U.S. patent application number 15/801552 was filed with the patent office on 2018-02-22 for compositions and methods comprising variant microbial proteases.
The applicant listed for this patent is DANISCO US INC.. Invention is credited to Joshua R. Basler, Luis G. Cascao-Pereira, David A. Estell, James T. Kellis, JR..
Application Number | 20180051269 15/801552 |
Document ID | / |
Family ID | 41009802 |
Filed Date | 2018-02-22 |
United States Patent
Application |
20180051269 |
Kind Code |
A1 |
Basler; Joshua R. ; et
al. |
February 22, 2018 |
COMPOSITIONS AND METHODS COMPRISING VARIANT MICROBIAL PROTEASES
Abstract
The present invention provides variant subtilisins and
compositions comprising at least one variant subtilisin set forth
herein, as well as methods for using these variants and
compositions. In some embodiments, the present invention provides
variant subtilisins suitable for laundry cleaning applications.
Inventors: |
Basler; Joshua R.; (Palo
Alto, CA) ; Cascao-Pereira; Luis G.; (Redwood City,
CA) ; Estell; David A.; (San Francisco, CA) ;
Kellis, JR.; James T.; (Woodside, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANISCO US INC. |
Palo Alto |
CA |
US |
|
|
Family ID: |
41009802 |
Appl. No.: |
15/801552 |
Filed: |
November 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14577443 |
Dec 19, 2014 |
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15801552 |
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14537761 |
Nov 10, 2014 |
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14577443 |
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12996564 |
Jun 14, 2011 |
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PCT/US2009/046066 |
Jun 3, 2009 |
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14537761 |
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61059695 |
Jun 6, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 304/21062 20130101;
C12N 9/52 20130101; C11D 11/0017 20130101; C11D 3/38681 20130101;
A23K 20/189 20160501; C12N 9/54 20130101; C11D 3/386 20130101 |
International
Class: |
C12N 9/54 20060101
C12N009/54; C11D 11/00 20060101 C11D011/00; A23K 20/189 20060101
A23K020/189; C11D 3/386 20060101 C11D003/386; C12N 9/52 20060101
C12N009/52 |
Claims
1. An isolated subtilisin variant comprising at least one set of
the following substitution sets: G97A-G128A-Y217Q,
G97A-L126A-G128A, G97A-L126A-G128A-Y217Q, G97A-L126A-Y217Q,
G97A-M124V-G128A, G97A-M124V-G128A-Y217Q, G97A-M124V-L126A,
G97A-M124V-L126A-G128A, G97A-M124V-L126A-Y217Q, G97A-M124V-Y217Q,
G97A-N123G-G128A, G97A-N123G-G128A-Y217Q, G97A-N123G-L126A,
G97A-N123G-L126A-G128A, G97A-N123G-L126A-Y217Q, G97A-N123G-M124V,
G97A-N123G-M124V-G128A, G97A-N123G-M124V-L126A,
G97A-N123G-M124V-Y217Q, G97A-N123G-Y217Q, L126A-G128A-Y217Q,
L96T-G128A-Y217Q, L96T-G97A-G128A, L96T-G97A-G128A-Y217Q,
L96T-G97A-L126A, L96T-G97A-L126A-G128A, L96T-G97A-L126A-Y217Q,
L96T-G97A-M124V, L96T-G97A-M124V-G128A, L96T-G97A-M124V-L126A,
L96T-G97A-M124V-Y217Q, L96T-G97A-N123G, L96T-G97A-N123G-G128A,
L96T-G97A-N123G-L126A, L96T-G97A-N123G-M124V,
L96T-G97A-N123G-Y217Q, L96T-G97A-Y217Q, L96T-L126A-G128A,
L96T-L126A-G128A-Y217Q, L96T-L126A-Y217Q, L96T-M124V-G128A,
L96T-M124V-G128A-Y217Q, L96T-M124V-L126A, L96T-M124V-L126A-G128A,
L96T-M124V-L126A-Y217Q, L96T-M124V-Y217Q, L96T-N123G-G128A,
L96T-N123G-G128A-Y217Q, L96T-N123G-L126A, L96T-N123G-L126A-G128A,
L96T-N123G-L126A-Y217Q, L96T-N123G-M124V, L96T-N123G-M124V-G128A,
L96T-N123G-M124V-L126A, L96T-N123G-M124V-Y217Q, L96T-N123G-Y217Q,
M124V-G128A-Y217Q, M124V-L126A-G128A, M124V-L126A-G128A-Y217Q,
M124V-L126A-Y217Q, N123G-G128A-Y217Q, N123G-L126A-G128A,
N123G-L126A-G128A-Y217Q, N123G-L126A-Y217Q, N123G-M124V-G128A,
N123G-M124V-G128A-Y217Q, N123G-M124V-L126A,
N123G-M124V-L126A-G128A, N123G-M124V-L126A-Y217Q,
N123G-M124V-Y217Q, N62Q-G128A-Y217Q, N62Q-G97A-G128A,
N62Q-G97A-G128A-Y217Q, N62Q-G97A-L126A, N62Q-G97A-L126A-G128A,
N62Q-G97A-L126A-Y217Q, N62Q-G97A-M124V, N62Q-G97A-M124V-G128A,
N62Q-G97A-M124V-L126A, N62Q-G97A-M124V-Y217Q, N62Q-G97A-N123G,
N62Q-G97A-N123G-G128A, N62Q-G97A-N123G-L126A,
N62Q-G97A-N123G-M124V, N62Q-G97A-N123G-Y217Q, N62Q-G97A-Y217Q,
N62Q-L126A-G128A, N62Q-L126A-G128A-Y217Q, N62Q-L126A-Y217Q,
N62Q-L96T-G128A, N62Q-L96T-G128A-Y217Q, N62Q-L96T-G97A,
N62Q-L96T-G97A-G128A, N62Q-L96T-G97A-L126A, N62Q-L96T-G97A-M124V,
N62Q-L96T-G97A-N123G, N62Q-L96T-G97A-Y217Q, N62Q-L96T-L126A,
N62Q-L96T-L126A-G128A, N62Q-L96T-L126A-Y217Q, N62Q-L96T-M124V,
N62Q-L96T-M124V-G128A, N62Q-L96T-M124V-L126A,
N62Q-L96T-M124V-Y217Q, N62Q-L96T-N123G, N62Q-L96T-N123G-G128A,
N62Q-L96T-N123G-L126A, N62Q-L96T-N123G-M124V,
N62Q-L96T-N123G-Y217Q, N62Q-L96T-Y217Q, N62Q-M124V-G128A,
N62Q-M124V-G128A-Y217Q, N62Q-M124V-L126A, N62Q-M124V-L126A-G128A,
N62Q-M124V-L126A-Y217Q, N62Q-M124V-Y217Q, N62Q-N123G-G128A,
N62Q-N123G-G128A-Y217Q, N62Q-N123G-L126A, N62Q-N123G-L126A-G128A,
N62Q-N123G-L126A-Y217Q, N62Q-N123G-M124V, N62Q-N123G-M124V-G128A,
N62Q-N123G-M124V-L126A, N62Q-N123G-M124V-Y217Q, and
N62Q-N123G-Y217Q, wherein said substitutions are at positions
equivalent to the positions of BPN' subtilisin set forth in SEQ ID
NO:1.
2. An isolated subtilisin variant comprising at least one set of
the following substitution sets: G97N-G128A-Y217M,
G97G-G128S-Y217E, G97A-G128A-Y217Q, G97M-G128S-Y217E,
G97A-G128S-Y217Q, G97D-G128S-Y217Q, G97M-G128G-Y217M,
G97G-G128S-Y217Q, G97S-G128S-Y217Q, G97G-G128A-Y217Q,
G97S-G128A-Y217E, G97A-G128S-Y217L, G97A-G128A-Y217N,
G97Q-G128S-Y217L, G97A-G128A-Y217M, G97A-G128A-Y217S,
G97D-G128A-Y217Q, G97M-G128S-Y217Q, G97Q-G128G-Y217D-S87Y,
G97S-G128A-Y217N, G97A-G128S-Y217T, G97D-G128S-Y217E,
G97D-G128A-Y217L, G97G-G128S-Y217E-S78P-A272T, G97T-G128S-Y217D,
G97D-G128A-Y217I, G97Q-G128S-Y217Q, G97G-G128A-Y217D,
G97Q-G128A-Y217N, G97S-G128A-Y217M, G97S-G128S-Y217N,
G97S-G128S-Y217M, G97E-G128S-Y217M, G97S-G128P-Y217Q,
G97T-G128S-Y217Q, G97D-G128S-Y217Q-A73T, G97E-G128S-Y217N,
G97G-G128A-Y217I, G97Q-G128A-Y217D, G97Q-G128S-Y217M,
G97R-G128T-Y217Q-S162P, G97S-G128S-Y217D, G97T-G128P-Y217I,
G97Q-G128G-Y217E, G97C-G128G-Y217N, G97D-G128S-Y217H,
G97M-G128S-Y217L, G97M-G128S-Y217N, G97S-G128S-Y217E,
G97M-G128S-Y217I, G97A-G128P-Y217A, G97R-G128S-Y217D,
G97D-G128A-Y217D, G97V-G128G-Y217D, G97V-G128G-Y217E,
G97A-G128G-Y217T, G97G-G128N-Y217L, G97D-G128A-Y217T,
G97M-G128A-Y217E, and G97M-G128A-Y217N, wherein said substitutions
are at positions equivalent to the positions of BPN' subtilisin set
forth in SEQ ID NO:1.
3. An isolated subtilisin variant comprising at least one set of
the following substitution sets: S24R-S87D-Q206E, P40E-A144K-K213L,
N61E-P129E-S159K, S87D-S162K-K265N, S87D-T242R-Q275E,
N61E-Q103E-N240K, S87D-T242R-K265N, N62R-K265N-Q275E,
P129E-S145D-N240K, P129E-P239R-K265N, Q103E-P129E-T242R,
P40E-N61E-S87D-S162K-T242R, S24R-N62R-S87D-S145D-K265N,
P40E-N62R-S87D-Q103E-S162K, S24R-P40E-S145D-S159K-K213L,
S24R-S87D-A144K-K265N-Q275E, N61E-P129E-S162K-K213L-N240K,
N61E-S145D-S162K-K213L-T242R, S87D-A144K-S145D-S159K-Q275E,
S24R-P129E-Q206E-N240K-K265N, N61E-Q103E-A144K-K213L-T242R,
N62R-S159K-Q206E-K265N-Q275E, S24R-Q103E-P129E-N240K-K265N,
N61E-Q103E-P129E-P239R-N240K, P129E-S145D-N240K-T242R-K265N,
Q103E-S162K-Q206E-K213L-P239R,
P40E-N61E-N62R-S87D-S159K-S162K-K265N,
S24R-P40E-N61E-A144K-Q206E-K213L-T242R,
P40E-N61E-S87D-P129E-S159K-S162K-T242R,
P40E-N62R-S87D-S145D-S159K-S162K-Q275E,
P40E-N62R-S87D-Q103E-A144K-S159K-Q275E,
P40E-N62R-S87D-S159K-S162K-K265N-Q275E,
P40E-N61E-P129E-A144K-S162K-K213L-N240K,
P40E-N61E-Q103E-A144K-S159K-S162K-Q275E,
P40E-N61E-Q103E-S159K-S162K-K213L-P239R,
P40E-N61E-Q103E-S159K-S162K-K213L-N240K,
N62R-S87D-P129E-S145D-S159K-S162K-Q275E,
S24R-N61E-Q103E-P129E-K213L-N240K-T242R,
P40E-N62R-S145D-S159K-S162K-Q206E-Q275E,
N62R-S87D-S145D-S159K-S162K-K265N-Q275E,
N61E-S87D-Q103E-S159K-S162K-K213L-T242R,
S24R-N61E-Q103E-P129E-Q206E-P239R-N240K,
S24R-N62R-P129E-S145D-P239R-K265N-Q275E,
S24R-N62R-P129E-Q206E-N240K-K265N-Q275E,
P40E-S145D-S159K-S162K-K213L-P239R-Q275E,
N61E-Q103E-A144K-Q206E-K213L-N240K-T242R,
S24R-Q103E-P129E-S145D-P239R-N240K-K265N,
N61E-Q103E-P129E-K213L-P239R-N240K-T242R,
N61E-Q103E-Q206E-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-S87D-Q103E-S159K-S162K-K213L-N240K,
N61E-N62R-S87D-Q103E-S159K-S162K-K213L-T242R-Q275E,
P40E-N62R-S87D-S145D-S159K-S162K-N240K-K265N-Q275E,
N62R-S87D-S145D-S159K-S162K-K213L-N240K-K265N-Q275E,
S24R-N61E-Q103E-P129E-Q206E-K213L-P239R-N240K-T242R,
S24R-N61E-Q103E-P129E-S145D-P239R-N240K-T242R-K265N,
N61E-S87D-Q103E-P129E-S159K-S162K-K213L-N240K-T242R,
P40E-N61E-Q103E-P129E-A144K-K213L-P239R-N240K-T242R,
S24R-Q103E-P129E-S145D-Q206E-P239R-N240K-T242R-K265N,
N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-S87D-Q103E-P129E-A144K-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-Q103E-P129E-A144K-K213L-P239R-N240K-T242R-K265N,
S24R-P40E-N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-Q103E-P129E-A144K-S145D-K213L-P239R-N240K-T242R,
S24R-N61E-S87D-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,
P40E-N61E-S87D-Q103E-S145D-S159K-S162K-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-Q103E-P129E-S162K-Q206E-K213L-P239R-N240K-T242R,
S24R-N61E-Q103E-P129E-A144K-S145D-Q206E-K213L-P239R-N240K-T242R,
P40E-N61E-Q103E-P129E-A144K-S162K-Q206E-K213L-P239R-N240K-T242R,
S24R-N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R-K265N,
and
S24R-P40E-N61E-S87D-Q103E-P129E-A144K-S162K-Q206E-K213L-P239R-N240K-T242R-
, wherein said substitutions are at positions equivalent to the
positions of BPN' subtilisin set forth in SEQ ID NO:1.
4. A subtilisin variant comprising the substitutions
G97A/G128A/Y217Q and further comprising at least one modification
of claim 3, and wherein the positions correspond to the positions
of BPN' subtilisin of SEQ ID NO:1.
5. An isolated subtilisin variant comprising at least one set of
the following substitution sets: S53G-F58G, S53G-S78N, S53G-Y104N,
S53G-I111V, S53G-A114G, S53G-N117S, S53G-S125A, S53G-S132N,
S53G-P239V, F58G-S78N, F58G-Y104N, F58G-I111V, F58G-A114G,
F58G-N117S, F58G-S125A, F58G-S132N, F58G-P239V, S78N-Y104N,
S78N-I111V, S78N-A114G, S78N-N117S, S78N-S125A, S78N-S132N,
S78N-P239V, Y104N-I111V, Y104N-A114G, Y104N-N117S, Y104N-S125A,
Y104N-S132N, Y104N-P239V, I111V-A114G, I111V-N117S, I111V-S125A,
I111V-S132N, I111V-P239V, A114G-N117S, A114G-S125A, A114G-S132N,
A114G-P239V, N117S-S125A, N117S-S132N, N117S-P239V, S125A-S132N,
S125A-P239V, S132N-P239V, S53G-F58G-S78N, S53G-F58G-Y104N,
S53G-F58G-I111V, S53G-F58G-A114G, S53G-F58G-N117S, S53G-F58G-S125A,
S53G-F58G-S132N, S53G-F58G-P239V, S53G-S78N-Y104N, S53G-S78N-I111V,
S53G-S78N-A114G, S53G-S78N-N117S, S53G-S78N-S125A, S53G-S78N-S132N,
S53G-S78N-P239V, S53G-Y104N-I111V, S53G-Y104N-A114G,
S53G-Y104N-N117S, S53G-Y104N-S125A, S53G-Y104N-S132N,
S53G-Y104N-P239V, S53G-I111V-A114G, S53G-I111V-N117S,
S53G-I111V-S125A, S53G-I111V-S132N, S53G-I111V-P239V,
S53G-A114G-N117S, S53G-A114G-S125A, S53G-A114G-S132N,
S53G-A114G-P239V, S53G-N117S-S125A, S53G-N117S-S132N,
S53G-N117S-P239V, S53G-S125A-S132N, S53G-S125A-P239V,
S53G-S132N-P239V, F58G-S78N-Y104N, F58G-S78N-I111V,
F58G-S78N-A114G, F58G-S78N-N117S, F58G-S78N-S125A, F58G-S78N-S132N,
F58G-S78N-P239V, F58G-Y104N-I111V, F58G-Y104N-A114G,
F58G-Y104N-N117S, F58G-Y104N-S125A, F58G-Y104N-S132N,
F58G-Y104N-P239V, F58G-I111V-A114G, F58G-I111V-N117S,
F58G-I111V-S125A, F58G-I111V-S132N, F58G-I111V-P239V,
F58G-A114G-N117S, F58G-A114G-S125A, F58G-A114G-S132N,
F58G-A114G-P239V, F58G-N117S-S125A, F58G-N117S-S132N,
F58G-N117S-P239V, F58G-S125A-S132N, F58G-S125A-P239V,
F58G-S132N-P239V, S78N-Y104N-I111V, S78N-Y104N-A114G,
S78N-Y104N-N117S, S78N-Y104N-S125A, S78N-Y104N-S132N,
S78N-Y104N-P239V, S78N-I111V-A114G, S78N-I111V-N117S,
S78N-I111V-S125A, S78N-I111V-S132N, S78N-I111V-P239V,
S78N-A114G-N117S, S78N-A114G-S125A, S78N-A114G-S132N,
S78N-A114G-P239V, S78N-N117S-S125A, S78N-N117S-S132N,
S78N-N117S-P239V, S78N-S125A-S132N, S78N-S125A-P239V,
S78N-S132N-P239V, Y104N-I111V-A114G, Y104N-I111V-N117S,
Y104N-I111V-S125A, Y104N-I111V-S132N, Y104N-I111V-P239V,
Y104N-A114G-N117S, Y104N-A114G-S125A, Y104N-A114G-S132N,
Y104N-A114G-P239V, Y104N-N117S-S125A, Y104N-N117S-S132N,
Y104N-N117S-P239V, Y104N-S125A-S132N, Y104N-S125A-P239V,
Y104N-S132N-P239V, I111V-A114G-N117S, I111V-A114G-S125A,
I111V-A114G-S132N, I111V-A114G-P239V, I111V-N117S-S125A,
I111V-N117S-S132N, I111V-N117S-P239V, I111V-S125A-S132N,
I111V-S125A-P239V, I111V-S132N-P239V, A114G-N117S-S125A,
A114G-N117S-S132N, A114G-N117S-P239V, A114G-S125A-S132N,
A114G-S125A-P239V, A114G-S132N-P239V,
N117S-S125A-S132N,N117S-S125A-P239V, N117S-S132N-P239V,
S125A-S132N-P239V, N76D-D120H-K213N-M222Q, wherein said
substitutions are at positions equivalent to the positions of BPN'
subtilisin set forth in SEQ ID NO:1.
6. A subtilisin variant comprising the substitutions
G97A/G128A/Y217Q and further comprising at least one modification
of claim 5, and wherein the positions correspond to the positions
of BPN' subtilisin of SEQ ID NO:1.
7. An isolated nucleic acid encoding a subtilisin variant set forth
in any of claims 1-6.
8. An expression vector comprising said nucleic acid of claim
7.
9. A host cell comprising said expression vector of claim 6.
10. A cleaning composition comprising at least one subtilisin
variant set forth in any of claims 1-7.
11. The cleaning composition of claim 10, wherein said cleaning
composition is a laundry detergent.
12. The laundry detergent of claim 11, wherein said laundry
detergent is a heavy duty liquid laundry detergent.
13. The cleaning composition of claim 10, wherein said cleaning
composition is a dish detergent.
14. The cleaning composition of any of claims 10-13, further
comprising one or more additional enzymes or enzyme derivatives
selected from the group consisting of hemicellulases, peroxidases,
proteases, cellulases, xylanases, lipases, phospholipases,
esterases, cutinases, pectinases, keratinases, reductases,
oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases,
tannases, pentosanases, malanases, .beta.-glucanases,
arabinosidases, hyaluronidase, chondroitinase, laccase, and
amylases, or mixtures thereof.
15. The cleaning composition of claim 10, further comprising at
least one stabilizing agent.
16. A cleaning composition comprising at least 0.0001 weight
percent of at least one subtilisin variant of any of claims 1-7,
and optionally, at least one suitable adjunct ingredient.
17. A method of cleaning, said method comprising the steps of: a)
contacting a surface and/or an article comprising a fabric with the
cleaning composition of any of claims 10-16; and b) optionally
washing and/or rinsing said surface or article.
18. An animal feed comprising at least one subtilisin variant of
any of claims 1-7.
19. A food processing composition comprising at least one
subtilisin variant of any of claims 1-7.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/577443, filed Dec. 19, 2014, which is a
continuation of U.S. patent application Ser. No. 14/537761, filed
Nov. 10, 2014, which is a divisional application of U.S. patent
application Ser. No. 12/996,564, filed Jun. 14, 2011, which is a
U.S. National Stage Application of International Application No.
PCT/US2009/046066, filed Jun. 3, 2009, which claims the benefit of
U.S. Provisional Application No. 61/059,695, filed Jun. 6, 2008,
which are herein incorporated by reference in their entireties.
SEQUENCE LISTING
[0002] The sequence listing submitted via EFS, in compliance with
37 C.F.R. .sctn. 1.52(e), is incorporated herein by reference. The
sequence listing text file submitted via EFS contains the file
"20171101_NB31178USCNT3_SeqLst.txt" created on Nov. 1, 2017, which
is 3 KB in size.
FIELD OF THE INVENTION
[0003] The present invention provides variant subtilisins and
compositions comprising at least one variant subtilisin set forth
herein, as well as methods for using these variants and
compositions. In particular, the present invention provides variant
proteases suitable for laundry cleaning applications.
BACKGROUND OF THE INVENTION
[0004] Serine proteases are a subgroup of carbonyl hydrolases
comprising a diverse class of enzymes having a wide range of
specificities and biological functions. Much research has been
conducted on the subtilisins, due largely to their usefulness in
cleaning and feed applications. Additional work has been focused on
the adverse environmental conditions (e.g., exposure to oxidative
agents, chelating agents, extremes of temperature and/or pH) which
can adversely impact the functionality of these enzymes in various
applications. Nonetheless, there remains a need in the art for
enzyme systems that are able to resist these adverse conditions and
retain or have improved activity over those currently known in the
art.
