U.S. patent application number 16/062911 was filed with the patent office on 2018-12-20 for polypeptides with endoglucanase activity and uses thereof.
The applicant listed for this patent is DANISCO US INC.. Invention is credited to Richard R. Bott, Neeraj Pandey, Sina Pricelius, Annapurna Sachan, Ajit Satapathy, Stepan Shipovskov.
Application Number | 20180362946 16/062911 |
Document ID | / |
Family ID | 59057760 |
Filed Date | 2018-12-20 |
United States Patent
Application |
20180362946 |
Kind Code |
A1 |
Pandey; Neeraj ; et
al. |
December 20, 2018 |
POLYPEPTIDES WITH ENDOGLUCANASE ACTIVITY AND USES THEREOF
Abstract
Disclosed herein are cellulase variants, or active fragments
thereof, and polynucleotides encoding same, wherein the cellulase
variants, or active fragments thereof, hav endoglucanase activity.
Also disclosed herein are compositions comprising said cellulase
variants, or active fragments thereof; vectors and/or host cells
comprising the polynucleotides encoding said cellulase variants, or
active fragments thereof; and methods for making and/or using said
cellulase variants, or active fragments thereof and/or compositions
containing same; wherein said cellulase variants, or active
fragments thereof, have endoglucanase activity.
Inventors: |
Pandey; Neeraj; (HARYANA,
IN) ; Bott; Richard R.; (Kirkland, WA) ;
Pricelius; Sina; (Arhus, DK) ; Sachan; Annapurna;
(Kanpur, IN) ; Shipovskov; Stepan; (Ega, DK)
; Satapathy; Ajit; (KARNATAKA, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANISCO US INC. |
Palo Alto |
CA |
US |
|
|
Family ID: |
59057760 |
Appl. No.: |
16/062911 |
Filed: |
December 16, 2016 |
PCT Filed: |
December 16, 2016 |
PCT NO: |
PCT/US2016/067223 |
371 Date: |
June 15, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62269678 |
Dec 18, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C11D 3/38645 20130101;
C12N 15/63 20130101; C12N 9/24 20130101; C12Y 302/01004 20130101;
C12N 9/2437 20130101 |
International
Class: |
C12N 9/42 20060101
C12N009/42; C12N 15/63 20060101 C12N015/63; C11D 3/386 20060101
C11D003/386 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 15, 2016 |
BD |
306/2016 |
Claims
1. A cellulase variant, or an active fragment thereof, comprising
an amino acid sequence comprising a substitution at one or more
positions selected from: (i) 4, 20, 23, 29, 32, 36, 44, 51, 77, 80,
87, 90, 97, 98, 99, 102, 112, 116, 135, 136, 142, 153, 154, 157,
161, 163, 192, 194, 204, 208, 210, 212, 216, 217, 221, 222, 225,
227, and 232; (ii) K4X, G/K20X, A/S23X, F29X, S32X, N/Q36X, K44X,
S51X, S77X, N80X, G87X, E90X, P97X, V98X, A99X, T102X, G112X,
T116X, S135X, P/5136X, A142X, G/H/S153X, Q154X, S157X, A161X,
K163X, L192X, A194X, G204X, V208X, T210X, P212X, Q216X, S217X,
S221X, S222X, S225X, K227X, and 5232X; (iii) X4V, X20N, X23L, X29W,
X32D/Y, X36T, X44V, X51T, X77K/M, X80S, X87A, X90A, X97S, X98G,
X99E/Y, X102K, X112S/T/V, X116V, X135T, X136E/K/S, X142D/E/P/Q,
X153D, X154E, X157D, X161E/P, X163V, X192V, X194S, X204S, X208H/K,
X210V, X212S, X216D/E/G/P/S/T/V, X217G/M, X221L/M, X222A, X225K,
X227R, and X232T; or (iv) K4V, G/K20L, A/S23L, F29W, S32D/Y,
N/Q36T, K44V, S51T, S77K/M, N80S, G87A, E90A, P97S, V98G, A99E/Y,
T102K, G112S/Y/V, T116V, S135T, P/S136E/K/S, A142D/E/P/Q,
G/H/S153D, Q154E, S157D, A161E/P, K163V, L192V, A194S, G204S,
V208H/K, T210V, P212S, Q216D/E/G/P/S/T/V, S217G/M, S221L/M, S222A,
S225K, K227R, and S232T, wherein said variant has endoglucanase
activity, and wherein the amino acid positions of the variant, or
active fragment thereof, are numbered by correspondence with the
amino acid sequence of SEQ ID NO:5.
2. The cellulase variant, or active fragment thereof, of claim 1,
wherein said one or more positions is selected from: (i) 142 and
216; (ii) A142X and Q216X; (iii) X142D/E/P/Q and X216D/E/G/P/S/T/V;
or (iv) A142D/E/P/Q, and Q216D/E/G/P/S/T/V.
3. The cellulase variant, or active fragment thereof, of any
preceding claim, wherein said one or more position is (i) 142, (ii)
216, or (iii) 142 and 216.
4. The cellulase variant, or active fragment thereof, of any
preceding claim, wherein said variant has at least one improved
property selected from improved thermostability, stability in the
presence of one or more protease, and stability in the presence of
one or more protease and one or more other detergent component when
compared to a parent or reference polypeptide.
5. The cellulase variant, or active fragment thereof, of any
preceding claim, wherein said variant comprises an amino acid
sequence having at least 70%, 75%, 80%, 82%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:5, 11, 14, 17, or 22.
6. The cellulase variant, or active fragment thereof, of any
preceding claim, wherein said variant is derived from a parent or
reference polypeptide selected from SEQ ID NOs:5, 11, 14, 17, and
22.
7. The cellulase variant, or active fragment thereof, of claim 6,
wherein the parent or reference polypeptide comprises (i) an amino
acid sequence having at least 70%, 75%, 80%, 82%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to amino acids 1-215 of
SEQ ID NO:5, or (ii) amino acids 1-215 of SEQ ID NO:5.
8. The cellulase variant, or active fragment thereof, of any
preceding claim, wherein the improved property is (i) improved
thermostability and wherein said variant, or active fragment
thereof, has a thermal PI that is greater than 1 or 1.1; (ii)
improved stability in the presence of one or more protease and
wherein said variant, or active fragment thereof, has a PI that is
greater than 1, 1.1, 1.5, or 2.0 when the stability of said
variant, or active fragment thereof is tested in the presence of
said protease; and/or (iii) improved stability in the presence of
one or more protease and one or more other detergent component, and
wherein said variant, or active fragment thereof, has a PI that is
greater than 1, 1.1, 1.5, 2.0, or 2.5 when the stability of said
variant, or active fragment thereof is tested in the presence of
said protease and said other detergent component.
9. The cellulase variant, or active fragment thereof, of claim 8,
wherein the PI is measured in accordance with the Cellulase
Activity Assay of Example 1.
10. The cellulase variant, or active fragment thereof, of 8 or 9,
wherein the other detergent component is a surfactant.
11. The cellulase variant, or active fragment thereof, of any
preceding claim, wherein said variant, or active fragment thereof,
comprises an amino acid sequence having: a) at least 70% amino acid
sequence identity to the amino acid sequence of SEQ ID NO:5, with
the proviso that the substitution is not A142P and/or Q216T and the
further proviso that (i) when the substitution is Q216S the
cellulase variant, or active fragment thereof, does not comprise
CN103343111-0003, CN103343111-0005, AGY80101, US20150299682-0004,
or WO2007071818-0012; or (ii) when the substitution is Q216G the
cellulase variant, or active fragment thereof, does not comprise
WO2014138983-0859; b) at least 75% amino acid sequence identity to
the amino acid sequence of SEQ ID NO:5, with the proviso that when
(i) the substitution is A142P the cellulase variant, or active
fragment thereof, does not comprise WO2012089024-0002,
WO9804663-AAW46618, GB2479462-AZN28533, WO2007071820-0019,
CN103343111-0005, CN103343111-0003, US20150299682-0004, AGY80101,
KOP50759, or CAD56665; ii) when the substitution is Q216T the
cellulase variant, or active fragment thereof, does not comprise
WO9743409-0066; or iii) when the substitution is Q216S the
cellulase variant, or active fragment thereof, does not comprise
CN103343111-0005, CN103343111-0003, US20150299682-0004, or
AGY80101; or c) at least 80% amino acid sequence identity to the
amino acid sequence of SEQ ID NO:5, with the proviso that when (i)
the substitution is A142P the cellulase variant, or active fragment
thereof, does not comprise WO2012089024-0002, WO9804663-AAW46618,
GB2479462-AZN28533, WO2007071820-0019, or CN103343111-0005; or ii)
when the substitution is Q216S the cellulase variant, or active
fragment thereof, does not comprise CN103343111-0005.
12. The cellulase variant, or active fragment thereof, of any
preceding claim, wherein said variant, or active fragment thereof,
is a family GH45 cellulase.
13. A composition comprising the cellulase variant, or active
fragment thereof, of any preceding claim.
14. The composition of claim 13, wherein said composition is
selected from an enzyme composition, detergent composition, and
fabric care composition.
15. The composition of claim 13 or 14, further comprising (i) one
or more other enzymes selected from the group consisting of acyl
transferases, amylases, alpha-amylases, beta-amylases,
alpha-galactosidases, arabinases, arabinosidases, aryl esterases,
beta-galactosidases, beta-glucanases, carrageenases, catalases,
chondroitinases, cutinases, endo-beta-mannanases,
exo-beta-mannanases, esterases, exo-mannanases, galactanases,
glucoamylases, hemicellulases, hyaluronidases, keratinases,
laccases, lactases, ligninases, lipases, lipolytic enzymes,
lipoxygenases, mannanases, metalloproteases, oxidases, pectate
lyases, pectin acetyl esterases, pectinases, pentosanases,
perhydrolases, peroxidases, phenoloxidases, phosphatases,
phospholipases, phytases, polygalacturonases, proteases,
pullulanases, reductases, rhamnogalacturonases, second cellulase,
beta-glucanases, tannases, transglutaminases, xylan
acetyl-esterases, xylanases, and xylosidases; (ii) one or more
surfactants; (iii) one or more ions selected from calcium and zinc;
(iv) one or more adjunct ingredients; (v) one or more stabilizers;
(vi) from about 0.001% to about 5.0 weight % of said cellulase
variant, or active fragment thereof of any one of claims 1-15;
(vii) one or more bleaching agents; and/or (viii) combinations
thereof.
16. The composition of any one of claims 13-15, wherein said
composition is a laundry detergent.
17. The composition of any one of claims 13-16, wherein the
composition is in a form selected from a liquid, a powder, a
granulated solid, a tablet, a sheet, and a unit dose.
18. The composition of any one of claims 13-17, wherein said
composition contains phosphate or is phosphate-free and/or contains
boron or is boron-free.
19. A polynucleotide comprising a nucleic acid sequence encoding
the cellulase variant, or active fragment thereof of any one of
claims 1-12.
20. An expression vector comprising the polynucleotide of claim
19.
21. A host cell comprising the expression vector of claim 20.
22. A method for producing the cellulase variant, or active
fragment thereof of any one of claims 1-12 comprising: (a) stably
transforming the host cell of claim 21 with the expression vector
of claim 20; (b) cultivating said transformed host cell under
conditions suitable for said host cell to produce said cellulase
variant or active fragment thereof; and (c) recovering said
cellulase variant or active fragment thereof.
23. Use of the cellulase variant, or active fragment thereof, of
any one of claims 1-12 in an enzyme composition, detergent
composition, fabric care composition, textile finishing process, or
paper and pulp process.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to and claims the benefit of
priority from U.S. Provisional Patent Application Ser. No.
62/269,678, filed Dec. 18, 2015, which is hereby incorporated
herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the sequence listing electronically submitted
with the application as an ASCII text file (Name:
NB40768WOPCT_ST25; Size: 67.5 KB; Created: Dec. 6, 2016) forms part
of the application and is hereby incorporated herein by reference
in its entirety.
[0003] Disclosed herein are cellulase variants, or active fragments
thereof, and polynucleotides encoding same, wherein the cellulase
variants, or active fragments thereof, have endoglucanase activity.
Also disclosed herein are compositions comprising said cellulase
variants, or active fragments thereof; vectors and/or host cells
comprising the polynucleotides encoding said cellulase variants, or
active fragments thereof; and methods for making and/or using said
cellulase variants, or active fragments thereof and/or compositions
containing same; wherein said cellulase variants, or active
fragments thereof, have endoglucanase activity.
[0004] Cellulase enzymes are glycoside hydrolase enzymes that
catalyze the hydrolysis of beta-1,4glycosidic linkages in cellulose
to break it down into monosaccharides or shorter polysaccharides
and oligosaccharides. Generally, cellulase enzymes contain a
cellulose binding module (CBM) and a catalytic domain that are
separated by a flexible spacer known as a "linker" or "linker
peptide". The catalytic domains of cellulases are classified by
both the Enzyme Commission (EC) and the Glycoside Hydrolase (GH)
family systems.
[0005] The Enzyme Commission has identified two classes of
cellulases, the endocellulases, which are classified as EC 3.2.1.4
enzymes and also referred to as endoglucanases, and the
exocellulases, which are classified as EC 3.2.1.91 enzymes and also
referred to as cellobiohydrolases or exoglucanase. The
endoglucanases randomly cleave internal bonds at amorphous sites
that create new chain ends, whereas the exoglucanases cleave two to
four units from the ends of the exposed chains created by the
endoglucanases. The GH family system, on the other hand, groups
cellulases based on enzyme structure and function resulting in a
number of GH Families including, for example, GH Family 5, 6, 7, 8,
9, 10, 12, 16, 18, 19, 26, 44, 45, 48, 51, 61, and 74.
[0006] Cellulases are known to be useful, for example, in detergent
compositions; for treating textiles; as animal feed additives; in
processing of paper and pulp for smoothing fiber, enhancing
drainage and de-inking; in the food industry for extracting and
clarifying juice from fruits and vegetables and for mashing; and in
reducing biomass to glucose that is then fermented and distilled to
make low CO.sub.2 cellulosic ethanol.
[0007] Cellulases are used in the textile industry to improve the
feel and/or appearance of cotton-containing fabric by, for example,
removing fuzz (untangled fiber ends that protrude from the surface
of yarn or fabric) and pills (bunches or balls of tangled fibers
that are held to the surface of fabric by one or more fibers), and
also helping to prevent pills, which make garments appear worn,
from forming through subsequent consumer wash and wear cycles. This
process is known as "depilling" or "biopolishing". Cellulases are
also used to impart, for example, a distressed or "stonewashed"
appearance to cotton-containing denim. This process is known as
"bio-stoning" and has largely replaced stones for generating the
soft, faded denim desired by consumers.
[0008] Cellulases are used in detergent compositions, for example,
to enhance soil removal, remove pills, brighten fabric colors, and
soften fabric. The detergent compositions to which cellulases are
added also often contain other enzymes, such as, for example,
proteases, making it important for the cellulase to be stable in
the presence of these other enzymes, as well as, other detergent
additives, such as, for example, surfactants. If the cellulase is
not stable, the protease, for example, will degrade the cellulase
over time negating the laundering benefits associated with the
cellulase. As a result, there remains a need in the art for
cellulases that are stable in the presence of one or more other
enzyme, such as, for example, protease and/or one or more other
detergent components, such as, for example, a surfactant.
DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 provides a plasmid map of pTTT-pyr2-stce1.
[0010] FIG. 2 provides a schematic of the main chain folding of the
truncated STCE1-WT cellulase, showing the beta subdomain (in
black), the loop subdomain (light lines), the alpha subdomain
(medium dark), and the C-terminal strand.
[0011] FIG. 3 provides a surface rendering of truncated STCE1-WT
cellulase displaying a view of the substrate binding cleft found to
occur in the groove formed between the beta (dark color) and alpha
(medium color) subdomains.
[0012] FIG. 4 provides a structural comparison of three GH45
cellulase family enzyme structures where the schematic of truncated
STCE1-WT is shown in black, the schematic for Humicola insolens
endoglucanase is shown in medium gray, and the schematic for
Melanocarpus albomyces endoglucanase is shown in light gray.
[0013] FIGS. 5A-5C depict the long term stability of STCE1-A142P,
STCE1-WT, and Benchmark cellulases in detergents containing a
protease.
[0014] FIGS. 6A-6C provides a MUSCLE multiple sequence alignment of
STCE1-WT cellulase and variants thereof with other GH45 cellulases
described in Example 7.
[0015] FIG. 7 provides a phylogenetic tree of STCE1-WT cellulase
and variants thereof with other GH45 cellulases described in
Example 7.
[0016] FIG. 8 provides a MUSCLE multiple sequence alignment of the
catalytic domain regions of the following GH45 cellulases: STCE-1
(SEQ ID NO:5), H. insolens (SEQ ID NO:11), N. crassa (SEQ ID NO:14)
and T. terrestris 120H (SEQ ID NO:17).
[0017] One embodiment is directed to a cellulase variant, or an
active fragment thereof, comprising an amino acid sequence
comprising a substitution at one or more positions selected from
(i) 4, 20, 23, 29, 32, 36, 44, 51, 77, 80, 87, 90, 97, 98, 99, 102,
112, 116, 135, 136, 142, 153, 154, 157, 161, 163, 192, 194, 204,
208, 210, 212, 216, 217, 221, 222, 225, 227, and 232; (ii) K4X,
G/K20X, A/S23X, F29X, S32X, N/Q36X, K44X, S51X, S77X, N80X, G87X,
E90X, P97X, V98X, A99X, T102X, G112X, T116X, S135X, P/5136X, A142X,
G/H/S153X, Q154X, S157X, A161X, K163X, L192X, A194X, G204X, V208X,
T210X, P212X, Q216X, S217X, S221X, S222X, S225X, K227X, and 5232X;
(iii) X4V, X20N, X23L, X29W, X32D/Y, X36T, X44V, X51T, X77K/M,
X80S, X87A, X90A, X97S, X98G, X99E/Y, X102K, X112S/T/V, X116V,
X135T, X136E/K/S, X142D/E/P/Q, X153D, X154E, X157D, X161E/P, X163V,
X192V, X194S, X204S, X208H/K, X210V, X212S, X216D/E/G/P/S/T/V,
X217G/M, X221L/M, X222A, X225K, X227R, and X232T; or (iv) K4V,
G/K20L, A/S23L, F29W, S32D/Y, N/Q36T, K44V, S51T, S77K/M, N80S,
G87A, E90A, P97S, V98G, A99E/Y, T102K, G112S/Y/V, T116V, S135T,
P/S136E/K/S, A142D/E/P/Q, G/H/S153D, Q154E, S157D, A161E/P, K163V,
L192V, A194S, G204S, V208H/K, T210V, P212S, Q216D/E/G/P/S/T/V,
S217G/M, S221L/M, S222A, S225K, K227R, and S232T, wherein said
variant has endoglucanase activity, and wherein the amino acid
positions of the variant, or active fragment thereof are numbered
by correspondence with the amino acid sequence of SEQ ID NO:5.
Another embodiment is directed to a cellulase variant, or active
fragment thereof, wherein said variant comprises an amino acid
sequence having at least 70%, 75%, 80%, 82%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:5, 11, 14, 17, 22, or amino
acids 1-215 of SEQ ID NO:5. In another embodiment, the cellulase
variant, or active fragment thereof is derived from a parent or
reference polypeptide selected from SEQ ID NOs:5, 11, 14, 17, and
22. In still yet another embodiment, the cellulase variant, or
active fragment thereof is a family GH45 cellulase.
[0018] In one embodiment, the cellulase variant, or active fragment
thereof, has one or more improved property selected from improved
thermostability, improved stability in the presence of one or more
other enzyme, and improved stability in the presence of one or more
other enzyme and one or more other detergent component. In another
embodiment, the other enzyme is protease and/or the other detergent
component is a surfactant. In some embodiments, the improved
property is improved when compared to a parent or reference
polypeptide. In other embodiments, the parent or reference
polypeptide comprises an amino acid sequence having at least 70%,
75%, 80%, 82% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% amino acid sequence identity to the amino acid sequence of SEQ
ID NO:5, 11, 14, 17, 22, or amino acids 1-215 of SEQ ID NO:5. In
still other embodiments, the parent or reference polypeptide
comprises SEQ ID NO:5 or amino acids 1-215 of SEQ ID NO:5.
[0019] A further embodiment is directed to a composition comprising
said cellulase variant, or active fragment thereof; a vector and/or
host cell comprising said cellulase variant, or active fragment
thereof; and a method for making and/or using said variant, or
active fragment thereof and/or said compositions containing such
variants, or active fragments thereof; wherein said cellulase
variant, or active fragment thereof, has endoglucanase
activity.
[0020] The features of the cellulase variants described herein make
them well-suited for use in detergent compositions, textile
processing, paper and pulp processing, and other industrial
applications, such as, for example, to impart soil release or
fabric care benefits and/or improve the feel and/or appearance of a
cotton-containing fabric.
[0021] The following terms are defined for clarity. Terms not
defined should be accorded their ordinary meaning as used in the
art. For example, technical and scientific terms not defined herein
have the same meaning as commonly understood by one of ordinary
skill in the art (See, e.g., Singleton and Sainsbury, Dictionary of
Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, N
Y 1994; and Hale and Marham, The Harper Collins Dictionary of
Biology, Harper Perennial, N Y 1991).
[0022] The singular terms "a," "an," and "the" include the plural
reference unless the context clearly indicates otherwise.
[0023] The term "about" when used in connection with a numerical
value refers to a range of -10% to +10% of the numerical value. For
instance, the phrase a "pH value of about 6" refers to pH values of
from 5.4 to 6.6.
[0024] The term "adjunct ingredient" when used in conjunction with
a detergent or fabric care composition means any liquid, solid or
gaseous material selected for the particular type of detergent or
fabric care composition desired and the form of the product (e.g.,
liquid, granule, powder, bar, paste, spray, tablet, gel, unit dose,
sheet, or foam composition), which materials are also preferably
compatible with the cellulase variant or active fragment thereof
used in the composition. In some embodiments, granular compositions
are in "compact" form, while in other embodiments, the liquid
compositions are in a "concentrated" form.
[0025] The term "cellulase variant" refers to a recombinant
polypeptide that is derived from a parent or reference polypeptide
by the substitution, addition, or deletion, of one or more amino
acids. A cellulase variant may differ from a parent polypeptide by
a small number of amino acid residues and may be defined by their
level of primary amino acid sequence homology/identity with a
parent polypeptide. For example, a cellulase variant has at least
70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence
identity with a parent (or reference) polypeptide.
[0026] The terms "detergent composition" and "detergent
formulation" refer to mixtures of chemical ingredients intended for
use in a wash medium to clean soiled objects. Detergent
compositions/formulations may include, for example, one or more
surfactant, hydrolytic enzyme, oxido-reductase, builder, bleaching
agent, bleach activator, bluing agent, fluorescent dye, caking
inhibitor, masking agent, enzyme activator, antioxidant, chelant,
polymer, foam regulator, fragrance, and solubilizer.
[0027] The term "derived from" encompasses the terms "originated
from," "obtained from," "obtainable from," "isolated from," and
"created from" and generally indicates that one specified material
find its origin in another specified material or has features that
can be described with reference to the another specified
material.
[0028] The term "effective amount" when used in conjunction with a
cellulase variant or active fragment thereof refers to the quantity
of cellulase variant or active fragment thereof needed to achieve
the desired level of endoglucanase activity in the specified
application or detergent or fabric care composition. Such effective
amounts are readily ascertained by one of ordinary skill in the art
and are based on many factors, such as the particular cellulase
variant or active fragment thereof that is used, the application,
the specific composition of the cleaning composition (including the
particular protease contained therein), and whether a liquid or dry
(e.g., granular, bar, powder, solid, liquid, tablet, gel, paste,
foam, sheet, or unit dose) composition is required.
[0029] The term "endoglucanase" refers to an endo-1,4-(1,3;
1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4), which
catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in
cellulose, cellulose derivatives (such as carboxymethyl cellulose
and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed
beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and
other plant material containing cellulosic components.
Endoglucanase activity can be determined according to the procedure
described in the examples.
[0030] The term "expression vector" refers to a DNA construct
containing a DNA sequence that encodes the specified polypeptide
and is operably linked to a suitable control sequence capable of
effecting the expression of the polypeptides in a suitable host.
Such control sequences include a promoter to effect transcription,
an optional operator sequence to control such transcription, a
sequence encoding suitable mRNA ribosome binding sites, and
sequences which control termination of transcription and
translation. The vector may be a plasmid, a phage particle, or
simply a potential genomic insert. Once transformed into a suitable
host, the vector may replicate and function independently of the
host genome, or may, in some instances, integrate into the genome
itself.
[0031] The term "fabric" refers to, for example, woven, knit, and
non-woven material, as well as staple fibers and filaments that can
be converted to, for example, yarns and woven, knit, and non-woven
fabrics. The term encompasses material made from natural, as well
as synthetic (e.g., manufactured) fibers.
[0032] The terms "fabric care composition" or "fabric care
formulation" refer to a composition/formulation containing a
cellulase variant, or active fragment thereof, described herein
that will, when added to a wash medium, remove pills and/or fuzz
from fabric; brighten fabric colors; and/or soften fabric.
[0033] The term "family GH45" refers to a polypeptide that is
classified as glycoside hydrolase Family 45 according to Henrissat
B., 1991, A classification of glycosyl hydrolases based on
amino-acid sequence similarities, Biochem. J. 280: 309-316, and
Henrissat and Bairoch, 1996, Updating the sequence-based
classification of glycosyl hydrolases, Biochem. J. 316: 695-696 and
classified based on structure and function relationships CMin the
Carbohydrate-Active enZYmes Database, CAZy (URL:
http://afmb.cnrs-mrs.fr/.about.cazy/CAZY/index.html).
