag gtg 1172 Val Thr Phe Gln Pro Cys Ser Asp Lys Ala Leu
Ser Asn Leu Lys Val 280 285 290 tac gtc gat gcg ttc cgc tcg atc tac
tcc atc aac agc ggg atc gcc 1220 Tyr Val Asp Ala Phe Arg Ser Ile
Tyr Ser Ile Asn Ser Gly Ile Ala 295 300 305 tcg aat gcg gcc gtt gct
acc ggc cgc tac ccc gag gac agc tac atg 1268 Ser Asn Ala Ala Val
Ala Thr Gly Arg Tyr Pro Glu Asp Ser Tyr Met 310 315 320 ggc gga aac
gtgagcgacc atttctgtgc gtacaccgcg gtcgcgttaa 1317 Gly Gly Asn 325
ctgagatgtt ctcctctcct gtag cca tgg tac ctc acc acc tcc gcc gtc 1368
Pro Trp Tyr Leu Thr Thr Ser Ala Val 330 335 gct gag cag ctc tac gat
gcg ctc att gtg tgg aac aag ctt ggc gcc 1416 Ala Glu Gln Leu Tyr
Asp Ala Leu Ile Val Trp Asn Lys Leu Gly Ala 340 345 350 ctg aac gtc
acg agc acc tcc ctc ccc ttc ttc cag cag ttc tcg tca 1464 Leu Asn
Val Thr Ser Thr Ser Leu Pro Phe Phe Gln Gln Phe Ser Ser 355 360 365
ggc gtc acc gtc ggc acc tat gcc tca tcc tcg tcc acc ttc aag acg
1512 Gly Val Thr Val Gly Thr Tyr Ala Ser Ser Ser Ser Thr Phe Lys
Thr 370 375 380 ctc act tcc gcc atc aag acc ttc gcc gac ggc ttc ctc
gcg gtc aac 1560 Leu Thr Ser Ala Ile Lys Thr Phe Ala Asp Gly Phe
Leu Ala Val Asn 385 390 395 gcc aag tac acg ccc tcg aac ggc ggc ctt
gct gaa cag tac agc cgg 1608 Ala Lys Tyr Thr Pro Ser Asn Gly Gly
Leu Ala Glu Gln Tyr Ser Arg 400 405 410 415 agc aac ggc tcg ccc gtc
agc gct gtg gac ctg acg tgg agc tat gct 1656 Ser Asn Gly Ser Pro
Val Ser Ala Val Asp Leu Thr Trp Ser Tyr Ala 420 425 430 gct gcc ctc
acg tcg ttt gct gcg cgc tca ggc aag acg tat gcg agc 1704 Ala Ala
Leu Thr Ser Phe Ala Ala Arg Ser Gly Lys Thr Tyr Ala Ser 435 440 445
tgg ggc gcg gcg ggt ttg act gtc ccg acg act tgc tcg ggg agt ggc
1752 Trp Gly Ala Ala Gly Leu Thr Val Pro Thr Thr Cys Ser Gly Ser
Gly 450 455 460 ggt gct ggg act gtg gcc gtc acc ttc aac gtg cag gcg
acc acc gtg 1800 Gly Ala Gly Thr Val Ala Val Thr Phe Asn Val Gln
Ala Thr Thr Val 465 470 475 ttc ggc g gtgagtacgc catcgtatgc
tactagggca gttactcata gcttgtcgga 1857 Phe Gly 480 cttgtag ag aac
att tac atc aca ggc tcg gtc ccc gct ctc cag aac 1905 Glu Asn Ile
Tyr Ile Thr Gly Ser Val Pro Ala Leu Gln Asn 485 490 495 tgg tcg ccc
gac aac gcg ctc atc ctc tca gcg gcc aac tac ccc act 1953 Trp Ser
Pro Asp Asn Ala Leu Ile Leu Ser Ala Ala Asn Tyr Pro Thr 500 505 510
tgg agc a gtacgtctga accgccttca gcctgcttca tacgttcgct gacatcgggc
2010 Trp Ser atccatctag tc acc gtg aac ctg ccg gcg agc acg acg atc
gag tac 2058 Ile Thr Val Asn Leu Pro Ala Ser Thr Thr Ile Glu Tyr
515 520 525 aag tac att cgc aag ttc aac ggc gcg gtc acc tgg gag tcc
gac ccg 2106 Lys Tyr Ile Arg Lys Phe Asn Gly Ala Val Thr Trp Glu
Ser Asp Pro 530 535 540 aac aac tcg atc acg acg ccc gcg agc ggc acg
ttc acc cag aac gac 2154 Asn Asn Ser Ile Thr Thr Pro Ala Ser Gly
Thr Phe Thr Gln Asn Asp 545 550 555 acc tgg cgg tag 2166 Thr Trp
Arg 560 37 579 PRT Trametes cingulata 37 Met Arg Phe Thr Leu Leu
Thr Ser Leu Leu Gly Leu Ala Leu Gly Ala -15 -10 -5 Phe Ala Gln Ser
Ser Ala Ala Asp Ala Tyr Val Ala Ser Glu Ser Pro -1 1 5 10 Ile Ala
Lys Ala Gly Val Leu Ala Asn Ile Gly Pro Ser Gly Ser Lys 15 20 25 30
Ser Asn Gly Ala Lys Ala Ser Asp Thr Pro Gly Ile Val Ile Ala Ser 35
40 45 Pro Ser Thr Ser Asn Pro Asn Tyr Leu Tyr Thr Trp Thr Arg Asp
Ser 50 55 60 Ser Leu Val Phe Lys Ala Leu Ile Asp Gln Phe Thr Thr
Gly Glu Asp 65 70 75 Thr Ser Leu Arg Thr Leu Ile Asp Glu Phe Thr
Ser Ala Glu Ala Ile 80 85 90 Leu Gln Gln Val Pro Asn Pro Ser Gly
Thr Val Ser Thr Gly Gly Leu 95 100 105 110 Gly Glu Pro Lys Phe Asn
