Stabilized enzyme compositions

Abstract

The present invention relates to methods for obtaining protease variants with improved properties. The present invention also relates to protease variants, compositions comprising the protease variants and methods of using the protease variants.

1 . The present invention relates to a method for improve binding of a protease to an inhibitor of the protease, comprising the steps of:
 a) identify the amino acid residues exposed to the inhibitor when the inhibitor binds to the protease,   b) modify the protease by modifying the amino acid residues identified in step a)   c) determine the binding constant Ki   d) select modified protease generated in step b)   
     
     
         2 . The method according to  claim 1 , wherein the improvement factor of the modified protease selected in step d) is above 1.0. 
     
     
         3 . The method of  claim 1 , wherein the subtilisin inhibitor is a peptide aldehyde, or a hydrosulfite adduct thereof; or a phenyl boronic acid, or a derivative thereof, such as 4-FPBA. 
     
     
         4 . The method of  claim 1 , wherein the inhibitor is a peptide aldehyde or ketone having the formula P-(A) y -L-(B) x —B 0 —R* or a hydrosulfite adduct of such aldehyde, wherein:
 a) R* is H (hydrogen), CH 3 , CX 3 , CHX 2 , or CH 2 X; 
 b) X is a halogen atom; 
 c) B 0  is a single amino acid residue with L- or D-configuration of the formula —NH—CH(R)—C(═O)—; 
 d) x is 1, 2 or 3; 
 e) B x  is independently a single amino acid residue, each connected to the next B or to B 0  via its C-terminal; 
 f) L is absent or independently a linker group of the formula —C(═O)—, —C(═O)—C(═O)—, —C(═S)—, —C(═S)—C(═S)— or —C(═S)—C(═O)—; 
 g) A is absent if L is absent or is independently a single amino acid residue connected to L via the N-terminal of the amino acid; 
 h) P is selected from the group consisting of hydrogen or if L is absent an N-terminal protection group; 
 i) y is 0, 1, or 2, 
 j) R is independently selected from the group consisting of C 1-6  alkyl, C 6-10  aryl or C 7-10  arylalkyl optionally substituted with one or more, identical or different, substituent's R′; 
 k) R′ is independently selected from the group consisting of halogen, —OH, —OR″, —SH, —SR″, —NH 2 , —NHR″, —NR″ 2 , —CO 2 H, —CONH 2 , —CONHR″, —CONR″ 2 , —NHC(═N)NH 2 ; and 
 l) R″ is a C 1-6  alkyl group. 
 m) x may be 1, 2 or 3. 
 
     
     
         5 . The method of  claim 4 , wherein the inhibitor is an aldehyde having the formula P—B 2 —B 1 —B 0 —H or an adduct having the formula P—B 2 —B 1 —N(H)—CHR—CHOH—SO 3 M, wherein
 a) H is hydrogen; 
 b) B 0  is a single amino acid residue with L- or D-configuration of the formula —NH—CH(R)—C(═O)—; 
 c) B 1  and B 2  are independently single amino acid residues; 
 d) R is independently selected from the group consisting of C 1-6  alkyl, C 6-10  aryl or C 7-10  arylalkyl optionally substituted with one or more, identical or different, substituent's R′; 
 e) R′ is independently selected from the group consisting of halogen, —OH, —OR″, —SH, —SR″, —NH 2 , —NHR″, —NR″ 2 , —CO 2 H, —CONH 2 , —CONHR″, —CONR″ 2 , —NHC(═N)NH 2 ; 
 f) R″ is a C 1-6  alkyl group; and 
 g) P is an N-terminal protection group. 
 
     
     
         6 . The method of  claim 4 , wherein R is such that B 0 =—NH—CH(R)—C(═O)— is Phe, Tyr or Leu; and B 1  is Ala, Gly or Val; and B 2  is Arg, Phe, Tyr or Trp. 
     
     
         7 . The method of  claim 4 , wherein x=2, L is absent, A is absent, and P is p-methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz). 
     
     
         8 . The method of  claim 4 , wherein the inhibitor is Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF 3 , Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF 3 , Moc-Val-Ala-Leu-CF 3 , Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Phe-CF 3 , Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H, MeO—CO-Val-Ala-Leu-H, MeNCO-Val-Ala-Leu-H, MeO—CO-Phe-Gly-Ala-Leu-H, MeO—CO-Phe-Gly-Ala-Phe-H, MeSO 2 -Phe-Gly-Ala-Leu-H, MeSO 2 -Val-Ala-Leu-H, PhCH 2 O—P(OH)(O)-Val-Ala-Leu-H, EtSO 2 -Phe-Gly-Ala-Leu-H, PhCH 2 SO 2 -Val-Ala-Leu-H, PhCH 2 O—P(OH)(O)-Leu-Ala-Leu-H, PhCH 2 O—P(OH)(O)-Phe-Ala-Leu-H, or MeO—P(OH)(O)-Leu-Gly-Ala-Leu-H or a hydrosulfite adduct of any of these, wherein Cbz is benzyloxycarbonyl and Moc is methoxycarbonyl. 
     
     
         9 . The method of  claim 4 , wherein the inhibitor is Cbz-Gly-Ala-Tyr-H or Moc-Val-Ala-Leu-H, or a hydrosulfite adduct thereof, wherein Cbz is benzyloxycarbonyl and Moc is methoxycarbonyl. 
     
     
         10 . The method according to  claim 1  wherein the protease is selected from a protease:
 a. having the amino acid sequence shown in SEQ ID NO: 1 or a protease having at least 60%, preferably at least 70%, preferably at least 80% or at least 90% sequence identity hereto, or 
 b. having the amino acid sequence shown in SEQ ID NO: 2 or a protease having at least 60%, preferably at least 70%, preferably at least 80% or at least 90% sequence identity hereto, or 
 c. having the amino acid sequence shown in SEQ ID NO: 3 or a protease having at least 60%, preferably at least 70%, preferably at least 80% or at least 90% sequence identity hereto, or 
 d. having the amino acid sequence shown in SEQ ID NO: 4 or a protease having at least 60%, preferably at least 70%, preferably at least 80% or at least 90% sequence identity hereto, or 
 e. having the amino acid sequence shown in SEQ ID NO: 5 or a protease having at least 60%, preferably at least 70%, preferably at least 80% or at least 90% sequence identity hereto, or 
 f. protease having the amino acid sequence shown in SEQ ID NO: 6 or a protease comprising compared to SEQ ID NO 1 an insertion of glutamic acid at the position corresponding to position 97 of SEQ ID NO 1 and having at least 60%, preferably at least 70%, preferably at least 80% or at least 90% sequence identity to SEQ ID NO 1. 
 
     
     
         11 . A protease obtained by the method of  claim 1 . 
     
     
         12 . The method according to  claim 1  wherein the modified protease variant has improved wash performance.