U.S. patent application number 16/456536 was filed with the patent office on 2019-10-17 for solubilized enzyme and uses thereof.
The applicant listed for this patent is XYLECO, INC.. Invention is credited to Natasha Kreder, Sean Landry, James Lynch, Thomas Craig Masterman, Marshall Medoff, Desiree Pangilinan, Aiichiro Yoshida.
Application Number | 20190316108 16/456536 |
Document ID | / |
Family ID | 55582048 |
Filed Date | 2019-10-17 |
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United States Patent
Application |
20190316108 |
Kind Code |
A1 |
Medoff; Marshall ; et
al. |
October 17, 2019 |
SOLUBILIZED ENZYME AND USES THEREOF
Abstract
The present invention relates to mixtures comprising a
polypeptide or a plurality of polypeptides having biomass-degrading
activity that is solubilized from an inclusion body, and retaining
biomass-degrading activity, and methods for producing and using the
same. The invention described herein provides methods for
increasing the yield of recombinant protein with biomass-degrading
activity that can be isolated from host cells.
Inventors: |
Medoff; Marshall;
(Brookline, MA) ; Kreder; Natasha; (Wakefield,
MA) ; Lynch; James; (Woburn, MA) ; Landry;
Sean; (Essex, MA) ; Yoshida; Aiichiro;
(Canton, MA) ; Pangilinan; Desiree; (Waltham,
MA) ; Masterman; Thomas Craig; (Rockport,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
XYLECO, INC. |
WAKEFIELD |
MA |
US |
|
|
Family ID: |
55582048 |
Appl. No.: |
16/456536 |
Filed: |
June 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14782205 |
Oct 2, 2015 |
10377999 |
|
|
PCT/US2015/052200 |
Sep 25, 2015 |
|
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16456536 |
|
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62055702 |
Sep 26, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/02 20130101;
C12Y 302/01021 20130101; C12N 9/2445 20130101; C12P 19/14
20130101 |
International
Class: |
C12N 9/42 20060101
C12N009/42; C12P 19/14 20060101 C12P019/14; C12P 19/02 20060101
C12P019/02 |
Claims
1. A mixture comprising a plurality of polypeptides having
biomass-degrading activity and a solubilizing agent, wherein the
polypeptides have at least 8-10% of the biomass-degrading activity
compared to a native polypeptide having biomass-degrading
activity.
2. The mixture of claim 1, further comprising one or more proteins
associated with an inclusion body.
3. The mixture of claim 1, wherein the mixture does not comprise
one or more proteins associated with an inclusion body.
4. The mixture of any of the preceding claims, further comprising
cellular debris, one or more ribosomal component, one or more host
protein, and/or host nucleic acid comprising DNA and/or RNA.
5. The mixture of any of the preceding claims, wherein the
biomass-degrading activity is cellobiase activity, ligninase
activity, endoglucanase activity, cellobiohydrolase activity, or
xylanase activity.
6. The mixture of any of the preceding claims, wherein the
polypeptide is partially unfolded, partially misfolded, or
partially denatured.
7. The mixture of claim 1, wherein the polypeptide comprises an
amino acid sequence with at least 90% identity to SEQ ID NO: 1.
8. The mixture of any of the preceding claims, wherein the
polypeptide comprises a Cel3A enzyme from T. reesei, or a
functional variant or fragment thereof.
9. The mixture of claim 8, wherein the Cel3A enzyme comprises the
amino acid sequence SEQ ID NO: 1, or an amino acid sequence with at
least 90% identity thereof.
10. The mixture any of the preceding claims, wherein the
polypeptide is encoded by a nucleic acid sequence comprising at
least 90% identity to SEQ ID NO: 2 or SEQ ID NO: 3.
11. The mixture of any of the preceding claims, wherein the
polypeptide is aglycosylated.
12. The mixture of any of claims 1-3 or 7, wherein the solubilizing
agent comprises urea, and optionally, is present at a concentration
between 0.2M-6M.
13. The mixture of any of the preceding claims, further comprising
at least one additional polypeptide having a biomass-degrading
activity or a microorganism that produces one or more enzymes
having a biomass-degrading activity.
14. The mixture of claim 13, wherein the additional polypeptide is
selected from a ligninase, an endoglucanase, a cellobiohydrolase, a
cellobiase, and a xylanase, or any combination thereof.
15. The mixture of claim 13 or 14, wherein the additional
polypeptide is selected from: a. a polypeptide comprising an amino
acid sequence with at least 90% identity to SEQ ID NO: 1; b. a
Cel3A enzyme from T. reesei, or a functional variant or fragment
thereof; or c. a polypeptide encoded by a nucleic acid sequence
comprising (e.g., consisting of) SEQ ID NO: 2 or SEQ ID NO: 3.
16. The mixture of any of claims 13-15, wherein the additional
polypeptide is aglycosylated.
17. The mixture of any of claims 13-15, wherein the additional
polypeptide is glycosylated.
18. A mixture comprising a plurality of polypeptides having an
amino acid sequence with at least 90% identity to SEQ ID NO: 1 and
a solubilizing agent, wherein the plurality of polypeptides have at
least 20%-40% of the activity of the native polypeptide comprising
SEQ ID NO: 1.
19. The mixture of claim 18, further comprising one or more
proteins associated with an inclusion body.
20. The mixture of claim 18, wherein the mixture does not comprise
one or more proteins associated with an inclusion body.
21. The mixture of any of claims 18-20, further comprising cellular
debris, one or more ribosomal component, one or more host protein,
and/or host nucleic acid comprising DNA and/or RNA.
22. The mixture of any of claims 18-21, wherein the polypeptide is
partially unfolded, partially misfolded, or partially
denatured.
23. The mixture any of claims 18-22, wherein the polypeptide is
encoded by a nucleic acid sequence comprising at least 90% identity
to SEQ ID NO: 2 or SEQ ID NO: 3.
24. The mixture of any of claims 18-23, wherein the polypeptide is
aglycosylated.
25. The mixture of any of claims 18-20, wherein the solubilizing
agent comprises urea, and optionally, is present at a concentration
between 0.2M-6M.
26. The mixture of any of claims 18-25, further comprising at least
one additional polypeptide having a biomass-degrading activity or a
microorganism that produces one or more enzymes having a
biomass-degrading activity.
27. The mixture of claim 26, wherein the additional polypeptide is
selected from a ligninase, an endoglucanase, a cellobiohydrolase, a
cellobiase, and a xylanase, or any combination thereof.
28. The mixture of claim 26 or 27, wherein the additional
polypeptide is selected from: a. a polypeptide comprising an amino
acid sequence with at least 90% identity to SEQ ID NO: 1; b. a
Cel3A enzyme from T. reesei, or a functional variant or fragment
thereof; or c. a polypeptide encoded by a nucleic acid sequence
comprising (e.g., consisting of) SEQ ID NO: 2 or SEQ ID NO: 3.
29. The mixture of any of claims 26-28 wherein the additional
polypeptide is aglycosylated.
30. The mixture of any of claims 26-28, wherein the additional
polypeptide is glycosylated.
31. A method for producing a mixture of any of claims 1-30
comprising contacting a cell expressing the polypeptide having
biomass-degrading activity, or lysate thereof, with a solubilizing
agent at a concentration suitable for solubilizing the
polypeptide.
32. The method of claim 31, further comprising lysing the cell to
obtain a lysate, separating a soluble fraction from an insoluble
fraction of the lysate, and resuspending the insoluble fraction in
the solubilizing agent.
33. The method of claim 31 or 32, wherein the solubilizing agent is
urea, and optionally, wherein the concentration of the solubilizing
agent is between 0.2M-6M.
34. The method of any of claims 31-33, wherein the
biomass-degrading activity is a cellobiase activity, a ligninase
activity, an endoglucanase activity, a cellobiohydrolase, or a
xylanase activity.
35. The method of any of claims 31-34, wherein the polypeptide
comprises an amino acid sequence with at least 90% identity to SEQ
ID NO: 1.
36. The method of any of claims 31-35, wherein the polypeptide
comprises a Cel3A from T. reesei, or a functional variant or
fragment thereof.
37. The method of any of claims 31-36 wherein the polypeptide is
aglycosylated.
38. A method for producing a polypeptide having biomass-degrading
activity comprising expressing the polypeptide in a cell and
contacting the cell or a lysate thereof with a solubilizing agent
at a concentration suitable for solubilizing the polypeptide.
39. A method for producing a polypeptide having biomass-degrading
activity comprising providing a cell that has been genetically
modified to produce at least one polypeptide having
biomass-degrading activity, wherein at least a portion of said
polypeptide having biomass-degrading activity is found in inclusion
bodies, and contacting the cell, or a lysate thereof containing the
inclusion bodies, with a solubilizing agent at a concentration
suitable for solubilizing the polypeptide.
40. The method of claim 38 or 39, wherein the solubilizing agent
comprises urea.
41. The method of any of claims 38-40, wherein the concentration of
the solubilizing agent is between 0.2M-6M.
42. The method of any of claims 38-41, further comprising lysing
the cell to obtain a lysate, separating a soluble fraction from an
insoluble fraction of the lysate, and resuspending the insoluble
fraction in the solubilizing agent.
43. The method of any of claims 38-42, wherein the
biomass-degrading activity is a cellobiase activity, a ligninase
activity, an endoglucanase activity, a cellobiohydrolase activity,
or a xylanase activity.
44. The method of any of claim 38 or 39, wherein the polypeptide
comprises an amino acid sequence with at least 90% identity to SEQ
ID NO: 1.
45. The method of any of claims 38-44, wherein the polypeptide
comprises a Cel3A from T. reesei, or a functional variant or
fragment thereof.
46. The method of any of claims 38-45, wherein the cell is a
prokaryotic or bacterial cell, e.g., E. coli cell, origami E. coli
cell.
47. The method of any of claims 38-46, wherein the polypeptide is
aglycosylated.
48. A method of producing a product from a biomass comprising
contacting a biomass with the mixture of any of claims 1-30, and,
optionally, a microorganism that produces one or more
biomass-degrading enzyme and/or an enzyme mixture comprising
biomass-degrading enzymes, under conditions suitable for the
production of the product.
49. The method of claim 48, further comprising treating the biomass
with an electron beam prior to contacting the biomass with the
mixture.
50. The method of claim 48 or 49, wherein the product is a sugar
product.
51. The method of claim 50, wherein the sugar product is glucose
and/or xylose.
52. The method of any of claims 48-51, further comprising isolating
the product.
53. The method of claim 52, wherein the isolating of the product
comprises precipitation, crystallization, chromatography,
centrifugation, and/or extraction.
54. The method of any of claims 48-53, wherein the enzyme mixture
comprises at least two of the enzymes selected from B2AF03, CIP1,
CIP2, Cel1a, Cel3a, Cel5a, Cel6a, Cel7a, Cel7b, Cel12a, Cel45a,
Cel74a, paMan5a, paMan26a, and Swollenin.
55. The method of any of claims 48-54, wherein the biomass
comprises one or more of an agricultural product or waste, a paper
product or waste, a forestry product, or a general waste, or any
combination thereof, wherein: a) an agricultural product or waste
comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay,
arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago,
sorghum, potato, sweet potato, taro, yams, beans, favas, lentils,
peas, grasses, switchgrass, miscanthus, cord grass, reed canary
grass, grain residues, canola straw, wheat straw, barley straw, oat
straw, rice straw, corn cobs, corn stover, corn fiber, coconut
hair, beet pulp, bagasse, soybean stover, grain residues, rice
hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a
combination thereof; b) a paper product or waste comprises paper,
pigmented papers, loaded papers, coated papers, filled papers,
magazines, printed matter, printer paper, polycoated paper,
cardstock, cardboard, paperboard, or paper pulp, or a combination
thereof; c) a forestry product comprises aspen wood, particle
board, wood chips, or sawdust, or a combination thereof; and d) a
general waste comprises manure, sewage, or offal, or a combination
thereof.
56. The method of any of claims 48-55, further comprises a step of
treating the biomass prior to introducing the microorganism or the
enzyme mixture to reduce the recalcitrance of the biomass, wherein
the treating comprises bombardment with electrons, sonication,
oxidation, pyrolysis, steam explosion, chemical treatment,
mechanical treatment, or freeze grinding.
57. The method of any of claims 48-56, wherein the microorganism
that produces a biomass-degrading enzyme is from species in the
genera selected from Bacillus, Coprinus, Myceliophthora,
Cephalosporium, Scytalidium, Penicillium, Aspergillus, Pseudomonas,
Humicola, Fusarium, Thielavia, Acremonium, Chrysosporium or
Trichoderma.
58. The method of any of claims 48-57, wherein the microorganism
that produces a biomass-degrading enzyme is selected from
Aspergillus, Humicola insolens (Scytalidium thermophilum) Coprinus
cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus
giganteus, Thielavia terrestris, Acremonium persicinum, Acremonium
acremonium, Acremonium brachypenium, Acremonium dichromosporum,
Acremonium obclavatum, Acremonium pinkertoniae, Acremonium
roseogriseum, Acremonium incoloratum, Acremonium furatum,
Chrysosporium lucknowense, Trichoderma viride, Trichoderma reesei,
or Trichoderma koningii.
59. The method of any of claims 48-58, wherein the microorganism
has been induced to produce biomass-degrading enzymes by combining
the microorganism with an induction biomass sample under conditions
suitable for increasing production of biomass-degrading enzymes
compared to an uninduced microorganism.
60. The method of any of claims 48-59, wherein the induction
biomass sample comprises one or more of an agricultural product or
waste, a paper product or waste, a forestry product, or a general
waste, or any combination thereof, wherein: a) an agricultural
product or waste comprises sugar cane jute, hemp, flax, bamboo,
sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava,
kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans,
favas, lentils, peas, grasses, switchgrass, miscanthus, cord grass,
reed canary grass, grain residues, canola straw, wheat straw,
barley straw, oat straw, rice straw, corn cobs, corn stover, corn
fiber, coconut hair, beet pulp, bagasse, soybean stover, grain
residues, rice hulls, oat hulls, wheat chaff, barley hulls, or
beeswing, or a combination thereof; b) a paper product or waste
comprises paper, pigmented papers, loaded papers, coated papers,
filled papers, magazines, printed matter, printer paper, polycoated
paper, cardstock, cardboard, paperboard, or paper pulp, or a
combination thereof; c) a forestry product comprises aspen wood,
particle board, wood chips, or sawdust, or a combination thereof;
and d) a general waste comprises manure, sewage, or offal, or a
combination thereof.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/782,205, filed Oct. 2, 2015, which is a national stage
application under 35 U.S.C. .sctn. 371 of International Application
No. PCT/US2015/052200, filed Sep. 25, 2015, which claims the
benefit of U.S. Provisional Application No. 62/055,702, filed Sep.
26, 2014; the entire contents of each of which are hereby
incorporated by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 1, 2015, is named X2002-7003WO_SL.txt and is 76,946 bytes
in size.
FIELD OF THE INVENTION
[0003] The present invention relates generally to mixtures
comprising a polypeptide having biomass-degrading activity
solubilized from inclusion bodies and having biomass-degrading
activity, and methods for producing the mixtures described herein.
The present invention also provides methods for using such
mixtures, e.g., to process biomass materials.
BACKGROUND OF THE INVENTION
[0004] Biomass-degrading enzymes, such as cellulases, xylanases,
and ligninases, are important for the degradation of biomass, such
as feedstock. Cellulosic and lignocellulosic materials are
produced, processed, and used in large quantities in a number of
applications. Often such materials are used once, and then
discarded as waste, or are simply considered to be wasted
materials, e.g., sewage, bagasse, sawdust, and stover.
SUMMARY OF THE INVENTION
[0005] High level of expression of recombinant proteins in host
cells such as E. coli can lead to accumulation of the recombinant
proteins into insoluble aggregates within the host cell. These
insoluble aggregates are called inclusion bodies and can also
contain other components, such as proteins endogenous to the host
cell, ribosomal components, nucleic acids, and cellular debris.
Solubilization of the recombinant proteins from the inclusion
bodies can be achieved through treatment with high concentrations
of a solubilizing agent such as urea, which disrupts hydrogen bonds
and hydrophobic interactions. However, treatment with a
solubilizing agent, such as urea, can result in denaturation of the
protein and loss of enzymatic activity. Thus, the aggregation of
recombinant proteins into inclusion bodies can reduce the yield of
recombinant protein with enzymatic activity that can be isolated
from the host cells.
[0006] The present invention is based, at least in part, on the
surprising discovery that a heterologously expressed cellobiase
that has been solubilized from inclusion bodies by solubilizing
agent, such as urea, retains cellobiase activity. Therefore, the
methods described herein for solubilization of heterologously
expressed cellobiase, or other biomass-degrading enzymes, are
useful for increasing the yield of the heterologously expressed
enzymes having biomass-degrading activity, e.g., by 30-40%.
Furthermore, the presence of the solubilizing agent, e.g., urea,
from the addition of the solubilized biomass-degrading enzyme,
e.g., cellobiase, does not adversely affect the saccharification
reaction for converting biomass to a sugar product and/or the yield
of products.
[0007] Accordingly, in one aspect, the disclosure features a
mixture comprising a polypeptide or a plurality of polypeptides
having a biomass-degrading activity and a solubilizing agent, e.g.,
urea, wherein the polypeptide or plurality thereof has at least
8-10% biomass-degrading activity compared the native
polypeptide.
[0008] In one embodiment, the mixture further comprises one or more
proteins associated with an inclusion body. Alternatively, in one
embodiment, the mixture does not comprise one or more proteins
associated with an inclusion body. In one embodiment, the mixture
further comprises cellular debris, one or more ribosomal component,
one or more host protein, e.g., protein endogenously expressed by
the host cell, and/or host nucleic acid, e.g., DNA and/or RNA.
[0009] In one embodiment, the biomass-degrading activity is
cellobiase activity, ligninase activity, endoglucanase activity,
cellobiohydrolase activity, or xylanase activity.
[0010] In one embodiment, the polypeptide is partially unfolded,
partially misfolded, or partially denatured.
[0011] In another aspect, the disclosure features a mixture
comprising a polypeptide or a plurality of polypeptides having an
amino acid sequence with at least 90% identity to SEQ ID NO: 1 and
a solubilizing agent, e.g., urea, wherein the polypeptide or
plurality thereof has at least 20% of the activity of the native
polypeptide, e.g., SEQ ID NO: 1 or Cel3a from T. reesei. For
example, the mixture further comprises one or more proteins
associated with an inclusion body. Alternatively, the mixture does
not comprise one or more proteins associated with an inclusion
body. The mixture may further comprise one or more of the
following: cellular debris, one or more ribosomal component, one or
more host protein, e.g., protein endogenously expressed by the host
cell, and/or host nucleic acid, e.g., DNA and/or RNA. The
polypeptide with at least 90% identity to SEQ ID NO: 1 may be
partially unfolded, partially misfolded, or partially
denatured.
[0012] In one embodiment, the polypeptide comprises an amino acid
sequence with at least 90% identity to SEQ ID NO: 1. In one
embodiment, the polypeptide comprises a Cel3A enzyme from T.
reesei, or a functional variant or fragment thereof. In one
embodiment, the Cel3A enzyme comprises (e.g., consists of) the
amino acid sequence SEQ ID NO: 1. In one embodiment, the
polypeptide is encoded by a nucleic acid sequence comprising (e.g.,
consisting of) at least 90% identity to SEQ ID NO: 2 or SEQ ID NO:
3.
[0013] In one embodiment, the polypeptide is aglycosylated.
[0014] In one embodiment, the solubilizing agent, e.g., urea, is
present in the mixture at a concentration between 0.2M-6M.
[0015] In one embodiment, the mixture further comprises at least
one additional polypeptide having a biomass-degrading activity or a
microorganism that produces one or more enzymes having a
biomass-degrading activity. In one embodiment, the additional
polypeptide is selected from a ligninase, an endoglucanase, a
cellobiohydrolase, a cellobiase, and a xylanase, or any combination
thereof. In one embodiment, the additional polypeptide is selected
from: [0016] a. a polypeptide comprising (e.g., consisting of) an
amino acid sequence with at least 90% identity to SEQ ID NO: 1;
[0017] b. a Cel3A enzyme from T. reesei, or a functional variant or
fragment thereof; or [0018] c. a polypeptide encoded by a nucleic
acid sequence comprising (e.g., consisting of) SEQ ID NO: 2 or SEQ
ID NO: 3.
[0019] In one embodiment, the additional polypeptide is
aglycosylated.
[0020] In one embodiment, the additional polypeptide is
glycosylated.
[0021] In one aspect, the disclosure features a method for
producing a mixture described herein comprising a polypeptide
having biomass-degrading activity, one or more proteins associated
with an inclusion body, and a solubilizing agent, e.g., urea,
wherein the method comprises contacting a cell expressing the
polypeptide having biomass-degrading activity, or lysate thereof,
with a solubilizing agent, e.g., urea, at a concentration suitable
for solubilizing the polypeptide. In one embodiment, the method
further comprises lysing the cell to obtain a lysate, separating a
soluble fraction from an insoluble fraction of the lysate, and
resuspending the insoluble fraction in the solubilizing agent,
e.g., urea. In one embodiment, the concentration of the
solubilizing agent, e.g., urea, is between 0.2M-6M, e.g., 6M.
[0022] In one embodiment, the biomass-degrading activity is a
cellobiase activity, a ligninase activity, an endoglucanase
activity, a cellobiohydrolase, or a xylanase activity.
[0023] In one embodiment, the polypeptide comprises an amino acid
sequence with at least 90% identity to SEQ ID NO: 1. In one
embodiment, the polypeptide comprises a Cel3A from T. reesei, or a
functional variant or fragment thereof.
[0024] In one embodiment, the polypeptide is aglycosylated.
[0025] In one aspect, the disclosure features a method for
producing a polypeptide having a biomass-degrading activity
comprising expressing the polypeptide in a cell and contacting the
cell or a lysate thereof with a solubilizing agent, e.g., urea, at
a concentration suitable for solubilizing the polypeptide.
[0026] In another aspect, the disclosure features a method for
producing a polypeptide having biomass-degrading activity
comprising providing a cell that has been genetically modified to
produce at least one polypeptide having biomass-degrading activity,
wherein at least a portion of said polypeptide having
biomass-degrading activity is found in inclusion bodies, and
contacting the cell, or a lysate thereof containing the inclusion
bodies, with a solubilizing agent, e.g., urea, at a concentration
suitable for solubilizing the polypeptide.
[0027] In one embodiment, the methods disclosed herein further
comprise lysing the cell to obtain a lysate, separating a soluble
fraction from an insoluble fraction of the lysate, and resuspending
the insoluble fraction in the solubilizing agent, e.g., urea. In
one embodiment, the concentration of the solubilizing agent, e.g.,
urea, is between 0.2M-6M, e.g., 6M.
[0028] In one embodiment, the biomass-degrading activity is a
cellobiase activity, a ligninase activity, an endoglucanase
activity, a cellobiohydrolase activity, or a xylanase activity.
[0029] In one embodiment, the aglycosylated polypeptide comprises
(e.g., consisting of) an amino acid sequence with at least 90%
identity to SEQ ID NO: 1. In one embodiment, the aglycosylated
polypeptide comprises a Cel3A from T. reesei, or a functional
variant or fragment thereof.
[0030] In one embodiment, the cell is a prokaryotic or bacterial
cell, e.g., E. coli cell, origami E. coli cell.
[0031] In one embodiment, the polypeptide is aglycosylated.
[0032] In one aspect, the disclosure features a method of producing
a product (e.g., hydrogen, a sugar, an alcohol) from a biomass (or
converting a biomass to a product) comprising contacting a biomass
with the mixture described herein comprising a polypeptide having
biomass-degrading activity, one or more proteins associated with an
inclusion body, and a solubilizing agent, e.g., urea, and,
optionally, with a microorganism that produces one or more
biomass-degrading enzyme and/or an enzyme mixture comprising
biomass-degrading enzymes, under conditions suitable for the
production of the product.
[0033] In one embodiment, the method further comprises a step of
treating the biomass with an electron beam prior to contacting the
biomass with the mixture described herein comprising a polypeptide
having biomass-degrading activity, one or more proteins associated
with an inclusion body, and a solubilizing agent, e.g., urea.
[0034] In one embodiment, the product is a sugar product. In one
embodiment, the sugar product is glucose and/or xylose.
[0035] In one embodiment, the method further comprises a step of
isolating the product. In one embodiment, the step of isolating the
product comprises precipitation, crystallization, chromatography,
centrifugation, and/or extraction.
[0036] In one embodiment, the enzyme mixture comprises at least two
of the enzymes selected from B2AF03, CIP1, CIP2, Cel1a, Cel3a,
Cel5a, Cel6a, Cel7a, Cel7b, Cel12a, Cel45a, Cel74a, paMan5a,
paMan26a, Swollenin.
[0037] In one embodiment, the biomass comprises starchy materials,
sugar cane, agricultural waste, paper, paper product, paper waste,
paper pulp, pigmented papers, loaded papers, coated papers, filled
papers, magazines, printed matter, printer paper, polycoated paper,
card stock, cardboard, paperboard, cotton, wood, particle board,
forestry wastes, sawdust, aspen wood, wood chips, grasses,
switchgrass, miscanthus, cord grass, reed canary grass, grain
residues, rice hulls, oat hulls, wheat chaff, barley hulls,
agricultural waste, silage, canola straw, wheat straw, barley
straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal,
abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa,
hay, coconut hair, sugar processing residues, bagasse, beet pulp,
agave bagasse, algae, seaweed, manure, sewage, offal, agricultural
or industrial waste, arracacha, buckwheat, banana, barley, cassava,
kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans,
favas, lentils, peas, or any combination thereof.
[0038] In one embodiment, the biomass comprises a starchy material
or a starchy material that includes a cellulosic component. In some
embodiments, the biomass comprises one or more of an agricultural
product or waste, a paper product or waste, a forestry product, or
a general waste, or any combination thereof; wherein: a) an
agricultural product or waste comprises sugar cane jute, hemp,
flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana,
barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato,
taro, yams, beans, favas, lentils, peas, grasses, switchgrass,
miscanthus, cord grass, reed canary grass, grain residues, canola
straw, wheat straw, barley straw, oat straw, rice straw, corn cobs,
corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean
stover, grain residues, rice hulls, oat hulls, wheat chaff, barley
hulls, or beeswing, or a combination thereof; b) a paper product or
waste comprises paper, pigmented papers, loaded papers, coated
papers, filled papers, magazines, printed matter, printer paper,
polycoated paper, cardstock, cardboard, paperboard, or paper pulp,
or a combination thereof; c) a forestry product comprises aspen
wood, particle board, wood chips, or sawdust, or a combination
thereof; and d) a general waste comprises manure, sewage, or offal,
or a combination thereof.
[0039] In one embodiment, the method further comprises a step of
treating the biomass prior to introducing the microorganism or the
enzyme mixture to reduce the recalcitrance of the biomass, e.g., by
treating the biomass with bombardment with electrons, sonication,
oxidation, pyrolysis, steam explosion, chemical treatment,
mechanical treatment, and/or freeze grinding.
[0040] In one embodiment, the microorganism that produces a
biomass-degrading enzyme is from species in the genera selected
from Bacillus, Coprinus, Myceliophthora, Cephalosporium,
Scytalidium, Penicillium, Aspergillus, Pseudomonas, Humicola,
Fusarium, Thielavia, Acremonium, Chrysosporium or Trichoderma. In
one embodiment, the microorganism that produces a biomass-degrading
enzyme is selected from Aspergillus, Humicola insolens (Scytalidium
thermophilum), Coprinus cinereus, Fusarium oxysporum,
Myceliophthora thermophila, Meripilus giganteus, Thielavia
terrestris, Acremonium persicinum, Acremonium acremonium,
Acremonium brachypenium, Acremonium dichromosporum, Acremonium
obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum,
Acremonium incoloratum, Acremonium furatum, Chrysosporium
lucknowense, Trichoderma viride, Trichoderma reesei, or Trichoderma
koningii.
[0041] In one embodiment, the microorganism has been induced to
produce biomass-degrading enzymes by combining the microorganism
with an induction biomass sample under conditions suitable for
increasing production of biomass-degrading enzymes compared to an
uninduced microorganism. In one embodiment, the induction biomass
sample comprises starchy materials, sugar cane, paper, paper
products, paper waste, paper pulp, pigmented papers, loaded papers,
coated papers, filled papers, magazines, printed matter, printer
paper, polycoated paper, card stock, cardboard, paperboard, cotton,
wood, particle board, forestry wastes, sawdust, aspen wood, wood
chips, grasses, switchgrass, miscanthus, cord grass, reed canary
grass, grain residues, rice hulls, oat hulls, wheat chaff, barley
hulls, agricultural waste, silage, canola straw, wheat straw,
barley straw, oat straw, rice straw, jute, hemp, flax, bamboo,
sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber,
alfalfa, hay, coconut hair, sugar processing residues, bagasse,
beet pulp, agave bagasse, algae, seaweed, manure, sewage, offal,
agricultural or industrial waste, arracacha, buckwheat, banana,
barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato,
taro, yams, beans, favas, lentils, peas, or any combination
thereof.
[0042] In one embodiment, the induction biomass comprises a starchy
material or a starchy material that includes a cellulosic
component. In some embodiments, the induction biomass comprises one
or more of an agricultural product or waste, a paper product or
waste, a forestry product, or a general waste, or any combination
thereof; wherein: a) an agricultural product or waste comprises
sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay,
arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago,
sorghum, potato, sweet potato, taro, yams, beans, favas, lentils,
peas, grasses, switchgrass, miscanthus, cord grass, reed canary
grass, grain residues, canola straw, wheat straw, barley straw, oat
straw, rice straw, corn cobs, corn stover, corn fiber, coconut
hair, beet pulp, bagasse, soybean stover, grain residues, rice
hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a
combination thereof; b) a paper product or waste comprises paper,
pigmented papers, loaded papers, coated papers, filled papers,
magazines, printed matter, printer paper, polycoated paper,
cardstock, cardboard, paperboard, or paper pulp, or a combination
thereof; c) a forestry product comprises aspen wood, particle
board, wood chips, or sawdust, or a combination thereof; and d) a
general waste comprises manure, sewage, or offal, or a combination
thereof.
[0043] In one embodiment, the present invention provides advantages
to current methods used in the art. These advantages include
providing access to insoluble enzymes that would normally be
discarded, increasing the yield of desired proteins that retain
enzyme activity, purified enzymes for cleaner downstream
processing, and organism selection (e.g., increase availability of
organisms that may have been previously excluded from use due to
propensity to develop inclusion bodies).
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 is a chromatogram showing the results of IMAC
purification of solubilized Cel3a. The purified solubilized Cel3a
peak is indicated by the arrow.
