CA2355579A1 - Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region - Google Patents

Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region Download PDF

Info

Publication number
CA2355579A1
CA2355579A1 CA002355579A CA2355579A CA2355579A1 CA 2355579 A1 CA2355579 A1 CA 2355579A1 CA 002355579 A CA002355579 A CA 002355579A CA 2355579 A CA2355579 A CA 2355579A CA 2355579 A1 CA2355579 A1 CA 2355579A1
Authority
CA
Canada
Prior art keywords
subtilase
amino acid
group
variant
acid residue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CA002355579A
Other languages
French (fr)
Other versions
CA2355579C (en
Inventor
Kim Andersen Vilbour
Frank Mikkelsen
Peter Hansen Kamp
Carsten Andersen
Mads Norregaard-Madsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of CA2355579A1 publication Critical patent/CA2355579A1/en
Application granted granted Critical
Publication of CA2355579C publication Critical patent/CA2355579C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

Abstract

Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid residue in position 100 of the active site loop (b) region from position 95 to 103. Variant subtilases exhibit improved wash performance in a detergent in comparison to its parent enzyme.

Claims (34)

1. A subtilase enzyme of the I-S1 and I-S2 sub-groups having at least one additional amino acid residue in position 100 of the active site loop (b) region from position 95 to 103, whereby said additional amino acid residue(s) correspond to the insertion of at least one amino acid residue between positions 100 and 101.
2. The isolated subtilase enzyme of claim 1, wherein said subtilase enzyme is a constructed variant having at least one inserted amino acid residue between positions 100 and 101 of a precursor subtilase.
3. The isolated subtilase enzyme of claim 1 or 2 selected from the group comprising X100X{A,T,G,S}, X100X{D,E,K,R}, X100X{H,V,C,N,Q}, and X100X{F,I,L,M,P,W,Y}
4. The isolated subtilase enzyme of claim 3, wherein said at least one additional or inserted amino acid residue is chosen from the group comprising: T, G, A, and S.
5. The isolated subtilase enzyme of claim 3, wherein said at least one additional or inserted amino acid residue is chosen from the group of charged amino acid residues comprising:
D,E,H,K, and R, more preferably D,E,K and R.
6. The isolated subtilase enzyme of claim 3, wherein said at least one additional or inserted amino acid residue is chosen from the group of hydrophilic amino acid residues comprising:
C,N,Q,S and T, more preferably N,Q,S and T.
7. The isolated subtilase enzyme of claim 3, wherein said at least one additional or inserted amino acid residue is chosen from the group of small hydrophobic amino acid residues comprising: A,G and V.
8. The isolated subtilase enzyme of claim 3, wherein said at least one additional or inserted amino acid residue is chosen from the group of large hydrophobic amino acid residues comprising: F,I,L,M,P,W and Y, more preferably F,I,L,M, and Y.
9. The isolated subtilase enzyme according to any of the preceding claims, wherein said at least one additional or inserted amino acid residue, comprises more than one additional or inserted amino acid residue in the active site loop (b).
10. The subtilase variant of any of the preceding claims, wherein said insertion(s) between positions 100 and 101 are combined with one or more further modification(s) in any other position (s).
11. The subtilase variant of claim 15, wherein said further modification(s) are in one or more of the positions 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222, 224, 235 and 274.
12. The subtilase variant of any of the preceding claims, wherein said modification(s) is/are combined with modification(s) in one or more of the positions 129, 131, 133 and 194.
13. The subtilase of any of the preceding claims, wherein the subtilase, or if the subtilase is a variant the parent subtilase, belongs to the sub-group I-S1.
14. The subtilase of claim 13, wherein the parent subtilase is chosen from the group comprising ABSS168, BASBPN, BSSDY, and BLSCAR, or functional variants thereof having retained the characteristic of sub-group I-S1.
15. The subtilase according to any of claims 1-14, wherein the subtilase, or if the subtilase is a variant, the parent subtilase belongs to the sub-group I-S2.
16. The subtilase of claim 15, wherein the parent subtilase is chosen from the group comprising BLS147, BLS309, BAPB92, TVTHER
and BYSYAB, or functional variants thereof having retained the characteristic of sub-group I-S2.
17. The isolated subtilase enzyme of claim 3, 15 or 16 selected from the group comprising G100GA, G100GT, G100GG, G100GS, G100GD, G100GE, G100GK, G100GR, G100GH, G100GV, G100GC, G100GN, G100GQ, G100GF, G100GI, G100GL, G100GM, G100GP, G100GW, and G100GY.
18. The subtilase variant of any of the claims 15 to 17, wherein said further modification(s) are chosen from the group comprising K27R, *36D, S57P, N76D, S87N, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Y167X, R170X, Q206E, N218S, M222S, M222A, T224S, K235L, and T274A.
19. The subtilase variant of any of the claims 15 to 17, wherein said further modification(s) are chosen from the group comprising S101G+V104N, S87N+S101G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I or N76D+V104A, or other combinations of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with any one or more of the substitutions, deletions and/or insertions mentioned in any of claims 1 to 14.
20. The subtilase variant of any of the claims 15 to 17, wherein said further modification(s) are chosen from the group further comprising P129K, P131H, A133P, A133D and A194P.
21. The variant according to any of the preceding claims comprising the modification chosen from the group comprising:
A98G+G100GA+S101A+S103T, and S99G+G100GGT+S101T
22. A subtilase belonging to the I-S1 sub-group having the amino acid sequence:
1 ~~ 10 20 30 A-Q-T-V-P-Y-G-I-P-L-I-K-A-D-K-V-Q-A-Q-G-F-K-G-A-N-V-K-V-A-V