SUMMARY OF THE INVENTION
[0005] The present invention provides variant subtilisins and
compositions comprising at least one variant subtilisin set forth
herein, as well as methods for using these variants and
compositions. In some embodiments, the present invention provides
variant proteases suitable for laundry cleaning applications.
[0006] The present invention provides isolated subtilisin variants
comprising at least one set of the following substitution sets:
G97A-G128A-Y217Q, G97A-L126A-G128A, G97A-L126A-G128A-Y217Q,
G97A-L126A-Y217Q, G97A-M124V-G128A, G97A-M124V-G128A-Y217Q,
G97A-M124V-L126A, G97A-M124V-L126A-G128A, G97A-M124V-L126A-Y217Q,
G97A-M124V-Y217Q, G97A-N123G-G128A, G97A-N123G-G128A-Y217Q,
G97A-N123G-L126A, G97A-N123G-L126A-G128A, G97A-N123G-L126A-Y217Q,
G97A-N123G-M124V, G97A-N123G-M124V-G128A, G97A-N123G-M124V-L126A,
G97A-N123G-M124V-Y217Q, G97A-N123G-Y217Q, L126A-G128A-Y217Q,
L96T-G128A-Y217Q, L96T-G97A-G128A, L96T-G97A-G128A-Y217Q,
L96T-G97A-L126A, L96T-G97A-L126A-G128A, L96T-G97A-L126A-Y217Q,
L96T-G97A-M124V, L96T-G97A-M124V-G128A, L96T-G97A-M124V-L126A,
L96T-G97A-M124V-Y217Q, L96T-G97A-N123G, L96T-G97A-N123G-G128A,
L96T-G97A-N123G-L126A, L96T-G97A-N123G-M124V,
L96T-G97A-N123G-Y217Q, L96T-G97A-Y217Q, L96T-L126A-G128A,
L96T-L126A-G128A-Y217Q, L96T-L126A-Y217Q, L96T-M124V-G128A,
L96T-M124V-G128A-Y217Q, L96T-M124V-L126A, L96T-M124V-L126A-G128A,
L96T-M124V-L126A-Y217Q, L96T-M124V-Y217Q, L96T-N123G-G128A,
L96T-N123G-G128A-Y217Q, L96T-N123G-L126A, L96T-N123G-L126A-G128A,
L96T-N123G-L126A-Y217Q, L96T-N123G-M124V, L96T-N123G-M124V-G128A,
L96T-N123G-M124V-L126A, L96T-N123G-M124V-Y217Q, L96T-N123G-Y217Q,
M124V-G128A-Y217Q, M124V-L126A-G128A, M124V-L126A-G128A-Y217Q,
M124V-L126A-Y217Q, N123G-G128A-Y217Q, N123G-L126A-G128A,
N123G-L126A-G128A-Y217Q, N123G-L126A-Y217Q, N123G-M124V-G128A,
N123G-M124V-G128A-Y217Q, N123G-M124V-L126A,
N123G-M124V-L126A-G128A, N123G-M124V-L126A-Y217Q,
N123G-M124V-Y217Q, N62Q-G128A-Y217Q, N62Q-G97A-G128A,
N62Q-G97A-G128A-Y217Q, N62Q-G97A-L126A, N62Q-G97A-L126A-G128A,
N62Q-G97A-L126A-Y217Q, N62Q-G97A-M124V, N62Q-G97A-M124V-G128A,
N62Q-G97A-M124V-L126A, N62Q-G97A-M124V-Y217Q, N62Q-G97A-N123G,
N62Q-G97A-N123G-G128A, N62Q-G97A-N123G-L126A,
N62Q-G97A-N123G-M124V, N62Q-G97A-N123G-Y217Q, N62Q-G97A-Y217Q,
N62Q-L126A-G128A, N62Q-L126A-G128A-Y217Q, N62Q-L126A-Y217Q,
N62Q-L96T-G128A, N62Q-L96T-G128A-Y217Q, N62Q-L96T-G97A,
N62Q-L96T-G97A-G128A, N62Q-L96T-G97A-L126A, N62Q-L96T-G97A-M124V,
N62Q-L96T-G97A-N123G, N62Q-L96T-G97A-Y217Q, N62Q-L96T-L126A,
N62Q-L96T-L126A-G128A, N62Q-L96T-L126A-Y217Q, N62Q-L96T-M124V,
N62Q-L96T-M124V-G128A, N62Q-L96T-M124V-L126A,
N62Q-L96T-M124V-Y217Q, N62Q-L96T-N123G, N62Q-L96T-N123G-G128A,
N62Q-L96T-N123G-L126A, N62Q-L96T-N123G-M124V,
N62Q-L96T-N123G-Y217Q, N62Q-L96T-Y217Q, N62Q-M124V-G128A,
N62Q-M124V-G128A-Y217Q, N62Q-M124V-L126A, N62Q-M124V-L126A-G128A,
N62Q-M124V-L126A-Y217Q, N62Q-M124V-Y217Q, N62Q-N123G-G128A,
N62Q-N123G-G128A-Y217Q, N62Q-N123G-L126A, N62Q-N123G-L126A-G128A,
N62Q-N123G-L126A-Y217Q, N62Q-N123G-M124V, N62Q-N123G-M124V-G128A,
N62Q-N123G-M124V-L126A, N62Q-N123G-M124V-Y217Q, and
N62Q-N123G-Y217Q, wherein the substitutions are at positions
equivalent to the positions of BPN' subtilisin set forth in SEQ ID
NO:1.
[0007] The present invention also provides isolated subtilisin
variants comprising at least one set of the following substitution
sets: G97N-G128A-Y217M, G97G-G128S-Y217E, G97A-G128A-Y217Q,
G97M-G128S-Y217E, G97A-G128S-Y217Q, G97D-G128S-Y217Q,
G97M-G128G-Y217M, G97G-G128S-Y217Q, G97S-G128S-Y217Q,
G97G-G128A-Y217Q, G97S-G128A-Y217E, G97A-G128S-Y217L,
G97A-G128A-Y217N, G97Q-G128S-Y217L, G97A-G128A-Y217M,
G97A-G128A-Y217S, G97D-G128A-Y217Q, G97M-G128S-Y217Q,
G97Q-G128G-Y217D-S87Y, G97S-G128A-Y217N, G97A-G128S-Y217T,
G97D-G128S-Y217E, G97D-G128A-Y217L, G97G-G128S-Y217E-S78P-A272T,
G97T-G128S-Y217D, G97D-G128A-Y217I, G97Q-G128S-Y217Q,
G97G-G128A-Y217D, G97Q-G128A-Y217N, G97S-G128A-Y217M,
G97S-G128S-Y217N, G97S-G128S-Y217M, G97E-G128S-Y217M,
G97S-G128P-Y217Q, G97T-G128S-Y217Q, G97D-G128S-Y217Q-A73T,
G97E-G128S-Y217N, G97G-G128A-Y217I, G97Q-G128A-Y217D,
G97Q-G128S-Y217M, G97R-G128T-Y217Q-S162P, G97S-G128S-Y217D,
G97T-G128P-Y217I, G97Q-G128G-Y217E, G97C-G128G-Y217N,
G97D-G128S-Y217H, G97M-G128S-Y217L, G97M-G128S-Y217N,
G97S-G128S-Y217E, G97M-G128S-Y217I, G97A-G128P-Y217A,
G97R-G128S-Y217D, G97D-G128A-Y217D, G97V-G128G-Y217D,
G97V-G128G-Y217E, G97A-G128G-Y217T, G97G-G128N-Y217L,
G97D-G128A-Y217T, G97M-G128A-Y217E, and G97M-G128A-Y217N, wherein
the substitutions are at positions equivalent to the positions of
BPN' subtilisin set forth in SEQ ID NO:1.
[0008] The present invention further provides isolated subtilisin
variants comprising at least one set of the following substitution
sets: S24R-S87D-Q206E, P40E-A144K-K213L, N61E-P129E-S159K,
S87D-S162K-K265N, S87D-T242R-Q275E, N61E-Q103E-N240K,
S87D-T242R-K265N, N62R-K265N-Q275E, P129E-S145D-N240K,
P129E-P239R-K265N, Q103E-P129E-T242R, P40E-N61E-S87D-S162K-T242R,
S24R-N62R-S87D-S145D-K265N, P40E-N62R-S87D-Q103E-S162K,
S24R-P40E-S145D-S159K-K213L, S24R-S87D-A144K-K265N-Q275E,
N61E-P129E-S162K-K213L-N240K, N61E-S145D-S162K-K213L-T242R,
S87D-A144K-S145D-S159K-Q275E, S24R-P129E-Q206E-N240K-K265N,
N61E-Q103E-A144K-K213L-T242R, N62R-S159K-Q206E-K265N-Q275E,
S24R-Q103E-P129E-N240K-K265N, N61E-Q103E-P129E-P239R-N240K,
P129E-S145D-N240K-T242R-K265N, Q103E-S162K-Q206E-K213L-P239R,
P40E-N61E-N62R-S87D-S159K-S162K-K265N,
S24R-P40E-N61E-A144K-Q206E-K213L-T242R,
P40E-N61E-S87D-P129E-S159K-S162K-T242R,
P40E-N62R-S87D-S145D-S159K-S162K-Q275E,
P40E-N62R-S87D-Q103E-A144K-S159K-Q275E,
P40E-N62R-S87D-S159K-S162K-K265N-Q275E,
P40E-N61E-P129E-A144K-S162K-K213L-N240K,
P40E-N61E-Q103E-A144K-S159K-S162K-Q275E,
P40E-N61E-Q103E-S159K-S162K-K213L-P239R,
P40E-N61E-Q103E-S159K-S162K-K213L-N240K,
N62R-S87D-P129E-S145D-S159K-S162K-Q275E,
S24R-N61E-Q103E-P129E-K213L-N240K-T242R,
P40E-N62R-S145D-S159K-S162K-Q206E-Q275E,
N62R-S87D-S145D-S159K-S162K-K265N-Q275E,
N61E-S87D-Q103E-S159K-S162K-K213L-T242R,
S24R-N61E-Q103E-P129E-Q206E-P239R-N240K,
S24R-N62R-P129E-S145D-P239R-K265N-Q275E,
S24R-N62R-P129E-Q206E-N240K-K265N-Q275E,
P40E-S145D-S159K-S162K-K213L-P239R-Q275E,
N61E-Q103E-A144K-Q206E-K213L-N240K-T242R,
S24R-Q103E-P129E-S145D-P239R-N240K-K265N,
N61E-Q103E-P129E-K213L-P239R-N240K-T242R,
N61E-Q103E-Q206E-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-S87D-Q103E-S159K-S162K-K213L-N240K,
N61E-N62R-S87D-Q103E-S159K-S162K-K213L-T242R-Q275E,
P40E-N62R-S87D-S145D-S159K-S162K-N240K-K265N-Q275E,
N62R-S87D-S145D-S159K-S162K-K213L-N240K-K265N-Q275E,
S24R-N61E-Q103E-P129E-Q206E-K213L-P239R-N240K-T242R,
S24R-N61E-Q103E-P129E-S145D-P239R-N240K-T242R-K265N,
N61E-S87D-Q103E-P129E-S159K-S162K-K213L-N240K-T242R,
P40E-N61E-Q103E-P129E-A144K-K213L-P239R-N240K-T242R,
S24R-Q103E-P129E-S145D-Q206E-P239R-N240K-T242R-K265N,
N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-S87D-Q103E-P129E-A144K-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-Q103E-P129E-A144K-K213L-P239R-N240K-T242R-K265N,
S24R-P40E-N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-Q103E-P129E-A144K-S145D-K213L-P239R-N240K-T242R,
S24R-N61E-S87D-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R,
P40E-N61E-S87D-Q103E-S145D-S159K-S162K-K213L-P239R-N240K-T242R,
S24R-P40E-N61E-Q103E-P129E-S162K-Q206E-K213L-P239R-N240K-T242R,
S24R-N61E-Q103E-P129E-A144K-S145D-Q206E-K213L-P239R-N240K-T242R,
P40E-N61E-Q103E-P129E-A144K-S162K-Q206E-K213L-P239R-N240K-T242R,
S24R-N61E-Q103E-P129E-A144K-Q206E-K213L-P239R-N240K-T242R-K265N,
and
S24R-P40E-N61E-S87D-Q103E-P129E-A144K-S162K-Q206E-K213L-P239R-N240K-T242R-
, wherein said substitutions are at positions equivalent to the
positions of BPN' subtilisin set forth in SEQ ID NO:1.
[0009] The present invention also provides subtilisin variants
comprising the substitutions G97A/G128A/Y217Q and further
comprising at least one of the above substitution sets, and wherein
the positions correspond to the positions of BPN' subtilisin of SEQ
ID NO:1.
[0010] The present invention further provides isolated subtilisin
variants comprising at least one set of the following substitution
sets: S53G-F58G, S53G-S78N, S53G-Y104N, S53G-I111V, S53G-A114G,
S53G-N117S, S53G-S125A, S53G-S132N, S53G-P239V, F58G-S78N,
F58G-Y104N, F58G-I111V, F58G-A114G, F58G-N117S, F58G-S125A,
F58G-S132N, F58G-P239V, S78N-Y104N, S78N-I111V, S78N-A114G,
S78N-N117S, S78N-S125A, S78N-S132N, S78N-P239V, Y104N-I111V,
Y104N-A114G, Y104N-N117S, Y104N-S125A, Y104N-S132N, Y104N-P239V,
I111V-A114G, I111V-N117S, I111V-S125A, I111V-S132N, I111V-P239V,
A114G-N117S, A114G-S125A, A114G-S132N, A114G-P239V, N117S-S125A,
N117S-S132N, N117S-P239V, S125A-S132N, S125A-P239V, S132N-P239V,
S53G-F58G-S78N,
[0011] S53G-F58G-Y104N, S53G-F58G-I111V, S53G-F58G-A114G,
S53G-F58G-N117S, S53G-F58G-S125A, S53G-F58G-S132N, S53G-F58G-P239V,
S53G-S78N-Y104N, S53G-S78N-I111V, S53G-S78N-A114G, S53G-S78N-N117S,
S53G-S78N-S125A, S53G-S78N-S132N, S53G-S78N-P239V,
S53G-Y104N-I111V, S53G-Y104N-A114G, S53G-Y104N-N117S,
S53G-Y104N-S125A, S53G-Y104N-S132N, S53G-Y104N-P239V,
S53G-I111V-A114G, S53G-I111V-N117S, S53G-I111V-S125A,
S53G-I111V-S132N, S53G-I111V-P239V, S53G-A114G-N117S,
S53G-A114G-S125A, S53G-A114G-S132N, S53G-A114G-P239V,
S53G-N117S-S125A, S53G-N117S-S132N, S53G-N117S-P239V,
S53G-S125A-S132N, S53G-S125A-P239V, S53G-S132N-P239V,
F58G-S78N-Y104N, F58G-S78N-I111V, F58G-S78N-A114G, F58G-S78N-N117S,
F58G-S78N-S125A, F58G-S78N-S132N, F58G-S78N-P239V,
F58G-Y104N-I111V, F58G-Y104N-A114G, F58G-Y104N-N117S,
F58G-Y104N-S125A, F58G-Y104N-S132N, F58G-Y104N-P239V,
F58G-I111V-A114G, F58G-I111V-N117S, F58G-I111V-S125A,
F58G-I111V-S132N, F58G-I111V-P239V, F58G-A114G-N117S,
F58G-A114G-S125A, F58G-A114G-S132N, F58G-A114G-P239V,
F58G-N117S-S125A, F58G-N117S-S132N, F58G-N117S-P239V,
F58G-S125A-S132N, F58G-S125A-P239V, F58G-S132N-P239V,
S78N-Y104N-I111V, S78N-Y104N-A114G, S78N-Y104N-N117S,
S78N-Y104N-S125A, S78N-Y104N-S132N, S78N-Y104N-P239V,
S78N-I111V-A114G, S78N-I111V-N117S, S78N-I111V-S125A,
S78N-I111V-S132N, S78N-I111V-P239V, S78N-A114G-N117S,
S78N-A114G-S125A, S78N-A114G-S132N, S78N-A114G-P239V,
S78N-N117S-S125A, S78N-N117S-S132N, S78N-N117S-P239V,
S78N-S125A-S132N, S78N-S125A-P239V, S78N-S132N-P239V,
Y104N-I111V-A114G, Y104N-I111V-N117S, Y104N-I111V-S125A,
Y104N-I111V-S132N, Y104N-I111V-P239V, Y104N-A114G-N117S,
Y104N-A114G-S125A, Y104N-A114G-S132N, Y104N-A114G-P239V,
Y104N-N117S-S125A, Y104N-N117S-S132N, Y104N-N117S-P239V,
Y104N-S125A-S132N, Y104N-S125A-P239V, Y104N-S132N-P239V,
I111V-A114G-N117S, I111V-A114G-S125A, I111V-A114G-S132N,
I111V-A114G-P239V, I111V-N1175-S125A, I111V-N117S-S132N,
I111V-N117S-P239V, 1111V-S125A-S132N, I111V-S125A-P239V,
I111V-S132N-P239V, A114G-N117S-S125A, A114G-N117S-S132N,
A114G-N117S-P239V, A114G-S125A-S132N, A114G-S125A-P239V,
A114G-S132N-P239V, N117S-S125A-S132N, N117S-S125A-P239V,
N117S-S132N-P239V, S125A-S132N-P239V, N76D-D120H-K213N-M222Q,
wherein wherein said substitutions are at positions equivalent to
the positions of BPN' subtilisin set forth in SEQ ID NO:1.
[0012] The present invention also provides subtilisin variants
comprising the substitutions G97A/G128A/Y217Q and further
comprising at least one of the above substitution sets, and wherein
the positions correspond to the positions of BPN' subtilisin of SEQ
ID NO:1.
[0013] The present invention also provides isolated nucleic acids
encoding the subtilisin variants set forth herein, as well as
expression vectors comprising these nucleic acids, and host cells
comprising these expression vectors.
[0014] The present invention further provides cleaning compositions
comprising at least one of the subtilisin variant provided herein.
In some embodiments, the cleaning compositions are laundry
detergents. In some additional embodiments, the laundry detergent
is a heavy duty liquid laundry detergent. In some further
embodiments, the cleaning composition is a dish detergent. In some
still further embodiments, the cleaning composition is a hard
surface cleaning composition. In some additional embodiments, the
cleaning compositions further comprise one or more additional
enzymes or enzyme derivatives selected from the group consisting of
hemicellulases, peroxidases, proteases, cellulases, xylanases,
lipases, phospholipases, esterases, cutinases, pectinases,
keratinases, reductases, oxidases, phenol oxidases, lipoxygenases,
ligninases, pullulanases, tannases, pentosanases, malanases,
.beta.-glucanases, arabinosidases, hyaluronidase, chondroitinase,
laccase, and amylases, or mixtures thereof. In some embodiments,
the cleaning compositions further comprise at least one stabilizing
agent. In some further embodiments, the cleaning compositions
comprise at least 0.0001 weight percent of at least one subtilisin
variant provided herein and optionally, a suitable adjunct
ingredient.
[0015] The present invention also provides methods for cleaning,
comprising the steps of: contacting a surface and/or an article
comprising a fabric with the cleaning compositions provided herein;
and optionally washing and/or rinsing the surface or article.
[0016] The present invention further provides animal feeds
comprising at least one subtilisin variant provided herein. The
present invention also provides food processing compositions
comprising at least one subtilisin variant provided herein.
DESCRIPTION OF THE INVENTION
[0017] The present invention provides variant subtilisins and
compositions comprising at least one variant subtilisin set forth
herein, as well as methods for using these variants and
compositions. In particular, the present invention provides variant
proteases suitable for laundry cleaning applications.
[0018] In some embodiments, the present invention provides means to
produce variant subtilisins with various commercial applications
where degradation or synthesis of polypeptides are desired,
including cleaning compositions, as well as feed components,
textile processing, leather finishing, grain processing, meat
processing, food processing, preparation of protein hydrolysates,
digestive aids, microbicidal compositions, bacteriostatic
compositions, fungistatic compositions, and personal care products
(e.g., oral care, hair care, and/or skin care).
[0019] The present invention further provides enzyme compositions
have comparable or improved wash performance, as compared to
presently used subtilisin proteases. In some embodiments, the
present invention provides cleaning compositions comprising at
least one subtilisin variant provided herein. In some embodiments,
the cleaning composition is a laundry detergent. In some
embodiments, the laundry detergent is a cold water detergent, a low
pH detergent, or a compact detergent. In additional embodiments,
the present invention provides methods for cleaning, comprising the
step of contacting a surface and/or an article comprising a fabric
with a cleaning composition comprising at least one subtilisin
variant.
Cleaning Compositions
[0020] The cleaning compositions of the present invention are
advantageously employed for example, in laundry applications.
Indeed, due to the unique advantages of increased effectiveness in
lower temperature solutions, the enzymes of the present invention
are ideally suited for laundry applications. Furthermore, the
enzymes of the present invention may be employed in both granular
and liquid compositions.
[0021] The variant proteases of the present invention also find use
laundry cleaning additive products. In some embodiments, low
temperature solution cleaning applications find use. The additive
product may be, in its simplest form, one or more proteases. In
some embodiments, the additive is packaged in dosage form for
addition to a cleaning process. Any suitable single dosage form
also finds use with the present invention, including but not
limited to pills, tablets, gelcaps, or other single dosage units
such as pre-measured powders or liquids. In some embodiments,
filler(s) or carrier material(s) are included to increase the
volume of such composition. Suitable filler or carrier materials
include, but are not limited to, various salts of sulfate,
carbonate and silicate as well as talc, clay and the like. Suitable
filler or carrier materials for liquid compositions include, but
are not limited to water or low molecular weight primary and
secondary alcohols including polyols and diols. Examples of such
alcohols include, but are not limited to, methanol, ethanol,
propanol and isopropanol. In some embodiments, the compositions
contain from about 5% to about 90% of such materials. Acidic
fillers find use to reduce pH. Alternatively, the cleaning additive
includes adjunct ingredients as more fully described below.