Ile Asp Glu Thr Ala Phe Thr Asp Ala Trp 115 120 125 Gly Arg Pro Gln
Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Ile Ile 130 135 140 Thr Tyr
Ala Asn Trp Leu Leu Asp Asn Lys Asn Thr Thr Tyr Val Thr 145 150 155
Asn Thr Leu Trp Pro Ile Ile Lys Leu Asp Leu Asp Tyr Val Ala Ser 160
165 170 Asn Trp Asn Gln Ser Thr Phe Asp Leu Trp Glu Glu Ile Asn Ser
Ser 175 180 185 190 Ser Phe Phe Thr Thr Ala Val Gln His Arg Ala Leu
Arg Glu Gly Ala 195 200 205 Thr Phe Ala Asn Arg Ile Gly Gln Thr Ser
Val Val Ser Gly Tyr Thr 210 215 220 Thr Gln Ala Asn Asn Leu Leu Cys
Phe Leu Gln Ala Ser Tyr Trp Asn 225 230 235 Pro Thr Gly Gly Tyr Ile
Thr Ala Asn Thr Gly Gly Gly Arg Ser Gly 240 245 250 Lys Asp Ala Asn
Thr Val Leu Thr Ser Ile His Thr Phe Asp Pro Ala 255 260 265 270 Ala
Gly Cys Asp Ala Val Thr Phe Gln Pro Cys Ser Asp Lys Ala Leu 275 280
285 Ser Asn Leu Lys Val Tyr Val Asp Ala Phe Arg Ser Ile Tyr Ser Ile
290 295 300 Asn Ser Gly Ile Ala Ser Asn Ala Ala Val Ala Thr Gly Arg
Tyr Pro 305 310 315 Glu Asp Ser Tyr Met Gly Gly Asn Pro Trp Tyr Leu
Thr Thr Ser Ala 320 325 330 Val Ala Glu Gln Leu Tyr Asp Ala Leu Ile
Val Trp Asn Lys Leu Gly 335 340 345 350 Ala Leu Asn Val Thr Ser Thr
Ser Leu Pro Phe Phe Gln Gln Phe Ser 355 360 365 Ser Gly Val Thr Val
Gly Thr Tyr Ala Ser Ser Ser Ser Thr Phe Lys 370 375 380 Thr Leu Thr
Ser Ala Ile Lys Thr Phe Ala Asp Gly Phe Leu Ala Val 385 390 395 Asn
Ala Lys Tyr Thr Pro Ser Asn Gly Gly Leu Ala Glu Gln Tyr Ser 400 405
410 Arg Ser Asn Gly Ser Pro Val Ser Ala Val Asp Leu Thr Trp Ser Tyr
415 420 425 430 Ala Ala Ala Leu Thr Ser Phe Ala Ala Arg Ser Gly Lys
Thr Tyr Ala 435 440 445 Ser Trp Gly Ala Ala Gly Leu Thr Val Pro Thr
Thr Cys Ser Gly Ser 450 455 460 Gly Gly Ala Gly Thr Val Ala Val Thr
Phe Asn Val Gln Ala Thr Thr 465 470 475 Val Phe Gly Glu Asn Ile Tyr
Ile Thr Gly Ser Val Pro Ala Leu Gln 480 485 490 Asn Trp Ser Pro Asp
Asn Ala Leu Ile Leu Ser Ala Ala Asn Tyr Pro
495 500 505 510 Thr Trp Ser Ile Thr Val Asn Leu Pro Ala Ser Thr Thr
Ile Glu Tyr 515 520 525 Lys Tyr Ile Arg Lys Phe Asn Gly Ala Val Thr
Trp Glu Ser Asp Pro 530 535 540 Asn Asn Ser Ile Thr Thr Pro Ala Ser
Gly Thr Phe Thr Gln Asn Asp 545 550 555 Thr Trp Arg 560 38 1740 DNA
Trametes cingulata misc_feature (1)..(1740) cDNA misc_feature
(55)..(1740) mature peptide coding region of cDNA misc_feature
(1435)..(1464) linker misc_feature (1465)..(1740) Binding domain 38
atgcgtttca cgctcctcac ctccctcctg ggcctcgccc tcggcgcgtt cgcgcagtcg
60 agtgcggccg acgcgtacgt cgcgtccgaa tcgcccatcg ccaaggcggg
tgtgctcgcc 120 aacatcgggc ccagcggctc caagtccaac ggagcaaagg
caagtgacac cccggcatcg 180 ntgattgcaa gtccgagcac atccaacccg
aactacctgt acacatggac gcgcgactcg 240 tccctcgtgt tcaaggcgct
catcgaccag ttcaccactg gcgaagatac ctcgctccga 300 actctgattg
acgagttcac ctcggcggag gccatactcc agcaggtgcc gaacccgagc 360
gggacagtca gcactggagg cctcggcgag cccaagttca acatcgacga gaccgcgttc
420 acggatgcct ggggtcgtcc tcagcgcgat ggtcccgctc tccgggcgac
tgccatcatc 480 acctacgcca actggctcct cgacaacaag aacacgacct
acgtgaccaa cactctctgg 540 cctatcatca agctcgacct cgactacgtc
gccagcaact ggaaccagtc cacgtttgat 600 ctctgggagg agattaactc
ctcgtcgttc ttcactaccg ccgtccagca ccgtgctctg 660 cgcgagggcg
cgactttcgc taatcgcatc ggacaaacct cggtggtcag cgggtacacc 720
acccaagcaa acaaccttct ctgcttcctg caggcatcgt actggaaccc caccggcggc
780 tatatcaccg caaacacggg cggcggccgc tctggcaagg acgcgaacac
cgttctcacg 840 tcgatccaca ccttcgaccc ggccgctgga tgcgacgctg
ttacgttcca gccgtgctcg 900 gacaaggcgc tgtcgaactt gaaggtgtac