[0045] FIG. 2 is a picture of an SDS-PAGE gel showing the proteins
in different fractions of the IMAC purification. Lane 1 shows the
molecular weight standards. Lane 2 shows purified Cel3a from the
soluble fraction. Lane 3 shows the flow through from IMAC
purification of the insoluble fraction. Lane 4 shows the purified
solubilized Cel3a from the insoluble fraction.
[0046] FIG. 3 is a graph comparing the cellobiase activity of
purified soluble Cel3a and purified solubilized Cel3a from the
insoluble fraction.
[0047] FIG. 4 is a graph comparing the cellobiase activity of
purified soluble Cel3a, the wash fraction of the insoluble
fraction, and Cel3a solubilized from the insoluble fraction without
purification.
DETAILED DESCRIPTION
Definitions
[0048] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains.
[0049] The term "a" and "an" refers to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
By way of example, "an element" means one element or more than one
element.
[0050] The term "aglycosylated", as used herein, refers to a
molecule, e.g., a polypeptide, that is not glycosylated (i.e., it
comprises a hydroxyl group or other functional group that is not
attached to a glycosylate group) at one or more sites which has a
glycan attached when the molecule is produced in its native
environment. In some embodiments, the aglycosylated molecule does
not have any attached glycans. In one embodiment, the molecule has
been altered or mutated such that the molecule cannot be
glycosylated, e.g., one or more glycosylation site is mutated such
that a glycan cannot be attached to the glycosylation site. In
another embodiment, an attached glycan can be removed from the
molecule, e.g., by an enzymatic process, e.g., by incubating with
enzymes that remove glycans or have deglycosylating activity. In
yet another embodiment, glycosylation of the molecule can be
inhibited, e.g., by use of a glycosylation inhibitor (that inhibits
a glycosylating enzyme). In another embodiment, the molecule, e.g.,
the polypeptide, can be produced by a host cell that does not
glycosylate, e.g., E. coli. For example, a Cel3A enzyme is
aglycosylated when one or more site in the protein that normally
has a glycan group attached to it when the Cel3A enzyme is produced
in T. reesei does not have a glycan attached at that site.
[0051] The term "biomass", as used herein, refers to any
non-fossilized, organic matter. The various types of biomass
include plant biomass (e.g., lignocellulosic and cellulosic
biomass), microbial biomass, animal biomass (any animal by-product,
animal waste, etc.) and municipal waste biomass (residential and
light commercial refuse with recyclables such as metal and glass
removed). Plant biomass refers to any plant-derived organic matter
(woody or non-woody). Plant biomass can include, but is not limited
to, agricultural or food crops (e.g., sugarcane, sugar beets or
corn kernels) or an extract therefrom (e.g., sugar from sugarcane
and corn starch from corn), agricultural crop wastes and residues
such as corn stover, wheat straw, rice straw, sugar cane bagasse,
and the like. Plant biomass further includes, but is not limited
to, trees, woody energy crops, wood wastes and residues such as
softwood forest thinnings, barky wastes, sawdust, paper and pulp
industry waste streams, wood fiber, and the like. Additionally,
grass crops, such as switchgrass and the like have potential to be
produced on a large-scale as another plant biomass source. For
urban areas, the best potential plant biomass feedstock includes
yard waste (e.g., grass clippings, leaves, tree clippings, and
brush) and vegetable processing waste.
[0052] The term "biomass degrading enzymes", as used herein, refers
to enzymes that break down components of the biomass matter
described herein into intermediates or final products. For example,
biomass-degrading enzymes include at least ligninases,
endoglucancases, cellobiases, xylanases, and cellobiohydrolases.
Biomass-degrading enzymes are produced by a wide variety of
microorganisms, and can be isolated from the microorganisms, such
as T. reesei.
[0053] The term "biomass degrading activity", as used herein,
refers to enzymatic activity that breaks down components of the
biomass matter described herein into intermediates or final
products. Biomass-degrading activity includes at least ligninase
activity, endoglucanase activity, cellobiase activity,
cellobiohydrolase activity, and xylanase activity. For example, a
polypeptide having biomass degrading activity is a cellobiase such
as Cel3a from T. reesei.
[0054] The term "cellobiase", as used herein, refers to an enzyme
that catalyzes the hydrolysis of a dimer, trimer, tetramer,
pentamer, hexamer, heptamer, octamer, or an oligomer of glucose, or
an oligomer of glucose and xylose, to glucose and/or xylose. For
example, the cellobiase is beta-glucosidase, which catalyzes
beta-1,4 bonds in cellobiose to release two glucose molecules.
[0055] The term "cellobiase activity", as used herein, refers to
the activity of a category of cellulases that catalyze the
hydrolysis of cellobiose to glucose, e.g., catalyzes the hydrolysis
of beta-D-glucose residues to release beta-D-glucose. Cellobiase
activity can be determined according to the assays described
herein, e.g., in Example 4. One unit of cellobiase activity can be
defined as [glucose] g/L/[Cel3a] g/L/30 minutes.
[0056] The term "cellobiohydrolase" as used herein, refers to an
enzyme that hydrolyzes glycosidic bonds in cellulose. For example,
the cellobiohydrolase is 1,4-beta-D-glucan cellobiohydrolase, which
catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in
cellulose, cellooligosaccharides, or any beta-1,4-linked glucose
containing polymer, releasing oligosaccharides from the polymer
chain.
[0057] The term "cellobiohydrolase activity", as used herein,
refers to the activity of an enzyme that catalyzes the hydrolysis
of glycosidic bonds in cellulose, specifically, the hydrolysis of
1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides,
or any beta-1,4-linked glucose-containing polymer, to release
cellobiose from the ends of the saccharide chain, e.g., from the
reducing or the non-reducing ends of the chain. Cellobiohydrolase
activity can be determined according to the assays described
herein. One unit of cellobiohydrolase activity can be defined, for
example, as the amount of enzyme that releases 1 .mu.M of glucose
equivalent from substrate (e.g., Avicel) per minute.
[0058] The term "endoglucanase" as used herein, refers to an enzyme
that catalyzes the hydrolysis of internal (3-1,4 glycosidic bonds.
For example, the endoglucanase is endo-1,4-(1,3; 1,4)-beta-D-glucan
4-glucanohydrolase, which catalyses endohydrolysis of
1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives
(such as carboxymethyl cellulose and hydroxyethyl cellulose),
lichenan, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal
beta-D-glucans or xyloglucans, and other plant material containing
cellulosic components.
[0059] The term "endoglucanase activity" as used herein, refers to
the activity of an enzyme that catalyzes the endohydrolysis of the
internal glycosidic bonds, e.g., internal beta-1,4 glycosidic
bonds, of cellulose, cellulose derivatives (such as carboxymethyl
cellulose and hydroxyethyl cellulose), lichenan, beta-1,4 bonds in
mixed beta-1,3 glucans such as cereal beta-D-glucans or
xyloglucans, and other plant material containing cellulosic
components. Endoglucanase activity can be determined according to
the assays described herein. One unit of endoglucanase activity can
be defined, for example, as the amount of enzyme that increases the
concentration of the reducing ends by 1 .mu.M from substrate per
minute.
[0060] The term "enzyme mixture" as used herein, refers to a
combination of at least two different enzymes, or two different
variants of an enzyme (e.g., a glycosylated and an aglycosylated
version of an enzyme). The enzyme mixture referred to herein
includes at least the aglycosylated polypeptide having cellobiase
activity described herein. In one embodiment, the enzyme mixture
includes one or more of a cellobiase, an endoglucanase, a
cellobiohydrolase, a ligninase, and/or a xylanase. In some
embodiments, the enzyme mixture includes a cell, e.g., a
microorganism, which expresses and, e.g., secretes, one or more of
the enzymes. For example, the enzyme mixture can include an
aglycosylated polypeptide described herein and a cell, e.g., a
microorganism, which expresses and, e.g., secretes, one or more
additional enzymes and/or variants of the polypeptide.
[0061] The term "inclusion body" as used herein, refers to
insoluble aggregates produced by a microorganism, e.g., a host
cell, containing one or more of the following: a heterologously
expressed polypeptide, e.g., a polypeptide having biomass-degrading
activity, cellular debris, one or more ribosomal component, one or
more protein endogenously expressed from the host cell, one or more
nucleic acids (RNA and/or DNA), or any combination thereof.
Inclusion bodies commonly occur in host cells, e.g., bacterial
cells, during high levels of expression of a recombinant protein.
The heterologously expressed polypeptides found in the inclusion
body may be partially unfolded, partially misfolded, or partially
denatured.
[0062] The term "ligninase" as used herein, refers to an enzyme
that catalyzes the breakdown of lignin, commonly found in the cell
walls of plants, such as by an oxidation reaction. Ligninases
include lignin-modifying enzymes, lignin peroxidases and
laccases.
[0063] The term "ligninase activity" as used herein, refers to the
activity of an enzyme that catalyzes the breakdown of lignin and
lignin-like polymers by an oxidation reaction. Ligninase activity
can be determined according to the assays described herein.
[0064] The terms "nucleic acid" or "polynucleotide" are used
interchangeable, and refer to deoxyribonucleic acids (DNA) or
ribonucleic acids (RNA) and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides that have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions),
alleles, orthologs, SNPs, and complementary sequences as well as
the sequence explicitly indicated. Specifically, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes
8:91-98 (1994)).
[0065] The term "operably linked", as used herein, refers to a
configuration in which a control or regulatory sequence is placed
at a position relative to a nucleic acid sequence that encodes a
polypeptide, such that the control sequence influences the
expression of a polypeptide (encoded by the DNA sequence). In an
embodiment, the control or regulatory sequence is upstream of a
nucleic acid sequence that encodes a polypeptide with cellobiase
activity. In an embodiment, the control or regulatory sequence is
downstream of a nucleic acid sequence that encodes a polypeptide
with cellobiase activity.
[0066] The terms "peptide," "polypeptide," and "protein" are used
interchangeably, and refer to a compound comprised of amino acid
residues covalently linked by peptide bonds. A protein or peptide
must contain at least two amino acids, and no limitation is placed
on the maximum number of amino acids that can comprise a protein's
or peptide's sequence. Polypeptides include any peptide or protein
comprising two or more amino acids joined to each other by peptide
bonds. "Polypeptides" include, for example, biologically active
fragments, substantially homologous polypeptides, oligopeptides,
homodimers, heterodimers, variants of polypeptides, modified
polypeptides, derivatives, analogs, fusion proteins, among others.
A polypeptide includes a natural peptide, a recombinant peptide, or
a combination thereof. A "plurality of polypeptides" refers to two
or more polypeptides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,
100, 200, or 500 or more polypeptides.
[0067] The term "promoter", as used herein, refers to a DNA
sequence recognized by the synthetic machinery of the cell, or
introduced synthetic machinery, required to initiate the specific
transcription of a polynucleotide sequence.
[0068] The term "regulatory sequence" or "control sequence", as
used interchangeably herein, refers to a nucleic acid sequence
which is required for expression of a nucleic acid product. In some
instances, this sequence may be a promoter sequence and in other
instances, this sequence may also include an enhancer sequence and
other regulatory elements which are required for expression of the
gene product. The regulatory/control sequence may, for example, be
one which expresses the nucleic acid product in a regulated manner,
e.g., inducible manner.
[0069] The term "constitutive" promoter refers to a nucleotide
sequence which, when operably linked with a polynucleotide which
encodes a polypeptide, causes the polypeptide to be produced in a
cell under most or all physiological conditions of the cell. In an
embodiment, the polypeptide is a polypeptide having cellobiase
activity.
[0070] The term "inducible" promoter refers to a nucleotide
sequence which, when operably linked with a polynucleotide which
encodes a polypeptide, causes the polypeptide to be produced in a
cell substantially only when an inducer which corresponds to the
promoter is present in the cell. In an embodiment, the polypeptide
is a polypeptide having cellobiase activity.
[0071] The term "repressible" promoter refers to a nucleotide
sequence, which when operably linked with a polynucleotide which
encodes a polypeptide, causes the polypeptide to be produced in a
cell substantially only until a repressor which corresponds to the
promoter is present in the cell. In an embodiment, the polypeptide
is a polypeptide having cellobiase activity.
[0072] The term "solubilizing agent" refers to an agent that has
the capacity for disrupting non-covalent bonds, e.g., hydrogen
bonds, hydrophobic interactions, van der Waals interactions,
dipole-dipole interactions, ionic interactions, pi-stacking, or any
combination thereof. The disruption of the non-covalent bonds leads
to the solubilization, or dissolution, of previously insoluble
matter into solution. Specifically, a solubilizing agent used
herein increases the ability of polypeptides having
biomass-degrading activity described herein that have aggregated
into inclusion bodies to dissolve into solution, e.g., water-based
solution or a buffer. Examples of suitable solubilizing agents are
described herein.
[0073] The term "xylanase" as used herein, refers to enzymes that
hydrolyze xylan-containing material. Xylan is polysaccharide
comprising units of xylose. A xylanase can be an endoxylanase, a
beta-xylosidase, an arabinofuranosidase, an alpha-glucuronidase, an
acetylxylan esterase, a feruloyl esterase, or an alpha-glucuronyl
esterase.
[0074] The term "xylanase activity" as used herein, refers to the
activity of enzymes that catalyze the endohydrolysis of
1,4-btea-D-xylosidic linkages in xylans and xylan-like polymers.
Xylanase activity can be determined according to the assays
described herein. One unit of xylanase activity will release 1
.mu.M of xylose equivalent from xylan per minute.
Description
[0075] High level of expression of recombinant proteins in host
cells such as E. coli often leads to accumulation of the
recombinant proteins into inactive, misfolded and insoluble
aggregates within the host cell. These insoluble aggregates are
called inclusion bodies and can also contain other components
endogenous to the host cell, such as protein, ribosomal components,
nucleic acids, and cellular debris. As much as 70-80% of proteins
produced by recombinant techniques can form inclusion bodies,
thereby significantly reducing the yield of active recombinant
protein that can be readily isolated from the host cells.
[0076] Solubilization of the recombinant proteins from the
inclusion bodies can be achieved through treatment with chaotropic
agents, e.g., high concentrations of urea, which disrupt hydrogen
bonds and hydrophobic interactions. However, such solubilization
processes often result in denaturation of the protein and loss of
native function or enzymatic activity. The soluble denatured
proteins can be refolded to their native state after removal of
chaotropic agents, however, refolding of recombinant proteins into
bioactive forms with enzymatic activity can be cumbersome, costly,
and result in low recovery of the final product.
[0077] The present invention is based, at least in part, on the
surprising discovery that a heterologously expressed cellobiase
that has been solubilized from inclusion bodies by urea retains
cellobiase activity. The recovery of heterologously expressed
cellobiase from the inclusion bodies increased the total yield of
cellobiase by 30-40%. Furthermore, the presence of the solubilizing
agent, e.g., urea, from the addition of the solubilized
biomass-degrading enzyme, e.g., cellobiase, does not adversely
affect the saccharification reaction for converting biomass to a
sugar product and/or the yield of products.
[0078] Accordingly, the present invention provides methods for
solubilizing a polypeptide having biomass-degrading activity from
inclusion bodies, where the resulting solubilized polypeptide
retains biomass-degrading activity, whereby the additional
processing steps of refolding the polypeptide and removing the
solubilizing agent is not required. The present invention provides
methods for increasing the recovery of heterologously-expressed
biomass-degrading enzymes from inclusion bodies, while retaining
enzymatic activity, and use of the recovered biomass-degrading
enzymes in methods for converting a biomass into products, e.g., by
saccharification.
Polypeptides Having Biomass-Degrading Activity
[0079] The present disclosure provides a polypeptide, a plurality
of polypeptides, having a biomass-degrading activity. In
embodiments, the polypeptide having biomass-degrading activity, or
plurality thereof, is present in a mixture with one or more
solubilizing agent. Some mixtures may also contain one or more
proteins associated with an inclusion body. In other embodiments,
the mixture does not contain one or more proteins associated with
the inclusion body, e.g., the polypeptide or plurality thereof
having biomass-degrading activity was purified from one or more
proteins associated with the inclusion body.
[0080] For example, the polypeptide has cellobiase activity,
ligninase activity, endoglucanase activity, cellobiohydrolase
activity, or xylanase activity.
[0081] In an embodiment, the polypeptide is a cellobiase. A
cellobiase is an enzyme that hydrolyzes beta-1,4 bonds in its
substrate, e.g., cellobiose, to release two glucose molecules.
Cellobiose is a water soluble 1,4-linked dimer of glucose. In an
embodiment, the polypeptide is Cel3a. Cel3a (also known as BglI) is
a cellobiase that was identified in Trichoderma reesei. The amino
acid sequence for Cel3a (GenBank Accession No. NW_006711153) is
provided below:
TABLE-US-00001 (SEQ ID NO: 1)
MGDSHSTSGASAEAVVPPAGTPWGTAYDKAKAALAKLNLQDKVGIVSGVG
WNGGPCVGNTSPASKISYPSLCLQDGPLGVRYSTGSTAFTPGVQAASTWD
VNLIRERGQFIGEEVKASGIHVILGPVAGPLGKTPQGGRNWEGFGVDPYL
TGIAMGQTINGIQSVGVQATAKHYILNEQELNRETISSNPDDRTLHELYT
WPFADAVQANVASVMCSYNKVNTTWACEDQYTLQTVLKDQLGFPGYVMTD
WNAQHTTVQSANSGLDMSMPGTDFNGNNRLWGPALTNAVNSNQVPTSRVD
DMVTRILAAWYLTGQDQAGYPSFNISRNVQGNHKTNVRAIARDGIVLLKN
DANILPLKKPASIAVVGSAAIIGNHARNSPSCNDKGCDDGALGMGWGSGA
VNYPYFVAPYDAINTRASSQGTQVTLSNTDNTSSGASAARGKDVAIVFIT
ADSGEGYITVEGNAGDRNNLDPWHNGNALVQAVAGANSNVIVVVHSVGAI
ILEQILALPQVKAVVWAGLPSQESGNALVDVLWGDVSPSGKLVYTIAKSP
NDYNTRIVSGGSDSFSEGLFIDYKHFDDANITPRYEFGYGLSYTKFNYSR
LSVLSTAKSGPATGAVVPGGPSDLFQNVATVTVDIANSGQVTGAEVAQLY
ITYPSSAPRTPPKQLRGFAKLNLTPGQSGTATFNIRRRDLSYWDTASQKW
VVPSGSFGISVGASSRDIRLTSTLSVAGSGS
[0082] In an embodiment, the polypeptide is a ligninase. A
ligninase is an enzyme that breaks down lignin, which is a complex
polymer of aromatic alcohols known as monolignols and plays an
integral part of the secondary cell walls of plants and some algae.
Ligninases include lignin peroxidases,
1,2-bis(3,4-dimethoxyphenyl)propane-1,3-diol:hydrogen-peroxide
oxidoreductase, diarylpropane oxygenase, ligninase I, diarylpropane
peroxidase, LiP, hydrogen-peroxide oxidoreductase
(C--C-bond-cleaving), and some laccases. Examples of ligninases
include CIP2 from Trichoderma reesei; LPOA, GLG2, GLG4, LIPA, GLG5,
GLG3, GLG6, and LIPB from Phanerochaete chrysosporium; ligninase-3
from Phelbia radiate; Ligninase A and B from Coriolus versicolor;
and LPG I and LPGIV Coriolus versicolor.
[0083] In an embodiment, the polypeptide is an endoglucanase. An
endoglucanase is an enzyme that catalyzes the hydrolysis of
cellulose. Specifically, the endoglucanases cleave the internal
bonds of the cellulose chain. Endoglucanases are produced by fungi,
bacteria, and protozoans. Endoglucanases are also known as beta-1-4
endoglucanase, 4-beta-D-glucan cellobiohydrolase,
exo-cellobiohydrolase, beta-1,4-glucan cellobiohydrolase,
beta-1,4-glucan cellobiosylhydrolase, 1,4-beta-glucan
cellobiosidase, exoglucanase, avicelase, CBH 1, C1 cellulase,
cellobiohydrolase I, cellobiohydrolase, exo-beta-1,4-glucan
cellobiohydrolase, 1,4-beta-D-glucan cellobiohydrolase, or
cellobiosidase. Examples of endoglucanases include Cel5A, Cel5B,
Cel7B, Cel12A, Cel45A, Cel61A, Cel61B, and Cel74A from Trichoderma
reesei.
[0084] In an embodiment, the polypeptide is a cellobiohydrolase,
also known as exoglucanase. A cellobiohydrolase catalyzes the
hydrolysis of 1-4-beta-D-glucosidic linkages in oligosaccharides
containing that linkage, e.g., cellulose and cellotetraose, thereby
releasing cellobiose from the non-reducing ends of the chains.
Examples of cellobiohydrolases include cellobiohydrolase I (CBHI)
and cellobiohydrolase II (CBHII) from Trichoderma reesei.
[0085] In an embodiment, the polypeptide is a xylanase. Xylanases
are also known as endo-(1-4)-beta-xylan 4-xylanohydrolase,
endo-1,4-xylanase, endo-1,4-beta-xylanase, beta-1,4-xylanase,
endo-1,4-beta-D-xylanase, 1,4-beta-xylan xylanohydrolase,
beta-xylanase, beta-1,4-xylan xylanohydrolase, beta-D-xylanase. A
xylanase breaks down a component of plant cell walls called
hemicellulose, e.g., degrades polysaccharides, such as xylan, e.g.,
beta-1,4-xylan, glucuronoxylan, arabinoxylan, glucomannan, and
xyloglucan, to release xylose. Examples of xylanases include Xyn1,
Xyn2, and Xyn3 from Trichoderma reesei; and TERTU_1599, TERTU_3603,
TERTU_2546, and TERTU_4506 from Terendinibacter turnerae T7901.
[0086] The present disclosure also provides functional variants of
a polypeptide having biomass-degrading activity described herein.
In an embodiment, a functional variant has an amino acid sequence
with at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% identity to a biomass-degrading
enzyme described herein, or a functional fragment thereof, e.g., at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% identity to a biomass-degrading
enzyme described herein, or a functional fragment thereof.
[0087] In an embodiment, a functional variant has an amino acid
sequence with at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 91%
identity, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identity
to Cel3a produced by T. reesei or SEQ ID NO: 1, or a functional
fragment thereof.
[0088] Percent identity in the context of two or more amino acid or
nucleic acid sequences, refers to two or more sequences that are
the same. Two sequences are "substantially identical" if two
sequences have a specified percentage of amino acid residues or
nucleotides that are the same (e.g., 60% identity, optionally 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identity over a specified region, or, when not
specified, over the entire sequence), when compared and aligned for
maximum correspondence over a comparison window, or designated
region as measured using one of the following sequence comparison
algorithms or by manual alignment and visual inspection.
Optionally, the identity exists over a region that is at least
about 50 nucleotides, 100 nucleotides, 150 nucleotides, in length.
More preferably, the identity exists over a region that is at least
about 200 or more amino acids, or at least about 500 or 1000 or
more nucleotides, in length.
[0089] For sequence comparison, one sequence typically acts as a
reference sequence, to which one or more test sequences are
compared. When using a sequence comparison algorithm, test and
reference sequences are entered into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. Default program parameters can
be used, or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters. Methods of alignment of
sequences for comparison are well known in the art. Optimal
alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith and Waterman, (1970) Adv.
Appl. Math. 2:482c, by the homology alignment algorithm of
Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search
for similarity method of Pearson and Lipman, (1988) Proc. Nat'l.
Acad. Sci. USA 85:2444, by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Brent et al., (2003) Current Protocols in Molecular
Biology).
[0090] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al.,
(1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J.
Mol. Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information.
[0091] Functional variants may comprise one or more mutations, such
that the variant retains biomass-degrading activity that is better
than the biomass-degrading activity of a biomass-degrading enzyme
described herein produced by the microorganism from which the
enzyme originates from. In an embodiment, the functional variant
has at least 10%, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, or at least 99% (e.g., at
least 80%, at least 85%, at least 90%, at least 95%, or at least
99%) of the biomass-degrading activity as a biomass-degrading
enzyme as produced by E. coli. In embodiments, the functional
variant has at least 200%, at least 300%, at least 400%, at least
500%, at least 1000% or more of the biomass-degrading activity as a
biomass-degrading enzyme produced by E. coli or the microorganism
from which the enzyme originates from. Biomass-degrading activity
can be tested using the functional assays described herein. In one
embodiment, the functional variant retains cellobiase activity that
is better than the cellobiase activity of Cel3a as produced by T.
reesei. In another embodiment, the functional variant has at least
10%, at least 20%, at least 30%, at least 40%, at least 50%, at
least 60%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, or at least 99% (e.g., at least 80%, at
least 85%, at least 90%, at least 95%, or at least 99%) of the
cellobiase activity as a Cel3a or enzyme comprising SEQ ID NO: 1 as
produced by E. coli. In embodiments, the functional variant has
increased biomass-degrading activity compared to a
biomass-degrading enzyme described herein, e.g., at least 200%, at
least 300%, at least 400%, at least 500%, at least 1000% or more of
the biomass-degrading activity of a biomass-degrading enzyme
described herein, e.g., cellobiase activity as a Cel3a or enzyme
comprising SEQ ID NO: 1 produced by E. coli or the microorganism
from which the enzyme originates from.
[0092] The mutations present in a functional variant include amino
acid substitutions, additions, and deletions. Mutations can be
introduced by standard techniques known in the art, such as
site-directed mutagenesis and PCR-mediated mutagenesis. The
mutation may be a conservative amino acid substitution, in which
the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the polypeptide having
cellobiase activity of the disclosure can be replaced with other
amino acids from the same side chain family, and the resultant
polypeptide retains cellobiase activity comparable (e.g., at least
80%, 85%, 90%, 95%, or 99% of the cellobiase activity) to that of
the wild-type polypeptide. Alternatively, the mutation may be an
amino acid substitution in which an amino acid residue is replaced
with an amino acid residue having a different side chain.
[0093] Such mutations may alter or affect various enzymatic
characteristics of the biomass-degrading enzyme, e.g., cellobiase,
ligninase, endoglucanase, or cellobiohydrolase. For example, such
mutations may alter or affect the biomass-degrading activity,
thermostability, optimal pH for reaction, enzyme kinetics, or
substrate recognition of the biomass-degrading enzyme. In some
embodiments, a mutation increases the biomass-degrading activity of
the variant in comparison to the biomass-degrading enzyme, e.g.,
cellobiase produced by T. reesei and/or SEQ ID NO: 1 produced in E.
coli. In some embodiments, a mutation increases or decreases the
thermostability of the variant in comparison to a wild-type biomass
degrading enzyme, e.g., a cellobiase and/or SEQ ID NO: 1 produced
in E. coli. In an embodiment, a mutation changes the pH range at
which the variant optimally performs the biomass-degrading reaction
in comparison to wild-type biomass-degrading enzyme, e.g.,
wild-type cellobiase and/or SEQ ID NO: 1 produced in E. coli. In an
embodiment, a mutation increases or decreases the kinetics of the
biomass-degrading reaction (e.g., k.sub.cat, K.sub.M or K.sub.D) in
comparison to wild-type biomass-degrading enzyme, e.g., wild-type
cellobiase and/or SEQ ID NO: 1 produced in E. coli. In an
embodiment, a mutation increases or decreases the ability of the
cellobiase to recognize or bind to the substrate (e.g., cellobiose)
in comparison to wild-type cellobiase and/or SEQ ID NO:1 produced
in E. coli.
[0094] The present invention also provides functional fragments of
a polypeptide having biomass-degrading activity, e.g., cellobiase
activity, as described herein, e.g., Cel3a or SEQ ID NO: 1. One of
ordinary skill in the art could readily envision that a fragment of
a polypeptide having biomass-degrading activity as described herein
that contains the functional domains responsible for enzymatic
activity would retain functional activity, e.g., biomass-degrading
activity, and therefore, such fragments are encompassed in the
present invention. In an embodiment, the functional fragment is at
least 700 amino acids, at least 650 amino acids, at least 600 amino
acids, at least 550 amino acids, at least 500 amino acids, at least
450 amino acids, at least 400 amino acids, at least 350 amino
acids, at least 300 amino acids, at least 250 amino acids, at least
200 amino acids, at least 150 amino acids, at least 100 amino
acids, or at least 50 amino acids in length. In an embodiment, the
functional fragment is 700 to 744 amino acids, 650 to 699 amino
acids, 600 to 649 amino acids, 550 to 599 amino acids, 500 to 549
amino acids, 450 to 499 amino acids, 400 to 449 amino acids, 350 to
399 amino acids, 300 to 349 amino acids, 250 to 299 amino acids,
200 to 249 amino acids, 150 to 199 amino acids, 100 to 149 amino
acids, or 50 to 99 amino acids. With regard to the ranges of amino
acid length described above, the lowest and highest values of amino
acid length are included within each disclosed range. In an
embodiment, the functional fragment has at least 10%, at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, or at least 99% of the biomass-degrading activity as a
wild-type biomass-degrading enzyme described herein, or the
biomass-degrading enzyme produced in E. coli. In an embodiment, the
functional fragment has at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, or at
least 99% of the cellobiase activity as wild-type Cel3a or the
polypeptide comprising SEQ ID NO: 1 produced in E. coli.
[0095] Assays for detecting cellobiase activity are known in the
art. For example, detection of the amount of glucose released from
cellobiose can be determined by incubating purified cellobiase with
substrate, e.g., cellobiose, D-(+)-cellobiose, and detecting the
resultant amount of free glucose after completion of the reaction.
The amount of free glucose can be determined using a variety of
methods known in the art. For example, dilutions of purified
cellobiase are prepared in a buffer containing 50 mM sodium
citrate, pH 5.0 NaOH. The cellobiose substrate is added to the
purified cellobiase in an amount such that the final concentration
of cellobiose in the reaction mixture is 30 mM. The reaction
mixture is incubated under conditions suitable for the reaction to
occur, e.g., in a shaker (700 rpm) at 48.degree. C. for 30 minutes.
To stop the reaction, the reaction mixture is heated for 5 minutes
at 100.degree. C. The reaction mixture is filtered through a 0.45
.mu.m filter and the filtrate is analyzed to quantify the amount of
glucose and/or cellobiase. A YSI instrument that measures analytes
such as glucose can be used to determine the concentration of
glucose produced from the reaction. Alternatively, UPLC (Ultra
Performance Liquid Chromatography) can be used to determine the
concentration of glucose and cellobiose from the reaction. This
assay can be formatted in a single reaction or in multiple reaction
formats, e.g., 96 well format. In some embodiments, the multiple
reaction format may be preferred to generate an activity curve
representing cellobiase activity with respect to different
concentrations of the purified cellobiase. The concentration of the
purified cellobiase can be determined using a standard Bradford
assay. Dilutions of the purified cellobiase assay are prepared,
e.g., 2-fold dilutions, and are aliquoted into a 96 well plate,
e.g., 12 wells of 2-fold dilutions. Cellobiose substrate is added
as previously described, such that the final concentration of
cellobiase in the reaction is 30 mM. The plate is sealed and
treated under conditions sufficient for the cellobiase reaction to
occur, and then under conditions to stop the reaction. The reaction
is then filtered through a 96 well format 0.45 .mu.m membrane
(e.g., Durapore) and analyzed by YSI and/or HPLC methods, e.g.,
UPLC.
