# US 2014/0154782 A1 · Compositions and Methods Comprising Serine Protease Variants

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- Number: US 2014/0154782 A1
- Document ID: US20140154782A1
- Status: Published application
- Kind: A1
- Application number: 14/115,801
- Filed: 2012-05-04
- Published: 2014-06-05
- Earliest claimed: 2011-05-05
- Assignees: Danisco US Inc.
- Inventors: Neelam S. Amin; Katherine Augustyn; Joshua R. Basler; Luis G. Cascao-Pereira; Katherine D. Collier; Edward M. Concar; David A. Estell; James T. Kellis, JR.; Euan John Magennis; Alexander Pisarchik; Ayrookaran J. Poulose; Philip Frank Souter; Glenn Steven Ward; Jian Yao
- Web page: https://patentgrep.com/patent/US20140154782A1

## CPC

- C12N 9/54: bacteria being Bacillus
- C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL

## Abstract

The present invention provides serine protease variants, more specifically subtilisin variants produced there from. Specifically, the present invention provides serine protease variants, more specifically subtilisin variants having one or more substitutions as compared to a reference serine protease. In addition, the present invention provides compositions comprising these serine protease variants, more specifically subtilisin variants. In some embodiments, the present invention provides cleaning compositions comprising at least one of these serine protease variants, more specifically subtilisin variants.

## Claims

### Claim 1

An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions at positions selected from T022R+V104I+Q245R, G020R+S024R+S101A, T022R+S103A+V104I, T022R+V104I+A232V, T022R+S103A+Q245R, T022R+A232V+Q245R, T022R+S103A+A232V, T022R+S103A+V104I+A232V, T022R+V104I+A232V+Q245R, T022R+S103A+V104I+Q245R, T022R+S103A+A232V+Q245R, T022R+S103A+V104I+A232V+Q245R, T022A+N076D+S103A+V104I+A232V+Q245R, T022A+N076D+S103A+V104I+A232V, T022A+N076D+V104I+A232V+Q245R, T022A+N076D+S103A+A232V+Q245R, T022A+N076D+S103A+V104I+Q245R, T022A+N076D+V104I+Q245R, T022A+N076D+A232V+Q245R, T022A+N076D+V104I+A232V, T022A+N076D+S103A+Q245R, T022A+N076D+S103A+A232V, T022A+N076D+S103A+V104I, T022A+N076D+S103A, T022A+N076D+V104I, T022A+N076D+A232V, T022A+N076D+Q245R, G020R+S101A+T213A, G020R+R045T+S101A, G020R+S101A+G211Q, S024R+S101A+T213A, G020R+T022W+S101A, S024R+S101A+G211Q, N043R+S101A+T213A, N043R+R045T+S101A, S024R+N043R+S101A, S024R+R045T+S101A, S101A+G211Q+T213A, G020R+N043R+S101A, R045T+S101A+T213A, T022W+S024R+S101A, T022R+S101G+V104I, T022W+S101A+T213A, T022R+S101G+Q245R, T022W+S101A+G211Q, G020R+S024R+Q245R, T022R+S101G+V104I+Q245R, T022R+S101G+A232V, T022R+S101G+S103A, T022R+S101G+S103A+V104I, T022R+S101G+S103A+Q245R, T022R+S101G+A232V+Q245R, T022R+S101G+V104I+A232V, G020R+5024R+S101A+T213A, G020R+S024R+T213AS101N+V104I+A232V, S101N+S103A+V104I, S101N+S103A+A232V, S101N+S103A+V104I+A232V, G020R+S103A+A232V, G020R+V104I+A232V, G020R+S103A+V104I+A232V, G020R+S103A+V104I, N018R+N076D+N116A, N018R+N076D+T213A, N018R+N076D+S101A, N018R+N076D+A215F, or N018R+N076D+G211Q, and wherein amino acid positions of the subtilisin variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

### Claim at position 2

2-16. (canceled)

### Claim 17 (depends on 1)

The isolated subtilisin variant of claim 1, wherein the subtilisin variant is a subtilisin variant of Bacillus lentus subtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36 protease comprises the amino acid sequence shown in SEQ ID NO:2.

### Claim 18 (depends on 1)

The isolated subtilisin variant of claim 1, wherein said subtilisin variant has at least 80% amino acid identity with said Bacillus lentus subtilisin GG36 protease comprising the amino acid sequence shown in SEQ ID NO:2.