[0034] The term "host cells" generally refers to prokaryotic or
eukaryotic hosts which are transformed or transfected with vectors
constructed using recombinant DNA techniques known in the art.
Transformed host cells are capable of either replicating vectors
encoding the protein variants or expressing the desired protein
variant. In the case of vectors which encode the pre- or pro-form
of the protein variant, such variants, when expressed, are
typically secreted from the host cell into the host cell
medium.
[0035] The term "hybridization" refers to the process by which a
strand of nucleic acid joins with a complementary strand through
base pairing, as known in the art.
[0036] The term "hybridization conditions" refers to the conditions
under which hybridization reactions are conducted. These conditions
are typically classified by degree of "stringency" of the
conditions under which hybridization is measured. The degree of
stringency can be based, for example, on the melting temperature
(T.sub.m) of the nucleic acid binding complex or probe. For
example, "maximum stringency" typically occurs at about
T.sub.m-5.degree. C. (5.degree. below the T.sub.m of the probe);
"high stringency" at about 5-10.degree. C. below the T.sub.m;
"intermediate stringency" at about 10-20.degree. C. below the
T.sub.m of the probe; and "low stringency" at about 20-25.degree.
C. below the T.sub.m. Alternatively, or in addition, hybridization
conditions can be based upon the salt or ionic strength conditions
of hybridization and/or one or more stringency washes, e.g.,
6.times.SSC=very low stringency; 3.times.SSC=low to medium
stringency; 1.times.SSC=medium stringency; and 0.5.times.SSC=high
stringency. Functionally, maximum stringency conditions may be used
to identify nucleic acid sequences having strict identity or
near-strict identity with the hybridization probe; while high
stringency conditions are used to identify nucleic acid sequences
having about 80% or more sequence identity with the probe. For
applications requiring high selectivity, it is typically desirable
to use relatively stringent conditions to form the hybrids (e.g.,
relatively low salt and/or high temperature conditions are
used).
[0037] The term "introduced" in the context of inserting a nucleic
acid sequence into a cell, means transformation, transduction, or
transfection. Means of transformation include protoplast
transformation, calcium chloride precipitation, electroporation,
naked DNA, and the like as known in the art. (See, Chang and Cohen
[1979] Mol. Gen. Genet. 168:111-115; Smith et al. [1986] Appl. Env.
Microbiol. 51:634; and the review article by Ferrari et al., in
Harwood, Bacillus, Plenum Publishing Corporation, pp. 57-72,
1989).
[0038] A nucleic acid or polynucleotide is "isolated" when it is at
least partially or completely separated from other components,
including but not limited to, for example, other proteins, nucleic
acids, and cells. Similarly, a polypeptide, protein or peptide is
"isolated" when it is at least partially or completely separated
from other components, including but not limited to, for example,
other proteins, nucleic acids, and cells. On a molar basis, an
isolated species is more abundant than are other species in a
composition. For example, an isolated species may comprise at least
about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% (on a molar basis) of all
macromolecular species present. Preferably, the species of interest
is purified to essential homogeneity (i.e., contaminant species
cannot be detected in the composition by conventional detection
methods). Purity and homogeneity can be determined using a number
of techniques well known in the art, such as agarose or
polyacrylamide gel electrophoresis of a nucleic acid or a protein
sample, respectively, followed by visualization upon staining. If
desired, a high-resolution technique, such as high performance
liquid chromatography (HPLC) or a similar means can be utilized for
purification of the material.
[0039] The term "other detergent component" refers to a non-enzyme
component that is added to a detergent composition or formulation,
such as, for example, a surfactant, oxido-reductase, builder,
bleaching agent, bleach activator, bluing agent, fluorescent dye,
caking inhibitor, masking agent, enzyme activator, antioxidant,
chelant, polymer, foam regulator, fragrance, and solubilizer.
[0040] The term "other enzyme" refers to a second, third, fourth,
etc enzyme that is added to a detergent composition, wherein the
first enzyme is a cellulase variant, or active fragment thereof
described herein. Examples of other enzymes include, for example,
acyl transferases, amylases, alpha-amylases, beta-amylases,
alpha-galactosidases, arabinases, arabinosidases, aryl esterases,
beta-galactosidases, beta-glucanases, carrageenases, catalases,
chondroitinases, cutinases, endo-beta-mannanases,
exo-beta-mannanases, esterases, exo-mannanases, galactanases,
glucoamylases, hemicellulases, hyaluronidases, keratinases,
laccases, lactases, ligninases, lipases, lipolytic enzymes,
lipoxygenases, mannanases, metalloproteases, oxidases, pectate
lyases, pectin acetyl esterases, pectinases, pentosanases,
perhydrolases, peroxidases, phenoloxidases, phosphatases,
phospholipases, phytases, polygalacturonases, proteases,
pullulanases, reductases, rhamnogalacturonases, second cellulase,
beta-glucanases, tannases, transglutaminases, xylan
acetyl-esterases, xylanases, xylosidases, and combinations
thereof.
[0041] The terms "polynucleotide" encompasses DNA, RNA,
heteroduplexes, and synthetic molecules capable of encoding a
polypeptide. Nucleic acids may be single-stranded or
double-stranded, and may have chemical modifications. The terms
"nucleic acid" and "polynucleotide" are used interchangeably.
Because the genetic code is degenerate, more than one codon may be
used to encode a particular amino acid, and the present
compositions and methods encompass nucleotide sequences which
encode a particular amino acid sequence. Unless otherwise
indicated, nucleic acid sequences are presented in a 5'-to-3'
orientation.
[0042] The term "polypeptide" refers to a molecule comprising a
plurality of amino acids linked through peptide bonds. The terms
"polypeptide," "peptide," and "protein" are used interchangeably.
Proteins may optionally be modified (e.g., glycosylated,
phosphorylated, acylated, farnesylated, prenylated, and sulfonated)
to add functionality. Where such amino acid sequences exhibit
activity, they may be referred to as an "enzyme". The conventional
one-letter or three-letter codes for amino acid residues are used,
with amino acid sequences being presented in the standard
amino-to-carboxy terminal orientation (i.e., N.fwdarw.C).
[0043] The term "recombinant," refers to genetic material (i.e.,
nucleic acids, the polypeptides they encode, and vectors and cells
comprising such polynucleotides) that has been modified to alter
its sequence or expression characteristics, such as, for example,
by mutating the coding sequence to produce an altered polypeptide,
fusing the coding sequence to that of another gene, placing a gene
under the control of a different promoter, expressing a gene in a
heterologous organism, expressing a gene at a decreased or elevated
level, and expressing a gene conditionally or constitutively in
manner different from its natural expression profile. Generally
recombinant nucleic acids, polypeptides, and cells based thereon,
have been manipulated by man such that they are not identical to
related nucleic acids, polypeptides, and/or cells found in
nature.
[0044] The term "second cellulase" refers to a second cellulase
enzyme that is added to a detergent composition, wherein the first
cellulase enzyme is a cellulase variant, or active fragment thereof
described herein. This second cellulase enzyme, includes, for
example, cellobiohydrolases, endoglucanases, xyloglucanases, and
combinations thereof.
[0045] The term "signal sequence" refers to a sequence of amino
acids bound to the N-terminal portion of a polypeptide, and which
facilitates the secretion of the mature form of the protein from
the cell. The mature form of the extracellular protein lacks the
signal sequence which is cleaved off during the secretion
process.
[0046] The term "surfactant" refers to any compound generally
recognized in the art as having surface active qualities.
Surfactants can include, for example, anionic, cationic, nonionic,
and zwitterionic compounds, which are further described,
herein.
[0047] The terms "thermostability" and "thermostable" refer to
cellulase variants that retain a specified amount of endoglucanase
activity after exposure to elevated temperatures over a given
period of time under conditions prevailing during cleaning, textile
treatment, or other process, for example, while exposed to elevated
temperatures. In some embodiments, the one or more cellulase
variant retains at least about 50%, about 60%, about 70%, about
75%, about 80%, about 85%, about 90%, about 92%, about 95%, about
96%, about 97%, about 98%, or about 99% endoglucanase activity
after exposure to elevated temperatures, for example, at least
about 50.degree. C., about 55.degree. C., about 60.degree. C.,
about 65.degree. C., or about 70.degree. C., over a given time
period, for example, at least about about 10 minutes, about 30
minutes, about 60 minutes, about 120 minutes, about 180 minutes,
about 240 minutes, about 300 minutes, etc.
[0048] The term "variant polynucleotide" refers to a polynucleotide
that encodes a cellulase variant, has a specified degree of
homology/identity with a parent polynucleotide, or hybridizes under
stringent conditions to a parent polynucleotide or the complement,
thereof. For example, a variant polynucleotide has at least 70%,
75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence
identity with a parent polynucleotide.
[0049] The terms, "wild-type" or "parental" with respect to a
polypeptide, refer to a naturally-occurring polypeptide that does
not include a man-made substitution, insertion, or deletion at one
or more amino acid positions. Similarly, the terms "wild-type" or
"parental,", with respect to a polynucleotide, refer to a
naturally-occurring polynucleotide that does not include a man-made
substitution, insertion, or deletion at one or more nucleosides. A
polynucleotide encoding a wild-type or parental polypeptide is,
however, not limited to a naturally-occurring polynucleotide, and
encompasses any polynucleotide encoding the wild-type or parental
polypeptide.
[0050] The term "naturally-occurring" refers to anything (e.g.,
polypeptide or nucleic acid sequences) that is found in nature.
Conversely, the term "non-naturally occurring" refers to anything
that is not found in nature (e.g., recombinant nucleic acids and
polypeptide sequences produced in the laboratory or modifications
of the wild-type sequence).
[0051] The term "reference", with respect to a polypeptide, refers
to a naturally-occurring polypeptide that does not include a
man-made substitution, insertion, or deletion at one or more amino
acid positions, as well as a naturally-occurring or synthetic
polypeptide that includes one or more man-made substitutions,
insertions, or deletions at one or more amino acid positions.
Similarly, the term "reference", with respect to a polynucleotide,
refers to a naturally-occurring polynucleotide that does not
include a man-made substitution, insertion, or deletion of one or
more nucleosides, as well as a naturally-occurring or synthetic
polynucleotide that includes one or more man-made substitutions,
insertions, or deletions at one or more nucleosides. For example, a
polynucleotide encoding a wild-type or parental polypeptide is not
limited to a naturally-occurring polynucleotide, and encompasses
any polynucleotide encoding the wild-type or parental
polypeptide.
[0052] The amino acid substitutions described herein use one or
more following nomenclatures: position or starting amino
acid:position:substituted amino acid(s). Reference to only a
position encompasses any starting amino acid that may be present in
a reference polypeptide, parent or wild-type molecule at that
position and any amino acid with which such starting amino acid may
be substituted (i.e., amino acid substitutions exclude the starting
amino acid of such reference polypeptide, parent or wild-type
molecule). Reference to a substituted amino acid or a starting
amino acid may be further expressed as several substituted amino
acids or several starting amino acids separated by a foreslash
("/"). For example, X130A/N-209-213 represents a three amino acid
substitution combination, wherein X is any starting amino acid at
position 130 that can be substituted with an alanine (A) or an
asparagine (N); 209 represents a position where any starting amino
acid can be substituted with an amino acid that is not the starting
amino acid; and 213 represents a position where any starting amino
acid can be substituted with an amino acid that is not the starting
amino acid. By way of further example, E/Q/S101F/G/H/T/V represents
five possible substitutions at position 101, wherein the starting
amino acid glutamate (E), glutamine (Q), or serine (S) can be
substituted with a phenylalanine (F), glycine (G), histidine (H),
threonine (T), or valine (V).
[0053] Sequence identity may be determined using known programs
such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See,
e.g., Altschul et al. [1990] J Mol. Biol. 215:403-410; Henikoff et
al. [1989] Proc. Natl. Acad. Sci. USA 89:10915; Karin et al. Proc.
Natl. Acad. Sci USA 90:5873; and Higgins et al. [1988] Gene
73:237-244). Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(NCBI). Databases may also be searched using FASTA (Pearson et al.
[1988] Proc. Natl. Acad. Sci. USA 85:2444-2448). One indication
that two polypeptides are substantially identical is that the first
polypeptide is immunologically cross-reactive with the second
polypeptide. Typically, polypeptides that differ by conservative
amino acid substitutions are immunologically cross-reactive. Thus,
a polypeptide is substantially identical to a second polypeptide,
for example, where the two peptides differ only by a conservative
substitution. Another useful algorithm for comparison of multiple
protein sequences is the MUSCLE program from Geneious software
(Biomatters Ltd.) (Robert C. Edgar. MUSCLE: multiple sequence
alignment with high accuracy and high throughput Nucl. Acids Res.
(2004) 32 (5): 1792-1797).
[0054] One embodiment is directed to a cellulase variant, or an
active fragment thereof, comprising an amino acid sequence
comprising a substitution at one or more positions selected from:
(i) 4, 20, 23, 29, 32, 36, 44, 51, 77, 80, 87, 90, 97, 98, 99, 102,
112, 116, 135, 136, 142, 153, 154, 157, 161, 163, 192, 194, 204,
208, 210, 212, 216, 217, 221, 222, 225, 227, and 232; (ii) K4X,
G/K20X, A/S23X, F29X, S32X, N/Q36X, K44X, S51X, S77X, N80X, G87X,
E90X, P97X, V98X, A99X, T102X, G112X, T116X, S135X, P/S136X, A142X,
G/H/S153X, Q154X, S157X, A161X, K163X, L192X, A194X, G204X, V208X,
T210X, P212X, Q216X, S217X, S221X, S222X, S225X, K227X, and S232X;
(iii) X4V, X20N, X23L, X29W, X32D/Y, X36T, X44V, X51T, X77K/M,
X80S, X87A, X90A, X97S, X98G, X99E/Y, X102K, X112S/T/V, X116V,
X135T, X136E/K/S, X142D/E/P/Q, X153D, X154E, X157D, X161E/P, X163V,
X192V, X194S, X204S, X208H/K, X210V, X212S, X216D/E/G/P/S/T/V,
X217G/M, X221L/M, X222A, X225K, X227R, and X232T; or (iv) K4V,
G/K20L, A/S23L, F29W, S32D/Y, N/Q36T, K44V, S51T, S77K/M, N80S,
G87A, E90A, P97S, V98G, A99E/Y, T102K, G112S/Y/V, T116V, S135T,
P/S136E/K/S, A142D/E/P/Q, G/H/S153D, Q154E, S157D, A161E/P, K163V,
L192V, A194S, G204S, V208H/K, T210V, P212S, Q216D/E/G/P/S/T/V,
S217G/M, S221L/M, S222A, S225K, K227R, and S232T, where the variant
has endoglucanase activity and the amino acid positions of the
variant, or active fragment thereof, are numbered by correspondence
with the amino acid sequence of SEQ ID NO:5. Another embodiment is
directed to a cellulase variant, or an active fragment thereof,
comprising an amino acid sequence comprising a substitution at one
or more positions selected from: (i) 4, 20, 23, 29, 32, 36, 44, 51,
77, 80, 87, 90, 97, 98, 99, 102, 112, 116, 135, 136, 153, 154, 157,
161, 163, 192, 194, 204, 208, 210, 212, 217, 221, 222, 225, 227,
and 232; (ii) K4X, G/K20X, A/S23X, F29X, S32X, N/Q36X, K44X, S51X,
S77X, N80X, G87X, E90X, P97X, V98X, A99X, T102X, G112X, T116X,
5135X, P/5136X, G/H/5153X, Q154X, S157X, A161X, K163X, L192X,
A194X, G204X, V208X, T210X, P212X, S217X, S221X, S222X, S225X,
K227X, and 5232X; (iii) X4V, X20N, X23L, X29W, X32D/Y, X36T, X44V,
X51T, X77K/M, X80S, X87A, X90A, X97S, X98G, X99E/Y, X102K,
X112S/T/V, X116V, X135T, X136E/K/S, X153D, X154E, X157D, X161E/P,
X163V, X192V, X194S, X204S, X208H/K, X210V, X212S, X217G/M,
X221L/M, X222A, X225K, X227R, and X232T; or (iv) K4V, G/K20L,
A/S23L, F29W, S32D/Y, N/Q36T, K44V, S51T, S77K/M, N80S, G87A, E90A,
P97S, V98G, A99E/Y, T102K, G112S/YN, T116V, S135T, P/S136E/K/S,
G/H/S153D, Q154E, S157D, A161E/P, K163V, L192V, A194S, G204S,
V208H/K, T210V, P212S, S217G/M, S221L/M, S222A, S225K, K227R, and
S232T, where the variant has endoglucanase activity and the amino
acid positions of the variant, or active fragment thereof, are
numbered by correspondence with the amino acid sequence of SEQ ID
NO:5.
[0055] Another embodiment is directed to a cellulase variant, or
active fragment thereof, wherein said variant comprises an amino
acid sequence having at least 70%, 75%, 80%, 82%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence
identity to the amino acid sequence of SEQ ID NO:5, 11, 14, 17, 22,
or amino acids 1-215 of SEQ ID NO:5.
[0056] Another further embodiment is directed to a cellulase
variant, or an active fragment thereof, comprising an amino acid
sequence comprising a substitution at one or more positions
selected from: (i) 142 and 216; (ii) A142X and Q216X; (iii)
X142D/E/P/Q and X216D/E/G/P/S/T/V; or (iv) A142D/E/P/Q, and
Q216D/E/G/P/S/T/V, where the variant has endoglucanase activity and
the amino acid positions of the variant, or active fragment
thereof, are numbered by correspondence with the amino acid
sequence of SEQ ID NO:5. A still further embodiment is directed to
a cellulase variant, or an active fragment thereof, comprising an
amino acid sequence comprising a substitution at one or more
positions selected from 142 and 216, where the variant has
endoglucanase activity and the amino acid positions of the variant,
or active fragment thereof, are numbered by correspondence with the
amino acid sequence of SEQ ID NO:5. Another still further
embodiment is directed to a cellulase variant, or an active
fragment thereof, comprising an amino acid sequence comprising a
substitution at one or more positions selected from A142X and
Q216X, where the variant has endoglucanase activity and the amino
acid positions of the variant, or active fragment thereof, are
numbered by correspondence with the amino acid sequence of SEQ ID
NO:5. An even still further embodiment is directed to a cellulase
variant, or an active fragment thereof, comprising an amino acid
sequence comprising a substitution at one or more positions
selected from X142D, X142E, X142P, X142Q, X216D, X216E, X216G,
X216P, X216S, X216T, and X216V, where the variant has endoglucanase
activity and the amino acid positions of the variant, or active
fragment thereof are numbered by correspondence with the amino acid
sequence of SEQ ID NO:5. A yet even still further embodiment is
directed to a cellulase variant, or an active fragment thereof,
comprising an amino acid sequence comprising a substitution at one
or more positions selected from A142D, A142E, A142P, A142Q, Q216D,
Q216E, Q216G, Q216P, Q216S, Q216T, and Q216V, where the variant has
endoglucanase activity and the amino acid positions of the variant,
or active fragment thereof, are numbered by correspondence with the
amino acid sequence of SEQ ID NO:5. A still even further embodiment
is directed to a cellulase variant, or an active fragment thereof,
comprising an amino acid sequence comprising a substitution at
position (i) 142, (ii) 216, or (iii) 142 and 216, where the variant
has endoglucanase activity and the amino acid positions of the
variant, or active fragment thereof, are numbered by correspondence
with the amino acid sequence of SEQ ID NO:5.
[0057] Another embodiment is directed to a cellulase variant, or an
active fragment thereof, comprising an amino acid sequence
comprising a substitution at position 142, where the variant has
endoglucanase activity and the amino acid positions of the variant,
or active fragment thereof, are numbered by correspondence with the
amino acid sequence of SEQ ID NO:5. A further embodiment is
directed to a cellulase variant, or an active fragment thereof,
comprising an amino acid sequence comprising a substitution at
position 142X, where the variant has endoglucanase activity and the
amino acid positions of the variant, or active fragment thereof,
are numbered by correspondence with the amino acid sequence of SEQ
ID NO:5. A still further embodiment is directed to a cellulase
variant, or an active fragment thereof, comprising an amino acid
sequence comprising a substitution at position X142D, X142E, X142P,
or X142Q, where the variant has endoglucanase activity and the
amino acid positions of the variant, or active fragment thereof are
numbered by correspondence with the amino acid sequence of SEQ ID
NO:5. A yet even still further embodiment is directed to a
cellulase variant, or an active fragment thereof, comprising an
amino acid sequence comprising a substitution at position A142D,
A142E, A142P, or A142Q, where the variant has endoglucanase
activity and the amino acid positions of the variant, or active
fragment thereof, are numbered by correspondence with the amino
acid sequence of SEQ ID NO:5.
[0058] A further embodiment is directed to a cellulase variant, or
an active fragment thereof, comprising an amino acid sequence
comprising a substitution at position 216, where the variant has
endoglucanase activity and the amino acid positions of the variant,
or active fragment thereof, are numbered by correspondence with the
amino acid sequence of SEQ ID NO:5. Another embodiment is directed
to a cellulase variant, or an active fragment thereof, comprising
an amino acid sequence comprising a substitution at position Q216X,
where the variant has endoglucanase activity and the amino acid
positions of the variant, or active fragment thereof, are numbered
by correspondence with the amino acid sequence of SEQ ID NO:5. A
still further embodiment is directed to a cellulase variant, or an
active fragment thereof, comprising an amino acid sequence
comprising a substitution at position X216D, X216E, X216G, X216P,
X216S, X216T, or X216V, where the variant has endoglucanase
activity and the amino acid positions of the variant, or active
fragment thereof are numbered by correspondence with the amino acid
sequence of SEQ ID NO:5. A yet even still further embodiment is
directed to a cellulase variant, or an active fragment thereof,
comprising an amino acid sequence comprising a substitution at
position Q216D, Q216E, Q216G, Q216P, Q216S, Q216T, or Q216V, where
the variant has endoglucanase activity and the amino acid positions
of the variant, or active fragment thereof, are numbered by
correspondence with the amino acid sequence of SEQ ID NO:5.
[0059] In another embodiment, the cellulase variant, or an active
fragment thereof, comprises an amino acid sequence comprising a
substitution at positions 142 and 216, where the variant has
endoglucanase activity and the amino acid positions of the variant,
or active fragment thereof, are numbered by correspondence with the
amino acid sequence of SEQ ID NO:5. Another embodiment is directed
to a cellulase variant, or an active fragment thereof, comprising
an amino acid sequence comprising a substitution at positions A142X
and Q216X, where the variant has endoglucanase activity and the
amino acid positions of the variant, or active fragment thereof,
are numbered by correspondence with the amino acid sequence of SEQ
ID NO:5. A still further embodiment is directed to a cellulase
variant, or an active fragment thereof, comprising an amino acid
sequence comprising a substitution at positions 142 and 216,
wherein position 142 is selected from X142D, X142E, X142P, and
X142Q, where the variant has endoglucanase activity and the amino
acid positions of the variant, or active fragment thereof are
numbered by correspondence with the amino acid sequence of SEQ ID
NO:5. An even still further embodiment is directed to a cellulase
variant, or an active fragment thereof, comprising an amino acid
sequence comprising a substitution at positions 142 and 216,
wherein position 216 is selected from X216D, X216E, X216G, X216P,
X216S, X216T, and X216V, where the variant has endoglucanase
activity and the amino acid positions of the variant, or active
fragment thereof, are numbered by correspondence with the amino
acid sequence of SEQ ID NO:5. An even still yet further embodiment
is directed to a cellulase variant, or an active fragment thereof,
comprising an amino acid sequence comprising an amino acid sequence
comprising a substitution at positions 142 and 216, wherein
position 142 is selected from A142D, A142E, A142P, and A142Q, where
the variant has endoglucanase activity and the amino acid positions
of the variant, or active fragment thereof, are numbered by
correspondence with the amino acid sequence of SEQ ID NO:5. In yet
another embodiment, the cellulase variant, or an active fragment
thereof, comprises an amino acid sequence comprising a substitution
at positions 142 and 216, wherein position 216 is selected from
Q216D, Q216E, Q216G, Q216P, Q216S, Q216T, and Q216V, where the
variant has endoglucanase activity and the amino acid positions of
the variant, or active fragment thereof, are numbered by
correspondence with the amino acid sequence of SEQ ID NO:5. In a
further embodiment, the cellulase variant, or active fragment
thereof, comprises an amino acid sequence comprising a substitution
at positions 142 and 216, wherein position 142 is selected from
X142D, X142E, X142P, and X142Q and position 216 is selected from
X216D, X216E, X216G, X216P, X216S, X216T, and X216V, where the
variant has endoglucanase activity and the amino acid positions of
the variant, or active fragment thereof are numbered by
correspondence with the amino acid sequence of SEQ ID NO:5. In a
still further embodiment, the cellulase variant, or active fragment
thereof, comprises an amino acid sequence comprising a substitution
at positions 142 and 216, wherein position 142 is selected from
A142D, A142E, A142P, and A142Q and position 216 is selected from
Q216D, Q216E, Q216G, Q216P, Q216S, Q216T, and Q216V, where the
variant has endoglucanase activity and the amino acid positions of
the variant, or active fragment thereof are numbered by
correspondence with the amino acid sequence of SEQ ID NO:5.
