Beta-lactamase variants

Abstract

The present invention relates to an isolated polypeptide having beta-lactamase activity and nucleic acid sequences encoding the polypeptide. The isolated polypeptide of the invention is a VIM-2 variant with improved properties such as improved protease stability, stability in intestinal medium, improved activity against one or more antibiotics, improved specific activity and/or improved production in a host cell.

What is claimed is: 
     
         1 . A polypeptide having beta-lactamase activity, which comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1, said polypeptide comprising:
 a substitution at each of positions 10 and 22;   a substitution at each of positions 10 and 34;   a substitution at each of positions 10 and 130;   a substitution at each of positions 10, 22 and 34;   a substitution at each of positions 10, 22 and 130;   a substitution at each of positions 10, 34 and 130; or   a substitution at each of positions 10, 22, 34 and 130,   wherein the positions correspond to the positions in the sequence represented in SEQ ID NO:1, and wherein said polypeptide has an improved property with respect to stability as compared with wild-type VIM-2 of SEQ ID NO:1.   
     
     
         2 . The polypeptide according to  claim 1 , which comprises at least one modification selected from the following:
 V10A;   Q22H or Q22N;   Q34R; and   E130D.   
     
     
         3 . The polypeptide according to  claim 1 , wherein the residue corresponding to the first residue of SEQ ID NO:1 is replaced with a methionine residue. 
     
     
         4 . The polypeptide according to  claim 1 , further comprising a signal peptide at its N-terminal end. 
     
     
         5 . The polypeptide according to  claim 1 , comprising a truncation at its N-terminal or C-terminal end as compared to the sequence shown in SEQ ID NO:1. 
     
     
         6 . The polypeptide according to  claim 5 , which comprises:
 a C-terminal truncation of residues 237-240 of SEQ ID NO:1;   a C-terminal truncation of residues 236-240 of SEQ ID NO:1; or   a C-terminal truncation of residues 235-240 of SEQ ID NO:1.   
     
     
         7 . The polypeptide according to  claim 1 , comprising or consisting of the amino acid sequence of any one of SEQ ID NO:4 to 23 or a fragment thereof having beta-lactamase activity. 
     
     
         8 . An isolated nucleic acid sequence comprising a nucleic acid sequence encoding the polypeptide according to  claim 1 . 
     
     
         9 . A nucleic acid construct comprising the nucleic acid sequence of  claim 8 , operably linked to one or more control sequences that direct the expression of the polypeptide in a suitable expression host. 
     
     
         10 . A recombinant host cell, comprising the nucleic acid construct of  claim 9 . 
     
     
         11 . A composition comprising the polypeptide of  claim 1 . 
     
     
         12 . The composition of  claim 11 , which is orally administrable and is able to release the polypeptide in a desired part of the intestine, in particular in the jejunum, the ileum, the caecum or the colon. 
     
     
         13 . A kit-of-parts comprising
 (a) the polypeptide of  claim 1  or the composition of  claim 11 ; and   (b) a beta-lactam antibiotic which is sensitive to said polypeptide of (a) or contained in the composition of (a);   for separate, sequential or simultaneous administration.   
     
     
         14 . The polypeptide of  claim 1 , for use as a medicament. 
     
     
         15 . A method for inactivating a beta-lactam antibiotic in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the polypeptide of  claim 1 , of the composition of  claim 11 , of the kit-of-parts of  claim 13  or of the recombinant host cell of  claim 10 . 
     
     
         16 . A method for treating a bacterial infection in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the polypeptide of  claim 1 , of the composition of  claim 11 , of the kit-of-parts of  claim 13  or of the host cell of  claim 10 , wherein said polypeptide or said composition or said host cell is used in combination with a beta-lactam antibiotic which is sensitive to said polypeptide. 
     
     
         17 . The polypeptide according to  claim 2 , wherein the residue corresponding to the first residue of SEQ ID NO:1 is replaced with a methionine residue. 
     
     
         18 . The polypeptide according to  claim 2 , further comprising a signal peptide at its N-terminal end. 
     
     
         19 . The polypeptide according to  claim 3 , further comprising a signal peptide at its N-terminal end. 
     
     
         20 . The polypeptide according to  claim 17 , further comprising a signal peptide at its N-terminal end.