gtcgatgcgt tccgctcgat ctactccatc 960 aacagcggga tcgcctcgaa
tgcggccgtt gctaccggcc gctaccccga ggacagctac 1020 atgggcggaa
acccatggta cctcaccacc tccgccgtcg ctgagcagct ctacgatgcg 1080
ctcattgtgt ggaacaagct tggcgccctg aacgtcacga gcacctccct ccccttcttc
1140 cagcagttct cgtcaggcgt caccgtcggc acctatgcct catcctcgtc
caccttcaag 1200 acgctcactt ccgccatcaa gaccttcgcc gacggcttcc
tcgcggtcaa cgccaagtac 1260 acgccctcga acggcggcct tgctgaacag
tacagccgga gcaacggctc gcccgtcagc 1320 gctgtggacc tgacgtggag
ctatgctgct gccctcacgt cgtttgctgc gcgctcaggc 1380 aagacgtatg
cgagctgggg cgcggcgggt ttgactgtcc cgacgacttg ctcggggagt 1440
ggcggtgctg ggactgtggc cgtcaccttc aacgtgcagg cgaccaccgt gttcggcgag
1500 aacatttaca tcacaggctc ggtccccgct ctccagaact ggtcgcccga
caacgcgctc 1560 atcctctcag cggccaacta ccccacttgg agcatcaccg
tgaacctgcc ggcgagcacg 1620 acgatcgagt acaagtacat tcgcaagttc
aacggcgcgg tcacctggga gtccgacccg 1680 aacaactcga tcacgacgcc
cgcgagcggc acgttcaccc agaacgacac ctggcggtag 1740 39 2182 DNA
Pachykytospora papyraceae CDS (1)..(159) misc_signal (1)..(54)
mat_peptide (55)..(2182) Intron (160)..(238) CDS (239)..(515)
Intron (516)..(565) misc_feature (523)..(524) n is a, c, g, or t
misc_feature (555)..(555) n is a, c, g, or t CDS (566)..(713)
misc_feature (613)..(613) n is a, c, g, or t misc_feature
(648)..(648) n is a, c, g, or t Intron (714)..(775) misc_feature
(727)..(727) n is a, c, g, or t misc_feature (753)..(753) n is a,
c, g, or t misc_feature (768)..(768) n is a, c, g, or t
misc_feature (770)..(770) n is a, c, g, or t CDS (776)..(935)
Intron (936)..(971) CDS (972)..(1274) Intron (1275)..(1333) CDS
(1334)..(1796) misc_feature (1736)..(1762) Linker misc_feature
(1763)..(2182) Binding domain Intron (1797)..(1875) CDS
(1876)..(1971) Intron (1972)..(2036) CDS (2037)..(2179) 39 atg cgc
ttc acc ctc ctc tcc tcc ctc gtc gcc ctc gcc acc ggc gcg 48 Met Arg
Phe Thr Leu Leu Ser Ser Leu Val Ala Leu Ala Thr Gly Ala -15 -10 -5
ttc acc cag acc agc cag gcc gac gcg tac gtc aag tcc gag ggc ccc 96
Phe Thr Gln Thr Ser Gln Ala Asp Ala Tyr Val Lys Ser Glu Gly Pro -1
1 5 10 atc gcg aag gcg ggc ctc ctc gcc aac atc ggg ccc agc ggc tcc
aag 144 Ile Ala Lys Ala Gly Leu Leu Ala Asn Ile Gly Pro Ser Gly Ser
Lys 15 20 25 30 tcg cac ggg gcg aag gtgcgcttct ctttttccca
ttctacgtcg cttaaagcgc 199 Ser His Gly Ala Lys 35 gctcatacat
gtgcatgacc gcgttccgcg tgcgcgcag gcc ggt ctc gtc gtc 253 Ala Gly Leu
Val Val 40 gcc ccc ccc agc acg tcg gac ccc gac tac gtc tac acc tgg
acg ctg 301 Ala Pro Pro Ser Thr Ser Asp Pro Asp Tyr Val Tyr Thr Trp
Thr Leu 45 50 55 gat tcg tca ctc gtc ttc aag act atc atc gac cag
ttc acc tcc ggg 349 Asp Ser Ser Leu Val Phe Lys Thr Ile Ile Asp Gln
Phe Thr Ser Gly 60 65 70 gaa gac act tcc ctc cgc aca ctc att gac
cag ttc act agc gcg gag 397 Glu Asp Thr Ser Leu Arg Thr Leu Ile Asp
Gln Phe Thr Ser Ala Glu 75 80 85 aag gac ctc cag cag acg tcc aac
cct agt ggc act gtt tcc acc ggc 445 Lys Asp Leu Gln Gln Thr Ser Asn
Pro Ser Gly Thr Val Ser Thr Gly 90 95 100 ggt ctc ggc gag ccc aag
ttc aac atc gat ggg tcc gcg ttc acc ggt 493 Gly Leu Gly Glu Pro Lys
Phe Asn Ile Asp Gly Ser Ala Phe Thr Gly 105 110 115 120 gcc tgg ggt
cgc cct cag cgc g gtatgcanna cagttgaagc ttgttaagcg 545 Ala Trp Gly
Arg Pro Gln Arg 125 cttacatgtn ttgtgtacag ac ggc cct gct ctc cgc
gcg act gct atc ata 597 Asp Gly Pro Ala Leu Arg Ala Thr Ala Ile Ile
130 135 gcc tac gct aac tgg ntg ctc gac aac aac aac ggc acg tct tac