[0060] In another embodiment, the cellulase variant, or active
fragment thereof, describe herein is derived from a parent or
reference polypeptide selected from SEQ ID NOs:5, 11, 14, 17, and
22. In a further embodiment, the cellulase variant, or active
fragment thereof, describe herein is derived from a parent or
reference polypeptide selected from SEQ ID NOs:11, 14, 17, and 22.
In yet another embodiment, the cellulase variant, or active
fragment thereof, describe herein is derived from the parent
polypeptide of SEQ ID NO:5. In a still further embodiment, the
cellulase variant, or active fragment thereof, describe herein is
derived from the reference polypeptide of amino acids 1-215 of SEQ
ID NO:5. In an even still further embodiment, the cellulase
variant, or active fragment thereof, describe herein is derived
from the parent polypeptide of SEQ ID NO:11. In still another
embodiment, the cellulase variant, or active fragment thereof,
describe herein is derived from the parent polypeptide of SEQ ID
NO:14. In yet still another embodiment, the cellulase variant, or
active fragment thereof, describe herein is derived from the parent
polypeptide of SEQ ID NO:17 or 22.
[0061] In a further embodiment, the cellulase variant, or active
fragment thereof, has one or more improved property selected from
improved thermostability, improved stability in the presence of one
or more other enzyme, and improved stability in the presence of one
or more other enzyme and one or more other detergent component. In
another embodiment, the other enzyme is protease and/or the other
detergent component is a surfactant. In some embodiments, the
improved property is improved when compared to a parent or
reference polypeptide. In other embodiments, the parent polypeptide
comprises an amino acid sequence having at least 70%, 75%, 80%,
82%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino
acid sequence identity to the amino acid sequence of SEQ ID NO:5,
11, 14, 17, or 22. In still other embodiments, the parent
polypeptide comprises an amino acid sequence having at least 70%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino
acid sequence identity to the amino acid sequence of SEQ ID NO:5.
In further embodiments, the parent polypeptide comprises an amino
acid sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the
amino acid sequence of SEQ ID NO:11. In yet further embodiments,
the parent polypeptide comprises an amino acid sequence having at
least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or 99% amino acid sequence identity to the amino acid sequence of
SEQ ID NO:14. In still further embodiments, the parent polypeptide
comprises an amino acid sequence having at least 70%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid
sequence identity to the amino acid sequence of SEQ ID NO:17 or 22.
In yet other embodiments, the reference polypeptide comprises an
amino acid sequence having at least 70%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity
to amino acids 1-215 of SEQ ID NO:5. In still other embodiments,
the parent polypeptide comprises SEQ ID NO:5. In yet other
embodiments, the parent polypeptide comprises SEQ ID NO:11. In
still other embodiments, the parent polypeptide comprises SEQ ID
NO:14. In even still other embodiments, the parent polypeptide
comprises SEQ ID NO:17 or 22. In yet still other embodiments, the
reference polypeptide comprises amino acids 1-215 of SEQ ID
NO:5.
[0062] In other embodiments, the improved property is improved
thermostability, and the variant, or active fragment thereof, has a
thermal PI that is greater than 1 or 1.1. In still other
embodiments, the improved property is improved stability in the
presence of one or more protease, and the variant, or active
fragment thereof, has a PI that is greater than 1, 1.1, 1.5, or 2.0
when the stability of said variant, or active fragment thereof is
tested in the presence of said protease. In an even further
embodiment, the improved property is improved stability in the
presence of one or more protease and one or more other detergent
component, and wherein said variant, or active fragment thereof,
has a PI that is greater than 1, 1.1, 1.5, 2.0, or 2.5 when the
stability of said variant, or active fragment thereof is tested in
the presence of said protease and said other detergent component.
In yet a further embodiment, the PI is measured in accordance with
the Cellulase Activity Assay of Example 1.
[0063] In one other embodiment, the cellulase variant, or active
fragment thereof comprises an amino acid sequence having at least
70%, 75%, 80%, 82%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% amino acid sequence identity to the amino acid sequence
of SEQ ID NO:5 or to amino acids 1-215 of SEQ ID NO:5. In a further
embodiment, the cellulase variant, or active fragment thereof,
comprises an amino acid sequence having at least 70% amino acid
sequence identity to the amino acid sequence of SEQ ID NO:5, with
the proviso that the substitution is not A142P or Q216T and the
further proviso that (i) when the substitution is Q216S the
cellulase variant, or active fragment thereof, does not comprise
CN103343111-0003, CN103343111-0005, AGY80101, US20150299682-0004,
or WO2007071818-0012; or (ii) when the substitution is Q216G the
cellulase variant, or active fragment thereof, does not comprise
WO2014138983-0859. In yet a further embodiment, the cellulase
variant, or active fragment thereof, comprises an amino acid
sequence having at least 70% amino acid sequence identity to the
amino acid sequence of SEQ ID NO:5, with the proviso that the
substitution is not A142P and is not Q216T and the further proviso
that (i) when the substitution is Q216S the cellulase variant, or
active fragment thereof, does not comprise CN103343111-0003,
CN103343111-0005, AGY80101, US20150299682-0004, or
WO2007071818-0012; or (ii) when the substitution is Q216G the
cellulase variant, or active fragment thereof, does not comprise
WO2014138983-0859. In a still further embodiment, the cellulase
variant, or active fragment thereof comprises an amino acid
sequence having at least 75% amino acid sequence identity to the
amino acid sequence of SEQ ID NO:5, with the proviso that when (i)
the substitution is A142P the cellulase variant, or active fragment
thereof, does not comprise WO2012089024-0002, WO9804663-AAW46618,
GB2479462-AZN28533, WO2007071820-0019, CN103343111-0005,
CN103343111-0003, U520150299682-0004, AGY80101, KOP50759, or
CAD56665; ii) when the substitution is Q216T the cellulase variant,
or active fragment thereof, does not comprise WO9743409-0066; or
iii) when the substitution is Q216S the cellulase variant, or
active fragment thereof, does not comprise CN103343111-0005,
CN103343111-0003, US20150299682-0004, or AGY80101. In yet still
another embodiment, the cellulase variant or active fragment
thereof comprises an amino acid sequence having at least 80% amino
acid sequence identity to the amino acid sequence of SEQ ID NO:5,
with the proviso that when (i) the substitution is A142P the
cellulase variant, or active fragment thereof, does not comprise
WO2012089024-0002, WO9804663-AAW46618, GB2479462-AZN28533,
WO2007071820-0019, or CN103343111-0005; or ii) when the
substitution is Q216S the cellulase variant, or active fragment
thereof, does not comprise CN103343111-0005. In an even still
further embodiment, the cellulase variant or active fragment
thereof comprises an amino acid sequence having at least 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence
identity to the amino acid sequence of SEQ ID NO:5.
[0064] In a further embodiment, the cellulase variant, or active
fragment thereof comprises an amino acid sequence having at least
82% amino acid sequence identity to amino acids 1-215 of SEQ ID
NO:5, with the proviso that (i) when the substitution is A142P the
cellulase variant, or active fragment thereof, does not comprise
US20150299682-0004, CN103343111-0005, AGY80101, WO2012089024-0004,
WO2012089024-0002, WO2004053039-0003, XP_003651003, CDF76465, or
XP_001903789; ii) when the substitution is A142G the cellulase
variant, or active fragment thereof, does not comprise
XP_001226436; iii) when the substitution is Q216T the cellulase
variant, or active fragment thereof, does not comprise
WO9743409-0066; or iv) when the substitution is Q216S the cellulase
variant, or active fragment thereof, does not comprise
US20150299682-0004, CN103343111-0005, or AGY80101. In a still
further embodiment, the cellulase variant, or active fragment
thereof, comprises an amino acid sequence having at least 85% amino
acid sequence identity to amino acids 1-215 of SEQ ID NO:5, with
the proviso that (i) when the substitution is A142P or Q216S the
cellulase variant, or active fragment thereof, does not comprise
US20150299682-0004, CN103343111-0005, or AGY80101; or ii) when the
substitution is A142G the cellulase variant, or active fragment
thereof, does not comprise XP_001226436. In yet an even still
further embodiment, the cellulase variant, or active fragment
thereof, comprises an amino acid sequence having at least 90%, 91%,
92%, 93%, 94%, 95%, 95%, 97%, 98%, or 99% amino acid sequence
identity to amino acids 1-215 of SEQ ID NO:5.
[0065] In one embodiment, the cellulase variants, or active
fragments thereof, described herein are family GH45 cellulases. In
some embodiments, the cellulase variants, or active fragments
thereof, described herein are isolated.
[0066] Further embodiments are directed to a polynucleotide that
encodes the cellulase variants, or active fragments thereof,
described herein. In one embodiment, the polynucleotide is
contained in an expression vector contained in a heterologous
organism. The polynucleotide may be operably-linked to regulatory
elements (e.g., a promoter, terminator, and enhancer) to assist in
expressing the encoded cellulase variants, or active fragments
thereof, described herein. In some embodiments, the cellulase
variant, or active fragment thereof, described herein is expressed
in a heterologous organism as a secreted polypeptide, in which
case, the compositions and method encompass a method for expressing
the variant or active fragment thereof as a secreted polypeptide in
a heterologous organism.
[0067] DNA that encodes a cellulase variant, or active fragment
thereof, described herein can be chemically synthesized from
published sequences or obtained directly from host cells harboring
the gene (e.g., by cDNA library screening or PCR
amplification).
[0068] Further embodiments are directed to methods of producing a
cellulase variant, or active fragment thereof, described herein
comprising: stably transforming a host cell with an expression
vector comprising a polynucleotide encoding the cellulase variant,
or active fragment thereof; culturing the transformed host cell
under suitable conditions to produce the cellulase variant, or
active fragment thereof; and recovering the cellulase variant, or
active fragment thereof.
[0069] In other embodiments, a cellulase variant, or active
fragment thereof, described herein is fused to a signal peptide for
directing the extracellular secretion of the variant, or active
fragment thereof. For example, in certain embodiments, the signal
peptide is the native signal peptide of the cellulase variant, or
active fragment thereof described herein. In other embodiments, the
signal peptide is a non-native signal peptide such as the B.
subtilis AprE signal peptide.
[0070] In some embodiments, the host cell in which the cellulase
variant, or active fragment thereof, described herein is expressed
in a heterologous organism, i.e., an organism other than
Staphylotrichum spp. Exemplary heterologous organisms, include, for
example, B. subtilis, B. licheniformis, B. lentus, B. brevis,
Geobacillus (formerly Bacillus) stearothermophilus, B.
alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.
lautus, B. megaterium, B. thuringiensis, S. lividans, S. murinus,
P. fluorescens, P. stutzerei, P. mirabilis, R. eutropha, S.
carnosus, L. lactis, E. coli, yeast (such as, for example,
Saccharomyces spp. or Schizosaccharomyces spp., e.g. S.
cerevisiae), C. lucknowense, and filamentous fungi such as
Aspergillus spp., e.g., A. oryzae or A. niger, H. grisea, H.
insolens, and T. reesei. Methods for transforming nucleic acids
into these organisms are well known in the art. A suitable
procedure for transformation of Aspergillus host cells is
described, for example, in EP238023. A suitable procedure for
transformation of Trichoderma host cells is described, for example,
in Steiger et al 2011, Appl. Environ. Microbiol. 77:114-121.
[0071] In some embodiments, the polynucleotide that encodes the
cellulase variant, or active fragment thereof has at least 60%,
65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid
sequence identity to the amino acid sequence of SEQ ID NO:3. In
some embodiments, the polynucleotide is codon-optimized for
expression in a different host, mutated to introduce cloning sites,
or otherwise altered to add functionality.
[0072] In some embodiments, expression vectors are provided in a
heterologous host cell suitable for expressing the cellulase
variant, or active fragment thereof, described herein, or suitable
for propagating the expression vector prior to introducing it into
a suitable host cell. In some embodiments, the polynucleotide is
included in an expression cassette and/or cloned into a suitable
expression vector by standard molecular cloning techniques. Such
expression cassettes or vectors contain sequences that assist
initiation and termination of transcription (e.g., promoters and
terminators), and generally contain a selectable marker
[0073] In some embodiments, a polynucleotide that encodes a
cellulase variant, or active fragment thereof, hybridizes to the
polynucleotide of SEQ ID NO:3 or the complement thereof under
specified hybridization conditions. The term "hybridization" refers
to the process by which a strand of nucleic acid joins with a
complementary strand through base pairing, as known in the art. The
term "hybridization conditions" refers to the conditions under
which hybridization reactions are conducted. These conditions are
typically classified by degree of "stringency" of the conditions
under which hybridization is measured. The degree of stringency can
be based, for example, on the melting temperature (T.sub.m) of the
nucleic acid binding complex or probe. For example, "maximum
stringency" typically occurs at about T.sub.m-5.degree. C.
(5.degree. below the T.sub.m of the probe); "high stringency" at
about 5-10.degree. C. below the T.sub.m; "intermediate stringency"
at about 10-20.degree. C. below the T.sub.m of the probe; and "low
stringency" at about 20-25.degree. C. below the T.sub.m.
Alternatively, or in addition, hybridization conditions can be
based upon the salt or ionic strength conditions of hybridization
and/or one or more stringency washes, e.g., 6.times.SSC=very low
stringency; 3.times.SSC=low to medium stringency;
1.times.SSC=medium stringency; and 0.5.times.SSC=high stringency.
Functionally, maximum stringency conditions may be used to identify
nucleic acid sequences having strict identity or near-strict
identity with the hybridization probe; while high stringency
conditions are used to identify nucleic acid sequences having about
80% or more sequence identity with the probe. For applications
requiring high selectivity, it is typically desirable to use
relatively stringent conditions to form the hybrids (e.g.,
relatively low salt and/or high temperature conditions are
used).
[0074] A further embodiment is directed to a composition comprising
one or more cellulase variant, or active fragment thereof, describe
herein. In another embodiment, the composition is selected from an
enzyme composition, detergent composition, and fabric care
composition. In some embodiments, the composition is an enzyme
composition. In other embodiments, the composition is a detergent
composition. In still other embodiments, the composition is a
fabric care composition. In yet other embodiments, the detergent
composition is a laundry detergent. In still other embodiments, the
laundry detergent is selected from heavy-duty liquid (HDL) laundry
detergent and heavy-duty dry (HDD) granular laundry detergent.
[0075] In some further embodiments, the composition is in a form
selected from a powder, liquid, granular, bar, solid, semi-solid,
gel, paste, emulsion, tablet, capsule, unit dose, sheet, and foam.
In even further embodiments, the composition is in a form selected
from a liquid, powder, granulated solid, tablet, sheet, and unit
dose. In some embodiments, the compositions described herein are
provided in unit dose form, including tablets, capsules, sachets,
pouches, sheets, and multi-compartment pouches. In some
embodiments, the unit dose format is designed to provide controlled
release of the ingredients within a multi-compartment pouch (or
other unit dose format). Suitable unit dose and controlled release
formats are known in the art (See e.g., EP2100949, EP2100947,
WO02/102955, WO04/111178, WO2013/165725, and U.S. Pat. Nos.
4,765,916 and 4,972,017). In some embodiments, the unit dose form
is provided by tablets wrapped with a water-soluble film or
water-soluble pouches.
[0076] In yet even further embodiments, the composition contains
phosphate or is phosphate-free and/or contains boron or is
boron-free. In still other embodiments, the composition contains
phosphate. In yet still other embodiments, the composition is
phosphate-free. In even still further embodiments, the composition
contains boron. In yet even still further embodiments, the
composition is boron-free.
[0077] In yet other embodiments, the composition further comprises
(i) one or more other enzymes selected from acyl transferases,
amylases, alpha-amylases, beta-amylases, alpha-galactosidases,
arabinases, arabinosidases, aryl esterases, beta-galactosidases,
beta-glucanases, carrageenases, catalases, chondroitinases,
cutinases, endo-beta-mannanases, exo-beta-mannanases, esterases,
exo-mannanases, galactanases, glucoamylases, hemicellulases,
hyaluronidases, keratinases, laccases, lactases, ligninases,
lipases, lipolytic enzymes, lipoxygenases, mannanases,
metalloproteases, oxidases, pectate lyases, pectin acetyl
esterases, pectinases, pentosanases, perhydrolases, peroxidases,
phenoloxidases, phosphatases, phospholipases, phytases,
polygalacturonases, proteases, pullulanases, reductases,
rhamnogalacturonases, second cellulase, beta-glucanases, tannases,
transglutaminases, xylan acetyl-esterases, xylanases, and
xylosidases; (ii) one or more surfactants; (iii) one or more ions
selected from calcium and zinc; (iv) one or more adjunct
ingredients; (v) one or more stabilizers; (vi) from about 0.001% to
about 5.0 weight % of the cellulase variant, or active fragment
thereof, described herein; (vii) one or more bleaching agents; or
(viii) combinations thereof.
[0078] In still further embodiments, the composition comprises one
or more other enzyme. In yet still further embodiments, the one or
more other enzyme is selected from acyl transferases, amylases,
alpha-amylases, beta-amylases, alpha-galactosidases, arabinases,
arabinosidases, aryl esterases, beta-galactosidases,
beta-glucanases, carrageenases, catalases, chondroitinases,
cutinases, endo-beta-mannanases, exo-beta-mannanases, esterases,
exo-mannanases, galactanases, glucoamylases, hemicellulases,
hyaluronidases, keratinases, laccases, lactases, ligninases,
lipases, lipolytic enzymes, lipoxygenases, mannanases,
metalloproteases, oxidases, pectate lyases, pectin acetyl
esterases, pectinases, pentosanases, perhydrolases, peroxidases,
phenoloxidases, phosphatases, phospholipases, phytases,
polygalacturonases, proteases, pullulanases, reductases,
rhamnogalacturonases, second cellulase, beta-glucanases, tannases,
transglutaminases, xylan acetyl-esterases, xylanases, and
xylosidases. In further embodiments, the compositions described
herein further comprise a protease, mannanase, and/or amylase.
[0079] In some embodiments, the composition further comprises one
or more surfactant. In some other embodiments, the surfactant is
selected from non-ionic, ampholytic, semi-polar, anionic, cationic,
zwitterionic, and combinations and mixtures thereof. In yet still
other embodiments, the surfactant is selected from anionic,
cationic, nonionic, and zwitterionic compounds. In some
embodiments, the composition comprises from about 0.1% to about
60%, about 1% to about 50%, or about 5% to about 40% surfactant by
weight of the composition. Exemplary surfactants include, but are
not limited to sodium dodecylbenzene sulfonate, C12-14 pareth-7,
C12-15 pareth-7, sodium C12-15 pareth sulfate, C14-15 pareth-4,
sodium laureth sulfate (e.g., Steol CS-370), sodium hydrogenated
cocoate, C.sub.12 ethoxylates (Alfonic 1012-6, Hetoxol LA7, Hetoxol
LA4), sodium alkyl benzene sulfonates (e.g., Nacconol 90G), and
combinations and mixtures thereof. Anionic surfactants include but
are not limited to linear alkylbenzenesulfonate (LAS),
alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate)
(AS), alcohol ethoxysulfate (AEOS or AES), secondary
alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,
alkyl- or alkenylsuccinic acid, or soap. Nonionic surfactants
include but are not limited to alcohol ethoxylate (AEO or AE),
carboxylated alcohol ethoxylates, nonylphenol ethoxylate,
alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty
acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy
alkyl fatty acid amide (e.g., as described in WO92/06154),
polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan
esters (e.g., TWEENs), polyoxyethylene alcohols, polyoxyethylene
isoalcohols, polyoxyethylene ethers (e.g., TRITONs and BRIJ),
polyoxyethylene esters, polyoxyethylene-p-tert-octylphenols or
octylphenyl-ethylene oxide condensates (e.g., NONIDET P40),
ethylene oxide condensates with fatty alcohols (e.g., LUBROL),
polyoxyethylene nonylphenols, polyalkylene glycols (SYNPERONIC
F108), sugar-based surfactants (e.g., glycopyranosides,
thioglycopyranosides), and combinations and mixtures thereof.
[0080] In a further embodiment, the detergent compositions
disclosed herein further comprise a surfactant mixture that
includes, but is not limited to 5-15% anionic surfactants, <5%
nonionic surfactants, cationic surfactants, phosphonates, soap,
enzymes, perfume, butylphenyl methylptopionate, geraniol, zeolite,
polycarboxylates, hexyl cinnamal, limonene, cationic surfactants,
citronellol, and benzisothiazolinone.
[0081] In other embodiments, the composition further comprises one
or more calcium and/or zinc ions.
[0082] In still other embodiments, the composition further
comprises one or more adjunct ingredients. In yet other
embodiments, the adjunct ingredient is selected from a bleach
activator, bleach catalyst, enzyme stabilizing system, chelant,
optical brightener, soil release polymer, dye transfer agent, dye
transfer inhibiting agent, catalytic material, hydrogen peroxide,
sources of hydrogen peroxide, preformed peracids, polymeric
dispersing agent, clay soil removal agent, structure elasticizing
agent, dispersant, suds suppressor, dye, perfume, colorant, filler
salt, hydrotrope, photoactivator, fluorescer, fabric conditioner,
hydrolyzable surfactant, solvent, preservative, anti-oxidant,
anti-shrinkage agent, anti-wrinkle agent, germicide, fungicide,
color speckle, anti-corrosion agent, alkalinity source,
solubilizing agent, carrier, processing aid, perfume, pigment, and
pH control agents (See, e.g., U.S. Pat. Nos. 6,610,642; 6,605,458;
5,705,464; 5,710,115; 5,698,504; 5,695,679; 5,686,014; and
5,646,101).
[0083] In another embodiment, the composition further comprises one
or more stabilizers. In another embodiment, the stabilizer is
selected from water-soluble sources of calcium and/or magnesium
ions; polysaccharides; and inorganic divalent metal salts,
including alkaline earth metals, such as calcium salts. In some
embodiments, the enzymes employed herein are stabilized by the
presence of water-soluble sources of zinc (II), calcium (II),
and/or magnesium (II) ions in the finished compositions, as well as
other metal ions (e.g., barium (II), scandium (II), iron (II),
manganese (II), aluminum (III), tin (II), cobalt (II), copper (II),
nickel (II), and oxovanadium (IV). Chlorides and sulfates also find
use in some embodiments. Examples of suitable oligosaccharides and
polysaccharides (e.g., dextrins) are known in the art (See, e.g.,
WO07/145964). In some embodiments, reversible protease inhibitors
also find use, such as boron-containing compounds (e.g., borate,
4-formyl phenyl boronic acid) and/or a tripeptide aldehyde find use
to further improve stability, as desired.
[0084] In yet another embodiment, the composition further comprises
an effective amount of a cellulase variant, or active fragment
thereof, described herein. In some embodiments, the effective
amount of a cellulase variant, or active fragment thereof, is from
about 0.00001% to about 10%, about 0.0001% to about 10%, about
0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to
about 0.5% of cellulase by weight of the composition. In other
embodiments, the effective amount of cellulase variant, or active
fragment thereof, is from about 0.001% to about 5.0 weight percent
composition. In still other embodiments, the effective amount of
cellulase variant, or active fragment thereof, is from about 0.001%
to about 4.5 weight percent composition. In still yet other
embodiments, the effective amount of cellulase variant, or active
fragment thereof, is from about 0.001% to about 4.0 weight percent
composition. In yet even other embodiments, the effective amount of
cellulase variant, or active fragment thereof, is from about 0.001%
to about 3.5, 3.6, 3.7, 3.8, or 3.9 weight percent composition.
[0085] In even still further embodiments, the composition further
comprises one or more bleaching agents. In yet another embodiment,
the bleaching agent is selected from inorganic and/or organic
bleaching compound(s). Inorganic bleaches may include, but are not
limited to perhydrate salts (e.g., perborate, percarbonate,
perphosphate, persulfate, and persilicate salts). In some
embodiments, inorganic perhydrate salts are alkali metal salts. In
some embodiments, inorganic perhydrate salts are included as the
crystalline solid, without additional protection, although in some
other embodiments, the salt is coated. Suitable salts include, for
example, those described in EP2100949.
[0086] In some embodiments, the compositions described herein
further comprises one or more detergent builders or builder
systems, complexing agents, polymers, foam boosters, suds
suppressors, anti-corrosion agents, soil-suspending agents,
anti-soil redeposition agents, dyes, bactericides, hydrotopes,
optical brighteners, fabric conditioners, and perfumes.
[0087] In some embodiments, the composition described herein
further comprises from about 1%, from about 3% to about 60%, or
from about 5% to about 40% builder by weight of the composition.
Builders may include, but are not limited to, the alkali metals,
ammonium and alkanolammonium salts of polyphosphates, alkali metal
silicates, alkaline earth and alkali metal carbonates,
aluminosilicates, polycarboxylate compounds, ether
hydroxypolycarboxylates, copolymers of maleic anhydride with
ethylene or vinyl methyl ether, 1,3,5-trihydroxy
benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid,
the various alkali metals, ammonium and substituted ammonium salts
of polyacetic acids such as ethylenediamine tetraacetic acid and
nitrilotriacetic acid, as well as polycarboxylates such as mellitic
acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic
acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic
acid, and soluble salts thereof.
[0088] In some embodiments, the builders form water-soluble
hardness ion complexes (e.g., sequestering builders), such as
citrates and polyphosphates (e.g., sodium tripolyphosphate and
sodium tripolyphospate hexahydrate, potassium tripolyphosphate, and
mixed sodium and potassium tripolyphosphate, etc.). Any suitable
builder can find use in the compositions described herein,
including those known in the art (See, e.g., EP 2100949).