L-D-T-G-I-Q-A-S-H-P-D-L-N-V-V-G-G-A-S-F-V-A-G-E-A-*-Y-N-T-D

G-N-G-H-G-T-H-V-A-G-T-V-A-A-L-D-N-T-T-G-V-L-G-V-A-P-S-V-S-L
100a 110 120 Y-A-V-K-V-L-N-S-S-G-X-S-G-T-Y-S-G-I-V-S-G-T-E-W-A-T-T-N-G-M-D

V-I-N-M-S-L-G-G-P-S-G-S-T-A-M-K-Q-A-V-D-N-A-Y-A-R-G-V-V-V-V

A-A-A-G-N-S-G-S-S-G-N-T-N-T-I-G-Y-P-A-K-Y-D-S-V-I-A-V-G-A-V

D-S-N-S-N-R-A-S-F-S-S-V-G-A-E-L-E-V-M-A-P-G-A-G-V-Y-S-T-Y-P

T-S-T-Y-A-T-L-N-G-T-S-M-A-S-P-H-V-A-G-A-A-A-L-I-L-S-K-H-P-N

L-S-A-S-Q-V-R-N-R-L-S-S-T-A-T-Y-L-G-S-S-F-Y-Y-G-K-G-L-I-N-V

E-A-A-A-Q
or a homologous subtilase having an amino acid sequence compris-ing a position 100a amino acid residue and exhibiting an iden-tity of more than 70%, 750, 80%, 85%, 90%, or 95% therewith.
23. A subtilase belonging to the I-S2 sub-group having the amino acid sequence:
.1 10 20 30 .A-Q-S-V-P-W-G-I-S-R-V-Q-A-P-A-A-H-N-R-G-L-T-G-S-G-V-K-V-A-V
. 40 50 60 .L-D-T-G-I-*-S-T-H-P-D-L-N-I-R-G-G-A-S-F-V-P-G-E-P-*-S-T-Q-D-. ~ 70 80 90 .G-N-G-H-G-T-H-V-A-G-T-I-A-A-L-N-N-S-I-G-V-L-G-V-A-P-S-A-E-L-. 100a 110 120 .Y-A-V-K-V-L-G-A-S-G-X-S-G-S-V-S-S-I-A-Q-G-L-E-W-A-G-N-N-G-M-H-. 130 140 150 .V-A-N-L-S-L-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S-R-G-V-L-V-V-. 160 170 180 .A-A-S-G-N-S-G-A-*-G-S-I-S-*-*-*-Y-P-A-R-Y-A-N-A-M-A-V-G-A-T-. 190 200 210 .D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I-V-A-P-G-V-N-V-Q-S-T-Y-P-. 220 230 240 .G-S-T-Y-A-S-L-N-G-T-S-M-A-T-P-H-V-A-G-A-A-A-L-V-K-Q-K-N-P-S-. 250 260 270 .W-S-N-V-Q-I-R-N-H-L-K-N-T-A-T-S-L-G-S-T-N-L-Y-G-S-G-L-V-N-A-. 275 .E-A-A-T-R
or a homologous subtilase having an amino acid sequence comprising a position 100a amino acid residue and exhibiting an identity of more than 70%, 75%, 80%, 85%, 90%, or 95% therewith.
24. The subtilase variant of claims 22 or 23, wherein X in position 100a is chosen from the group comprising T, A, G, S, and P.
25. An isolated DNA sequence encoding a subtilase or a subtilase variant of any of the claims 1 to 24.
26. An expression vector comprising an isolated DNA sequence of claim 25.
27. A microbial host cell transformed with an expression vector of claim 26.
28. The microbial host of claim 27, which is a bacterium, preferably a Bacillus, especially B. lentus.
29. The microbial host of claim 27, which is a fungus or yeast, preferably a filamentous fungus, especially an Aspergillus.
30. A method for producing a subtilase or a subtilase variant of any of claims 1 to 24, wherein a host of any of claims 27 to 29 is cultured under conditions conducive to the expression and secretion of said variant, and the variant is recovered.
31. A composition comprising a subtilase or a subtilase variant according to any of claims 1 to 24.
32. The composition according to claim 31, which additionally comprises a cellulase, lipase, cutinase, oxidoreductase, another protease, or an amylase.
33. The composition according to claim 31 or 32, wherein the composition is a detergent composition.
34. Use of a subtilase or a subtilase variant according to any of claims 1 to 24 or an enzyme composition according to claims 31 or 32 in a laundry and/or a dishwash detergent.
CA2355579A 1998-12-18 1999-12-20 Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region Expired - Fee Related CA2355579C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DKPA199801673 1998-12-18
DKPA199801673 1998-12-18
PCT/DK1999/000715 WO2000037624A1 (en) 1998-12-18 1999-12-20 Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region