[0022] The present cleaning compositions and cleaning additives
require an effective amount of at least one of the protease
variants provided herein, alone or in combination with other
proteases and/or additional enzymes. The required level of enzyme
is achieved by the addition of one or more protease variants of the
present invention. Typically the present cleaning compositions
comprise at least about 0.0001 weight percent, from about 0.0001 to
about 1, from about 0.001 to about 0.5, or even from about 0.01 to
about 0.1 weight percent of at least one of the variant proteases
of the present invention.
[0023] The cleaning compositions herein are typically formulated
such that, during use in aqueous cleaning operations, the wash
water will have a pH of from about 5.0 to about 11.5 or even from
about 7.5 to about 10.5. Liquid product formulations are typically
formulated to have a neat pH from about 3.0 to about 9.0 or even
from about 3 to about 5. Granular laundry products are typically
formulated to have a pH from about 9 to about 11. 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.
[0024] Suitable low pH cleaning compositions typically have a neat
pH of from about 3 to about 5, and are typically free of
surfactants that hydrolyze in such a pH environment. Such
surfactants include sodium alkyl sulfate surfactants that comprise
at least one ethylene oxide moiety or even from about 1 to about 16
moles of ethylene oxide. Such cleaning compositions typically
comprise a sufficient amount of a pH modifier, such as sodium
hydroxide, monoethanolamine or hydrochloric acid, to provide such
cleaning composition with a neat pH of from about 3 to about 5.
Such compositions typically comprise at least one acid stable
enzyme. In some embodiments, the compositions are liquids, while in
other embodiments, they are solids. The pH of such liquid
compositions is typically measured as a neat pH. The pH of such
solid compositions is measured as a 10% solids solution of said
composition wherein the solvent is distilled water. In these
embodiments, all pH measurements are taken at 20.degree. C.
[0025] In some embodiments, when the variant protease(s) is/are
employed in a granular composition or liquid, it is desirable for
the variant protease to be in the form of an encapsulated particle
to protect the variant protease from other components of the
granular composition during storage. In addition, encapsulation is
also a means of controlling the availability of the variant
protease during the cleaning process. In some embodiments,
encapsulation enhances the performance of the variant protease(s)
and/or additional enzymes. In this regard, the variant proteases of
the present invention are encapsulated with any suitable
encapsulating material known in the art. In some embodiments, the
encapsulating material typically encapsulates at least part of the
catalyst for the variant protease(s) of the present invention.
Typically, the encapsulating material is water-soluble and/or
water-dispersible. In some embodiments, the encapsulating material
has a glass transition temperature (Tg) of 0.degree. C. or higher.
Glass transition temperature is described in more detail in WO
97/11151. The encapsulating material is selected from consisting of
carbohydrates, natural or synthetic gums, chitin, chitosan,
cellulose and cellulose derivatives, silicates, phosphates,
borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes,
and combinations thereof. When the encapsulating material is a
carbohydrate, it is typically selected from monosaccharides,
oligosaccharides, polysaccharides, and combinations thereof.
Typically, the encapsulating material is a starch. Suitable
starches are described in EP 0 922 499; U.S. Pat. No. 4,977,252;
U.S. Pat. No. 5,354,559, and U.S. Pat. No. 5,935,826. In some
embodiments, the encapsulating material is a microsphere made from
plastic such as thermoplastics, acrylonitrile, methacrylonitrile,
polyacrylonitrile, polymethacrylonitrile and mixtures thereof;
commercially available microspheres that find use include those
supplied by EXPANCEL.RTM. (Stockviksverken, Sweden), and PM 6545,
PM 6550, PM 7220, PM 7228, EXTENDOSPHERES.RTM., LUXSIL.RTM.,
Q-CEL.RTM., and SPHERICEL.RTM. (PQ Corp., Valley Forge, PA)>
[0026] As described herein, the variant proteases of the present
invention find particular use in laundry detergents. These
applications place enzymes under various environmental stresses.
The variant proteases of the present invention provide advantages
over many currently used enzymes, due to their stability under
various conditions.
[0027] Indeed, there are a variety of wash conditions including
varying detergent formulations, wash water volumes, wash water
temperatures, and lengths of wash time, to which proteases involved
in washing are exposed. In addition, detergent formulations used in
different geographical areas have different concentrations of their
relevant components present in the wash water. For example, a
European detergent typically has about 4500-5000 ppm of detergent
components in the wash water, while a Japanese detergent typically
has approximately 667 ppm of detergent components in the wash
water. In North America, particularly the United States, detergents
typically have about 975 ppm of detergent components present in the
wash water.
[0028] A low detergent concentration system includes detergents
where less than about 800 ppm of detergent components are present
in the wash water. Japanese detergents are typically considered low
detergent concentration system as they have approximately 667 ppm
of detergent components present in the wash water.
[0029] A medium detergent concentration includes detergents where
between about 800 ppm and about 2000 ppm of detergent components
are present in the wash water. North American detergents are
generally considered to be medium detergent concentration systems
as they have approximately 975 ppm of detergent components present
in the wash water. Brazil typically has approximately 1500 ppm of
detergent components present in the wash water.
[0030] A high detergent concentration system includes detergents
where greater than about 2000 ppm of detergent components are
present in the wash water. European detergents are generally
considered to be high detergent concentration systems as they have
approximately 4500-5000 ppm of detergent components in the wash
water.
[0031] Latin American detergents are generally high suds phosphate
builder detergents and the range of detergents used in Latin
America can fall in both the medium and high detergent
concentrations as they range from 1500 ppm to 6000 ppm of detergent
components in the wash water. As mentioned above, Brazil typically
has approximately 1500 ppm of detergent components present in the
wash water. However, other high suds phosphate builder detergent
geographies, not limited to other Latin American countries, may
have high detergent concentration systems up to about 6000 ppm of
detergent components present in the wash water.
[0032] In light of the foregoing, it is evident that 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.
[0033] The concentrations of the typical wash solutions are
determined empirically. For example, in the U.S., a typical washing
machine holds a volume of about 64.4 L of wash solution.
Accordingly, in order to obtain a concentration of about 975 ppm of
detergent within the wash solution about 62.79 g of detergent
composition must be added to the 64.4 L of wash solution. This
amount is the typical amount measured into the wash water by the
consumer using the measuring cup provided with the detergent.
[0034] As a further example, different geographies use different
wash temperatures. The temperature of the wash water in Japan is
typically less than that used in Europe. For example, the
temperature of the wash water in North America and Japan is
typically between 10 and 30.degree. C. (e.g., about 20.degree. C.),
whereas the temperature of wash water in Europe is typically
between 30 and 60.degree. C. (e.g., about 40.degree. C.).
[0035] 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 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
[0036] European water hardness is typically greater than 10.5 (for
example 10.5-20.0) 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 3 to10 grains, 3-8 grains or
about 6 grains. Japanese water hardness is typically lower than
North American water hardness, usually less than 4, for example 3
grains per gallon mixed Ca.sup.2+/Mg.sup.2+.
[0037] Accordingly, in some embodiments, the present invention
provides variant proteases that show surprising wash performance in
at least one set of wash conditions (e.g., water temperature, water
hardness, and/or detergent concentration). In some embodiments, the
variant proteases of the present invention are comparable in wash
performance to other subtilisin proteases. In some embodiments, the
variant proteases of the present invention exhibit enhanced wash
performance as compared to subtilisin proteases currently
commercially available. Thus, in some preferred embodiments of the
present invention, the variant proteases provided herein exhibit
enhanced oxidative stability, enhanced thermal stability, and/or
enhanced chelator stability. In addition, the variant proteases of
the present invention find use in cleaning compositions that do not
include detergents, again either alone or in combination with
builders and stabilizers.
[0038] In some embodiments of the present invention, the cleaning
compositions comprise at least one variant protease of the present
invention at a level from about 0.00001% to about 10% by weight of
the composition and the balance (e.g., about 99.999% to about
90.0%) comprising cleaning adjunct materials by weight of
composition. In other aspects of the present invention, the
cleaning compositions of the present invention comprises at least
one variant protease at a level of 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% by weight of the composition and the balance of the
cleaning composition (e.g., about 99.9999% to about 90.0%, about
99.999% to about 98%, about 99.995% to about 99.5% by weight)
comprising cleaning adjunct materials.
[0039] In some embodiments, preferred cleaning compositions
comprise one or more additional enzymes or enzyme derivatives which
provide cleaning performance and/or fabric care benefits, in
addition to one or more of the variant proteases provided herein.
Such enzymes include, but are not limited to other proteases,
lipases, cutinases, amylases, cellulases, peroxidases, oxidases
(e.g. laccases), and/or mannanases.
[0040] Any other suitable protease finds use in the compositions of
the present invention. Suitable proteases include those of animal,
vegetable or microbial origin. In some particularly preferred
embodiments, microbial proteases are used. In some embodiments,
chemically or genetically modified mutants are included. In some
embodiments, the protease is a serine protease, preferably an
alkaline microbial protease or a trypsin-like protease. Examples of
alkaline proteases include subtilisins, especially those derived
from Bacillus (e.g., subtilisin, lentus, amyloliquefaciens,
subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin
168). Additional examples include those mutant proteases described
in U.S. Pat. Nos. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and
6,482,628, all of which are incorporated herein by reference.
Additional protease examples include, but are not limited to
trypsin (e.g., of porcine or bovine origin), and the Fusarium
protease described in WO 89/06270. Preferred commercially available
protease enzymes include MAXATASE.RTM., MAXACAL.TM., MAXAPEM.TM.,
OPTICLEAN.RTM., OPTIMASE.RTM., PROPERASE.RTM., PURAFECT.RTM. and
PURAFECT.RTM. OXP (Genencor); ALCALASE.RTM., SAVINASE.RTM.,
PRIMASE.RTM., DURAZYM.TM., RELASE.RTM. and ESPERASE.RTM.
(Novozymes); and BLAP.TM. (Henkel Kommanditgesellschaft auf Aktien,
Duesseldorf, Germany. Various proteases are described in
WO95/23221, WO 92/21760, and U.S. Pat. Nos. 5,801,039, 5,340,735,
5,500,364, 5,855,625, U.S. Pat. Nos. RE 34,606, 5,955,340,
5,700,676, 6,312,936, and 6,482,628, and various other patents.
[0041] In addition, any suitable lipase finds use in the present
invention. Suitable lipases include, but are not limited to those
of bacterial or fungal origin. Chemically or genetically modified
mutants are encompassed by the present invention. Examples of
useful lipases include Humicola lanuginosa lipase (See e.g., EP 258
068, and EP 305 216), Rhizomucor miehei lipase (See e.g., EP 238
023), Candida lipase, such as C. antarctica lipase (e.g., the C.
antarctica lipase A or B; See e.g., EP 214 761), a Pseudomonas
lipase such as P. alcaligenes and P. pseudoalcaligenes lipase (See
e.g., EP 218 272), P. cepacia lipase (See e.g., EP 331 376), P.
stutzeri lipase (See e.g., GB 1,372,034), P. fluorescens lipase,
Bacillus lipase (e.g., B. subtilis lipase [Dartois et al., Biochem.
Biophys. Acta 1131:253-260 [1993]); B. stearothermophilus lipase
[See e.g., JP 64/744992]; and B. pumilus lipase [See e.g., WO
91/16422]).
[0042] Furthermore, a number of cloned lipases find use in some
embodiments of the present invention, including but not limited to
Penicillium camembertii lipase (See, Yamaguchi et al., Gene
103:61-67 [1991]), Geotricum candidum lipase (See, Schimada et al.,
J. Biochem., 106:383-388 [1989]), and various Rhizopus lipases such
as R. delemar lipase (See, Hass et al., Gene 109:117-113 [1991]), a
R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem.
56:716-719 [1992]) and R. oryzae lipase.
[0043] Other types of lipolytic enzymes such as cutinases also find
use in some embodiments of the present invention, including but not
limited to the cutinase derived from Pseudomonas mendocina (See, WO
88/09367), and the cutinase derived from Fusarium solani pisi (See,
WO 90/09446).
[0044] Additional suitable lipases include commercially available
lipases such as M1 LIPASE.TM., LUMA FAST.TM., and LIPOMAX.TM.
(Genencor); LIPOLASE.RTM. and LIPOLASE.RTM. ULTRA (Novozymes); and
LIPASE P.TM. "Amano" (Amano Pharmaceutical Co. Ltd., Japan).
[0045] In some embodiments of the present invention, the cleaning
compositions of the present invention further comprise lipases at a
level from about 0.00001% to about 10% of additional lipase by
weight of the composition and the balance of cleaning adjunct
materials by weight of composition. In other aspects of the present
invention, the cleaning compositions of the present invention also
comprise, lipases at a level of 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% lipase by weight of the composition.
[0046] Any amylase (alpha and/or beta) suitable for use in alkaline
solutions also find use in some embodiments of the present
invention. Suitable amylases include, but are not limited to those
of bacterial or fungal origin. Chemically or genetically modified
mutants are included in some embodiments. Amylases that find use in
the present invention, include, but are not limited to
.alpha.-amylases obtained from B. licheniformis (See e.g., GB
1,296,839). Commercially available amylases that find use in the
present invention include, but are not limited to DURAMYL.RTM.,
TERMAMYL.RTM., FUNGAMYL.RTM. and BAN.TM. (Novozymes) and
RAPIDASE.RTM. and MAXAMYL.RTM. P (Genencor).
[0047] In some embodiments of the present invention, the cleaning
compositions of the present invention further comprise amylases at
a level from about 0.00001% to about 10% of additional amylase by
weight of the composition and the balance of cleaning adjunct
materials by weight of composition. In other aspects of the present
invention, the cleaning compositions of the present invention also
comprise, amylases at a level of 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% amylase by weight of the composition.
[0048] In some further embodiments, any suitable cellulase finds
used in the cleaning compositions of the present invention.
Suitable cellulases include, but are not limited to those of
bacterial or fungal origin. Chemically or genetically modified
mutants are included in some embodiments. Suitable cellulases
include, but are not limited to Humicola 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., EP 0 495 257).
Commercially available cellulases that find use in the present
include, but are not limited to CELLUZYME.RTM. (Novozymes), 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
cleaning compositions of the present invention further comprise
cellulases at a level from about 0.00001% to about 10% of
additional cellulase by weight of the composition and the balance
of cleaning adjunct materials by weight of composition. In other
aspects of the present invention, the cleaning compositions of the
present invention also comprise cellulases at a level of 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% cellulase by weight of the
composition.
[0049] Any mannanase suitable for use in detergent compositions
also finds use in the present invention. Suitable mannanases
include, but are not limited to those of bacterial or fungal
origin. Chemically or genetically modified mutants are included in
some embodiments. Various mannanases are known which find use in
the present invention (See e.g., U.S. Pat. No. 6,566,114, U.S. Pat.
No.6,602,842, and US Pat. No. 6,440,991, all of which are
incorporated herein by reference). In some embodiments, the
cleaning compositions of the present invention further comprise
mannanases at a level from about 0.00001% to about 10% of
additional mannanase by weight of the composition and the balance
of cleaning adjunct materials by weight of composition. In other
aspects of the present invention, the cleaning compositions of the
present invention also comprise, mannanases at a level of 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% mannanase by weight of the
composition.
[0050] In some embodiments, peroxidases are used in combination
with hydrogen peroxide or a source thereof (e.g., a percarbonate,
perborate or persulfate) in the compositions of the present
invention. 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., WO 94/12621 and WO 95/01426). 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 cleaning compositions of the present invention
further comprise peroxidase and/or oxidase enzymes at a level from
about 0.00001% to about 10% of additional peroxidase and/or oxidase
by weight of the composition and the balance of cleaning adjunct
materials by weight of composition. In other aspects of the present
invention, the cleaning compositions of the present invention also
comprise peroxidase and/or oxidase enzymes at a level of 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% peroxidase and/or oxidase
enzymes by weight of the composition.
[0051] In some embodiments, additional enzymes find use, including
but not limited to perhydrolases (See e.g., WO 05/056782). In
addition, in some particularly preferred embodiments, mixtures of
the above mentioned enzymes are encompassed herein, in particular
one or more additional protease, amylase, lipase, mannanase, and/or
at least one cellulase. Indeed, it is contemplated that various
mixtures of these enzymes will find use in the present invention.
It is also contemplated that the varying levels of the variant
protease(s) and one or more additional enzymes may both
independently range to about 10%, the balance of the cleaning
composition being cleaning adjunct materials. The specific
selection of cleaning adjunct materials are readily made by
considering the surface, item, or fabric to be cleaned, and the
desired form of the composition for the cleaning conditions during
use (e.g., through the wash detergent use).
[0052] Examples of suitable cleaning adjunct materials include, but
are not limited to, surfactants, builders, bleaches, bleach
activators, bleach catalysts, other enzymes, enzyme stabilizing
systems, chelants, optical brighteners, soil release polymers, dye
transfer agents, dispersants, suds suppressors, dyes, perfumes,
colorants, filler salts, hydrotropes, photoactivators, fluorescers,
fabric conditioners, hydrolyzable surfactants, preservatives,
anti-oxidants, anti-shrinkage agents, anti-wrinkle agents,
germicides, fungicides, color speckles, silvercare, anti-tarnish
and/or anti-corrosion agents, alkalinity sources, solubilizing
agents, carriers, processing aids, pigments, 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, all of
which are incorporated herein by reference). Embodiments of
specific cleaning composition materials are exemplified in detail
below. In embodiments in which the cleaning adjunct materials are
not compatible with the variant proteases of the present invention
in the cleaning compositions, then suitable methods of keeping the
cleaning adjunct materials and the protease(s) separated (i.e., not
in contact with each other) until combination of the two components
is appropriate are used. Such separation methods include any
suitable method known in the art (e.g., gelcaps, encapulation,
tablets, physical separation, etc.).
[0053] By way of example, several cleaning compositions wherein the
variant proteases of the present invention find use are described
in greater detail below. In embodiments in which the cleaning
compositions of the present invention are formulated as
compositions suitable for use in laundry machine washing method(s),
the compositions of the present invention preferably contain at
least one surfactant and at least one builder compound, as well as
one or more cleaning adjunct materials preferably selected from
organic polymeric compounds, bleaching agents, additional enzymes,
suds suppressors, dispersants, lime-soap dispersants, soil
suspension and anti-redeposition agents and corrosion inhibitors.
In some embodiments, laundry compositions also contain softening
agents (i.e., as additional cleaning adjunct materials). The
compositions of the present invention also find use detergent
additive products in solid or liquid form. Such additive products
are intended to supplement and/or boost the performance of
conventional detergent compositions and can be added at any stage
of the cleaning process. In some embodiments, the density of the
laundry detergent compositions herein ranges from about 400 to
about 1200 g/liter, while in other embodiments, it ranges from
about 500 to about 950 g/liter of composition measured at
20.degree. C.
[0054] In some embodiments, various cleaning compositions such as
those provided in U.S. Pat. No. 6,605,458 find use with the variant
proteases of the present invention. Thus, in some embodiments, the
compositions comprising at least one variant protease of the
present invention is a compact granular fabric cleaning
composition, while in other embodiments, the composition is a
granular fabric cleaning composition useful in the laundering of
colored fabrics, in further embodiments, the composition is a
granular fabric cleaning composition which provides softening
through the wash capacity, in additional embodiments, the
composition is a heavy duty liquid fabric cleaning composition. In
some embodiments, the compositions comprising at least one variant
protease of the present invention are fabric cleaning compositions
such as those described in U.S. Pat. Nos. 6,610,642 and 6,376,450.
In addition, the variant proteases of the present invention find
use in granular laundry detergent compositions of particular
utility under European or Japanese washing conditions (See e.g.,
U.S. Pat. No. 6,610,642).
[0055] The cleaning compositions of the present invention are
formulated into any suitable form and prepared by any process
chosen by the formulator, non-limiting examples of which are
described in U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005,
5,569,645, 5,565,422, 5,516,448, 5,489,392, and 5,486,303, all of
which are incorporated herein by reference. When a low pH cleaning
composition is desired, the pH of such composition is adjusted via
the addition of a material such as monoethanolamine or an acidic
material such as HCl.
[0056] While not essential for the purposes of the present
invention, the non-limiting list of adjuncts illustrated
hereinafter are suitable for use in the instant cleaning
compositions. In some embodiments, these adjuncts are incorporated
for example, to assist or enhance cleaning performance, for
treatment of the substrate to be cleaned, or to modify the
aesthetics of the cleaning composition as is the case with
perfumes, colorants, dyes or the like. It is understood that such
adjuncts are in addition to the variant proteases of the present
invention. The precise nature of these additional components, and
levels of incorporation thereof, will depend on the physical form
of the composition and the nature of the cleaning operation for
which it is to be used. Suitable adjunct materials include, but are
not limited to, surfactants, builders, chelating agents, dye
transfer inhibiting agents, deposition aids, dispersants,
additional enzymes, and enzyme stabilizers, catalytic materials,
bleach activators, bleach boosters, hydrogen peroxide, sources of
hydrogen peroxide, preformed peracids, polymeric dispersing agents,
clay soil removal/anti-redeposition agents, brighteners, suds
suppressors, dyes, perfumes, structure elasticizing agents, fabric
softeners, carriers, hydrotropes, processing aids and/or pigments.
In addition to the disclosure below, suitable examples of such
other adjuncts and levels of use are found in U.S. Pat. Nos.
5,576,282, 6,306,812, and 6,326,348, that are incorporated by
reference. The aforementioned adjunct ingredients may constitute
the balance of the cleaning compositions of the present
invention.
[0057] In some embodiments, the cleaning compositions according to
the present invention comprise a surfactant or surfactant system
wherein the surfactant is selected from nonionic surfactants,
anionic surfactants, cationic surfactants, ampholytic surfactants,
zwitterionic surfactants, semi-polar nonionic surfactants and
mixtures thereof.
[0058] In some additional embodiments, the cleaning compositions of
the present invention comprise one or more detergent builders or
builder systems. Builders include, but are not limited to, the
alkali metal, ammonium and alkanolammonium salts of polyphosphates,
alkali metal silicates, alkaline earth and alkali metal carbonates,
aluminosilicate builders 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 metal, 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.
[0059] In some further embodiments, the cleaning compositions
herein contain a chelating agent. Suitable chelating agents include
copper, iron and/or manganese chelating agents and mixtures
thereof.
[0060] In some still further embodiments, the cleaning compositions
provided herein contain a deposition aid. Suitable deposition aids
include, polyethylene glycol, polypropylene glycol,
polycarboxylate, soil release polymers such as polytelephthalic
acid, clays such as Kaolinite, montmorillonite, atapulgite, illite,
bentonite, halloysite, and mixtures thereof.