gtc 645 Ala Tyr Ala Asn Trp Xaa Leu Asp Asn Asn Asn Gly Thr Ser Tyr
Val 140 145 150 acn aac acc ctc tgg ccc atc atc aag ctt gac ttg gac
tac acc cag 693 Thr Asn Thr Leu Trp Pro Ile Ile Lys Leu Asp Leu Asp
Tyr Thr Gln 155 160 165 170 aac aac tgg aac cag tcg ac gtaagttcat
tatnccagct ttggctgtta 743 Asn Asn Trp Asn Gln Ser Thr 175
gaactgcatn gatcctcatg tcttntnccc ag g ttc gac ctt tgg gag gag gtc
797 Phe Asp Leu Trp Glu Glu Val 180 aac tcc tcc tct ttc ttc acg act
gcc gtc cag cac cgt gct ctc cgc 845 Asn Ser Ser Ser Phe Phe Thr Thr
Ala Val Gln His Arg Ala Leu Arg 185 190 195 200 gag ggt atc gcc ttc
gcg aag aag atc ggc caa acg tcg gtc gtg agc 893 Glu Gly Ile Ala Phe
Ala Lys Lys Ile Gly Gln Thr Ser Val Val Ser 205 210 215 ggc tac acc
acg cag gcg acc aac ctt ctc tgc ttc ctg cag 935 Gly Tyr Thr Thr Gln
Ala Thr Asn Leu Leu Cys Phe Leu Gln 220 225 230 gtcagtacgc
atgtgcagca cgccttctgg ctatag ctt aac ccg tgt tcc gca 989 Leu Asn
Pro Cys Ser Ala 235 tct tcg cag tcg tac tgg aac ccc tcg ggc ggc tat
gtc act gcg aac 1037 Ser Ser Gln Ser Tyr Trp Asn Pro Ser Gly Gly
Tyr Val Thr Ala Asn 240 245 250 aca ggc ggc ggc cgg tcc ggc aag gac
tcg aac acc gtc ctg acc tcg 1085 Thr Gly Gly Gly Arg Ser Gly Lys
Asp Ser Asn Thr Val Leu Thr Ser 255 260 265 atc cac acc ttc gac ccc
gcc gct ggc tgc gac gcc gcg acg ttc cag 1133 Ile His Thr Phe Asp
Pro Ala Ala Gly Cys Asp Ala Ala Thr Phe Gln 270 275 280 ccg tgc tct
gac aag gcc ctg tcc aac ctt aag gtc tac gtc gac tcg 1181 Pro Cys
Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Tyr Val Asp Ser 285 290 295
300 ttc cgt tcc atc tac tcc atc aac agt ggc atc acc tcc aac gcc gct
1229 Phe Arg Ser Ile Tyr Ser Ile Asn Ser Gly Ile Thr Ser Asn Ala
Ala 305 310 315 gtc gct gtt ggc cgc tac ccc gag gat gtg tac tac aac
ggc aac 1274 Val Ala Val Gly Arg Tyr Pro Glu Asp Val Tyr Tyr Asn
Gly Asn 320 325 330 gtgagttccg tgtcccctgc atcattgtca acagcagaaa
ctgaatccca tccgcgtag 1333 ccc tgg tgc ctc tcc acg tcc gcc gtc gct
gag cag ctc tac gac gcg 1381 Pro Trp Cys Leu Ser Thr Ser Ala Val
Ala Glu Gln Leu Tyr Asp Ala 335 340 345 atc atc gtc tgg aac aag ctc
ggc tcg ctc gaa gtg acg agc acc tcg 1429 Ile Ile Val Trp Asn Lys
Leu Gly Ser Leu Glu Val Thr Ser Thr Ser 350 355 360 ctc gcg ttc ttc
aag cag ctc tcc tcg gat gcc gcc gtc ggc acc tac 1477 Leu Ala Phe
Phe Lys Gln Leu Ser Ser Asp Ala Ala Val Gly Thr Tyr 365 370 375 tcg
tcc tcg tcc gcg acg ttc aag acg ctc acc gcg gcc gcg aag acg 1525
Ser Ser Ser Ser Ala Thr Phe Lys Thr Leu Thr Ala Ala Ala Lys Thr 380
385 390 395 ctc gcg gat ggc ttc ctc gct gtg aac gcg aag tac acg ccc
tcg aac 1573 Leu Ala Asp Gly Phe Leu Ala Val Asn Ala Lys Tyr Thr
Pro Ser Asn 400 405 410 ggc ggc ctc gcg gag cag ttc agc aag agc aac
ggc tcg ccg ctc agc 1621 Gly Gly Leu Ala Glu Gln Phe Ser Lys Ser
Asn Gly Ser Pro Leu Ser 415 420 425 gcc gtc gac ctc acg tgg agc tac
gcc gcc gcg ctc acg tcc ttt gcc 1669 Ala Val Asp Leu Thr Trp Ser
Tyr Ala Ala Ala Leu Thr Ser Phe Ala 430 435 440 gcg cgt gag ggc aag
acc ccc gcg agc tgg ggc gct gcg ggc ctc acc 1717 Ala Arg Glu Gly
Lys Thr Pro Ala Ser Trp Gly Ala Ala Gly Leu Thr 445 450 455 gtg ccg
tcg acg tgc tcg ggt aac gcg ggc ccc agc gtg aag gtg acg 1765 Val
Pro Ser Thr Cys Ser Gly Asn Ala Gly Pro Ser Val Lys Val Thr 460 465
470 475 ttc aac gtc cag gct acg act acc ttc ggc g gtcagtcctc
ttctccaact 1816 Phe Asn Val Gln Ala Thr Thr Thr Phe Gly 480 485
cgtttcggtc ggtgatgttg agcattcgtc tgacgtgtgt gtgttactgc tgcttgcag
1875 ag aac atc tac atc acc ggt aac acc gct gcg ctc cag aac tgg tcg
