Novel Subtilases

Abstract

The present invention relates to methods for producing variants of a parent TY145 subtilase and of a parent BPN′ subtilase and to TY145 and BPN′ variants having altered properties as compared to the parent TY145/BPN′ subtilase.

1 . A TY145 like subtilase variant which is at least 63% homologous to the sequence of SEQ ID NO:1, preferably at least 65%, at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence of SEQ ID NO:1, which variant comprises:
 (1) an alteration in one or more positions located at a distance of not more than 10 Å to one of the ion-binding sites of TY145, wherein the positions, as specified in SEQ ID NO:1, located at a distance of not more than 10 Å to:
 a) the Weak ion-binding site are: 154, 155, 158, 164, 165, 166, 167, 168, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 211, 220, 221, 222, 223, 224, 225, 226, 227, 228, 277, 281 and 305, 
 b) the Near ion-binding site are: 185, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 277, 281, 299, 300, 301, 304, 305, 
 c) the Far ion-binding site are: 193, 198, 199, 201, 202, 204, 216, 217, 219, 226, 227, 228, 229, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306 and 307; 
   (2) one or more alterations in one or more of the positions contained in the following highly mobile regions:
 84, 85, 86, 87 and 88, 
 108, 109, 110, 111, 112, 113, 114, 115, 116 and 117, 
 141, 142, 143, 144, 145 and 146, 
 150, 151 and 152, 
 169, 170 and 171, 
 200 and 201, 
 211, 212, 213, 214, 215, 216, 217, 218, 219 and 220, 
 242 and 243, 268, 269 and 270; 
   (3) one or more alterations in one or more of the positions contained in the following mobile regions:
 1, 2, 3, 4, 5, 6 and 7, 
 17, 18, 19, 20, 21, 22 and 23, 
 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, 
 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 and 69, 
 84, 85, 86, 87, 88, 89, 90, 91 and 92, 
 107, 108, 109 and 110, 
 239, 240, 241, 242 and 243 
 265 and 266, 
 wherein said alterations preferably are introduced in one or both of the regions 57-69 and 84-92; 
   (4) an alteration in one or more of the positions contained in the following regions:
 16, 17, 18, 19, 20, 21 and 22, 
 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73, 
 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 and 131, 
 40, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 and 161, 
 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293 and 294, 
 wherein such alterations preferably are made in one or both the regions 40-73 and 140-161, preferably in the sub-regions 65-73 and 140-150; 
   (5) an alteration in one or more of the following positions:
 35, 36, 70, 72, 106, 109, 110, 111, 112, 113, 114, 117, 139, 140, 141, 142, 143, 
 144, 145, 147, 150, 167, 168, 169, 170, 171, 172, 173, 174, 177, 180, 207, 239, 247, 
 148, 149, 150, 151 and 252 of SEQ ID NO:1; or 
   (6) an alteration in one or more of the following positions: V31, V38, T79, V80, L81, V188, T254, wherein preferred variants comprise one or more of V31I, T79S and V80A.   
     
     
         2 . The TY145 like subtilase variant according to  claim 1  wherein the alterations are one or more of the substitutions I220S,T,D,E; T215S,D,E; G298A,S,T,D,E; G296A,S,T,D,E; V185T,D,E; I221N,D,T,E; V185P and I293P. 
     
     
         3 . The TY145 like subtilase variant according to  claim 1 , in which one or more ion-binding sites have been removed, wherein said variant comprises one or both of the alterations
 a) deletion of the region K290-D300, or at least one deletion of one amino acid residue in the region K290-D300 of SEQ ID NO:1 and subsequent insertion of one or more amino acid residues, preferably insertion between 1289 and Y301 of the sequence GDS (SEQ ID NO: 49) or DST (SEQ ID NO: 50), and preferably further comprising the substitution S303Y,   b) deletion of the region N212-R224, or at least one deletion of one amino acid residue in the region N212-R224 of SEQ ID NO:1 and subsequent insertion of one or more amino acid residues, preferably insertion of a proline residue or an alanine residue between G211 and D225.   
     
     
         4 . A TY145 like subtilase variant comprising the introduction of a ion-binding site corresponding to the Strong ion-binding site of the Subtilisin family subtilases, wherein said variant has a deletion of the region H83-G90, or at least one deletion of one amino acid residue in the region H83-G90, of SEQ ID NO:1 and subsequent insertion of one or more amino acid residues, preferably insertion of the sequence LNNSIG (SEQ ID NO: 48) between residues A82 and V91. 
     
