Modified beta-lactamases and methods and uses related thereto

Abstract

The present invention relates to pharmaceuticals and modified beta-lactamases. Specifically, the invention relates to novel recombinant beta-lactamases and pharmaceutical compositions comprising the beta-lactamases. Also, the present invention relates to methods for modifying a beta-lactamase, producing the beta-lactamase and treating or preventing beta-lactam antibiotic induced adverse effects. Furthermore, the present invention relates to the beta-lactamase for use as a medicament and to the use of the beta-lactamase in the manufacture of a medicament for treating or preventing beta-lactam antibiotics induced adverse effects. Still further, the invention relates to a polynucleotide and a host cell comprising the polynucleotide.

1 . A beta-lactamase comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 and having a hydrophilic amino acid residue at a position of SEQ ID NO: 1 corresponding to position 276 according to Ambler classification, or a variant or fragment thereof. 
     
     
         2 . A method of modifying a beta-lactamase comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1, characterized in that an amino acid of the beta-lactamase at a position of SEQ ID NO: 1 corresponding to position 276 according to Ambler classification is replaced with a hydrophilic amino acid. 
     
     
         3 . The beta-lactamase according to  claim 1  or the method according to  claim 2 , wherein the beta-lactamase comprises an amino acid sequence having at least 68% sequence identity with SEQ ID NO:1, or a variant or fragment thereof having beta-lactamase activity and including a hydrophilic amino acid residue other than aspartic acid (D) at a position corresponding to position 276 according to Ambler classification. 
     
     
         4 . The beta-lactamase according to  claim 1  or the method according to  claim 2 , wherein the beta-lactamase comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO:1, and wherein the hydrophilic amino acid residue at a position corresponding to position 276 according to Ambler classification is other than aspartic acid (D). 
     
     
         5 . The beta-lactamase according to  claim 1  or the method according to  claim 2 , characterized in that the hydrophilic amino acid is selected from polar and positively charged hydrophilic amino acids from the group consisting of arginine (R), histidine (H) and lysine (K). 
     
     
         6 . The beta-lactamase according to  claim 1  or the method according to  claim 2 , characterized in that the hydrophilic amino acid is selected from polar and neutral of charge hydrophilic amino acids from the group consisting of asparagine (N), glutamine (Q), serine (S) and threonine (T). 
     
     
         7 . The beta-lactamase according to  claim 6 , characterized in that the amino acid at the position of SEQ ID NO: 1 corresponding to position 276 is asparagine. 
     
     
         8 . The beta-lactamase according to  claim 5 , characterized in that the amino acid at the position of SEQ ID NO: 1 corresponding to position 276 is arginine. 
     
     
         9 . The beta-lactamase or the method according to any one of the previous claims, characterized in that the hydrophilic amino acid at the position of SEQ ID NO: 1 corresponding to position 276 locates in an alpha helix. 
     
     
         10 . The beta-lactamase or the method according to any one of the previous claims, characterized in that the beta-lactamase further comprises at least one amino acid selected from the group consisting of Leu220 and Arg244 according to Ambler classification. 
     
     
         11 . The beta-lactamase or the method according to any one of the previous claims, characterized in that the beta-lactamase hydrolyses penicillins and/or cephalosporins. 
     
     
         12 . The beta-lactamase or the method according to any one of the previous claims, characterized in that the beta-lactamase has improved catalytic efficiency on cephalosporins compared to wild type beta-lactamases. 
     
     
         13 . The beta-lactamase or the method according to  claim 11  or  12 , characterized in that the cephalosporins are selected from the group consisting of cefoperazone, ceftriaxone and cefazoline. 
     
     
         14 . A method of producing the beta-lactamase according to any one of  claim 1  or  3 - 13 , characterized in that the method comprises the following steps:
 v) providing a gene encoding the beta-lactamase according to any one of  claim 1  or  3 - 13 ; 
 vi) transforming a host cell with the gene; 
 vii) obtaining a host cell that produces the beta-lactamase; 
 viii) recovering the beta-lactamase. 
 
     
     
         15 . A method of treating or preventing beta-lactam antibiotic induced adverse effects in the gastro-intestinal tract, characterized by administering beta-lactamase according to any one of  claim 1  or  3 - 13  simultaneously or sequentially with a beta-lactam antibiotic to a subject. 
     
     
         16 . A polynucleotide, characterized in that it comprises a sequence of any one of SEQ ID NO:s 2 or 4 or a degenerate thereof, or it encodes the beta-lactamase according to any one of  claim 1  or  3 - 13 . 
     
     
         17 . A host cell comprising the polynucleotide according to  claim 16 . 
     
     
         18 . A pharmaceutical composition comprising the beta-lactamase according to any one of  claim 1  or  3 - 13 . 
     
     
         19 . The beta-lactamase according to any one of  claim 1  or  3 - 13  for use as a medicament. 
     
     
         20 . Use of the beta-lactamase according to any one of  claim 1  or  3 - 13  in the manufacture of a medicament for treating or preventing beta-lactam antibiotics induced adverse effects in the gastro-intestinal tract. 
     
     
         21 . The method according to  claim 15  or the use according to  claim 20 , characterized in that the beta-lactamase(s) is administered orally. 
     
     
         22 . The method according to  claim 15  or the use according to  claim 20 , characterized in that the beta-lactamase(s) is administered directly to the gastro-intestine of a patient. 
     
     
         23 . The method according to any one of  claim 15  or  21 - 22  or the use according to any one of  claims 20 - 22 , characterized in that the beta-lactamase(s) and the beta-lactam antibiotic are administered together in the form of an enteric coated pellet to a subject.