[0096] This activity assay can also be used to determine the
concentration, or titer, of a cellobiase in a sample with unknown
concentration by generating a standard curve of activity of known
concentrations of the cellobiase to extrapolate the concentration
for the unknown concentration sample. For example, two-fold serial
dilutions of a known concentration of the cellobiase are prepared
in one row of a 96 well plate, e.g., 12 two-fold serial dilutions.
The other rows contain two-fold serial dilutions of other remaining
samples whose titer is to be determined, e.g., the crude lysate
sample or solubilized inclusion body sample. The dilutions are
incubated with a D-(+)-Cellobiose (Fluka) substrate solution in 50
mM sodium citrate monobasic buffer at pH 5.0, at 48.degree. C. for
30 minutes. After 30 minutes, the samples are heated to 100.degree.
C. for 10 minutes to stop the reaction. Samples are analyzed for
glucose and cellobiose using the YSI Biochemistry analyser (YSI
Life Sciences) and/or HPLC methods. Using the samples of known
concentration, a standard curve is generated using the data points
within the linear range of the assay. The cellobiase activity
detected from the samples with unknown titer can be compared to the
standard curve to determine the titer of cellobiase in these
sample.
[0097] Units of activity are only relative if calculated using
values within the linear range of the assay. The linear range of
the assay is defined as using glucose values that are less than 30%
of the original soluble substrate load. In addition, glucose values
lower than 0.05 g/L are omitted due to instrumentation reporting
levels. One unit of cellobiase activity is defined as the amount of
glucose per the amount of Cel3a per 30 minutes:
[Glucose]g/L/[Cel3a]g/L/30 min.
[0098] In other embodiments, a colorimetric/fluorometric assay can
be used. The purified cellobiase is incubated with substrate
cellobiose under conditions for the reaction to occur. Detection of
the product glucose is as follows. Glucose oxidase is added to the
mixture, which oxidizes glucose (the product) to gluconic acid and
hydrogen peroxide. Peroxidase and o-dianisidine is then added.
O-dianisidine reacts with the hydrogen peroxide in the presence of
peroxidase to form a colored product. Sulfuric acid is added, which
reacts with the oxidized o-dianisidine reacts to form a more stable
colored product. The intensity of the color when measured, e.g., by
spectrophotometer or colorimeter, e.g., at 540 nm, is directly
proportional to the glucose concentration. Such
colorimetric/fluorometric glucose assays are commercially
available, for example from Sigma Aldrich, Catalog No. GAGO-20.
[0099] Assays for detecting ligninase activity are known in the
art. Ligninase activity can be measured by determining the rate of
oxidation of veratryl alcohol to veratrylaldehyde (abbreviated as
VAO for veratryl alcohol oxidation). Reaction mixtures are
prepared, and contain dilutions of enzyme, 2 mM veratryl alcohol,
0.4 mM H.sub.2O.sub.2 and either 20 or 100 mM sodium tartrate, pH
2.9 in a final volume of 0.5 ml. The reactions were started by
H.sub.2O.sub.2 addition and were monitored by spectrophotometry at
310 nm. Protein was determined according to Bradford, M. M., (1976)
Anal. Biochem. 72:248-254, using bovine serum albumin (Sigma
Chemical Co., St. Louis, Mo.) as standard or by using the 409 nm
absorbance of a protein solution and calculating protein amount
from the extinction coefficient of ligninase.
[0100] Assays for detecting endoglucanase activity are known in the
art. For example, endoglucanase activity can be determined by
measuring the hydrolysis of substrate carboxymethyl cellulose (CMC)
and quantifying the concentration of reducing end by BCA method, in
which the total concentration of reducing ends is exhibited by a
color change of the sample solution in proportion to the
concentration of the reducing ends. First, the polypeptide having
biomass-degrading activity is diluted in a 50 mM citrate buffer at
pH 4.8. CMC solution (0.05% w/v CMC in the sodium citrate buffer)
is added to a reaction tube and equilibrated at 50 C. The diluted
enzyme samples are added to the reaction and incubated at 50 C for
10 minutes. BCA reagents are added and incubated at 75 C for 30
minutes. The absorbance is read at 560 nm after subtracting the
readings for the enzyme blanks and the substrate blank. Enzyme
activity can be calculated based on a linear range between reducing
end concentration and enzyme concentrations. Other endoglucanase
activity assays are known in the art, for example, by determining a
reduction in substrate viscosity (Zhang et al., Biotechnol Adv,
2006, 24:452-481).
[0101] Assays for detecting cellobiohydrolase activity are known in
the art. Cellobiohydrolase activity can be determined by measuring
soluble substrate released from substrate Avicel in a
phenol-sulfuric assay. An Avicel solution (1.25% w/v in acetate
buffer) is aliquoted into reaction tubes, and dilutions of the
enzyme is prepared. Both substrate and enzyme solutions are
equilibrated at 50 C. The diluted enzyme solutions are added to the
substrate and incubated for a time sufficient for the reaction to
occur, e.g., at 50 C for 2 hours. The reactions are stopped by
submerging the samples into an ice cold water bath. The samples are
centrifuged to separate the samples into a soluble and insoluble
fraction. The total concentration of soluble sugars in the soluble
fraction is determined by phenol-sulfuric assay. Specifically, an
aliquot of the soluble fraction is mixed with 5% phenol, and
concentrated sulfuric acid is added. The reaction is cooled to room
temperature (about 20-30 minutes), and absorbance of the samples
are read at 490 nm. The enzyme activity is calculated on the basis
of a linear relationship between total soluble sugar release and
the enzyme dilution. Other cellobiohydrolase activity assays are
described in Zhang et al., Biofuels: Methods and Protocols, Vol.
581, pages 213-231.
[0102] Assays for detecting xylanase activity are known in the art.
Xylanase activity can be determined by measuring the level of
xylose released from a xylan substrate by a colorimetric assay.
Xylan substrate is prepared as a 1.0% w/v solution in 50 mM sodium
acetate buffer, pH 4.5. Dilutions of the enzyme of prepared. Xylan
and the enzyme dilutions are mixed, and incubated under conditions
sufficient for the reaction to occur, e.g., 30 C for 10 minutes.
Then a solution containing 16 mM copper sulfate, 1.3M sodium
sulfate, 226 mM sodium carbonate, 190 mM sodium bicarbonate, and 43
mM sodium potassium tartrate is added to the reaction. The reaction
is then boiled for 10 minutes, and allowed to cool to room
temperature. A solution containing 40 mM molybdic acid, 19 mM
arsenic acid, and 756 mM sulfuric acid is added. The reaction is
shaken or vortexed until the foaming stops and any preceiptate
present is dissolved. The reaction is centrifuged to clarify, then
the solutions are ready by spectrophotometer at 540 nM, and enzyme
activity is calculated on the basis of a linear relationship
between total soluble sugar release and the enzyme dilution.
Aglycosylated Polypeptides
[0103] Any of the polypeptides having biomass-degrading activity
described herein, e.g., cellobiase activity, can be glycosylated or
aglycosylated. An aglycosylated polypeptide having
biomass-degrading activity may be solubilized from an inclusion
body, as described herein. Alternatively, an aglycosylated
polypeptide having biomass-degrading activity may be added to a
mixture comprising a polypeptide having biomass-degrading activity
that has been solubilized from an inclusion body, in which the
polypeptide that was solubilized from an inclusion body can be
glycosylated or aglycosylated.
[0104] Glycosylation is the enzymatic process by which a
carbohydrate is attached to a glycosyl acceptor, e.g., the nitrogen
of arginine or asparginine side chains or the hydroxyl oxygen of
serine, threonine, or tyrosine side chains. There are two types of
glycosylation: N-linked and O-linked glycosylation. N-linked
glycosylation occurs at consensus site Asn-X-Ser/Thr, wherein the X
can be any amino acid except a proline. O-linked glycosylation
occurs at Ser/Thr residues. Glycosylation sites can be predicted
using various algorithms known in the art, such as Prosite,
publicly available by the Swiss Institute of Bioinformatics, and
NetNGlyc 1.0 or NetOGlyc 4.0, publicly available by the Center for
Biological Sequence Analysis.
[0105] The present invention provides methods for producing an
aglycosylated polypeptide having biomass-degrading activity. In one
embodiment, the nucleic acid encoding the polypeptide has been
altered or mutated such that the polypeptide cannot be
glycosylated, e.g., one or more glycosylation sites are mutated
such that a glycan cannot be attached to the glycosylation site.
For example, an aglycosylated polypeptide having biomass-degrading
activity encoded by a nucleic acid sequence described herein
contains one or more mutations at one or more glycosylation sites
have been mutated such that a glycan can no longer be attached or
linked to the glycosylation site. In another example, the
polypeptide having biomass-degrading activity encoded by a nucleic
acid sequence described herein contains one or more mutations
proximal to one or more glycosylation sites that have been mutated
such that a glycan can no longer be attached or linked to the
glycosylation site. For example, the mutation proximal to a
glycosylation site mutates the consensus motif recognized by the
glycosylating enzyme, or changes the conformation of the
polypeptide such that the polypeptide cannot be glycosylated, e.g.,
the glycoslation site is hidden or steric hindrance due to the new
conformation prevents the glycosylating enzymes from accessing the
glycosylation site. A mutation proximal to a glycosylation site in
the polypeptide having biomass-degrading activity is directly
adjacent to, or at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
15, at least 20, at least 30 or at least 40 amino acids from the
glycosylation site that, as a result of the proximal mutation, will
not be glycosylated.
[0106] In an embodiment, one or more of the following glycosylation
sites of a cellobiase, e.g., Cel3a, or SEQ ID NO: 1, are mutated:
the threonine at amino acid position 78, the threonine at amino
acid position 241, the serine at amino acid position 343, the
serine at amino acid position 450, the threonine at amino acid
position 599, the serine at amino acid position 616, the threonine
at amino acid position 691, the serine at amino acid position 21,
the threonine at amino acid position 24, the serine at amino acid
position 25, the serine at amino acid position 28, the threonine at
amino acid position 38, the threonine at amino acid position 42,
the threonine at amino acid position 303, the serine at amino acid
position at 398, the serine at amino acid position 435, the serine
at amino acid position 436, the threonine at amino acid position
439, the threonine at amino acid position 442, the threonine at
amino acid position 446, the serine at amino acid position 451, the
serine at amino acid position 619, the serine at amino acid
position 622, the threonine at amino acid position 623, the serine
at amino acid position 626, or the threonine at amino acid position
630, or any combination thereof. In embodiments, the glycosylation
site is mutated from a serine or threonine to an alanine. For
example, the aglycosylated polypeptide described herein has one or
more of the following mutations: T78A, T241A, S343A, S450A, T599A,
S616A, T691A, S21A, T24A, S25A, S28A, T38A, T42A, T303A, T398A,
S435A, S436A, T439A, T442A, T446A, S451A, S619A, S622A, T623A,
S626A, or T630A, or any combination thereof. Alternatively, one or
more amino acids proximal to the glycosylation sites described
above are mutated.
[0107] Assays to detect whether a polypeptide is modified by a
glycan (e.g., whether the polypeptide is glycosylated or
aglycosylated) are known in the art. The polypeptide can be
purified or isolated and can be stained for detection and
quantification of glycan moieties, or the polypeptide can be
analyzed by mass spectrometry, and compared to a corresponding
reference polypeptide. The reference polypeptide has the same
primary sequence as the test polypeptide (of which the
glycosylation state is to be determined), but is either
glycosylated or aglycosylated.
[0108] The aglycosylated polypeptides described herein may have
increased biomass-degrading activity, e.g., cellobiase activity,
compared to a corresponding glycosylated polypeptide, e.g.,
glycosylated Cel3a polypeptide. For example, the aglycosylated
polypeptide having biomass-degrading activity, e.g., cellobiase
activity, has at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, or 200% biomass-degrading activity,
e.g., cellobiase activity, compared to the glycosylated
polypeptide.
Nucleic Acids, Expression Vectors and Host Cells
[0109] A polypeptide having biomass-degrading activity as described
herein is expressed in host cells. In one embodiment, an expression
vector comprising a nucleic acid sequence encoding any of the
polypeptides described herein having biomass-degrading activity is
introduced into a host cell, and the host cell is cultured under
conditions appropriate for expression of the polypeptide having
biomass-degrading activity. In embodiments, the expression of the
polypeptide having biomass-degrading activity is at a level such
that inclusion bodies, or aggregates comprising the polypeptide
having biomass-degrading activity, are formed. Also described
herein are methods for expressing and isolating the soluble
polypeptide having biomass-degrading activity expressed in the host
cell. Methods for solubiziling and isolating the polypeptide having
biomass-degrading activity from the inclusion bodies is described
further in the section titled "Solubilization from Inclusion
Bodies".
[0110] The present invention also provides a nucleic acid sequence
encoding a polypeptide having biomass-degrading activity. In an
embodiment, the nucleic acid sequence encodes a ligninase, an
endoglucanase, a cellobiohydrolase, a xylanase, or a cellobiase
described herein. The nucleic acid sequence encoding a polypeptide
having biomass-degrading activity can be codon-optimized for
increased expression in host cells. Codon optimization includes
changing the nucleic acid sequence to take into consideration
factors including codon usage bias, cryptic splicing sites, mRNA
secondary structure, premature polyA sites, interaction of codon
and anti-codon, and RNA instability motifs, to increase expression
of the encoded polypeptide in the host. Various algorithms and
commercial services for codon-optimization are known and available
in the art.
[0111] In an embodiment, the nucleic acid sequence encodes a Cel3a
enzyme from T. reesei with the amino acid sequence described
herein, e.g., SEQ ID NO: 1. In an embodiment, the nucleic acid
sequence that encodes Cel3a is provided below:
TABLE-US-00002 (SEQ ID NO: 2)
ATGCGTTACCGAACAGCAGCTGCGCTGGCACTTGCCACTGGGCCCTTTGC
TAGGGCAGACAGTCACTCAACATCGGGGGCCTCGGCTGAGGCAGTTGTAC
CTCCTGCAGGGACTCCATGGGGAACCGCGTACGACAAGGCGAAGGCCGCA
TTGGCAAAGCTCAATCTCCAAGATAAGGTCGGCATCGTGAGCGGTGTCGG
CTGGAACGGCGGTCCTTGCGTTGGAAACACATCTCCGGCCTCCAAGATCA
GCTATCCATCGCTATGCCTTCAAGACGGACCCCTCGGTGTTCGATACTCG
ACAGGCAGCACAGCCTTTACGCCGGGCGTTCAAGCGGCCTCGACGTGGGA
TGTCAATTTGATCCGCGAACGTGGACAGTTCATCGGTGAGGAGGTGAAGG
CCTCGGGGATTCATGTCATACTTGGTCCTGTGGCTGGGCCGCTGGGAAAG
ACTCCGCAGGGCGGTCGCAACTGGGAGGGCTTCGGTGTCGATCCATATCT
CACGGGCATTGCCATGGGTCAAACCATCAACGGCATCCAGTCGGTAGGCG
TGCAGGCGACAGCGAAGCACTATATCCTCAACGAGCAGGAGCTCAATCGA
GAAACCATTTCGAGCAACCCAGATGACCGAACTCTCCATGAGCTGTATAC
TTGGCCATTTGCCGACGCGGTTCAGGCCAATGTCGCTTCTGTCATGTGCT
CGTACAACAAGGTCAATACCACCTGGGCCTGCGAGGATCAGTACACGCTG
CAGACTGTGCTGAAAGACCAGCTGGGGTTCCCAGGCTATGTCATGACGGA
CTGGAACGCACAGCACACGACTGTCCAAAGCGCGAATTCTGGGCTTGACA
TGTCAATGCCTGGCACAGACTTCAACGGTAACAATCGGCTCTGGGGTCCA
GCTCTCACCAATGCGGTAAATAGCAATCAGGTCCCCACGAGCAGAGTCGA
CGATATGGTGACTCGTATCCTCGCCGCATGGTACTTGACAGGCCAGGACC
AGGCAGGCTATCCGTCGTTCAACATCAGCAGAAATGTTCAAGGAAACCAC
AAGACCAATGTCAGGGCAATTGCCAGGGACGGCATCGTTCTGCTCAAGAA
TGACGCCAACATCCTGCCGCTCAAGAAGCCCGCTAGCATTGCCGTCGTTG
GATCTGCCGCAATCATTGGTAACCACGCCAGAAACTCGCCCTCGTGCAAC
GACAAAGGCTGCGACGACGGGGCCTTGGGCATGGGTTGGGGTTCCGGCGC
CGTCAACTATCCGTACTTCGTCGCGCCCTACGATGCCATCAATACCAGAG
CGTCTTCGCAGGGCACCCAGGTTACCTTGAGCAACACCGACAACACGTCC
TCAGGCGCATCTGCAGCAAGAGGAAAGGACGTCGCCATCGTCTTCATCAC
CGCCGACTCGGGTGAAGGCTACATCACCGTGGAGGGCAACGCGGGCGATC
GCAACAACCTGGATCCGTGGCACAACGGCAATGCCCTGGTCCAGGCGGTG
GCCGGTGCCAACAGCAACGTCATTGTTGTTGTCCACTCCGTTGGCGCCAT
CATTCTGGAGCAGATTCTTGCTCTTCCGCAGGTCAAGGCCGTTGTCTGGG
CGGGTCTTCCTTCTCAGGAGAGCGGCAATGCGCTCGTCGACGTGCTGTGG
GGAGATGTCAGCCCTTCTGGCAAGCTGGTGTACACCATTGCGAAGAGCCC
CAATGACTATAACACTCGCATCGTTTCCGGCGGCAGTGACAGCTTCAGCG
AGGGACTGTTCATCGACTATAAGCACTTCGACGACGCCAATATCACGCCG
CGGTACGAGTTCGGCTATGGACTGTCTTACACCAAGTTCAACTACTCACG
CCTCTCCGTCTTGTCGACCGCCAAGTCTGGTCCTGCGACTGGGGCCGTTG
TGCCGGGAGGCCCGAGTGATCTGTTCCAGAATGTCGCGACAGTCACCGTT
GACATCGCAAACTCTGGCCAAGTGACTGGTGCCGAGGTAGCCCAGCTGTA
CATCACCTACCCATCTTCAGCACCCAGGACCCCTCCGAAGCAGCTGCGAG
GCTTTGCCAAGCTGAACCTCACGCCTGGTCAGAGCGGAACAGCAACGTTC
AACATCCGACGACGAGATCTCAGCTACTGGGACACGGCTTCGCAGAAATG
GGTGGTGCCGTCGGGGTCGTTTGGCATCAGCGTGGGAGCGAGCAGCCGGG
ATATCAGGCTGACGAGCACTCTGTCGGTAGCG
The codon-optimized nucleic acid sequence that encodes Cel3a is
provided below:
TABLE-US-00003 (SEQ ID NO: 3)
ATGCGTTATCGTACAGCCGCAGCCCTGGCACTGGCCACAGGTCCGTTCGC
ACGTGCCGATAGTCACAGTACCAGCGGTGCCAGCGCAGAAGCCGTGGTTC
CGCCGGCAGGCACACCGTGGGGCACAGCCTATGATAAAGCCAAAGCCGCC
CTGGCCAAGCTGAATCTGCAGGATAAAGTGGGCATCGTGAGTGGCGTGGG
CTGGAACGGTGGTCCGTGCGTTGGCAACACCAGCCCGGCAAGCAAGATCA
GCTATCCGAGCTTATGCCTGCAGGATGGTCCGCTGGGCGTGCGCTATAGC
ACCGGTAGTACCGCCTTTACACCTGGTGTGCAGGCCGCCAGTACCTGGGA
CGTTAACCTGATCCGCGAACGTGGCCAATTTATCGGCGAAGAAGTTAAAG
CCAGCGGCATTCATGTTATTCTGGGTCCGGTGGCCGGTCCTCTGGGTAAA
ACCCCGCAGGGCGGCCGTAATTGGGAAGGCTTCGGCGTTGATCCGTATTT
AACCGGCATCGCAATGGGCCAGACCATTAATGGCATCCAGAGCGTGGGTG
TTCAAGCCACCGCCAAACACTACATATTAAACGAACAGGAACTGAATCGT
GAAACCATCAGCAGCAATCCGGATGATCGCACCCTGCATGAGCTGTATAC
ATGGCCTTTTGCCGACGCAGTTCAGGCCAACGTGGCAAGTGTGATGTGTA
GCTATAACAAGGTGAACACCACCTGGGCCTGCGAAGACCAGTACACCCTG
CAGACCGTTTTAAAAGACCAACTGGGCTTCCCTGGTTACGTGATGACAGA
TTGGAATGCCCAGCACACAACCGTTCAGAGCGCAAACAGTGGCCTGGATA
TGAGCATGCCGGGCACCGACTTCAACGGCAATAATCGTCTGTGGGGTCCG
GCACTGACCAATGCCGTTAACAGCAACCAGGTGCCGACCAGTCGTGTGGA
CGATATGGTTACCCGTATTCTGGCCGCCTGGTACCTGACAGGTCAAGACC
AGGCCGGCTACCCGAGCTTCAACATCAGCCGCAACGTGCAGGGTAATCAC
AAGACCAACGTTCGCGCAATCGCACGCGATGGTATCGTGCTGTTAAAGAA
CGATGCCAACATTCTGCCGCTGAAAAAACCGGCCAGCATCGCCGTTGTTG
GTAGCGCAGCCATCATTGGCAACCACGCCCGTAACAGTCCGAGCTGCAAT
GATAAAGGCTGTGACGACGGTGCCCTGGGCATGGGTTGGGGTAGTGGTGC
CGTGAACTACCCGTATTTCGTGGCCCCGTACGACGCCATTAACACCCGTG
CAAGTAGCCAGGGTACCCAGGTTACCCTGAGCAACACCGACAACACAAGC
AGCGGTGCCAGTGCAGCACGTGGTAAGGATGTGGCCATCGTGTTCATCAC
CGCCGACAGCGGCGAAGGCTACATTACCGTGGAGGGTAATGCCGGTGATC
GCAATAATCTGGACCCGTGGCATAACGGCAACGCCCTGGTTCAGGCAGTG
GCAGGCGCAAATAGCAACGTGATCGTTGTGGTGCATAGCGTGGGTGCCAT
CATTCTGGAGCAGATCCTGGCCCTGCCGCAAGTTAAGGCAGTTGTGTGGG
CAGGTCTGCCGAGCCAAGAAAGTGGCAATGCCCTGGTGGACGTTCTGTGG
GGCGATGTTAGTCCGAGCGGCAAGCTGGTGTATACAATCGCCAAGAGCCC
GAACGACTATAACACCCGCATCGTTAGCGGCGGCAGTGATAGCTTCAGCG
AGGGCCTGTTTATCGACTACAAGCATTTCGATGATGCCAATATTACCCCG
CGCTACGAATTTGGTTATGGCCTGAGCTATACCAAGTTCAACTACAGCCG
CCTGAGCGTTTTAAGTACCGCCAAGAGTGGTCCGGCAACAGGTGCCGTGG
TTCCTGGTGGTCCGAGTGATCTGTTTCAGAATGTGGCCACCGTGACCGTG
GATATCGCCAACAGTGGTCAGGTTACCGGCGCCGAAGTGGCACAGCTGTA
CATCACCTATCCGAGCAGTGCACCGCGCACCCCGCCGAAACAGCTGCGTG
GCTTCGCCAAATTAAACCTGACCCCGGGCCAGAGCGGTACAGCAACCTTC
AATATTCGCCGCCGTGATCTGAGCTATTGGGACACCGCCAGCCAAAAATG
GGTGGTGCCGAGCGGCAGCTTTGGCATTAGTGTGGGTGCAAGTAGCCGCG
ACATTCGCTTAACAAGCACCCTGAGTGTTGCC
[0112] In an embodiment, the nucleic acid sequence encoding a Cel3a
enzyme or functional variant thereof comprises at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2.
In an embodiment, the nucleic acid sequence encoding a Cel3a enzyme
or functional variant thereof comprises at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% identity to SEQ ID NO:2 or SEQ ID NO:
3.
[0113] Also provided herein is a nucleic acid sequence encoding an
aglycosylated polypeptide having biomass-degrading activity
described herein, e.g., Cel3a polypeptide, in which one or more
glycoslyation sites present in the polypeptide has been mutated
such that a glycan can no longer be attached or linked to the
glycosylation site. In another embodiment, the nucleic acid
sequence described herein encoding an aglycosylated polypeptide,
e.g., a Cel3a polypeptide, as described above, in which one or more
mutations proximal to one or more glycosylation sites present in
the polypeptide has been mutated such that a glycan can no longer
be attached or linked to the glycosylation site, as previously
described.
[0114] The techniques used to isolate or clone a nucleic acid
sequence 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 nucleic acid sequences 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
amplification procedures such as ligase chain reaction (LCR),
ligated activated transcription (LAT) and nucleotide sequence-based
amplification (NASBA) may be used. The nucleic acid sequence may be
cloned from a strain of Trichoderma reesei, e.g., wild-type T.
reesei, or T. reesei RUTC30, or another or related organism and
thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the nucleic acid sequence.
[0115] The nucleic acid sequence may be obtained by standard
cloning procedures used in genetic engineering to relocate the
nucleic acid sequence from its natural location to a different site
where it will be reproduced. The cloning procedures may involve
excision and isolation of a desired fragment comprising the
nucleotide sequence encoding the polypeptide, insertion of the
fragment into a vector molecule, and incorporation of the
recombinant vector into a host cell where multiple copies or clones
of the nucleotide sequence will be replicated. The nucleotide
sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic
origin, or any combinations thereof.
[0116] As used herein, an "expression vector" is a nucleic acid
construct for introducing and expressing a nucleic acid sequence of
interest into a host cell. In some embodiments, the vector
comprises a suitable control sequence operably linked to and
capable of effecting the expression of the polypeptide encoded by
the nucleic acid sequence described herein. The control sequence
may be an appropriate promoter sequence, recognized by a host cell
for expression of the nucleic acid sequence. In an embodiment, the
nucleic acid sequence of interest is a nucleic acid sequence
encoding a polypeptide having biomass-degrading activity, e.g.,
cellobiase activity, as described herein.
[0117] A promoter in the expression vector of described herein can
include promoters obtained from genes encoding extracellular or
intracellular polypeptides either homologous or heterologous to the
host cell, mutant promoters, truncated promoters, and hybrid
promoters.
[0118] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
bacterial host cell are the promoters obtained from the E. coli lac
operon, E. coli tac promoter (hybrid promoter, DeBoer et al, PNAS,
1983, 80:21-25), E. coli rec A, E. coli araBAD, E. coli tetA, and
prokaryotic beta-lactamase. Other examples of suitable promoters
include viral promoters, such as promoters from bacteriophages,
including a T7 promoter, a T5 promoter, a T3 promoter, an M13
promoter, and a SP6 promoter. In some embodiments, more than one
promoter controls the expression of the nucleic acid sequence of
interest, e.g., an E. coli lac promoter and a T7 promoter. Further
promoters that may be suitable for use in the present invention are
described in "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242:74-94, and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 1989. In some preferred
embodiments, the promoter is inducible, where the addition of a
molecule stimulates the transcription and expression of the
downstream reading frame.
[0119] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
eukaryotic host cell, e.g., in a fungal or yeast cell are promoters
obtained from the genes of Trichoderma Reesei, methanol-inducible
alcohol oxidase (AOX promoter), Aspergillus nidulans tryptophan
biosynthesis (trpC promoter), Aspergillus niger var. awamori
flucoamylase (glaA), Saccharomyces cerevisiae galactokinase (GALl),
or Kluyveromyces lactis Plac4-PBI promoter.
[0120] A control sequence present in the expression vector
described herein may also be a signal sequence 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, e.g., a secretion signal sequence. The signal sequence may
be an endogenous signal sequence, e.g., where the signal sequence
is present at the N-terminus of the wild-type polypeptide when
endogenously expressed by the organism from which the polypeptide
of interest originates from. The signal sequence may be a foreign,
or heterologous, signal peptide, in which the signal sequence is
from a different organism or a different polypeptide than that of
the polypeptide of interest being expressed. Any signal sequence
which directs the expressed polypeptide into the secretory pathway
of a host cell may be used in the present invention. Typically,
signal sequences are composed of between 6 and 136 basic and/or
hycrophobic amino acids.
[0121] Examples of signal sequences suitable for the present
invention include the signal sequence from Saccharomyces cerevisiae
alpha-factor.
[0122] Fusion tags may also be used in the expression vector
described herein to facilitate the detection and purification of
the expressed polypeptide. Examples of suitable fusion tags include
His-tag (e.g., 3.times. His, 6.times. His (SEQ ID NO: 22), or
8.times. His (SEQ ID NO: 21)), GST-tag, HSV-tag, S-tag, T7 tag.
Other suitable fusion tags include myc tag, hemagglutinin (HA) tag,
and fluorescent protein tags (e.g., green fluorescent protein). The
fusion tag is typically operably linked to the N or C terminus of
the polypeptide to be expressed. In some embodiments, there may be
a linker region between the fusion tag sequence and the N-terminus
or C-terminus of the polypeptide to be expressed. In an embodiment,
the linker region comprises a sequence between 1 to 20 amino acids,
that does not affect or alter the expression or function of the
expressed polypeptide.
[0123] Utilization of the fusion tags described herein allows
detection of the expressed protein, e.g., by western blot by using
antibodies that specifically recognize the tag. The tags also
allows for purification of the expressed polypeptide from the host
cell, e.g., by affinity chromatography. For example, an expressed
polypeptide fused to a His-tag can be purified by using nickel
affinity chromatography. The His tag has affinity for the Nickel
ions, and a nickel column will retain the his-tagged polypeptide,
while allowing all other proteins and cell debris to flow through
the column. Elution of the His-tagged polypeptide using an elution
buffer, e.g., containing imidazole, releases the His-tagged
polypeptide from the column, resulting in substantially purified
polypeptide.
[0124] The expression vector described herein may further comprise
a selectable marker gene to enable isolation of a genetically
modified microbe transformed with the construct as is commonly
known to those of skill in the art. The selectable marker gene may
confer resistance to an antibiotic or the ability to grow on medium
lacking a specific nutrient to the host organism that otherwise
could not grow under these conditions. The present invention is not
limited by the choice of selectable marker gene, and one of skill
in the art may readily determine an appropriate gene. For example,
the selectable marker gene may confer resistance to ampicillin,
chloramphenicol, tetracycline, kanamycin, hygromycin, phleomycin,
geneticin, or G418, or may complement a deficiency of the host
microbe in one of the trp, arg, leu, pyr4, pyr, ura3, ura5, his, or
ade genes or may confer the ability to grow on acetamide as a sole
nitrogen source.