[0061] In some additional embodiments, the cleaning compositions of
the present invention also include 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.
[0062] In some still additional embodiments, the cleaning
compositions of the present invention also contain dispersants.
Suitable water-soluble organic materials include 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.
[0063] In some particularly preferred embodiments, the cleaning
compositions comprise one or more detergent enzymes which provide
cleaning performance and/or fabric care benefits. Examples of
suitable enzymes include, but are not limited to, hemicellulases,
peroxidases, proteases, cellulases, xylanases, lipases,
phospholipases, esterases, cutinases, pectinases, keratinases,
reductases, oxidases, phenol oxidases, lipoxygenases, ligninases,
pullulanases, tannases, pentosanases, malanases, .beta.-glucanases,
arabinosidases, hyaluronidase, chondroitinase, laccase, and
amylases, or mixtures thereof. A typical combination is a cocktail
of conventional applicable enzymes including at least one protease,
at least one lipase, at least one cutinase, and/or at least one
cellulase in conjunction with at least one amylase.
[0064] In some further embodiments, the enzymes used in the
cleaning compositions are stabilized any suitable technique. In
some embodiments, the enzymes employed herein are stabilized by the
presence of water-soluble sources of calcium and/or magnesium ions
in the finished compositions that provide such ions to the
enzymes.
[0065] In some still further embodiments, the cleaning compositions
of the present invention include catalytic metal complexes. One
type of metal-containing bleach catalyst is a catalyst system
comprising a transition metal cation of defined bleach catalytic
activity, such as copper, iron, titanium, ruthenium, tungsten,
molybdenum, or manganese cations, an auxiliary metal cation having
little or no bleach catalytic activity, such as zinc or aluminum
cations, and a sequestrate having defined stability constants for
the catalytic and auxiliary metal cations, particularly
ethylenediaminetetraacetic acid, ethylenediaminetetra
(methylenephosphonic acid) and water-soluble salts thereof. Such
catalysts are disclosed in U.S. Pat. No. 4,430,243.
[0066] In some embodiments, the compositions herein are catalyzed
by means of a manganese compound. Such compounds and levels of use
are well known in the art and include, for example, the
manganese-based catalysts disclosed in U.S. Pat. No. 5,576,282. In
addition, cobalt bleach catalysts useful herein are known, and are
described, for example, in U.S. Pat. Nos. 5,597,936, and 5,595,967.
Such cobalt catalysts are readily prepared by known procedures,
such as taught for example in U.S. Pat. Nos. 5,597,936, and
5,595,967. In some embodiments, the compositions provided herein
also suitably include a transition metal complex of a
macropolycyclic rigid ligand (i.e., "MRL"). As a practical matter,
and not by way of limitation, the compositions and cleaning
processes herein are adjustable, to provide on the order of at
least one part per hundred million of the active MRL species in the
aqueous washing medium, and will preferably provide from about
0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to
about 10 ppm, and most preferably from about 0.1 ppm to about 5
ppm, of the MRL in the wash liquor. Preferred transition-metals in
the instant transition-metal bleach catalyst include manganese,
iron and chromium. Preferred MRLs herein are a special type of
ultra-rigid ligand that is cross-bridged such as
5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitable
transition metal MRLs are readily prepared by known procedures,
such as taught for example in WO 00/332601, and U.S. Pat. No.
6,225,464.
[0067] As indicated above, the cleaning compositions of the present
invention are formulated into any suitable form and prepared by any
process chosen by the formulator, non-limiting examples of which
are described in U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005,
5,569,645, 5,516,448, 5,489,392, and 5,486,303, all of which are
incorporated herein by reference.
[0068] The cleaning compositions disclosed herein of find use in
cleaning a situs (e.g., a surface, dishware, or fabric). Typically,
at least a portion of the situs is contacted with an embodiment of
the present cleaning composition, in neat form or diluted in a wash
liquor, and then the situs is optionally washed and/or rinsed. For
purposes of the present invention, "washing" includes but is not
limited to, scrubbing, and mechanical agitation. In some
embodiments, the cleaning compositions are typically employed at
concentrations of from about 500 ppm to about 15,000 ppm in
solution. When the wash solvent is water, the water temperature
typically ranges from about 5.degree. C. to about 90.degree. C.
and, when the situs comprises a fabric, the water to fabric mass
ratio is typically from about 1:1 to about 30:1.
Definitions
[0069] Unless otherwise indicated, the practice of the present
invention involves conventional techniques commonly used in
molecular biology, protein engineering, microbiology, and
recombinant DNA, which are within the skill of the art. Such
techniques are known to those of skill in the art and are described
in numerous texts and reference works (See e.g., Sambrook et al.,
"Molecular Cloning: A Laboratory Manual", Second Edition (Cold
Spring Harbor), [1989]); and Ausubel et al., "Current Protocols in
Molecular Biology" [1987]). All patents, patent applications,
articles and publications mentioned herein, both supra and infra,
are hereby expressly incorporated herein by reference.
[0070] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention pertains. There are various dictionaries available and
known to those in the art that provide definitions of these terms.
Although any methods and materials similar or equivalent to those
described herein find use in the practice of the present invention,
the preferred methods and materials are described herein.
Accordingly, the terms defined immediately below are more fully
described by reference to the Specification as a whole. Also, as
used herein, the singular "a", "an" and "the" includes the plural
reference unless the context clearly indicates otherwise. Numeric
ranges are inclusive of the numbers defining the range. Unless
otherwise indicated, nucleic acids are written left to right in 5'
to 3' orientation; amino acid sequences are written left to right
in amino to carboxy orientation, respectively. It is to be
understood that this invention is not limited to the particular
methodology, protocols, and reagents described, as these may vary,
depending upon the context they are used by those of skill in the
art.
[0071] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of protein
purification, molecular biology, microbiology, recombinant DNA
techniques and protein sequencing, all of which are within the
skill of those in the art.
[0072] Furthermore, the headings provided herein are not
limitations of the various aspects or embodiments of the invention
which can be had by reference to the specification as a whole.
Accordingly, the terms defined immediately below are more fully
defined by reference to the specification as a whole. Nonetheless,
in order to facilitate understanding of the invention, a number of
terms are defined below.
[0073] As used herein, the terms "protease," and "proteolytic
activity" refer to a protein or peptide exhibiting the ability to
hydrolyze peptides or substrates having peptide linkages. Many well
known procedures exist for measuring proteolytic activity (Kalisz,
"Microbial Proteinases," In: Fiechter (ed.), Advances in
Biochemical Engineering/Biotechnology, [1988]). For example,
proteolytic activity may be ascertained by comparative assays which
analyze the respective protease's ability to hydrolyze a commercial
substrate. Exemplary substrates useful in the analysis of protease
or proteolytic activity, include, but are not limited to di-methyl
casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine
elastin (Sigma E-1625), and bovine keratin (ICN Biomedical 902111).
Colorimetric assays utilizing these substrates are well known in
the art (See e.g., WO 99/34011; and U.S. Pat. No. 6,376,450, both
of which are incorporated herein by reference). The pNA assay (See
e.g., Del Mar et al., Anal. Biochem., 99:316-320 [1979]) also finds
use in determining the active enzyme concentration for fractions
collected during gradient elution. This assay measures the rate at
which p-nitroaniline is released as the enzyme hydrolyzes the
soluble synthetic substrate,
succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide
(sAAPF-pNA). The rate of production of yellow color from the
hydrolysis reaction is measured at 410 nm on a spectrophotometer
and is proportional to the active enzyme concentration. In
addition, absorbance measurements at 280 nm can be used to
determine the total protein concentration. The active
enzyme/total-protein ratio gives the enzyme purity.
[0074] As used herein, "the genus Bacillus" includes all species
within the genus "Bacillus," as known to those of skill in the art,
including but not limited to B. subtilis, B. licheniformis, B.
lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.
amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.
coagulans, B. circulans, B. lautus, and B. thuringiensis. It is
recognized that the genus Bacillus continues to undergo taxonomical
reorganization. Thus, it is intended that the genus include species
that have been reclassified, including but not limited to such
organisms as B. stearothermophilus, which is now named "Geobacillus
stearothermophilus." The production of resistant endospores in the
presence of oxygen is considered the defining feature of the genus
Bacillus, although this characteristic also applies to the recently
named Alicyclobacillus, Amphibacillus, Aneurinibacillus,
Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,
Halobacillus, Paenibacillus, Salibacillus, Thermobacillus,
Ureibacillus, and Virgibacillus.
[0075] The terms "polynucleotide" and "nucleic acid", used
interchangeably herein, refer to a polymeric form of nucleotides of
any length, either ribonucleotides or deoxyribonucleotides. These
terms include, but are not limited to, a single-, double- or
triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a
polymer comprising purine and pyrimidine bases, or other natural,
chemically, biochemically modified, non-natural or derivatized
nucleotide bases. The following are non-limiting examples of
polynucleotides: genes, gene fragments, chromosomal fragments,
ESTs, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes, and primers. In some embodiments,
polynucleotides comprise modified nucleotides, such as methylated
nucleotides and nucleotide analogs, uracyl, other sugars and
linking groups such as fluororibose and thioate, and nucleotide
branches. In alternative embodiments, the sequence of nucleotides
is interrupted by non-nucleotide components.
[0076] As used herein, the terms "DNA construct" and "transforming
DNA" are used interchangeably to refer to DNA used to introduce
sequences into a host cell or organism. The DNA may be generated in
vitro by PCR or any other suitable technique(s) known to those in
the art. In particularly preferred embodiments, the DNA construct
comprises a sequence of interest (e.g., as an incoming sequence).
In some embodiments, the sequence is operably linked to additional
elements such as control elements (e.g., promoters, etc.). The DNA
construct may further comprise a selectable marker. It may further
comprise an incoming sequence flanked by homology boxes. In a
further embodiment, the transforming DNA comprises other
non-homologous sequences, added to the ends (e.g., stuffer
sequences or flanks). In some embodiments, the ends of the incoming
sequence are closed such that the transforming DNA forms a closed
circle. The transforming sequences may be wild-type, mutant or
modified. In some embodiments, the DNA construct comprises
sequences homologous to the host cell chromosome. In other
embodiments, the DNA construct comprises non-homologous sequences.
Once the DNA construct is assembled in vitro it may be used to: 1)
insert heterologous sequences into a desired target sequence of a
host cell, and/or 2) mutagenize a region of the host cell
chromosome (i.e., replace an endogenous sequence with a
heterologous sequence), 3) delete target genes, and/or 4) introduce
a replicating plasmid into the host.
[0077] As used herein, the terms "expression cassette" and
"expression vector" refer to nucleic acid constructs generated
recombinantly or synthetically, with a series of specified nucleic
acid elements that permit transcription of a particular nucleic
acid in a target cell. The recombinant expression cassette can be
incorporated into a plasmid, chromosome, mitochondrial DNA, plastid
DNA, virus, or nucleic acid fragment. Typically, the recombinant
expression cassette portion of an expression vector includes, among
other sequences, a nucleic acid sequence to be transcribed and a
promoter. In preferred embodiments, expression vectors have the
ability to incorporate and express heterologous DNA fragments in a
host cell. Many prokaryotic and eukaryotic expression vectors are
commercially available. Selection of appropriate expression vectors
is within the knowledge of those of skill in the art. The term
"expression cassette" is used interchangeably herein with "DNA
construct," and their grammatical equivalents. Selection of
appropriate expression vectors is within the knowledge of those of
skill in the art.
[0078] As used herein, the term "vector" refers to a polynucleotide
construct designed to introduce nucleic acids into one or more cell
types. Vectors include cloning vectors, expression vectors, shuttle
vectors, plasmids, cassettes and the like. In some embodiments, the
polynucleotide construct comprises a DNA sequence encoding the
protease (e.g., precursor or mature protease) that is operably
linked to a suitable prosequence (e.g., secretory, etc.) capable of
effecting the expression of the DNA in a suitable host.
[0079] As used herein, the term "plasmid" refers to a circular
double-stranded (ds) DNA construct used as a cloning vector, and
which forms an extrachromosomal self-replicating genetic element in
some eukaryotes or prokaryotes, or integrates into the host
chromosome.
[0080] As used herein in the context of introducing a nucleic acid
sequence into a cell, the term "introduced" refers to any method
suitable for transferring the nucleic acid sequence into the cell.
Such methods for introduction include but are not limited to
protoplast fusion, transfection, transformation, conjugation, and
transduction (See e.g., Ferrari et al.,"Genetics," in Hardwood et
al, (eds.), Bacillus, Plenum Publishing Corp., pages 57-72,
[1989]).
[0081] As used herein, the terms "transformed" and "stably
transformed" refers to a cell that has a non-native (heterologous)
polynucleotide sequence integrated into its genome or as an
episomal plasmid that is maintained for at least two
generations.
[0082] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA encoding a secretory leader (i.e., a signal peptide),
is operably linked to DNA for a polypeptide if it is expressed as a
preprotein that participates in the secretion of the polypeptide; a
promoter or enhancer is operably linked to a coding sequence if it
affects the transcription of the sequence; or a ribosome binding
site is operably linked to a coding sequence if it is positioned so
as to facilitate translation. Generally, "operably linked" means
that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading phase.
However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, the synthetic oligonucleotide adaptors or
linkers are used in accordance with conventional practice.
[0083] As used herein the term "gene" refers to a polynucleotide
(e.g., a DNA segment), that encodes a polypeptide and includes
regions preceding and following the coding regions as well as
intervening sequences (introns) between individual coding segments
(exons).
[0084] As used herein, "homologous genes" refers to a pair of genes
from different, but usually related species, which correspond to
each other and which are identical or very similar to each other.
The term encompasses genes that are separated by speciation (i.e.,
the development of new species) (e.g., orthologous genes), as well
as genes that have been separated by genetic duplication (e.g.,
paralogous genes).
[0085] As used herein, proteins are defined as having a common
"fold" if they have the same major secondary structures in the same
arrangement and with the same topological connections. Different
proteins with the same fold often have peripheral elements of
secondary structure and turn regions that differ in size and
conformation. In some cases, these differing peripheral regions may
comprise half the structure. Proteins placed together in the same
fold category do not necessarily have a common evolutionary origin
(e.g., structural similarities arising from the physics and
chemistry of proteins favoring certain packing arrangements and
chain topologies).
[0086] As used herein, "homology" refers to sequence similarity or
identity, with identity being preferred. This homology is
determined using standard techniques known in the art (See e.g.,
Smith and Waterman, Adv. Appl. Math., 2:482 [1981]; Needleman and
Wunsch, J. Mol. Biol., 48:443 [1970]; Pearson and Lipman, Proc.
Natl. Acad. Sci. USA 85:2444 [1988]; programs such as GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package
(Genetics Computer Group, Madison, Wis.); and Devereux et al.,
Nucl. Acid Res., 12:387-395 [1984]).
[0087] As used herein, an "analogous sequence" is one wherein the
function of the gene is essentially the same as the gene based on
the wild-type subtilisin protease. Additionally, analogous genes
include at least about 45%, about 50%, about 55%, about 60%, about
65%, about 70%, about 75%, about 80%, about 85%, about 90%, about
95%, about 97%, about 98%, about 99%, or about 100% sequence
identity with the sequence of the wild-type subtilisin protease.
Alternately, analogous sequences have an alignment of between about
70 to about 100% of the genes found in the B. amyloliquefaciens
subtilisin protease region. In additional embodiments more than one
of the above properties applies to the sequence. Analogous
sequences are determined by known methods of sequence alignment. A
commonly used alignment method is BLAST, although as indicated
above and below, there are other methods that also find use in
aligning sequences.
[0088] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pair-wise alignments. It can also plot a tree
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng and Doolittle (Feng and Doolittle, J. Mol. Evol., 35:351-360
[1987]). The method is similar to that described by Higgins and
Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUP
parameters including a default gap weight of 3.00, a default gap
length weight of 0.10, and weighted end gaps.
[0089] Another example of a useful algorithm is the BLAST
algorithm, described by Altschul et al., (Altschul et al., J. Mol.
Biol., 215:403-410, [1990]; and Karlin et al., Proc. Natl. Acad.
Sci. USA 90:5873-5787 [1993]). A particularly useful BLAST program
is the WU-BLAST-2 program (See, Altschul et al., Meth. Enzymol.,
266:460-480 [1996]). WU-BLAST-2 uses several search parameters,
most of which are set to the default values. The adjustable
parameters are set with the following values: overlap span=1,
overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2
parameters are dynamic values and are established by the program
itself depending upon the composition of the particular sequence
and composition of the particular database against which the
sequence of interest is being searched. However, the values may be
adjusted to increase sensitivity. A % amino acid sequence identity
value is determined by the number of matching identical residues
divided by the total number of residues of the "longer" sequence in
the aligned region. The "longer" sequence is the one having the
most actual residues in the aligned region (gaps introduced by
WU-Blast-2 to maximize the alignment score are ignored).
[0090] Thus, "percent (%) nucleic acid sequence identity" is
defined as the percentage of nucleotide residues in a candidate
sequence that are identical with the nucleotide residues of the
starting sequence (i.e., the sequence of interest). A preferred
method utilizes the BLASTN module of WU-BLAST-2 set to the default
parameters, with overlap span and overlap fraction set to 1 and
0.125, respectively.
[0091] As used herein, "recombinant" includes reference to a cell
or vector, that has been modified by the introduction of a
heterologous nucleic acid sequence or that the cell is derived from
a cell so modified. Thus, for example, recombinant cells express
genes that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all as a result of deliberate human intervention. "Recombination,"
"recombining," and generating a "recombined" nucleic acid are
generally the assembly of two or more nucleic acid fragments
wherein the assembly gives rise to a chimeric gene.
[0092] In some preferred embodiments, mutant DNA sequences are
generated with site saturation mutagenesis in at least one codon.
In another preferred embodiment, site saturation mutagenesis is
performed for two or more codons. In a further embodiment, mutant
DNA sequences have more than about 50%, more than about 55%, more
than about 60%, more than about 65%, more than about 70%, more than
about 75%, more than about 80%, more than about 85%, more than
about 90%, more than about 95%, or more than about 98% homology
with the wild-type sequence. In alternative embodiments, mutant DNA
is generated in vivo using any known mutagenic procedure such as,
for example, radiation, nitrosoguanidine and the like. The desired
DNA sequence is then isolated and used in the methods provided
herein.
[0093] As used herein, the terms "amplification" and "gene
amplification" refer to a process by which specific DNA sequences
are disproportionately replicated such that the amplified gene
becomes present in a higher copy number than was initially present
in the genome. In some embodiments, selection of cells by growth in
the presence of a drug (e.g., an inhibitor of an inhibitable
enzyme) results in the amplification of either the endogenous gene
encoding the gene product required for growth in the presence of
the drug or by amplification of exogenous (i.e., input) sequences
encoding this gene product, or both.
[0094] "Amplification" is a special case of nucleic acid
replication involving template specificity. It is to be contrasted
with non-specific template replication (i.e., replication that is
template-dependent but not dependent on a specific template).
Template specificity is here distinguished from fidelity of
replication (i.e., synthesis of the proper polynucleotide sequence)
and nucleotide (ribo- or deoxyribo-) specificity. Template
specificity is frequently described in terms of "target"
specificity. Target sequences are "targets" in the sense that they
are sought to be sorted out from other nucleic acid. Amplification
techniques have been designed primarily for this sorting out.
[0095] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0096] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0097] As used herein, the term "target," when used in reference to
the polymerase chain reaction, refers to the region of nucleic acid
bounded by the primers used for polymerase chain reaction. Thus,
the "target" is sought to be sorted out from other nucleic acid
sequences. A "segment" is defined as a region of nucleic acid
within the target sequence.
[0098] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the methods of U.S. Pat. Nos. 4,683,195 4,683,202, and
4,965,188, hereby incorporated by reference, which include methods
for increasing the concentration of a segment of a target sequence
in a mixture of genomic DNA without cloning or purification. This
process for amplifying the target sequence consists of introducing
a large excess of two oligonucleotide primers to the DNA mixture
containing the desired target sequence, followed by a precise
sequence of thermal cycling in the presence of a DNA polymerase.
The two primers are complementary to their respective strands of
the double stranded target sequence. To effect amplification, the
mixture is denatured and the primers then annealed to their
complementary sequences within the target molecule. Following
annealing, the primers are extended with a polymerase so as to form
a new pair of complementary strands. The steps of denaturation,
primer annealing and polymerase extension can be repeated many
times (i.e., denaturation, annealing and extension constitute one
"cycle"; there can be numerous "cycles") to obtain a high
concentration of an amplified segment of the desired target
sequence. The length of the amplified segment of the desired target
sequence is determined by the relative positions of the primers
with respect to each other, and therefore, this length is a
controllable parameter. By virtue of the repeating aspect of the
process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified".
[0099] As used herein, the term "amplification reagents" refers to
those reagents (deoxyribonucleotide triphosphates, buffer, etc.),
needed for amplification except for primers, nucleic acid template
and the amplification enzyme. Typically, amplification reagents
along with other reaction components are placed and contained in a
reaction vessel (test tube, microwell, etc.).
[0100] As used herein, the term "RT-PCR" refers to the replication
and amplification of RNA sequences. In this method, reverse
transcription is coupled to PCR, most often using a one enzyme
procedure in which a thermostable polymerase is employed, as
described in U.S. Pat. No. 5,322,770, herein incorporated by
reference. In RT-PCR, the RNA template is converted to cDNA due to
the reverse transcriptase activity of the polymerase, and then
amplified using the polymerizing activity of the polymerase (i.e.,
as in other PCR methods).
[0101] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0102] A "restriction site" refers to a nucleotide sequence
recognized and cleaved by a given restriction endonuclease and is
frequently the site for insertion of DNA fragments. In certain
embodiments of the invention restriction sites are engineered into
the selective marker and into 5' and 3' ends of the DNA
construct.
[0103] "Homologous recombination" means the exchange of DNA
fragments between two DNA molecules or paired chromosomes at the
site of identical or nearly identical nucleotide sequences. In a
preferred embodiment, chromosomal integration is homologous
recombination.
[0104] As used herein "amino acid" refers to peptide or protein
sequences or portions thereof. The terms "protein," "peptide," and
"polypeptide" are used interchangeably.