1922 Glu Asn Ile Tyr Ile Thr Gly Asn Thr Ala Ala Leu Gln Asn Trp
Ser 490 495 500 ccc gat aac gcg ctc ctc ctc tct gct gac aag tac ccc
acc tgg agc a 1971 Pro Asp Asn Ala Leu Leu Leu Ser Ala Asp Lys Tyr
Pro Thr Trp Ser 505 510 515 gtacgtgtca tctcatctcc agcctctcat
attacgttgt ttgctcatct gcatgtgctt 2031 cgcag tc acg ctc gac ctc ccc
gcg aac acc gtc gtc gag tac aaa tac 2080 Ile Thr Leu Asp Leu Pro
Ala Asn Thr Val Val Glu Tyr Lys Tyr 520 525 530 atc cgc aag ttc aac
ggc cag gtc acc tgg gaa tcg gac ccc aac aac 2128 Ile Arg Lys Phe
Asn Gly Gln Val Thr Trp Glu Ser Asp Pro Asn Asn 535 540 545 tcg atc
acg acg ccc gcc gac ggt acc ttc acc cag aac gac acc tgg 2176 Ser
Ile Thr Thr Pro Ala Asp Gly Thr Phe Thr Gln Asn Asp Thr Trp 550 555
560 cgg tga 2182 Arg 565 40 583 PRT Pachykytospora papyraceae
misc_feature (144)..(144) The 'Xaa' at location 144 stands for Met,
Val, or Leu. 40 Met Arg Phe Thr Leu Leu Ser Ser Leu Val Ala Leu Ala
Thr Gly Ala -15 -10 -5 Phe Thr Gln Thr Ser Gln Ala Asp Ala Tyr Val
Lys Ser Glu Gly Pro -1 1 5 10 Ile Ala Lys Ala Gly Leu Leu Ala Asn
Ile Gly Pro Ser Gly Ser Lys 15 20 25 30 Ser His Gly Ala Lys Ala Gly
Leu Val Val Ala Pro Pro Ser Thr Ser 35 40 45 Asp Pro Asp Tyr Val
Tyr Thr Trp Thr Leu Asp Ser Ser Leu Val Phe 50 55 60 Lys Thr Ile
Ile Asp Gln Phe Thr Ser Gly Glu Asp Thr Ser Leu Arg 65 70 75 Thr
Leu Ile Asp Gln Phe Thr Ser Ala Glu Lys Asp Leu Gln Gln Thr 80 85
90 Ser Asn Pro Ser Gly Thr Val Ser Thr Gly Gly Leu Gly Glu Pro Lys
95 100 105 110 Phe Asn Ile Asp Gly Ser Ala Phe Thr Gly Ala Trp Gly
Arg Pro Gln 115 120 125 Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Ile
Ile Ala Tyr Ala Asn 130 135 140 Trp Xaa Leu Asp Asn Asn Asn Gly Thr
Ser Tyr Val Thr Asn Thr Leu 145 150 155 Trp Pro Ile Ile Lys Leu Asp
Leu Asp Tyr Thr Gln Asn Asn Trp Asn 160 165 170 Gln Ser Thr Phe Asp
Leu Trp Glu Glu Val Asn Ser Ser Ser Phe Phe 175 180 185 190 Thr Thr
Ala Val Gln His Arg Ala Leu Arg Glu Gly Ile Ala Phe Ala 195 200 205
Lys Lys Ile Gly Gln Thr Ser Val Val Ser Gly Tyr Thr Thr Gln Ala 210
215 220 Thr Asn Leu Leu Cys Phe Leu Gln Leu Asn Pro Cys Ser Ala Ser
Ser 225 230 235 Gln Ser Tyr Trp Asn Pro Ser Gly Gly Tyr Val Thr Ala
Asn Thr Gly 240 245 250 Gly Gly Arg Ser Gly Lys Asp Ser Asn Thr Val
Leu Thr Ser Ile His 255 260 265 270 Thr Phe Asp Pro Ala Ala Gly Cys
Asp Ala Ala Thr Phe Gln Pro Cys 275 280 285 Ser Asp Lys Ala Leu Ser
Asn Leu Lys Val Tyr Val Asp Ser Phe Arg 290 295 300 Ser Ile Tyr Ser
Ile Asn Ser Gly Ile Thr Ser Asn Ala Ala Val Ala 305 310 315 Val Gly
Arg Tyr Pro Glu Asp Val Tyr Tyr Asn Gly Asn Pro Trp Cys 320 325 330
Leu Ser Thr Ser Ala Val Ala Glu Gln Leu Tyr Asp Ala Ile Ile Val 335
340 345 350 Trp Asn Lys Leu Gly Ser Leu Glu Val Thr Ser Thr Ser Leu
Ala Phe 355 360 365 Phe Lys Gln Leu Ser Ser Asp Ala Ala Val Gly Thr
Tyr Ser Ser Ser 370 375 380 Ser Ala Thr Phe Lys Thr Leu Thr Ala Ala
Ala Lys Thr Leu Ala Asp 385 390 395 Gly Phe Leu Ala Val Asn Ala Lys
Tyr Thr Pro Ser Asn Gly Gly Leu 400 405 410 Ala Glu Gln Phe Ser Lys
Ser Asn Gly Ser Pro Leu Ser Ala Val Asp 415 420 425 430 Leu Thr Trp
Ser Tyr Ala Ala Ala Leu Thr Ser Phe Ala Ala Arg Glu 435 440 445 Gly
Lys Thr Pro Ala Ser Trp Gly Ala Ala Gly Leu Thr Val Pro Ser 450 455
460 Thr Cys Ser Gly Asn Ala Gly Pro Ser Val Lys Val Thr Phe Asn Val
465 470 475 Gln Ala Thr Thr Thr Phe Gly Glu Asn Ile Tyr Ile Thr Gly
Asn Thr 480 485 490 Ala Ala Leu Gln Asn Trp Ser Pro Asp Asn Ala Leu
Leu Leu Ser Ala 495 500 505 510 Asp Lys Tyr Pro Thr Trp Ser Ile Thr