[0125] The expression vector described herein may further comprise
other nucleic acid sequences, e.g., additional control sequences,
as is commonly known to those of skill in the art, for example,
transcriptional terminators, synthetic sequences to link the
various other nucleic acid sequences together, origins of
replication, ribosome binding sites, a multiple cloning site (or
polylinker site), a polyadenylation signal and the like. The
ribosomal binding site suitable for the expression vector depends
on the host cell used, for example, for expression in a prokaryotic
host cell, a prokaryotic RBS, e.g., a T7 phage RBS can be used. A
multiple cloning site, or polylinker site, contains one or more
restriction enzyme sites that are preferably not present in the
remaining sequence of the expression vector. The restriction enzyme
sites are utilized for the insertion of a nucleic acid sequence
encoding a polypeptide having cellobiase activity or other desired
control sequences. The practice of the present invention is not
limited by the presence of any one or more of these other nucleic
acid sequences, e.g., other control sequences.
[0126] Examples of suitable expression vectors for use in the
present invention include vectors for expression in prokaryotes,
e.g., bacterial expression vectors. A bacterial expression vector
suitable for use in the present invention in the pET vector
(Novagen), which contains the following: a viral T7 promoter which
is specific to only T7 RNA polymerase (not bacterial RNA
polymerase) and also does not occur anywhere in the prokaryotic
genome, a lac operator comprising a lac promoter and coding
sequence for the lac repressor protein (lacI gene), a polylinker,
an f1 origin of replication (so that a single-stranded plasmid can
be produced when co-infected with M13 helper phage), an ampicillin
resistance gene, and a ColE1 origin of replication (Blaber, 1998).
Both the promoter and the lac operator are located 5', or upstream,
of the polylinker in which the nucleic acid sequence encoding a
polypeptide described herein is inserted. The lac operator confers
inducible expression of the nucleic acid sequence encoding a
polypeptide having cellobiase activity. Addition of IPTG (Isopropyl
3-D-1-thiogalactopyranoside), a lactose metabolite, triggers
transcription of the lac operon and induces protein expression of
the nucleic acid sequence under control of the lac operator. Use of
this system requires the addition of T7 RNA polymerase to the host
cell for vector expression. The T7 RNA polymerase can be introduced
via a second expression vector, or a host cell strain that is
genetically engineered to express T7 RNA polymerase can be
used.
[0127] An exemplary expression vector for use with the invention is
a pET vector, commercially available from Novagen. The pET
expression system is described in U.S. Pat. Nos. 4,952,496;
5,693,489; and 5,869,320. In one embodiment, the pET vector is a
pET-DUET vector, e.g., pET-Duet1, commercially available from
Novagen. Other vectors suitable for use in the present invention
include vectors containing His-tag sequences, such as those
described in U.S. Pat. Nos. 5,310,663 and 5,284,933; and European
Patent No. 282042.
[0128] The present invention also relates to a host cell comprising
the nucleic acid sequence or expression vector of the invention,
which are used in the recombinant production of the polypeptides
having biomass-degrading activity.
[0129] An expression vector comprising a nucleic acid sequence of
the present invention is introduced into a host cell so that the
vector is maintained (e.g., by chromosomal integration or as a
self-replicating extra-chromosomal vector) such that the
polypeptide is expressed.
[0130] The host cell may be a prokaryote or a eukaryote. The host
cell may be a bacteria, such as an E. coli strain, e.g., K12
strains NovaBlue, NovaBlue T1R, JM109, and DH5a. Preferably, the
bacteria cell has the capability to fold, or partially fold,
exogenously expressed proteins, such as E. coli Origami strains,
e.g., Origami B, Origami B (DE3), Origami 2, and Origami 2(DE3)
strains. In some embodiments, it may be preferred to use a host
cell that is deficient for glycosylation, or has an impaired
glycosylation pathway such that proteins expressed by the host cell
are not significantly glycosylated.
[0131] The host cell may be a yeast or a filamentous fungus,
particularly those classified as Ascomycota. Genera of yeasts
useful as host microbes for the expression of modified TrCel3A
beta-glucosidases of the present invention include Saccharomyces,
Pichia, Hansenula, Kluyveromyces, Yarrowia, and Arxula. Genera of
fungi useful as microbes for the expression of the polypeptides of
the present invention include Trichoderma, Hypocrea, Aspergillus,
Fusarium, Humicola, Neurospora, Chrysosporium, Myceliophthora,
Thielavia, Sporotrichum and Penicillium. For example, the host cell
may be Pichia pastoris. For example, the host cell may be an
industrial strain of Trichoderma reesei, or a mutant thereof, e.g.,
T. reesei RUTC30. Typically, the host cell is one which does not
express a parental biomass-degrading enzyme, e.g., cellobiase or
Cel3a.
[0132] The selection of the particular host cell, e.g., bacterial
cell or a fungal cell, depends on the expression vector (e.g., the
control sequences) and/or the method utilized for producing an
aglycosylated polypeptide of the invention, as described in further
detail below.
[0133] The expression vector of the invention may be introduced
into the host cell by any number of methods known by one skilled in
the art of microbial transformation, including but not limited to,
transformation, treatment of cells with CaCl.sub.2,
electroporation, biolistic bombardment, lipofection, and
PEG-mediated fusion of protoplasts (e.g. White et al., WO
2005/093072, which is incorporated herein by reference). After
selecting the recombinant host cells containing the expression
vector (e.g., by selection utilizing the selectable marker of the
expression vector), the recombinant host cells may be cultured
under conditions that induce the expression of the polypeptide
having biomass-degrading activity of the invention.
[0134] Methods for recovering the soluble polypeptides having
biomass-degrading activity expressed from prokaryote and eukaryote
cells are known in the art. In embodiments, the method for
recovering the polypeptide comprises collecting the cells, e.g., by
centrifugation or filtration, and lysing the cells, e.g., by
mechanical, chemical, or enzymatic means. For example, cells can be
physically broken apart, e.g., by sonication, milling (shaking with
beads), or shear forces. Cell membranes can be treated such that
they are permeabilized such that the contents of the cells are
released, such as treatment with detergents, e.g., Triton, NP-40,
or SDS. Cells with cell walls, e.g., bacterial cells, can be
permeabilized using enzymes, such as a lysozyme or lysonase. Any
combination of the mechanical, chemical, and enzymatic techniques
described above are also suitable for recovering expressed
polypeptides of interest from the host cell in the context of this
invention. For example, when expressing a polypeptide having
biomass-degrading activity described herein in a bacterial cell,
e.g., an E. coli cell, the cell is typically collected by
centrifuging and pelleting the cell culture, and lysed by
resuspending the cell pellet in a lysis buffer containing lysozyme.
To ensure complete lysis, the resuspended cells are subjected to
one of the following methods: sonication, milling, or
homogenization. After centrifugation, the soluble polypeptides
having biomass-degrading activity are present in the supernatant,
while the insoluble polypeptides having biomass-degrading activity,
e.g., in inclusion bodies, are found in the pellet. Methods for
recovering the insoluble polypeptides having biomass-degrading
activity, e.g., from inclusion bodies, are described further below
in the section titled "Solubilization from Inclusion Bodies".
[0135] The soluble polypeptides having biomass-degrading activity
can then be purified or isolated from the cell lysate using
standard methods known in the art. For polypeptides having
biomass-degrading activity comprising a tag, e.g., a His tag,
affinity chromatography can be used to separate the soluble
polypeptides from the remainder of the soluble fraction of the
lysate.
[0136] In one embodiment, the host cell expressing a polypeptide
having biomass-degrading activity described herein is not lysed
before addition to the biomass for the saccharification reaction.
In some instances, the methods for lysing host cells and extracting
the polypeptides having biomass-degrading activity can result in
protein denaturation and/or decreased enzyme activity, which leads
to increased cost of downstream processing. Thus, the present
invention also provides methods for directly adding the host cells
expressing an aglycosylated polypeptide having biomass-degrading
activity described herein to the biomass prior to the
saccharification step.
[0137] In an embodiment, the host cell, e.g., the E. coli cell,
expressing a polypeptide having biomass-degrading activity
described herein is isolated, e.g., by centrifugation, and added to
the saccharification reaction, e.g., the saccharification reactor
containing biomass. The cells are lysed by a combination of shear
from the biomass, the impellers, and the increased temperature. In
an embodiment, the culture of host cell, e.g., the E. coli cell,
expressing the polypeptide having biomass-degrading activity
described herein is added directly from the fermentation tank
directly to the saccharification tank and eliminating the need to
pellet cells by centrifugation. In an embodiment, the polypeptide
is glycosylated or aglycosylated.
Solubilization from Inclusion Bodies
[0138] In embodiments, a cell, e.g., a microorganism disclosed
herein, has been genetically modified using methods described
herein to produce at least one polypeptide having a
biomass-degrading activity. At least a portion, e.g., at least 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, of the polypeptide
having biomass-degrading activity is found in inclusion bodies in
the genetically modified cell. Disclosed herein are methods for
solubilizing the at least one polypeptide having biomass-degrading
activity from the inclusion bodies.
[0139] Inclusion bodies are insoluble aggregates in host cells
comprising heterologously expressed proteins, e.g., a polypeptide
having a biomass-degrading activity, when expressed at high levels.
Inclusion bodies can be found in the nucleus or the cytoplasm.
Inclusion bodies can also contain other components, such as other
proteins endogenous to the host cell, e.g., host proteins,
ribosomal components, nucleic acids (e.g., RNA and/or DNA), and
cellular debris (e.g., cell wall debris, lipids, metabolites).
Proteins endogenous to the host cell includes any protein that is
encoded by genomic DNA of the host cell. The protein endogenous to
the host cell may be localized to the cytoplasm, or may interact
with the heterologously expressed protein. Examples of ribosomal
components that can be found in an inclusion body include
ribosomes, fragments of ribosomal subunits (e.g., 50 S subunit or
30 S subunit), partially translated polypeptides, transfer RNA,
elongation factors, and/or messenger RNA. Examples of nucleic acids
that can be found in an inclusion body include genomic DNA of the
host, exogenous DNA (e.g., from a plasmid or expression vector
introduced into the host cell), messenger RNA, transfer RNA,
ribosomal RNA, or any fragments thereof. Examples of cellular
debris that can be found in an inclusion body include cell wall or
membrane debris (e.g., fragments or components of the cell wall or
membrane), nuclear membrane debris (e.g., fragments or components
of the nuclear membrane), fragments or components of other host
organelles, endotoxins, lipids, and/or metabolites.
[0140] Additional methods for reducing aggregation of inclusion
bodies include sonication, incubation at varying temperatures,
acid/base treatment, protease treatment, electrical treatment,
mechanical treatment, and addition of organisms that produce
proteases. For example, the cells or lysates thereof containing
inclusion bodies are incubated at temperatures ranging from
-20.degree. C. to 0.degree. C., 0.degree. C. to 4.degree. C.,
4.degree. C. to 20.degree. C., 20.degree. C. to 40.degree. C., and
40.degree. C. to 80.degree. C.
[0141] To isolate the inclusion bodies, the host cell expressing a
polypeptide having biomass-degrading activity is first lysed, using
standard methods in the art, such as lysis by lysozyme or other
denaturing agents, ultrasound treatment, sonication, or high
pressure homogenization. The host cells are lysed under conditions
that do not lead to solubilization of an inclusion body. Inclusion
bodies are isolated from the host cell using techniques known in
the art. For example, the cell lysate is separated such that the
inclusion bodies containing polypeptides having biomass-degrading
activity and other insoluble matter are present in an insoluble
fraction, while the soluble fraction contains the soluble
polypeptides having biomass-degrading activity. Such separation can
be accomplished through centrifugation, whereby the inclusion
bodies are found in the pellet, e.g., the insoluble fraction, and
the soluble polypeptides are found in the supernatant, e.g., the
soluble fraction. Other methods suitable for separation of an
insoluble fraction from the soluble fraction include
filtration.
[0142] Solubilization of the inclusion bodies to release a
polypeptide having biomass-degrading activity comprises adding a
solubilizing agent to the insoluble fraction or inclusion bodies.
In some embodiments a solubilizing agent can be an agent that
prevents protein aggregation or precipitation, or dissolves protein
aggregates. In some embodiments, the solubilizing agent includes an
agent that disrupts van der Waals interactions, hydrophobic
interactions, hydrogen bonding, dipole-dipole interactions, ionic
interactions, pi stacking, or any combination thereof.
[0143] In some embodiments, the solubilizing agent can be an agent
that disrupts hydrophobic interactions, e.g., such as a detergent.
Exemplary detergents include nonionic, zwitterionic, anionic and
cationic detergents. In some embodiments, the solubilizing agent
can be nonionic, e.g., NP-40 and Triton X-100. In some embodiments,
the solubilizing agent can be zwitterionic, e.g., CHAPS and
sulfobetaines, e.g., SB3-10 or ASB 14. In some embodiments, the
protein agent can be anionic, e.g., sodium dodecyl sulfate
(SDS).
[0144] In some embodiments, the solubilizing agent can be an agent
that reduces disulfide bonds, e.g., a thiol reducing agent.
Exemplary thiol reducing agents include 2-mercaptoethanol .beta.ME
and dithiothreitol (DTT). In some embodiments, the solubilizing
agent that reduces disulfide bonds can be a phosphine, e.g.,
tributylphosphine (TBP) or triscarboxyethylphosphine (TCEP).
[0145] In some embodiments, the solubilizing agent can be an agent
that disrupts hydrogen bonding and hydrophobic interactions. In
some embodiments, the solubilizing agent can be a chaotropic
compound, e.g., urea and substituted ureas (e.g., thiourea), and
guanidinium hydrochloride.
[0146] In some embodiments, the solubilizing agent can be an agent
that is a nonpolar solvent. Nonpolar solvents contain bonds between
atoms with similar electronegativities, such as carbon and
hydrogen, and have very low dielectric constants. For example,
nonpolar solvents have a dielectric constant of less than 5.
Examples of nonpolar solvents include pentane, hexane, cyclohexane,
benzene, toluene, chloroform, diethyl ether.
[0147] In some embodiments, the solubilizing agent can be an agent
that is a polar solvent. Polar solvents are characterized by having
large dipole moments (or "partial charges"); they contain bonds
between atoms with very different electronegativities, such as
oxygen and hydrogen. In one embodiment, the polar solvents suitable
for use in the invention herein have a dielectric constant of at
least 5, or at least 20. In a preferred embodiment, the polar
solvents have a high dielectric constant, e.g., a dielectric
constant greater than 25. In some embodiments, the solubilizing
agent is a protic polar solvent, which has O--H or N--H bonds, have
high dielectric constants, e.g., greater than 20, greater than 25,
and are good hydrogen bond donors, e.g., formic acid, n-butanol,
isopropanol, n-propanol, ethanol, methanol, or nitromethane. In
some embodiments, the solubilizing agent can be an aprotic polar
solvent, which lack O--H or N--H bonds, and has a dielectric
constant between 5 and 20, e.g., dimethylsulfoxide (DMSO),
dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate,
acetone, dimethylformamide (DMF), or acetonitrile (MeCN).
[0148] In some embodiments, the solubilizing agent can be an agent
that has a positive charge, which may be suitable for disrupting
ionic interactions of a net negatively charged molecule. Exemplary
positively charged solubilizing agents include N-methyl
D-glucamine, choline, arginine, lysine, procaine, tromethamine
(TRIS), spermine, N-methyl-morpholine, glucosamine, N,N-bis
2-hydroxyethyl glycine, diazabicycloundecene, creatine, arginine
ethyl ester, amantadine, rimantadine, ornithine, taurine, and
citrulline. Cationic moieties may additionally include sodium,
potassium, calcium, magnesium, ammonium, monoethanolamine,
diethanolamine, triethanolamine, tromethamine, lysine, histidine,
arginine, morpholine, methylglucamine, and glucosamine.
[0149] In some embodiments, the solubilizing agent can be an agent
that has a negative charge, which may be suitable for disrupting
ionic interactions of a net positively charged molecule. Exemplary
negatively charged solubilizing agents include acetate, propionate,
butyrate, pentanoate, hexanoate, heptanoate, levulinate, chloride,
bromide, iodide, citrate, succinate, maleate, glycolate gluconate,
glucuronate, 3-hydroxyisobutyrate, 2-hydroxyisobutyrate, lactate,
malate, pyruvate, fumarate, tartarate, tartronate, nitrate,
phosphate, benzene sulfonate, methane sulfonate, sulfate,
sulfonate, acetic acid, adamantoic acid, alpha keto glutaric acid,
D- or L-aspartic acid, benzensulfonic acid, benzoic acid,
10-camphorsulfunic acid, citric acid, 1,2-ethanedisulfonic acid,
fumaric acid, D-gluconic acid, D-glucuronic acid, glucaric acid, D-
or L-glutamic acid, glutaric acid, glycolic acid, hippuric acid,
hydrobromic acid, hydrochloric acid, 1-hydroxyl-2-napthoic acid,
lactobioinic acid, maleic acid, L-malic acid, mandelic acid,
methanesulfonic acid, mucic acid, 1,5 napthalenedisulfonic acid
tetrahydrate, 2-napthalenesulfonic acid, nitric acid, oleic acid,
pamoic acid, phosphoric acid, p-toluenesulfonic acid hydrate,
D-saccharide acid monopotassium salt, salicyclic acid, stearic
acid, succinic acid, sulfuric acid, tannic acid, and D- or
L-tartaric acid.
[0150] The solubilizing agent is added to an inclusion body, or a
fraction containing inclusion bodies, at a sufficient concentration
to solubilize a polypeptide having biomass degrading activity from
the inclusion body, for example, at a concentration of about
0.01-10M, about 0.05-10M, about 0.1-10M, about 0.2-10M, about
0.5-10M, about 1-10M, about 2-10M, about 5-10M, about 8-10M, about
0.01-6M, about 0.05-6M, about 0.1-6M, about 0.2-6M, about 0.5-6M,
about 1-6M, about 2-6M, about 4-6M, or about 5-6M. In an
embodiment, the solubilizing agent is added to an inclusion body,
or a fraction containing inclusion bodies, at a concentration of
about 0.01M, about 0.02M, about 0.05M, about 0.1M, about 0.2M,
about 0.5M, about 1M, about 2M, about 3M, about 4M, about 5M, about
6M, about 7M, about 8M, about 9M, or about 10M.
[0151] In a preferred embodiment, the solubilizing agent is urea,
and is added to an inclusion body, or a fraction containing
inclusion bodies, at a concentration of about 0.01-10M, about
0.05-10M, about 0.1-10M, about 0.2-10M, about 0.5-10M, about 1-10M,
about 2-10M, about 5-10M, about 8-10M, about 0.01-6M, about
0.05-6M, about 0.1-6M, about 0.2-6M, about 0.5-6M, about 1-6M,
about 2-6M, about 4-6M, or about 5-6M. In an embodiment, urea is
added to an inclusion body, or a fraction containing inclusion
bodies, at a concentration of about 0.01M, about 0.02M, about
0.05M, about 0.1M, about 0.2M, about 0.5M, about 1M, about 2M,
about 3M, about 4M, about 5M, about 6M, about 7M, about 8M, about
9M, or about 10M. In a preferred embodiment, the urea is added to
an inclusion body, or a fraction containing inclusion bodies, at a
concentration of 6M.
[0152] After solubilization using a solubilizing agent, the
resulting mixture contains a solubilized polypeptide having
biomass-degrading activity, as described herein. The resulting
mixture can be used directly in an enzymatic processes, such as a
reaction for producing products, e.g., a saccharification reaction,
as described in further detail in the section titled "Methods of
Producing Products Using Solubilized Enzymes". In this embodiment,
the mixture may contain other components of the inclusion body,
such as other proteins endogenous to the host cell, ribosomal
components, nucleic acids (e.g., RNA and/or DNA), and cellular
debris (e.g., cell wall debris, lipids, metabolites).
[0153] In other embodiments, the resulting mixture containing a
solubilized polypeptide having biomass-degrading activity is
further processed to purify or isolate the solubilized polypeptide
having biomass-degrading activity from the other solubilized
components of the inclusion bodies. Suitable methods for isolating
or purifying the solubilized polypeptide having biomass-degrading
activity include affinity purification techniques. For example, the
polypeptide having biomass-degrading activity preferably contains a
tag or fusion peptide that can be utilized for affinity
purification. In an embodiment, the polypeptide having
biomass-degrading activity contains a His-tag, e.g., an 8.times.
His tag (SEQ ID NO: 21), and the solubilized polypeptide having
biomass-degrading activity can be purified using nickel affinity
chromatography, e.g., an immobilized metal ion affinity
chromatography (IMAC) system. In one embodiment, all purification
steps occur in the presence of the solubilizing agent used to
solubilize the polypeptide from an inclusion body, including in the
washing and elution steps of the purification process. Accordingly,
in one embodiment, the resulting purified solubilized polypeptide
having biomass-degrading activity also contains a solubilizing
agent.
[0154] The present invention provides a mixture comprising a
polypeptide having biomass-degrading activity and a solubilizing
agent. The mixture is obtained through the solubilization of
inclusion bodies, as described above. The resulting mixture may
also further comprise one or more proteins associated with the
inclusion bodies. The solubilized polypeptide having
biomass-degrading activity may also be purified by affinity
purification techniques. In this case, the resulting mixture does
not comprise one or more proteins associated with the inclusion
bodies. The mixture can comprise other components found in
inclusion bodies, such as other proteins endogenous to the host
cell, ribosomal components, nucleic acids (e.g., RNA and/or DNA),
and cellular debris (e.g., cell wall debris, lipids,
metabolites).
[0155] In an embodiment, the solubilized polypeptide having
biomass-degrading activity can be partially unfolded, partially
misfolded, or partially denatured. In an embodiment, the
solubilized polypeptide having biomass-degrading activity has at
least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at
least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at
least 8-10%, at least 15%, at least 20%, at least 25%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or at least
100% biomass-degrading activity compared to the native polypeptide
having biomass-degrading activity. In an embodiment, the
solubilized polypeptide having biomass-degrading activity has about
1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90%,
1-100%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%,
10-90%, 10-100%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%,
20-90%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%,
30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-100%, 50-60%,
50-70%, 50-80%, 50-90%, 50-100%, 60-70%, 60-80%, 60-90%, 60-100%,
70-80%, 70-90%, 70-100%, 80-90%, 80-100%, or 90-100% of the
biomass-degrading activity compared to the native polypeptide
having biomass-degrading activity. In a preferred embodiment, the
solubilized polypeptide having biomass-degrading activity has at
least 8-10% of the activity of the native polypeptide. The native
polypeptide having biomass-degrading activity refers to, e.g., the
corresponding polypeptide having biomass-degrading activity
isolated from the soluble fraction, the corresponding polypeptide
having biomass-degrading activity that is properly folded in its
native form (thereby having 100% biomass-degrading activity), or
the corresponding polypeptide having biomass-degrading activity
endogenously expressed from the microorganism from which the
polypeptide originates from. Biomass-degrading activity can be
determined by any of the assays described herein, e.g., a ligninase
activity assay, an endoglucanase activity assay, a
cellobiohydrolase activity assay, a cellobiase activity assay, or a
xylanase activity assay.
[0156] In one aspect, the mixture comprises a polypeptide having
cellobiase activity, e.g., a Cel3a or a functional variant thereof
from T. reesei, e.g., a polypeptide with at least 90% identity to
SEQ ID NO: 1, and a solubilizing agent, e.g., urea. In one
embodiment, the mixture comprises a polypeptide having at least 90%
identity to SEQ ID NO: 1 and a solubilizing agent, e.g., urea,
wherein the polypeptide has at least 20% of the cellobiase activity
compared to the native polypeptide, e.g., SEQ ID NO: 1 or Cel3a
from T. reesei. The mixture is obtained through the solubilization
of inclusion bodies, as described above. The resulting mixture may
also further comprise one or more proteins associated with the
inclusion bodies. The solubilized polypeptide having cellobiase
activity, e.g., a polypeptide with at least 90% identity to SEQ ID
NO: 1, may also be purified by affinity purification techniques. In
this case, the resulting mixture does not comprise one or more
proteins associated with the inclusion bodies. The mixture can
comprise other components found in inclusion bodies, such as other
proteins endogenous to the host cell, ribosomal components, nucleic
acids (e.g., RNA and/or DNA), and cellular debris (e.g., cell wall
debris, lipids, metabolites). In an embodiment, the solubilizing
agent is urea, and the urea is present at 0.2-6M.
[0157] In an embodiment, the solubilized polypeptide having
cellobiase activity, or at least 90% identity with SEQ ID NO: 1,
can be partially unfolded, partially misfolded, or partially
denatured. In an embodiment, the solubilized polypeptide having
cellobiase activity, or at least 90% identity with SEQ ID NO: 1,
has at least 1%, at least 2%, at least 3%, at least 4%, at least
5%, at least 6%, at least 7%, at least 8%, at least 9%, at least
10%, at least 8-10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, or at
least 100% cellobiase activity compared to the native polypeptide.
In an embodiment, the solubilized polypeptide having cellobiase
activity, or at least 90% identity with SEQ ID NO: 1, has about
1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90%,
1-100%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%,
10-90%, 10-100%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%,
20-90%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%,
30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-100%, 50-60%,
50-70%, 50-80%, 50-90%, 50-100%, 60-70%, 60-80%, 60-90%, 60-100%,
70-80%, 70-90%, 70-100%, 80-90%, 80-100%, or 90-100% of the
cellobiase activity compared to the native polypeptide. The native
polypeptide is, for example, SEQ ID NO: 1 that is properly folded
(e.g., 100% folded), Cel3a that is isolated from T. reesei, or a
functional variant thereof. Cellobiase activity can be measured
using the assays described herein, and can be quantified as the
concentration of glucose (g/L) released after 30 minutes or the %
of cellobiose converted to glucose in 30 minutes.
Methods for Producing Aglycosylated Polypeptides
[0158] The present invention further provides methods for producing
an aglycosylated polypeptide having biomass-degrading activity in a
host cell, wherein the host cell, or lysate thereof, is treated
with a solubilizing agent at a concentration suitable for
solubilizing the aglycosylated polypeptide, as described herein.
The method comprises culturing the host cell expressing the
polypeptide having biomass-degrading activity under conditions
suitable for the expression of the polypeptide. The method may also
comprise recovering the aglycosylated polypeptide having
biomass-degrading activity from the host cell. In the methods
described in further detail below, the polypeptide having
biomass-degrading activity has, e.g., ligninase activity,
endoglucanase activity, cellobiohydrolase activity, cellobiase
activity, or xylanase activity. In an embodiment, the polypeptide
having cellobiase activity comprises a Cel3a from T. reesei, or a
functional fragment thereof. In another embodiment, the polypeptide
having cellobiase activity comprises SEQ ID NO: 1.
Using a Host Cell Deficient for Glycosylation
[0159] In embodiments, the expression vector comprises a nucleic
acid sequence encoding a polypeptide having biomass-degrading
activity described herein operably linked to a fusion tag is
introduced to and expressed in a cell that does not significantly
glycosylate proteins expressed in the cell, e.g., a bacterial host
cell. The recombinant host cell is cultured under conditions for
expression of the polypeptide, resulting in the production of an
aglycosylated polypeptide having biomass-degrading activity. The
aglycosylated polypeptide can be purified or isolated from the host
cell using affinity chromatography methods for the fusion tag as
described herein.
[0160] For example, in this embodiment, the expression vector
contains a lac operator and a T7 promoter upstream of the nucleic
acid sequence encoding a polypeptide having biomass-degrading
activity, and the host cell has the capacity to express T7 RNA
polymerase. Expression of the polypeptide having biomass-degrading
activity is induced by addition of IPTG. Preferably, the host cell
is an E. coli cell, preferably an E. coli Origami cell. In this
embodiment, the fusion tag is a His-tag, and the purification of
the expressed aglycosylated polypeptide comprises nickel affinity
chromatography.
Using a Host Cell with the Capacity for Glycosylation
[0161] In another embodiment, an expression vector comprising a
nucleic acid sequence encoding a polypeptide comprising one or more
glycosylation site mutations such that the polypeptide is not
glycosylated, as described herein, is expressed in a host cell,
wherein the host cell is capable of glycosylating proteins
expressed within the cell, e.g., a yeast or fungal host cell.
Alternatively, the host cell is not capable of glycosylating
proteins expressed within the cell, e.g., a bacterial host cell. In
this embodiment, the polypeptide is operably linked to a fusion
tag. The aglycosylated polypeptide can be purified or isolated from
the bacterial host cell using affinity chromatography methods for
the fusion tag as described herein.
[0162] In yet another embodiment, an expression vector comprising a
nucleic acid sequence encoding a polypeptide having
biomass-degrading activity described herein is expressed in a host
cell, wherein the host cell is capable of glycosylating proteins
expressed within the cell. The cells are cultured under conditions
sufficient for expression and glycosylation of the polypeptide. In
this embodiment, the polypeptide is operably linked to a fusion
tag. The glycosylated polypeptide can be purified or isolated from
the bacterial host cell using affinity chromatography methods for
the fusion tag as described herein. After purification from the
host cells and other endogenous host enzymes, e.g., glycosylation
enzymes, the glycans of the isolated glycosylated polypeptide can
be removed by incubation with deglycosylating enzymes.
Deglycosylating enzymes include PNGase F, PNGase A, EndoH
(endoglycosidase H), EndoS (endoglycosidase S), EndoD
(endoglycosidase D), EndoF (endoglycosidase F), EndoF1
(endoglycosidase F1), or EndoF2 (endoglycosidase F2). Protein
deglycosylation mixes containing enzymes sufficient for the
complete removal of glycans are commercially available, e.g., from
New England Biolabs. The isolated polypeptide is incubated with one
or more deglycosylating enzyme under conditions sufficient for the
removal of all of the glycans from the polypeptide. Other methods
are known in the art for removing glycans from a polypeptide, e.g.,
-elimination with mild alkali or mild hydrazinolysis. Assessment of
the glycosylation state of the polypeptide can be determined using
methods for staining and visualization of glycans known in the art,
or mass spectrometry.
[0163] In yet another embodiment, an expression vector comprising a
nucleic acid sequence encoding a polypeptide having
biomass-degrading activity described herein is expressed in a host
cell, wherein the host cell is capable of glycosylating proteins
expressed within the cell. The cells are cultured under conditions
sufficient for expression of the polypeptide, but in the presence
of glycosylation inhibitors. The glycosylation inhibitors are
present at a concentration and for a sufficient time such that the
expressed polypeptides are aglycosylated. In this embodiment, the
polypeptide is operably linked to a fusion tag. The resulting
aglycosylated polypeptide can be purified or isolated from the
bacterial host cell using affinity chromatography methods for the
fusion tag as described herein.
[0164] Examples of suitable glycosylation inhibitors for use in
this embodiment include tunicamycin, Benzyl-GalNAc (Benzyl
2-acetamido-2-deoxy-.alpha.-D-galactopyranoside),
2-Fluoro-2-deoxy-D-glucose, and 5'CDP (5' cytidylate diphosphate).