[0105] As used herein, "protein of interest" and "polypeptide of
interest" refer to a protein/polypeptide that is desired and/or
being assessed. In some embodiments, the protein of interest is
expressed intracellularly, while in other embodiments, it is a
secreted polypeptide. In particularly preferred embodiments, these
enzymes include the serine proteases of the present invention. In
some embodiments, the protein of interest is a secreted polypeptide
which is fused to a signal peptide (i.e., an amino-terminal
extension on a protein to be secreted). Nearly all secreted
proteins use an amino-terminal protein extension which plays a
crucial role in the targeting to and translocation of precursor
proteins across the membrane. This extension is proteolytically
removed by a signal peptidase during or immediately following
membrane transfer.
[0106] A polynucleotide is said to "encode" an RNA or a polypeptide
if, in its native state or when manipulated by methods known to
those of skill in the art, it can be transcribed and/or translated
to produce the RNA, the polypeptide or a fragment thereof. The
anti-sense strand of such a nucleic acid is also said to encode the
sequences. As is known in the art, a DNA can be transcribed by an
RNA polymerase to produce RNA, but an RNA can be reverse
transcribed by reverse transcriptase to produce a DNA. Thus a DNA
can encode a RNA and vice versa.
[0107] "Host strain" or "host cell" refers to a suitable host for
an expression vector comprising DNA according to the present
invention.
[0108] An enzyme is "overexpressed" in a host cell if the enzyme is
expressed in the cell at a higher level that the level at which it
is expressed in a corresponding wild-type cell.
[0109] The terms "protein" and "polypeptide" are used
interchangeability herein. The 3-letter code for amino acids as
defined in conformity with the IUPAC-IUB Joint Commission on
Biochemical Nomenclature (JCBN) is used through out this
disclosure. It is also understood that a polypeptide may be coded
for by more than one nucleotide sequence due to the degeneracy of
the genetic code. A "prosequence" is an amino acid sequence between
the signal sequence and mature protease that is necessary for the
secretion of the protease. Cleavage of the pro sequence results in
a mature active protease.
[0110] The term "signal sequence" or "signal peptide" refers to any
sequence of nucleotides and/or amino acids which may participate in
the secretion of the mature or precursor forms of the protein. This
definition of signal sequence is a functional one, meant to include
all those amino acid sequences encoded by the N-terminal portion of
the protein gene, which participate in the effectuation of the
secretion of protein. They are often, but not universally, bound to
the N-terminal portion of a protein or to the N-terminal portion of
a precursor protein. The signal sequence may be endogenous or
exogenous. The signal sequence may be that normally associated with
the protein (e.g., protease), or may be from a gene encoding
another secreted protein. One exemplary exogenous signal sequence
comprises the first seven amino acid residues of the signal
sequence from Bacillus subtilis subtilisin fused to the remainder
of the signal sequence of the subtilisin from Bacillus lentus (ATCC
21536).
[0111] The term "hybrid signal sequence" refers to signal sequences
in which part of sequence is obtained from the expression host
fused to the signal sequence of the gene to be expressed. In some
embodiments, synthetic sequences are utilized.
[0112] The term "mature" form of a protein or peptide refers to the
final functional form of the protein or peptide. For example, the
mature form of the BPN' subtilisin protease of the present
invention includes at least the amino acid sequence identical to
residue positions 1-275 of SEQ ID NO:1, as set forth below:
TABLE-US-00002 (SEQ ID NO: 1)
AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASM
VPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADG
SGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVV
AAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMA
PGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSL
ENTTTKLGDSFYYGKGLINVQAAAQ
[0113] The term "precursor" form of a protein or peptide refers to
a mature form of the protein having a prosequence operably linked
to the amino or carbonyl terminus of the protein. The precursor may
also have a "signal" sequence operably linked, to the amino
terminus of the prosequence. The precursor may also have additional
polynucleotides that are involved in post-translational activity
(e.g., polynucleotides cleaved therefrom to leave the mature form
of a protein or peptide).
[0114] "Naturally occurring enzyme" refers to an enzyme having the
unmodified amino acid sequence identical to that found in nature.
Naturally occurring enzymes include native enzymes, those enzymes
naturally expressed or found in the particular microorganism.
[0115] The terms "derived from" and "obtained from" refer to not
only a protease produced or producible by a strain of the organism
in question, but also a protease encoded by a DNA sequence isolated
from such strain and produced in a host organism containing such
DNA sequence. Additionally, the term refers to a protease which is
encoded by a DNA sequence of synthetic and/or cDNA origin and which
has the identifying characteristics of the protease in
question.
[0116] A "derivative" within the scope of this definition generally
retains the characteristic proteolytic activity observed in the
wild-type, native or parent form to the extent that the derivative
is useful for similar purposes as the wild-type, native or parent
form. Functional derivatives of serine protease encompass naturally
occurring, synthetically or recombinantly produced peptides or
peptide fragments which have the general characteristics of the
serine protease of the present invention.
[0117] The term "functional derivative" refers to a derivative of a
nucleic acid which has the functional characteristics of a nucleic
acid which encodes serine protease. Functional derivatives of a
nucleic acid which encode serine protease of the present invention
encompass naturally occurring, synthetically or recombinantly
produced nucleic acids or fragments and encode serine protease
characteristic of the present invention. Wild type nucleic acid
encoding serine proteases according to the invention include
naturally occurring alleles and homologues based on the degeneracy
of the genetic code known in the art.
[0118] The term "identical" in the context of two nucleic acids or
polypeptide sequences refers to the residues in the two sequences
that are the same when aligned for maximum correspondence, as
measured using one of the following sequence comparison or analysis
algorithms.
[0119] The term "optimal alignment" refers to the alignment giving
the highest percent identity score.
[0120] "Percent sequence identity," "percent amino acid sequence
identity," "percent gene sequence identity," and/or "percent
nucleic acid/polynucleotide sequence identity," with respect to two
amino acid, polynucleotide and/or gene sequences (as appropriate),
refer to the percentage of residues that are identical in the two
sequences when the sequences are optimally aligned. Thus, 80% amino
acid sequence identity means that 80% of the amino acids in two
optimally aligned polypeptide sequences are identical.
[0121] The phrase "substantially identical" in the context of two
nucleic acids or polypeptides thus refers to a polynucleotide or
polypeptide that comprising at least about 70% sequence identity,
preferably at least about 75%, preferably at least about 80%,
preferably at least about 85%, preferably at least about 90%,
preferably at least about 95% , preferably at least about 97% ,
preferably at least about 98%, and preferably at least about 99%
sequence identity as compared to a reference sequence using the
programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard
parameters. 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 indication that two nucleic acid sequences are
substantially identical is that the two molecules hybridize to each
other under stringent conditions (e.g., within a range of medium to
high stringency).
[0122] The phrase "equivalent," in this context, refers to serine
proteases enzymes that are encoded by a polynucleotide capable of
hybridizing to the polynucleotide encoding the protease of
interest, under conditions of medium to maximum stringency. For
example, being equivalent means that an equivalent mature serine
protease comprises at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
91%, at least about 92%, at least about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%,and/or at least about 99% sequence identity to the mature
subtilisin protease having the amino acid sequence of SEQ ID
NO:1.
[0123] The term "isolated" or "purified" refers to a material that
is removed from its original environment (e.g., the natural
environment if it is naturally occurring). For example, the
material is said to be "purified" when it is present in a
particular composition in a higher or lower concentration than
exists in a naturally occurring or wild type organism or in
combination with components not normally present upon expression
from a naturally occurring or wild type organism. For example, a
naturally-occurring polynucleotide or polypeptide present in a
living animal is not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the coexisting materials
in the natural system, is isolated. In some embodiments, such
polynucleotides are part of a vector, and/or such polynucleotides
or polypeptides are part of a composition, and still be isolated in
that such vector or composition is not part of its natural
environment. In preferred embodiments, a nucleic acid or protein is
said to be purified, for example, if it gives rise to essentially
one band in an electrophoretic gel or blot.
[0124] The term "isolated", when used in reference to a DNA
sequence, refers to a DNA sequence that has been removed from its
natural genetic milieu and is thus free of other extraneous or
unwanted coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (See e.g., Dynan and
Tijan, Nature 316:774-78 [1985]). The term "an isolated DNA
sequence" is alternatively referred to as "a cloned DNA
sequence".
[0125] The term "isolated," when used in reference to a protein,
refers to a protein that is found in a condition other than its
native environment. In a preferred form, the isolated protein is
substantially free of other proteins, particularly other homologous
proteins. An isolated protein is more than about 10% pure,
preferably more than about 20% pure, and even more preferably more
than about 30% pure, as determined by SDS-PAGE. Further aspects of
the invention encompass the protein in a highly purified form
(i.e., more than about 40% pure, more than about 60% pure, more
than about 80% pure, more than about 90% pure, more than about 95%
pure, more than about 97% pure, and even more than about 99% pure),
as determined by SDS-PAGE.
[0126] As used herein, the term, "combinatorial mutagenesis" refers
to methods in which libraries of variants of a starting sequence
are generated. In these libraries, the variants contain one or
several mutations chosen from a predefined set of mutations. In
addition, the methods provide means to introduce random mutations
which were not members of the predefined set of mutations. In some
embodiments, the methods include those set forth in U.S. patent
application Ser. No. 09/699.250, filed Oct. 26, 2000, hereby
incorporated by reference. In alternative embodiments,
combinatorial mutagenesis methods encompass commercially available
kits (e.g., QuikChange.RTM. Multisite, Stratagene, San Diego,
Calif.).
[0127] As used herein, the term "library of mutants" refers to a
population of cells which are identical in most of their genome but
include different homologues of one or more genes. Such libraries
can be used, for example, to identify genes or operons with
improved traits.
[0128] As used herein, the term "starting gene" refers to a gene of
interest that encodes a protein of interest that is to be improved
and/or changed using the present invention.
[0129] As used herein, the term "multiple sequence alignment"
("MSA") refers to the sequences of multiple homologs of a starting
gene that are aligned using an algorithm (e.g., Clustal W).
[0130] As used herein, the terms "consensus sequence" and
"canonical sequence" refer to an archetypical amino acid sequence
against which all variants of a particular protein or sequence of
interest are compared. The terms also refer to a sequence that sets
forth the nucleotides that are most often present in a DNA sequence
of interest. For each position of a gene, the consensus sequence
gives the amino acid that is most abundant in that position in the
MSA.
[0131] As used herein, the term "consensus mutation" refers to a
difference in the sequence of a starting gene and a consensus
sequence. Consensus mutations are identified by comparing the
sequences of the starting gene and the consensus sequence resulting
from an MSA. In some embodiments, consensus mutations are
introduced into the starting gene such that it becomes more similar
to the consensus sequence. Consensus mutations also include amino
acid changes that change an amino acid in a starting gene to an
amino acid that is more frequently found in an MSA at that position
relative to the frequency of that amino acid in the starting gene.
Thus, the term consensus mutation comprises all single amino acid
changes that replace an amino acid of the starting gene with an
amino acid that is more abundant than the amino acid in the
MSA.
[0132] As used herein, the term "enhanced combinatorial consensus
mutagenesis library" refers to a CCM library that is designed and
constructed based on screening and/or sequencing results from an
earlier round of CCM mutagenesis and screening. In some
embodiments, the enhanced CCM library is based on the sequence of
an initial hit resulting from an earlier round of CCM. In
additional embodiments, the enhanced CCM is designed such that
mutations that were frequently observed in initial hits from
earlier rounds of mutagenesis and screening are favored. In some
preferred embodiments, this is accomplished by omitting primers
that encode performance-reducing mutations or by increasing the
concentration of primers that encode performance-enhancing
mutations relative to other primers that were used in earlier CCM
libraries.
[0133] As used herein, the term "performance-reducing mutations"
refer to mutations in the combinatorial consensus mutagenesis
library that are less frequently found in hits resulting from
screening as compared to an unscreened combinatorial consensus
mutagenesis library. In preferred embodiments, the screening
process removes and/or reduces the abundance of variants that
contain "performance-reducing mutations."
[0134] As used herein, the term "functional assay" refers to an
assay that provides an indication of a protein's activity. In
particularly preferred embodiments, the term refers to assay
systems in which a protein is analyzed for its ability to function
in its usual capacity. For example, in the case of enzymes, a
functional assay involves determining the effectiveness of the
enzyme in catalyzing a reaction.
[0135] As used herein, the term "target property" refers to the
property of the starting gene that is to be altered. It is not
intended that the present invention be limited to any particular
target property. However, in some preferred embodiments, the target
property is the stability of a gene product (e.g., resistance to
denaturation, proteolysis or other degradative factors), while in
other embodiments, the level of production in a production host is
altered. Indeed, it is contemplated that any property of a starting
gene will find use in the present invention.
[0136] The term "property" or grammatical equivalents thereof in
the context of a nucleic acid, as used herein, refer to any
characteristic or attribute of a nucleic acid that can be selected
or detected. These properties include, but are not limited to, a
property affecting binding to a polypeptide, a property conferred
on a cell comprising a particular nucleic acid, a property
affecting gene transcription (e.g., promoter strength, promoter
recognition, promoter regulation, enhancer function), a property
affecting RNA processing (e.g., RNA splicing, RNA stability, RNA
conformation, and post-transcriptional modification), a property
affecting translation (e.g., level, regulation, binding of mRNA to
ribosomal proteins, post-translational modification). For example,
a binding site for a transcription factor, polymerase, regulatory
factor, etc., of a nucleic acid may be altered to produce desired
characteristics or to identify undesirable characteristics.
[0137] The term "property" or grammatical equivalents thereof in
the context of a polypeptide (including proteins), as used herein,
refer to any characteristic or attribute of a polypeptide that can
be selected or detected. These properties include, but are not
limited to oxidative stability, substrate specificity, catalytic
activity, thermal stability, alkaline stability, pH activity
profile, resistance to proteolytic degradation, K.sub.M, k.sub.cat,
k.sub.cat/k.sub.M ratio, protein folding, inducing an immune
response, ability to bind to a ligand, ability to bind to a
receptor, ability to be secreted, ability to be displayed on the
surface of a cell, ability to oligomerize, ability to signal,
ability to stimulate cell proliferation, ability to inhibit cell
proliferation, ability to induce apoptosis, ability to be modified
by phosphorylation or glycosylation, and/or ability to treat
disease, etc.
[0138] The terms "modified sequence" and "modified genes" are used
interchangeably herein to refer to a sequence that includes a
deletion, insertion or interruption of naturally occurring nucleic
acid sequence. In some preferred embodiments, the expression
product of the modified sequence is a truncated protein (e.g., if
the modification is a deletion or interruption of the sequence). In
some particularly preferred embodiments, the truncated protein
retains biological activity. In alternative embodiments, the
expression product of the modified sequence is an elongated protein
(e.g., modifications comprising an insertion into the nucleic acid
sequence). In some embodiments, an insertion leads to a truncated
protein (e.g., when the insertion results in the formation of a
stop codon). Thus, an insertion may result in either a truncated
protein or an elongated protein as an expression product.
[0139] As used herein, the terms "mutant sequence" and "mutant
gene" are used interchangeably and refer to a sequence that has an
alteration in at least one codon occurring in a host cell's
wild-type sequence. The expression product of the mutant sequence
is a protein with an altered amino acid sequence relative to the
wild-type. The expression product may have an altered functional
capacity (e.g., enhanced enzymatic activity).
[0140] The terms "mutagenic primer" or "mutagenic oligonucleotide"
(used interchangeably herein) are intended to refer to
oligonucleotide compositions which correspond to a portion of the
template sequence and which are capable of hybridizing thereto.
With respect to mutagenic primers, the primer will not precisely
match the template nucleic acid, the mismatch or mismatches in the
primer being used to introduce the desired mutation into the
nucleic acid library. As used herein, "non-mutagenic primer" or
"non-mutagenic oligonucleotide" refers to oligonucleotide
compositions which will match precisely to the template nucleic
acid. In one embodiment of the invention, only mutagenic primers
are used. In another preferred embodiment of the invention, the
primers are designed so that for at least one region at which a
mutagenic primer has been included, there is also non-mutagenic
primer included in the oligonucleotide mixture. By adding a mixture
of mutagenic primers and non-mutagenic primers corresponding to at
least one of the mutagenic primers, it is possible to produce a
resulting nucleic acid library in which a variety of combinatorial
mutational patterns are presented. For example, if it is desired
that some of the members of the mutant nucleic acid library retain
their precursor sequence at certain positions while other members
are mutant at such sites, the non-mutagenic primers provide the
ability to obtain a specific level of non-mutant members within the
nucleic acid library for a given residue. The methods of the
invention employ mutagenic and non-mutagenic oligonucleotides which
are generally between 10-50 bases in length, more preferably about
15-45 bases in length. However, it may be necessary to use primers
that are either shorter than 10 bases or longer than 50 bases to
obtain the mutagenesis result desired. With respect to
corresponding mutagenic and non-mutagenic primers, it is not
necessary that the corresponding oligonucleotides be of identical
length, but only that there is overlap in the region corresponding
to the mutation to be added. Primers may be added in a pre-defined
ratio according to the present invention. For example, if it is
desired that the resulting library have a significant level of a
certain specific mutation and a lesser amount of a different
mutation at the same or different site, by adjusting the amount of
primer added, it is possible to produce the desired biased library.
Alternatively, by adding lesser or greater amounts of non-mutagenic
primers, it is possible to adjust the frequency with which the
corresponding mutation(s) are produced in the mutant nucleic acid
library.
[0141] As used herein, the phrase "contiguous mutations" refers to
mutations which are presented within the same oligonucleotide
primer. For example, contiguous mutations may be adjacent or nearby
each other, however, they will be introduced into the resulting
mutant template nucleic acids by the same primer.
[0142] The terms "wild-type sequence," or "wild-type gene" are used
interchangeably herein, to refer to a sequence that is native or
naturally occurring in a host cell. In some embodiments, the
wild-type sequence refers to a sequence of interest that is the
starting point of a protein engineering project. The wild-type
sequence may encode either a homologous or heterologous protein. A
homologous protein is one the host cell would produce without
intervention. A heterologous protein is one that the host cell
would not produce but for the intervention. Unless otherwise
indicated, the amino acid position numbers refer to those assigned
to the mature Bacillus amyloliquefaciens subtilisin sequence of SEQ
ID NO:1. The invention, however, is not limited to the mutation of
this particular subtilisin but extends to precursor proteases
containing amino acid residues at positions which are "equivalent"
to the particular identified residues in the Bacillus
amyloliquefaciens subtilisin.
[0143] As used herein, the terms "protease variant," "subtilisin
variant," "subtilisin protease variant," are used in reference to
proteases that are similar to a wild-type subtilisin or a parent
protease (e.g., subtilisin) used as a starting point in protein
engineering. These variants are similar to the wild-type or other
parent in their function, but have mutations in their amino acid
sequence that make them different in sequence from the wild-type or
parent protease (e.g., subtilisin).
[0144] As used herein, the terms "modification" and "mutation"
refers to any change or alteration in an amino acid sequence. It is
intended that the term encompass substitutions, deletions,
insertions, and/or replacement of amino acid side chains in an
amino acid sequence of interest (e.g., a subtilisin sequence). It
is also intended that the term encompass chemical modification of
an amino acid sequence of interest (e.g., a subtilisin
sequence).
[0145] The term "oxidation stable" refers to proteases of the
present invention that retain a specified amount of enzymatic
activity over a given period of time under conditions prevailing
during the proteolytic, hydrolyzing, cleaning or other process of
the invention, for example while exposed to or contacted with
bleaching agents or oxidizing agents. In some embodiments, the
proteases retain 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% proteolytic activity after
contact with a bleaching or oxidizing agent over a given time
period, for example, at least about 1 minute, about 3 minutes,
about 5 minutes, about 8 minutes, about 12 minutes, about 16
minutes, about 20 minutes, etc. In some embodiments, the stability
is measured as described in the Examples.
[0146] The term "chelator stable" refers to proteases of the
present invention that retain a specified amount of enzymatic
activity over a given period of time under conditions prevailing
during the proteolytic, hydrolyzing, cleaning or other process of
the invention, for example while exposed to or contacted with
chelating agents. In some embodiments, the proteases retain 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% proteolytic activity after contact with a
chelating agent over a given time period, for example, at least
about 10 minutes, about 20 minutes, about 40 minutes, about 60
minutes, about 100 minutes, etc. In some embodiments, the chelator
stability is measured as described in the Examples.
[0147] The terms "thermally stable" and "thermostable" refer to
proteases of the present invention that retain a specified amount
of enzymatic activity after exposure to identified temperatures
over a given period of time under conditions prevailing during the
proteolytic, hydrolyzing, cleaning or other process of the
invention, for example while exposed altered temperatures. Altered
temperatures include increased or decreased temperatures. In some
embodiments, the proteases retain 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% proteolytic
activity after exposure to altered temperatures over a given time
period, for example, at least about 60 minutes, about 120 minutes,
about 180 minutes, about 240 minutes, about 300 minutes, etc. In
some embodiments, the thermostability is determined as described in
the Examples.
[0148] The term "enhanced stability" in the context of an
oxidation, chelator, thermal and/or pH stable protease refers to a
higher retained proteolytic activity over time as compared to other
serine proteases (e.g., subtilisin proteases) and/or wild-type
enzymes.
[0149] The term "diminished stability" in the context of an
oxidation, chelator, thermal and/or pH stable protease refers to a
lower retained proteolytic activity over time as compared to other
serine proteases (e.g., subtilisin proteases) and/or wild-type
enzymes.
[0150] The term "cleaning activity" refers to the cleaning
performance achieved by the protease under conditions prevailing
during the proteolytic, hydrolyzing, cleaning or other process of
the invention. In some embodiments, cleaning performance is
determined by the application of various cleaning assays concerning
enzyme sensitive stains, for example grass, blood, milk, or egg
protein as determined by various chromatographic,
spectrophotometric or other quantitative methodologies after
subjection of the stains to standard wash conditions. Exemplary
assays include, but are not limited to those described in WO
99/34011, and U.S. Pat. No. 6,605,458 (both of which are herein
incorporated by reference), as well as those methods included in
the Examples.
[0151] As used herein, "cleaning compositions" and "cleaning
formulations" refer to compositions that find use in the removal of
undesired compounds from items to be cleaned, such as fabric,
dishes, contact lenses, other solid substrates, hair (shampoos),
skin (soaps and creams), teeth (mouthwashes, toothpastes) etc. The
term encompasses any materials/compounds selected for the
particular type of cleaning composition desired and the form of the
product (e.g., liquid, gel, granule, or spray composition), as long
as the composition is compatible with the perhydrolase and other
enzyme(s) used in the composition. The specific selection of
cleaning composition materials are readily made by considering the
surface, item or fabric to be cleaned, and the desired form of the
composition for the cleaning conditions during use.