[0089] In an even further embodiment, the pH of the composition is
neutral to basic. The laundry compositions described herein are
typically formulated such that, during use in aqueous conditions,
the wash water will have a pH of from about 3.0 to about 11. Liquid
products are typically formulated to have a neat pH from about 5.0
to about 9.0. Granular products are typically formulated to have a
pH from about 8.0 to about 11.0. Techniques for controlling pH at
recommended usage levels include the use of buffers, alkalis,
acids, etc., and are well known to those skilled in the art.
[0090] The term "granular composition" refers to a conglomeration
of discrete solid, macroscopic particles. Powders are a special
class of granular material due to their small particle size, which
makes them more cohesive and more easily suspended.
[0091] Concentrations of detergent compositions in typical wash
solutions throughout the world varies from less than about 800 ppm
of detergent composition ("low detergent concentration
geographies"), for example about 667 ppm in Japan, to between about
800 ppm to about 2000 ppm ("medium detergent concentration
geographies"), for example about 975 ppm in U.S. and about 1500 ppm
in Brazil, to greater than about 2000 ppm ("high detergent
concentration geographies"), for example about 4500 ppm to about
5000 ppm in Europe and about 6000 ppm in high suds phosphate
builder geographies.
[0092] In some embodiments, the detergent compositions described
herein may be utilized at a temperature of from about 10.degree. C.
to about 60.degree. C., or from about 20.degree. C. to about
60.degree. C., or from about 30.degree. C. to about 60.degree. C.,
from about 40.degree. C. to about 60.degree. C., from about
40.degree. C. to about 55.degree. C., or all ranges within
10.degree. C. to 60.degree. C. In some embodiments, the detergent
compositions described herein are used in "cold water washing" at
temperatures of from about 10.degree. C. to about 40.degree. C., or
from about 20.degree. C. to about 30.degree. C., from about
15.degree. C. to about 25.degree. C., from about 15.degree. C. to
about 35.degree. C., or all ranges within 10.degree. C. to
40.degree. C.
[0093] As a further example, different geographies typically have
different water hardness. Water hardness is usually described in
terms of the grains per gallon mixed Ca.sup.2+/Mg.sup.2+. Hardness
is a measure of the amount of calcium (Ca.sup.2+) and magnesium
(Mg.sup.2+) in the water. Most water in the United States is hard,
but the degree of hardness varies. Moderately hard (60-120 ppm) to
hard (121-181 ppm) water has 60 to 181 parts per million (parts per
million converted to grains per U.S. gallon is ppm # divided by
17.1 equals grains per gallon) of hardness minerals.
TABLE-US-00001 TABLE II Water Hardness Levels Water Grains per
gallon Parts per million Soft less than 1.0 less than 17 Slightly
hard 1.0 to 3.5 17 to 60 Moderately hard 3.5 to 7.0 60 to 120 Hard
7.0 to 10.5 120 to 180 Very hard greater than 10.5 greater than
180
[0094] European water hardness is typically greater than about 10.5
(for example about 10.5 to about 20.0) grains per gallon mixed
Ca.sup.2+/Mg.sup.2+ (e.g., about 15 grains per gallon mixed
Ca.sup.2+/Mg.sup.2+). North American water hardness is typically
greater than Japanese water hardness, but less than European water
hardness. For example, North American water hardness can be between
about 3 to about 10 grains, about 3 to about 8 grains or about 6
grains. Japanese water hardness is typically lower than North
American water hardness, usually less than about 4, for example
about 3 grains per gallon mixed Ca.sup.2+/Mg.sup.2+.
[0095] In some embodiments, the detergent compositions described
herein further comprise a protease. In some embodiments the
composition comprises from about 0.00001% to about 10% protease by
weight of the composition. In another embodiment, the cleaning
composition comprises from about 0.0001% to about 10%, about 0.001%
to about 5%, about 0.001% to about 2%, or about 0.005% to about
0.5% protease by weight of the composition.
[0096] In one embodiment, the protease is a serine protease.
Suitable proteases include those of animal, vegetable or microbial
origin. In some embodiments, the protease is a microbial protease.
In other embodiments, the protease is a chemically or genetically
modified mutant. In another embodiment, the protease is an alkaline
microbial protease or a trypsin-like protease. Exemplary alkaline
proteases include subtilisins derived from, for example, Bacillus
(e.g., subtilisin, lentus, amyloliquefaciens, subtilisin Carlsberg,
subtilisin 309, subtilisin 147 and subtilisin 168). Exemplary
additional proteases include but are not limited to those described
in WO9221760, WO9523221, WO2008010925, WO09149200, WO09149144,
WO09149145, WO 10056640, WO10056653, WO20100566356, WO11072099,
WO201113022, WO11140364, WO 12151534, WO2015038792, WO2015089447,
WO2015089441, WO2015/143360, WO2016 061438, WO2016069548,
WO2016069544, WO2016069557, WO2016069563, WO2016 069569,
WO2016069552, WO2016145428, WO2016183509, US Publ. No. 20080090747,
U.S. Pat. No. 5,801,039, U.S. Pat. No. 5,340,735, U.S. Pat. No.
5,500,364, U.S. Pat. No. 5,855,625, U.S. Pat. No. RE34606, U.S.
Pat. No. 5,955,340, U.S. Pat. No. 5,700,676, U.S. Pat. No.
6,312,936, U.S. Pat. No. 6,482,628, U.S. Pat. No. 8,530,219, U.S.
Provisional Appl Nos. 62/331,282, 62/332,417, 62/343,618, and
62/351,649, and International Appl No. PCT/US2016/038245, as well
as metalloproteases described in WO1999014341, WO1999033960,
WO1999014342, WO1999 034003, WO2007044993, WO2009058303,
WO2009058661, WO2014071410, WO2014 194032, WO2014194034,
WO2014194054, and WO2014 194117. Exemplary proteases also include,
but are not limited to trypsin (e.g., of porcine or bovine origin)
and the Fusarium protease described in WO8906270.
[0097] Exemplary commercial proteases include, but are not limited
to MAXATASE.RTM., MAXACAL.TM., MAXAPEM.TM., OPTICLEAN.RTM.,
OPTIMASE.RTM., PROPERASE.RTM., PURAFECT.RTM., PURAFECT.RTM. OXP,
PURAMAX.TM., EXCELLASE.TM., PREFERENZ.TM. proteases (e.g. P100,
P110, P280), EFFECTENZ.TM. proteases (e.g. P1000, P1050, P2000),
EXCELLENZ.TM. proteases (e.g. P1000), ULTIMASE.RTM., and
PURAFAST.TM. (DuPont); ALCALASE.RTM., ALCALASE.RTM. ULTRA,
BLAZE.RTM., BLAZE.RTM. EVITY.RTM., BLAZE.RTM. EVITY.RTM. 16L,
CORONASE.RTM., SAVINASE.RTM., SAVINASE.RTM. ULTRA, SAVINASE.RTM.
EVITY.RTM., SAVINASE.RTM. EVERTS.RTM., PRIMASE.RTM., DURAZYM.TM.,
POLARZYME.RTM., OVOZYME.RTM., KANNASE.RTM., LIQUANASE.RTM.,
LIQUANASE EVERIS.RTM., NEUTRASE.RTM., PROGRESS UNO.RTM.,
RELASE.RTM. and ESPERASE.RTM. (Novozymes); BLAP.TM. and BLAP.TM.
variants (Henkel); LAVERGY.TM. PRO 104 L (BASF). and KAP (B.
alkalophilus subtilisin (Kao)).
[0098] In some embodiments, the detergent compositions described
herein further comprise a suitable amylase. In one embodiment, the
composition comprises from about 0.00001% to about 10%, about
0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to
about 2%, or about 0.005% to about 0.5% amylase by weight of the
composition. Any amylase (e.g., alpha and/or beta) suitable for use
in alkaline solutions may be useful to include in such composition.
An exemplary amylase can be a chemically or genetically modified
mutant. Exemplary amylases include, but are not limited amylases
described in GB1296839, WO91 00353, WO9402597, WO94183314,
WO9510603, WO9526397, WO9535382, WO9605295, WO9623873, WO9623874,
WO9630481, WO9710342, WO9741213, WO9743424, WO98 13481, WO9826078,
WO9902702, WO9909183, WO9919467, WO9923211, WO9929876, WO9942567,
WO9943793, WO9943794, WO9946399, WO0029560, WO0060058, WO00 60059,
WO0060060, WO0114532, WO0134784, WO0164852, WO0166712, WO0188107,
WO0196537, WO02092797, WO0210355, WO0231124, WO2004055178,
WO2004113551, WO2005001064, WO2005003311, WO2005018336, WO
2005019443, WO2005066338, WO 2006002643, WO2006012899,
WO2006012902, WO2006 031554, WO2006063594, WO2006 066594,
WO2006066596, WO2006136161, WO2008 000825, WO2008088493, WO2008
092919, WO2008101894, WO2008112459, WO2009 061380, WO2009061381,
WO2009 100102, WO2009140504, WO2009149419, WO2010 059413,
WO2010088447, WO2010 091221, WO2010104675, WO2010 115021,
WO10115028, WO2010117511, WO2011076123, WO2011076897, WO2011080352,
WO2011080353, WO2011080354, WO2011082425, WO 2011082429,
WO2011087836, WO2011098531, WO2013063460, WO2013184577, WO2014
099523, WO2014164777, and WO2015077126. Exemplary commercial
amylases include, but are not limited to AMPLIFY.RTM., AMPLIFY
PRIME.RTM., BAN.TM. DURAMYL.RTM., TERMAMYL.RTM., TERMAMYL.RTM.
ULTRA, FUNGAMYL.RTM., STAINZYME.RTM., STAINZYME PLUS.RTM.,
STAINZYME ULTRA.RTM., and STAINZYME EVITY.RTM. (Novozymes);
EFFECTENZ.TM. S 1000, POWERASE.TM., PREFERENZ.TM. S 100,
PREFERENZ.TM. S 110, EXCELLENZ.TM. S 2000, RAPIDASE.RTM. and
MAXAMYL.RTM. P (DuPont).
[0099] In some embodiments, the detergent compositions described
herein further comprise a suitable pectin degrading enzyme. As used
herein, "pectin degrading enzyme(s)" encompass arabinanase (EC
3.2.1.99), galactanases (EC 3.2.1.89), polygalacturonase (EC
3.2.1.15) exo-polygalacturonase (EC 3.2.1.67),
exo-poly-alpha-galacturonidase (EC 3.2.1.82), pectin lyase (EC
4.2.2.10), pectin esterase (EC 3.2.1.11), pectate lyase (EC
4.2.2.2), exo-polygalacturonate lyase (EC 4.2.2.9) and
hemicellulases such as endo-1,3-.beta.-xylosidase (EC 3.2.1.32),
xylan-1,4-.beta.-xylosidase (EC 3.2.1.37) and
.alpha.-L-arabinofuranosidase (EC 3.2.1.55). Pectin degrading
enzymes are natural mixtures of the above mentioned enzymatic
activities. Pectin enzymes therefore include the pectin
methylesterases which hdyrolyse the pectin methyl ester linkages,
polygalacturonases which cleave the glycosidic bonds between
galacturonic acid molecules, and the pectin transeliminases or
lyases which act on the pectic acids to bring about non-hydrolytic
cleavage of .alpha.-1,4 glycosidic linkages to form unsaturated
derivatives of galacturonic acid.
[0100] Suitable pectin degrading enzymes include those of plant,
fungal, or microbial origin. In some embodiments, chemically or
genetically modified mutants are included. In some embodiments, the
pectin degrading enzymes are alkaline pectin degrading enzymes,
i.e., enzymes having an enzymatic activity of at least 10%, at
least 25%, or at least 40% of their maximum activity at a pH of
from about 7.0 to about 12. In certain other embodiments, the
pectin degrading enzymes are enzymes having their maximum activity
at a pH of from about 7.0 to about 12. Alkaline pectin degrading
enzymes are produced by alkalophilic microorganisms e.g.,
bacterial, fungal, and yeast microorganisms such as Bacillus
species. In some embodiments, the microorganisms are B. firmus, B.
circulans, and B. subtilis as described in JP 56131376 and JP
56068393. Alkaline pectin decomposing enzymes may include but are
not limited to galacturn-1,4-.alpha.-galacturonase (EC 3.2.1.67),
polygalacturonase activities (EC 3.2.1.15, pectin esterase (EC
3.1.1.11), pectate lyase (EC 4.2.2.2) and their iso enzymes.
Alkaline pectin decomposing enzymes can be produced by the Erwinia
species. In some embodiments, the alkaline pectin decomposing
enzymes are produced by E. chrysanthemi, E. carotovora, E.
amylovora, E. herbicola, and E. dissolvens as described in JP
59066588, JP 63042988, and in World J. Microbiol. Microbiotechnol.
(8, 2, 115-120) 1992. In certain other embodiments, the alkaline
pectin enzymes are produced by Bacillus species as disclosed in JP
73006557 and Agr. Biol. Chem. (1972), 36 (2) 285-93. In some
embodiments, the detergent compositions described herein further
comprise about 0.00001% to about 10%, about 0.0001% to about 10%,
about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005%
to about 0.5% of pectin degrading enzyme by weight of the
composition.
[0101] In some embodiments, the detergent compositions described
herein further comprise a suitable mannanase enzyme. In one
embodiment, the composition comprises from about 0.00001% to about
10%, about 0.0001% to about 10%, about 0.001% to about 5%, about
0.001% to about 2%, or about 0.005% to about 0.5% mannanase by
weight composition. An exemplary mannanase can be a chemically or
genetically modified mutant. Exemplary mannanases include, but are
not limited to, those of bacterial or fungal origin, such as, for
example, as is described in WO 2016/007929; U.S. Pat. Nos.
6,566,114; 6,602,842; and 6,440,991; and International Patent Appl
Nos: PCT/US2016/060850 and PCT/US2016/060844 filed Nov. 7, 2016.
Exemplary commercial mannanases include, but are not limited to
MANNAWAY.RTM. (Novozymes) and EFFECTENZ.TM. M 1000, PREFERENZ.RTM.
M 100, MANNASTAR.RTM., and PURABRITE.TM. (DuPont).
[0102] In some further embodiments, the detergent compositions
described herein further comprise a suitable second cellulase.
Suitable second cellulases include, but are not limited to those of
bacterial or fungal origin. Chemically or genetically modified
mutants are included in some embodiments. Suitable second
cellulases include, but are not limited to H. insolens cellulases
(See, e.g., U.S. Pat. No. 4,435,307). Especially suitable
cellulases are the cellulases having color care benefits (See,
e.g., EP0495257). Commercially available second cellulases include,
but are not limited to ENDOLASE.RTM., CELLUCLEAN.RTM.,
CELLUZYME.RTM., CAREZYME.RTM., RENOZYME.RTM., and CAREZYME.RTM.
PREMIUM (Novozymes A/S, Denmark), PURADEX.RTM., (DuPont), and
KAC-500(B).TM. (Kao Corporation). In some embodiments, cellulases
are incorporated as portions or fragments of mature wild-type or
variant cellulases, wherein a portion of the N-terminus is deleted
(See, e.g., U.S. Pat. No. 5,874,276). In some embodiments, the
detergent compositions described herein further comprise from about
0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to
about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5%
of cellulase by weight of the composition.
[0103] In still further embodiments, the detergent compositions
described herein further comprise a suitable lipase. In some
embodiments, the composition comprises from about 0.00001% to about
10%, about 0.0001% to about 10%, about 0.001% to about 5%, about
0.001% to about 2%, or about 0.005% to about 0.5% lipase by weight
composition. An exemplary lipase can be a chemically or genetically
modified mutant. Exemplary lipases include, but are not limited to,
e.g., bacterial or fungal origin, such as, e.g., H. lanuginosa
lipase (See, e.g., EP258068, and EP305216), R. miehei lipase (See,
e.g., EP 238 023), Candida lipase, such as C. antarctica lipase
(e.g., C. antarctica lipase A or B; see, e.g., EP214761),
Pseudomonas lipases such as P. alcaligenes lipase and P.
pseudoalcaligenes lipase (See, e.g., EP218272), P. cepacia lipase
(See, e.g., EP331376), P. stutzeri lipase (See, e.g., GB
1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B.
subtilis lipase [Dartois et al., (1993) Biochem. Biophys. Acta
1131:253-260]; B. stearothermophilus lipase [See, e.g., JP
64/744992]; and B. pumilus lipase [See, e.g., WO91/16422]).
Exemplary cloned lipases include, but are not limited to P.
camembertii lipase (See, Yamaguchi et al., [1991] Gene 103:61-67),
G. candidum lipase (See, Schimada et al., [1989] J. Biochem.
106:383-388), and various Rhizopus lipases such as R. delemar
lipase (See, Hass et al., [1991] Gene 109:117-113), R. niveus
lipase (Kugimiya et al., [1992] Biosci. Biotech. Biochem.
56:716-719), and R. oryzae lipase. Other types of suitable
lipolytic enzymes include cutinases such as, for example, cutinase
derived from P. mendocina (See, WO88/09367) and from F. solani pisi
(See, WO90/09446). Exemplary commercial lipases include, but are
not limited to M1 LIPASE.TM., LUMA FAST.TM., and LIPOMAX.TM.
(DuPont); LIPEX.RTM., LIPOCLEAN.RTM., LIPOLASE.RTM. and
LIPOLASE.RTM. ULTRA (Novozymes); and LIPASE P.TM. (Amano
Pharmaceutical Co. Ltd).
[0104] In some embodiments, detergent compositions described herein
further comprise peroxidases in combination with hydrogen peroxide
or a source thereof (e.g., a percarbonate, perborate or
persulfate). In some alternative embodiments, oxidases are used in
combination with oxygen. Both types of enzymes are used for
"solution bleaching" (i.e., to prevent transfer of a textile dye
from a dyed fabric to another fabric when the fabrics are washed
together in a wash liquor), preferably together with an enhancing
agent (See, e.g., WO94/12621 and WO95/01426). Suitable
peroxidases/oxidases include, but are not limited to those of
plant, bacterial or fungal origin. Chemically or genetically
modified mutants are included in some embodiments. In some
embodiments, the detergent compositions further comprise from about
0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to
about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% of
peroxidase and/or oxidase by weight of composition.
[0105] In some embodiments, detergent compositions described herein
further comprise additional enzymes, including but not limited to
perhydrolases (See, e.g., WO 05/056782).
[0106] In some embodiments, the detergent compositions described
herein further comprise at least one chelating agent. Suitable
chelating agents may include, but are not limited to copper, iron,
and/or manganese chelating agents, and mixtures thereof. In
embodiments in which at least one chelating agent is used, the
detergent compositions comprise from about 0.1% to about 15% or
even from about 3.0% to about 10% chelating agent by weight of
composition.
[0107] In some still further embodiments, the detergent
compositions described herein further comprise at least one
deposition aid. Suitable deposition aids include, but are not
limited to, polyethylene glycol, polypropylene glycol,
polycarboxylate, soil release polymers such as polytelephthalic
acid, clays such as kaolinite, montmorillonite, atapulgite, illite,
bentonite, halloysite, and mixtures thereof.
[0108] In some embodiments, the detergent compositions described
herein further comprise at least one anti-redeposition agent. In
some embodiments, the anti-redeposition agent is a non-ionic
surfactant, such as, for example, described in EP2100949.
[0109] In some embodiments, the detergent compositions described
herein further comprise one or more dye transfer inhibiting agents.
Suitable polymeric dye transfer inhibiting agents include, but are
not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide
polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,
polyvinyloxazolidones, and polyvinylimidazoles, or mixtures
thereof. In some embodiments, the detergent compositions described
herein comprise from about 0.0001% to about 10%, from about 0.01%
to about 5%, or even from about 0.1% to about 3% dye transfer
inhibiting agent by weight of composition.
[0110] In some embodiments, the detergent compositions described
herein further comprise one or more silicates. In some such
embodiments, sodium silicates (e.g., sodium disilicate, sodium
metasilicate, and crystalline phyllosilicates) find use. In some
embodiments, the detergent compositions described herein comprise
from about 1% to about 20% or from about 5% to about 15% silicate
by weight of the composition.
[0111] In yet further embodiments, the detergent compositions
described herein further comprise one or more dispersant. Suitable
water-soluble organic materials include, but are not limited to the
homo- or co-polymeric acids or their salts, in which the
polycarboxylic acid comprises at least two carboxyl radicals
separated from each other by not more than two carbon atoms.
[0112] In yet further embodiments, the detergent compositions
described herein further comprise one or more bleach activator
and/or bleach catalyst. Bleach activators are typically organic
peracid precursors that enhance the bleaching action in the course
of cleaning at temperatures of 60.degree. C. and below. Bleach
activators suitable for use herein include compounds which, under
perhydrolysis conditions, give aliphatic peroxycarboxylic acids
having preferably from about 1 to about 10 carbon atoms, in
particular from about 2 to about 4 carbon atoms, and/or optionally
substituted perbenzoic acid. Suitable bleach activators include,
for example, those described in EP2100949. Bleach catalysts
typically include, for example, manganese triazacyclononane and
related complexes, and cobalt, copper, manganese, and iron
complexes, as well as those described in U.S. Pat. Nos. 4,246,612;
5,227,084; 4,810,410; and WO99/06521 and EP2100949.
[0113] In some embodiments, fabric is exposed to a cellulase
variant, or active fragment thereof, described herein prior to
being worn such that removal of soil subsequently adhered to the
fabric is improved in the first and/or subsequent two, three, or
more wash cycles over soiled fabric that is not exposed to the
cellulase variant, or active fragment thereof, described herein
prior to being worn. In other embodiments, fabric is exposed to the
cellulase variant, or active fragment thereof, described herein
after being worn such that the removal of soil subsequently adhered
to the fabric is improved in the first and/or subsequent two,
three, or more wash cycles over soiled fabric that is not
subsequently exposed to the cellulase variant, or active fragment
thereof, described herein. In still further embodiments, cellulase
variant, or active fragment thereof, described herein finds use in
a detergent composition, a textile finishing process, or a paper
and pulp process.
[0114] Yet another embodiment is directed to enhancing the feel
and/or appearance and/or providing color enhancement and/or a stone
washed appearance to a cellulose containing textile material (such
as, for example, cotton, flax, ramie, jute, viscose, modified
viscose fibers, lyocell and cupro) comprising treating the material
with an effective amount of a cellulase variant, or active fragment
thereof, described herein or a composition comprising an effective
amount of a cellulase variant, or active fragment thereof,
described herein. A still further embodiment is directed to a
method for reducing color redeposition during the stone washing of
colored fabrics comprising contacting the fabric with an effective
amount of a cellulase variant, or active fragment thereof,
described herein or a composition comprising an effective amount of
a cellulase variant, or active fragment thereof, described herein
under conditions sufficient to impart a stone-washed appearance to
the fabric. In still another embodiment, a cellulose containing
textile material treated with an effective amount of a cellulase
variant, or active fragment thereof, described herein or a
composition comprising an effective amount of a cellulase variant,
or active fragment thereof, described herein retains substantially
all of the material's tensile strength as compared to an untreated
cellulose containing textile material. In yet still another
embodiment, a cellulose containing textile material treated with an
effective amount of a cellulase variant, or active fragment
thereof, described herein or a composition comprising an effective
amount of a cellulase variant, or active fragment thereof,
described herein retains 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, and 100% of the material's tensile
strength as compared to an untreated cellulose containing textile
material.
[0115] Other aspects and embodiments of the present compositions
and methods will be apparent from the foregoing description and
following examples. Various alternative embodiments beyond those
described herein can be employed in practicing the invention
without departing from the spirit and scope of the invention.
Accordingly, the claims, and not the specific embodiments described
herein, define the scope of the invention and as such methods and
structures within the scope of the claims and their equivalents are
covered thereby.
EXAMPLES
[0116] The following examples are provided to demonstrate and/or
illustrate certain aspects of the present disclosure and should not
be construed to limit the scope of the disclosure or any
subsequently claimed invention.
Example 1
Assays
[0117] The following assays are standard assays used in the
examples described below. Occasionally specific protocols called
for deviations from these standard assays. In those cases,
deviations from these standard assay protocols below are identified
in the examples.
Performance Index
[0118] The performance index (PI) of an enzyme compares the
performance of the variant (measured value) with a parent or
reference polypeptide (theoretical or measured value) at the same
protein concentration. Theoretical concentrations for the parent or
reference polypeptide can be calculated using the parameters
extracted from a Langmuir fit of a standard curve of the parent
enzyme. A PI that is greater than 1 (PI>1) indicates improved
performance by a variant as compared to the parent or reference
polypeptide, while a PI of 1 (PI=1) identifies a variant that
performs the same as the parent or reference polypeptide, and a PI
that is less than 1 (PI<1) identifies a variant that does not
perform as well as the parent or reference polypeptide. For
example, the STCE1 wild-type (STCE1-WT) mature protein set forth as
SEQ ID NO:5 is the parent of the STCE1-A142P variant set forth as
SEQ ID NO:8.
Protein Determination Assay (A280)
[0119] Absorbance at 280 nm was measured for purified enzyme
samples in microtiter plates (Costar 3635, Sigma/Aldrich, USA)
using a SpectraMax plate reader (Molecular Devices, USA). The
responses of the STCE1-WT standards (standard protein ranges 50 ppm
to 1000 ppm) were used to plot a standard curve. Absorbance values
of variant samples were then interpolated from those graphs.
Protein Determination Assay (HPLC)
[0120] For high resolution concentration determinations, high
performance liquid chromatography (HPLC) method was performed on
protein samples. An Agilent 1290 U (HPLC) equipped with a Zorbax
C-3 column was used for protein quantitation. Samples were eluted
from the column using a gradient of 0.1% trifluoroacetic acid (TFA)
in water and 0.07% TFA in acetonitrile. Absorbance was measured at
220 nm, and peaks were integrated using ChemStation software
(Agilent Technologies, USA). The protein concentrations of the
samples were calculated based on a standard curve of the purified
STCE1-WT mature protein (SEQ ID NO:5).