     
         5 . A Subtilisin family subtilase variant in which the Strong ion-binding site has been removed, wherein said variant comprises deletion of the region L75-G80 (BPN′ numbering), or at least one deletion of one amino acid residue in the region L75-G80 (BPN′ numbering), or a corresponding region in another Subtilisin family subtilase, and subsequent insertion of one or more amino acid residues, preferably insertion of the sequence GGSNG (SEQ ID NO: 51) of positions 84-88 of TY145 (SEQ ID NO:1) between A74 and V81, and preferably further comprising one or both of the substitutions L80Y and Q2A,N. 
     
     
         6 . The TY145 like subtilase variant according to  claim 1 , comprising the alterations D115P and/or T269P. 
     
     
         7 . The TY145 like subtilase variant according to  claim 1 , comprising the substitution D116H,K,R and/or Q18P. 
     
     
         8 . The TY145 like subtilase variant according to  claim 1 , comprising one or more disulfide bridges introduced by one or more of the following modifications: G26C+A95C; A167C+T254C: R203C+G292C; V228C+A284C, wherein the positions corresponds to the positions in SEQ ID NO:1 
     
     
         9 . The TY145 like subtilase variant according to  claim 1 , comprising an alteration of an Asn-Gly sequence by deletion or substitution of at least one of the Asn or Gly residues, preferably the Asn residue. 
     
     
         10 . The variant of  claim 9  comprising substitution of the Asn and/or Gly residue with an amino acid residue selected from the group consisting of A, Q, S, P, T and Y. 
     
     
         11 . The variant of  claim 10 , wherein the substitution is performed in one or more of the following positions:
   B. sphaericus:  198-199, 240-241   TY145: 87-88, 109-110, 199-200   TA41: 83-84, 198-199   TA39: 88-89, 198-199.   
     
     
         12 . The TY145 like subtilase variant according to  claim 1 , comprising an alteration of a tyrosine residue by deletion or substitution, preferably to phenylalanine. 
     
     
         13 . The variant of  claim 12 , wherein the substitution is performed in one or more of the following positions:
   B. sphaericus:  14, 91, 102, 112, 155, 157, 172, 179, 201, 206, 211, 218, 235, 239, 243, 292, 300,   TY145: 15, 39, 92, 103, 113, 156, 158, 202, 219, 240, 244, 287, 301, 307,   TA41: 15, 91, 102, 112, 155, 157, 179, 201, 218, 235, 243,   TA39: 15, 61, 91, 102, 112, 155, 157, 173, 179, 201, 211, 218, 235, 243, 267, 281, 284, 292, 293, 296.   
     
     
         14 . A TY145 like subtilase variant comprising an alteration of a methionine residue by deletion or substitution, preferably to a serine or alanine residue. 
     
     
         15 . The variant of  claim 14 , wherein the substitution is performed in one or more of the following positions:
   B. sphaericus:  138, 251,   TY145: 139, 252,   TA41: 1, 138, 251,   TA39: 1, 138, 251.   
     