[0152] The terms further refer to any composition that is suited
for cleaning, bleaching, disinfecting, and/or sterilizing any
object and/or surface. It is intended that the terms include, but
are not limited to detergent compositions (e.g., liquid and/or
solid laundry detergents and fine fabric detergents; hard surface
cleaning formulations, such as for glass, wood, ceramic and metal
counter tops and windows; carpet cleaners; oven cleaners; fabric
fresheners; fabric softeners; and textile and laundry pre-spotters,
as well as dish detergents).
[0153] Indeed, the term "cleaning composition" as used herein,
includes unless otherwise indicated, granular or powder-form
all-purpose or heavy-duty washing agents, especially cleaning
detergents; liquid, gel or paste-form all-purpose washing agents,
especially the so-called heavy-duty liquid (HDL) types; liquid
fine-fabric detergents; hand dishwashing agents or light duty
dishwashing agents, especially those of the high-foaming type;
machine dishwashing agents, including the various tablet, granular,
liquid and rinse-aid types for household and institutional use;
liquid cleaning and disinfecting agents, including antibacterial
hand-wash types, cleaning bars, mouthwashes, denture cleaners, car
or carpet shampoos, bathroom cleaners; hair shampoos and
hair-rinses; shower gels and foam baths and metal cleaners; as well
as cleaning auxiliaries such as bleach additives and "stain-stick"
or pre-treat types.
[0154] As used herein, the terms "detergent composition" and
"detergent formulation" are used in reference to mixtures which are
intended for use in a wash medium for the cleaning of soiled
objects. In some preferred embodiments, the term is used in
reference to laundering fabrics and/or garments (e.g., "laundry
detergents"). In alternative embodiments, the term refers to other
detergents, such as those used to clean dishes, cutlery, etc.
(e.g., "dishwashing detergents"). It is not intended that the
present invention be limited to any particular detergent
formulation or composition. Indeed, it is intended that in addition
to perhydrolase, the term encompasses detergents that contain
surfactants, transferase(s), hydrolytic enzymes, oxido reductases,
builders, bleaching agents, bleach activators, bluing agents and
fluorescent dyes, caking inhibitors, masking agents, enzyme
activators, antioxidants, and solubilizers.
[0155] The term "cleaning effective amount" of a protease refers to
the quantity of protease described hereinbefore that achieves a
desired level of enzymatic activity in a specific cleaning
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 protease used, the cleaning application, the
specific composition of the cleaning composition, and whether a
liquid or dry (e.g., granular, bar) composition is required,
etc.
[0156] The term "cleaning adjunct materials," as used herein, means
any liquid, solid or gaseous material selected for the particular
type of cleaning composition desired and the form of the product
(e.g., liquid, granule, powder, bar, paste, spray, tablet, gel; or
foam composition), which materials are also preferably compatible
with the protease enzyme 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.
[0157] The term "enhanced performance" in the context of cleaning
activity refers to an increased or greater cleaning activity of
certain enzyme sensitive stains such as egg, milk, grass or blood,
as determined by usual evaluation after a standard wash cycle
and/or multiple wash cycles.
[0158] The term "diminished performance" in the context of cleaning
activity refers to an decreased or lesser cleaning activity of
certain enzyme sensitive stains such as egg, milk, grass or blood,
as determined by usual evaluation after a standard wash cycle.
[0159] The term "comparative performance" in the context of
cleaning activity refers to at least about 60%, at least about 70%,
at least about 80% at least about 90%, or at least about 95% of the
cleaning activity of a comparative subtilisin protease (e.g.,
commercially available proteases), including but not limited to
OPTIMASE.TM. protease (Genencor), PURAFECT.TM. protease products
(Genencor), SAVINASE.TM. protease (Novozymes), BPN'-variants (See
e.g., U.S. Pat. No. Re 34,606), RELASE.TM., DURAZYME.TM.,
EVERLASE.TM., KANNASE.TM. protease (Novozymes), MAXACAL.TM.,
MAXAPEM.TM., PROPERASE.TM. proteases (Genencor; See also, U.S. Pat.
No. Re 34,606, and U.S. Pat. Nos. 5,700,676; 5,955,340; 6,312,936;
and 6,482,628), and B. lentus variant protease products (e.g.,
those described in WO 92/21760, WO 95/23221 and/or WO 97/07770).
Exemplary subtilisin protease variants include, but are not limited
to those having substitutions or deletions at residue positions
equivalent to positions 76, 101, 103, 104, 120, 159, 167, 170, 194,
195, 217, 232, 235, 236, 245, 248, and/or 252 of BPN'. Cleaning
performance can be determined by comparing the proteases of the
present invention with those subtilisin proteases in various
cleaning assays concerning enzyme sensitive stains such as grass,
blood or milk as determined by usual spectrophotometric or
analytical methodologies after standard wash cycle conditions.
[0160] As used herein, "fabric cleaning compositions" include hand
and machine laundry detergent compositions including laundry
additive compositions and compositions suitable for use in the
soaking and/or pretreatment of stained fabrics (e.g., clothes,
linens, and other textile materials).
[0161] The "compact" form of the cleaning compositions herein is
best reflected by density and, in terms of composition, by the
amount of inorganic filler salt. Inorganic filler salts are
conventional ingredients of detergent compositions in powder form.
In conventional detergent compositions, the filler salts are
present in substantial amounts, typically about 17 to about 35% by
weight of the total composition. In contrast, in compact
compositions, the filler salt is present in amounts not exceeding
about 15% of the total composition. In some embodiments, the filler
salt is present in amounts that do not exceed about 10%, or more
preferably, about 5%, by weight of the composition. In some
embodiments, the inorganic filler salts are selected from the
alkali and alkaline-earth-metal salts of sulfates and chlorides. A
preferred filler salt is sodium sulfate.
[0162] As used herein, the term "surfactant" refers to any compound
generally recognized in the art as having surface active qualities.
Surfactants generally include anionic, cationic, nonionic, and
zwitterionic compounds, which are further described, herein.
Experimental
[0163] The present invention is described in further detail in the
following Examples which are not in any way intended to limit the
scope of the invention as claimed. The following Examples are
offered to illustrate, but not to limit the claimed invention.
[0164] In the experimental disclosure which follows, the following
abbreviations apply: PI (proteinase inhibitor), ppm (parts per
million); M (molar); mM (millimolar); .mu.M (micromolar); nM
(nanomolar); mol (moles); mmol (millimoles); .mu.mol (micromoles);
nmol (nanomoles); gm (grams); mg (milligrams); .mu.g (micrograms);
pg (picograms); L (liters); ml and mL (milliliters); .mu.l and
.mu.L (microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers); U (units); V (volts); MW (molecular
weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s)
(hour/hours); .degree. C. (degrees Centigrade); QS (quantity
sufficient); ND (not done); NA (not applicable); rpm (revolutions
per minute); H.sub.2O (water); dH.sub.2O (deionized water); (HCl
(hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobase
pair); kD (kilodaltons); cDNA (copy or complementary DNA); DNA
(deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double
stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA
(ribonucleic acid); MgCl.sub.2 (magnesium chloride); NaCl (sodium
chloride); w/v (weight to volume); v/v (volume to volume); g
(gravity); OD (optical density); Dulbecco's phosphate buffered
solution (DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract,
10 mM NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto Tryptone,
24 g/l glycerol, 2.31 g/l KH.sub.2PO.sub.4, and 12.54 g/l
K.sub.2HPO.sub.4); OD.sub.280 (optical density at 280 nm);
OD.sub.600 (optical density at 600 nm); A.sub.405 (absorbance at
405 nm); Vmax (the maximum initial velocity of an enzyme catalyzed
reaction); PAGE (polyacrylamide gel electrophoresis); PBS
(phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate
buffer, pH 7.2]); PBST (PBS+0.25% TWEEN.RTM. 20); PEG (polyethylene
glycol); PCR (polymerase chain reaction); RT-PCR (reverse
transcription PCR); SDS (sodium dodecyl sulfate); Tris
(tris(hydroxymethyl)aminomethane); HEPES
(N[2-Hydroxyethyl]piperazine-N42-ethanesulfonic acid]); HBS (HEPES
buffered saline); Tris-HCl
(tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine
(N-[tris-(hydroxymethyl)-methyl]-glycine); CHES
(2-(N-cyclo-hexylamino) ethane-sulfonic acid); TAPS
(3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid);
CAPS (3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl
sulfoxide); DTT (1,4-dithio-DL-threitol); SA (sinapinic acid
(s,5-dimethoxy-4-hydroxy cinnamic acid); TCA (trichloroacetic
acid); Glut and GSH (reduced glutathione); GSSG (oxidized
glutathione); TCEP (Tris[2-carboxyethyl] phosphine); Ci (Curies);
mCi (milliCuries); .mu.Ci (microCuries); HPLC (high pressure liquid
chromatography); RP-HPLC (reverse phase high pressure liquid
chromatography); TLC (thin layer chromatography); MALDI-TOF
(matrix-assisted laser desorption/ionization-time of flight); Ts
(tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et (ethyl), Me
(methyl); Taq (Thermus aquaticus DNA polymerase); Klenow (DNA
polymerase I large (Klenow) fragment); EGTA (ethylene
glycol-bis(.beta.-aminoethyl ether) N, N, N', N'-tetraacetic acid);
EDTA (ethylenediaminetetracetic acid); bla (.beta.-lactamase or
ampicillin-resistance gene); HDL (high density liquid); MJ Research
(MJ Research, Reno, Nev.); Baseclear (Baseclear BV, Inc., Leiden,
the Netherlands); PerSeptive (PerSeptive Biosystems, Framingham,
Mass.); ThermoFinnigan (ThermoFinnigan, San Jose, Calif.); Argo
(Argo BioAnalytica, Morris Plains, N.J.); Seitz EKS (SeitzSchenk
Filtersystems GmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East
Hills, N.Y.); Spectrum (Spectrum Laboratories, Dominguez Rancho,
Calif.); Molecular Structure (Molecular Structure Corp., Woodlands,
Tex.); Accelrys (Accelrys, Inc., San Diego, Calif.); Chemical
Computing (Chemical Computing Corp., Montreal, Canada); New
Brunswick (New Brunswick Scientific, Co., Edison, N.J.); CFT
(Center for Test Materials, Vlaardingen, the Netherlands); Test
Fabrics (Test Fabrics, Inc., West Pittiston, Pa.), Procter &
Gamble (Procter & Gamble, Inc., Cincinnati, Ohio); GE
Healthcare (GE Healthcare, Chalfont St. Giles, United Kingdom);
OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (Megazyme
International Ireland Ltd., Bray Business Park, Bray, Co., Wicklow,
Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP
Kelco, Wilmington, Del.); Corning (Corning Life Sciences, Corning,
N.Y.); (NEN (NEN Life Science Products, Boston, Mass.); Pharma AS
(Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway);
Bio-Synthesis (Bio-Synthesis, Lewisville, Tex.); ATCC (American
Type Culture Collection, Rockville, Md.); Gibco/BRL (Gibco/BRL,
Grand Island , N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.);
Pharmacia (Pharmacia Biotech, Piscataway, N.J.); NCBI (National
Center for Biotechnology Information); Applied Biosystems (Applied
Biosystems, Foster City, Calif.); BD Biosciences and/or Clontech
(BD Biosciences CLONTECH Laboratories, Palo Alto, Calif.); Operon
Technologies (Operon Technologies, Inc., Alameda, Calif.); MWG
Biotech (MWG Biotech, High Point, N.C.); Oligos Etc (Oligos Etc.
Inc, Wilsonville, Oreg.); Bachem (Bachem Bioscience, Inc , King of
Prussia, Pa.); Difco (Difco Laboratories, Detroit, Mich.);
Mediatech (Mediatech, Herndon, Va.; Santa Cruz (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.); Oxoid (Oxoid Inc.,
Ogdensburg, N.Y.); Worthington (Worthington Biochemical Corp.,
Freehold, N.J.); GIBCO BRL or Gibco BRL (Life Technologies, Inc.,
Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.);
Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp.,
San Diego, Calif.); NEB (New England Biolabs, Ipswich, Mass.);
Sigma (Sigma Chemical Co., St. Louis, Mo.); Pierce (Pierce
Biotechnology, Rockford, Ill.); Takara (Takara Bio Inc. Otsu,
Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science
(EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia,
Calif.); Biodesign (Biodesign Intl., Saco, Me.); Aptagen (Aptagen,
Inc., Herndon, Va.); Sorvall (Sorvall brand, from Kendro Laboratory
Products, Asheville, N.C.); United States Testing (United States
Testing Co., Hoboken, N.J.);Molecular Devices (Molecular Devices,
Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems,
Minneapolis, Minn.); Stratagene (Stratagene Cloning Systems, La
Jolla, Calif.); Marsh (Marsh Biosciences, Rochester, N.Y.); Geneart
(Geneart GmbH, Regensburg, Germany); DNA2.0 (DNA2.0, Menlo Park,
Calif.); Gene Oracle (Gene Oracle, Mountain View, Calif.); Bio-Tek
(Bio-Tek Instruments, Winooski, Vt.); Biacore (Biacore, Inc.,
Piscataway, N.J.); PeproTech (PeproTech, Rocky Hill, N.J.); SynPep
(SynPep, Dublin, Calif.); New Objective (New Objective brand;
Scientific Instrument Services, Inc., Ringoes, N.J.); Waters
(Waters, Inc., Milford, Mass.); Matrix Science (Matrix Science,
Boston, Mass.); Dionex (Dionex, Corp., Sunnyvale, Calif.); Monsanto
(Monsanto Co., St. Louis, Mo.); Wintershall (Wintershall AG,
Kassel, Germany); BASF (BASF Co., Florham Park, N.J.); Huntsman
(Huntsman Petrochemical Corp., Salt Lake City, Utah); Enichem
(Enichem Iberica, Barcelona, Spain); Fluka Chemie AG (Fluka Chemie
AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV, Delft,
the Netherlands); Dow Corning (Dow Corning Corp., Midland, Mich.);
and Microsoft (Microsoft, Inc., Redmond, Wash.).
EXAMPLE 1
Assays
[0165] In the following Examples, various assays were used as set
forth below for ease in reading. Any deviations from the protocols
provided below are indicated in the Examples.
A. TCA Assay for Protein Content Determination in 96-Well
Microtiter Plates
[0166] For BPN' (e.g., reference protease) and BPN' variants, this
assay was started using filtered culture supernatant from
microtiter plates grown 3-4 days at 33.degree. C. with shaking at
230 rpm and humidified aeration. A fresh 96-well flat bottom
microtiter plate (MTP) was used for the assay. First, 100
.mu.L/well of 0.25 N HCl was placed in each well. Then, 50 .mu.L of
filtered culture broth was added. The light scattering/absorbance
at 405 nm (use 5 sec mixing mode in the plate reader) was then
determined, in order to provide the "blank" reading. For the test,
100 .mu.L/well of 15% (w/v) trichloroacetic acid (TCA) was placed
in the plates and incubated between 5 and 30 min at room
temperature. The light scattering/absorbance at 405 nm (use 5 sec
mixing mode in the plate reader) was then determined.
[0167] The equipment used was a Biomek FX Robot (Beckman Coulter)
and a SpectraMAX (type 340; Molecular Devices) MTP Reader; the
MTP's were from Costar (type 9017). The equipment used was a Biomek
FX Robot (Beckman Coulter) and a SpectraMAX type 340 (Molecular
Devices) MTP Reader; and the MTPs were type 9017 (Costar).
[0168] The calculations were performed by subtracting the blank (no
TCA) from the test reading with TCA to provide a relative measure
of the protein content in the samples. If desired, a standard curve
can be created by calibrating the TCA readings with AAPF assays of
clones with known conversion factors. However, the TCA results are
linear with respect to protein concentration from 50 to 500 protein
per ml (ppm) and can thus be plotted directly against enzyme
performance for the purpose of choosing good-performing variants.
The turbidity/light scatter increase in the samples correlates to
the total amount of precipitable protein in the culture
supernatant.
B. AAPF Protease Assay in 96-Well Microtiter Plates
[0169] In order to determine the protease activity of the proteases
and variants thereof of the present invention, the hydrolysis of
N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide
(suc-AAPF-pNA) was measured. The reagent solutions used were: 100
mM Tris/HCl, pH 8.6, containing 0.005% TWEEN.RTM.-80 (Tris dilution
buffer); 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl.sub.2
and 0.005% TWEEN.RTM.-80 (Tris/Ca buffer); and 160 mM suc-AAPF-pNA
in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare a
suc-AAPF-pNA working solution, 1 ml suc-AAPF-pNA stock solution was
added to 100 ml Tris/Ca buffer and mixed well for at least 10
seconds. The assay was performed by adding 10 .mu.l of diluted
protease solution to each well, immediately followed by the
addition of 190 .mu.l 1 mg/ml suc-AAPF-pNA working solution. The
solutions were mixed for 5 sec., and the absorbance change in
kinetic mode (20 readings in 5 minutes) was read at 410 nm in an
MTP reader, at 25.degree. C. The protease activity was expressed as
AU (activity=.DELTA.ODmin.sup.-1 ml.sup.-1).
C. Surfactant and Chelant Stability Assays
[0170] LAS and LAS/EDTA stability was measured after incubation of
the test protease in the presence of LAS and LAS/EDTA respectively,
as a function of residual activity determined using the AAPF
assay.
LAS Stability Method
Reagents:
[0171] Dodecylbenzenesulfonate, Sodium salt (=LAS): Sigma D-2525
[0172] TWEEN.RTM.-80: Sigma P-8074 [0173] TRIS buffer (free acid):
Sigma T-1378); 6.35 g is dissolved in about 960 ml water; pH is
adjusted to 8.2 with 4N HCl. Final concentration of TRIS is 52.5
mM. [0174] LAS stock solution: Prepare a 10.5% LAS solution in MQ
water (=10.5 g per 100 ml MQ) [0175] TRIS buffer-100 mM/pH 8.6 (100
mM Tris/0.005% Tween.RTM.-80) [0176] TRIS-Ca buffer, pH 8.6 (100 mM
Tris/10 mM CaCl2/0.005% Tween-.RTM.80)
Hardware:
[0176] [0177] Flat bottom MTPs (Costar No. 9017) [0178] Biomek FX
[0179] ASYS Multipipettor [0180] Spectramax MTP Reader [0181] iEMS
Incubator/Shaker [0182] Innova 4330 Incubator/Shaker [0183] Biohit
multichannel pipette [0184] BMG Thermostar Shaker
[0185] A 0.063% LAS solution was prepared in 52.5 mM Tris buffer pH
8.2. The suc-AAPF-pNA working solution was prepared by adding 1 ml
of 100 mg/ml suc-AAPF-pNA stock solution (in DMSO) to 100 ml (100
mM) TRIS buffer, pH 8.6. To dilute the supernatants, flat-bottomed
plates were filled with dilution buffer and an aliquot of the
supernatant was added and mixed well. The dilution ratio depended
on the concentration of the protease controls in the growth plates
(AAPF activity). The desired protein concentration was 80 ppm.
[0186] Ten .mu.l of the diluted supernatant were added to 190 .mu.l
A 0.063% LAS buffer/well. The MTP was covered with tape, shaken for
a few seconds and placed in an incubator (Innova 4230) at
45.degree. C., for 60 minutes at 200 rpm agitation. The initial
activity (t=10 minutes) was determined after 10 minutes of
incubation by transferring 10 .mu.l of the mixture in each well to
a fresh MTP containing 190 .mu.l suc-AAPF-pNA work solution. These
solutions were mixed well and the AAPF activity was measured using
a MTP Reader (20 readings in 5 minutes and 25.degree. C.).
[0187] The final activity (t=60 minutes) was determined by removing
another 10 .mu.l of solution from the incubating plate after 60
minutes of incubation. The AAPF activity was then determined as
described above. The stability of the samples was determined by
calculating the ration of the residual and initial AAPF activity as
follows:
[0188] Residual Activity (%)=[t-60 value]*100/[t-10 value].
LAS/EDTA Stability Method
[0189] The stability of protease variants in the presence of a
representative anionic surfactant (LAS=linear alkylbene sulfonate,
sodium dodecylbenzenesulfonate-DOBS) and di-sodium EDTA was
measured after incubation under defined conditions and the residual
activity was determined using the AAPF assay. The reagents used
were dodecyllbenzene sulfonate, sodium salt (DOBS, Sigma No.
D-2525), TWEEN.RTM.-80 (Sigma No. P-8074), di-sodium EDTA
(Siegfried Handel No. 164599-02), HEPES (Sigma No. H-7523),
unstress buffer: 50 mM HEPES (11.9 g/l)+0.005% TWEEN.RTM.-80, pH
8.0, Stress buffer: 50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1
g/l), 10 mM EDTA (3.36 g/l), pH 8.0, reference protease and
protease variant culture supernatants, containing 200-400 .mu.g/ml
protein. The equipment used was V- or U-bottom MTP as dilution
plates (Greiner 651101 and 650161 respectively), F-bottom MTP
(Corning 9017) for unstress and LAS/EDTA buffer as well as for
suc-AAPF-pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus
384 MTP Reader (Molecular Devices), iEMS Incubator/Shaker (1 mm
amplitude) (Thermo Electron Corporation), sealing tape: Nunc
(236366)
[0190] The iEMS incubator/shaker (Thermo/Labsystems) was set at
29.degree. C. Culture supernatants were diluted into plates
containing unstress buffer to a concentration of .about.25 ppm
(master dilution plate). 20 .mu.l of sample from the master
dilution plate was added to plates containing 180 .mu.l unstress
buffer to give a final incubation concentration of 2.5 ppm. The
contents were mixed and kept at room temperature and a AAPF assay
was performed on this plate. 20 .mu.l of sample from the master
dilution plate was also added to plates containing 180 .mu.l A
stress buffer (50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10
mM EDTA (3.36 g/l), pH 8.0). The solutions were mixed and
immediately placed in 29.degree. C. iEMS shaker for 30 min at 400
rpm. Following 30 minutes of incubation, a AAPF assay was performed
on the stress plate. The stability of the samples was determined by
calculating the ration of the residual and initial AAPF activity as
follows: Residual Activity (%)=[mOD.min-1 stressed]*100/[mOD. min-1
unstressed].