Cellulase Activity Assay Using PAHBAH Reagent
[0121] The activity of cellulase enzymes was tested on
carboxymethyl cellulose (CMC) substrate using the 4-hydroxybenzoic
acid hydrazide (PAHBAH) reagent. The enzymatic activity was based
on the hydrolysis of CMC substrate into reducing sugars followed by
the detection of the new reducing ends by the PAHBAH reagent. The
reagent PAHBAH stock solution (Sigma-Aldrich, USA) was prepared as
5% solution in 0.5 N hydrochloric acid (HCL). The PAHBAH reagent
was further diluted in 0.5 M sodium hydroxide (NaOH) in 1:4 ratios
to make up a final 1% solution. 1% CMC substrate was made in one of
three different buffer solutions: 50 mM HEPES buffer solution, pH
8.2; 50 mM sodium acetate (NaOAc) buffer solution, pH 5; and 50 mM
CAPS buffer solution, pH 10.8. The enzyme samples, adjusted to
either pH 5 (4 pH 8.2 (6 .mu.L) or pH 10 (8 .mu.L), were added to a
microtiter plates (MTP) containing 40 .mu.l of the 1% CMC solution
and incubated at 40.degree. C. in iEMS.TM. Microplate
Incubator/Shaker HT (ThermoFisher Scientific Inc, USA) for 15 min;
then 8 .mu.l of enzyme-substrate mix was transferred to a new MTP
containing 20 .mu.l of 1% PAHBAH per well and incubated at
95.degree. C. in a thermocycler machine (Eppendorf.TM.
Mastercycler.TM. pro PCR system, Thermo Fisher Scientific, USA) for
5 min. Finally, 20 .mu.l of each reaction solution was transferred
to a fresh MTP and the absorbance was measured at 410 nm using a
SpectraMax plate reader.
Stability Measurement Using Cellulase Activity Assay
[0122] Cellulase enzyme samples were tested for stability in the
presence and absence of protease under various stress conditions
set forth on Table 1 by measuring residual activity. The stressed
and unstressed cellulase activities were measured using the
cellulase activity assay described herein. The protease used in the
assays was commercially available BPN'-Y217L subtilisin, which is
forth below as SEQ ID NO:7.
[0123] The stability assay conditions that were used are described
in Table 1. Prior to using commercially available detergent in the
stability assays, the enzymes contained in the detergent was
inactivated by heating the detergent samples in a water bath set to
100.degree. C. for 2 hrs.
TABLE-US-00002 TABLE 1 Conditions for cellulase stability assays
using CMC substrate Stress temperature (.degree. C.) Measurement
Condition and incubation time Thermostability of STCE1-WT 50 mM
HEPES buffer pH 8.2 80.degree. C. for 10 min and variants thereof
Stability of STCE1-WT and 1500 ppm BPN'-Y217L protease 65.degree.
C. for 37 min variants thereof in the presence in 50 mM HEPES
buffer pH 8.2 of Protease Stability of H. insolens, 1500 ppm
BPN'-Y217L protease 65.degree. C. for 15 min N. crassa, T.
terrestris and in 50 mM HEPES buffer pH 8.2 variants thereof
Stability of STCE1-WT and 1% OMO Klein & Krachtig* plus
37.degree. C. for 24 hr variants thereof in the presence 1000 ppm
BPN'-Y217L protease of detergent and protease in 50 mM HEPES buffer
pH 8.2 Stability of H. insolens and 1% OMO Klein & Krachtig*
plus 37.degree. C. for 36 hr variants thereof in the presence 1000
ppm BPN'-Y217L protease of detergent and protease in 50 mM HEPES
buffer pH 8.2 Stability of N. crassa and 1% OMO Klein &
Krachtig* plus 37.degree. C. for 2 hr variants thereof in the
presence 1000 ppm BPN'-Y217L protease of detergent and protease in
50 mM HEPES buffer pH 8.2 Stability of T. terrestris and 1% OMO
Klein & Krachtig* plus 37.degree. C. for 4 hr variants thereof
in the presence 1000 ppm BPN'-Y217L protease of detergent and
protease in 50 mM HEPES buffer pH 8.2 *Heavy-duty liquid laundry
detergent commercially sold by Unilever
[0124] For the unstressed test conditions, the enzyme samples were
not incubated prior to assaying for activity.
[0125] For the stressed test conditions, the enzyme samples were
prepared in PCR/MTP plates, sealed, and incubated at the elevated
temperatures and under the conditions described in Table 1 using:
i) an Eppendorf.TM. 384 MasterCycler.TM. Pro Thermocycler to
measure thermostability, and stability in the presence of protease
(ii) an iEMS incubator to measure stability in the presence of
detergent and protease. After the various incubation periods, the
samples were assayed for cellulase activity.
[0126] Stressed and unstressed activities were measured by the
cellulase activity assay described above. The residual activity of
the enzyme sample tested in each assay was calculated as the mean
of three replicates with blank subtracted. The relative residual
activity of each enzyme sample was calculated as the ratio between
residual activity and unstressed activity. The relative residual
activity percentage was calculated by multiplying the relative
residual activity number by 100. The PI of each STCE1-WT variant
was calculated as the ratio between relative residual activity for
the variant and relative residual activity for the STCE1-WT
cellulase.
Example 2
Expression of Various G1145 Cellulases and Variants Thereof
[0127] The gene encoding the STCE1-WT cellulase (previously
described as a family 45 glycoside hydrolase in the publication,
Koga, J., Y. Baba, A. Shimonaka, T. Nishimura, S. Hanamura and T.
Kono (2008), "Purification and characterization of a new family 45
endoglucanase, STCE1, from Staphylotrichum coccosporum and its
overproduction in Humicola insolens." Appl. Environ. Microbiol.
74(13): 4210-4217) was selected as a template for generating the
parent cellulase and variants thereof. The genes encoding the
Humicola insolens-WT cellulase (accession number CAA01574.1), and
the Neurospora crassa-WT cellulase (accession number XP_957107.1)
were also selected as templates for generating the parent
cellulases and variants thereof. In addition, a one amino acid
variant of the Thielavia terrestris-WT cellulase (accession number
XP_003651003.1) was also selected as template for generating the
parent cellulase and variants thereof. The modified Thielavia
terrestris cellulase has a histidine (H) at position 120 of the
mature cellulase protein sequence and is describe here as Thielavia
terrestris 120H.
[0128] The stce1, T. terrestris and N. crassa genes, encoding the
wildtype (WT) form of the proteins (also referred to herein as the
parent), contained an intron within the coding DNA that was removed
using molecular biology techniques known in the art. The resulting
genes were sub-cloned into the expression vector pTTT-pyr2
utilizing standard reagents. The pTTTpyr2 vector is similar to the
pTTTpyrG vector described previously, for example, in
WO2011153449A1 except that the A. nidulans pyrG gene is replaced
with the H. jecorina pyr2 gene. The pTTT-pyr2 expression vector
contained the T. reesei cbhI-derived promoter (cbhI) and cbhI
terminator regions allowing for a strong inducible expression of
the gene of interest, the A. nidulans amdS and pyr2 selective
markers conferring growth of transformants on acetamide as a sole
nitrogen source, and the T. reesei telomere regions allowing for
non-chromosomal plasmid maintenance in a fungal cell.
[0129] A map of the pTTT-pyr2 based vector containing the stce1-WT
cellulase gene (SEQ ID NO:3), pTTT-pyr2-stce1 is shown in FIG. 1.
Similarly built vectors (pTTT-pyr2-H. insolens, pTTT-pyr2-N.
crassa, and pTTT-pyr2-T. terrestris) were prepared for the
expression of the H. insolens, N. crassa and T. terrestris
cellulases.
[0130] Using molecular biology techniques known in the art, stce1,
H. insolens, N. crassa and T. terrestris single amino acid
substitutions were designed by introducing the codon sequence for
the desired amino acid change in the base plasmids pTTT-pyr2-stce1,
pTTT-pyr2-H. insolens, pTTT-pyr2-N. crassa, and pTTT-pyr2-T.
terrestris. Protoplasts of a suitable Trichoderma reesei host
strains were transformed by the PEG based protocol described in
U.S. Pat. No. 8,679,792. After growth of transformants, spores from
each well were pooled and re-patched using minimal medium
containing acetamide as a sole nitrogen source. Upon sporulation,
spores were harvested and used for inoculation in a Aachen medium.
Cultures were incubated for 6 days at 28.degree. C., 80% humidity
and 50 mm shaking throw in a Multitron shaker incubator (Infors,
Switzerland). The culture broth was filtered to collect the
clarified supernatant, which was stored at -20.degree. C.
[0131] The nucleotide sequence of the stce1-WT gene in the
pTTT-pyr2-STCE1 vector is set forth as SEQ ID NO:3 (951 base
pairs). The amino acid sequence of the translation product of the
stce1-WT gene is set forth as SEQ ID NO:4 (316 amino acids),
wherein the N-terminal 21 amino acids constitute the signal
peptide. The amino acid sequence of the mature form of STCE1-WT
protein is set forth as SEQ ID NO:5 (295 amino acids), wherein the
C-terminal 37 amino acids constitute the carbohydrate binding
module (CBM).
[0132] The STCE1-WT and variant proteins were purified by ammonium
sulfate precipitation, adding 3M (NH.sub.4).sub.2SO.sub.4 to
clarified culture broth in 1:1 dilution and using a Relisorb OC 400
resin (Resindon, Italy) in 20 mM KH.sub.2PO.sub.4, pH 6.0. The
resin was washed with 20 mM KH.sub.2PO.sub.4 pH 6.0+0.5M
(NH.sub.4).sub.2SO.sub.4 buffer and the STCE1 proteins were eluted
in 20 mM KH.sub.2PO.sub.4 pH 6.0 buffer and refrigerated until
assayed.
[0133] The nucleotide sequence of the H. insolens-WT gene in the
pTTT-pyr2-H. insolens vector is set forth as SEQ ID NO:9. The amino
acid sequence of the translation product of the H. insolens-WT gene
is set forth as SEQ ID NO:10. The amino acid sequence of the mature
H. insolens-WT cellulase is set forth as SEQ ID NO:11.
[0134] The nucleotide sequence of the N. crassa-WT gene in the
pTTT-pyr2-N. crassa vector is set forth as SEQ ID NO:12. The amino
acid sequence of the translation product of the N. crassa-WT gene
is set forth as SEQ ID NO:13. The amino acid sequence of the mature
N. crassa-WT cellulase is set forth as SEQ ID NO:14.
[0135] The nucleotide sequence of the T. terrestris-120H gene in
the pTTT-pyr2-T. terrestris vector is set forth as SEQ ID NO:15.
The amino acid sequence of the translation product of the T.
terrestris-120H gene is set forth as SEQ ID NO:16. The amino acid
sequence of the mature T. terrestris-120H cellulase is set forth as
SEQ ID NO:17.
Example 3
Crystal Structure Determination and Unique Features of STCE1-WT
Cellulase
Crystallization of STCE1-WT Protein
[0136] The mature STCE1-WT protein (SEQ ID NO:5) was found
refractory to crystallization. Purified STCE1-WT protein was
digested by papain enzyme, which clipped the protein backbone
following the G214 position in the linker domain of the mature
STCE1-WT protein (SEQ ID NO:5). The amino acid sequence of the
clipped STCE1-WT protein, also referred to as truncated STCE1-WT,
obtained by papain digestion is set forth as SEQ ID NO:6 (214 amino
acids).
[0137] This truncated STCE1-WT protein was crystallized using the
hanging drop method from a solution of protein stock at a
concentration of 25.5 mg/mL in 50 mM IVIES pH 6.3. Aliquots of 2.5
mL of the protein stock and 2.5 mL of the crystallization solution
(1.79M ammonium sulfate, 95 mM Tris pH 8.5 and 91 mM sodium iodide)
were mixed on a plastic coverslip and inverted and sealed on a
chamber containing the crystallization solution in a Linbro
6.times.4 culture plate. Crystals grew in the space group P43212
with unit cell dimensions; a=39.4 .ANG., b=39.4 .ANG., and c=228.65
.ANG.. Data were collected on the native crystal to a resolution of
2.0 .ANG. and the structure of STCE1-WT was determined by molecular
replacement using a related protein, Melanocarpus albomyces
endoglucanase (pdb ID 10A7) as the phasing model. The crystal data
collected for STCE1-WT is set forth in Table 2.
TABLE-US-00003 TABLE 2 Statistics of truncated STCE1-WT data
Wavelength 1.54 .ANG. Space group P4.sub.32.sub.12 Molecules in
asymmetric unit 1 Unit cell dimensions 39.4, 39.4, 228.65 .ANG.
Resolution 2.0 Unique reflections 13244 Multiplicity 6.4
Completeness 94% R.sub.merge 0.06 I/.sigma..sub.I 21
[0138] The model was fitted in the resulting electron density using
the program COOT as described in Emsley et al (Emsley, P., B.
Lohkamp, W. G. Scott and K. Cowtan (2010). "Features and
development of Coot." Acta Crystallogr D Biol Crystallogr 66(Pt 4):
486-501). After fitting and refitting adjustments, the coordinates
were refined using the REFMAC program with standard defaults in the
CCP4 software suite. The statistics of the structural model are
presented in Table 3.
TABLE-US-00004 TABLE 3 Statistics of the refined model R work 0.196
R free 0.262 No. protein residues 214 No. atoms 1674 rmsd Bond
lengths 0.015 .ANG. rmsd bond angles 1.79.degree.
Structure Determination of Truncated STCE1-WT
[0139] FIG. 2 depicts the structure of the catalytic core of
truncated STCE1-WT cellulase. The catalytic core can be visualized
as consisting of three subdomains: a twisted beta barrel domain
formed by strands belonging to residues 1-9, 61-121, and 180-185;
an alpha helical domain formed by residues 125-172; and an extended
loop domain formed by residues 10-60. The C-terminal segment, which
would extend into the linker and CBM in the full length molecule,
extends over the beta subdomain and will be considered part of it
for the remainder of this discussion.
[0140] FIG. 3 shows a surface rendering, depicting the substrate
binding cleft formed between the beta barrel and alpha helical
subdomains. The substrate binding cleft is found to occur in the
groove formed between the beta and alpha subdomains. The crystal
structure includes part of the linker domain that would connect the
catalytic core with the CBM. This partial linker extends over the
central beta strand formed by residues 100-107 which itself is part
of the beta barrel subdomain.
[0141] The structure of the catalytic core of truncated STCE1-WT
cellulase is homologous to the catalytic core of two other known
GH45 family enzymes, the Melanocarpus albomyces endoglucanase (pdb
ID 10A7) and the Endoglucanase V from Humicola insolens (pdb ID
3ENG). As shown in FIG. 4, the overall protein folding is highly
similar among the three structures, including the distribution of
the six disulfide bridges. It can be seen that the overall folding
of these enzymes is highly conserved, even though the catalytic
core of STCE1-WT shares only 75.2% and 80.8% sequence identity,
respectively, with the catalytic cores of the other two GH45
cellulases.
Example 4
Evaluation of Protease Labile Sites in STCE1-WT Cellulase
[0142] In order to identify protease labile sites, STCE1-WT
cellulase (SEQ ID NO:5) was incubated with commercially available
BPN'-Y217L protease (10:1 ratio) at 37.degree. C. The amino acid
sequence of the commercially available BPN'-Y217L is set forth as
SEQ ID NO:7.
[0143] Protease treated samples of the cellulase were withdrawn at
differing time points and assayed for cellulase activity. The
incubation reactions were stopped by using IN HCl, and the sample
was filtered to collect soluble fraction. The samples were further
digested with trypsin to obtain appropriate sized fragments for
mass spectrometric analysis. Tryptic digests were subjected to LC
MS/MS using the Proxeon Easy-nano system (Thermo Scientific, FL,
USA). Trypsin is known to cleave polypeptide backbones C-terminal
to positively charged amino acid residues, lysine (K) or arginine
(R). In addition to the expected cleavage pattern due to trypsin
action on STCE1-WT (SEQ ID NO:5), the BPN'-Y217L cleavage pattern
identified cleavage sites at A142 and Q216.
[0144] In view of the above, single amino acid variants were
generated for the A142 and Q216 sites and these variants were
assayed under various conditions to measure improvements in
properties valuable for laundry cellulases. In particular, the
stability of these variant cellulase and STCE1-WT cellulase were
measured under the test conditions described in Example 1
including: thermostability, stability in the presence of a
protease, and stability in the presence of protease and detergent.
The relative activity and stability performance results are shown
in Table 4, reported as performance index (PI) of variants versus
STCE1-WT (SEQ ID NO:5). These results indicate that replacing
alanine at position 142 with an amino acid selected from D, E, P
and Q yielded variants with improved stability. Likewise, replacing
glutamine at position 216 with an amino acid selected from D, E, G,
P, S, T and V also yielded variants with improved stability when
compared to STCE1-WT (SEQ ID NO:5).
TABLE-US-00005 TABLE 4 Relative Performance Compared to STCE1-WT,
Reported as PI Stability Performance CMC Activity, Detergent (OMO)
Position Variant pH 8.2 and Protease Protease Thermal 142 A142D 0.9
2.7 1.7 1.0 142 A142E 1.0 2.9 1.9 1.0 142 A142P 0.9 3.0 2.2 1.0 142
A142Q 1.1 0.8 1.1 0.9 216 Q216D 1.0 0.9 1.0 1.2 216 Q216E 1.0 0.8
1.2 0.5 216 Q216G 1.0 0.8 1.2 1.0 216 Q216P 1.0 1.2 1.0 1.0 216
Q216S 1.1 1.0 0.9 1.0 216 Q216T 1.2 1.1 1.1 1.0 216 Q216V 1.1 1.5
1.1 1.1
Example 5
Long Term Stability Analysis of the STCE1-A142P Variant
Cellulase
[0145] The STCE1-A142P variant (SEQ ID NO:8) was tested for long
term stability in various liquid detergents containing a protease
relative to two other cellulases, the STCE1-WT parent and a
commercial Benchmark cellulase (Carezyme.RTM. 4500L) (Novozymes AS,
Denmark). The amino acid sequence of the mature form of the
STCE1-A142P variant is set forth as SEQ ID NO:8 (295 amino
acids).
[0146] The in-detergent stability of the STCE1-A142P variant was
tested in commercial Heavy Duty Liquid (HDL) detergents: OMO Color
Klein & Krachtig (Unilever), Persil Color-Gel (Henkel) and
Ariel Actilift.TM. Excel Gel (Procter and Gamble). All commercial
detergents were purchased in the Netherlands in 2014. For the
detergent resistance stability tests in Persil and Ariel, the
enzymes present in the detergents were heat inactivated as
described in Example 1. The STCE1-A142P variant, STCE1-WT parent,
or Benchmark was added to cooled detergent to provide a final
concentration of 200 ppm in detergent. To determine the level of
cellulase resistance to proteolytic inactivation, commercially
available BPN'-Y217L subtilisin protease was added to the cooled
cellulase containing detergent to final concentration of 1000 ppm
in detergent.
[0147] Another subset of stability tests were carried out in
commercial OMO Color Klein & Krachtig detergent wherein the
detergent was not heat inactivated and thus contained the protease
already present in the commercially available detergent. The
STCE1-WT parent, STCE1-A142P variant, or commercial Benchmark
cellulase was added to this detergent to a final concentration of
200 ppm in detergent. All detergent samples were incubated at
30.degree. C. in an incubator. To determine remaining cellulase
activity, aliquots were removed from each detergent reaction sample
at different time intervals and tested using the AZCL-HE-Cellulose
substrate (Cellazyme C tablet, Megazyme International). Briefly,
100 .mu.l of sample was added to 1.9 ml of 50 mM
Acetate/Bis-Tris/HEPES/CHES, pH 8.0 buffer and mixed well. 500
.mu.l of sample in substrate was pre-incubated at 40.degree. C. for
2 min followed by addition of a Cellazyme C tablet and incubated
for another 10 min at 40.degree. C. Following this step, the
reaction was stopped by adding 10 ml of 2% Tris (pH 12.0) solution.
Resultant solution was then filtered through a Whatman number #1
filter paper and absorbance measured in SpectraMax plate reader at
590 nm wavelength. The resultant data was calculated as percent
remaining cellulase activity, with day zero activity considered as
100%. The results are shown in FIGS. 5A-5C.
[0148] As FIGS. 5A-5C show, the STCE1-A142P variant has remarkable
stability in the detergents tested as compared to the STCE1-WT and
Benchmark cellulases.
Example 6
Performance of Cellulases in a Liquid Detergent
[0149] Performance tests were carried out in commercial Kirkland
Signature.TM. liquid detergent (Sun Products Corp., USA). Detergent
was heat inactivated at 80.degree. C. for 3 hrs, followed by
addition of 1% boric acid, 2% glycerol and 0.02% CaCl.sub.2) after
cooling. To this detergent stabilizer mix, either STCE1-WT,
Benchmark (Carezyme.RTM. 4500L), or STCE1-A142P variant cellulase
was added along with commercially available proteases: BPN'-Y217L
or Properase.RTM. (DuPont, USA) protease. All samples were
incubated at 37.degree. C. in an incubator up to 6 weeks. Aliquots
of the samples were taken every couple of weeks and were tested for
wash performance on cellulase-sensitive stains. 2 g/L of liquid
detergent was added per wash cycle in a Tergotometer (India
Tergotometer, India). To a total of 1.5 L wash liquor, cellulase
and either commercially available BPN'-Y217L or Properase.RTM.
protease were added such that their final concentrations in wash
liquor were 3 ppm each. To mimic depilling performance six swatches
of pre-pilled homogenous size (127 mm.times.127 mm) brushed
interlock fabric were added to the Tergotometer pot, filled with
1.49 L of deionized water and 7.5 ml of Ca.sup.2+Mg.sup.2+ (4:1)
liquid in an overall 250 ppm water hardness. The wash cycle was
carried out at a temperature of 30.degree. C., for 90 min. After
single wash cycle, the swatches were rinsed in the running water
for 10 min and dry-spun in Front Load Washing Machine (Bosch). The
swatches were cooled to room temperature before fuzz and pill
measurement readings were taken as Panel Unit Scores (PUS).
[0150] PUS were measured using a Multiray device (Videometer A/S
Denmark) with six readings taken per swatch with and the average of
thirty-six readings being used as the PUS for each sample. These
readings are based on a calibration model generated by treating
pre-pilled swatches with varying units of cellulase enzyme to
generate a standard curve with readings between 1 to 5 PUS
measured, depending on the fuzz and pills present on the surface of
the fabric.
[0151] The higher the PUS (3-5), the better the performance or
longer stability of the cellulase enzyme in the presence of
protease; whereas, low PUS (1-2) indicate little or no improvement
in cellulase performance in the presence of protease. Results are
shown in Table 5. As indicated from the performance results shown
on Table 6, the Day 0 wash results shows that the STCE1-A142P
variant has similar depilling performance when compared with
STCE1-WT and Benchmark. The wash result of aged samples shows that
the STCE1-A142P variant retains a markedly higher depilling
performance than STCE1-WT and Benchmark in Kirkland Signature
UltraClean HE Liquid laundry detergent (Kirkland HDL, purchased in
Costco, USA) when stored at 37.degree. C. in the presence of two
commercially available proteases commonly used in detergent
applications, BPN'-Y217L and Properase.RTM..
TABLE-US-00006 TABLE 5 Measure of cellulase depilling activity in
Kirkland HDL detergent Incubation time (weeks) Enzyme(s) 0 2 6 No
Enzyme 1.5 1.5 1.7 STCE1-A142P variant cellulase 4.5 4.6 4.4
STCE1-WT cellulase 4.6 4.6 4.4 Benchmark cellulase 4.4 4.4 4.2
STCE1-A142P cellulase + 4.6 3.5 3.1 BPN'-Y217L protease STCE1-WT
cellulase + 4.3 2.0 1.5 BPN'-Y217L protease Benchmark cellulase +
4.4 2.5 1.6 BPN'-Y217L protease STCE1-A142P variant cellulase + 4.5
3.8 3.7 Properase .RTM. protease STCE1-WT cellulase + 4.4 3.5 2.5
Properase .RTM. protease Benchmark cellulase + 4.4 3.0 2.2
Properase .RTM. protease
Example 7
Comparison of STCE1-WT Cellulase to Related Molecules and
Identification of Homologous Cellulases
[0152] Related proteins were identified by a BLAST search (as
described in Altschul, S. F., T. L. Madden, A. A. Schaffer, J.
Zhang, Z. Zhang, W. Miller and D. J. Lipman (1997), "Gapped BLAST
and PSI-BLAST: a new generation of protein database search
programs." Nucleic Acids Res 25 (17): 3389-3402) against the NCBI
non-redundant protein database using the mature STCE1-WT protein
sequence (SEQ ID NO:5) and a subset of the results are shown in
Table 6. A similar search was run against the Genome Quest Patent
database with search parameters set to default values using the
mature protein amino acid sequences for STCE1-WT cellulase (SEQ ID
NO:5) as the query sequence, and a subset are shown in Table 7.
TABLE-US-00007 TABLE 6 NCBI Protein Sequence Search of STCE1 WT
Protein Sequence Accession Subject Alignment Number % ID Organism
Length Length BAG69187 100.0 Staphylotrichum coccosporum 316 295
KOP50759 79.6 Madurella mycetomatis 238 211 CDF76466 79.4
Scytalidium indonesiacum 277 218 CDF76460 78.9 Chaetomium
senegalense 190 190 XP_006694935 78.6 Chaetomium thermophilum var.
