Subtilase variants

Abstract

The present invention relates to novel subtilase variants exhibiting alterations relative to the parent subtilase in one or more properties including: Wash performance, thermal stability, storage stability or catalytic activity. The variants of the invention are suitable for use in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dishwash compositions, including automatic dishwash compositions.

1 . A subtilase variant comprising at least one or more of the following alterations:
 X11{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V}, deletion, insertion;   X30{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V}, deletion, insertion;   X31{R,N,D,Q,E,H,K,M,P,W,Y}, deletion, insertion;   X32{A,R,D,C,E,G,H,I,L,K,M,F,P,T,W,Y,V}, insertion;   X34{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V}, deletion;   X65{A,R,C,G,H,I,L,K,M,F,T,W,Y,V}, deletion, insertion;   X66{A,R,C,H,I,L,K,M,F,T,W,Y,V}, deletion, insertion;   X67{A,R,N,Q,G,H,I,K,M,F,P,S,T,W,Y,V};   X68{R,N,D,Q,E,G,H,I,L,K,F,P,S,T,W,Y,V}, deletion, insertion;   X69{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V}, deletion, insertion;   X70G;   X71T;   X77N;   X83G, insertion;   X84V;   X90{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V}, deletion, insertion;   X95{R,K,F,W,Y,V}, deletion;   X107I   X110G, insertion;   X121V, insertion;   X122 insertion;   X123N;   X125S;   X150{A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V}, deletion, insertion;   X152{A,R,N,D,Q,E,H,K,F,P,W,Y,V};   X153{R,N,D,Q,E,G,H,I,L,K,M,F,T,W,Y,V};   X154{A,R,G,H,I,L,M,F,W,Y,V};   X164{A,R,H,T,W};   X165{R,L,F,W,Y};   X166{W,Y}, insertion;   X175{R,N,D,Q,E,G,H,K,M,F,P,T,W,Y,V};   X177{R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V}, deletion, insertion;   X178{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V}, deletion, insertion;   X180{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V}, deletion, insertion;   X199{R,L,K,F,W,Y,V}, deletion, insertion;   X200{A,R,I,L,K,M,F,W,Y,V}, deletion, insertion;   X201{A,R,H,I,L,K,M,F,P,T,W,Y,V}, deletion, insertion;   X202{A,R,C,G,H,I,L,K,M,F,T,W,Y,V};   X207{A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V};   X220T, insertion;   X223A, insertion;   X225P, insertion;   X226H, insertion;   X227V, insertion;   X228A, insertion;   X229G, insertion;   X230 insertion;   X231A, insertion;   X253{A,R,N,D,Q,E,G,H,I,L,M,F,S,T,W,Y,V}, deletion, insertion;   X264{A,R,N,Q,G,H,I,L,M,F,S,T,W,Y,V}, deletion, insertion;   X266{A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V}, deletion, insertion; wherein   (a) the alteration(s) are independently, as specified,
 (i) a substitution of the amino acid that occupies the position with a different amino acid, 
 (ii) a deletion of the amino acid which occupies the position, or 
 (iii) an insertion of at least one additional amino acid downstream of the amino acid that occupies the position, 
   (b) the variant has protease activity, and   (c) each position corresponds to a position of the amino acid sequence of subtilisin BPN′, shown in  FIG. 1 .   
     
     
         2 . The variant according to  claim 1 , wherein the parent subtilase belongs to the sub-group I-S1. 
     
     
         3 . The variant according to  claim 2 , wherein the parent subtilase is selected from the group consisting of subtilisin I168, subtilisin BPN═, subtilisin DY, and subtilisin Carlsberg. 
     
     
         4 . The variant according to  claim 1 , wherein the parent subtilase belongs to the sub-group I-S2. 
     
     
         5 . The variant according to  claim 4 , wherein the parent subtilase is selected from the group consisting of subtilisin 147, subtilisin 309, subtilisin PB92, and subtilisin YaB. 
     
     
         6 . The variant according to  claim 4 , wherein the variant is N76D+M175ML+A194P+A230V; N76D+M175MI+A194P+A230V. 
     
     
         7 . The variant according to  claim 6 , wherein the variant further comprises at least one modification in the positions 27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 120, 123, 159, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245, 248, 252 or 274 (wherein the positions correspond to BASBPN numbering). 
     
     
         8 . (canceled) 
     
     
         9 . An isolated DNA sequence encoding a subtilase variant as defined in  claim 1 . 
     
     
         10 . An expression vector comprising the isolated DNA sequence of  claim 9 . 
     
     
         11 . A microbial host cell transformed with the expression vector of  claim 10 . 
     
     
         12 . A microbial host cell according to  claim 11 , which is a bacterium,  Bacillus,  or  B. lentus.    
     
     
         13 . A microbial host cell according to  claim 11 , which is fungus, yeast, filamentous fungus, or  Aspergillus.    
     
     
         14 . A method for producing a subtilase variant, comprising:
 (a) culturing a host of  claim 11  under conditions conducive to the expression and secretion of the variant; and   (b) recovering the variant.   
     
     
         15 . A cleaning or detergent composition comprising: the variant as defined in  claim 1 , and a surfactant. 
     
     
         16 . A composition according to  claim 15 , which additionally comprises a cellulase, a lipase, a cutinase, an oxidoreductase, another protease, an amylase or a mixture thereof. 
     
     
         17 . (canceled)