D. Cleaning Performance Assays
[0191] The stain removal performance of the protease variants was
determined in commercially available detergents. Heat inactivation
of commercial detergent formulas serves to destroy the enzymatic
activity of any protein components while retaining the properties
of non-enzymatic components. Thus this method was suitable for
preparing commercially purchased detergents for use in testing the
enzyme variants of the present invention.
Microswatches:
[0192] Microswatches of 1/4'' circular diameter were ordered and
delivered by CFT (Vlaardingen, The Netherlands). Single
microswatches or two microswatches were placed vertically in each
well of a 96-well MTP to expose the whole surface area (i.e., not
flat on the bottom of the well).
BMI Microswatch Assay
[0193] Microswatches containing blood milk and ink (BMI) of 0.25
inch circular diameter were obtained from CFT. Before cutting of
the swatches, the fabric (EMPA 116) was washed with water. One
microswatch was vertically placed in each well of a 96-well
microtiter plate in order to expose the whole surface area (i.e.,
not flat on the bottom of the well). The desired detergent solution
was prepared as described herein. After equilibrating the
Thermomixer at 25.degree. C., 190 .mu.l of detergent solution was
added to each well of the MTP, containing microswatches. To this
mixture, 10 .mu.l of the diluted enzyme solution was added so that
the final enzyme concentration was 1 .mu.g/ml (determined from BCA
assay). The MTP was sealed with tape and placed in the incubator
for 30 minutes, with agitation at 1400 rpm. Following incubation
under the appropriate conditions, 100 .mu.l of the solution from
each well was transferred into a fresh MTP. The new MTP containing
100 .mu.l of solution/well was read at 405 nm using a MTP
SpectraMax reader. Blank controls, as well as a control containing
two microswatches and detergent but no enzyme were also
included.
"Pre-Washed" Swatch
[0194] This type of microswatch was pre-washed in deionised water
for 20 minutes at ambient temperature. After the pre-washing step,
the swatches were put on top of paper towels to dry. The air-dried
swatches were then punched using a 1/4'' circular die on an
expulsion press. Finally two microswatches were put into each well
of a 96-well MTP vertically to expose the whole surface area (i.e.
not flat on the bottom of the well).
Detergents
[0195] For North American (NA) and Western European (WE) heavy duty
liquid laundry (HDL) detergents, heat inactivation was performed by
placing pre-weighed liquid detergent (in a glass bottle) in a water
bath at 95.degree. C. for 2 hours. The detergents were purchased
from local supermarket stores. Both un-heated and heated detergents
were assayed within 5 minutes of dissolving the detergent to
accurately determine percentage deactivated. Enzyme activity was
tested by the AAPF assay.
[0196] For testing of enzyme activity in heat-inactivated
detergents, working solutions of detergents were made from the heat
inactivated stocks. Appropriate amounts of water hardness (6 gpg)
and buffer were added to the detergent solutions to match the
desired conditions. The solutions were mixed by vortexing or
inverting the bottles.
Enzymes and Equipment
[0197] Samples of reference serine proteases variants thereof were
obtained from filtered culture broth of cultures grown in MTP
plates. The equipment used was a Biomek FX Robot (Beckman Coulter),
a SpectraMAX MTP Reader (type 340; Molecular Devices), an iEMS
incubator/shaker (Thermo/Labsystems); F-bottom MTPs (Costar type
9017 used for reading reaction plates after incubation); and
V-bottom MTPs (Greiner 651101 used for pre-dilution of
supernatant). In this assay, the proteases hydrolyze the substrate
and liberate pigment and insoluble particles from the substrate.
Thus the rate of turbidity is a measure of enzyme activity.
[0198] The stain removal performance of reference serine proteases
and variants therefrom on microswatches was determined on a MTP
scale in commercially available heat-inactivated detergent. The
reagents used were: 5 mM HEPES, pH 8.0 or 5 mM MOPS, pH 7 buffer,
3:1 Ca: Mg for medium water hardness. (CaCl.sub.2: MgCl26H2O);
15000 grains per gallon (gpg) stock diluted to 6 gpg, 2 BMI
(blood/milk/ink) swatches per plate: EMPA-116 BMI cotton swatches
processed by CFT: pre-rinsed and punched 2 swatches per well, and
heat inactivated TIDE.RTM. 2X Cold off-the-shelf detergent in which
lack of protease activity was confirmed.
TABLE-US-00003 TABLE 1-2 Working Detergent Solutions Temp Detergent
Detergent (C.) g/L pH Buffer gpg Protease TIDE .RTM. 2X Cold 16
0.98 8 5 mM 6 BPN' HEPES TIDE .RTM. 2X Cold 32 0.98 8 5 mM 6 BPN'
HEPES TIDE .RTM. 2X Cold 16 0.98 7 5 mM 6 BPN' MOPS
[0199] The incubator was set at the desired temperature (16.degree.
C. or 32.degree. C.). 10 .mu.L samples from the master dilution
plate of .about.10 ppm enzyme was added to BMI 2-swatch plates with
190 .mu.L working detergent solutions listed above. The volume was
adjusted to give final concentration of 0.5 ppm for variants in the
assay plates. The plates were immediately transferred to iEMS
incubators and incubated for 30 minutes with 1400 rpm shaking at
given temperature. Following incubation, 100 .mu.L of supernatant
was transferred into a new 96-well plate and the absorbance was
measured in MTP Reader at 405 nm and/or 600 nm. Control wells,
containing 1 or 2 microswatches and detergent without the addition
of protease samples were also included in the test. The measurement
at 405 nm provides a higher value and tracks pigment removal, while
the measurement at 600 nm tracks turbidity and cleaning.
Calculation of the Stain Removal Activity for All Microswatch Assay
Methods:
[0200] The absorbance value obtained was corrected for the blank
value (substrate without enzyme), providing a measure of hydrolytic
activity. For each sample (variant) the performance index was
calculated. The performance index compares the performance of the
variant (actual value) and the standard enzyme (theoretical value)
at the same protein concentration. In addition, the theoretical
values can be calculated, using the parameters of the Langmuir
equation of the standard enzyme.
E. Relative Specific Activity of Proteases and Variants Thereof
[0201] In order to discriminate the protease variants, the relative
specific activity was calculated using suc-AAPF-pNA as a substrate,
which enabled the comparison and ranking of the variants versus the
wild-type or standard protease. The specific activity on the
suc-AAPF-pNA substrate was determined by dividing the proteolytic
activity by the measured TCA-values of each sample, using the
assays described above. Using these values, the relative specific
activity was calculated (specific activity of variant/specific
activity of reference protease).
F. Performance Index
[0202] The performance index compares the performance of the
variant (actual value) and the standard or reference protease
(theoretical value) at the same protein concentration. In addition,
the theoretical values can be calculated, using the parameters of
the binding curve (i.e., Langmuir equation) of the standard
protease. A performance index (PI) that is greater than 1 (PI>1)
identifies a better variant as compared to the standard (e.g.,
wild-type), while a PI of 1 (PI=1) identifies a variant that
performs the same as the standard, and a PI that is less than 1
(PI<1) identifies a variant that performs worse than the
standard. Thus, the PI identifies winners, as well as variants that
are less desirable for use under certain circumstances.
EXAMPLE 2
Stain Removal Performance of BPN' Multiple Mutation Library (MML)
Variants
[0203] BPN' multiple mutation libraries (or combinatorial
libraries) were produced by Geneart or DNA 2.0, using BPN' as the
parent protein. Protein concentration of culture supernatants was
determined by TCA precipitation as described in Example 1. The
stain removal performance of the variants was tested in laundry
applications on EMPA 116 swatches (BMI stain, CFT) at pH
8/16.degree. C., pH 7/16.degree. C. and pH 8/32.degree. C. using
methods described in Example 1, with the following modifications.
The test detergent used was heat inactivated TIDE.RTM. 2X Cold
detergent (Procter & Gamble), prepared as described in Example
1. Heat inactivation of commercial detergent formulas serves to
destroy the endogenous enzymatic activity of any protein components
while retaining the properties of non0enzymatic components.
[0204] Heat inactivation of the detergents was performed by placing
pre-weighed amounts of liquid detergent (in a glass bottle) in a
water bath at 95.degree. C. for 2 hours. The detergent was
purchased from local supermarket stores. Both unheated and heated
detergents were assayed within 5 minutes of dissolving the
detergent, in order to accurately determine percentage deactivated.
Enzyme activity was tested by AAPF assay. Functionality of BPN'
variants was quantified as a performance index (Pi) (i.e., the
ratio of performance of a variant relative to parent BPN'). Results
are shown in Table 2-1. BPN' variants showing a Pi value greater
than or equal to 0.5 for one or more BMI stain removal performance
tests and/or TCA precipitation showed improved cleaning benefits
and/or expression. Performance indices less than or equal to 0.05
were fixed to 0.05 and indicated in bold italics in the table. For
every variant with a TCA protein performance index less than or
equal to 0.05, all values were fixed at 0.05.
TABLE-US-00004 TABLE 2-1 P.sub.i Values of BPN' Variants Tested for
Expression (TCA) and Stain Removal Performance (BMI pH 8/16.degree.
C., BMI pH 7/16.degree. C., and BMI pH 8/32.degree. C.) BMI BMI BMI
pH 8/ pH 7/ pH 8/ Variant Code TCA 16.degree. C. 16.degree. C.
32.degree. C. Parent BPN' 1.00 1.00 1.00 1.00 FNA (BPN'Y217L) 1.01
1.12 1.08 1.08 G97A-G128A-Y217Q 1.33 1.4 1.24 1.23 G97A-L126A-G128A
0.56 0.83 0.85 0.89 G97A-L126A-G128A-Y217Q 0.45 0.57 0.79 0.79
G97A-L126A-Y217Q 1.11 1.32 1.24 1.23 G97A-M124V-G128A 0.63 1.16 1.1
1.07 G97A-M124V-G128A-Y217Q 0.52 1.12 1.04 1.06 G97A-M124V-L126A
1.63 1.29 1.21 1.18 G97A-M124V-L126A-G128A 0.66 1.04 1.15 0.95
G97A-M124V-L126A-Y217Q 1.79 1.35 1.22 1.16 G97A-M124V-Y217Q 0.72
G97A-N123G-G128A 0.5 0.54 0.55 0.55 G97A-N123G-G128A-Y217Q 0.36 0.5
0.59 0.53 G97A-N123G-L126A 0.64 0.46 0.4 0.65
G97A-N123G-L126A-G128A 0.47 0.12 G97A-N123G-L126A-Y217Q 0.62 0.24
0.38 0.43 G97A-N123G-M124V 0.38 0.38 0.34 0.53
G97A-N123G-M124V-G128A 0.36 G97A-N123G-M124V-L126A 0.58 0.4 0.4
0.49 G97A-N123G-M124V-Y217Q G97A-N123G-Y217Q 0.55 1.35 1.24 1.13
L126A-G128A-Y217Q 0.56 0.8 0.84 0.79 L96T-G128A-Y217Q 0.3 0.28 0.32
0.58 L96T-G97A-G128A 0.39 0.41 0.3 0.54 L96T-G97A-G128A-Y217Q 0.5
0.21 0.34 0.5 L96T-G97A-L126A 0.47 0.16 0.1 0.32
L96T-G97A-L126A-G128A 0.43 0.1 0.19 0.09 L96T-G97A-L126A-Y217Q 0.59
0.11 L96T-G97A-M124V 0.49 1.17 1.03 1.02 L96T-G97A-M124V-G128A 0.54
0.19 0.31 0.32 L96T-G97A-M124V-L126A 0.88 0.34 0.52 0.57
L96T-G97A-M124V-Y217Q 0.43 1.08 1.06 0.95 L96T-G97A-N123G 0.35 0.13
0.09 0.21 L96T-G97A-N123G-G128A 0.44 0.06 L96T-G97A-N123G-L126A
0.48 L96T-G97A-N123G-M124V 0.49 0.22 0.28 0.24
L96T-G97A-N123G-Y217Q 0.49 0.22 0.07 L96T-G97A-Y217Q 0.46 1.3 1.11
1.08 L96T-L126A-G128A 0.56 L96T-L126A-G128A-Y217Q 0.42 0.06
L96T-L126A-Y217Q 0.51 0.11 0.06 0.36 L96T-M124V-G128A 0.49 0.59
0.53 0.67 L96T-M124V-G128A-Y217Q 0.42 0.34 0.54 0.51
L96T-M124V-L126A 0.68 0.62 0.6 0.79 L96T-M124V-L126A-G128A 0.48
0.08 0.09 L96T-M124V-L126A-Y217Q 0.73 0.43 0.53 0.67
L96T-M124V-Y217Q 0.51 1.23 1.06 1.03 L96T-N123G-G128A 0.48
L96T-N123G-G128A-Y217Q 0.49 0.09 0.08 L96T-N123G-L126A 0.43
L96T-N123G-L126A-G128A 0.48 L96T-N123G-L126A-Y217Q 0.35
L96T-N123G-M124V 0.51 0.07 L96T-N123G-M124V-G128A 0.41 0.06
L96T-N123G-M124V-L126A 0.42 0.06 0.14 L96T-N123G-M124V-Y217Q 0.48
0.06 0.12 0.14 L96T-N123G-Y217Q 0.45 0.15 0.27 M124V-G128A-Y217Q
0.77 1.27 1.18 1.03 M124V-L126A-G128A 0.72 1.12 1.1 1.02
M124V-L126A-G128A-Y217Q 0.52 1.21 1.22 1.12 M124V-L126A-Y217Q 1.71
1.39 1.16 1.17 N123G-G128A-Y217Q 0.13 1 1.25 1.08 N123G-L126A-G128A
0.49 0.13 0.17 0.33 N123G-L126A-G128A-Y217Q 0.39 0.13 0.11 0.21
N123G-L126A-Y217Q 0.91 0.37 0.43 0.55 N123G-M124V-G128A 0.6 0.13
0.38 N123G-M124V-G128A-Y217Q 0.63 N123G-M124V-L126A 0.81 0.48 0.44
0.62 N123G-M124V-L126A-G128A 0.46 0.24 0.47 0.44
N123G-M124V-L126A-Y217Q 0.71 0.35 0.44 0.48 N123G-M124V-Y217Q 0.6
0.35 0.42 0.51 N62Q-G128A-Y217Q 0.69 1.3 1.2 1.13 N62Q-G97A-G128A
0.69 1.15 1.14 1.07 N62Q-G97A-G128A-Y217Q 0.67 1.15 1.17 0.98
N62Q-G97A-L126A 0.94 0.74 0.74 0.96 N62Q-G97A-L126A-G128A 0.47 0.23
0.28 N62Q-G97A-L126A-Y217Q 0.87 0.58 0.77 0.72 N62Q-G97A-M124V 1.17
1.19 1.08 1.11 N62Q-G97A-M124V-G128A 0.57 0.52 0.62 0.64
N62Q-G97A-M124V-L126A 1.21 1.03 1.08 1.03 N62Q-G97A-M124V-Y217Q
0.94 1.21 1.14 1.04 N62Q-G97A-N123G 0.37 0.58 0.56 0.58
N62Q-G97A-N123G-G128A 0.44 0.08 0.09 N62Q-G97A-N123G-L126A 0.53
0.07 0.06 N62Q-G97A-N123G-M124V 0.37 0.07 N62Q-G97A-N123G-Y217Q
0.51 0.72 0.8 0.72 N62Q-G97A-Y217Q 1.72 1.38 1.18 1.32
N62Q-L126A-G128A 0.48 0.12 0.13 0.36 N62Q-L126A-G128A-Y217Q 0.47
0.08 0.27 0.15 N62Q-L126A-Y217Q 0.99 0.93 0.95 1 N62Q-L96T-G128A
0.48 0.2 0.1 0.31 N62Q-L96T-G128A-Y217Q 0.9 0.11 0.26 0.27
N62Q-L96T-G97A 0.48 0.88 0.82 0.9 N62Q-L96T-G97A-G128A 0.53 0.15
0.2 0.25 N62Q-L96T-G97A-L126A 0.58 0.16 0.14 N62Q-L96T-G97A-M124V
0.64 0.68 0.74 0.72 N62Q-L96T-G97A-N123G 0.37 0.09 0.25 0.14
N62Q-L96T-G97A-Y217Q 0.57 0.85 0.74 0.83 N62Q-L96T-L126A 0.5
N62Q-L96T-L126A-G128A 0.51 0.09 0.2 0.18 N62Q-L96T-L126A-Y217Q 0.43
0.19 0.23 N62Q-L96T-M124V 0.44 0.78 0.72 0.79 N62Q-L96T-M124V-G128A
0.74 0.14 0.11 N62Q-L96T-M124V-L126A 0.7 0.25 0.3 0.44
N62Q-L96T-M124V-Y217Q 0.55 0.73 0.78 0.76 N62Q-L96T-N123G 0.36 0.06
N62Q-L96T-N123G-G128A 0.44 N62Q-L96T-N123G-L126A 0.42 0.14 0.18
N62Q-L96T-N123G-M124V 0.41 N62Q-L96T-N123G-Y217Q 0.5 0.08
N62Q-L96T-Y217Q 0.58 0.86 0.64 0.85 N62Q-M124V-G128A 0.46 0.75 0.71
0.71 N62Q-M124V-G128A-Y217Q 0.45 0.43 0.59 0.46 N62Q-M124V-L126A
1.62 1.2 1.06 1.09 N62Q-M124V-L126A-G128A 0.51 0.23 0.45 0.42
N62Q-M124V-L126A-Y217Q 0.97 0.09 N62Q-M124V-Y217Q 1.04 1.22 1.09
0.89 N62Q-N123G-G128A 0.41 0.06 N62Q-N123G-G128A-Y217Q 0.43 0.13
0.09 N62Q-N123G-L126A 0.55 0.14 N62Q-N123G-L126A-G128A 0.5
N62Q-N123G-L126A-Y217Q 0.54 0.09 N62Q-N123G-M124V 0.48
N62Q-N123G-M124V-G128A 0.45 N62Q-N123G-M124V-L126A 0.34 0.11 0.07
N62Q-N123G-M124V-Y217Q 0.41 0.08 0.1 N62Q-N123G-Y217Q 0.49 0.97 0.8
0.92
EXAMPLE 3
Saturation Libraries at 97-128-217 and Additional Mutations
[0205] Saturation libraries at positions 97-128-217 in BPN'
(parent) were produced by DNA 2.0. Protein concentration of culture
supernatants was determined by TCA precipitation as described in
Example 1. The stain removal performance of the variants was tested
in laundry applications on EMPA 116 swatches (BMI stain, CFT) at
pH8/16.degree. C. using methods described in Example 1, as
described in Example 2. Enzyme activity was tested by AAPF assay as
described in Example 1. Functionality of BPN' variants was
quantified as a performance index (Pi) (i.e., the ratio of
performance of a variant relative to FNA). Results are shown in
Table 3-1. BPN' variants showing a Pi value greater than or equal
to 0.5 for BMI stain removal performance test and/or TCA
precipitation showed improved cleaning benefits and/or
expression.
TABLE-US-00005 TABLE 3-1 P.sub.i Values of BPN' Variants Tested for
Protein Determination (TCA) and Stain Removal Performance (BMI pH
8/16.degree. C.) Variant BMI pH Number Variants TCA 8 16.degree. C.
1 G97N-G128A-Y217M 1.09 1.43 2 G97G-G128S-Y217E 1.53 1.39 3
G97A-G128A-Y217Q 1.34 1.36 4 G97M-G128S-Y217E 1.20 1.35 5
G97A-G128S-Y217Q 1.90 1.33 6 G97D-G128S-Y217Q 1.55 1.33 7
G97M-G128G-Y217M 1.61 1.33 8 G97G-G128S-Y217Q 1.64 1.32 9
G97S-G128S-Y217Q 1.52 1.32 10 G97G-G128A-Y217Q 1.33 1.30 11
G97S-G128A-Y217E 1.03 1.30 12 G97A-G128S-Y217L 2.18 1.29 13
G97A-G128A-Y217N 1.22 1.28 14 G97Q-G128S-Y217L 1.89 1.28 15
G97A-G128A-Y217M 1.45 1.28 16 G97A-G128A-Y217S 1.35 1.27 17
G97D-G128A-Y217Q 1.14 1.27 18 G97M-G128S-Y217Q 0.99 1.27 19
G97Q-G128G-Y217D-S87Y 1.45 1.27 20 G97S-G128A-Y217N 1.09 1.27 21
G97A-G128S-Y217T 1.61 1.27 22 G97D-G128S-Y217E 1.01 1.27 23
G97D-G128A-Y217L 1.38 1.26 24 G97G-G128S-Y217E-S78P-A272T 1.00 1.26
25 G97T-G128S-Y217D 1.13 1.26 26 G97D-G128A-Y217I 0.99 1.26 27
G97Q-G128S-Y217Q 1.59 1.26 28 G97G-G128A-Y217D 1.12 1.25 29
G97Q-G128A-Y217N 1.09 1.25 30 G97S-G128A-Y217M 1.41 1.25 31
G97S-G128S-Y217N 1.55 1.25 32 G97S-G128S-Y217M 1.53 1.25 33
G97E-G128S-Y217M 1.58 1.24 34 G97S-G128P-Y217Q 0.99 1.24 35
G97T-G128S-Y217Q 1.06 1.24 36 G97D-G128S-Y217Q-A73T 1.18 1.23 37
G97E-G128S-Y217N 1.24 1.23 38 G97G-G128A-Y217I 1.51 1.23 39
G97Q-G128A-Y217D 1.14 1.23 40 G97Q-G128S-Y217M 1.98 1.23 41
G97R-G128T-Y217Q-S162P 0.68 1.23 42 G97S-G128S-Y217D 1.50 1.23 43
G97T-G128P-Y217I 1.30 1.23 44 G97Q-G128G-Y217E 1.58 1.23 45
G97C-G128G-Y217N 1.26 1.22 46 G97D-G128S-Y217H 1.49 1.22 47
G97M-G128S-Y217L 1.02 1.22 48 G97M-G128S-Y217N 0.98 1.21 49
G97S-G128S-Y217E 0.59 1.21 50 G97M-G128S-Y217I 1.11 1.19 51
G97A-G128P-Y217A 0.84 1.18 52 G97R-G128S-Y217D 0.95 1.16 53
G97D-G128A-Y217D 0.75 1.15 54 G97V-G128G-Y217D 0.71 1.14 55
G97V-G128G-Y217E 0.72 1.13 56 G97A-G128G-Y217T 1.29 1.12 57
G97G-G128N-Y217L 0.82 1.12 58 G97D-G128A-Y217T 0.69 1.11 59
G97M-G128A-Y217E 0.64 1.11 60 G97M-G128A-Y217N 0.58 1.06 FNA
G97G-G128G-Y217L 1.00 1.00
EXAMPLE 4
Additional Library Designs and Stain Removal Performance of
Variants
[0206] Additional BPN' multiple mutation libraries were produced by
Geneart or Gene Oracle, using BPN': G97A-G128A-Y217Q protein as the
parent molecule. Results of experiments conducted to determine
stain removal activity (microswatch assay to determine stain
removal performance in laundry applications using EMPA 116 swatches
(BMI stain, CFT Vlaardingen) (BMI pH8, BMI pH7, BMI 32.degree. C.),
protein determination by TCA precipitation, and LAS/EDTA stability
(tests of properties of interest) of BPN'variants are shown in
Tables 4-1, 4-2, 4-3, and 4-4.