314 294 thermophilum DSM 1495 AGY80101 78.6 Chaetomium thermophilum
293 294 CAD56665 75.5 Melanocarpus albomyces 235 212 CAA01574 73.6
Humicola insolens 305 298 BAA74956 73.2 Humicola grisea var.
thermoidea 305 295 XP_003651003 71.7 Thielavia terrestris NRRL 8126
299 293 CDF76465 70.8 Remersonia thermophila 260 271 XP_001903789
68.9 Podospora anserina S mat+ 302 293 KFH43153 68.3 Acremonium
chrysogenum ATCC 11550 232 205 XP_001226436 67.8 Chaetomium
globosum CBS 148.51 308 301 CEP15980 66.5 Parasitella parasitica
393 200 XP_957107 66.1 Neurospora crassa 293 292 XP_008593736 65.8
Beauveria bassiana ARSEF 2860 258 202 XP_009853913 65.8 Neurospora
tetrasperma FGSC 2509 293 292 KGQ12157 65.7 Beauveria bassiana D1-5
232 207 EQB52129 65.4 Colletotrichum gloeosporioides Cg-14 229 205
XP_007840161 65.0 Pestalotiopsis fici W106-1 233 206 XP_008020030
65.0 Setosphaeria turcica Et28A 227 203 XP_001547700 58.4 Botrytis
cinerea T4 355 233 P45699 48.2 Fusarium oxysporum 376 353
TABLE-US-00008 TABLE 7 Genome Quest Search for Homologs of STCE1-WT
Protein Sequence Patent No/GENESEQ .TM. Subject Alignment
Identifier % ID Organism Length Length WO2011002063-0006 100 S.
coccosporum IFO 31817 295 295 US20140051147-0038 100 S. coccosporum
IFO 31817 296 295 US20140051147-0040 100 S. coccosporum IFO 31817
299 295 WO2012106824-0018 100 S. coccosporum 316 295
US20140051147-0004 100 S. coccosporum IFO 31817 298 294
WO2005054475-AEA35116 99.66 Fungi 317 296 WO2005056787-AEB69298
99.33 Unidentified 297 297 CN103343111- BBB37873 85.05 C.
thermophilum 217 214 CN105155324-BCN40565 84.58 C. thermophilum 217
214 WO2012089024-0002 82.55 T. terrestris, T. terrestris NRRL 8126
237 212 Synthetic WO2016090474-0358 81.36 C. olivicolor 309 295
WO9804663-AAW46618 81.22 H. insolens 234 213 GB2479462-AZN28533
80.75 H. insolens 213 213 WO2007071820-0019 80.75 A. thermophilum
235 213 US20150299682-0004 80 T. hyrcaniae 305 295 CN103343111-
BBB37871 79.59 C. thermophilum 314 294 WO2015109405-0384 79.32 C.
thermophilum 310 295 WO2016090474-0328 77.71 C. olivicolor 335 314
WO2009085859-0065 76.42 A. oryzae, H. insolens A. oryzae Chimeric
1097 229 Synthetic CN101784659-0080 76.42 rice koji mold 1097 229
WO2009085935-0075 76.42 A. oryzae, rice koji mold, A. oryzae 1097
229 Synthetic, A. oryzae H. insolens Chimeric Synthetic, A. oryzae
Chimeric Unidentified, A. oryzae Synthetic Unidentified
WO9743409-0066 75.93 H. nigrescens, H. insolens Chimeric, 306 295
H. nigrescens, Hybrid WO2016090472-0739 75.59 Melanocarpus
albomyces 235 213 WO2014086976-0036 74.06 Artificial Sequence,
Synthetic Unidentified 278 293 WO2012089024-0004 74.06 T.
terrestris, T. terrestris NRRL 8126 299 293 Synthetic
WO9407998-ABB04129 73.99 H. insolens Synthetic 284 296
CN103184163-BAY17845 73.99 H. insolens Synthetic 305 296
WO2007071818-0012 73.9 S. coccosporum, A. thermophilum 297 295
WO2004053039-0003 73.72 T. terrestris, T. terrestis, IAMA.A,
locally 299 293 born shuttle spore shell US20050121156-0006 73.65
H. insolens 285 296 WO9811239-0043 73.65 H. insolens 286 296
WO9407998-ABB04141 73.65 H. insolens Synthetic 284 296
WO9407998-ABB04140 73.65 H. insolens Synthetic 284 296
WO2012106824-0007 73.65 H. insolens 284 296 WO9407998-ABB04135
73.31 H. insolens Synthetic 284 296 WO9743409-0056 73.65 H.
insolens; DSM 1800, 305 296 B. amyloliquefaciens WO9218599-0001
73.65 empty 304 296 EP0633311-0002 73.65 H. insolens 305 296
WO9218598-0002 73.65 empty 304 296 JP2000217583-0031 73.65 H.
insolens 304 296 WO2012106824-0016 73.65 H. grisea var. thermoidea
305 296 WO2013181760-0760 73.65 S. thermophilum; CBS 625.91 305 296
WO9803640-0003 73.65 H. insolens 305 296 WO2008151999-0002 73.65 H.
insolens, H. jecorina, T. reesei, Artificial 305 296 Sequence
WO2005056787-0002 73.65 H. insolens MN200-1, H. insolens, 289 296
Unidentified CN103184163-0001 73.65 H. insolens 318 296
WO0240997-0224 73.41 H. insolens 276 267 WO2005054475-AEA35112 73.4
Fungi 286 297 IN2003CH00919-0009 73.38 empty 299 293 CN1198939-0004
73.31 empty 286 296 WO9407998-ABB04139 73.31 H. insolens Synthetic
284 296 WO9407998-ABB04131 73.31 H. insolens Synthetic 284 296
WO9407998-ABB04132 73.31 H. insolens Synthetic 284 296
WO9407998-ABB04137 73.31 H. insolens Synthetic 284 296
WO9407998-ABB04138 73.31 H. insolens Synthetic 284 296
WO9407998-ABB04133 72.97 H. insolens Synthetic 284 296
JP2003070489-0002 73.31 H. insolens 305 296 CN1151762-0007 73.31
empty 304 296 CN1231689-0001 73.31 empty 304 296 WO9218688-0001
73.31 empty 303 296 CN1230988-0001 73.31 empty 305 296
CN1230988-0002 73.31 empty 304 296 CN1231689-0002 73.31 empty 302
296 EP0959128-0003 73.06 empty 306 297 CN101955921-1027 73.04 empty
297 293 WO9407998-ABB04146 72.97 H. insolens Synthetic 284 296
WO9219726- AAR28818 72.97 H. insolens 305 296 WO9218688- AAR28300
72.97 H. insolens; DSM 1800 305 296 CN1151762-0008 72.97 empty 303
296 WO9617994- AAG78352 72.97 H. insolens Synthetic 305 296
WO9617994- AAG78356 72.97 H. insolens 305 296 WO9617994- AAG78358
72.97 H. insolens Synthetic 305 296 WO9617994- AAG78355 72.64 H.
insolens Synthetic 305 296 WO9617994- AAG78353 72.64 H. insolens
Synthetic 305 296 WO9617994- AAG78357 72.64 H. insolens Synthetic
305 296 WO9617994- AAG78354 72.64 H. insolens Synthetic 305 296
WO9617994- AAG78360 72.64 H. insolens Synthetic 305 296
WO2007071820-0017 72.52 A. thermophilum 264 262 WO9617994- AAG78359
72.3 H. insolens Synthetic 305 296 WO9407998-ABB04147 71.62 H.
insolens Synthetic 284 296 WO2014138983-0859 71.33 T. australiensis
293 293 US7361487-0006 71.28 A. thermophilum; strain ALK04245 315
296 C. thermophilum; strain ALKO4265 Chimeric Synthetic
WO2007071820-0041 71.28 A. thermophilum, C. thermophilum Chimeric
311 296 WO9407998-ABB04136 70.95 H. insolens Synthetic 267 296
WO9617994- AAG78361 70.95 H. insolens Synthetic 305 296
WO2012101206-0036 70.48 R. thermophila 260 271 CN1230995-0003 70.27
A. thermophilum Synthetic, A. thermophilum 293 296
WO2007071820-0040 70.07 S. indonesiacum, Scytalidium sp.; 300 294
indonesiacum WO2012101206-0046 69.65 277 257 WO2012106824-0020
69.28 P. anserina 302 293 WO9407998-ABB04143 69.26 H. insolens
Synthetic 284 296 WO9743409-0068 69.15 Cylindrocarpon; sp. H.
insolens Chimeric, 306 295 Hybrid WO9407998-ABB04145 68.92 H.
insolens Synthetic 284 296 WO2007071820-0039 68.71 H. jecorina A.
thermophilum Chimeric, 298 294 A. thermophilum WO2014026630-0002
68.49 S. fimicola, ATCC 52644 294 292 WO2014035458-0326 68.44 C.
globosum 308 301 WO2014101753-0002 68.03 H. hyalothermophila,
Humicola sp.; 286 294 CBS454.80 WO2012106824-0027 67.81 N. crassa
293 292 WO9743409-0072 67.8 C. rosea f. catenulata, H. insolens
Chimeric, 304 295 Hybrid WO9743409-0070 67.34 F. anguioides H.
insolens Chimeric, 308 297 F. anguioides, Hybrid WO9743409-0074
65.54 T. roseum, T. roseum H. insolens Chimeric, 307 296 Hybrid
Example 8
Alignment of GH45 Cellulases with STCE1-WT
[0153] An alignment of the amino acid sequences of the following
cellulases S_coccosporum_BAG69187.1(SEQ ID NO:1), STCE1-WT (SEQ ID
NO:5), STCE1-A142P (SEQ ID NO:8), Madurella mycetomatis (NCBI
Accession No: KOP50759)(SEQ ID NO:2), S_indonesiacum (NCBI
Accession No: CDF76466.1)(SEQ ID NO:29), C_thermophilum (NCBI
Accession NO: AGY80101.1)(SEQ ID NO:19), M_albomyces (NCBI
Accession No: CAD56665)(SEQ ID NO:20), H_insolens (NCBI Accession
No: CAA01574.1)(SEQ ID NO:10), H_grisea (NCBI Accession
No:BAA74956.1)(SEQ ID NO:23), T_terrestris (NCBI Accession No:
XP_003651003.1)(SEQ ID NO:22), Podospora anserina S mat+(NCBI
Accession No: XP_001903789; GenBank accession number
CAP61565.1)(SEQ ID NO:25), Acremonium chrysogenum (NCBI Accession
NO: KFH43153)(SEQ ID NO:26), Neurospora crassa (NCBI Accession No:
XP_957107)(SEQ ID NO:13), SEQ ID NO:4 from US20150299682 (SEQ ID
NO:18), SEQ ID NO:66 from WO9743409 (Genome Quest Identifier:
CAB42308) (SEQ ID NO:21), SEQ ID NO:6 from U.S. Pat. No. 7,361,487
(Genome Quest Identifier: ACE10216.1) (SEQ ID NO:24), SEQ ID NO:72
from WO9743409 (Genome Quest Identifier: CAB42311) (SEQ ID NO:27),
and SEQ ID NO:74 from WO9743409 (Genome Quest Identifier: CAB42312)
(SEQ ID NO:28) is shown in FIG. 6A-C. The sequences were aligned
with default parameters using the MUSCLE program from Geneious
software (Biomatters Ltd.) (Robert C. Edgar. MUSCLE: multiple
sequence alignment with high accuracy and high throughput Nucl.
Acids Res. (2004) 32 (5): 1792-1797). A phylogenetic tree for amino
acid sequences of the mature forms of the subtilisins from FIG. 6
was built using the Geneious Tree builder program and is shown in
FIG. 7.
Example 9
Identification of Additional Substitutions in STCE-1 Cellulase that
Impart Improved Stability Under Multiple Conditions
[0154] Additional substitutions on the STCE-1 cellulase backbone
were generated using methods described in Example 2. Similarly to
the evaluations described in Example 4, the activity and stability
of the singly substituted cellulase variants was evaluated as
described in Example 1. The relative activity and stability
performance results are shown in Table 8, reported as performance
index (PI) of variants versus STCE1-WT (SEQ ID NO:5).
TABLE-US-00009 TABLE 8 STCE-1 Cellulase Variants Showing Improved
Activity and/or Improved Stability Performance Across Multiple
Conditions. CMC Stability Performance Activity, Detergent (OMO)
Position Variant pH 8.2 and protease Protease Thermal 4 K4V 1.0 1.3
1.4 1.1 20 G20N 1.2 1.2 1.3 0.8 23 S23L 1.2 1.5 1.2 0.9 29 F29W 1.1
1.6 1.2 1.0 32 S32D 1.1 2.2 1.7 0.9 32 S32Y 1.2 1.4 1.2 0.8 36 Q36T
1.1 1.8 1.6 1.1 44 K44V 1.1 1.5 1.0 1.1 51 S51T 1.2 2.0 1.2 1.1 77
S77M 1.0 1.5 1.3 1.1 77 S77K 1.1 1.3 1.4 1.1 80 N80S 1.2 1.3 1.3
1.1 87 G87A 1.1 1.3 1.3 1.2 90 E90A 1.2 1.2 0.9 1.0 97 P97S 1.4 2.2
1.0 1.1 98 V98G 1.1 1.7 1.2 1.1 99 A99Y 1.1 1.7 1.2 1.0 99 A99E 1.0
1.5 1.3 1.3 102 T102K 1.0 1.1 1.1 1.3 112 G112T 1.2 1.8 1.0 1.1 112
G112S 1.2 1.8 1.4 1.2 112 G112V 1.2 2.1 1.3 1.2 116 T116V 1.2 1.2
1.0 1.0 135 S135T 1.0 2.6 2.0 1.1 136 P136E 1.0 2.0 1.9 1.1 136
P136K 0.9 1.8 2.0 1.0 136 P136S 1.1 2.4 1.6 0.9 153 S153D 1.2 2.0
1.4 1.1 154 Q154E 1.0 2.6 1.9 1.0 157 S157D 1.1 1.8 1.1 1.1 161
A161P 0.9 2.0 1.7 0.9 161 A161E 1.2 2.2 1.4 1.2 163 K163V 1.2 2.3
1.4 1.1 192 L192V 1.4 1.1 1.1 1.0 194 A194S 1.4 1.5 1.3 1.1 204
G204S 1.0 1.8 1.1 1.0 208 V208H 1.2 1.9 1.0 0.9 208 V208K 1.1 1.5
1.1 0.9 210 T210V 1.2 1.9 1.0 1.0 212 P212S 1.2 2.0 0.7 1.0 217
S217G 1.1 1.4 0.9 1.5 217 S217M 1.2 1.1 1.2 1.1 221 S221L 0.9 1.7
0.9 1.0 221 S221M 1.1 1.2 1.1 1.4 222 S222A 0.9 2.0 1.1 1.0 225
S225K 1.1 1.5 1.1 1.0 227 K227R 1.1 1.1 1.3 1.1 232 S232T 1.1 1.0
1.2 0.8
Example 10
Evaluation of Mutations that Improve Stability Across GH45
Cellulases
[0155] FIG. 8 provides a MUSCLE multiple sequence alignment of the
catalytic domains of the following GH45 cellulases: STCE1-WT (SEQ
ID NO: 5), H. insolens (SEQ ID NO: 11), N. crassa (SEQ ID NO:14)
and T. terrestris 120H (SEQ ID NO:17). Single amino acid
substitutions at positions that correspond to the positions (based
on the multiple sequence alignment set forth in FIG. 8) that were
shown to improve the stability of the STCE1-WT cellulase (as shown
in Example 9) were evaluated in the related GH45 cellulases: H.
insolens-WT (SEQ ID NO:11), N. crassa-WT (SEQ ID NO:14), and T.
terrestris-120H (SEQ ID NO:17). The stability performance of these
other GH45 variant cellulases and associated parents were measured
under the test conditions described in Example 1, the results are
shown in Tables 10, 11 and 12, reported as performance index (PI)
of variants versus parent. Substituting amino acids at positions in
other endoglucanases (e.g., H. insolens, N. crassa and T.
terrestris) at positions that correspond to the positions that
improved the stability of STCE1-WT yielded numerous GH45 variants
with improved stability.
TABLE-US-00010 TABLE 10 Relative Performance Compared to H.
insolens-WT, Reported as PI H. insolens enzyme, Sequence numbering
sequence numbering relative to mature relative to H. insolens
STCE1-WT Stability WT (SEQ ID NO: 11) (SEQ ID NO: 5) Detergent
Protease H. insolens-WT -- 1.0 1.0 (parent) K20N K20N 1.4 0.3 Q36T
Q36T 1.6 0.9 G113T G112T 2.0 1.3 N154D H153D 2.0 0.9 A162P A161P
2.0 1.8
TABLE-US-00011 TABLE 11 Relative Performance Compared to N.
crassa-WT, Reported as PI N. crassa enzyme, sequence numbering
Sequence numbering relative to mature relative to mature N. crassa
WT STCE1-WT Stability (SEQ ID NO: 14) (SEQ ID NO: 5) Detergent
Protease N. crassa-WT -- 1.0 1.0 (parent) N38T N36T 5.0 35 G114T
G112T 2.0 27 S155D G153D 40 0 Q156E Q154E 81 0 A163P A161P 15
64
TABLE-US-00012 TABLE 12 Relative Performance Compared to T.
terrestris-WT, Reported as PI T. terrestris enzyme, sequence
numbering Sequence numbering relative to mature relative to mature
T. terrestris 120H STCE1-WT Stability (SEQ ID NO: 17) (SEQ ID NO:
5) Detergent Protease T. terrestris-120H -- 1.0 1.0 (parent) A25L
A23L 1.7 0 Q38T Q36T 1.7 110 G114T G112T 1.6 0 S137T S135T 1.0 48
S138E S136E 1.7 0 Q156E Q154E 17 840
Sequence CWU 1
1
291316PRTStaphylotrichum coccosporum 1Met Arg Ser Ser Pro Val Leu
Arg Thr Ala Leu Ala Ala Ala Leu Pro 1 5 10 15 Leu Ala Ala Leu Ala
Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro
Ser Cys Ser Trp Pro Gly Lys Ala Ser Val Asn Gln Pro 35 40 45 Val
Phe Ala Cys Ser Ala Asn Phe Gln Arg Ile Ser Asp Pro Asn Val 50 55
60 Lys Ser Gly Cys Asp Gly Gly Ser Ala Tyr Ala Cys Ala Asp Gln Thr
65 70 75 80 Pro Trp Ala Val Asn Asp Asn Phe Ser Tyr Gly Phe Ala Ala
Thr Ser 85 90 95 Ile Ser Gly Gly Asn Glu Ala Ser Trp Cys Cys Gly
Cys Tyr Glu Leu 100 105 110 Thr Phe Thr Ser Gly Pro Val Ala Gly Lys
Thr Met Val Val Gln Ser 115 120 125 Thr Ser Thr Gly Gly Asp Leu Gly
Thr Asn His Phe Asp Leu Ala Met 130 135 140 Pro Gly Gly Gly Val Gly
Ile Phe Asp Gly Cys Ser Pro Gln Phe Gly 145 150 155 160 Gly Leu Ala
Gly Asp Arg Tyr Gly Gly Val Ser Ser Arg Ser Gln Cys 165 170 175 Asp
Ser Phe Pro Ala Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe Asp 180 185
190 Trp Phe Lys Asn Ala Asp Asn Pro Thr Phe Thr Phe Arg Gln Val Gln
195 200 205 Cys Pro Ser Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn
Asp Asp 210 215 220 Gly Asn Phe Pro Val Phe Thr Pro Pro Ser Gly Gly
Gln Ser Ser Ser 225 230 235 240 Ser Ser Ser Ser Ser Ser Ala Lys Pro
Thr Ser Thr Ser Thr Ser Thr 245 250 255 Thr Ser Thr Lys Ala Thr Ser
Thr Thr Ser Thr Ala Ser Ser Gln Thr 260 265 270 Ser Ser Ser Thr Gly
Gly Gly Cys Ala Ala Gln Arg Trp Ala Gln Cys 275 280 285 Gly Gly Ile
Gly Phe Ser Gly Cys Thr Thr Cys Val Ser Gly Thr Thr 290 295 300 Cys
Asn Lys Gln Asn Asp Trp Tyr Ser Gln Cys Leu 305 310 315
2238PRTMadurella mycetomatis 2Met His Ser Ala Pro Val Leu Arg Ala
Ala Leu Ile Ala Thr Leu Pro 1 5 10 15 Leu Ala Ala His Ala Ala Ser
Gly Ser Gly Gln Ser Thr Arg Tyr Trp 20 25 30 Asp Cys Cys Lys Pro
Ser Cys Ser Trp Pro Gly Lys Ala Pro Val Asn 35 40 45 Gln Pro Val
Phe Ala Cys Asp Ala Asn Phe Gln Arg Ile Asn Asp Phe 50 55 60 Gly
Ala Thr Ser Gly Cys Asp Gly Gly Ser Ala Tyr Ser Cys Ala Asp 65 70
75 80 His Ala Pro Trp Ala Ile Asn Asp Asn Leu Ser Tyr Gly Phe Ala
Ala 85 90 95 Thr Ala Leu Ser Gly Glu Thr Glu Ser Ser Trp Cys Cys
Ala Cys Tyr 100 105 110 Ala Leu Thr Phe Thr Ser Gly Pro Val Ala Gly
Lys Thr Met Val Val 115 120 125 Gln Ser Thr Ser Thr Gly Gly Asp Leu
Gly Ser Asn His Phe Asp Leu 130 135 140 Asn Ile Pro Gly Gly Gly Val
Gly Leu Phe Asp Gly Cys Thr Pro Gln 145 150 155 160 Phe Gly Gly Leu
Pro Gly Ala Gln Tyr Gly Gly Ile Ser Asp Arg Ser 165 170 175 Gln Cys
Ser Ser Phe Pro Ser Gln Leu Gln Pro Gly Cys Asn Trp Arg 180 185 190
Phe Asp Trp Phe Met Asn Ala Asp Asn Pro Ser Phe Thr Phe Asp Gln 195
200 205 Val Gln Cys Pro Asp Glu Leu Val Ala Arg Thr Gly Cys Arg Arg
Ser 210 215 220 Asp Asp Ala Asn Phe Pro Ala Phe Ser Pro Pro Ser Arg
Leu 225 230 235 3951DNAStaphylotrichum coccosporum 3atgcgttcct
cccccgtcct ccgcacggcc ctggccgctg ccctccccct ggccgccctc 60gctgccgatg
gcaagtcgac ccgctactgg gactgttgca agccgtcgtg ctcgtggccc
120ggcaaggcct cggtgaacca gcccgtcttc gcctgcagcg ccaacttcca
gcgcatcagc 180gaccccaacg tcaagtcggg ctgcgacggc ggctccgcct
acgcctgcgc cgaccagacc 240ccgtgggccg tcaacgacaa cttctcgtac
ggcttcgccg ccacgtccat ctcgggcggc 300aacgaggcct cgtggtgctg
tggctgctac gagctgacct tcacctcggg ccccgtcgct 360ggcaagacca
tggttgtcca gtccacctcg accggcggcg acctcggcac caaccacttc
420gacctggcca tgcccggtgg tggtgtcggc atcttcgacg gctgctcgcc
ccagttcggc 480ggcctcgccg gcgaccgcta cggcggcgtc tcgtcgcgca
gccagtgcga ctcgttcccc 540gccgccctca agcccggctg ctactggcgc
ttcgactggt tcaagaacgc cgacaacccg 600accttcacct tccgccaggt
ccagtgcccg tcggagctcg tcgcccgcac cggctgccgc 660cgcaacgacg
acggcaactt ccccgtcttc acccctccct cgggcggtca gtcctcctcg
720tcttcctcct ccagcagcgc caagcccacc tccacctcca cctcgaccac
ctccaccaag 780gctacctcca ccacctcgac cgcctccagc cagacctcgt
cgtccaccgg cggcggctgc 840gccgcccagc gctgggcgca gtgcggcggc
atcgggttct cgggctgcac cacgtgcgtc 900agcggcacca cctgcaacaa
gcagaacgac tggtactcgc agtgcctttg a 9514316PRTStaphylotrichum
coccosporum 4Met Arg Ser Ser Pro Val Leu Arg Thr Ala Leu Ala Ala
Ala Leu Pro 1 5 10 15 Leu Ala Ala Leu Ala Ala Asp Gly Lys Ser Thr
Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser Cys Ser Trp Pro Gly
Lys Ala Ser Val Asn Gln Pro 35 40 45 Val Phe Ala Cys Ser Ala Asn
Phe Gln Arg Ile Ser Asp Pro Asn Val 50 55 60 Lys Ser Gly Cys Asp
Gly Gly Ser Ala Tyr Ala Cys Ala Asp Gln Thr 65 70 75 80 Pro Trp Ala
Val Asn Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thr Ser 85 90 95 Ile
Ser Gly Gly Asn Glu Ala Ser Trp Cys Cys Gly Cys Tyr Glu Leu 100 105
110 Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser
115 120 125 Thr Ser Thr Gly Gly Asp Leu Gly Thr Asn His Phe Asp Leu
Ala Met 130 135 140 Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Ser
Pro Gln Phe Gly 145 150 155 160 Gly Leu Ala Gly Asp Arg Tyr Gly Gly