Publications (2)

Publication Number Publication Date
CA2355579A1 true CA2355579A1 (en) 2000-06-29
CA2355579C CA2355579C (en) 2011-11-22

Family

ID=8107093

Family Applications (1)

Application Number Title Priority Date Filing Date
CA2355579A Expired - Fee Related CA2355579C (en) 1998-12-18 1999-12-20 Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region

Country Status (7)

Country Link
JP (1) JP4647788B2 (en)
KR (1) KR100660799B1 (en)
CN (1) CN1302106C (en)
AU (1) AU773327B2 (en)
BR (1) BRPI9916350B1 (en)
CA (1) CA2355579C (en)
WO (1) WO2000037624A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69034159T2 (en) * 1989-06-26 2005-02-17 Unilever N.V. Enzymatic detergent compositions
DK316989D0 (en) * 1989-06-26 1989-06-26 Novo Nordisk As ENZYMES
US6599730B1 (en) * 1994-05-02 2003-07-29 Procter & Gamble Company Subtilisin 309 variants having decreased adsorption and increased hydrolysis
US6455295B1 (en) * 1995-03-08 2002-09-24 The Procter & Gamble Company Subtilisin Carlsberg variants having decreased adsorption and increased hydrolysis
HUP9901725A3 (en) * 1995-05-05 2001-09-28 Unilever Nv Subtilisin variants
ATE338823T1 (en) * 1997-06-04 2006-09-15 Genecor Int Inc PROTEASE FOR THOROUGH CLEANING AND/OR STAIN AND FILM REDUCTION AND COMPOSITIONS THEREOF
EP1032655B1 (en) * 1997-11-21 2005-06-29 Novozymes A/S Protease variants and compositions

Also Published As

Publication number Publication date
BR9916350A (en) 2001-09-18
JP4647788B2 (en) 2011-03-09
JP2002533077A (en) 2002-10-08
AU1772900A (en) 2000-07-12
BRPI9916350B1 (en) 2016-04-12
KR100660799B1 (en) 2006-12-26
CN1333825A (en) 2002-01-30
WO2000037624A1 (en) 2000-06-29
KR20010093172A (en) 2001-10-27
AU773327B2 (en) 2004-05-20
CN1302106C (en) 2007-02-28
CA2355579C (en) 2011-11-22

Similar Documents

Publication Publication Date Title
CA2355574A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region
CA2310454A1 (en) Protease variants and compositions
CA2355580A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in the active site loop region
JP2002515221A5 (en)
CA2301851A1 (en) Protease variants and compositions
CA2270180A1 (en) Subtilase variants and compositions
EP1794297B1 (en) Subtilases
EP1794295B1 (en) Subtilases
US8658410B2 (en) Subtilases
CA2355619A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region
CA2374350A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 97 and 98
CA2355655A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region
CA2355969A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region
CA2355576A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region
CA2355579A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region
EP1183343B2 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 125 and 126
AU773674B2 (en) Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid residue in an active site loop region
EP1183341B2 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 127 and 128
EP1803817A1 (en) Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid residue in an active site loop region
EP1141259A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region

Legal Events

Date Code Title Description
EEER Examination request
MKLA Lapsed

Effective date: 20171220