Leu Asp Leu Pro Ala Asn Thr 515 520 525 Val Val Glu Tyr Lys Tyr Ile
Arg Lys Phe Asn Gly Gln Val Thr Trp 530 535 540 Glu Ser Asp Pro Asn
Asn Ser Ile Thr Thr Pro Ala Asp Gly Thr Phe 545 550 555 Thr Gln Asn
Asp Thr Trp Arg 560 565 41 1752 DNA Pachykytospora papyraceae
misc_feature (1)..(54) Signal misc_feature (55)..(1752) mature
peptide coding cDNA misc_feature (1450)..(1476) Linker misc_feature
(1477)..(1752) Binding domain 41 atgcgcttca ccctcctctc ctccctcgtc
gccctcgcca ccggcgcgtt cacccagacc 60 agccaggccg acgcgtacgt
caagtccgag ggccccatcg cgaaggcggg cctcctcgcc 120 aacatcgggc
ccagcggctc caagtcgcac ggggcgaagg ccggtctcgt cgtcgccccc 180
cccagcacgt cggaccccga ctacgtctac acctggacgc tggattcgtc actcgtcttc
240 aagactatca tcgaccagtt cacctccggg gaagacactt ccctccgcac
actcattgac 300 cagttcacta gcgcggagaa ggacctccag cagacgtcca
accctagtgg cactgtttcc 360 accggcggtc tcggcgagcc caagttcaac
atcgatgggt ccgcgttcac cggtgcctgg 420 ggtcgccctc agcgcgacgg
ccctgctctc cgcgcgactg ctatcatagc ctacgctaac 480 tggntgctcg
acaacaacaa cggcacgtct tacgtcacya acaccctctg gcccatcatc 540
aagcttgact tggactacac ccagaacaac tggaaccagt cgacgttcga cctttgggag
600 gaggtcaact cctcctcttt cttcacgact gccgtccagc accgtgctct
ccgcgagggt 660 atcgccttcg cgaagaagat cggccaaacg tcggtcgtga
gcggctacac cacgcaggcg 720 accaaccttc tctgcttcct gcagcttaac
ccgtgttccg catcttcgca gtcgtactgg 780 aacccctcgg gcggctatgt
cactgcgaac acaggcggcg gccggtccgg caaggactcg 840 aacaccgtcc
tgacctcgat ccacaccttc gaccccgccg ctggctgcga cgccgcgacg 900
ttccagccgt gctctgacaa ggccctgtcc aaccttaagg tctacgtcga ctcgttccgt
960 tccatctact ccatcaacag tggcatcacc tccaacgccg ctgtcgctgt
tggccgctac 1020 cccgaggatg tgtactacaa cggcaacccc tggtgcctct
ccacgtccgc cgtcgctgag 1080 cagctctacg acgcgatcat cgtctggaac
aagctcggct cgctcgaagt gacgagcacc 1140 tcgctcgcgt tcttcaagca
gctctcctcg gatgccgccg tcggcaccta ctcgtcctcg 1200 tccgcgacgt
tcaagacgct caccgcggcc gcgaagacgc tcgcggatgg cttcctcgct 1260
gtgaacgcga agtacacgcc ctcgaacggc ggcctcgcgg agcagttcag caagagcaac
1320 ggctcgccgc tcagcgccgt cgacctcacg tggagctacg ccgccgcgct
cacgtccttt 1380 gccgcgcgtg agggcaagac ccccgcgagc tggggcgctg
cgggcctcac cgtgccgtcg 1440 acgtgctcgg gtaacgcggg ccccagcgtg
aaggtgacgt tcaacgtcca ggctacgact 1500 accttcggcg agaacatcta
catcaccggt aacaccgctg cgctccagaa ctggtcgccc 1560 gataacgcgc
tcctcctctc tgctgacaag taccccacct ggagcatcac gctcgacctc 1620
cccgcgaaca ccgtcgtcga gtacaaatac atccgcaagt tcaacggcca ggtcacctgg
1680
gaatcggacc ccaacaactc gatcacgacg cccgccgacg gtaccttcac ccagaacgac
1740 acctggcggt ga 1752 42 1623 DNA Leucopaxillus giganteus CDS
(1)..(1620) cDNA misc_signal (1)..(51) mat_peptide (52)..(1620)
misc_feature (1306)..(1338) linker misc_feature (1339)..(1620)
binding domain misc_feature (1339)..(1620) binding domain 42 atg
cat ttc tct gtc ctc tcc gta ttt ctc gcg att agt tct gct tgg 48 Met
His Phe Ser Val Leu Ser Val Phe Leu Ala Ile Ser Ser Ala Trp -15 -10
-5 gct cag tct agc gca gtc gat gcc tat ctc gct ctc gaa tcc tcc gtc
96 Ala Gln Ser Ser Ala Val Asp Ala Tyr Leu Ala Leu Glu Ser Ser Val
-1 1 5 10 15 gcc aag gcc ggg ttg ctc gcc aac att ggc cca tct ggt
tca aag tct 144 Ala Lys Ala Gly Leu Leu Ala Asn Ile Gly Pro Ser Gly
Ser Lys Ser 20 25 30 tcg ggt gcc aag tct ggg att gtc att gcg tcg
cct tcg cat agc aac 192 Ser Gly Ala Lys Ser Gly Ile Val Ile Ala Ser
Pro Ser His Ser Asn 35 40 45 cct gac tac ctg ttc acc tgg acc cgc
gat tct tcg ctt gtg ttc cag 240 Pro Asp Tyr Leu Phe Thr Trp Thr Arg