[0207] The results were obtained using the methods described in
Example 1, with the following modifications for the stain removal
performance assay. The test detergent used was heat inactivated
TIDE.RTM. 2X Cold detergent (Procter & Gamble, Cincinnati,
Ohio, USA).
[0208] Heat inactivation of commercial detergent formulas serves to
destroy the endogenous enzymatic activity of any protein components
while retaining the properties of nonenzymatic components. Heat
inactivation of the detergents was performed by placing pre-weighed
amounts of liquid detergent (in a glass bottle) in a water bath at
95.degree. C. for 2 hours. The detergent was purchased from local
supermarket stores. Both unheated and heated detergents were
assayed within 5 minutes of dissolving the detergent to accurately
determine percentage deactivated. Enzyme activity was tested by
AAPF assay. As described throughout herein, functionality of BPN'
variants was quantified as a performance index (Pi), which is the
ratio of performance of a variant to parent protein BPN':
G97A-G128A-Y217Q. BPN': G97A-G128A-Y217Q variants showing a Pi
value greater or equal than 0.5 for BMI stain removal performance
and/or TCA precipitation showed improved cleaning benefits and/or
expression. Performance indices less than or equal to 0.05 were
fixed to 0.05 and indicated in bold italics in the table.
TABLE-US-00006 TABLE 4-1 Stain removal performance of multiple
mutation variants of BPN': G97A-G128A-Y217Q Parent BMI BMI BMI pH
pH 32.degree. Variant 8 7 C. TCA S145D 1.06 1.03 1.18 0.96 P239R
1.03 0.98 1.11 0.67 N61E P129E S162K K213L N240K 1.02 0.93 1.05
0.98 N61E 1.02 0.98 1.09 1.33 P40E A144K K213L 1.01 0.92 0.91 0.80
P129E 0.99 1.01 1.03 1.04 N61E P129E S159K 0.99 0.98 1.07 1.19
K213L 0.98 1.00 1.03 1.06 S87D 0.98 1.03 1.02 0.75 Q206E 0.97 0.96
0.98 1.01 S24R P40E S145D S159K K213L 0.97 0.96 0.98 0.91 K265N
0.96 1.00 1.00 0.93 S24R 0.96 0.87 0.99 0.99 P40E 0.96 0.96 0.96
0.80 Q275E 0.95 1.01 0.95 0.99 P129E S145D N240K 0.95 0.97 0.98
0.82 A144K 0.95 0.87 0.95 0.93 S159K 0.94 0.90 0.96 1.03 S162K 0.94
0.92 1.01 0.96 N240K 0.94 0.92 1.03 0.71 S24R S87D Q206E 0.90 0.95
0.98 0.83 S87D S162K K265N 0.89 0.96 0.93 0.64 N61E S145D S162K
K213L T242R 0.88 0.95 0.89 0.72 S87D A144K S145D S159K Q275E 0.88
0.91 0.96 0.72 S24R S87D A144K K265N Q275E 0.88 0.94 0.93 0.55
T242R 0.87 0.84 0.83 0.53 P40E N61E P129E A144K S162K 0.85 0.88
0.89 0.64 K213L N240K S24R P40E N61E A144K Q206E 0.85 0.81 0.91
0.45 K213L T242R P129E P239R K265N 0.83 0.93 0.86 0.59 S24R P129E
Q206E N240K K265N 0.81 0.86 0.82 0.63 P40E S145D S159K S162K K213L
0.78 0.82 0.83 0.47 P239R Q275E Q103E 0.67 0.84 0.72 0.46 S87D
T242R Q275E 0.60 0.74 0.62 0.38 P129E S145D N240K T242R K265N 0.58
0.72 0.68 0.37 N62R 0.58 0.63 0.64 0.44 P40E N61E S87D S162K T242R
0.55 0.62 0.58 0.29 P40E N61E S87D P129E S159K 0.52 0.47 0.57 0.27
S162K T242R P40E N61E Q103E A144K S159K 0.44 0.50 0.53 0.30 S162K
Q275E N61E Q103E N240K 0.44 0.62 0.51 0.42 S87D T242R K265N 0.42
0.60 0.48 0.28 Q103E S162K Q206E K213L P239R 0.42 0.53 0.46 0.31
P40E N61E Q103E S159K S162K 0.39 0.43 0.50 0.25 K213L P239R P40E
N61E Q103E S159K S162K 0.38 0.40 0.49 0.26 K213L N240K N61E Q103E
A144K K213L T242R 0.36 0.38 0.49 0.27 N62R K265N Q275E 0.29 0.42
0.32 0.38 N62R S159K Q206E K265N Q275E 0.26 0.35 0.33 0.35 S24R
Q103E P129E N240K K265N 0.24 0.29 0.27 0.24 N62R S87D P129E S145D
S159K 0.24 0.25 0.33 0.30 S162K Q275E Q103E P129E T242R 0.23 0.28
0.31 0.28 S24R N61E Q103E P129E K213L 0.22 0.15 0.27 0.25 N240K
T242R P40E N62R S145D S159K S162K 0.21 0.24 0.31 0.32 Q206E Q275E
S24R P40E N61E S87D Q103E 0.19 0.21 0.25 0.23 S159K S162K K213L
N240K N62R S87D S145D S159K S162K 0.19 0.22 0.19 0.33 K265N Q275E
P40E N61E N62R S87D S159K 0.18 0.19 0.21 0.28 S162K K265N N61E S87D
Q103E S159K S162K 0.17 0.13 0.17 0.25 K213L T242R N61E Q103E P129E
P239R N240K 0.14 0.18 0.23 0.33 P40E N62R S87D S145D S159K 0.14
0.14 0.27 0.33 S162K Q275E S24R N62R S87D S145D K265N 0.13 0.22
0.16 0.32 N62R S87D S145D S159K S162K 0.11 0.11 0.12 0.32 K213L
N240K K265N Q275E S24R N61E Q103E P129E 0.09 0.26 Q206E P239R N240K
N61E Q103E A144K Q206E K213L 0.07 0.15 0.31 N240K T242R S24R Q103E
P129E S145D 0.06 0.26 P239R N240K K265N S24R N61E Q103E P129E Q206E
0.43 K213L P239R N240K T242R N61E Q103E P129E K213L 0.38 P239R
N240K T242R P40E N62R S87D Q103E 0.25 A144K S159K Q275E N61E N62R
S87D Q103E S159K 0.33 S162K K213L T242R Q275E S24R P40E N61E Q103E
0.36 P129E A144K K213L P239R N240K T242R K265N S24R Q103E P129E
S145D Q206E 0.38 P239R N240K T242R K265N P40E N62R S87D Q103E S162K
0.29 N61E Q103E Q206E K213L P239R 0.32 N240K T242R S24R P40E N61E
Q103E 0.37 P129E A144K Q206E K213L P239R N240K T242R S24R N61E
Q103E P129E 0.38 A144K S145D Q206E K213L P239R N240K T242R S24R
N61E Q103E P129E S145D 0.35 P239R N240K T242R K265N S24R P40E N61E
Q103E 0.34 P129E A144K S145D K213L P239R N240K T242R N61E S87D
Q103E P129E S159K 0.25 S162K K213L N240K T242R S24R P40E N61E S87D
Q103E 0.35 P129E A144K S162K Q206E K213L P239R N240K T242R S24R
N61E S87D Q103E 0.34 P129E A144K Q206E K213L P239R N240K T242R S24R
N62R P129E S145D 0.28 P239R K265N Q275E P40E N61E S87D Q103E S145D
S159K 0.30 S162K K213L P239R N240K T242R P40E N61E Q103E P129E
A144K S162K 0.37 Q206E K213L P239R N240K T242R S24R N62R P129E
Q206E N240K 0.28 K265N Q275E P40E N62R S87D S145D S159K 0.27 S162K
N240K K265N Q275E P40E N62R S87D S159K S162K 0.06 0.06 0.30 K265N
Q275E S24R P40E N61E Q103E 0.37 P129E S162K Q206E K213L P239RN240K
T242R N61E Q103E P129E A144K Q206E 0.34 K213L P239R N240K T242R
S24R P40E N61E S87D Q103E 0.33 P129E A144K K213L P239R N240K T242R
P40E N61E Q103E P129E A144K 0.34 K213L P239R N240K T242R S24R N61E
Q103E P129E A144K 0.43 Q206E K213L P239R N240K T242R K265N
[0209] The LAS/EDTA stability of the BPN' multiple mutation
libraries was tested and compared to BPN': G97A-G128A-Y217Q. The
LAS/EDTA assay was performed as described in LAS Stability assay in
Example 1 except that the stress buffer contained 0.1% LAS+10mM
EDTA and the stress plates were incubated at40.degree. C. for 30
minutes. The functionality of BPN' variants was quantified as a
performance index (Pi), which is the ratio of performance of a
variant to parent protein BPN': G97A-G128A-Y217Q. The results are
shown in Table 4-2.
TABLE-US-00007 TABLE 4-2 Stain removal performance of multiple
mutation variants of BPN': G97A-G128A-Y217Q Parent BMI BMI LAS/
Variant Mutations pH 8 pH 7 TCA EDTA LHS1 S53G 1.05 1.03 1.18 0.98
LHS2 F58G 0.38 0.43 0.50 0.99 LHS3 S78N 0.99 1.00 1.00 1.50 LHS4
Y104N 0.36 0.53 0.73 1.49 LHS5 I111V 0.91 0.95 0.75 1.19 LHS6 A114G
0.71 0.72 0.60 0.93 LHS7 N117S 0.80 0.88 0.66 0.97 LHS8 S125A 0.73
0.79 0.85 0.97 LHS9 S132N 0.86 0.80 0.59 1.03 LHS10 P239V 0.42 0.43
0.47 1.22 LHS11 S53G F58G 0.67 0.71 0.49 0.75 LHS12 S53G S78N 0.98
0.99 1.10 1.44 LHS13 S53G Y104N 0.35 0.56 0.71 1.47 LHS14 S53G
I111V 0.99 0.99 0.83 1.20 LHS15 S53G A114G 0.85 0.91 0.64 1.15
LHS16 S53G N117S 0.93 1.00 0.75 1.30 LHS17 S53G S125A 0.71 0.76
0.85 0.87 LHS18 S53G S132N 0.93 0.93 0.74 1.03 LHS19 S53G P239V
0.52 0.59 0.46 1.12 LHS20 F58G S78N 0.44 0.49 0.50 1.32 LHS21 F58G
Y104N 0.56 1.67 LHS22 F58G I111V 0.15 0.19 0.52 1.33 LHS23 F58G
A114G 0.59 1.59 LHS24 F58G N117S 0.09 0.09 0.53 1.47 LHS25 F58G
S125A 0.56 1.56 LHS26 F58G S132N 0.07 0.07 0.49 1.17 LHS27 F58G
P239V 0.52 1.64 LHS28 S78N Y104N 0.37 0.56 0.67 1.71 LHS29 S78N
I111V 0.92 0.92 0.71 1.53 LHS30 S78N A114G 0.74 0.75 0.48 1.49
LHS31 S78N N117S 0.83 0.87 0.61 1.51 LHS32 S78N S125A 0.77 0.79
0.89 1.33 LHS33 S78N S132N 0.81 0.76 0.60 1.38 LHS34 S78N P239V
0.40 0.47 0.50 1.66 LHS35 Y104N I111V 0.21 0.31 0.64 1.55 LHS36
Y104N A114G 0.06 0.59 1.70 LHS37 Y104N N117S 0.08 0.14 0.45 1.72
LHS38 Y104N S125A 0.06 0.09 0.64 1.77 LHS39 Y104N S132N 0.98 1.07
0.74 1.26 LHS40 Y104N P239V 0.05 0.69 1.82 LHS41 I111V A114G 0.15
0.16 0.43 1.24 LHS42 I111V N117S 0.45 0.43 0.51 1.12 LHS43 I111V
S125A 0.37 0.47 0.62 1.21 LHS44 I111V S132N 0.53 0.58 0.41 1.00
LHS45 I111V P239V 0.24 0.31 0.40 1.29 LHS46 A114G N117S 0.14 0.15
0.48 1.37 LHS47 A114G S125A 0.20 0.29 0.58 1.41 LHS48 A114G S132N
0.24 0.25 0.50 1.14 LHS49 A114G P239V 0.07 0.48 1.48 LHS50 N117S
S125A 0.31 0.39 0.48 1.27 LHS51 N117S S132N 0.35 0.35 0.45 1.12
LHS52 N117S P239V 0.15 0.19 0.38 1.32 LHS53 S125A S132N 0.38 0.51
0.47 1.03 LHS54 S125A P239V 0.09 0.12 0.47 1.59 LHS55 S132N P239V
0.16 0.22 0.45 1.29 LHS56 S53G F58G S78N 0.72 0.76 0.56 1.48 LHS57
S53G F58G Y104N 0.52 1.78 LHS58 S53G F58G I111V 0.35 0.43 0.38 1.16
LHS59 S53G F58G A114G 0.10 0.14 0.46 1.37 LHS60 S53G F58G N117S
0.22 0.30 0.41 1.19 LHS61 S53G F58G S125A 0.18 0.23 0.44 1.46 LHS62
S53G F58G S132N 0.29 0.32 0.41 1.01 LHS63 S53G F58G P239V 0.06 0.08
0.49 1.54 LHS64 S53G S78N Y104N 0.43 0.63 0.76 1.80 LHS65 S53G S78N
I111V 0.97 1.01 0.89 1.69 LHS66 S53G S78N A114G 0.85 0.89 0.60 1.63
LHS67 S53G S78N N117S 0.94 0.92 0.69 1.61 LHS68 S53G S78N S125A
0.78 0.82 0.90 1.37 LHS69 S53G S78N S132N 0.96 0.93 0.75 1.48 LHS70
S53G S78N P239V 0.62 0.66 0.44 1.59 LHS71 S53G Y104N I111V 0.25
0.41 0.63 1.73 LHS72 S53G Y104N A114G 0.08 0.09 0.53 1.75 LHS73
S53G Y104N N117S 0.12 0.50 1.68 LHS74 S53G Y104N S125A 0.20 0.23
0.50 1.20 LHS75 S53G Y104N S132N 0.94 1.00 1.21 1.10 LHS76 S53G
Y104N P239V 0.54 1.63 LHS77 S53G I111V A114G 0.48 0.51 0.47 1.25
LHS78 S53G I111V N117S 0.66 0.74 0.48 1.17 LHS79 S53G I111V S125A
0.49 0.64 0.72 1.27 LHS80 S53G I111V S132N 0.78 0.80 0.54 1.19
LHS81 S53G I111V P239V 0.32 0.38 0.43 1.26 LHS82 S53G A114G N117S
0.16 0.29 0.47 1.19 LHS83 S53G A114G S125A 0.32 0.44 0.50 1.19
LHS84 S53G A114G S132N 0.44 0.50 0.40 1.05 LHS85 S53G A114G P239V
0.15 0.31 0.44 1.42 LHS86 S53G N117S S125A 0.50 0.56 0.54 1.08
LHS87 S53G N117S S132N 0.54 0.59 0.49 1.15 LHS88 S53G N117S P239V
0.28 0.41 0.50 1.31 LHS89 S53G S125A S132N 0.50 0.55 0.67 LHS90
S53G S125A P239V 0.06 0.12 0.59 LHS91 S53G S132N P239V 0.27 0.33
0.49 LHS92 F58G S78N Y104N 0.67 LHS93 F58G S78N I111V 0.17 0.18
0.56 LHS94 F58G S78N A114G 0.07 0.67 LHS95 F58G S78N N117S 0.11
0.12 0.64 LHS96 F58G S78N S125A 0.11 0.15 0.65 LHS97 F58G S78N
S132N 0.06 0.12 0.57 LHS98 F58G S78N P239V 0.60 LHS99 F58G Y104N
I111V 0.57 LHS100 F58G Y104N A114G 0.59 LHS101 F58G Y104N N117S
0.61 LHS102 F58G Y104N S125A 0.61 LHS103 F58G Y104N S132N 0.19 0.28
0.48 LHS104 F58G Y104N P239V 0.05 0.62 LHS105 F58G I111V A114G 0.62
LHS106 F58G I111V N117S 0.58 LHS107 F58G I111V S125A 0.53 LHS108
F58G I111V S132N 0.52 LHS109 F58G I111V P239V 0.55 LHS110 F58G
A114G N117S 0.64 LHS111 F58G A114G S125A 0.62 LHS112 F58G A114G
S132N 0.58 LHS113 F58G A114G P239V 0.45 LHS114 F58G N117S S125A
0.60 LHS115 F58G N117S S132N 0.55 LHS116 F58G N117S P239V 0.52
LHS117 F58G S125A S132N 0.62 LHS118 F58G S125A P239V 0.62 LHS119
F58G S132N P239V 0.65 LHS120 S78N Y104N I111V 0.33 0.44 0.63 LHS121
S78N Y104N A114G 0.68 LHS122 S78N Y104N N117S 0.13 0.60 LHS123 S78N
Y104N S125A 0.06 0.69 LHS124 S78N Y104N S132N 1.02 1.00 0.77 LHS125
S78N Y104N P239V 0.05 0.65 LHS126 S78N I111V A114G 0.35 0.36 0.60
LHS127 S78N I111V N117S 0.52 0.53 0.54 LHS128 S78N I111V S125A 0.51
0.57 0.67 LHS129 S78N I111V S132N 0.50 0.46 0.49 LHS130 S78N I111V
P239V 0.27 0.29 0.55 LHS131 S78N A114G N117S 0.11 0.14 0.50 LHS132
S78N A114G S125A 0.27 0.27 0.51 LHS133 S78N A114G S132N 0.23 0.21
0.56 LHS134 S78N A114G P239V 0.12 0.11 0.54 LHS135 S78N N117S S125A
0.39 0.48 0.62 LHS136 S78N N117S S132N 0.42 0.47 0.55 LHS137 S78N
N117S P239V 0.09 0.05 0.53 LHS138 S78N S125A S132N 0.46 0.47 0.56
LHS139 S78N S125A P239V 0.06 0.08 0.64 LHS140 S78N S132N P239V 0.21
0.16 0.45 LHS141 Y104N I111V A114G 0.06 0.63 LHS142 Y104N I111V
N117S 0.05 0.05 0.57 LHS143 Y104N I111V S125A 0.05 0.09 0.66 LHS144
Y104N I111V S132N 0.87 0.91 0.60 LHS145 Y104N I111V P239V 0.61
LHS146 Y104N A114G N117S 0.53 LHS147 Y104N A114G S125A 0.50 LHS148
Y104N A114G S132N 0.32 0.45 0.50 LHS149 Y104N A114G P239V 0.05 0.05
0.62 LHS150 Y104N N117S S125A 0.05 0.56 LHS151 Y104N N117S S132N
0.53 0.63 0.54 LHS152 Y104N N117S P239V 0.06 0.66 LHS153 Y104N
S125A S132N 0.30 0.41 0.69 LHS154 Y104N S125A P239V 0.54 LHS155
Y104N S132N P239V 0.25 0.36 0.50 LHS156 I111V A114G N117S 0.05 0.54
LHS157 I111V A114G S125A 0.05 0.06 0.59 LHS158 I111V A114G S132N
0.05 0.58 LHS159 I111V A114G P239V 0.63 LHS160 I111V N117S S125A
0.10 0.10 0.66 LHS161 I111V N117S S132N 0.55 LHS162 I111V N117S
P239V 0.55 LHS163 I111V S125A S132N 0.14 0.56 LHS164 I111V S125A
P239V 0.60 LHS165 I111V S132N P239V 0.57 LHS166 A114G N117S S125A
0.57 LHS167 A114G N117S S132N 0.07 0.52 LHS168 A114G N117S P239V
0.67 LHS169 A114G S125A S132N 0.59 LHS170 A114G S125A P239V 0.63
LHS171 A114G S132N P239V 0.55 LHS172 N117S S125A S132N 0.05 0.56
LHS173 N117S S125A P239V 0.56 LHS174 N117S S132N P239V 0.57 LHS175
S125A S132N P239V 0.55 LHS176 N76D D120H K213N M222Q 0.22 0.31
0.68
[0210] The LAS/EDTA stability of the BPN' triple variants was
tested and compared to BPN'-Y217L. The LAS/EDTA assay was performed
as described in the "LAS Stability Assay" section of Example 1,
except that the stress buffer contained 0.1% LAS+10 mM EDTA and the
stress plates were incubated at 35.degree. C. for 25 minutes. The
functionality of BPN' variants was quantified as a performance
index (Pi), which is the ratio of performance of a variant to
parent protein BPN'-Y217L. Results are shown in Table 4-3.
TABLE-US-00008 TABLE 4-3 LAS/EDTA Stability Results for BPN'
Variants Pi Variant LAS/EDTA N62Q-G97A-Y217Q 1.26 G97A-N123G-Y217Q
0.95 G97A-G128A-Y217Q 1.24 FNA: G97G-G128G- 1.00 Y217L
[0211] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Those of skill in the art readily
appreciate that the present invention is well adapted to carry out
the objects and obtain the ends and advantages mentioned, as well
as those inherent therein. The compositions and methods described
herein are representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
It is readily apparent to one skilled in the art that varying
substitutions and modifications may be made to the invention
disclosed herein without departing from the scope and spirit of the
invention.
[0212] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by herein.
[0213] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not excised material is
specifically recited herein.
Sequence CWU 1
1
21275PRTBacillus amyloliquefaciens 1Ala 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 Tyr 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 275
24PRTArtificialsynthetic reagent 2Ala Ala Pro Phe 1
* * * * *