     
         16 . A BPN′ like subtilase variant comprising one or more of the following alterations:
 a) deletion of residues PSPSATLEQAVN (SEQ ID NO: 23) (positions 129-140) in Savinase (BPN′ numbering) and subsequent insertion of residues SAKDSLIASAVD (SEQ ID NO: 22) (positions 144-155) from TY145 between S128 and S141 in Savinase;   b) deletion of residues SGNSGAGSISYPARYA (SEQ ID NO: 25) (positions 153-172) in Savinase (BPN′ numbering) and subsequent insertion of residues AGNSGSGSNTIGFPGGLV (SEQ ID NO: 24)(positions 168-185) from TY145 between A152 and N173 in Savinase;   c) deletion of residues VNVQSTYPGSTYASLN (SEQ ID NO: 27) (positions 203-218) in Savinase (BPN′ numbering) and subsequent insertion of residues ASVESTWYTGGYNTIS (SEQ ID NO: 26) (positions 233-248) from TY145 between G202 and G219 in Savinase;   d) V28I,A,L; I35V,A,L; T71S; I72A,G,V; A73L,G; M175V,A; and T224S,A, wherein preferred variants of Savinase comprises one or more of the substitutions V28I, I35V, T71S, I72A, A73L, M175V and T224S (BPN′ numbering), especially variants comprising the combinations V28I+I35V, V28I+T71S, V28I+I72A, V28I+A73L, V28I+M175V, I35V+T71S, I35V+I72A, I35V+A73L, I35V+A73L, I35V+M175V, T71S+I72A, T71S+A73L, T71S+A73L, T71S+M175V, I72A+A73L, I72A+A73L, I72A+M175V, A73L+M175V;   e) deletion of residues LSLGSPS (SEQ ID NO: 29) (positions 124-130) in Savinase (BPN′ numbering) and subsequent insertion of residues MSLGSSG (SEQ ID NO: 28) (positions 138-144) from TA39 subtilase between N123 and P131 in Savinase;   f) deletion of residues LSLGSPSPSATL (SEQ ID NO: 31) (positions 124-135) in Savinase variant V104S (BPN′ numbering) and subsequent insertion of residues MSLGSSGESSLI (SEQ ID NO: 30) (positions 138-149) from TA39 subtilase between N123 and E136 in Savinase variant V104S;   g) deletion of residues VQAPAAHN (SEQ ID NO: 33) (positions 11-18) in Savinase (BPN′ numbering) and subsequent insertion of residues NNSSITQT (SEQ ID NO: 32) (positions 16-23) from TA39 subtilase between R10 and R19 in Savinase;   h) deletion of residues VPG*EPST (SEQ ID NO: 35) (positions 51-58) in Savinase (BPN′ numbering) and subsequent insertion of residues TVGTTYTN (SEQ ID NO: 34 positions 56-63 from TA39 subtilase between F50 and Q59 in Savinase;   i) deletion of residues GN (positions 61-62) in Savinase (BPN′ numbering) and subsequent insertion of residues RQ (positions 69-70) from TA39 subtilase between D60 and G63 in Savinase;   j) deletion of residues PSPSATL (SEQ ID NO: 37) (positions 129-135) in Savinase (BPN′ numbering) and subsequent insertion of residues SGESSLI (SEQ ID NO: 36) (positions 143-149) from TA39 subtilase between S128 and E136 in Savinase;   k) deletion of residues YPGSTYASL (SEQ ID NO: 39) (positions 209-217) in Savinase (BPN′ numbering) and subsequent insertion of residues WFDGGYATI (SEQ ID NO: 38) (positions 238-246) from TA39 subtilase between T208 and N218 in Savinase;   l) deletion of residues VPG*EPST (SEQ ID NO: 41) (positions 51-58) in Savinase (BPN′ numbering) and subsequent insertion of residues TVGTNFTD (SEQ ID NO: 40) (positions 56-63) from TA41 subtilase between F50 and Q59 in Savinase;   m) deletion of residues ALNNSI (SEQ ID NO: 43) (positions 74-79) in Savinase (BPN′ numbering) and subsequent insertion of residues NGGTGS (SEQ ID NO: 42) (positions 83-88) from TA41 subtilase between A73 and G80 in Savinase;   n) deletion of residues ASGSGSV (SEQ ID NO: 45) (positions 98-104) in Savinase (BPN′ numbering) and subsequent insertion of residues DDGSGYA (SEQ ID NO: 44) (positions 107-113) from TA41 subtilase between G97 and S105 in Savinase; or   o) deletion of residues KQKNPSW (SEQ ID NO: 47) (positions 235-241) in Savinase (BPN′ numbering) and subsequent insertion of residues WAQSPAA (SEQ ID NO: 46) (positions 264-270) from TA41 subtilase between V234 and S242 in Savinase.   
     
     
         17 . An isolated nucleic acid sequence comprising a nucleic acid sequence, which encodes for the subtilase or subtilase variant defined or produced in  claim 16 . 
     
     
         18 . An isolated nucleic acid sequence according to  claim 17 , wherein the nucleic acid sequence is selected form the group consisting of:
 a) a nucleic acid sequence having at least 40% homology with the nucleic acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21, and   b) a nucleic acid sequence which hybridizes under low stringency conditions, preferably under medium stringency conditions, in particular under high stringency conditions, with   c) a complementary strand of the nucleic acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21, or   d) a subsequence of any of a), b) or c) of at least 100 nucleotides.   
     
     
         19 . An isolated nucleic acid sequence according to  claim 18 , wherein the nucleic acid sequence has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleic acid sequence shown in SEQ ID NO:20 or SEQ ID NO:21. 
     
     
         20 . An isolated nucleic acid construct comprising a nucleic acid sequence as defined in accordance with  claim 19 , operably linked to one or more control sequences capable of directing the expression of the polypeptide in a suitable expression host. 
     
     
         21 . A recombinant host cell comprising the nucleic acid construct of  claim 19 . 
     
     
         22 . A method for producing the subtilase or subtilase variant defined or produced in  claim 1 , the method comprising:
 a) cultivating the recombinant host cell of  claim 21  under conditions conducive to the production of the subtilase variant; and   b) recovering the variant.   
     
     
         23 . A detergent composition comprising a subtilase or subtilase variant defined or produced in  claim 1 .