Asp Ser Ser Leu Val Phe Gln 50 55 60 act atc atc aac cag ttc acg
ttg gga cac gac aat agt ttg agg cct 288 Thr Ile Ile Asn Gln Phe Thr
Leu Gly His Asp Asn Ser Leu Arg Pro 65 70 75 gag att gac aat ttt
gtt gat tcc caa agg aag atc caa caa gtc tca 336 Glu Ile Asp Asn Phe
Val Asp Ser Gln Arg Lys Ile Gln Gln Val Ser 80 85 90 95 aac cct tcg
gga act gtt agt tct ggc ggc ctt ggc gag ccc aag ttc 384 Asn Pro Ser
Gly Thr Val Ser Ser Gly Gly Leu Gly Glu Pro Lys Phe 100 105 110 aat
atc gac gaa acc gcc ttt aca ggg gca tgg ggc aac aca tcc tac 432 Asn
Ile Asp Glu Thr Ala Phe Thr Gly Ala Trp Gly Asn Thr Ser Tyr 115 120
125 gtc acc aac acc cta tgg ccc atc atc aaa ttg gac ctc gac tac gtc
480 Val Thr Asn Thr Leu Trp Pro Ile Ile Lys Leu Asp Leu Asp Tyr Val
130 135 140 gcg tcc aac tgg aac cag act ggt ttc gat ttg tgg gaa gaa
gta tcc 528 Ala Ser Asn Trp Asn Gln Thr Gly Phe Asp Leu Trp Glu Glu
Val Ser 145 150 155 tct tct tcc ttc ttc act act gcg gtt caa cac cgc
tcc ctt cgc caa 576 Ser Ser Ser Phe Phe Thr Thr Ala Val Gln His Arg
Ser Leu Arg Gln 160 165 170 175 ggt gct tcc cta gcc act gcc att gga
caa acc tct gtc gtt cct ggc 624 Gly Ala Ser Leu Ala Thr Ala Ile Gly
Gln Thr Ser Val Val Pro Gly 180 185 190 tac acc acc cag gcc aac aat
ata ctc tgc ttt caa cag tcc tac tgg 672 Tyr Thr Thr Gln Ala Asn Asn
Ile Leu Cys Phe Gln Gln Ser Tyr Trp 195 200 205 aac tca gct ggg tat
atg act gcc aat acc gga ggc ggg cgt tct ggg 720 Asn Ser Ala Gly Tyr
Met Thr Ala Asn Thr Gly Gly Gly Arg Ser Gly 210 215 220 aaa gac gcc
aac acc gtc ctc aca agt att cac aca ttc gat ccc gat 768 Lys Asp Ala
Asn Thr Val Leu Thr Ser Ile His Thr Phe Asp Pro Asp 225 230 235 gcc
ggc tgc gat tcc atc act ttc caa cct tgt tca gac cgt gcg ctc 816 Ala
Gly Cys Asp Ser Ile Thr Phe Gln Pro Cys Ser Asp Arg Ala Leu 240 245
250 255 atc aac ctt gtc aca tac gtc aat gca ttc cga agc atc tac gct
atc 864 Ile Asn Leu Val Thr Tyr Val Asn Ala Phe Arg Ser Ile Tyr Ala
Ile 260 265 270 aac gcg ggc atc gct aat aac caa ggc gtt gcc act ggt
agg tat cct 912 Asn Ala Gly Ile Ala Asn Asn Gln Gly Val Ala Thr Gly
Arg Tyr Pro 275 280 285 gaa gat ggc tac atg ggc gga aac ctc tac tac
gct ctc tcc act tgg 960 Glu Asp Gly Tyr Met Gly Gly Asn Leu Tyr Tyr
Ala Leu Ser Thr Trp 290 295 300 aag aaa cat agc tcc ctc acc att acg
gcg aca tca caa cct ttt ttc 1008 Lys Lys His Ser Ser Leu Thr Ile
Thr Ala Thr Ser Gln Pro Phe Phe 305 310 315 gcg ctc ttc tcg ccg ggt
gtt gct act ggc aca tat gcg tcc tct acg 1056 Ala Leu Phe Ser Pro
Gly Val Ala Thr Gly Thr Tyr Ala Ser Ser Thr 320 325 330 335 act acc
tat gct aca ctt act act gct att cag aat tac gcg gat agc 1104 Thr
Thr Tyr Ala Thr Leu Thr Thr Ala Ile Gln Asn Tyr Ala Asp Ser 340 345
350 ttc atc gct gtc gtg gct aag tat acg cct gcc aat ggc gga ctg gcg
1152 Phe Ile Ala Val Val Ala Lys Tyr Thr Pro Ala Asn Gly Gly Leu
Ala 355 360 365 gaa cag tac agc agg agt aac ggt ttg ccc gtt agt gcc
gtt gat tta 1200 Glu Gln Tyr Ser Arg Ser Asn Gly Leu Pro Val Ser
Ala Val Asp Leu 370 375 380 act tgg agc tat gcc gct ctc ttg acg gcg
gct gat gcg cga gcg ggg 1248 Thr Trp Ser Tyr Ala Ala Leu Leu Thr
Ala Ala Asp Ala Arg Ala Gly 385 390 395 cta aca ccc gct gca tgg gga
gca gcg ggg ttg acc gtg cca agc act 1296 Leu Thr Pro Ala Ala Trp
Gly Ala Ala Gly Leu Thr Val Pro Ser Thr 400 405 410 415 tgc tct act
ggg ggt ggt tca aac cca ggt ggt gga ggg tcg gtc tct 1344 Cys Ser
Thr Gly Gly Gly Ser Asn Pro Gly Gly Gly Gly Ser Val Ser 420 425 430
gtt acg ttc aat gtt caa gct aca acc acc ttt ggt gaa aac att ttt