### Claim 19 (depends on 1)

The isolated subtilisin variant of claim 1, wherein the total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease.

### Claim at position 6

20-23. (canceled)

### Claim 24 (depends on 19)

The isolated subtilisin variant of claim 19, wherein the total net charge is obtained by one or more substitutions selected from: A001E, V004E, R010H/A, Q012E, A015D, N018D, R019H/S, V026D, K027E, N043D, R045C/T/S/P, S049D, V051E, G061E, N062D/E, N076D, N077D, S078D, S087D, A098E, S101D, S106E, G115E, G118D, N123D, S128D, P129E, S130D/E, S132D, A158E, G159D/E, S160D, S166D, R170T, N183D, N184D, N185E, R186H, S188D/E, A194E, A200D, Y209E, A215D, N204D, S212D, L217D/E, N218D, A230E, K235F, N237D, K237E, N238D, S240D, N243D, Q245D, R247L, N248D/E, K251C, N263D, N269D/E, A272D, A273E, or R275H/S, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

### Claim 25 (depends on 19)

The isolated subtilisin variant of claim 19, wherein the total net charge is obtained by one or more substitutions selected from: A001R, V004R, Q012R, P014R, H017R, N018R/K, G020K/R, T022R/K, S024R/K, G025R, D032I, D041K/L/N, N043R/K, G046R, A048R, F050R/K, P055R, S056R/K, T057R, Q059K, G061R, N076K, S078R, P086R, S087R, E089G/P/I, G097R, S099R, Q109R, G115R, G118R, S132K, S144R, G159R/K, D181C/S/T, L196K, N204K, Q206R, K235R, Q236R/K, K237R, N238R, S240R, W241R, S242R/K, V244R, Q245R/K, N248R, H249R, N252R/K, S256R, G258R, T260K, N269R/K, or E271F/H/T/L/W/R/S/I/A/GN, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:1.

### Claim 26 (depends on 1)

The isolated subtilisin variant of claim 1, wherein the subtilisin variant has one or more of the following characteristics: a) a Test Method 2 performance index of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 10, from 1.1 to about 8, or even from 1.1 to about 5; b) a Test Method 3 performance index of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 10, from 1.1 to about 8, or even from 1.1 to about 5; c) a Test Method 4 performance index of at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.0 to about 10, from 1.0 to about 8, or even from 1.0 to about 5; and/or d) a Test Method 6 performance index of at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.0 to about 10, from 1.0 to about 8, or even from 1.0 to about 5; and/or e) Test Method 7 performance index of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 15, from 1.1 to about 10, or even from 1.1 to about 7.

### Claim at position 10

27-34. (canceled)

### Claim 35 (depends on 1)

A composition comprising at least one subtilisin variant of claim 1, wherein said composition is not a fabric and home care product.

### Claim 36 (depends on 35)

The composition of claim 35, wherein said composition is a cleaning composition.

### Claim 37 (depends on 36)

The composition of claim 36, wherein said cleaning composition is a granular, powder, solid, bar, liquid, tablet, gel, or paste composition.

### Claim 38 (depends on 35)

The composition of claim 35, further comprising at least one bleaching agent.

### Claim 39 (depends on 35)

The composition of claim 35, wherein said cleaning composition is phosphate-free.

### Claim 40 (depends on 35)

The composition of claim 35, wherein said cleaning composition contains phosphate.

### Claim 41 (depends on 35)

The composition of claim 35, further comprising at least one additional enzyme.

### Claim 42 (depends on 41)

The composition of claim 41, wherein said at least one additional enzyme is selected from hemicellulases, cellulases, peroxidases, proteases, metalloproteases, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, and amylases, or any combination thereof.

### Claim 43 (depends on 35)

The composition of claim 35, wherein said subtilisin variant is not a cold water protease variant.

### Claim 44 (depends on 1)

A method of cleaning, comprising contacting a surface or an item with a cleaning composition comprising at least one subtilisin variant of claim 1.

### Claim 45 (depends on 35)

A method of cleaning comprising contacting a surface or an item with a cleaning composition set forth in claim 35.

### Claim at position 22

46-51. (canceled)

### Claim 52 (depends on 44)

The method of claim 44, wherein said subtilisin variant is not a cold water protease variant.