Val Ser Ser Arg Ser Gln Cys 165 170 175 Asp Ser Phe Pro Ala Ala Leu
Lys Pro Gly Cys Tyr Trp Arg Phe Asp 180 185 190 Trp Phe Lys Asn Ala
Asp Asn Pro Thr Phe Thr Phe Arg Gln Val Gln 195 200 205 Cys Pro Ser
Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp Asp 210 215 220 Gly
Asn Phe Pro Val Phe Thr Pro Pro Ser Gly Gly Gln Ser Ser Ser 225 230
235 240 Ser Ser Ser Ser Ser Ser Ala Lys Pro Thr Ser Thr Ser Thr Ser
Thr 245 250 255 Thr Ser Thr Lys Ala Thr Ser Thr Thr Ser Thr Ala Ser
Ser Gln Thr 260 265 270 Ser Ser Ser Thr Gly Gly Gly Cys Ala Ala Gln
Arg Trp Ala Gln Cys 275 280 285 Gly Gly Ile Gly Phe Ser Gly Cys Thr
Thr Cys Val Ser Gly Thr Thr 290 295 300 Cys Asn Lys Gln Asn Asp Trp
Tyr Ser Gln Cys Leu 305 310 315 5295PRTStaphylotrichum coccosporum
5Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys 1
5 10 15 Ser Trp Pro Gly Lys Ala Ser Val Asn Gln Pro Val Phe Ala Cys
Ser 20 25 30 Ala Asn Phe Gln Arg Ile Ser Asp Pro Asn Val Lys Ser
Gly Cys Asp 35 40 45 Gly Gly Ser Ala Tyr Ala Cys Ala Asp Gln Thr
Pro Trp Ala Val Asn 50 55 60 Asp Asn Phe Ser Tyr Gly Phe Ala Ala
Thr Ser Ile Ser Gly Gly Asn 65 70 75 80 Glu Ala Ser Trp Cys Cys Gly
Cys Tyr Glu Leu Thr Phe Thr Ser Gly 85 90 95 Pro Val Ala Gly Lys
Thr Met Val Val Gln Ser Thr Ser Thr Gly Gly 100 105 110 Asp Leu Gly
Thr Asn His Phe Asp Leu Ala Met Pro Gly Gly Gly Val 115 120 125 Gly
Ile Phe Asp Gly Cys Ser Pro Gln Phe Gly Gly Leu Ala Gly Asp 130 135
140 Arg Tyr Gly Gly Val Ser Ser Arg Ser Gln Cys Asp Ser Phe Pro Ala
145 150 155 160 Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe Asp Trp Phe
Lys Asn Ala 165 170 175 Asp Asn Pro Thr Phe Thr Phe Arg Gln Val Gln
Cys Pro Ser Glu Leu 180 185 190 Val Ala Arg Thr Gly Cys Arg Arg Asn
Asp Asp Gly Asn Phe Pro Val 195 200 205 Phe Thr Pro Pro Ser Gly Gly
Gln Ser Ser Ser Ser Ser Ser Ser Ser 210 215 220 Ser Ala Lys Pro Thr
Ser Thr Ser Thr Ser Thr Thr Ser Thr Lys Ala 225 230 235 240 Thr Ser
Thr Thr Ser Thr Ala Ser Ser Gln Thr Ser Ser Ser Thr Gly 245 250 255
Gly Gly Cys Ala Ala Gln Arg Trp Ala Gln Cys Gly Gly Ile Gly Phe 260
265 270 Ser Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Asn Lys Gln
Asn 275 280 285 Asp Trp Tyr Ser Gln Cys Leu 290 295
6214PRTStaphylotrichum coccosporum 6Ala Asp Gly Lys Ser Thr Arg Tyr
Trp Asp Cys Cys Lys Pro Ser Cys 1 5 10 15 Ser Trp Pro Gly Lys Ala
Ser Val Asn Gln Pro Val Phe Ala Cys Ser 20 25 30 Ala Asn Phe Gln
Arg Ile Ser Asp Pro Asn Val Lys Ser Gly Cys Asp 35 40 45 Gly Gly
Ser Ala Tyr Ala Cys Ala Asp Gln Thr Pro Trp Ala Val Asn 50 55 60
Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thr Ser Ile Ser Gly Gly Asn 65
70 75 80 Glu Ala Ser Trp Cys Cys Gly Cys Tyr Glu Leu Thr Phe Thr
Ser Gly 85 90 95 Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr
Ser Thr Gly Gly 100 105 110 Asp Leu Gly Thr Asn His Phe Asp Leu Ala
Met Pro Gly Gly Gly Val 115 120 125 Gly Ile Phe Asp Gly Cys Ser Pro
Gln Phe Gly Gly Leu Ala Gly Asp 130 135 140 Arg Tyr Gly Gly Val Ser
Ser Arg Ser Gln Cys Asp Ser Phe Pro Ala 145 150 155 160 Ala Leu Lys
Pro Gly Cys Tyr Trp Arg Phe Asp Trp Phe Lys Asn Ala 165 170 175 Asp
Asn Pro Thr Phe Thr Phe Arg Gln Val Gln Cys Pro Ser Glu Leu 180 185
190 Val Ala Arg Thr Gly Cys Arg Arg Asn Asp Asp Gly Asn Phe Pro Val
195 200 205 Phe Thr Pro Pro Ser Gly 210 7338PRTartificial
sequencevariant of BPN' protein 7Ala Gln Ser Val Pro Tyr Gly Val
Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr
Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp
Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met
Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60
Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly 65
70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys
Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile
Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val Ile
Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys
Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val
Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser
Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val
Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185
190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr
195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Leu Asn Gly Thr Ser Met
Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser
Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser
Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr
Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln Lys Ser
Phe Pro Glu Val Val Gly Lys Thr Val Asp Gln 275 280 285 Ala Arg Glu
Tyr Phe Thr Leu His Tyr Pro Gln Tyr Asp Val Tyr Phe 290 295 300 Leu
Pro Glu Gly Ser Pro Val Thr Leu Asp Leu Arg Tyr Asn Arg Val 305 310
315 320 Lys Val Phe Tyr Asn Pro Gly Thr Asn Val Val Asn His Val Pro
His 325 330 335 Val Gly 8295PRTartificial sequencemature form of
the STCE1-A142P variant 8Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp
Cys Cys Lys Pro Ser Cys 1 5 10 15 Ser Trp Pro Gly Lys Ala Ser Val
Asn Gln Pro Val Phe Ala Cys Ser 20 25 30 Ala Asn Phe Gln Arg Ile
Ser Asp Pro Asn Val Lys Ser Gly Cys Asp 35 40 45 Gly Gly Ser Ala
Tyr Ala Cys Ala Asp Gln Thr Pro Trp Ala Val Asn 50 55 60 Asp Asn
Phe Ser Tyr Gly Phe Ala Ala Thr Ser Ile Ser Gly Gly Asn 65 70 75 80
Glu Ala Ser Trp Cys Cys Gly Cys Tyr Glu Leu Thr Phe Thr Ser Gly 85
90 95 Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr Gly
Gly 100 105 110 Asp Leu Gly Thr Asn His Phe Asp Leu Ala Met Pro Gly
Gly Gly Val 115 120 125 Gly Ile Phe Asp Gly Cys Ser Pro Gln Phe Gly
Gly Leu Pro Gly Asp 130 135 140 Arg Tyr Gly Gly Val Ser Ser Arg Ser
Gln Cys Asp Ser Phe Pro Ala 145 150 155 160 Ala Leu Lys Pro Gly Cys
Tyr Trp Arg Phe Asp Trp Phe Lys Asn Ala 165 170 175 Asp Asn Pro Thr
Phe Thr Phe Arg Gln Val Gln Cys Pro Ser Glu Leu 180 185 190 Val Ala
Arg Thr Gly Cys Arg Arg Asn Asp Asp Gly Asn Phe Pro Val 195 200 205
Phe Thr Pro Pro Ser Gly Gly Gln Ser Ser Ser Ser Ser Ser Ser Ser 210
215 220 Ser Ala Lys Pro Thr Ser Thr Ser Thr Ser Thr Thr Ser Thr Lys
Ala 225 230 235 240 Thr Ser Thr Thr Ser Thr Ala Ser Ser Gln Thr Ser
Ser Ser Thr Gly 245 250 255 Gly Gly Cys Ala Ala Gln Arg Trp Ala Gln
Cys Gly Gly Ile Gly Phe 260 265 270 Ser Gly Cys Thr Thr Cys Val Ser
Gly Thr Thr Cys Asn Lys Gln Asn 275 280 285 Asp Trp Tyr Ser Gln Cys
Leu 290 295 91060DNAHumicola insolens 9ggatccaaga tgcgttcctc
ccccctcctc ccgtccgccg ttgtggccgc cctgccggtg 60ttggcccttg ccgctgatgg
caggtccacc cgctactggg actgctgcaa gccttcgtgc 120ggctgggcca
agaaggctcc cgtgaaccag cctgtctttt cctgcaacgc caacttccag
180cgtatcacgg acttcgacgc caagtccggc tgcgagccgg gcggtgtcgc
ctactcgtgc 240gccgaccaga ccccatgggc tgtgaacgac gacttcgcgc
tcggttttgc tgccacctct 300attgccggca gcaatgaggc gggctggtgc
tgcgcctgct acgagctcac cttcacatcc 360ggtcctgttg ctggcaagaa
gatggtcgtc cagtccacca gcactggcgg tgatcttggc 420agcaaccact
tcgatctcaa catccccggc ggcggcgtcg gcatcttcga cggatgcact
480ccccagttcg gcggtctgcc cggccagcgc tacggcggca tctcgtcccg
caacgagtgc 540gatcggttcc ccgacgccct caagcccggc tgctactggc
gcttcgactg gttcaagaac 600gccgacaatc
cgagcttcag cttccgtcag gtccagtgcc cagccgagct cgtcgctcgc
660accggatgcc gccgcaacga cgacggcaac ttccctgccg tccagatccc
ctccagcagc 720accagctctc cggtcaacca gcctaccagc accagcacca
cgtccacctc caccacctcg 780agcccgccag tccagcctac gactcccagc
ggctgcactg ctgagaggtg ggctcagtgc 840ggcggcaatg gctggagcgg
ctgcaccacc tgcgtcgctg gcagcacttg cacgaagatt 900aatgactggt
accatcagtg cctgtagacg cagggcagct tgagggcctt actggtggcc
960gcaacgaaat gacactccca atcactgtat tagttcttgt acataatttc
gtcatccctc 1020cagggattgt cacataaatg caatgaggaa caatgagtac
106010305PRTHumicola insolens 10Met Arg Ser Ser Pro Leu Leu Pro Ser
Ala Val Val Ala Ala Leu Pro 1 5 10 15 Val Leu Ala Leu Ala Ala Asp
Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser Cys
Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35 40 45 Val Phe Ser
Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50 55 60 Lys
Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln 65 70
75 80 Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala
Thr 85 90 95 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala
Cys Tyr Glu 100 105 110 Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys
Lys Met Val Val Gln 115 120 125 Ser Thr Ser Thr Gly Gly Asp Leu Gly
Ser Asn His Phe Asp Leu Asn 130 135 140 Ile Pro Gly Gly Gly Val Gly
Ile Phe Asp Gly Cys Thr Pro Gln Phe 145 150 155 160 Gly Gly Leu Pro
Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165 170 175 Cys Asp
Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180 185 190
Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195
200 205 Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn
Asp 210 215 220 Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser
Thr Ser Ser 225 230 235 240 Pro Val Asn Gln Pro Thr Ser Thr Ser Thr
Thr Ser Thr Ser Thr Thr 245 250 255 Ser Ser Pro Pro Val Gln Pro Thr
Thr Pro Ser Gly Cys Thr Ala Glu 260 265 270 Arg Trp Ala Gln Cys Gly
Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys 275 280 285 Val Ala Gly Ser
Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys 290 295 300 Leu 305
11284PRTHumicola insolens 11Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp
Cys Cys Lys Pro Ser Cys 1 5 10 15 Gly Trp Ala Lys Lys Ala Pro Val
Asn Gln Pro Val Phe Ser Cys Asn 20 25 30 Ala Asn Phe Gln Arg Ile
Thr Asp Phe Asp Ala Lys Ser Gly Cys Glu 35 40 45 Pro Gly Gly Val
Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala Val 50 55 60 Asn Asp
Asp Phe Ala Leu Gly Phe Ala Ala Thr Ser Ile Ala Gly Ser 65 70 75 80
Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu Leu Thr Phe Thr Ser 85
90 95 Gly Pro Val Ala Gly Lys Lys Met Val Val Gln Ser Thr Ser Thr
Gly 100 105 110 Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn Ile Pro
Gly Gly Gly 115 120 125 Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
Gly Gly Leu Pro Gly 130 135 140 Gln Arg Tyr Gly Gly Ile Ser Ser Arg
Asn Glu Cys Asp Arg Phe Pro 145 150 155 160 Asp Ala Leu Lys Pro Gly
Cys Tyr Trp Arg Phe Asp Trp Phe Lys Asn 165 170 175 Ala Asp Asn Pro
Ser Phe Ser Phe Arg Gln Val Gln Cys Pro Ala Glu 180 185 190 Leu Val
Ala Arg Thr Gly Cys Arg Arg Asn Asp Asp Gly Asn Phe Pro 195 200 205
Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser Pro Val Asn Gln Pro 210
215 220 Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr Ser Ser Pro Pro
Val 225 230 235 240 Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu Arg
Trp Ala Gln Cys 245 250 255 Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr
Cys Val Ala Gly Ser Thr 260 265 270 Cys Thr Lys Ile Asn Asp Trp Tyr
His Gln Cys Leu 275 280 12882DNANeurospora crassa 12atgcgctcct
ccactattct gcaaaccggg ctagtggccg ctcttccttt cgccgtccag 60gctgcttccg
gatccggcca gtccaccaga tattgggact gctgcaaacc atcttgctcc
120tggtccggca aggctcctgt caaccgaccc gtcctcgctt gcgacgcaaa
caacaacccc 180ctgagcgacg ccagtgtcaa gtctggatgt gatggcggtt
ctgcatacac ctgtgccaac 240aactcaccat gggcggtgaa cgaccagctc
tcctacggct ttgccgccac gaaactcagt 300ggtggaaccg agtcatcttg
gtgctgtgcc tgttatgccc ttaccttcac ttcgggccct 360gttgctggca
agaccttggt cgttcagtct accagtaccg gcggtgatct cggctccaac
420cacttcgata tcaacatgcc cggcggcggc gtcggcctgt ttgatggatg
taaacgacag 480ttcggcggtc tccccggcgc tcaatatggc ggcatcagct
cccgcagcca gtgcgactcg 540ttccctgccg ctctcaagcc cggttgccag
tggcgcttcg actggttcca gaacgccgat 600aacccgaact tcaccttcaa
gcaggtccaa tgcccatccg agctcacatc ccgcaccggc 660tgcaagcgaa
acgacgactc ccaattccct gtcttcactc cgccctctgg tggaggcagt
720aacccctcta ctccgacaac ccctccctct tcaggcggcg gttccggatg
tacagcggat 780aaatacgctc aatgtggtgg ctcggggtgg tctggctgca
ccaactgccc gtctggatcg 840acctgcaaga ctatcaacga ttattaccat
cagtgtgcct ga 88213293PRTNeurospora crassa 13Met Arg Ser Ser Thr
Ile Leu Gln Thr Gly Leu Val Ala Ala Leu Pro 1 5 10 15 Phe Ala Val
Gln Ala Ala Ser Gly Ser Gly Gln Ser Thr Arg Tyr Trp 20 25 30 Asp
Cys Cys Lys Pro Ser Cys Ser Trp Ser Gly Lys Ala Pro Val Asn 35 40
45 Arg Pro Val Leu Ala Cys Asp Ala Asn Asn Asn Pro Leu Ser Asp Ala
50 55 60 Ser Val Lys Ser Gly Cys Asp Gly Gly Ser Ala Tyr Thr Cys
Ala Asn 65 70 75 80 Asn Ser Pro Trp Ala Val Asn Asp Gln Leu Ser Tyr
Gly Phe Ala Ala 85 90 95 Thr Lys Leu Ser Gly Gly Thr Glu Ser Ser
Trp Cys Cys Ala Cys Tyr 100 105 110 Ala Leu Thr Phe Thr Ser Gly Pro
Val Ala Gly Lys Thr Leu Val Val 115 120 125 Gln Ser Thr Ser Thr Gly
Gly Asp Leu Gly Ser Asn His Phe Asp Ile 130 135 140 Asn Met Pro Gly
Gly Gly Val Gly Leu Phe Asp Gly Cys Lys Arg Gln 145 150 155 160 Phe
Gly Gly Leu Pro Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg Ser 165 170
175 Gln Cys Asp Ser Phe Pro Ala Ala Leu Lys Pro Gly Cys Gln Trp Arg
180 185 190 Phe Asp Trp Phe Gln Asn Ala Asp Asn Pro Asn Phe Thr Phe
Lys Gln 195 200 205 Val Gln Cys Pro Ser Glu Leu Thr Ser Arg Thr Gly
Cys Lys Arg Asn 210 215 220 Asp Asp Ser Gln Phe Pro Val Phe Thr Pro
Pro Ser Gly Gly Gly Ser 225 230 235 240 Asn Pro Ser Thr Pro Thr Thr
Pro Pro Ser Ser Gly Gly Gly Ser Gly 245 250 255 Cys Thr Ala Asp Lys
Tyr Ala Gln Cys Gly Gly Ser Gly Trp Ser Gly 260 265 270 Cys Thr Asn
Cys Pro Ser Gly Ser Thr Cys Lys Thr Ile Asn Asp Tyr 275 280 285 Tyr
His Gln Cys Ala 290 14272PRTNeurospora crassa 14Ala Ser Gly Ser Gly
Gln Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro 1 5 10 15 Ser Cys Ser
Trp Ser Gly Lys Ala Pro Val Asn Arg Pro Val Leu Ala 20 25 30 Cys
Asp Ala Asn Asn Asn Pro Leu Ser Asp Ala Ser Val Lys Ser Gly 35 40
45 Cys Asp Gly Gly Ser Ala Tyr Thr Cys Ala Asn Asn Ser Pro Trp Ala
50 55 60 Val Asn Asp Gln Leu Ser Tyr Gly Phe Ala Ala Thr Lys Leu
Ser Gly 65 70 75 80 Gly Thr Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala
Leu Thr Phe Thr 85 90 95 Ser Gly Pro Val Ala Gly Lys Thr Leu Val
Val Gln Ser Thr Ser Thr 100 105 110 Gly Gly Asp Leu Gly Ser Asn His
Phe Asp Ile Asn Met Pro Gly Gly 115 120 125 Gly Val Gly Leu Phe Asp
Gly Cys Lys Arg Gln Phe Gly Gly Leu Pro 130 135 140 Gly Ala Gln Tyr
Gly Gly Ile Ser Ser Arg Ser Gln Cys Asp Ser Phe 145 150 155 160 Pro
Ala Ala Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp Trp Phe Gln 165 170
175 Asn Ala Asp Asn Pro Asn Phe Thr Phe Lys Gln Val Gln Cys Pro Ser
180 185 190 Glu Leu Thr Ser Arg Thr Gly Cys Lys Arg Asn Asp Asp Ser
Gln Phe 195 200 205 Pro Val Phe Thr Pro Pro Ser Gly Gly Gly Ser Asn
Pro Ser Thr Pro 210 215 220 Thr Thr Pro Pro Ser Ser Gly Gly Gly Ser
Gly Cys Thr Ala Asp Lys 225 230 235 240 Tyr Ala Gln Cys Gly Gly Ser
Gly Trp Ser Gly Cys Thr Asn Cys Pro 245 250 255 Ser Gly Ser Thr Cys
Lys Thr Ile Asn Asp Tyr Tyr His Gln Cys Ala 260 265 270
15897DNAThielavia terrestris 15atgcgctcta ctcccgttct tcgcacaacc
ctggccgctg cacttcctct ggtcgcctcc 60gcggccagtg gcagtggcca gtccacgaga
tactgggact gctgcaagcc gtcgtgcgct 120tggcccggga aggccgccgt
cagccaaccg gtctacgcgt gcgatgccaa cttccagcgc 180ctgtccgact
tcaatgtcca gtcgggctgc aacggcggct cggcctactc ctgcgccgac
240cagactccct gggcggtgaa cgacaatctc gcctacggct tcgccgcgac
gagcatcgcc 300ggcgggtccg aatcctcgtg gtgctgcgcc tgctacgcgc
tcaccttcac ttccggtccc 360gtcgccggca agacaatggt ggtgcagtca
acgagcactg gcggcgacct gggaagtaac 420catttcgata tcgccatgcc
cggcggcggc gtgggcatct tcaacggctg cagctcgcag 480ttcggcggcc
tccccggcgc tcaatacggc ggcatttcgt cgcgcgacca gtgcgattcc
540ttccccgcgc cgctcaagcc cggctgccag tggcggtttg actggttcca
gaacgccgac 600aacccgacgt tcacgttcca gcaggtgcag tgccccgccg
agatcgttgc ccgctccggc 660tgcaagcgca acgacgactc cagcttcccc
gtcttcaccc ccccaagcgg tggcaacggt 720ggcaccggga cgcccacgtc
gactgcgcct gggtcgggcc agacgtctcc cggcggcggc 780agtggctgca
cgtctcagaa gtgggctcag tgcggtggca tcggcttcag cggatgcacc
840acctgtgtct ctggcaccac ctgccagaag ttgaacgact actactcgca gtgcctc
89716299PRTThielavia terrestris 16Met Arg Ser Thr Pro Val Leu Arg
Thr Thr Leu Ala Ala Ala Leu Pro 1 5 10 15 Leu Val Ala Ser Ala Ala
Ser Gly Ser Gly Gln Ser Thr Arg Tyr Trp 20 25 30 Asp Cys Cys Lys
Pro Ser Cys Ala Trp Pro Gly Lys Ala Ala Val Ser 35 40 45 Gln Pro
Val Tyr Ala Cys Asp Ala Asn Phe Gln Arg Leu Ser Asp Phe 50 55 60
Asn Val Gln Ser Gly Cys Asn Gly Gly Ser Ala Tyr Ser Cys Ala Asp 65
70 75 80 Gln Thr Pro Trp Ala Val Asn Asp Asn Leu Ala Tyr Gly Phe
Ala Ala 85 90 95 Thr Ser Ile Ala Gly Gly Ser Glu Ser Ser Trp Cys
Cys Ala Cys Tyr 100 105 110 Ala Leu Thr Phe Thr Ser Gly Pro Val Ala
Gly Lys Thr Met Val Val 115 120 125 Gln Ser Thr Ser Thr Gly Gly Asp
Leu Gly Ser Asn His Phe Asp Ile 130 135 140 Ala Met Pro Gly Gly Gly
Val Gly Ile Phe Asn Gly Cys Ser Ser Gln 145 150 155 160 Phe Gly Gly
Leu Pro Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg Asp 165 170 175 Gln
Cys Asp Ser Phe Pro Ala Pro Leu Lys Pro Gly Cys Gln Trp Arg 180 185
190 Phe Asp Trp Phe Gln Asn Ala Asp Asn Pro Thr Phe Thr Phe Gln Gln
195 200 205 Val Gln Cys Pro Ala Glu Ile Val Ala Arg Ser Gly Cys Lys
Arg Asn 210 215 220 Asp Asp Ser Ser Phe Pro Val Phe Thr Pro Pro Ser
Gly Gly Asn Gly 225 230 235 240 Gly Thr Gly Thr Pro Thr Ser Thr Ala
Pro Gly Ser Gly Gln Thr Ser 245 250 255 Pro Gly Gly Gly Ser Gly Cys
Thr Ser Gln Lys Trp Ala Gln Cys Gly 260 265 270 Gly Ile Gly Phe Ser
Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys 275 280 285 Gln Lys Leu
Asn Asp Tyr Tyr Ser Gln Cys Leu 290 295 17278PRTThielavia
terrestris 17Ala Ser Gly Ser Gly Gln Ser Thr Arg Tyr Trp Asp Cys
Cys Lys Pro 1 5 10 15 Ser Cys Ala Trp Pro Gly Lys Ala Ala Val Ser
Gln Pro Val Tyr Ala 20 25 30 Cys Asp Ala Asn Phe Gln Arg Leu Ser
Asp Phe Asn Val Gln Ser Gly 35 40 45 Cys Asn Gly Gly Ser Ala Tyr
Ser Cys Ala Asp Gln Thr Pro Trp Ala 50 55 60 Val Asn Asp Asn Leu
Ala Tyr Gly Phe Ala Ala Thr Ser Ile Ala Gly 65 70 75 80 Gly Ser Glu
Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr 85 90 95 Ser
Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Ser Thr 100 105
110 Gly Gly Asp Leu Gly Ser Asn His Phe Asp Ile Ala Met Pro Gly Gly
115 120 125 Gly Val Gly Ile Phe Asn Gly Cys Ser Ser Gln Phe Gly Gly
Leu Pro 130 135 140 Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg Asp Gln
Cys Asp Ser Phe 145 150 155 160 Pro Ala Pro Leu Lys Pro Gly Cys Gln
Trp Arg Phe Asp Trp Phe Gln 165 170 175 Asn Ala Asp Asn Pro Thr Phe
