Compositions and methods comprising serine protease variants

Patent metadata

Field	Value
Publication number	US-9856466-B2
Application number	US-201214115801-A
Country	US
Kind code	B2
Filing date	May 4, 2012
Priority date	May 5, 2011
Publication date	Jan 2, 2018
Grant date	Jan 2, 2018

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.

First claim

We 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+S101G+Q245R, wherein said subtilisin variant has at least 80% amino acid identity with a Bacillus lentus subtilisin GG36 protease comprising the amino acid sequence shown in SEQ ID NO:2, 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.

2. 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.

3. 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.

4. The isolated subtilisin variant of claim 3 , 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, 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.

5. The isolated subtilisin variant of claim 3 , 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/G/V, 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.

6. 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.

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

8. The composition of claim 7 , wherein said composition is a cleaning composition.

9. The composition of claim 8 , wherein said cleaning composition is a granular, powder, solid, bar, liquid, tablet, gel, or paste composition.

10. The composition of claim 7 , further comprising at least one bleaching agent.

11. The composition of claim 7 , wherein said cleaning composition is phosphate-free.

12. The composition of claim 7 , wherein said cleaning composition contains phosphate.

13. The composition of claim 7 , further comprising at least one additional enzyme.

14. The composition of claim 13 , 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.

15. The composition of claim 7 , wherein said subtilisin variant is not a cold water protease variant, wherein said cold water protease 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.

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

17. A method of cleaning comprising contacting a surface or an item with a cleaning composition set forth in claim 7 .

18. The method of claim 16 , wherein said subtilisin variant is not a cold water protease variant, wherein said cold water protease 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 abou

Assignees

• Amin Neelam S
• Augustyn Katherine
• Basler Joshua R
• Cascao-Pereira Luis G
• Collier Katherine D
• Concar Edward M
• Estell David A
• Kellis Jr James T
• Magennis Euan John
• Pisarchik Alexander
• Poulose Ayrookaran J
• Souter Philip Frank
• Ward Glenn Steven
• Yao Jian
• Danisco Us Inc

Inventors

• Amin Neelam S
• Augustyn Katherine
• Basler Joshua R
• Cascao-Pereira Luis G
• Collier Katherine D
• Concar Edward M
• Estell David A
• Kellis Jr James T
• Magennis Euan John
• Pisarchik Alexander
• Poulose Ayrookaran J
• Souter Philip Frank
• Ward Glenn Steven
• Yao Jian

Classifications

• C12N9/54 Primary

  bacteria being Bacillus · CPC title

• C11D3/386

  Preparations containing enzymes {, e.g. protease or amylase} · CPC title