Subtilase Variants and Polynucleotides Encoding Same

Patent metadata

Field	Value
Publication number	US-2016137998-A1
Application number	US-201414899583-A
Country	US
Kind code	A1
Filing date	Jun 27, 2014
Priority date	Jun 27, 2013
Publication date	May 19, 2016
Grant date	—

Abstract

The present invention relates to novel subtilase variants exhibiting increased stability and optionally on par or improved wash performance. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention.

First claim

1 . A subtilase variant having protease activity and comprises the double substitution 194P+261D and wherein the positions correspond to the positions of the mature polypeptide of SEQ ID NO: 2.

2 . The variant according to claim 1 which further comprises one or more alterations from the group consisting of 3{F, I, L, V, Y}, 9{A, G, M, T}, 18{A, D, E, G, M, S, T}, 40{D, E}, 43{D, E}, 45{D, E}, 58{F, Y}, 59{D, E}, 61{D, E}, 72{L, V}, 76{D, E}, 99{A, D, E, M, T}, 104{F, Y}, 105{D, E}, 120{D, E, I, L, V}, 130*, 133{D, E}, 141{F, Y}, 182{D, E}, 183{D, E}, 184{D, E}, 205{I, L}, 206{D, E}, 212{D, E}, 218{D, E}, 224{A, G, M, S}, 225{A, G, M, S, T}, 228{G, M, S, T}, 236{D, E}, 245{H, K}, 259{D, E} and 262{F, W, Y}.

3 . The variant of claim 1 , wherein the double substitution is A194P+N261D.

4 . The variant of claim 1 wherein the variant subtilase is:
a) a polypeptide that has at least 60% but less than 100% sequence identity to the mature polypeptide of the parent subtilase;
b) a polypeptide that is encoded by a polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of parent subtilase or
(ii) the full-length complement of (i); or

c) a polypeptide that is encoded by a polynucleotide having at least 60% but less than 100% sequence identity to the mature polypeptide coding sequence of parent subtilase.

5 . The variant of claim 1 wherein the variant subtilase is:
a) a polypeptide that has at least 60% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 4;
b) a polypeptide that is encoded by a polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 3 or
(ii) the full-length complement of (i); or

c) a polypeptide that is encoded by a polynucleotide having at least 60% but less than 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3.

6 . The variant of claim 1 , wherein the total number of alterations is between 3 and 30 alterations.

7 . (canceled)

8 . The variant of claim 1 , wherein the variant consists of 150 to 350 amino acids.

9 . The variant of claim 1 , wherein the variant comprises one or more of the alterations selected from the group consisting of:
3F+194P+261D; 3I+194P+261D; 3L+194P+261D; 3V+194P+261D; 3Y+194P+261D; 9A+194P+261D; 9G+194P+261D; 9M+194P+261D; 9T+194P+261D; 9+194P+261D; 18A+194P+261D; 18D+194P+261D; 18E+194P+261D; 18G+194P+261D; 18M+194P+261D; 18S+194P+261D; 18T+194P+261D; 40D+194P+261D; 40E+194P+261D; 43D+194P+261D; 43E+194P+261D; 45D+194P+261D; 45E+194P+261D; 58F+194P+261D; 58Y+194P+261D; 59D+194P+261D; 59E+194P+261D; 61D+194P+261D; 61E+194P+261D; 72L+194P+261D; 72V+194P+261D; 76D+194P+261D; 76E+194P+261D; 99A+194P+261D; 99D+194P+261D; 99E+194P+261D; 99M+194P+261D; 99T+194P+261D; 104F+194P+261D; 104Y+194P+261D; 105D+194P+261D; 105E+194P+261D; 120D+194P+261D; 120E+194P+261D; 120I+194P+261D; 120L+194P+261D; 120V+194P+261D; 130*+194P+261D; 133D+194P+261D; 133E+194P+261D; 141F+194P+261D; 141Y+194P+261D; 182D+194P+261D; 182E+194P+261D; 183D+194P+261D; 183E+194P+261D; 184D+194P+261D; 184E+194P+261D; 194P+205I+261D; 194P+205L+261D; 194P+206D+261D; 194P+206E+261D; 194P+212D+261D; 194P+212E+261D; 194P+218D+261D; 194P+218E+261D; 194P+224A+261D; 194P+224G+261D; 194P+224M+261D; 194P+224S+261D; 194P+225A+261D; 194P+225G+261D; 194P+225M+261D; 194P+225S+261D; 194P+225T+261D; 194P+228G+261D; 194P+228M+261D; 194P+228S+261D; 194P+228T+261D; 194P+236D+261D; 194P+236E+261D; 194P+245H+261D; 194P+245K+261D; 194P+259D+261D; 194P+259E+261D; 194P+261 D+262F; 194P+261 D+262W; and 194P+261 D+262Y.

10 . The variant of claim 1 , comprising a substitution, deletion and/or insertion at one or more positions selected from the group consisting of positions: 4, 9, 12, 14, 15, 40, 63, 68, 79, 84, 86, 88, 92, 98, 101, 132, 146, 188, 217, 245, 255 and 270 wherein the position corresponds to the position of the mature polypeptide of SEQ ID NO: 2.

11 . The variant of claim 1 , which has an improved stability, preferably improved in wash stability, compared to the parent or compared to a reference protease.

12 . The variant of claim 11 , which has an improved wash performance compared to the parent or compared to a reference protease.

13 . A detergent composition comprising a variant according to claim 1 .

14 . The detergent composition of claim 13 further comprising one or more detergent components.

15 . The detergent composition according to claim 13 further comprising one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes, haloperoxygenases, catalases and mannanases, or any mixture thereof.

16 . The detergent composition according to claim 13 in form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

17 - 18 . (canceled)

Assignees

• Novozymes As

Inventors

• Nielsen Jens Erik
• Micheelsen Pernille Ollendorf
• Knotzel Jurgen Carsten Franz
• Hockauf Maria Norman
• Beier Lars
• Bauer Michael
• Johansen Annette Helle
• Christensen Lars Lehmann
• Rannes Julie Billie

Classifications

• C12N9/54 Primary

  bacteria being Bacillus · CPC title

• C11D3/386 Primary

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

• C11D3/38681

  Chemically modified or immobilised enzymes · CPC title

• C12N9/6424

  Serine endopeptidases (3.4.21) · CPC title

• C12Y304/21062

  Subtilisin (3.4.21.62) · CPC title