Thr Phe Gln Gln Val Gln Cys Pro Ala 180 185 190 Glu Ile Val Ala Arg
Ser Gly Cys Lys Arg Asn Asp Asp Ser Ser Phe 195 200 205 Pro Val Phe
Thr Pro Pro Ser Gly Gly Asn Gly Gly Thr Gly Thr Pro 210 215 220 Thr
Ser Thr Ala Pro Gly Ser Gly Gln Thr Ser Pro Gly Gly Gly Ser 225 230
235 240 Gly Cys Thr Ser Gln Lys Trp Ala Gln Cys Gly Gly Ile Gly Phe
Ser 245 250 255 Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gln Lys
Leu Asn Asp 260 265 270 Tyr Tyr Ser Gln Cys Leu 275
18305PRTThielavia hyrcaniae 18Met Arg Ser Thr Pro Val Leu Arg Thr
Ala Ile Ala Ala Ala Leu Pro 1 5 10 15 Leu Val Ala Phe Ala Ala Asp
Gly Lys Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser Cys
Ala Trp Ser Gly Lys Ala Ala Val Ser Ala Pro 35 40 45 Val Tyr Ala
Cys Ser Ala Asn Phe Gln Arg Leu Ser Asp Pro Asn Ala 50 55 60 Lys
Ser Gly Cys Asp Gly Gly Ser Ala Tyr Thr Cys Ala Asp Gln Thr 65 70
75 80 Pro Trp Ala Ile Asn Asp Asn Leu Ser Tyr Gly Phe Ala Ala Thr
Ser 85 90 95 Ile Ser Gly Gly Ser Glu Ala Ser Trp Cys Cys Ala Cys
Tyr Glu Leu 100 105 110 Thr Phe Asn Ser Gly Pro Val Ala Gly Lys Lys
Met Val Val Gln Ser 115 120 125 Thr Ser Thr Gly Gly Asp Leu Gly Thr
Asn His Phe Asp Leu Asn Ile 130 135 140 Pro Gly Gly Gly Val Gly Leu
Phe Asp Gly Cys Lys Asn Gln Phe Gly 145 150 155 160 Gly Leu Pro Gly
Ala Gln Tyr Gly Gly Ile Ser Ser Arg Ser Gln Cys 165 170 175 Asp Ser
Phe Pro Glu Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe Asp 180 185
190 Trp Phe Gln Asn Ala Asp Asn Pro Thr Phe Thr Phe Arg Gln Val Gln
195 200 205 Cys Pro Ser Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn
Asp Asp 210 215 220 Ser Ser Phe Pro Val Phe Thr Pro Gly Thr Ser Gly
Ser Ser Ser Thr 225 230 235 240 Ala Lys Pro Ala Ser Ser Ser Thr Arg
Ala Thr Ser Thr Lys Thr Ser 245 250 255 Ala Pro Ala Thr Gln Thr Ser
Ser Thr Gly Gly Gly Cys Val Ala Gln 260 265 270 Lys Trp Ala Gln Cys
Gly Gly Ser Gly Phe Ser Gly Cys Thr Thr Cys 275 280 285 Ala Ala Gly
Ser Thr Cys Thr Lys Gln Asn Asp Tyr Tyr Ser Gln Cys 290 295 300 Leu
305 19293PRTChaetomium thermophilum 19Ala Asp Gly Lys Ser Thr Arg
Tyr Trp Asp Cys Cys Lys Pro Ser Cys 1 5 10 15 Ser Trp Pro Gly Lys
Ala Ala Val Ser Gln Pro Val Phe Ala Cys Asp 20 25 30 Arg Asn Phe
Asn Arg Ile Tyr Asp Phe Gly Ala Lys Ser Gly Cys Glu 35 40 45 Gly
Gly Pro Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala Val Asn 50 55
60 Asp Gln Phe Ser Tyr Gly Phe Ala Ala Thr Asn Ile Ala Gly Gly Asn
65 70 75 80 Glu Ala Ser Trp Cys Cys Ala Cys Tyr Lys Leu Thr Phe Thr
Ser Gly 85 90 95 Pro Val Ala Gly Lys Val Met Val Val Gln Ser Thr
Ser Thr Gly Gly 100 105 110 Asp Leu Gly Asn Asn His Phe Asp Leu Asn
Ile Pro Gly Gly Gly Val 115 120 125 Gly Ile Phe Asp Gly Cys Thr Pro
Gln Phe Gly Gly Leu Pro Gly Glu 130 135 140 Arg Tyr Gly Gly Ile Ser
Ser Arg Ser Gln Cys Asp Ser Phe Pro Asp 145 150 155 160 Ala Leu Lys
Pro Gly Cys Tyr Trp Arg Phe Asp Trp Phe Leu Asn Ala 165 170 175 Asp
Asn Pro Asn Phe Thr Phe Glu Arg Val Gln Cys Pro Ser Glu Leu 180 185
190 Val Ala Arg Thr Gly Cys Lys Arg Asn Asp Asp Gly Asn Tyr Pro Val
195 200 205 Phe Thr Pro Pro Ser Gly Asp Ser Pro Ser Ser Ser Ser Ala
Ala Pro 210 215 220 Thr Ser Thr Ser Thr Ser Gln Gln Pro Gln Gln Pro
Thr Ser Ser Ser 225 230 235 240 Ser Gln Ala Ser Val Pro Thr Ser Asn
Pro Gly Gly Cys Thr Ser Gln 245 250 255 Lys Trp Ala Gln Cys Gly Gly
Ile Gly Phe Thr Gly Cys Thr Thr Cys 260 265 270 Val Ser Gly Thr Thr
Cys Thr Lys Leu Asn Asp Trp Tyr Ser Gln Cys 275 280 285 Thr Met Ile
Asn Leu 290 20235PRTMelanocarpus albomyces 20Met Arg Ser Thr Pro
Val Leu Arg Ala Leu Leu Ala Ala Ala Leu Pro 1 5 10 15 Leu Gly Ala
Leu Ala Ala Asn Gly Gln Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys
Lys Pro Ser Cys Gly Trp Arg Gly Lys Gly Pro Val Asn Gln Pro 35 40
45 Val Tyr Ser Cys Asp Ala Asn Phe Gln Arg Ile His Asp Phe Asp Ala
50 55 60 Val Ser Gly Cys Glu Gly Gly Pro Ala Phe Ser Cys Ala Asp
His Ser 65 70 75 80 Pro Trp Ala Ile Asn Asp Asn Leu Ser Tyr Gly Phe
Ala Ala Thr Ala 85 90 95 Leu Ser Gly Gln Thr Glu Glu Ser Trp Cys
Cys Ala Cys Tyr Ala Leu 100 105 110 Thr Phe Thr Ser Gly Pro Val Ala
Gly Lys Thr Met Val Val Gln Ser 115 120 125 Thr Ser Thr Gly Gly Asp
Leu Gly Ser Asn His Phe Asp Leu Asn Ile 130 135 140 Pro Gly Gly Gly
Val Gly Leu Phe Asp Gly Cys Thr Pro Gln Phe Gly 145 150 155 160 Gly
Leu Pro Gly Ala Arg Tyr Gly Gly Ile Ser Ser Arg Gln Glu Cys 165 170
175 Asp Ser Phe Pro Glu Pro Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp
180 185 190 Trp Phe Gln Asn Ala Asp Asn Pro Ser Phe Thr Phe Glu Arg
Val Gln 195 200 205 Cys Pro Glu Glu Leu Val Ala Arg Thr Gly Cys Arg
Arg His Asp Asp 210 215 220 Gly Gly Phe Ala Val Phe Lys Ala Pro Ser
Ala 225 230 235 21306PRTunknownHumicola sp. 21Pro Phe Met Met Val
Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln 1 5 10 15 Val Ala Ala
Pro Ala Phe Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp 20 25 30 Asp
Cys Cys Lys Pro Ser Cys Ser Trp Pro Gly Lys Ala Leu Val Asn 35 40
45 Gln Pro Val Tyr Ala Arg Asn Ala Asn Phe Gln Arg Ile Thr Asp Pro
50 55 60 Asn Ala Lys Ser Gly Cys Asp Gly Gly Ser Ala Phe Ser Cys
Ala Asp 65 70 75 80 Gln Thr Pro Trp Ala Val Ser Asp Asp Phe Ala Tyr
Gly Phe Ala Ala 85 90 95 Thr Ala Leu Ala Gly Gln Ser Glu Ser Ser
Trp Cys Cys Ala Cys Tyr 100 105 110 Glu Leu Thr Phe Thr Ser Gly Pro
Val Ala Gly Lys Lys Met Ala Val 115 120 125 Gln Ser Thr Ser Thr Gly
Gly Asp Leu Gly Ser Asn His Phe Asp Leu 130 135 140 Asn Met Pro Gly
Gly Gly Val Gly Ile Phe Asp Gly Cys Ser Pro Gln 145 150 155 160 Val
Gly Gly Leu Ala Gly Gln Arg Tyr Gly Gly Val Ser Ser Arg Ser 165 170
175 Glu Cys Asp Ser Phe Pro Ala Ala Leu Lys Pro Gly Cys Tyr Trp Arg
180 185 190 Tyr Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe
Arg Gln 195 200 205 Val Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly
Cys Arg Arg Asn 210 215 220 Asp Asp Gly Asn Phe Pro Ala Val Gln Ile
Pro Ser Ser Ser Thr Ser 225 230 235 240 Ser Pro Val Asn Gln Pro Thr
Ser Thr Ser Thr Thr Ser Thr Ser Thr 245 250 255 Thr Ser Ser Pro Pro
Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala 260 265 270 Glu Arg Trp
Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr 275 280 285 Cys
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln 290 295
300 Cys Leu 305 22299PRTThielavia terrestris NRRL 8126 22Met Arg
Ser Thr Pro Val Leu Arg Thr Thr Leu Ala Ala Ala Leu Pro 1 5 10 15
Leu Val Ala Ser Ala Ala Ser Gly Ser Gly Gln Ser Thr Arg Tyr Trp 20
25 30 Asp Cys Cys Lys Pro Ser Cys Ala Trp Pro Gly Lys Ala Ala Val
Ser 35 40 45 Gln Pro Val Tyr Ala Cys Asp Ala Asn Phe Gln Arg Leu
Ser Asp Phe 50 55 60 Asn Val Gln Ser Gly Cys Asn Gly Gly Ser Ala
Tyr Ser Cys Ala Asp 65 70 75 80 Gln Thr Pro Trp Ala Val Asn Asp Asn
Leu Ala Tyr Gly Phe Ala Ala 85 90 95 Thr Ser Ile Ala Gly Gly Ser
Glu Ser Ser Trp Cys Cys Ala Cys Tyr 100 105 110 Ala Leu Thr Phe Thr
Ser Gly Pro Val Ala Gly Lys Thr Met Val Val 115 120 125 Gln Ser Thr
Ser Thr Gly Gly Asp Leu Gly Ser Asn Gln Phe Asp Ile 130 135 140 Ala
Met Pro Gly Gly Gly Val Gly Ile Phe Asn Gly Cys Ser Ser Gln 145 150
155 160 Phe Gly Gly Leu Pro Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg
Asp 165 170 175 Gln Cys Asp Ser Phe Pro Ala Pro Leu Lys Pro Gly Cys
Gln Trp Arg 180 185 190 Phe Asp Trp Phe Gln Asn Ala Asp Asn Pro Thr
Phe Thr Phe Gln Gln 195 200 205 Val Gln Cys Pro Ala Glu Ile Val Ala
Arg Ser Gly Cys Lys Arg Asn 210 215 220 Asp Asp Ser Ser Phe Pro Val
Phe Thr Pro Pro Ser Gly Gly Asn Gly 225 230 235 240 Gly Thr Gly Thr
Pro Thr Ser Thr Ala Pro Gly Ser Gly Gln Thr Ser 245 250 255 Pro Gly
Gly Gly Ser Gly Cys Thr Ser Gln Lys Trp Ala Gln Cys Gly 260 265 270
Gly Ile Gly Phe Ser Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys 275
280 285 Gln Lys Leu Asn Asp Tyr Tyr Ser Gln Cys Leu 290 295
23305PRTHumicola grisea 23Met Arg Ser Ser Pro Leu Leu Pro Ser Asp
Val Val Ala Ala Leu Pro 1 5 10 15 Val Leu Ala Leu Ala Ala Asp Gly
Lys Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser Cys Gly
Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35 40 45 Val Phe Ser Cys
Asn Ala Asn Phe Gln Arg Leu Thr Asp Phe Asp Ala 50 55 60 Lys Ser
Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln 65 70 75 80
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr 85
90 95 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr
Glu 100 105 110 Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met
Val Val Gln 115 120 125 Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn
His Phe Asp Leu Asn 130 135 140 Ile Pro Gly Gly Gly Val Gly Ile Phe
Asp Gly Cys Thr Pro Gln Phe 145 150 155 160 Gly Gly Leu Pro Gly Gln
Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165 170 175 Cys Asp Arg Phe
Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180 185 190 Asp Trp
Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200 205
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210
215 220 Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser
Ser 225 230 235 240 Pro Val Gly Gln Pro Thr Ser Thr Ser Thr Thr Ser
Thr Ser Thr Thr 245 250 255 Ser Ser Pro Pro Val Gln Pro Thr Thr Pro
Ser Gly Cys Thr Ala Glu 260 265 270 Arg Trp Ala Gln Cys Gly Gly Asn
Gly Trp Ser Gly Cys Thr Thr Cys 275 280 285 Val Ala Gly Ser Thr Cys
Thr Lys Ile Asn Asp Trp Tyr His Gln Cys 290 295 300 Leu 305
24315PRTunknownAcremonium sp. 24Met Arg Ser Ser Pro Phe Leu Arg Ala
Ala Leu Ala Ala Ala Leu Pro 1 5 10 15 Leu Ser Ala His Ala Leu Asp
Gly Lys Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser Cys
Gly Trp Ala Gly Lys Ala Ser Val Asn Gln Pro 35 40 45 Val Phe Ser
Cys Ser Ala Asp Trp Gln Arg Ile Ser Asp Phe Asn Ala 50 55 60 Lys
Ser Gly Cys Asp Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gln Thr 65 70
75 80 Pro Trp Ala Val Asn Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thr
Ala 85 90 95 Ile Ala Gly Gly Ser Glu Ser Ser Trp Cys Cys Ala Cys
Tyr Ala Leu 100 105 110 Thr Phe Asn Ser Gly Pro Val Ala Gly Lys Thr
Met Val Val Gln Ser 115 120 125 Thr Ser Thr Gly Gly Asp Leu Gly Ser
Asn Gln Phe Asp Leu Ala Ile 130 135 140 Pro Gly Gly Gly Val Gly Ile
Phe Asn Gly Cys Ala Ser Gln Phe Gly 145 150 155 160 Gly Leu Pro Gly
Ala Gln Tyr Gly Gly Ile Ser Asp Arg Ser Gln Cys 165 170 175 Ser Ser
Phe Pro Ala Pro Leu Gln Pro Gly Cys Gln Trp Arg Phe Asp 180 185 190
Trp Phe Gln Asn Ala Asp Asn Pro Thr Phe Thr Phe Gln Arg Val Gln 195
200 205 Cys Pro Ser Glu Leu Thr Ser Arg Thr Gly Cys Lys Arg Asp Asp
Asp 210 215 220 Ala Ser Tyr Pro Val Phe Asn Pro Pro Ser Val Pro Gly
Leu Asp Gly 225 230 235 240 Ser Asn Pro Gly Asn Pro Thr Thr Thr Val
Val Pro Pro Ala Ser Thr 245 250 255 Ser Thr Ser Arg Pro Thr Ser Ser
Thr Ser Ser Pro Val Ser Thr Pro 260 265 270 Thr Gly Gln Pro Gly Gly
Cys Thr Thr Gln Lys Trp Gly Gln Cys Gly 275 280 285 Gly Ile Gly Tyr
Thr Gly Cys Thr Asn Cys Val Ala Gly Thr Thr Cys 290 295 300 Thr Gln
Leu Asn Pro Trp Tyr Ser Gln Cys Leu 305 310 315 25302PRTPodospora
anserina 25Met Arg Ser Ser Thr Val Leu Gln Thr Ser Leu Leu Ala Val
Leu Pro 1 5 10 15 Leu Ala Val Gln Ala Gln Gly Ala Ser Gly Ser Gly
Lys Ser Thr Arg 20 25 30 Tyr Trp Asp Cys Cys Lys Pro Ser Cys Ala
Trp Pro Gly Lys Ala Ala 35 40 45 Val Asn Arg Pro Val Phe Ala Cys
Asp Ala Asn Phe Gln Arg Ile Ser 50 55 60 Asp Ser Gly Val Ala Ser
Gly Cys Asn Gly Gly Ser Ala Tyr Ser Cys 65 70 75 80 Ala Asp His Ser
Ala Trp Ala Ile Asn Asp Asn Leu Ser Tyr Gly Phe 85 90 95 Ala Ala
Thr Ala Leu Ser Gly Gly Ser Glu Ala Ser Trp Cys Cys Ala 100 105 110
Cys Tyr Glu Leu Thr Phe Thr Asp Gly Pro Val Ala Gly Lys Lys Met 115
120 125 Val Val Gln Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His
Phe 130 135 140 Asp Leu Asn Ile Pro Gly Gly Gly Val Gly Leu Phe Asp
Gly Cys Lys 145 150 155 160 Pro Gln Phe Gly Gly Leu Pro Gly Ala Thr
Tyr Gly Gly Ile Ser Asp 165 170 175 Arg Ser Gln Cys Ala Ser Phe Pro
Asp Ala Leu Lys Pro Gly Cys Asn 180 185 190 Trp Arg Phe Asp Trp Phe
Lys Asn Ala Asp Asn Pro Ser Phe Thr Phe 195 200 205 Arg Gln Val Gln
Cys Pro Ser Glu Leu Thr Ala Arg Ser Gly Cys Lys 210 215 220 Arg Asp
Asp Asp Ser Arg Phe Pro Val Phe Ser Pro Pro Gly Gly Gly 225 230 235
240 Ser Gln Pro Gln Pro Gln Pro Thr Ser Ser Ala Ala Gln Asn Pro Asn
245 250 255 Pro Thr Pro Ser Ala Ala Pro Gly Gly Cys Arg Ala Ala Lys
Tyr Ala 260 265 270 Gln Cys Gly Gly Gln Gly Phe Thr Gly Cys Thr Thr
Cys Glu Ala Gly 275 280 285 Ser Thr Cys Thr Ala Ser Asn Gln Trp Tyr
Ser Gln Cys Leu 290 295 300 26232PRTAcremonium chrysogenum
ATCC11550 26Met Thr Pro Ser Val Phe Lys Ile Ala Leu Ala Ala Ala Ser
Val Ala 1 5 10 15 Ala Pro Ala Leu Ala Ala Glu Gly Ala Gly Lys Ser
Thr Arg Tyr Trp 20 25 30 Asp Cys Cys Lys Pro Ser Cys Ala Trp Pro
Glu Lys Ala Asp Val Ser 35 40 45 Ala Pro Ala Ala Val Cys Asn Ala
Asp Asn Thr Pro Leu Asn Asp Pro 50 55 60 Leu Ala Asp Ser Gly Cys
Asp Gly Gly Pro Ala Phe Thr Cys Ala Asn 65 70 75 80 Tyr Ser Pro Trp
Ala Val Asn Asp Asn Leu Ala Tyr Gly Phe Ala Ala 85 90 95 Thr Ala
Ile Asn
Gly Gly Ser Glu Ala Ser Trp Cys Cys Ala Cys Tyr 100 105 110 Arg Leu
Thr Phe Thr Asp Gly Pro Val Ala Gly Lys Thr Met Ile Val 115 120 125
Gln Ser Thr Asn Thr Gly Gly Asp Ile Ser Asn Asn His Phe Asp Ile 130
135 140 Leu Met Pro Gly Gly Gly Val Gly Leu Phe Asp Gly Cys Thr Pro
Gln 145 150 155 160 Tyr Gly Gly Ile Pro Gly Ala Gln Tyr Gly Gly Val
Ser Ser Arg Glu 165 170 175 Glu Cys Glu Gln Met Pro Glu Ala Leu Lys
Glu Gly Cys Phe Trp Arg 180 185 190 Phe Asp Trp Phe Ala Asn Ala Asp
Asn Pro Asn Leu Asn Phe Glu Gln 195 200 205 Val Gln Cys Pro Ser Glu
Ile Thr Ala Ile Ser Gly Cys Thr Arg Ser 210 215 220 Asp Asp Gly Asn
Phe Pro Ala Ala 225 230 27304PRTunknownHumicola sp. 27Met Met Val
Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala 1 5 10 15 Ala
Pro Ala Phe Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20 25
30 Cys Lys Pro Ser Cys Ala Trp Ser Gly Lys Ala Ser Val Ser Ser Pro
35 40 45 Val Arg Thr Cys Asp Ala Asn Asn Ser Pro Leu Ser Asp Val
Asp Ala 50 55 60 Lys Ser Ala Cys Asp Gly Gly Val Ala Tyr Thr Cys
Ser Asn Asn Ala 65 70 75 80 Pro Trp Ala Val Asn Asp Asn Leu Ser Tyr
Gly Phe Ala Ala Thr Ala 85 90 95 Ile Asn Gly Gly Ser Glu Ser Ser
Trp Cys Cys Ala Cys Tyr Lys Leu 100 105 110 Thr Phe Thr Ser Gly Pro
Ala Ser Gly Lys Val Met Val Val Gln Ser 115 120 125 Thr Asn Thr Gly
Tyr Asp Leu Ser Asn Asn His Phe Asp Ile Leu Met 130 135 140 Pro Gly
Gly Gly Val Gly Ala Phe Asp Gly Cys Ser Arg Gln Tyr Gly 145 150 155
160 Ser Ile Pro Gly Glu Arg Tyr Gly Gly Val Thr Ser Arg Asp Gln Cys
165 170 175 Asp Gln Met Pro Ser Ala Leu Lys Gln Gly Cys Tyr Trp Arg
Phe Asp 180 185 190 Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe
Arg Gln Val Gln 195 200 205 Cys Pro Ala Glu Leu Val Ala Arg Thr Gly
Cys Arg Arg Asn Asp Asp 210 215 220 Gly Asn Phe Pro Ala Val Gln Ile
Pro Ser Ser Ser Thr Ser Ser Pro 225 230 235 240 Val Asn Gln Pro Thr
Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr Ser 245 250 255 Ser Pro Pro
Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu Arg 260 265 270 Trp
Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys Val 275 280
285 Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys Leu
290 295 300 28307PRTunknownHumicola sp. 28Pro Phe Met Met Val Ala
Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln 1 5 10 15 Val Ala Ala Pro
Ala Phe Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp 20 25 30 Asp Cys
Cys Lys Pro Ser Cys Ser Trp Pro Asp Lys Ala Pro Val Gly 35 40 45
Ser Pro Val Gly Thr Cys Asp Ala Gly Asn Ser Pro Leu Gly Asp Pro 50
55 60 Leu Ala Lys Ser Gly Cys Glu Gly Gly Pro Ser Tyr Thr Cys Ala
Asn 65 70 75 80 Tyr Gln Pro Trp Ala Val Asn Asp Gln Leu Ala Tyr Gly
Phe Ala Ala 85 90 95 Thr Ala Ile Asn Gly Gly Thr Glu Asp Ser Trp
Cys Cys Ala Cys Tyr 100 105 110 Lys Leu Thr Phe Thr Asp Gly Pro Ala
Ser Gly Lys Thr Met Ile Val 115 120 125 Gln Ser Thr Asn Thr Gly Gly
Asp Leu Ser Asp Asn His Phe Asp Leu 130 135 140 Leu Ile Pro Gly Gly
Gly Val Gly Ile Phe Asp Gly Cys Thr Ser Gln 145 150 155 160 Tyr Gly
Gln Ala Leu Pro Gly Ala Gln Tyr Gly Gly Val Ser Ser Arg 165 170 175
Ala Glu Cys Asp Gln Met Pro Glu Ala Ile Lys Ala Gly Cys Gln Trp 180
185 190 Arg Tyr Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe
Arg 195 200 205 Gln Val Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly
Cys Arg Arg 210 215 220 Asn Asp Asp Gly Asn Phe Pro Ala Val Gln Ile
Pro Ser Ser Ser Thr 225 230 235 240 Ser Ser Pro Val Asn Gln Pro Thr
Ser Thr Ser Thr Thr Ser Thr Ser 245 250 255 Thr Thr Ser Ser Pro Pro
Val Gln Pro Thr Thr Pro Ser Gly Cys Thr 260 265 270 Ala Glu Arg Trp
Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr 275 280 285 Thr Cys
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His 290 295 300
Gln Cys Leu 305 29277PRTScytalidium indonesiacum 29Met Arg Ser Ser
Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro 1 5 10 15 Val Leu
Ala Leu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys 20 25 30
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35
40 45 Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Leu Tyr Asp Phe Asp
Ala 50 55 60 Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys
Ala Asp Gln 65 70 75 80 Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe
Gly Phe Ala Ala Thr 85 90 95 Ser Ile Ala Gly Ser Asn Glu Ala Gly
Trp Cys Cys Ala Cys Tyr Glu 100 105 110 Leu Thr Phe Thr Ser Gly Pro
Val Ala Gly Lys Lys Met Val Val Gln 115 120 125 Ser Thr Ser Thr Gly
Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135 140 Ile Pro Gly
Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe 145 150 155 160
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165
170 175 Cys Glu Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg
Phe 180 185 190 Asp Trp Phe Lys Asn Ala Asp Asn Pro Asn Phe Ser Phe
Arg Gln Val 195 200 205 Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly
Cys Arg Arg Asn Asp 210 215 220 Asp Gly Asn Phe Pro Ala Val Gln Ile
Pro Ser Ser Ser Thr Ser Ser 225 230 235 240 Pro Arg Ile Gln Asn Leu
Gly Ser Thr Arg Met Leu Ser Arg Ser Arg 245 250 255 Arg Arg Arg Pro
Ala Gly Gly Ser Gln Leu Ala Ala Ala Gly Gly Val 260 265 270 Arg Gly
Gly Ala Pro 275
* * * * *
References