In some embodiments, a combination of glycosylation inhibitors is
used. Preferably, the concentration of glycosylation inhibitors
used in this embodiment is sufficient to inhibit glycosylation of
the polypeptide, but do not cause cytotoxicity or inhibition of
protein expression of the host cell.
Methods of Converting Biomass into Products
[0165] The present invention provides methods and compositions for
converting or processing a biomass into products, using an
aglycosylated polypeptide having cellobiase activity, as described
herein. Methods for converting a biomass to products, such as sugar
products, are known in the art, for example, as described in US
Patent Application 2014/0011258, the contents of which are
incorporated by reference in its entirety. Briefly, a biomass is
optimally pretreated, e.g., to reduce the recalcitrance, and
saccharified by a saccharification process that involves incubating
the treated biomass with biomass-degrading, or cellulolytic,
enzymes to produce sugars (e.g., glucose and/or xylose). The sugar
products can then be further processed to produce a final product,
e.g., by fermentation or distillation. Final products include
alcohols (e.g., ethanol, isobutanol, or n-butanol), sugar alcohols
(e.g., erythritol, xylitol, or sorbitol), or organic acids (e.g.,
lactic acid, pyurvic acid, succinic acid).
[0166] Using the processes described herein, the biomass material
can be converted to one or more products, such as energy, fuels,
foods and materials. Specific examples of products include, but are
not limited to, hydrogen, sugars (e.g., glucose, xylose, arabinose,
mannose, galactose, fructose, cellobiose, disaccharides,
oligosaccharides and polysaccharides), alcohols (e.g., monohydric
alcohols or dihydric alcohols, such as ethanol, n-propanol,
isobutanol, sec-butanol, tert-butanol or n-butanol), hydrated or
hydrous alcohols (e.g., containing greater than 10%, 20%, 30% or
even greater than 40% water), biodiesel, organic acids,
hydrocarbons (e.g., methane, ethane, propane, isobutene, pentane,
n-hexane, biodiesel, bio-gasoline and mixtures thereof).
co-products (e.g., proteins, such as cellulolytic proteins
(enzymes) or single cell proteins), and mixtures of any of these in
any combination or relative concentration, and optionally in
combination with any additives (e.g., fuel additives). Other
examples include carboxylic acids, salts of a carboxylic acid, a
mixture of carboxylic acids and salts of carboxylic acids and
esters of carboxylic acids (e.g., methyl, ethyl and n-propyl
esters), ketones (e.g., acetone), aldehydes (e.g., acetaldehyde),
alpha and beta unsaturated acids (e.g., acrylic acid) and olefins
(e.g., ethylene). Other alcohols and alcohol derivatives include
propanol, propylene glycol, 1,4-butanediol, 1,3-propanediol, sugar
alcohols and polyols (e.g., glycol, glycerol, erythritol, threitol,
arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, iditol,
inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol,
maltotetraitol, and polyglycitol and other polyols), and methyl or
ethyl esters of any of these alcohols. Other products include
methyl acrylate, methylmethacrylate, lactic acid, citric acid,
formic acid, acetic acid, propionic acid, butyric acid, succinic
acid, valeric acid, caproic acid, 3-hydroxypropionic acid, palmitic
acid, stearic acid, oxalic acid, malonic acid, glutaric acid, oleic
acid, linoleic acid, glycolic acid, gamma-hydroxybutyric acid, and
mixtures thereof, salts of any of these acids, mixtures of any of
the acids and their respective salts.
Biomass
[0167] The biomass to be processed using the methods described
herein is a starchy material and/or a cellulosic material
comprising cellulose, e.g., a lignocellulosic material. The biomass
may also comprise hemicellulose and/or lignin. The biomass can
comprise one or more of an agricultural product or waste, a paper
product or waste, a forestry product, or a general waste, or any
combination thereof. An agricultural product or waste comprises
material that can be cultivated, harvested, or processed for use or
consumption, e.g., by humans or animals, or any intermediate,
byproduct, or waste that is generated from the cultivation,
harvest, or processing methods. Agricultural products or waste
include, but are not limited to, sugar cane, jute, hemp, flax,
bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley,
cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro,
yams, beans, favas, lentils, peas, grasses, switchgrass,
miscanthus, cord grass, reed canary grass, grain residues, canola
straw, wheat straw, barley straw, oat straw, rice straw, corn cobs,
corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean
stover, grain residues, rice hulls, oat hulls, wheat chaff, barley
hulls, or beeswing, or a combination thereof. A paper product or
waste comprises material that is used to make a paper product, any
paper product, or any intermediate, byproduct or waste that is
generated from making or breaking down the paper product. Paper
products or waste include, but are not limited to, paper, pigmented
papers, loaded papers, coated papers, corrugated paper, filled
papers, magazines, printed matter, printer paper, polycoated paper,
cardstock, cardboard, paperboard, or paper pulp, or a combination
thereof. A forestry product or waste comprises material that is
produced by cultivating, harvesting, or processing of wood, or any
intermediate, byproduct, or waste that is generated from the
cultivation, harvest, or processing of the wood. Forestry products
or waste include, but are not limited to, aspen wood, wood from any
genus or species of tree, particle board, wood chips, or sawdust,
or a combination thereof. A general waste includes, but is not
limited to, manure, sewage, or offal, or a combination thereof.
[0168] The biomass may include, but is not limited to starchy
materials, sugar cane, agricultural waste, paper, paper products,
paper waste, paper pulp, pigmented papers, loaded papers, coated
papers, filled papers, magazines, printed matter, printer paper,
polycoated paper, card stock, cardboard, paperboard, cotton, wood,
particle board, forestry wastes, sawdust, aspen wood, wood chips,
grasses, switchgrass, miscanthus, cord grass, reed canary grass,
grain residues, rice hulls, oat hulls, wheat chaff, barley hulls,
agricultural waste, silage, canola straw, wheat straw, barley
straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal,
abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa,
hay, coconut hair, sugar processing residues, bagasse, beet pulp,
agave bagasse, algae, seaweed, plankton manure, sewage, offal,
agricultural or industrial waste, arracacha, buckwheat, banana,
barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato,
taro, yams, beans, favas, lentils, peas, or mixtures of any of
these. In a preferred embodiment, the biomass comprises agriculture
waste, such as corn cobs, e.g., corn stover. In another embodiment,
the biomass comprises grasses.
[0169] In one embodiment, the biomass is treated prior to contact
with the compositions described herein. For example, the biomass is
treated to reduce the recalcitrance of the biomass, to reduce its
bulk density, and/or increase its surface area. Suitable biomass
treatment process may include, but are not limited to: bombardment
with electrons, sonication, oxidation, pyrolysis, steam explosion,
chemical treatment, mechanical treatment, and freeze grinding.
Preferably, the treatment method is bombardment with electrons.
[0170] In some embodiments, electron bombardment is performed until
the biomass receives a total dose of at least 0.5 Mrad, e.g. at
least 5, 10, 20, 30, or at least 40 Mrad. In some embodiments, the
treatment is performed until the biomass receives a dose a of from
about 0.5 Mrad to about 150 Mrad, about 1 Mrad to about 100 Mrad,
about 5 Mrad to about 75 Mrad, about 2 Mrad to about 75 Mrad, about
10 Mrad to about 50 Mrad, e.g., about 5 Mrad to about 50 Mrad,
about 20 Mrad to about 40 Mrad, about 10 Mrad to about 35 Mrad, or
from about 20 Mrad to about 30 Mrad. In some implementations, a
total dose of 25 to 35 Mrad is preferred, applied ideally over a
couple of seconds, e.g., at 5 Mrad/pass with each pass being
applied for about one second. Applying a dose of greater than 7 to
9 Mrad/pass can in some cases cause thermal degradation of the
feedstock material.
[0171] The biomass material (e.g., plant biomass, animal biomass,
paper, and municipal waste biomass) can be used as feedstock to
produce useful intermediates and products such as organic acids,
salts of organic acids, anhydrides, esters of organic acids and
fuels, e.g., fuels for internal combustion engines or feedstocks
for fuel cells. Systems and processes are described herein that can
use as feedstock cellulosic and/or lignocellulosic materials that
are readily available, but often can be difficult to process, e.g.,
municipal waste streams and waste paper streams, such as streams
that include newspaper, kraft paper, corrugated paper or mixtures
of these.
[0172] In order to convert the feedstock to a form that can be
readily processed, the glucan- or xylan-containing cellulose in the
feedstock can be hydrolyzed to low molecular weight carbohydrates,
such as sugars, by a saccharifying agent, e.g., an enzyme or acid,
a process referred to as saccharification. The low molecular weight
carbohydrates can then be used, for example, in an existing
manufacturing plant, such as a single cell protein plant, an enzyme
manufacturing plant, or a fuel plant, e.g., an ethanol
manufacturing facility.
[0173] The feedstock can be hydrolyzed using an enzyme, e.g., by
combining the materials and the enzyme in a solvent, e.g., in an
aqueous solution. The enzymes can be made/induced according to the
methods described herein.
[0174] Specifically, the enzymes can be supplied by organisms that
are capable of breaking down biomass (such as the cellulose and/or
the lignin portions of the biomass), or that contain or manufacture
various cellulolytic enzymes (cellulases), ligninases or various
small molecule biomass-degrading metabolites. These enzymes may be
a complex of enzymes that act synergistically to degrade
crystalline cellulose or the lignin portions of biomass. Examples
of cellulolytic enzymes include: endoglucanases,
cellobiohydrolases, and cellobiases (beta-glucosidases).
[0175] During saccharification a cellulosic substrate can be
initially hydrolyzed by endoglucanases at random locations
producing oligomeric intermediates. These intermediates are then
substrates for exo-splitting glucanases such as cellobiohydrolase
to produce cellobiose from the ends of the cellulose polymer.
Cellobiose is a water-soluble 1,4-linked dimer of glucose. Finally,
cellobiase cleaves cellobiose to yield glucose. The efficiency
(e.g., time to hydrolyze and/or completeness of hydrolysis) of this
process depends on the recalcitrance of the cellulosic
material.
Saccharification
[0176] The reduced-recalcitrance biomass is treated with the
biomass-degrading enzymes discussed above, generally by combining
the reduced-recalcitrance biomass and the biomass-degrading enzymes
in a fluid medium, e.g., an aqueous solution. In some cases, the
feedstock is boiled, steeped, or cooked in hot water prior to
saccharification, as described in U.S. Pat. App. Pub. 2012/0100577
A1 by Medoff and Masterman, published on Apr. 26, 2012, the entire
contents of which are incorporated herein.
[0177] Provided herein are mixtures of enzymes that are capable of
degrading the biomass, e.g., an enzyme mixture of biomass-degrading
enzymes, for use in the saccharification process described
herein.
[0178] The saccharification process can be partially or completely
performed in a tank (e.g., a tank having a volume of at least 4000
L, 40,000 L, 500,000 L, 2,000,000 L, 4,000,000 L, or 6,000,000 L or
more) in a manufacturing plant, and/or can be partially or
completely performed in transit, e.g., in a rail car, tanker truck,
or in a supertanker or the hold of a ship. The time required for
complete saccharification will depend on the process conditions and
the biomass material and enzyme used. If saccharification is
performed in a manufacturing plant under controlled conditions, the
cellulose may be substantially entirely converted to sugar, e.g.,
glucose in about 12-96 hours. If saccharification is performed
partially or completely in transit, saccharification may take
longer.
[0179] In a preferred embodiment, the saccharification reaction
occurs at a pH optimal for the enzymatic reactions to occur, e.g.,
at the pH optimal for the activity of the biomass-degrading
enzymes. Preferably, the pH of the saccharification reaction is at
pH 4-4.5. In a preferred embodiment, the saccharification reaction
occurs at a temperature optimal for the enzymatic reactions to
occur, e.g., at the temperature optimal for the activity of the
biomass-degrading enzymes. Preferably, the temperature of the
saccharification reaction is at 42.degree. C. -52.degree. C.
[0180] It is generally preferred that the tank contents be mixed
during saccharification, e.g., using jet mixing as described in
International App. No. PCT/US2010/035331, filed May 18, 2010, which
was published in English as WO 2010/135380 and designated the
United States, the full disclosure of which is incorporated by
reference herein.
[0181] The addition of surfactants can enhance the rate of
saccharification. Examples of surfactants include non-ionic
surfactants, such as a Tween.RTM. 20 or Tween.RTM. 80 polyethylene
glycol surfactants, ionic surfactants, or amphoteric
surfactants.
[0182] It is generally preferred that the concentration of the
sugar solution resulting from saccharification be relatively high,
e.g., greater than 5%, 7.5%, 10%, 10.5%, or greater than 40%, or
greater than 50, 60, 70, or even greater than 80% by weight. Water
may be removed, e.g., by evaporation, to increase the concentration
of the sugar solution. This reduces the volume to be shipped, and
also inhibits microbial growth in the solution.
[0183] Alternatively, sugar solutions of lower concentrations may
be used, in which case it may be desirable to add an antimicrobial
additive, e.g., a broad spectrum antibiotic, in a low
concentration, e.g., 50 to 150 ppm. Other suitable antibiotics
include amphotericin B, ampicillin, chloramphenicol, ciprofloxacin,
gentamicin, hygromycin B, kanamycin, neomycin, penicillin,
puromycin, streptomycin. Antibiotics will inhibit growth of
microorganisms during transport and storage, and can be used at
appropriate concentrations, e.g., between 15 and 10,000 ppm by
weight, e.g., between 25 and 500 ppm, or between 50 and 150 ppm. If
desired, an antibiotic can be included even if the sugar
concentration is relatively high. Alternatively, other additives
with anti-microbial of preservative properties may be used.
Preferably the antimicrobial additive(s) are food-grade.
[0184] A relatively high concentration solution can be obtained by
limiting the amount of water added to the biomass material with the
enzyme. The concentration can be controlled, e.g., by controlling
how much saccharification takes place. For example, concentration
can be increased by adding more biomass material to the solution.
In order to keep the sugar that is being produced in solution, a
surfactant can be added, e.g., one of those discussed above.
Solubility can also be increased by increasing the temperature of
the solution. For example, the solution can be maintained at a
temperature of 40-50.degree. C., 60-80.degree. C., or even
higher.
[0185] In the processes described herein, for example after
saccharification, sugars (e.g., glucose and xylose) can be
isolated. For example, sugars can be isolated by precipitation,
crystallization, chromatography (e.g., simulated moving bed
chromatography, high pressure chromatography), centrifugation.
extraction, any other isolation method known in the art, and
combinations thereof.
Mixtures for Use in Saccharification
[0186] In an aspect, the present invention features a mixture for
use in a saccharification comprising a polypeptide having
biomass-degrading activity that has been solubilized from an
inclusion body, as described herein, one or more proteins
associated with an inclusion body, and a solubilizing agent. In an
embodiment, the mixture may contain other components of the
inclusion body, such as other proteins endogenous to the host cell,
ribosomal components, nucleic acids (e.g., RNA and/or DNA), and
cellular debris (e.g., cell wall debris, lipids, metabolites). The
polypeptide having biomass-degrading activity can be glycosylated
or aglycosylated.
[0187] In one embodiment, the mixture comprises a polypeptide
having cellobiase activity and urea. In an embodiment, the urea is
present at 0.2-6M. In one embodiment, the mixture comprises a Cel3a
from T. reesei, or a functional variant or a fragment thereof. In
one embodiment, the mixture comprises a polypeptide comprising at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity to a Cel3a from T. reesei.
In another embodiment, the mixture comprises a polypeptide
comprising at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID
NO: 1. The polypeptide having cellobiase activity can be
glycosylated or aglycosylated.
[0188] In embodiments, the mixture described herein further
comprises at least one additional enzyme derived from a
microorganism, wherein the additional enzyme has biomass or
cellulose-based material-degrading activity. For example, the
additional enzyme is a ligninase, an endoglucanase, a
cellobiohydrolase, a xylanase, or a cellobiase. In an embodiment,
the mixture further comprises one or more ligninase, one or more
endogluconase, one or more cellobiohydrolase, one or more xylanase,
or one or more cellobiase. In embodiments, the additional
biomass-degrading enzyme is glycosylated. In embodiments, the
enzyme mixture further comprises at least 2, at least 3, at least
4, at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, or at least 20 or more additional biomass-degrading
enzymes described herein. Typical primary amino acid sequences for
several biomass-degrading enzymes are shown below.
[0189] For example, the mixture further comprises a mixture of
additional biomass-degrading enzymes produced by a microorganism,
e.g., a fungal cell, such as wild-type T. reesei, or a mutant
thereof, e.g., T. Reesei RUTC30. In an embodiment, the additional
biomass-degrading enzymes are isolated from the microorganisms. In
an embodiment, the mixture comprises one or more of the following
biomass-degrading enzymes: B2AF03, CIP1, CIP2, Cel1a, Cel3a, Cel5a,
Cel6a, Cel7a, Cel7b, Cel12a, Cel45a, Cel74a, paMan5a, paMan26a, or
Swollenin, or any combination thereof. The additional
biomass-degrading enzymes, e.g., listed above, can be endogenously
expressed and isolated from the microorganism, e.g., fungal cell,
from which the enzyme originates from (listed below in Table 1).
Alternatively, the additional biomass-degrading enzymes, e.g.,
listed above, can be heterologously expressed using similar methods
of expression in a host cell described herein, and isolated from
the host cells. In an embodiment, the heterologously expressed
additional biomass-degrading enzymes are tagged with a His tag at
the C or N terminus of the enzyme and are isolated using nickel
affinity chromatography techniques known in the art. For example,
the additional biomass-degrading enzymes are selected from Table 1
below.
TABLE-US-00004 TABLE 1 Examples of Additional Biomass-Degrading
Enzymes MW, no Protein kDa AA's th. pI no. Cysteines Organism
B2AF03 87.1 800 5.94 10 Podospora anserina CIP1 32.9 316 4.93 8
Trichoderma reesei CIP2 48.2 460 7.0 12 Trichoderma reesei Cel1a
52.2 466 5.3 5 Trichoderma reesei Cel3a 78.4 744 6.3 6 Trichoderma
reesei Cel5a 44.1 418 4.9 12 Trichoderma reesei Cel6a 49.6 471 5.1
12 Trichoderma reesei Cel7a 54.1 514 4.6 24 Trichoderma reesei
Cel7b 48.2 459 4.7 22 Trichoderma reesei Cel12a 25.1 234 6.6 2
Trichoderma reesei Cel45a 24.4 242 4.2 16 Trichoderma reesei Cel74a
87.1 838 5.4 4 Trichoderma reesei paMan5a 41.1 373 7.0 6 Podospora
anserina paMan26a 51.7 469 4.7 1 Podospora anserina Swollenin 51.5
493 4.8 28 Trichoderma reesei
[0190] The amino acid sequences for the biomass-degrading enzymes
listed in Table 1 are provided below.
TABLE-US-00005 B2AF03 (Podospora anserina) (SEQ ID NO: 6)
MKSSVFWGASLTSAVVRAIDLPFQFYPNCVDDLLSTNQVCNTTLSPPERAAALVAALTPEEKLQNIVSK
SLGAPRIGLPAYNWWSEALHGVAYAPGTQFWQGDGPFNSSTSFPMPLLMAATFDDELLEKIAEVIGIEG
RAFGNAGFSGLDYWTPNVNPFKDPRWGRGSETPGEDVLLVKRYAAAMIKGLEGPVPEKERRVVATCKHY
AANDFEDWNGATRHNFNAKISLQDMAEYYFMPFQQCVRDSRVGSIMCAYNAVNGVPSCASPYLLQTILR
EHWNWTEHNNYITSDCEAVLDVSLNHKYAATNAEGTAISFEAGMDTSCEYEGSSDIPGAWSQGLLKEST
VDRALLRLYEGIVRAGYFDGKQSLYSSLGWADVNKPSAQKLSLQAAVDGTVLLKNDGTLPLSDLLDKSR
PKKVAMIGFWSDAKDKLRGGYSGTAAYLHTPAYAASQLGIPFSTASGPILHSDLASNQSWTDNAMAAAK
DADYILYFGGIDTSAAGETKDRYDLDWPGAQLSLINLLTTLSKPLIVLQMGDQLDNTPLLSNPKINAIL
WANWPGQDGGTAVMELVTGLKSPAGRLPVTQYPSNFTELVPMTDMALRPSAGNSQLGRTYRWYKTPVQA
FGFGLHYTTFSPKFGKKFPAVIDVDEVLEGCDDKYLDTCPLPDLPVVVENRGNRTSDYVALAFVSAPGV
GPGPWPIKTLGAFTRLRGVKGGEKREGGLKWNLGNLARHDEEGNTVVYPGKYEVSLDEPPKARLRFEIV
RGGKGKGKVKGKGKAAQKGGVVLDRWPKPPKGQEPPAIERV C1P1 (Trichoderma reesei)
(SEQ ID NO: 7)
MVRRTALLALGALSTLSMAQISDDFESGWDQTKWPISAPDCNQGGTVSLDTTVAHSGSNSMKVVGGPNG
YCGHIFFGTTQVPTGDVYVRAWIRLQTALGSNHVTFIIMPDTAQGGKHLRIGGQSQVLDYNRESDDATL
PDLSPNGIASTVTLPTGAFQCFEYHLGTDGTIETWLNGSLIPGMTVGPGVDNPNDAGWTRASYIPEITG
VNFGWEAYSGDVNTVWFDDISIASTRVGCGPGSPGGPGSSTTGRSSTSGPTSTSRPSTTIPPPTSRTTT
ATGPTQTHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL C1P2 (Trichoderma reesei)
(SEQ ID NO: 8)
MASRFFALLLLAIPIQAQSPVWGQCGGIGWSGPTTCVGGATCVSYNPYYSQCIPSTQASSSIASTTLVT
SFTTTTATRTSASTPPASSTGAGGATCSALPGSITLRSNAKLNDLFTMFNGDKVTTKDKFSCRQAEMSE
LIQRYELGTLPGRPSTLTASFSGNTLTINCGEAGKSISFTVTITYPSSGTAPYPAIIGYGGGSLPAPAG
VAMINFNNDNIAAQVNTGSRGQGKFYDLYGSSHSAGAMTAWAWGVSRVIDALELVPGARIDTTKIGVTG
CSRNGKGAMVAGAFEKRIVLTLPQESGAGGSACWRISDYLKSQGANIQTASEIIGEDPWFSTTFNSYVN
QVPVLPFDHHSLAALIAPRGLFVIDNNIDWLGPQSCFGCMTAAHMAWQALGVSDHMGYSQIGAHAHCAF
PSNQQSQLTAFVQKFLLGQSTNTAIFQSDFSANQSQWIDWTTPTLS Cel1a (Trichoderma
reesei) (SEQ ID NO: 9)
MLPKDFQWGFATAAYQIEGAVDQDGRGPSIWDTFCAQPGKIADGSSGVTACDSYNRTAEDIALLKSLGA
KSYRFSISWSRIIPEGGRGDAVNQAGIDHYVKFVDDLLDAGITPFITLFHWDLPEGLHQRYGGLLNRTE
FPLDFENYARVMFRALPKVRNWITFNEPLCSAIPGYGSGTFAPGRQSTSEPWTVGHNILVAHGRAVKAY
RDDFKPASGDGQIGIVLNGDFTYPWDAADPADKEAAERRLEFFTAWFADPIYLGDYPASMRKQLGDRLP
TFTPEERALVHGSNDFYGMNHYTSNYIRHRSSPASADDTVGNVDVLFTNKQGNCIGPETQSPWLRPCAA
GFRDFLVWISKRYGYPPIYVTENGTSIKGESDLPKEKILEDDFRVKYYNEYIRAMVTAVELDGVNVKGY
FAWSLMDNFEWADGYVTRFGVTYVDYENGQKRFPKKSAKSLKPLFDELIAAA Cel3a
(Trichoderma reesei) (SEQ ID NO: 10)
MRYRTAAALALATGPFARADSHSTSGASAEAVVPPAGTPWGTAYDKAKAALAKLNLQDKVGIVSGVGWN
GGPCVGNTSPASKISYPSLCLQDGPLGVRYSTGSTAFTPGVQAASTWDVNLIRERGQFIGEEVKASGIH
VILGPVAGPLGKTPQGGRNWEGFGVDPYLTGIAMGQTINGIQSVGVQATAKHYILNEQELNRETISSNP
DDRTLHELYTWPFADAVQANVASVMCSYNKVNTTWACEDQYTLQTVLKDQLGFPGYVMTDWNAQHTTVQ
SANSGLDMSMPGTDFNGNNRLWGPALTNAVNSNQVPTSRVDDMVTRILAAWYLTGQDQAGYPSFNISRN
VQGNHKTNVRAIARDGIVLLKNDANILPLKKPASIAVVGSAAIIGNHARNSPSCNDKGCDDGALGMGWG
SGAVNYPYFVAPYDAINTRASSQGTQVTLSNTDNTSSGASAARGKDVAIVFITADSGEGYITVEGNAGD
RNNLDPWHNGNALVQAVAGANSNVIVVVHSVGAIILEQILALPQVKAVVWAGLPSQESGNALVDVLWGD
VSPSGKLVYTIAKSPNDYNTRIVSGGSDSFSEGLFIDYKHFDDANITPRYEFGYGLSYTKFNYSRLSVL
STAKSGPATGAVVPGGPSDLFQNVATVTVDIANSGQVTGAEVAQLYITYPSSAPRTPPKQLRGFAKLNL
TPGQSGTATFNIRRRDLSYWDTASQKWVVPSGSFGISVGASSRDIRLTSTLSVA Cel5a
(Trichoderma reesei) (SEQ ID NO: 11)
MNKSVAPLLLAASILYGGAAAQQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRP
PSGPTTTTRATSTSSSTPPTSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQM
QHFVNDDGMTIFRLPVGWQYLVNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQ
GGPTNAQFTSLWSQLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDW
QSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQ
AILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSC
LARK Cel6a (Trichoderma reesei) (SEQ ID NO: 12)
MIVGILTTLATLATLAASVPLEERQACSSVWGQCGGQNWSGPTCCASGSTCVYSNDYYSQCLPGAASSS
SSTRAASTTSRVSPTTSRSSSATPPPGSTTTRVPPVGSGTATYSGNPFVGVTPWANAYYASEVSSLAIP
SLTGAMATAAAAVAKVPSFMWLDTLDKTPLMEQTLADIRTANKNGGNYAGQFVVYDLPDRDCAALASNG
EYSIADGGVAKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINYAVTQ
LNLPNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSPRALRGLATNVANYNGWNITSPPSYTQGN
AVYNEKLYIHAIGPLLANHGWSNAFFITDQGRSGKQPTGQQQWGDWCNVIGTGFGIRPSANTGDSLLDS
FVWVKPGGECDGTSDSSAPRFDSHCALPDALQPAPQAGAWFQAYFVQLLTNANPSFL Cel7a
(Trichoderma reesei) (SEQ ID NO: 13)
MYRKLAVISAFLATARAQSACTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYD
GNTWSSTLCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQKNVGARLYLMASDTTYQEFT
LLGNEFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLKFINGQANVE
GWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNRYGGTCDPD
GCDWNPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNELND
DYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLWLDSTYPTNETSSTPGAVRGSC
STSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGNPSGGNPPGGNPPGTTTTRRPATTTGSSPGPTQSHY
GQCGGIGYSGPTVCASGTTCQVLNPYYSQCL Cel7b (Trichoderma reesei) (SEQ ID
NO: 14)
MAPSVTLPLTTAILAIARLVAAQQPGTSTPEVHPKLTTYKCTKSGGCVAQDTSVVLDWNYRWMHDANYN
SCTVNGGVNTTLCPDEATCGKNCFIEGVDYAASGVTTSGSSLTMNQYMPSSSGGYSSVSPRLYLLDSDG
EYVMLKLNGQELSFDVDLSALPCGENGSLYLSQMDENGGANQYNTAGANYGSGYCDAQCPVQTWRNGTL
NTSHQGFCCNEMDILEGNSRANALTPHSCTATACDSAGCGFNPYGSGYKSYYGPGDTVDTSKTFTIITQ
FNTDNGSPSGNLVSITRKYQQNGVDIPSAQPGGDTISSCPSASAYGGLATMGKALSSGMVLVFSIWNDN
SQYMNWLDSGNAGPCSSTEGNPSNILANNPNTHVVFSNIRWGDIGSTTNSTAPPPPPASSTTFSTTRRS
STTSSSPSCTQTHWGQCGGIGYSGCKTCTSGTTCQYSNDYYSQCL Cel12a (Trichoderma
reesei) (SEQ ID NO: 15)
MKFLQVLPALIPAALAQTSCDQWATFTGNGYTVSNNLWGASAGSGFGCVTAVSLSGGASWHADWQWSGG
QNNVKSYQNSQIAIPQKRTVNSISSMPTTASWSYSGSNIRANVAYDLFTAANPNHVTYSGDYELMIWLG
KYGDIGPIGSSQGTVNVGGQSWTLYYGYNGAMQVYSFVAQTNTTNYSGDVKNFFNYLRDNKGYNAAGQY
VLSYQFGTEPFTGSGTLNVASWTASIN Cel45a (Trichoderma reesei) (SEQ ID NO:
16)
MKATLVLGSLIVGAVSAYKATTTRYYDGQEGACGCGSSSGAFPWQLGIGNGVYTAAGSQALFDTAGASW
CGAGCGKCYQLTSTGQAPCSSCGTGGAAGQSIIVMVTNLCPNNGNAQWCPVVGGTNQYGYSYHFDIMAQ
NEIFGDNVVVDFEPIACPGQAASDWGTCLCVGQQETDPTPVLGNDTGSTPPGSSPPATSSSPPSGGGQQ
TLYGQCGGAGWTGPTTCQAPGTCKVQNQWYSQCLP Cel74a (Trichoderma reesei)
(SEQ ID NO: 17)
MKVSRVLALVLGAVIPAHAAFSWKNVKLGGGGGFVPGIIFHPKTKGVAYARTDIGGLYRLNADDSWTAV
TDGIADNAGWHNWGIDAVALDPQDDQKVYAAVGMYTNSWDPSNGAIIRSSDRGATWSFTNLPFKVGGNM
PGRGAGERLAVDPANSNIIYFGARSGNGLWKSTDGGVTFSKVSSFTATGTYIPDPSDSNGYNSDKQGLM
WVTFDSTSSTTGGATSRIFVGTADNITASVYVSTNAGSTWSAVPGQPGKYFPHKAKLQPAEKALYLTYS
DGTGPYDGTLGSVWRYDIAGGTWKDITPVSGSDLYFGFGGLGLDLQKPGTLVVASLNSWWPDAQLFRST
DSGTTWSPIWAWASYPTETYYYSISTPKAPWIKNNFIDVTSESPSDGLIKRLGWMIESLEIDPTDSNHW
LYGTGMTIFGGHDLTNWDTRHNVSIQSLADGIEEFSVQDLASAPGGSELLAAVGDDNGFTFASRNDLGT
SPQTVWATPTWATSTSVDYAGNSVKSVVRVGNTAGTQQVAISSDGGATWSIDYAADTSMNGGTVAYSAD
GDTILWSTASSGVQRSQFQGSFASVSSLPAGAVIASDKKTNSVFYAGSGSTFYVSKDTGSSFTRGPKLG
SAGTIRDIAAHPTTAGTLYVSTDVGIFRSTDSGTTFGQVSTALTNTYQIALGVGSGSNWNLYAFGTGPS
GARLYASGDSGASWTDIQGSQGFGSIDSTKVAGSGSTAGQVYVGTNGRGVFYAQGTVGGGTGGTSSSTK
QSSSSTSSASSSTTLRSSVVSTTRASTVTSSRTSSAAGPTGSGVAGHYAQCGGIGWTGPTQCVAPYVCQ
KQNDYYYQCV paMan5a (Podospora anserina) (SEQ ID NO: 18)
MKGLFAFGLGLLSLVNALPQAQGGGAAASAKVSGTRFVIDGKTGYFAGTNSYWIGFLTNNRDVDTTLDH
IASSGLKILRVWGFNDVNNQPSGNTVWFQRLASSGSQINTGPNGLQRLDYLVRSAETRGIKLIIALVNY
WDDFGGMKAYVNAFGGTKESWYTNARAQEQYKRYIQAVVSRYVNSPAIFAWELANEPRCKGCNTNVIFN
WATQISDYIRSLDKDHLITLGDEGFGLPGQTTYPYQYGEGTDFVKNLQIKNLDFGTFHMYPGHWGVPTS
FGPGWIKDHAAACRAAGKPCLLEEYGYESDRCNVQKGWQQASRELSRDGMSGDLFWQWGDQLSTGQTHN
DGFTIYYGSSLATCLVTDHVRAINALPA paMan26a (Podospora anserina) (SEQ ID
NO: 19)
MVKLLDIGLFALALASSAVAKPCKPRDGPVTYEAEDAILTGTTVDTAQVGYTGRGYVTGFDEGSDKITF
QISSATTKLYDLSIRYAAIYGDKRTNVVLNNGAVSEVFFPAGDSFTSVAAGQVLLNAGQNTIDIVNNWG
WYLIDSITLTPSAPRPPHDINPNLNNPNADTNAKKLYSYLRSVYGNKIISGQQELHHAEWIRQQTGKTP
ALVAVDLMDYSPSRVERGTTSHAVEDAIAHHNAGGIVSVLWHWNAPVGLYDTEENKWWSGFYTRATDFD
IAATLANPQGANYTLLIRDIDAIAVQLKRLEAAGVPVLWRPLHEAEGGWFWWGAKGPEPAKQLWDILYE
RLTVHHGLDNLIWVWNSILEDWYPGDDTVDILSADVYAQGNGPMSTQYNELIALGRDKKMIAAAEVGAA
PLPGLLQAYQANWLWFAVWGDDFINNPSWNTVAVLNEIYNSDYVLTLDEIQGWRS Swollenin
(Trichoderma reesei) (SEQ ID NO: 20)
MAGKLILVALASLVSLSIQQNCAALFGQCGGIGWSGTTCCVAGAQCSFVNDWYSQCLASTGGNPPNGTT
SSSLVSRTSSASSSVGSSSPGGNSPTGSASTYTTTDTATVAPHSQSPYPSIAASSCGSWTLVDNVCCPS
YCANDDTSESCSGCGTCTTPPSADCKSGTMYPEVHHVSSNESWHYSRSTHFGLTSGGACGFGLYGLCTK
GSVTASWTDPMLGATCDAFCTAYPLLCKDPTGTTLRGNFAAPNGDYYTQFWSSLPGALDNYLSCGECIE
LIQTKPDGTDYAVGEAGYTDPITLEIVDSCPCSANSKWCCGPGADHCGEIDFKYGCPLPADSIHLDLSD
IAMGRLQGNGSLTNGVIPTRYRRVQCPKVGNAYIWLRNGGGPYYFALTAVNTNGPGSVTKIEIKGADTD
NWVALVHDPNYTSSRPQERYGSWVIPQGSGPFNLPVGIRLTSPTGEQIVNEQAIKTFTPPATGDPNFYY
IDIGVQFSQN
[0191] Other examples of suitable biomass-degrading enzymes for use
in the enzyme mixture of the present invention include the enzymes
from species in the genera Bacillus, Coprinus, Myceliophthora,
Cephalosporium, Scytalidium, Penicillium, Aspergillus, Pseudomonas,
Humicola, Fusarium, Thielavia, Acremonium, Chrysosporium and
Trichoderma, especially those produced by a strain selected from
the species Aspergillus (see, e.g., EP Pub. No. 0 458 162),
Humicola insolens (reclassified as Scytalidium thermophilum, see,
e.g., U.S. Pat. No. 4,435,307), Coprinus cinereus, Fusarium
oxysporum, Myceliophthora thermophila, Meripilus giganteus,
Thielavia terrestris, Acremonium sp. (including, but not limited
to, A. persicinum, A. acremonium, A. brachypenium, A.