1392 Val Thr Phe Asn Val Gln Ala Thr Thr Thr Phe Gly Glu Asn Ile
Phe 435 440 445 ttg acc ggc tcg atc aac gag tta gct aac tgg tct cct
gat aat gct 1440 Leu Thr Gly Ser Ile Asn Glu Leu Ala Asn Trp Ser
Pro Asp Asn Ala 450 455 460 ctc gcc ctc tct gcg gcc aat tat ccc acc
tgg agc ata acc gtc aac 1488 Leu Ala Leu Ser Ala Ala Asn Tyr Pro
Thr Trp Ser Ile Thr Val Asn 465 470 475 gtt ccc gca agc act acg atc
caa tac aag ttt atc cgt aaa ttc aac 1536 Val Pro Ala Ser Thr Thr
Ile Gln Tyr Lys Phe Ile Arg Lys Phe Asn 480 485 490 495 gga gcc atc
acc tgg gag tcc gac ccg aat agg cag atc aca acg ccg 1584 Gly Ala
Ile Thr Trp Glu Ser Asp Pro Asn Arg Gln Ile Thr Thr Pro 500 505 510
tct tcg gga agt ttt gtc cag aat gac tcg tgg aag tag 1623 Ser Ser
Gly Ser Phe Val Gln Asn Asp Ser Trp Lys 515 520 43 540 PRT
Leucopaxillus giganteus 43 Met His Phe Ser Val Leu Ser Val Phe Leu
Ala Ile Ser Ser Ala Trp -15 -10 -5 Ala Gln Ser Ser Ala Val Asp Ala
Tyr Leu Ala Leu Glu Ser Ser Val -1 1 5 10 15 Ala Lys Ala Gly Leu
Leu Ala Asn Ile Gly Pro Ser Gly Ser Lys Ser 20 25 30 Ser Gly Ala
Lys Ser Gly Ile Val Ile Ala Ser Pro Ser His Ser Asn 35 40 45 Pro
Asp Tyr Leu Phe Thr Trp Thr Arg Asp Ser Ser Leu Val Phe Gln 50 55
60 Thr Ile Ile Asn Gln Phe Thr Leu Gly His Asp Asn Ser Leu Arg Pro
65 70 75 Glu Ile Asp Asn Phe Val Asp Ser Gln Arg Lys Ile Gln Gln
Val Ser 80 85 90 95 Asn Pro Ser Gly Thr Val Ser Ser Gly Gly Leu Gly
Glu Pro Lys Phe 100 105 110 Asn Ile Asp Glu Thr Ala Phe Thr Gly Ala
Trp Gly Asn Thr Ser Tyr 115 120 125 Val Thr Asn Thr Leu Trp Pro Ile
Ile Lys Leu Asp Leu Asp Tyr Val 130 135 140 Ala Ser Asn Trp Asn Gln
Thr Gly Phe Asp Leu Trp Glu Glu Val Ser 145 150 155 Ser Ser Ser Phe
Phe Thr Thr Ala Val Gln His Arg Ser Leu Arg Gln 160 165 170 175 Gly
Ala Ser Leu Ala Thr Ala Ile Gly Gln Thr Ser Val Val Pro Gly 180 185
190 Tyr Thr Thr Gln Ala Asn Asn Ile Leu Cys Phe Gln Gln Ser Tyr Trp
195 200 205 Asn Ser Ala Gly Tyr Met Thr Ala Asn Thr Gly Gly Gly Arg
Ser Gly 210 215 220 Lys Asp Ala Asn Thr Val Leu Thr Ser Ile His Thr
Phe Asp Pro Asp 225 230 235 Ala Gly Cys Asp Ser Ile Thr Phe Gln Pro
Cys Ser Asp Arg Ala Leu 240 245 250 255 Ile Asn Leu Val Thr Tyr Val
Asn Ala Phe Arg Ser Ile Tyr Ala Ile 260 265 270 Asn Ala Gly Ile Ala
Asn Asn Gln Gly Val Ala Thr Gly Arg Tyr Pro 275 280 285 Glu Asp Gly
Tyr Met Gly Gly Asn Leu Tyr Tyr Ala Leu Ser Thr Trp 290 295 300 Lys
Lys His Ser Ser Leu Thr Ile Thr Ala Thr Ser Gln Pro Phe Phe 305 310
315 Ala Leu Phe Ser Pro Gly Val Ala Thr Gly Thr Tyr Ala Ser Ser Thr
320 325 330 335 Thr Thr Tyr Ala Thr Leu Thr Thr Ala Ile Gln Asn Tyr
Ala Asp Ser 340 345 350 Phe Ile Ala Val Val Ala Lys Tyr Thr Pro Ala
Asn Gly Gly Leu Ala 355 360 365 Glu Gln Tyr Ser Arg Ser Asn Gly Leu
Pro Val Ser Ala Val Asp Leu 370 375 380 Thr Trp Ser Tyr Ala Ala Leu
Leu Thr Ala Ala Asp Ala Arg Ala Gly 385 390 395 Leu Thr Pro Ala Ala
Trp Gly Ala Ala Gly Leu Thr Val Pro Ser Thr 400 405 410 415 Cys Ser
Thr Gly Gly Gly Ser Asn Pro Gly Gly Gly Gly Ser Val Ser 420 425 430
Val Thr Phe Asn Val Gln Ala Thr Thr Thr Phe Gly Glu Asn Ile Phe 435
440 445 Leu Thr Gly Ser Ile Asn Glu Leu Ala Asn Trp Ser Pro Asp Asn
Ala 450 455 460 Leu Ala Leu Ser Ala Ala Asn Tyr Pro Thr Trp Ser Ile
Thr Val Asn 465 470 475 Val Pro Ala Ser Thr Thr Ile Gln Tyr Lys Phe
Ile Arg Lys Phe Asn 480 485 490 495 Gly Ala Ile Thr Trp Glu Ser Asp
Pro Asn Arg Gln Ile Thr Thr Pro 500 505 510 Ser Ser Gly Ser Phe Val
Gln Asn Asp Ser Trp Lys 515 520
* * * * *
References