Alkaline protease variants

• US 8,778,650 B2
• Filed: 04/26/2010
• Issued: 07/15/2014
• Est. Priority Date: 04/30/2009
• Status: Active Grant

• Current Assignee: KAO Corporation
• Original Assignee: KAO Corporation
• Inventors: Tohata, Masatoshi, Okuda, Mitsuyoshi, Sato, Tsuyoshi, Endo, Keiji, Saeki, Katsuhisa
• Primary Examiner(s): Swope, Sheridan
• Application Number:
  US13/266,525
• Publication Number:
  US 20120058928A1
• Time in Patent Office: 1,541 Days
• Field of Search: None
• US Class Current: 435/221
• CPC Class Codes: C11D 3/386 Preparations containing enzymes , e.g. protease or amylase
• CPC Class Codes: C11D 3/38618 Protease or amylase in liquid compositions only
• CPC Class Codes: C11D 3/38681 Chemically modified or immobilised enzymes
• CPC Class Codes: C12N 9/54 bacteria being Bacillus

Abstract

The invention is directed to an alkaline protease that has the amino acid sequence of SEQ ID NO: 2 or of an amino acid sequence having an identity of 90% or more thereto, except that one or more amino acid residues at positions selected from (a) position 6, (b) position 15, (c) position 16, (d) position 65, (e) position 66, (f) position 82, (g) position 83, (h) position 204, (i) position 319, and (j) position 337 of SEQ ID NO: 2, or at positions corresponding thereto, are substituted with certain other amino acid residues. The invention is also directed to detergent compositions and culture medium that contain the protease.

Claims

1. A recombinant protein comprising (a) an amino acid sequence consisting of SEQ ID NO:

2 or (b) an amino acid sequence having 95% or more identity with SEQ ID NO;

2, in which, in both (a) and (b), glutamine at SEQ ID NO;

2 position 204 or at a position corresponding thereto is substituted with;

glutamic acid, aspartic acid, cysteine, valine, threonine, proline, histidine, isoleucine, tryptophan, serine, asparagine, lysine or arginine,

and wherein the amino acid sequence has alkaline protease activity to cleave the substrate Glt-Ala-Ala-Pro-Leu-pNA to release p-nitroaniline.

2. The recombinant protein according to claim 1, wherein said protein comprises said (a), an amino acid sequence consisting of the amino acid sequence of SEQ ID NO:

2 in which glutamine at SEQ ID NO;

2 position 204 is substituted with;

glutamic acid, aspartic acid, cysteine, valine, threonine, proline, histidine, isoleucine, tryptophan, serine, asparagine, lysine or arginine.

15. The recombinant alkaline protease according to claim 1, wherein one or more of SEQ ID NO:

2 positions selected from positions 6, 65, 66, 82, 83, 319 and 337, or positions corresponding thereto, are also substituted.

3. A detergent composition comprising a protein comprising (a) an amino acid sequence consisting of SEQ ID NO:

2 or (b) an amino acid sequence having 95% or more identity with SEQ ID NO;

2, in which, in both (a) and (b), glutamine at SEQ ID NO;

2 position 204 or at the position corresponding thereto is substituted with;

glutamic acid, aspartic acid, cysteine, valine, threonine, proline, histidine, isoleucine, tryptophan, serine, asparagine, lysine or arginine,

and wherein the amino acid sequence has alkaline protease activity to cleave the substrate Glt-Ala-Ala-Pro-Leu-pNA to release p-nitroaniline.

4. The detergent composition according to claim 3, which comprises an anionic surfactant.

5. A recombinant protein comprising an amino acid sequence consisting of an amino acid sequence having 95% or more identity with the amino acid sequence of SEQ ID NO:

2, and in which the amino acid at SEQ ID NO;

2 position 204, or at the position corresponding thereto, is aspartic acid, and wherein the amino acid sequence has alkaline protease activity to cleave the substrate Glt-Ala-Ala-Pro-Leu-pNA to release p-nitroaniline.

6. The recombinant protein of claim 5, wherein the identity is 96% or more.

7. The recombinant protein of claim 6, wherein the identity is 97% or more.

8. The recombinant protein of claim 7, wherein the identity is 98% or more.

9. The recombinant protein of claim 8, wherein the identity is 99% or more.

16. A detergent composition comprising the recombinant protein of claim 5.

10. Culture medium from the culture of an isolated transformed host cell that contains a recombinant protein produced by the host cell, wherein the amino acid sequence of the recombinant protein comprises an amino acid sequence consisting of a sequence having 95% or more identity with SEQ ID NO:

2, and in which the amino acid at SEQ ID NO;

2 position 204, or the position corresponding thereto, is aspartic acid, and wherein the amino acid sequence has alkaline protease activity to cleave the substrate Glt-Ala-Ala-Pro-Leu-pNA to release p-nitroaniline.

11. The culture medium of claim 10, wherein the identity is 96% or more.

12. The culture medium of claim 11, wherein the identity is 97% or more.

13. The culture medium of claim 12, wherein the identity is 98% or more.

14. The culture medium of claim 13, wherein the identity is 99% or more.