dichromosporum, A. obclavatum, A. pinkertoniae, A. roseogriseum, A.
incoloratum, and A. furatum). Preferred strains include Humicola
insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthora
thermophila CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp.
CBS 478.94, Acremonium sp. CBS 265.95, Acremonium persicinum CBS
169.65, Acremonium acremonium AHU 9519, Cephalosporium sp. CBS
535.71, Acremonium brachypenium CBS 866.73, Acremonium
dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74,
Acremonium pinkertoniae CBS 157.70, Acremonium roseogriseum CBS
134.56, Acremonium incoloratum CBS 146.62, and Acremonium furatum
CBS 299.70H. Biomass-degrading enzymes may also be obtained from
Chrysosporium, preferably a strain of Chrysosporium lucknowense.
Additional strains that can be used include, but are not limited
to, Trichoderma (particularly T. viride, T. reesei, and T.
koningii), alkalophilic Bacillus (see, for example, U.S. Pat. No.
3,844,890 and EP Pub. No. 0 458 162), and Streptomyces (see, e.g.,
EP Pub. No. 0 458 162).
[0192] In embodiments, the microorganism is induced to produce the
biomass-degrading enzymes described herein under conditions
suitable for increasing production of biomass-degrading enzymes
compared to an uninduced microorganism. For example, an induction
biomass sample comprising biomass as described herein is incubated
with the microorganism to increase production of the
biomass-degrading enzymes. Further description of the induction
process can be found in US 2014/0011258, the contents of which are
hereby incorporated by reference in its entirety.
[0193] The biomass-degrading enzymes produced and/or secreted by
the aforementioned microorganisms can be isolated and added to the
mixture of the present invention, or directly to the
saccharification reaction. Alternatively, in one embodiment, the
aforementioned microorganisms or host cells expressing the
biomass-degrading enzymes described herein and above are not lysed
before addition to the saccharification reaction.
[0194] In an embodiment, an enzyme mixture comprising the host cell
expressing one or more additional biomass-degrading enzymes as
described herein can be used with the mixture comprising the
solubilized polypeptide having biomass-degrading activity described
herein.
[0195] Use of the mixture described herein comprising a polypeptide
having biomass-degrading activity solubilized from inclusion bodies
and a solubilizing agent does not inhibit, prevent or decrease the
yield of sugar products from saccharification compared to
saccharification without addition of the solubilized polypeptide.
In some embodiments, the yield of sugar products increases upon use
of the mixture described herein comprising a polypeptide having
biomass-degrading activity solubilized from inclusion bodies and a
solubilizing agent. The yield of sugar products increases at least
5%, at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
100% compared to when the standard mixture of biomass-degrading
enzymes is added to the saccharification without the mixture
containing solubilized polypeptide and solubilized agent.
Further Processing
[0196] Further processing steps may be performed on the sugars
produced by saccharification to produce alternative products. For
example, the sugars can be hydrogenated, fermented, or treated with
other chemicals to produce other products.
[0197] Glucose can be hydrogenated to sorbitol. Xylose can be
hydrogenated to xylitol. Hydrogenation can be accomplished by use
of a catalyst (e.g., Pt/gamma-Al.sub.2O.sub.3, Ru/C, Raney Nickel,
or other catalysts know in the art) in combination with H.sub.2
under high pressure (e.g., 10 to 12000 psi). The sorbitol and/or
xylitol products can be isolated and purified using methods known
in the art.
[0198] Sugar products from saccharification can also be fermented
to produce alcohols, sugar alcohols, such as erythritol, or organic
acids, e.g., lactic, glutamic or citric acids or amino acids.
[0199] Yeast and Zymomonas bacteria, for example, can be used for
fermentation or conversion of sugar(s) to alcohol(s). Other
microorganisms are discussed below. The optimum pH for
fermentations is about pH 4 to 7. For example, the optimum pH for
yeast is from about pH 4 to 5, while the optimum pH for Zymomonas
is from about pH 5 to 6. Typical fermentation times are about 24 to
168 hours (e.g., 24 to 96 hrs) with temperatures in the range of
20.degree. C. to 40.degree. C. (e.g., 26.degree. C. to 40.degree.
C.), however thermophilic microorganisms prefer higher
temperatures.
[0200] In some embodiments, e.g., when anaerobic organisms are
used, at least a portion of the fermentation is conducted in the
absence of oxygen, e.g., under a blanket of an inert gas such as
N.sub.2, Ar, He, CO.sub.2 or mixtures thereof. Additionally, the
mixture may have a constant purge of an inert gas flowing through
the tank during part of or all of the fermentation. In some cases,
anaerobic conditions can be achieved or maintained by carbon
dioxide production during the fermentation and no additional inert
gas is needed.
[0201] In some embodiments, all or a portion of the fermentation
process can be interrupted before the low molecular weight sugar is
completely converted to a product (e.g., ethanol). The intermediate
fermentation products include sugar and carbohydrates in high
concentrations. The sugars and carbohydrates can be isolated via
any means known in the art. These intermediate fermentation
products can be used in preparation of food for human or animal
consumption. Additionally or alternatively, the intermediate
fermentation products can be ground to a fine particle size in a
stainless-steel laboratory mill to produce a flour-like
substance.
[0202] Jet mixing may be used during fermentation, and in some
cases saccharification and fermentation are performed in the same
tank.
[0203] Nutrients for the microorganisms may be added during
saccharification and/or fermentation, for example the food-based
nutrient packages described in U.S. Pat. App. Pub. 2012/0052536,
filed Jul. 15, 2011, the complete disclosure of which is
incorporated herein by reference.
[0204] "Fermentation" includes the methods and products that are
disclosed in U.S. Prov. App. No. 61/579,559, filed Dec. 22, 2012,
and U.S. Prov. App. No. 61/579,576, filed Dec. 22, 2012, the
contents of both of which are incorporated by reference herein in
their entirety.
[0205] Mobile fermenters can be utilized, as described in
International App. No. PCT/US2007/074028 (which was filed Jul. 20,
2007, was published in English as WO 2008/011598 and designated the
United States), the contents of which is incorporated herein in its
entirety. Similarly, the saccharification equipment can be mobile.
Further, saccharification and/or fermentation may be performed in
part or entirely during transit.
[0206] The microorganism(s) used in fermentation can be
naturally-occurring microorganisms and/or engineered
microorganisms. For example, the microorganism can be a bacterium
(including, but not limited to, e.g., a cellulolytic bacterium), a
fungus, (including, but not limited to, e.g., a yeast), a plant, a
protist, e.g., a protozoa or a fungus-like protest (including, but
not limited to, e.g., a slime mold), or an algae. When the
organisms are compatible, mixtures of organisms can be
utilized.
[0207] Suitable fermenting microorganisms have the ability to
convert carbohydrates, such as glucose, fructose, xylose,
arabinose, mannose, galactose, oligosaccharides or polysaccharides
into fermentation products. Fermenting microorganisms include
strains of the genus Saccharomyces spp. (including, but not limited
to, S. cerevisiae (baker's yeast), S. distaticus, S. uvarum), the
genus Kluyveromyces, (including, but not limited to, K. marxianus,
K. fragilis), the genus Candida (including, but not limited to, C.
pseudotropicalis, and C. brassicae), Pichia stipitis (a relative of
Candida shehatae), the genus Clavispora (including, but not limited
to, C. lusitaniae and C. opuntiae), the genus Pachysolen
(including, but not limited to, P. tannophilus), the genus
Bretannomyces (including, but not limited to, e.g., B. clausenii
(Philippidis, G. P., 1996, Cellulose bioconversion technology, in
Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,
ed., Taylor & Francis, Washington, D.C., 179-212)). Other
suitable microorganisms include, for example, Zymomonas mobilis,
Clostridium spp. (including, but not limited to, C. thermocellum
(Philippidis, 1996, supra), C. saccharobutylacetonicum, C.
saccharobutylicum, C. Puniceum, C. beijernckii, and C.
acetobutylicum), Moniliella pollinis, Moniliella megachiliensis,
Lactobacillus spp. Yarrowia lipolytica, Aureobasidium sp.,
Trichosporonoides sp., Trigonopsis variabilis, Trichosporon sp.,
Moniliellaacetoabutans sp., Typhula variabilis, Candida magnoliae,
Ustilaginomycetes sp., Pseudozyma tsukubaensis, yeast species of
genera Zygosaccharomyces, Debaryomyces, Hansenula and Pichia, and
fungi of the dematioid genus Torula.
[0208] For instance, Clostridium spp. can be used to produce
ethanol, butanol, butyric acid, acetic acid, and acetone.
Lactobacillus spp. can be used to produce lactic acid.
[0209] Many such microbial strains are publicly available, either
commercially or through depositories such as the ATCC (American
Type Culture Collection, Manassas, Va., USA), the NRRL
(Agricultural Research Sevice Culture Collection, Peoria, Ill.,
USA), or the DSMZ (Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH, Braunschweig, Germany), to name a few.
[0210] Commercially available yeasts include, for example, Red
Star.RTM./Lesaffre Ethanol Red (available from Red Star/Lesaffre,
USA), FALI.RTM. (available from Fleischmann's Yeast, a division of
Burns Philip Food Inc., USA), SUPERSTART.RTM. (available from
Alltech, now Lalemand), GERT STRAND.RTM. (available from Gert
Strand AB, Sweden) and FERMOL.RTM. (available from DSM
Specialties).
[0211] Many microorganisms that can be used to saccharify biomass
material and produce sugars can also be used to ferment and convert
those sugars to useful products.
[0212] After fermentation, the resulting fluids can be distilled
using, for example, a "beer column" to separate ethanol and other
alcohols from the majority of water and residual solids. The vapor
exiting the beer column can be, e.g., 35% by weight ethanol and can
be fed to a rectification column. A mixture of nearly azeotropic
(92.5%) ethanol and water from the rectification column can be
purified to pure (99.5%) ethanol using vapor-phase molecular
sieves. The beer column bottoms can be sent to the first effect of
a three-effect evaporator. The rectification column reflux
condenser can provide heat for this first effect. After the first
effect, solids can be separated using a centrifuge and dried in a
rotary dryer. A portion (25%) of the centrifuge effluent can be
recycled to fermentation and the rest sent to the second and third
evaporator effects. Most of the evaporator condensate can be
returned to the process as fairly clean condensate with a small
portion split off to waste water treatment to prevent build-up of
low-boiling compounds.
[0213] Other types of chemical transformation of the products from
the processes described herein can be used, for example, production
of organic sugar derived products such (e.g., furfural and
furfural-derived products). Chemical transformations of sugar
derived products are described in U.S. Prov. App. No. 61/667,481,
filed Jul. 3, 2012, the disclosure of which is incorporated herein
by reference in its entirety.
EXAMPLES
[0214] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0215] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples specifically point out various aspects
of the present invention, and are not to be construed as limiting
in any way the remainder of the disclosure.
Example 1: Expression of Cel3a-C'his in E. coli
[0216] The mature sequence for Cel3a (amino acids 20-744) was
synthesized and codon-optimized for E. coli expression by Genewiz.
The Cel3a-C'His referred to in the following examples refers to the
codon-optimized mature sequence for Cel3a (aas 20-744) with an
8.times. His (SEQ ID NO: 21) tag at the C-terminus. The below
primers were used to clone the Cel3a-C'His into pET-Duet (Novagen,
Catalog No. 71146):
TABLE-US-00006 Forward (SEQ ID NO: 4)
5'-CATGCCATGGGCGATAGTCACAGTACCAGC Reverse (SEQ ID NO: 5)
3'-CCCAAGCTTTCATTAGTGATGATGATGATGATGATGATGGCTGCCGC
TGCCGGCAACACTCAGGGTGC
(NcoI and HindIII sites are underlined; start and stop codons are
in bold; the polyhistidine (8-His (SEQ ID NO: 21) tag; and
glycine-serine (GSGS (SEQ ID NO: 23)) linker are italicized.) The
Amplification reaction was performed using PfuUltra II Fusion HS
Polymerase (Agilent, Catalog No. 600672).
[0217] The amplified DNA was cloned by restriction digestion using
NcoI restriction enzyme (New England Biolabs, R3193) and HindIII
restriction enzyme (New England Biolabs, R3104) under conditions
suggested by the manufacturer. The digested amplified DNA was
ligated into the NcoI-HindIII sites in the pETDuet vector using T4
DNA ligase (New England Biolabs, M0202), followed by transformation
of E. coli cloning host Top 10 One Shot (Invitrogen). Plasmid
purification was carried out using Qiagen's plasmid purification
kit.
[0218] The Cel3A-C'His constructs were transformed into the E. coli
expression host Origami B (DE3) (EMD Millipore, Catalog No. 70837)
and streaked on plates containing LB medium and 100 .mu.g/ml
ampicillin (Fisher Scientific, Catalog No. BP1760), 15 .mu.g/ml
kanamysin (Fisher Scientific, Catalog No. BP906) and 12.5 .mu.g/ml
tetracycline (Fisher Scientified, Catalog No. BP912). Colonies
carrying the recombinant DNA were picked from plates for the
inoculation of 2 ml starter cultures, and grown overnight at
37.degree. C., then subsequently used to inoculate 100 ml of LB
media containing the appropriate antibiotics. Cultures were grown
at 37.degree. C. until OD600 reached 0.8.
[0219] To induce protein expression, 500 .mu.M IPTG
(Isopropyl-b-D-thiogalactopyranoside; Fisher Scientific, Catalog
No. BP1755) was added. The expression culture was further grown for
another 4 hours at 37.degree. C. The cells were harvested by
centrifugation at 4200 at room temperature (RT) for 30 minutes
using the Sorvall St16 rotor TX400.
Example 2: Solubilization of Cel3a from the Insoluble Fraction
[0220] An E. coli culture expressing an enzyme having
biomass-degrading activity, Cel3a, was cultured and enzyme
expression was induced, as described in Example 1. Isolation of
Cel3a tagged with a His tag at the C-terminus (Cel3a-C'His) from
the soluble and insoluble fraction was performed as follows. The
cell culture was centrifuged at 4200 rpm for 30 minutes. The
supernatant was discarded and the cell pellet was re-suspended in
lysis buffer with 1 mg/mL lysozyme. Lysonase, e.g., 10 .mu.l, of
Lysonase Bioprocessing Reagent (EMD Millipore 71320) per gram of
cell paste was added and the sample was incubated for 1 hour at
ambient temperature. After 1 hour, the sample was sonicated for a
total of 2 minutes in 30 second intervals. Following sonication,
the sample was centrifuged for 30 minutes at 10000 rpm. The
supernatant, or soluble fraction, contains solubilized Cel3a, while
the remaining pellet, or insoluble fraction, contains inclusion
bodies and insoluble Cel3a.
[0221] The insoluble fraction was re-suspended in buffer containing
a solubilizing agent for 15 minutes and vortexed at room
temperature, specifically, 6M Urea pH 8 IMAC binding buffer. The
sample was then filtered through a 0.45 .mu.m membrane to prepare
for IMAC purification. The amount of 6M Urea pH 8 IMAC binding
buffer added was proportional to the amount of cell mass, e.g., 2
or 3 volumes of buffer to 1 volume of cell mass. The amount of
binding buffer is increased as the cell mass increases in order to
make filtering of the sample possible.
Example 3: Purification of Cel3a
[0222] Purification of Soluble Cel3a
[0223] The soluble fraction from Example 2 was transferred to a
fresh tube containing 100 l of pre-equilibrated Bio-Scale.TM.
Profinity (Biorad) Ni-charged IMAC resin slurry (BioRad, Catalog
No. 732-4614). The native binding buffer contained 50 mM Tris HCl
pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 5 .mu.M imidazole. The
protein was batch-bound for 1 hour at room temperature (RT), and
then washed with native buffer containing 25 .mu.M imidazole. The
protein was eluted in 300 .mu.l of native buffer containing 200
.mu.M imidazole.
[0224] Purification of Insoluble Cel3a
[0225] A Bio-Scale.TM. Mini Profinity IMAC 5 mL cartridge (BioRad,
Catalog No. 732-4614) was equilibrated with 5 column volumes of 6M
Urea pH8 IMAC binding buffer at a flow rate of 5 mL/min. After
column equilibration the resuspended insoluble fraction from
Example 2 was loaded at a flow rate of 1 mL/min. The column then
received a 15 column volume wash of the 6M Urea pH8 IMAC binding
buffer at a flow rate of 5 mL/min. The solubilized Cel3a was then
eluted from the column with 10 column volumes of 6M Urea pH 4 IMAC
elution buffer at a flow rate of 5 mL/min. The resulting
solubilized Cel3a sample contains 6M urea.
[0226] IMAC chromatography analysis was performed (using IMAC
columns, Bio-Scale.TM. Mini Profinity.TM. IMAC Cartridges 5 mL,
Catalog #732-4614), and as shown in FIG. 1, purified solubilized
Cel3a was detected.
[0227] SDS-PAGE analysis was performed to assess the amount of
Cel3a from the purification described above in the following
fractions: purified soluble Cel3a (lane 2 in FIG. 2); flow through
from the IMAC purification of the insoluble fraction (lane 3 in
FIG. 2); and the purified Cel3a from the insoluble fraction
(solubilized Cel3a) (lane 4 in FIG. 2). As shown in FIG. 2, Cel3a
was successfully isolated from the inclusion bodies of the
insoluble fraction using the methods described above.
Example 4: Analysis of Cellobiase Activity of Solubilized Cel3a
[0228] Cel3a was purified using IMAC techniques, as described in
Example 3. Prior to performing the activity assay, the amount
(titer) of purified Cel3a was determined using Bradford assay
and/or the nanodrop. For nanodrop quantification, the molar
extinction coefficient was estimated by inserting the amino acid
sequence of the target form of Cel3a into the ExPASy ProtParam
online tool.
[0229] For the activity assay, two fold serial dilutions of samples
containing purified Cel3a were prepared using 50 mM sodium citrate,
pH 5.0 NaOH as buffer. Dilutions were aliquoted across one row of a
96 well plate. Dilutions were incubated with a D-(+)-Cellobiose
(Fluka) substrate solution in 50 mM sodium citrate monobasic buffer
at pH 5.0, at 48.degree. C. for 30 minutes. The plates were
immediately sealed using an adhesive plate seal and placed on a
microplate incubator shaker set at 48.degree. C., 700 rpm. After 30
minutes, the samples were heated on a heating dry bath for 5
minutes at 100.degree. C. to stop the reaction. The plate was then
filtered through a 96 well format 0.45 m Durapore membrane. The
filtrate samples were analysed for glucose and cellobiose using the
YSI Biochemistry analyser (YSI Life Sciences) and/or HPLC (UPLC)
methods. The cellobiase activity of the dilutions of purified Cel3a
from the soluble and insoluble fractions was plotted on a graph and
the results are shown in FIG. 3. FIG. 3 shows that the solubilized
Cel3a has cellobiase activity, even in the presence of urea.
[0230] Cellobiase activity was also assessed for solubilized Cel3a
that has not been purified by the IMAC purification methods
described in Example 3. The cell pellet of cells expressing Cel3a
was washed with lysis buffer before solubilising with the
solubilizing buffer containing 6M urea. Cellobiase activity of the
crude lysate sample containing solubilized Cel3a in 6M urea buffer
is assayed by the cellobiase assay described above. The percentage
of cellobiose converted to glucose in 30 minutes was compared
between soluble Cel3a, the soluble wash, and the solubilized Cel3a
from crude lysate (FIG. 4). The solubilized Cel3a without
purification also possessed cellobiase activity.
EQUIVALENTS
[0231] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific aspects, it is apparent
that other aspects and variations of this invention may be devised
by others skilled in the art without departing from the true spirit
and scope of the invention. The appended claims are intended to be
construed to include all such aspects and equivalent variations.
