Functional characterisation of a metagenome derived family VIII esterase with a deacetylation activity on ß-lactam antibiotics

Abstract

Family VIII esterases represent a poorly characterised esterase family, with high sequence identity to class C b-lactamases, peptidases and penicillin binding protein. In this study we report on the metagenomic screening and biochemical characterisation of a novel esterase (Est22) derived from an acidic Leachate environment. The enzyme is 423 amino acids in length and contained 22aa signal peptide. Analysis of the Est22 primary structure revealed the presence of N-terminus S-x-x-K sequence, which is highly conserved in class C ß-lactamases, peptidases as well as carboxylesterases belonging to family VIII. Phylogenetic analysis using representative sequences from class C ß-lactamases and family VIII esterases indicated that Est22 clustered mainly with family VIII esterases. Substrate specificity profiling using p-nitrophenyl esters (C2-16) indicated that Est22 preferred shorter chain p-nitrophenyl esters (C2-C5), a characteristic typical of true carboxylesterase. In addition of hydrolysing nitrocefin, Est22 also hydrolysed first generation cephalosporin derivatives. Detailed selectivity study using cephalosporin revealed that Est22 selectively hydrolyse the ester bond of a cephalosporin derivatives leaving the amide bond of the ß-lactam ring intact. The selective nature of Est22 makes this enzyme potential candidate for use in the synthesis and modification cephalosporin based molecules.