Sequence CWU 1
1
231731PRTTrichoderma reesei 1Met Gly Asp Ser His Ser Thr Ser Gly
Ala Ser Ala Glu Ala Val Val1 5 10 15Pro Pro Ala Gly Thr Pro Trp Gly
Thr Ala Tyr Asp Lys Ala Lys Ala 20 25 30Ala Leu Ala Lys Leu Asn Leu
Gln Asp Lys Val Gly Ile Val Ser Gly 35 40 45Val Gly Trp Asn Gly Gly
Pro Cys Val Gly Asn Thr Ser Pro Ala Ser 50 55 60Lys Ile Ser Tyr Pro
Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly Val65 70 75 80Arg Tyr Ser
Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln Ala Ala 85 90 95Ser Thr
Trp Asp Val Asn Leu Ile Arg Glu Arg Gly Gln Phe Ile Gly 100 105
110Glu Glu Val Lys Ala Ser Gly Ile His Val Ile Leu Gly Pro Val Ala
115 120 125Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg Asn Trp Glu
Gly Phe 130 135 140Gly Val Asp Pro Tyr Leu Thr Gly Ile Ala Met Gly
Gln Thr Ile Asn145 150 155 160Gly Ile Gln Ser Val Gly Val Gln Ala
Thr Ala Lys His Tyr Ile Leu 165 170 175Asn Glu Gln Glu Leu Asn Arg
Glu Thr Ile Ser Ser Asn Pro Asp Asp 180 185 190Arg Thr Leu His Glu
Leu Tyr Thr Trp Pro Phe Ala Asp Ala Val Gln 195 200 205Ala Asn Val
Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Thr Thr 210 215 220Trp
Ala Cys Glu Asp Gln Tyr Thr Leu Gln Thr Val Leu Lys Asp Gln225 230
235 240Leu Gly Phe Pro Gly Tyr Val Met Thr Asp Trp Asn Ala Gln His
Thr 245 250 255Thr Val Gln Ser Ala Asn Ser Gly Leu Asp Met Ser Met
Pro Gly Thr 260 265 270Asp Phe Asn Gly Asn Asn Arg Leu Trp Gly Pro
Ala Leu Thr Asn Ala 275 280 285Val Asn Ser Asn Gln Val Pro Thr Ser
Arg Val Asp Asp Met Val Thr 290 295 300Arg Ile Leu Ala Ala Trp Tyr
Leu Thr Gly Gln Asp Gln Ala Gly Tyr305 310 315 320Pro Ser Phe Asn
Ile Ser Arg Asn Val Gln Gly Asn His Lys Thr Asn 325 330 335Val Arg
Ala Ile Ala Arg Asp Gly Ile Val Leu Leu Lys Asn Asp Ala 340 345
350Asn Ile Leu Pro Leu Lys Lys Pro Ala Ser Ile Ala Val Val Gly Ser
355 360 365Ala Ala Ile Ile Gly Asn His Ala Arg Asn Ser Pro Ser Cys
Asn Asp 370 375 380Lys Gly Cys Asp Asp Gly Ala Leu Gly Met Gly Trp
Gly Ser Gly Ala385 390 395 400Val Asn Tyr Pro Tyr Phe Val Ala Pro
Tyr Asp Ala Ile Asn Thr Arg 405 410 415Ala Ser Ser Gln Gly Thr Gln
Val Thr Leu Ser Asn Thr Asp Asn Thr 420 425 430Ser Ser Gly Ala Ser
Ala Ala Arg Gly Lys Asp Val Ala Ile Val Phe 435 440 445Ile Thr Ala
Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly Asn Ala 450 455 460Gly
Asp Arg Asn Asn Leu Asp Pro Trp His Asn Gly Asn Ala Leu Val465 470
475 480Gln Ala Val Ala Gly Ala Asn Ser Asn Val Ile Val Val Val His
Ser 485 490 495Val Gly Ala Ile Ile Leu Glu Gln Ile Leu Ala Leu Pro
Gln Val Lys 500 505 510Ala Val Val Trp Ala Gly Leu Pro Ser Gln Glu
Ser Gly Asn Ala Leu 515 520 525Val Asp Val Leu Trp Gly Asp Val Ser
Pro Ser Gly Lys Leu Val Tyr 530 535 540Thr Ile Ala Lys Ser Pro Asn
Asp Tyr Asn Thr Arg Ile Val Ser Gly545 550 555 560Gly Ser Asp Ser
Phe Ser Glu Gly Leu Phe Ile Asp Tyr Lys His Phe 565 570 575Asp Asp
Ala Asn Ile Thr Pro Arg Tyr Glu Phe Gly Tyr Gly Leu Ser 580 585
590Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Ser Val Leu Ser Thr Ala Lys
595 600 605Ser Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly Pro Ser
Asp Leu 610 615 620Phe Gln Asn Val Ala Thr Val Thr Val Asp Ile Ala
Asn Ser Gly Gln625 630 635 640Val Thr Gly Ala Glu Val Ala Gln Leu
Tyr Ile Thr Tyr Pro Ser Ser 645 650 655Ala Pro Arg Thr Pro Pro Lys
Gln Leu Arg Gly Phe Ala Lys Leu Asn 660 665 670Leu Thr Pro Gly Gln
Ser Gly Thr Ala Thr Phe Asn Ile Arg Arg Arg 675 680 685Asp Leu Ser
Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val Val Pro Ser 690 695 700Gly
Ser Phe Gly Ile Ser Val Gly Ala Ser Ser Arg Asp Ile Arg Leu705 710
715 720Thr Ser Thr Leu Ser Val Ala Gly Ser Gly Ser 725
73022232DNATrichoderma reesei 2atgcgttacc gaacagcagc tgcgctggca
cttgccactg ggccctttgc tagggcagac 60agtcactcaa catcgggggc ctcggctgag
gcagttgtac ctcctgcagg gactccatgg 120ggaaccgcgt acgacaaggc
gaaggccgca ttggcaaagc tcaatctcca agataaggtc 180ggcatcgtga
gcggtgtcgg ctggaacggc ggtccttgcg ttggaaacac atctccggcc
240tccaagatca gctatccatc gctatgcctt caagacggac ccctcggtgt
tcgatactcg 300acaggcagca cagcctttac gccgggcgtt caagcggcct
cgacgtggga tgtcaatttg 360atccgcgaac gtggacagtt catcggtgag
gaggtgaagg cctcggggat tcatgtcata 420cttggtcctg tggctgggcc
gctgggaaag actccgcagg gcggtcgcaa ctgggagggc 480ttcggtgtcg
atccatatct cacgggcatt gccatgggtc aaaccatcaa cggcatccag
540tcggtaggcg tgcaggcgac agcgaagcac tatatcctca acgagcagga
gctcaatcga 600gaaaccattt cgagcaaccc agatgaccga actctccatg
agctgtatac ttggccattt 660gccgacgcgg ttcaggccaa tgtcgcttct
gtcatgtgct cgtacaacaa ggtcaatacc 720acctgggcct gcgaggatca
gtacacgctg cagactgtgc tgaaagacca gctggggttc 780ccaggctatg
tcatgacgga ctggaacgca cagcacacga ctgtccaaag cgcgaattct
840gggcttgaca tgtcaatgcc tggcacagac ttcaacggta acaatcggct
ctggggtcca 900gctctcacca atgcggtaaa tagcaatcag gtccccacga
gcagagtcga cgatatggtg 960actcgtatcc tcgccgcatg gtacttgaca
ggccaggacc aggcaggcta tccgtcgttc 1020aacatcagca gaaatgttca
aggaaaccac aagaccaatg tcagggcaat tgccagggac 1080ggcatcgttc
tgctcaagaa tgacgccaac atcctgccgc tcaagaagcc cgctagcatt
1140gccgtcgttg gatctgccgc aatcattggt aaccacgcca gaaactcgcc
ctcgtgcaac 1200gacaaaggct gcgacgacgg ggccttgggc atgggttggg
gttccggcgc cgtcaactat 1260ccgtacttcg tcgcgcccta cgatgccatc
aataccagag cgtcttcgca gggcacccag 1320gttaccttga gcaacaccga
caacacgtcc tcaggcgcat ctgcagcaag aggaaaggac 1380gtcgccatcg
tcttcatcac cgccgactcg ggtgaaggct acatcaccgt ggagggcaac
1440gcgggcgatc gcaacaacct ggatccgtgg cacaacggca atgccctggt
ccaggcggtg 1500gccggtgcca acagcaacgt cattgttgtt gtccactccg
ttggcgccat cattctggag 1560cagattcttg ctcttccgca ggtcaaggcc
gttgtctggg cgggtcttcc ttctcaggag 1620agcggcaatg cgctcgtcga
cgtgctgtgg ggagatgtca gcccttctgg caagctggtg 1680tacaccattg
cgaagagccc caatgactat aacactcgca tcgtttccgg cggcagtgac
1740agcttcagcg agggactgtt catcgactat aagcacttcg acgacgccaa
tatcacgccg 1800cggtacgagt tcggctatgg actgtcttac accaagttca
actactcacg cctctccgtc 1860ttgtcgaccg ccaagtctgg tcctgcgact
ggggccgttg tgccgggagg cccgagtgat 1920ctgttccaga atgtcgcgac
agtcaccgtt gacatcgcaa actctggcca agtgactggt 1980gccgaggtag
cccagctgta catcacctac ccatcttcag cacccaggac ccctccgaag
2040cagctgcgag gctttgccaa gctgaacctc acgcctggtc agagcggaac
agcaacgttc 2100aacatccgac gacgagatct cagctactgg gacacggctt
cgcagaaatg ggtggtgccg 2160tcggggtcgt ttggcatcag cgtgggagcg
agcagccggg atatcaggct gacgagcact 2220ctgtcggtag cg
223232232DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 3atgcgttatc gtacagccgc agccctggca
ctggccacag gtccgttcgc acgtgccgat 60agtcacagta ccagcggtgc cagcgcagaa
gccgtggttc cgccggcagg cacaccgtgg 120ggcacagcct atgataaagc
caaagccgcc ctggccaagc tgaatctgca ggataaagtg 180ggcatcgtga
gtggcgtggg ctggaacggt ggtccgtgcg ttggcaacac cagcccggca
240agcaagatca gctatccgag cttatgcctg caggatggtc cgctgggcgt
gcgctatagc 300accggtagta ccgcctttac acctggtgtg caggccgcca
gtacctggga cgttaacctg 360atccgcgaac gtggccaatt tatcggcgaa
gaagttaaag ccagcggcat tcatgttatt 420ctgggtccgg tggccggtcc
tctgggtaaa accccgcagg gcggccgtaa ttgggaaggc 480ttcggcgttg
atccgtattt aaccggcatc gcaatgggcc agaccattaa tggcatccag
540agcgtgggtg ttcaagccac cgccaaacac tacatattaa acgaacagga
actgaatcgt 600gaaaccatca gcagcaatcc ggatgatcgc accctgcatg
agctgtatac atggcctttt 660gccgacgcag ttcaggccaa cgtggcaagt
gtgatgtgta gctataacaa ggtgaacacc 720acctgggcct gcgaagacca
gtacaccctg cagaccgttt taaaagacca actgggcttc 780cctggttacg
tgatgacaga ttggaatgcc cagcacacaa ccgttcagag cgcaaacagt
840ggcctggata tgagcatgcc gggcaccgac ttcaacggca ataatcgtct
gtggggtccg 900gcactgacca atgccgttaa cagcaaccag gtgccgacca
gtcgtgtgga cgatatggtt 960acccgtattc tggccgcctg gtacctgaca
ggtcaagacc aggccggcta cccgagcttc 1020aacatcagcc gcaacgtgca
gggtaatcac aagaccaacg ttcgcgcaat cgcacgcgat 1080ggtatcgtgc
tgttaaagaa cgatgccaac attctgccgc tgaaaaaacc ggccagcatc
1140gccgttgttg gtagcgcagc catcattggc aaccacgccc gtaacagtcc
gagctgcaat 1200gataaaggct gtgacgacgg tgccctgggc atgggttggg
gtagtggtgc cgtgaactac 1260ccgtatttcg tggccccgta cgacgccatt
aacacccgtg caagtagcca gggtacccag 1320gttaccctga gcaacaccga
caacacaagc agcggtgcca gtgcagcacg tggtaaggat 1380gtggccatcg
tgttcatcac cgccgacagc ggcgaaggct acattaccgt ggagggtaat
1440gccggtgatc gcaataatct ggacccgtgg cataacggca acgccctggt
tcaggcagtg 1500gcaggcgcaa atagcaacgt gatcgttgtg gtgcatagcg
tgggtgccat cattctggag 1560cagatcctgg ccctgccgca agttaaggca
gttgtgtggg caggtctgcc gagccaagaa 1620agtggcaatg ccctggtgga
cgttctgtgg ggcgatgtta gtccgagcgg caagctggtg 1680tatacaatcg
ccaagagccc gaacgactat aacacccgca tcgttagcgg cggcagtgat
1740agcttcagcg agggcctgtt tatcgactac aagcatttcg atgatgccaa
tattaccccg 1800cgctacgaat ttggttatgg cctgagctat accaagttca
actacagccg cctgagcgtt 1860ttaagtaccg ccaagagtgg tccggcaaca
ggtgccgtgg ttcctggtgg tccgagtgat 1920ctgtttcaga atgtggccac
cgtgaccgtg gatatcgcca acagtggtca ggttaccggc 1980gccgaagtgg
cacagctgta catcacctat ccgagcagtg caccgcgcac cccgccgaaa
2040cagctgcgtg gcttcgccaa attaaacctg accccgggcc agagcggtac
agcaaccttc 2100aatattcgcc gccgtgatct gagctattgg gacaccgcca
gccaaaaatg ggtggtgccg 2160agcggcagct ttggcattag tgtgggtgca
agtagccgcg acattcgctt aacaagcacc 2220ctgagtgttg cc
2232430DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4catgccatgg gcgatagtca cagtaccagc
30568DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5cgtgggactc acaacggccg tcgccgtcgg tagtagtagt
agtagtagta gtgattactt 60tcgaaccc 686800PRTPodospora anserina 6Met
Lys Ser Ser Val Phe Trp Gly Ala Ser Leu Thr Ser Ala Val Val1 5 10
15Arg Ala Ile Asp Leu Pro Phe Gln Phe Tyr Pro Asn Cys Val Asp Asp
20 25 30Leu Leu Ser Thr Asn Gln Val Cys Asn Thr Thr Leu Ser Pro Pro
Glu 35 40 45Arg Ala Ala Ala Leu Val Ala Ala Leu Thr Pro Glu Glu Lys
Leu Gln 50 55 60Asn Ile Val Ser Lys Ser Leu Gly Ala Pro Arg Ile Gly
Leu Pro Ala65 70 75 80Tyr Asn Trp Trp Ser Glu Ala Leu His Gly Val
Ala Tyr Ala Pro Gly 85 90 95Thr Gln Phe Trp Gln Gly Asp Gly Pro Phe
Asn Ser Ser Thr Ser Phe 100 105 110Pro Met Pro Leu Leu Met Ala Ala
Thr Phe Asp Asp Glu Leu Leu Glu 115 120 125Lys Ile Ala Glu Val Ile
Gly Ile Glu Gly Arg Ala Phe Gly Asn Ala 130 135 140Gly Phe Ser Gly
Leu Asp Tyr Trp Thr Pro Asn Val Asn Pro Phe Lys145 150 155 160Asp
Pro Arg Trp Gly Arg Gly Ser Glu Thr Pro Gly Glu Asp Val Leu 165 170
175Leu Val Lys Arg Tyr Ala Ala Ala Met Ile Lys Gly Leu Glu Gly Pro
180 185 190Val Pro Glu Lys Glu Arg Arg Val Val Ala Thr Cys Lys His
Tyr Ala 195 200 205Ala Asn Asp Phe Glu Asp Trp Asn Gly Ala Thr Arg
His Asn Phe Asn 210 215 220Ala Lys Ile Ser Leu Gln Asp Met Ala Glu
Tyr Tyr Phe Met Pro Phe225 230 235 240Gln Gln Cys Val Arg Asp Ser
Arg Val Gly Ser Ile Met Cys Ala Tyr 245 250 255Asn Ala Val Asn Gly
Val Pro Ser Cys Ala Ser Pro Tyr Leu Leu Gln 260 265 270Thr Ile Leu
Arg Glu His Trp Asn Trp Thr Glu His Asn Asn Tyr Ile 275 280 285Thr
Ser Asp Cys Glu Ala Val Leu Asp Val Ser Leu Asn His Lys Tyr 290 295
300Ala Ala Thr Asn Ala Glu Gly Thr Ala Ile Ser Phe Glu Ala Gly
Met305 310 315 320Asp Thr Ser Cys Glu Tyr Glu Gly Ser Ser Asp Ile
Pro Gly Ala Trp 325 330 335Ser Gln Gly Leu Leu Lys Glu Ser Thr Val
Asp Arg Ala Leu Leu Arg 340 345 350Leu Tyr Glu Gly Ile Val Arg Ala
Gly Tyr Phe Asp Gly Lys Gln Ser 355 360 365Leu Tyr Ser Ser Leu Gly
Trp Ala Asp Val Asn Lys Pro Ser Ala Gln 370 375 380Lys Leu Ser Leu
Gln Ala Ala Val Asp Gly Thr Val Leu Leu Lys Asn385 390 395 400Asp
Gly Thr Leu Pro Leu Ser Asp Leu Leu Asp Lys Ser Arg Pro Lys 405 410
415Lys Val Ala Met Ile Gly Phe Trp Ser Asp Ala Lys Asp Lys Leu Arg
420 425 430Gly Gly Tyr Ser Gly Thr Ala Ala Tyr Leu His Thr Pro Ala
Tyr Ala 435 440 445Ala Ser Gln Leu Gly Ile Pro Phe Ser Thr Ala Ser
Gly Pro Ile Leu 450 455 460His Ser Asp Leu Ala Ser Asn Gln Ser Trp
Thr Asp Asn Ala Met Ala465 470 475 480Ala Ala Lys Asp Ala Asp Tyr
Ile Leu Tyr Phe Gly Gly Ile Asp Thr 485 490 495Ser Ala Ala Gly Glu
Thr Lys Asp Arg Tyr Asp Leu Asp Trp Pro Gly 500 505 510Ala Gln Leu
Ser Leu Ile Asn Leu Leu Thr Thr Leu Ser Lys Pro Leu 515 520 525Ile
Val Leu Gln Met Gly Asp Gln Leu Asp Asn Thr Pro Leu Leu Ser 530 535
540Asn Pro Lys Ile Asn Ala Ile Leu Trp Ala Asn Trp Pro Gly Gln
Asp545 550 555 560Gly Gly Thr Ala Val Met Glu Leu Val Thr Gly Leu
Lys Ser Pro Ala 565 570 575Gly Arg Leu Pro Val Thr Gln Tyr Pro Ser
Asn Phe Thr Glu Leu Val 580 585 590Pro Met Thr Asp Met Ala Leu Arg
Pro Ser Ala Gly Asn Ser Gln Leu 595 600 605Gly Arg Thr Tyr Arg Trp
Tyr Lys Thr Pro Val Gln Ala Phe Gly Phe 610 615 620Gly Leu His Tyr
Thr Thr Phe Ser Pro Lys Phe Gly Lys Lys Phe Pro625 630 635 640Ala
Val Ile Asp Val Asp Glu Val Leu Glu Gly Cys Asp Asp Lys Tyr 645 650
655Leu Asp Thr Cys Pro Leu Pro Asp Leu Pro Val Val Val Glu Asn Arg
660 665 670Gly Asn Arg Thr Ser Asp Tyr Val Ala Leu Ala Phe Val Ser
Ala Pro 675 680 685Gly Val Gly Pro Gly Pro Trp Pro Ile Lys Thr Leu
Gly Ala Phe Thr 690 695 700Arg Leu Arg Gly Val Lys Gly Gly Glu Lys
Arg Glu Gly Gly Leu Lys705 710 715 720Trp Asn Leu Gly Asn Leu Ala
Arg His Asp Glu Glu Gly Asn Thr Val 725 730 735Val Tyr Pro Gly Lys
Tyr Glu Val Ser Leu Asp Glu Pro Pro Lys Ala 740 745 750Arg Leu Arg
Phe Glu Ile Val Arg Gly Gly Lys Gly Lys Gly Lys Val 755 760 765Lys
Gly Lys Gly Lys Ala Ala Gln Lys Gly Gly Val Val Leu Asp Arg 770 775
780Trp Pro Lys Pro Pro Lys Gly Gln Glu Pro Pro Ala Ile Glu Arg
Val785 790 795 8007316PRTTrichoderma reesei 7Met Val Arg Arg Thr
Ala Leu Leu Ala Leu Gly Ala Leu Ser Thr Leu1 5 10 15Ser Met Ala Gln
Ile Ser Asp Asp Phe Glu Ser Gly Trp Asp Gln Thr 20 25 30Lys Trp Pro
Ile Ser Ala Pro Asp Cys Asn Gln Gly Gly Thr Val Ser 35 40 45Leu Asp
Thr Thr Val Ala His Ser Gly Ser Asn Ser Met Lys Val Val 50 55 60Gly
Gly Pro Asn Gly Tyr Cys Gly His Ile Phe Phe Gly Thr Thr Gln65 70 75
80Val Pro Thr Gly Asp Val Tyr Val Arg Ala Trp Ile Arg Leu Gln Thr
85 90 95Ala Leu Gly Ser Asn His Val Thr Phe Ile Ile Met Pro Asp Thr
Ala 100 105
110Gln Gly Gly Lys His Leu Arg Ile Gly Gly Gln Ser Gln Val Leu Asp
115 120 125Tyr Asn Arg Glu Ser Asp Asp Ala Thr Leu Pro Asp Leu Ser
Pro Asn 130 135 140Gly Ile Ala Ser Thr Val Thr Leu Pro Thr Gly Ala
Phe Gln Cys Phe145 150 155 160Glu Tyr His Leu Gly Thr Asp Gly Thr
Ile Glu Thr Trp Leu Asn Gly 165 170 175Ser Leu Ile Pro Gly Met Thr
Val Gly Pro Gly Val Asp Asn Pro Asn 180 185 190Asp Ala Gly Trp Thr
Arg Ala Ser Tyr Ile Pro Glu Ile Thr Gly Val 195 200 205Asn Phe Gly
Trp Glu Ala Tyr Ser Gly Asp Val Asn Thr Val Trp Phe 210 215 220Asp
Asp Ile Ser Ile Ala Ser Thr Arg Val Gly Cys Gly Pro Gly Ser225 230
235 240Pro Gly Gly Pro Gly Ser Ser Thr Thr Gly Arg Ser Ser Thr Ser
Gly 245 250 255Pro Thr Ser Thr Ser Arg Pro Ser Thr Thr Ile Pro Pro
Pro Thr Ser 260 265 270Arg Thr Thr Thr Ala Thr Gly Pro Thr Gln Thr
His Tyr Gly Gln Cys 275 280 285Gly Gly Ile Gly Tyr Ser Gly Pro Thr
Val Cys Ala Ser Gly Thr Thr 290 295 300Cys Gln Val Leu Asn Pro Tyr
Tyr Ser Gln Cys Leu305 310 3158460PRTTrichoderma reesei 8Met Ala
Ser Arg Phe Phe Ala Leu Leu Leu Leu Ala Ile Pro Ile Gln1 5 10 15Ala
Gln Ser Pro Val Trp Gly Gln Cys Gly Gly Ile Gly Trp Ser Gly 20 25
30Pro Thr Thr Cys Val Gly Gly Ala Thr Cys Val Ser Tyr Asn Pro Tyr
35 40 45Tyr Ser Gln Cys Ile Pro Ser Thr Gln Ala Ser Ser Ser Ile Ala
Ser 50 55 60Thr Thr Leu Val Thr Ser Phe Thr Thr Thr Thr Ala Thr Arg
Thr Ser65 70 75 80Ala Ser Thr Pro Pro Ala Ser Ser Thr Gly Ala Gly
Gly Ala Thr Cys 85 90 95Ser Ala Leu Pro Gly Ser Ile Thr Leu Arg Ser
Asn Ala Lys Leu Asn 100 105 110Asp Leu Phe Thr Met Phe Asn Gly Asp
Lys Val Thr Thr Lys Asp Lys 115 120 125Phe Ser Cys Arg Gln Ala Glu
Met Ser Glu Leu Ile Gln Arg Tyr Glu 130 135 140Leu Gly Thr Leu Pro
Gly Arg Pro Ser Thr Leu Thr Ala Ser Phe Ser145 150 155 160Gly Asn
Thr Leu Thr Ile Asn Cys Gly Glu Ala Gly Lys Ser Ile Ser 165 170
175Phe Thr Val Thr Ile Thr Tyr Pro Ser Ser Gly Thr Ala Pro Tyr Pro
180 185 190Ala Ile Ile Gly Tyr Gly Gly Gly Ser Leu Pro Ala Pro Ala
Gly Val 195 200 205Ala Met Ile Asn Phe Asn Asn Asp Asn Ile Ala Ala
Gln Val Asn Thr 210 215 220Gly Ser Arg Gly Gln Gly Lys Phe Tyr Asp
Leu Tyr Gly Ser Ser His225 230 235 240Ser Ala Gly Ala Met Thr Ala
Trp Ala Trp Gly Val Ser Arg Val Ile 245 250 255Asp Ala Leu Glu Leu
Val Pro Gly Ala Arg Ile Asp Thr Thr Lys Ile 260 265 270Gly Val Thr
Gly Cys Ser Arg Asn Gly Lys Gly Ala Met Val Ala Gly 275 280 285Ala
Phe Glu Lys Arg Ile Val Leu Thr Leu Pro Gln Glu Ser Gly Ala 290 295
300Gly Gly Ser Ala Cys Trp Arg Ile Ser Asp Tyr Leu Lys Ser Gln
Gly305 310 315 320Ala Asn Ile Gln Thr Ala Ser Glu Ile Ile Gly Glu
Asp Pro Trp Phe 325 330 335Ser Thr Thr Phe Asn Ser Tyr Val Asn Gln
Val Pro Val Leu Pro Phe 340 345 350Asp His His Ser Leu Ala Ala Leu
Ile Ala Pro Arg Gly Leu Phe Val 355 360 365Ile Asp Asn Asn Ile Asp
Trp Leu Gly Pro Gln Ser Cys Phe Gly Cys 370 375 380Met Thr Ala Ala
His Met Ala Trp Gln Ala Leu Gly Val Ser Asp His385 390 395 400Met
Gly Tyr Ser Gln Ile Gly Ala His Ala His Cys Ala Phe Pro Ser 405 410
415Asn Gln Gln Ser Gln Leu Thr Ala Phe Val Gln Lys Phe Leu Leu Gly
420 425 430Gln Ser Thr Asn Thr Ala Ile Phe Gln Ser Asp Phe Ser Ala
Asn Gln 435 440 445Ser Gln Trp Ile Asp Trp Thr Thr Pro Thr Leu Ser
450 455 4609466PRTTrichoderma reesei 9Met Leu Pro Lys Asp Phe Gln
Trp Gly Phe Ala Thr Ala Ala Tyr Gln1 5 10 15Ile Glu Gly Ala Val Asp
Gln Asp Gly Arg Gly Pro Ser Ile Trp Asp 20 25 30Thr Phe Cys Ala Gln
Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly Val 35 40 45Thr Ala Cys Asp
Ser Tyr Asn Arg Thr Ala Glu Asp Ile Ala Leu Leu 50 55 60Lys Ser Leu
Gly Ala Lys Ser Tyr Arg Phe Ser Ile Ser Trp Ser Arg65 70 75 80Ile
Ile Pro Glu Gly Gly Arg Gly Asp Ala Val Asn Gln Ala Gly Ile 85 90
95Asp His Tyr Val Lys Phe Val Asp Asp Leu Leu Asp Ala Gly Ile Thr
100 105 110Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Glu Gly Leu
His Gln 115 120 125Arg Tyr Gly Gly Leu Leu Asn Arg Thr Glu Phe Pro
Leu Asp Phe Glu 130 135 140Asn Tyr Ala Arg Val Met Phe Arg Ala Leu
Pro Lys Val Arg Asn Trp145 150 155 160Ile Thr Phe Asn Glu Pro Leu
Cys Ser Ala Ile Pro Gly Tyr Gly Ser 165 170 175Gly Thr Phe Ala Pro
Gly Arg Gln Ser Thr Ser Glu Pro Trp Thr Val 180 185 190Gly His Asn
Ile Leu Val Ala His Gly Arg Ala Val Lys Ala Tyr Arg 195 200 205Asp
Asp Phe Lys Pro Ala Ser Gly Asp Gly Gln Ile Gly Ile Val Leu 210 215
220Asn Gly Asp Phe Thr Tyr Pro Trp Asp Ala Ala Asp Pro Ala Asp
Lys225 230 235 240Glu Ala Ala Glu Arg Arg Leu Glu Phe Phe Thr Ala
Trp Phe Ala Asp 245 250 255Pro Ile Tyr Leu Gly Asp Tyr Pro Ala Ser
Met Arg Lys Gln Leu Gly 260 265 270Asp Arg Leu Pro Thr Phe Thr Pro
Glu Glu Arg Ala Leu Val His Gly 275 280 285Ser Asn Asp Phe Tyr Gly
Met Asn His Tyr Thr Ser Asn Tyr Ile Arg 290 295 300His Arg Ser Ser
Pro Ala Ser Ala Asp Asp Thr Val Gly Asn Val Asp305 310 315 320Val
Leu Phe Thr Asn Lys Gln Gly Asn Cys Ile Gly Pro Glu Thr Gln 325 330
335Ser Pro Trp Leu Arg Pro Cys Ala Ala Gly Phe Arg Asp Phe Leu Val
340 345 350Trp Ile Ser Lys Arg Tyr Gly Tyr Pro Pro Ile Tyr Val Thr
Glu Asn 355 360 365Gly Thr Ser Ile Lys Gly Glu Ser Asp Leu Pro Lys
Glu Lys Ile Leu 370 375 380Glu Asp Asp Phe Arg Val Lys Tyr Tyr Asn
Glu Tyr Ile Arg Ala Met385 390 395 400Val Thr Ala Val Glu Leu Asp
Gly Val Asn Val Lys Gly Tyr Phe Ala 405 410 415Trp Ser Leu Met Asp
Asn Phe Glu Trp Ala Asp Gly Tyr Val Thr Arg 420 425 430Phe Gly Val
Thr Tyr Val Asp Tyr Glu Asn Gly Gln Lys Arg Phe Pro 435 440 445Lys
Lys Ser Ala Lys Ser Leu Lys Pro Leu Phe Asp Glu Leu Ile Ala 450 455
460Ala Ala46510744PRTTrichoderma reesei 10Met Arg Tyr Arg Thr Ala
Ala Ala Leu Ala Leu Ala Thr Gly Pro Phe1 5 10 15Ala Arg Ala Asp Ser
His Ser Thr Ser Gly Ala Ser Ala Glu Ala Val 20 25 30Val Pro Pro Ala
Gly Thr Pro Trp Gly Thr Ala Tyr Asp Lys Ala Lys 35 40 45Ala Ala Leu
Ala Lys Leu Asn Leu Gln Asp Lys Val Gly Ile Val Ser 50 55 60Gly Val
Gly Trp Asn Gly Gly Pro Cys Val Gly Asn Thr Ser Pro Ala65 70 75
80Ser Lys Ile Ser Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly
85 90 95Val Arg Tyr Ser Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln
Ala 100 105 110Ala Ser Thr Trp Asp Val Asn Leu Ile Arg Glu Arg Gly
Gln Phe Ile 115 120 125Gly Glu Glu Val Lys Ala Ser Gly Ile His Val
Ile Leu Gly Pro Val 130 135 140Ala Gly Pro Leu Gly Lys Thr Pro Gln
Gly Gly Arg Asn Trp Glu Gly145 150 155 160Phe Gly Val Asp Pro Tyr
Leu Thr Gly Ile Ala Met Gly Gln Thr Ile 165 170 175Asn Gly Ile Gln
Ser Val Gly Val Gln Ala Thr Ala Lys His Tyr Ile 180 185 190Leu Asn
Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asn Pro Asp 195 200
205Asp Arg Thr Leu His Glu Leu Tyr Thr Trp Pro Phe Ala Asp Ala Val
210 215 220Gln Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val
Asn Thr225 230 235 240Thr Trp Ala Cys Glu Asp Gln Tyr Thr Leu Gln
Thr Val Leu Lys Asp 245 250 255Gln Leu Gly Phe Pro Gly Tyr Val Met
Thr Asp Trp Asn Ala Gln His 260 265 270Thr Thr Val Gln Ser Ala Asn
Ser Gly Leu Asp Met Ser Met Pro Gly 275 280 285Thr Asp Phe Asn Gly
Asn Asn Arg Leu Trp Gly Pro Ala Leu Thr Asn 290 295 300Ala Val Asn
Ser Asn Gln Val Pro Thr Ser Arg Val Asp Asp Met Val305 310 315
320Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gly Gln Asp Gln Ala Gly
325 330 335Tyr Pro Ser Phe Asn Ile Ser Arg Asn Val Gln Gly Asn His
Lys Thr 340 345 350Asn Val Arg Ala Ile Ala Arg Asp Gly Ile Val Leu
Leu Lys Asn Asp 355 360 365Ala Asn Ile Leu Pro Leu Lys Lys Pro Ala
Ser Ile Ala Val Val Gly 370 375 380Ser Ala Ala Ile Ile Gly Asn His
Ala Arg Asn Ser Pro Ser Cys Asn385 390 395 400Asp Lys Gly Cys Asp
Asp Gly Ala Leu Gly Met Gly Trp Gly Ser Gly 405 410 415Ala Val Asn
Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Ile Asn Thr 420 425 430Arg
Ala Ser Ser Gln Gly Thr Gln Val Thr Leu Ser Asn Thr Asp Asn 435 440
445Thr Ser Ser Gly Ala Ser Ala Ala Arg Gly Lys Asp Val Ala Ile Val
450 455 460Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu
Gly Asn465 470 475 480Ala Gly Asp Arg Asn Asn Leu Asp Pro Trp His
Asn Gly Asn Ala Leu 485 490 495Val Gln Ala Val Ala Gly Ala Asn Ser
Asn Val Ile Val Val Val His 500 505 510Ser Val Gly Ala Ile Ile Leu
Glu Gln Ile Leu Ala Leu Pro Gln Val 515 520 525Lys Ala Val Val Trp
Ala Gly Leu Pro Ser Gln Glu Ser Gly Asn Ala 530 535 540Leu Val Asp
Val Leu Trp Gly Asp Val Ser Pro Ser Gly Lys Leu Val545 550 555
560Tyr Thr Ile Ala Lys Ser Pro Asn Asp Tyr Asn Thr Arg Ile Val Ser
565 570 575Gly Gly Ser Asp Ser Phe Ser Glu Gly Leu Phe Ile Asp Tyr
Lys His 580 585 590Phe Asp Asp Ala Asn Ile Thr Pro Arg Tyr Glu Phe
Gly Tyr Gly Leu 595 600 605Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu
Ser Val Leu Ser Thr Ala 610 615 620Lys Ser Gly Pro Ala Thr Gly Ala
Val Val Pro Gly Gly Pro Ser Asp625 630 635 640Leu Phe Gln Asn Val
Ala Thr Val Thr Val Asp Ile Ala Asn Ser Gly 645 650 655Gln Val Thr
Gly Ala Glu Val Ala Gln Leu Tyr Ile Thr Tyr Pro Ser 660 665 670Ser
Ala Pro Arg Thr Pro Pro Lys Gln Leu Arg Gly Phe Ala Lys Leu 675 680
685Asn Leu Thr Pro Gly Gln Ser Gly Thr Ala Thr Phe Asn Ile Arg Arg
690 695 700Arg Asp Leu Ser Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val
Val Pro705 710 715 720Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Ser
Ser Arg Asp Ile Arg 725 730 735Leu Thr Ser Thr Leu Ser Val Ala
74011418PRTTrichoderma reesei 11Met Asn Lys Ser Val Ala Pro Leu Leu
Leu Ala Ala Ser Ile Leu Tyr1 5 10 15Gly Gly Ala Ala Ala Gln Gln Thr
Val Trp Gly Gln Cys Gly Gly Ile 20 25 30Gly Trp Ser Gly Pro Thr Asn
Cys Ala Pro Gly Ser Ala Cys Ser Thr 35 40 45Leu Asn Pro Tyr Tyr Ala
Gln Cys Ile Pro Gly Ala Thr Thr Ile Thr 50 55 60Thr Ser Thr Arg Pro
Pro Ser Gly Pro Thr Thr Thr Thr Arg Ala Thr65 70 75 80Ser Thr Ser
Ser Ser Thr Pro Pro Thr Ser Ser Gly Val Arg Phe Ala 85 90 95Gly Val
Asn Ile Ala Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr 100 105
110Cys Val Thr Ser Lys Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly Ser
115 120 125Asn Asn Tyr Pro Asp Gly Ile Gly Gln Met Gln His Phe Val
Asn Asp 130 135 140Asp Gly Met Thr Ile Phe Arg Leu Pro Val Gly Trp
Gln Tyr Leu Val145 150 155 160Asn Asn Asn Leu Gly Gly Asn Leu Asp
Ser Thr Ser Ile Ser Lys Tyr 165 170 175Asp Gln Leu Val Gln Gly Cys
Leu Ser Leu Gly Ala Tyr Cys Ile Val 180 185 190Asp Ile His Asn Tyr
Ala Arg Trp Asn Gly Gly Ile Ile Gly Gln Gly 195 200 205Gly Pro Thr
Asn Ala Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala Ser 210 215 220Lys
Tyr Ala Ser Gln Ser Arg Val Trp Phe Gly Ile Met Asn Glu Pro225 230
235 240His Asp Val Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Glu Val
Val 245 250 255Thr Ala Ile Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile
Ser Leu Pro 260 265 270Gly Asn Asp Trp Gln Ser Ala Gly Ala Phe Ile
Ser Asp Gly Ser Ala 275 280 285Ala Ala Leu Ser Gln Val Thr Asn Pro
Asp Gly Ser Thr Thr Asn Leu 290 295 300Ile Phe Asp Val His Lys Tyr
Leu Asp Ser Asp Asn Ser Gly Thr His305 310 315 320Ala Glu Cys Thr
Thr Asn Asn Ile Asp Gly Ala Phe Ser Pro Leu Ala 325 330 335Thr Trp
Leu Arg Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly 340 345
350Gly Gly Asn Val Gln Ser Cys Ile Gln Asp Met Cys Gln Gln Ile Gln
355 360 365Tyr Leu Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly
Trp Gly 370 375 380Ala Gly Ser Phe Asp Ser Thr Tyr Val Leu Thr Glu
Thr Pro Thr Gly385 390 395 400Ser Gly Asn Ser Trp Thr Asp Thr Ser
Leu Val Ser Ser Cys Leu Ala 405 410 415Arg Lys12471PRTTrichoderma
reesei 12Met Ile Val Gly Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr
Leu Ala1 5 10 15Ala Ser Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser
Val Trp Gly 20 25 30Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys
Cys Ala Ser Gly 35 40 45Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser
Gln Cys Leu Pro Gly 50 55 60Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala
Ala Ser Thr Thr Ser Arg65 70 75 80Val Ser Pro Thr Thr Ser Arg Ser
Ser Ser Ala Thr Pro Pro Pro Gly 85 90 95Ser Thr Thr Thr Arg Val Pro
Pro Val Gly Ser Gly Thr Ala Thr Tyr 100 105 110Ser Gly Asn Pro Phe
Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr 115 120 125Ala Ser Glu
Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met 130 135 140Ala
Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu145 150
155 160Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp
Ile
165 170 175Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe
Val Val 180 185 190Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala
Ser Asn Gly Glu 195 200 205Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys
Tyr Lys Asn Tyr Ile Asp 210 215 220Thr Ile Arg Gln Ile Val Val Glu
Tyr Ser Asp Ile Arg Thr Leu Leu225 230 235 240Val Ile Glu Pro Asp
Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr 245 250 255Pro Lys Cys
Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 260 265 270Ala
Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 275 280
285Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala
290 295 300Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg
Ala Leu305 310 315 320Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn
Gly Trp Asn Ile Thr 325 330 335Ser Pro Pro Ser Tyr Thr Gln Gly Asn
Ala Val Tyr Asn Glu Lys Leu 340 345 350Tyr Ile His Ala Ile Gly Pro
Leu Leu Ala Asn His Gly Trp Ser Asn 355 360 365Ala Phe Phe Ile Thr
Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 370 375 380Gln Gln Gln
Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly385 390 395
400Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val
405 410 415Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser
Ser Ala 420 425 430Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala
Leu Gln Pro Ala 435 440 445Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr
Phe Val Gln Leu Leu Thr 450 455 460Asn Ala Asn Pro Ser Phe Leu465
47013514PRTTrichoderma reesei 13Met Tyr Arg Lys Leu Ala Val Ile Ser
Ala Phe Leu Ala Thr Ala Arg1 5 10 15Ala Gln Ser Ala Cys Thr Leu Gln
Ser Glu Thr His Pro Pro Leu Thr 20 25 30Trp Gln Lys Cys Ser Ser Gly
Gly Thr Cys Thr Gln Gln Thr Gly Ser 35 40 45Val Val Ile Asp Ala Asn
Trp Arg Trp Thr His Ala Thr Asn Ser Ser 50 55 60Thr Asn Cys Tyr Asp
Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp65 70 75 80Asn Glu Thr
Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala 85 90 95Ser Thr
Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe 100 105
110Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met
115 120 125Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn
Glu Phe 130 135 140Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly
Leu Asn Gly Ala145 150 155 160Leu Tyr Phe Val Ser Met Asp Ala Asp
Gly Gly Val Ser Lys Tyr Pro 165 170 175Thr Asn Thr Ala Gly Ala Lys
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 180 185 190Cys Pro Arg Asp Leu
Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly 195 200 205Trp Glu Pro
Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly 210 215 220Ser
Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu225 230
235 240Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys
Glu 245 250 255Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr
Gly Gly Thr 260 265 270Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr
Arg Leu Gly Asn Thr 275 280 285Ser Phe Tyr Gly Pro Gly Ser Ser Phe
Thr Leu Asp Thr Thr Lys Lys 290 295 300Leu Thr Val Val Thr Gln Phe
Glu Thr Ser Gly Ala Ile Asn Arg Tyr305 310 315 320Tyr Val Gln Asn
Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 325 330 335Ser Tyr
Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu 340 345
350Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln
355 360 365Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser
Leu Trp 370 375 380Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser
Thr Tyr Pro Thr385 390 395 400Asn Glu Thr Ser Ser Thr Pro Gly Ala
Val Arg Gly Ser Cys Ser Thr 405 410 415Ser Ser Gly Val Pro Ala Gln
Val Glu Ser Gln Ser Pro Asn Ala Lys 420 425 430Val Thr Phe Ser Asn
Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 435 440 445Pro Ser Gly
Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr 450 455 460Thr
Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln465 470
475 480Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr
Val 485 490 495Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr
Tyr Ser Gln 500 505 510Cys Leu14459PRTTrichoderma reesei 14Met Ala
Pro Ser Val Thr Leu Pro Leu Thr Thr Ala Ile Leu Ala Ile1 5 10 15Ala
Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu Val 20 25
30His Pro Lys Leu Thr Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val
35 40 45Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Met
His 50 55 60Asp Ala Asn Tyr Asn Ser Cys Thr Val Asn Gly Gly Val Asn
Thr Thr65 70 75 80Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys Asn Cys
Phe Ile Glu Gly 85 90 95Val Asp Tyr Ala Ala Ser Gly Val Thr Thr Ser
Gly Ser Ser Leu Thr 100 105 110Met Asn Gln Tyr Met Pro Ser Ser Ser
Gly Gly Tyr Ser Ser Val Ser 115 120 125Pro Arg Leu Tyr Leu Leu Asp
Ser Asp Gly Glu Tyr Val Met Leu Lys 130 135 140Leu Asn Gly Gln Glu
Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro145 150 155 160Cys Gly
Glu Asn Gly Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly 165 170
175Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr
180 185 190Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr
Leu Asn 195 200 205Thr Ser His Gln Gly Phe Cys Cys Asn Glu Met Asp
Ile Leu Glu Gly 210 215 220Asn Ser Arg Ala Asn Ala Leu Thr Pro His
Ser Cys Thr Ala Thr Ala225 230 235 240Cys Asp Ser Ala Gly Cys Gly
Phe Asn Pro Tyr Gly Ser Gly Tyr Lys 245 250 255Ser Tyr Tyr Gly Pro
Gly Asp Thr Val Asp Thr Ser Lys Thr Phe Thr 260 265 270Ile Ile Thr
Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu 275 280 285Val
Ser Ile Thr Arg Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser 290 295
300Ala Gln Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser
Ala305 310 315 320Tyr Gly Gly Leu Ala Thr Met Gly Lys Ala Leu Ser
Ser Gly Met Val 325 330 335Leu Val Phe Ser Ile Trp Asn Asp Asn Ser
Gln Tyr Met Asn Trp Leu 340 345 350Asp Ser Gly Asn Ala Gly Pro Cys
Ser Ser Thr Glu Gly Asn Pro Ser 355 360 365Asn Ile Leu Ala Asn Asn
Pro Asn Thr His Val Val Phe Ser Asn Ile 370 375 380Arg Trp Gly Asp
Ile Gly Ser Thr Thr Asn Ser Thr Ala Pro Pro Pro385 390 395 400Pro
Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr 405 410
415Thr Ser Ser Ser Pro Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly
420 425 430Gly Ile Gly Tyr Ser Gly Cys Lys Thr Cys Thr Ser Gly Thr
Thr Cys 435 440 445Gln Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu 450
45515234PRTTrichoderma reesei 15Met Lys Phe Leu Gln Val Leu Pro Ala
Leu Ile Pro Ala Ala Leu Ala1 5 10 15Gln Thr Ser Cys Asp Gln Trp Ala
Thr Phe Thr Gly Asn Gly Tyr Thr 20 25 30Val Ser Asn Asn Leu Trp Gly
Ala Ser Ala Gly Ser Gly Phe Gly Cys 35 40 45Val Thr Ala Val Ser Leu
Ser Gly Gly Ala Ser Trp His Ala Asp Trp 50 55 60Gln Trp Ser Gly Gly
Gln Asn Asn Val Lys Ser Tyr Gln Asn Ser Gln65 70 75 80Ile Ala Ile
Pro Gln Lys Arg Thr Val Asn Ser Ile Ser Ser Met Pro 85 90 95Thr Thr
Ala Ser Trp Ser Tyr Ser Gly Ser Asn Ile Arg Ala Asn Val 100 105
110Ala Tyr Asp Leu Phe Thr Ala Ala Asn Pro Asn His Val Thr Tyr Ser
115 120 125Gly Asp Tyr Glu Leu Met Ile Trp Leu Gly Lys Tyr Gly Asp
Ile Gly 130 135 140Pro Ile Gly Ser Ser Gln Gly Thr Val Asn Val Gly
Gly Gln Ser Trp145 150 155 160Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala
Met Gln Val Tyr Ser Phe Val 165 170 175Ala Gln Thr Asn Thr Thr Asn
Tyr Ser Gly Asp Val Lys Asn Phe Phe 180 185 190Asn Tyr Leu Arg Asp
Asn Lys Gly Tyr Asn Ala Ala Gly Gln Tyr Val 195 200 205Leu Ser Tyr
Gln Phe Gly Thr Glu Pro Phe Thr Gly Ser Gly Thr Leu 210 215 220Asn
Val Ala Ser Trp Thr Ala Ser Ile Asn225 23016242PRTTrichoderma
reesei 16Met Lys Ala Thr Leu Val Leu Gly Ser Leu Ile Val Gly Ala
Val Ser1 5 10 15Ala Tyr Lys Ala Thr Thr Thr Arg Tyr Tyr Asp Gly Gln
Glu Gly Ala 20 25 30Cys Gly Cys Gly Ser Ser Ser Gly Ala Phe Pro Trp
Gln Leu Gly Ile 35 40 45Gly Asn Gly Val Tyr Thr Ala Ala Gly Ser Gln
Ala Leu Phe Asp Thr 50 55 60Ala Gly Ala Ser Trp Cys Gly Ala Gly Cys
Gly Lys Cys Tyr Gln Leu65 70 75 80Thr Ser Thr Gly Gln Ala Pro Cys
Ser Ser Cys Gly Thr Gly Gly Ala 85 90 95Ala Gly Gln Ser Ile Ile Val
Met Val Thr Asn Leu Cys Pro Asn Asn 100 105 110Gly Asn Ala Gln Trp
Cys Pro Val Val Gly Gly Thr Asn Gln Tyr Gly 115 120 125Tyr Ser Tyr
His Phe Asp Ile Met Ala Gln Asn Glu Ile Phe Gly Asp 130 135 140Asn
Val Val Val Asp Phe Glu Pro Ile Ala Cys Pro Gly Gln Ala Ala145 150
155 160Ser Asp Trp Gly Thr Cys Leu Cys Val Gly Gln Gln Glu Thr Asp
Pro 165 170 175Thr Pro Val Leu Gly Asn Asp Thr Gly Ser Thr Pro Pro
Gly Ser Ser 180 185 190Pro Pro Ala Thr Ser Ser Ser Pro Pro Ser Gly
Gly Gly Gln Gln Thr 195 200 205Leu Tyr Gly Gln Cys Gly Gly Ala Gly
Trp Thr Gly Pro Thr Thr Cys 210 215 220Gln Ala Pro Gly Thr Cys Lys
Val Gln Asn Gln Trp Tyr Ser Gln Cys225 230 235 240Leu
Pro17838PRTTrichoderma reesei 17Met Lys Val Ser Arg Val Leu Ala Leu
Val Leu Gly Ala Val Ile Pro1 5 10 15Ala His Ala Ala Phe Ser Trp Lys
Asn Val Lys Leu Gly Gly Gly Gly 20 25 30Gly Phe Val Pro Gly Ile Ile
Phe His Pro Lys Thr Lys Gly Val Ala 35 40 45Tyr Ala Arg Thr Asp Ile
Gly Gly Leu Tyr Arg Leu Asn Ala Asp Asp 50 55 60Ser Trp Thr Ala Val
Thr Asp Gly Ile Ala Asp Asn Ala Gly Trp His65 70 75 80Asn Trp Gly
Ile Asp Ala Val Ala Leu Asp Pro Gln Asp Asp Gln Lys 85 90 95Val Tyr
Ala Ala Val Gly Met Tyr Thr Asn Ser Trp Asp Pro Ser Asn 100 105
110Gly Ala Ile Ile Arg Ser Ser Asp Arg Gly Ala Thr Trp Ser Phe Thr
115 120 125Asn Leu Pro Phe Lys Val Gly Gly Asn Met Pro Gly Arg Gly
Ala Gly 130 135 140Glu Arg Leu Ala Val Asp Pro Ala Asn Ser Asn Ile
Ile Tyr Phe Gly145 150 155 160Ala Arg Ser Gly Asn Gly Leu Trp Lys
Ser Thr Asp Gly Gly Val Thr 165 170 175Phe Ser Lys Val Ser Ser Phe
Thr Ala Thr Gly Thr Tyr Ile Pro Asp 180 185 190Pro Ser Asp Ser Asn
Gly Tyr Asn Ser Asp Lys Gln Gly Leu Met Trp 195 200 205Val Thr Phe
Asp Ser Thr Ser Ser Thr Thr Gly Gly Ala Thr Ser Arg 210 215 220Ile
Phe Val Gly Thr Ala Asp Asn Ile Thr Ala Ser Val Tyr Val Ser225 230
235 240Thr Asn Ala Gly Ser Thr Trp Ser Ala Val Pro Gly Gln Pro Gly
Lys 245 250 255Tyr Phe Pro His Lys Ala Lys Leu Gln Pro Ala Glu Lys
Ala Leu Tyr 260 265 270Leu Thr Tyr Ser Asp Gly Thr Gly Pro Tyr Asp
Gly Thr Leu Gly Ser 275 280 285Val Trp Arg Tyr Asp Ile Ala Gly Gly
Thr Trp Lys Asp Ile Thr Pro 290 295 300Val Ser Gly Ser Asp Leu Tyr
Phe Gly Phe Gly Gly Leu Gly Leu Asp305 310 315 320Leu Gln Lys Pro
Gly Thr Leu Val Val Ala Ser Leu Asn Ser Trp Trp 325 330 335Pro Asp
Ala Gln Leu Phe Arg Ser Thr Asp Ser Gly Thr Thr Trp Ser 340 345
350Pro Ile Trp Ala Trp Ala Ser Tyr Pro Thr Glu Thr Tyr Tyr Tyr Ser
355 360 365Ile Ser Thr Pro Lys Ala Pro Trp Ile Lys Asn Asn Phe Ile
Asp Val 370 375 380Thr Ser Glu Ser Pro Ser Asp Gly Leu Ile Lys Arg
Leu Gly Trp Met385 390 395 400Ile Glu Ser Leu Glu Ile Asp Pro Thr
Asp Ser Asn His Trp Leu Tyr 405 410 415Gly Thr Gly Met Thr Ile Phe
Gly Gly His Asp Leu Thr Asn Trp Asp 420 425 430Thr Arg His Asn Val
Ser Ile Gln Ser Leu Ala Asp Gly Ile Glu Glu 435 440 445Phe Ser Val
Gln Asp Leu Ala Ser Ala Pro Gly Gly Ser Glu Leu Leu 450 455 460Ala
Ala Val Gly Asp Asp Asn Gly Phe Thr Phe Ala Ser Arg Asn Asp465 470
475 480Leu Gly Thr Ser Pro Gln Thr Val Trp Ala Thr Pro Thr Trp Ala
Thr 485 490 495Ser Thr Ser Val Asp Tyr Ala Gly Asn Ser Val Lys Ser
Val Val Arg 500 505 510Val Gly Asn Thr Ala Gly Thr Gln Gln Val Ala
Ile Ser Ser Asp Gly 515 520 525Gly Ala Thr Trp Ser Ile Asp Tyr Ala
Ala Asp Thr Ser Met Asn Gly 530 535 540Gly Thr Val Ala Tyr Ser Ala
Asp Gly Asp Thr Ile Leu Trp Ser Thr545 550 555 560Ala Ser Ser Gly
Val Gln Arg Ser Gln Phe Gln Gly Ser Phe Ala Ser 565 570 575Val Ser
Ser Leu Pro Ala Gly Ala Val Ile Ala Ser Asp Lys Lys Thr 580 585
590Asn Ser Val Phe Tyr Ala Gly Ser Gly Ser Thr Phe Tyr Val Ser Lys
595 600 605Asp Thr Gly Ser Ser Phe Thr Arg Gly Pro Lys Leu Gly Ser
Ala Gly 610 615 620Thr Ile Arg Asp Ile Ala Ala His Pro Thr Thr Ala
Gly Thr Leu Tyr625 630 635 640Val Ser Thr Asp Val Gly Ile Phe Arg
Ser Thr Asp Ser Gly Thr Thr 645 650 655Phe Gly Gln Val Ser Thr Ala
Leu Thr Asn Thr Tyr Gln Ile Ala Leu 660 665 670Gly Val Gly Ser Gly
Ser Asn Trp Asn Leu Tyr Ala Phe Gly Thr Gly 675 680 685Pro Ser Gly
Ala Arg Leu Tyr Ala Ser Gly Asp Ser Gly Ala Ser Trp 690 695 700Thr
Asp Ile Gln Gly
Ser Gln Gly Phe Gly Ser Ile Asp Ser Thr Lys705 710 715 720Val Ala
Gly Ser Gly Ser Thr Ala Gly Gln Val Tyr Val Gly Thr Asn 725 730
735Gly Arg Gly Val Phe Tyr Ala Gln Gly Thr Val Gly Gly Gly Thr Gly
740 745 750Gly Thr Ser Ser Ser Thr Lys Gln Ser Ser Ser Ser Thr Ser
Ser Ala 755 760 765Ser Ser Ser Thr Thr Leu Arg Ser Ser Val Val Ser
Thr Thr Arg Ala 770 775 780Ser Thr Val Thr Ser Ser Arg Thr Ser Ser
Ala Ala Gly Pro Thr Gly785 790 795 800Ser Gly Val Ala Gly His Tyr
Ala Gln Cys Gly Gly Ile Gly Trp Thr 805 810 815Gly Pro Thr Gln Cys
Val Ala Pro Tyr Val Cys Gln Lys Gln Asn Asp 820 825 830Tyr Tyr Tyr
Gln Cys Val 83518373PRTPodospora anserina 18Met Lys Gly Leu Phe Ala
Phe Gly Leu Gly Leu Leu Ser Leu Val Asn1 5 10 15Ala Leu Pro Gln Ala
Gln Gly Gly Gly Ala Ala Ala Ser Ala Lys Val 20 25 30Ser Gly Thr Arg
Phe Val Ile Asp Gly Lys Thr Gly Tyr Phe Ala Gly 35 40 45Thr Asn Ser
Tyr Trp Ile Gly Phe Leu Thr Asn Asn Arg Asp Val Asp 50 55 60Thr Thr
Leu Asp His Ile Ala Ser Ser Gly Leu Lys Ile Leu Arg Val65 70 75
80Trp Gly Phe Asn Asp Val Asn Asn Gln Pro Ser Gly Asn Thr Val Trp
85 90 95Phe Gln Arg Leu Ala Ser Ser Gly Ser Gln Ile Asn Thr Gly Pro
Asn 100 105 110Gly Leu Gln Arg Leu Asp Tyr Leu Val Arg Ser Ala Glu
Thr Arg Gly 115 120 125Ile Lys Leu Ile Ile Ala Leu Val Asn Tyr Trp
Asp Asp Phe Gly Gly 130 135 140Met Lys Ala Tyr Val Asn Ala Phe Gly
Gly Thr Lys Glu Ser Trp Tyr145 150 155 160Thr Asn Ala Arg Ala Gln
Glu Gln Tyr Lys Arg Tyr Ile Gln Ala Val 165 170 175Val Ser Arg Tyr
Val Asn Ser Pro Ala Ile Phe Ala Trp Glu Leu Ala 180 185 190Asn Glu
Pro Arg Cys Lys Gly Cys Asn Thr Asn Val Ile Phe Asn Trp 195 200
205Ala Thr Gln Ile Ser Asp Tyr Ile Arg Ser Leu Asp Lys Asp His Leu
210 215 220Ile Thr Leu Gly Asp Glu Gly Phe Gly Leu Pro Gly Gln Thr
Thr Tyr225 230 235 240Pro Tyr Gln Tyr Gly Glu Gly Thr Asp Phe Val
Lys Asn Leu Gln Ile 245 250 255Lys Asn Leu Asp Phe Gly Thr Phe His
Met Tyr Pro Gly His Trp Gly 260 265 270Val Pro Thr Ser Phe Gly Pro
Gly Trp Ile Lys Asp His Ala Ala Ala 275 280 285Cys Arg Ala Ala Gly
Lys Pro Cys Leu Leu Glu Glu Tyr Gly Tyr Glu 290 295 300Ser Asp Arg
Cys Asn Val Gln Lys Gly Trp Gln Gln Ala Ser Arg Glu305 310 315
320Leu Ser Arg Asp Gly Met Ser Gly Asp Leu Phe Trp Gln Trp Gly Asp
325 330 335Gln Leu Ser Thr Gly Gln Thr His Asn Asp Gly Phe Thr Ile
Tyr Tyr 340 345 350Gly Ser Ser Leu Ala Thr Cys Leu Val Thr Asp His
Val Arg Ala Ile 355 360 365Asn Ala Leu Pro Ala 37019469PRTPodospora
anserina 19Met Val Lys Leu Leu Asp Ile Gly Leu Phe Ala Leu Ala Leu
Ala Ser1 5 10 15Ser Ala Val Ala Lys Pro Cys Lys Pro Arg Asp Gly Pro
Val Thr Tyr 20 25 30Glu Ala Glu Asp Ala Ile Leu Thr Gly Thr Thr Val
Asp Thr Ala Gln 35 40 45Val Gly Tyr Thr Gly Arg Gly Tyr Val Thr Gly
Phe Asp Glu Gly Ser 50 55 60Asp Lys Ile Thr Phe Gln Ile Ser Ser Ala
Thr Thr Lys Leu Tyr Asp65 70 75 80Leu Ser Ile Arg Tyr Ala Ala Ile
Tyr Gly Asp Lys Arg Thr Asn Val 85 90 95Val Leu Asn Asn Gly Ala Val
Ser Glu Val Phe Phe Pro Ala Gly Asp 100 105 110Ser Phe Thr Ser Val
Ala Ala Gly Gln Val Leu Leu Asn Ala Gly Gln 115 120 125Asn Thr Ile
Asp Ile Val Asn Asn Trp Gly Trp Tyr Leu Ile Asp Ser 130 135 140Ile
Thr Leu Thr Pro Ser Ala Pro Arg Pro Pro His Asp Ile Asn Pro145 150
155 160Asn Leu Asn Asn Pro Asn Ala Asp Thr Asn Ala Lys Lys Leu Tyr
Ser 165 170 175Tyr Leu Arg Ser Val Tyr Gly Asn Lys Ile Ile Ser Gly
Gln Gln Glu 180 185 190Leu His His Ala Glu Trp Ile Arg Gln Gln Thr
Gly Lys Thr Pro Ala 195 200 205Leu Val Ala Val Asp Leu Met Asp Tyr
Ser Pro Ser Arg Val Glu Arg 210 215 220Gly Thr Thr Ser His Ala Val
Glu Asp Ala Ile Ala His His Asn Ala225 230 235 240Gly Gly Ile Val
Ser Val Leu Trp His Trp Asn Ala Pro Val Gly Leu 245 250 255Tyr Asp
Thr Glu Glu Asn Lys Trp Trp Ser Gly Phe Tyr Thr Arg Ala 260 265
270Thr Asp Phe Asp Ile Ala Ala Thr Leu Ala Asn Pro Gln Gly Ala Asn
275 280 285Tyr Thr Leu Leu Ile Arg Asp Ile Asp Ala Ile Ala Val Gln
Leu Lys 290 295 300Arg Leu Glu Ala Ala Gly Val Pro Val Leu Trp Arg
Pro Leu His Glu305 310 315 320Ala Glu Gly Gly Trp Phe Trp Trp Gly
Ala Lys Gly Pro Glu Pro Ala 325 330 335Lys Gln Leu Trp Asp Ile Leu
Tyr Glu Arg Leu Thr Val His His Gly 340 345 350Leu Asp Asn Leu Ile
Trp Val Trp Asn Ser Ile Leu Glu Asp Trp Tyr 355 360 365Pro Gly Asp
Asp Thr Val Asp Ile Leu Ser Ala Asp Val Tyr Ala Gln 370 375 380Gly
Asn Gly Pro Met Ser Thr Gln Tyr Asn Glu Leu Ile Ala Leu Gly385 390
395 400Arg Asp Lys Lys Met Ile Ala Ala Ala Glu Val Gly Ala Ala Pro
Leu 405 410 415Pro Gly Leu Leu Gln Ala Tyr Gln Ala Asn Trp Leu Trp
Phe Ala Val 420 425 430Trp Gly Asp Asp Phe Ile Asn Asn Pro Ser Trp
Asn Thr Val Ala Val 435 440 445Leu Asn Glu Ile Tyr Asn Ser Asp Tyr
Val Leu Thr Leu Asp Glu Ile 450 455 460Gln Gly Trp Arg
Ser46520493PRTTrichoderma reesei 20Met Ala Gly Lys Leu Ile Leu Val
Ala Leu Ala Ser Leu Val Ser Leu1 5 10 15Ser Ile Gln Gln Asn Cys Ala
Ala Leu Phe Gly Gln Cys Gly Gly Ile 20 25 30Gly Trp Ser Gly Thr Thr
Cys Cys Val Ala Gly Ala Gln Cys Ser Phe 35 40 45Val Asn Asp Trp Tyr
Ser Gln Cys Leu Ala Ser Thr Gly Gly Asn Pro 50 55 60Pro Asn Gly Thr
Thr Ser Ser Ser Leu Val Ser Arg Thr Ser Ser Ala65 70 75 80Ser Ser
Ser Val Gly Ser Ser Ser Pro Gly Gly Asn Ser Pro Thr Gly 85 90 95Ser
Ala Ser Thr Tyr Thr Thr Thr Asp Thr Ala Thr Val Ala Pro His 100 105
110Ser Gln Ser Pro Tyr Pro Ser Ile Ala Ala Ser Ser Cys Gly Ser Trp
115 120 125Thr Leu Val Asp Asn Val Cys Cys Pro Ser Tyr Cys Ala Asn
Asp Asp 130 135 140Thr Ser Glu Ser Cys Ser Gly Cys Gly Thr Cys Thr
Thr Pro Pro Ser145 150 155 160Ala Asp Cys Lys Ser Gly Thr Met Tyr
Pro Glu Val His His Val Ser 165 170 175Ser Asn Glu Ser Trp His Tyr
Ser Arg Ser Thr His Phe Gly Leu Thr 180 185 190Ser Gly Gly Ala Cys
Gly Phe Gly Leu Tyr Gly Leu Cys Thr Lys Gly 195 200 205Ser Val Thr
Ala Ser Trp Thr Asp Pro Met Leu Gly Ala Thr Cys Asp 210 215 220Ala
Phe Cys Thr Ala Tyr Pro Leu Leu Cys Lys Asp Pro Thr Gly Thr225 230
235 240Thr Leu Arg Gly Asn Phe Ala Ala Pro Asn Gly Asp Tyr Tyr Thr
Gln 245 250 255Phe Trp Ser Ser Leu Pro Gly Ala Leu Asp Asn Tyr Leu
Ser Cys Gly 260 265 270Glu Cys Ile Glu Leu Ile Gln Thr Lys Pro Asp
Gly Thr Asp Tyr Ala 275 280 285Val Gly Glu Ala Gly Tyr Thr Asp Pro
Ile Thr Leu Glu Ile Val Asp 290 295 300Ser Cys Pro Cys Ser Ala Asn
Ser Lys Trp Cys Cys Gly Pro Gly Ala305 310 315 320Asp His Cys Gly
Glu Ile Asp Phe Lys Tyr Gly Cys Pro Leu Pro Ala 325 330 335Asp Ser
Ile His Leu Asp Leu Ser Asp Ile Ala Met Gly Arg Leu Gln 340 345
350Gly Asn Gly Ser Leu Thr Asn Gly Val Ile Pro Thr Arg Tyr Arg Arg
355 360 365Val Gln Cys Pro Lys Val Gly Asn Ala Tyr Ile Trp Leu Arg
Asn Gly 370 375 380Gly Gly Pro Tyr Tyr Phe Ala Leu Thr Ala Val Asn
Thr Asn Gly Pro385 390 395 400Gly Ser Val Thr Lys Ile Glu Ile Lys
Gly Ala Asp Thr Asp Asn Trp 405 410 415Val Ala Leu Val His Asp Pro
Asn Tyr Thr Ser Ser Arg Pro Gln Glu 420 425 430Arg Tyr Gly Ser Trp
Val Ile Pro Gln Gly Ser Gly Pro Phe Asn Leu 435 440 445Pro Val Gly
Ile Arg Leu Thr Ser Pro Thr Gly Glu Gln Ile Val Asn 450 455 460Glu
Gln Ala Ile Lys Thr Phe Thr Pro Pro Ala Thr Gly Asp Pro Asn465 470
475 480Phe Tyr Tyr Ile Asp Ile Gly Val Gln Phe Ser Gln Asn 485
490218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 8xHis tag 21His His His His His His His His1
5226PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 22His His His His His His1 5234PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Gly
Ser Gly Ser1
* * * * *