title: Functional characterisation of a metagenome derived family VIII esterase with a deacetylation activity on β-lactam antibiotics.
abstract: Family VIII esterases represent a poorly characterised esterase family, with high sequence identity to class C β-lactamases, peptidases and penicillin binding proteins. This study reports on the metagenomic library 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 22 aa signal peptide. The Est22 primary structure revealed the presence of N-terminus S-x-x-K sequence, which is also highly conserved in class C β-lactamases, peptidases as well as carboxylesterases belonging to family VIII. Phylogenetic analysis using the representative sequences from class C β-lactamases and family VIII esterases indicated that Est22 is a member of family VIII esterases. Substrate specificity profiling using p-nitrophenyl esters (C2-C16) indicated that Est22 preferred shorter chain p-nitrophenyl esters (C2-C5), a characteristic that is typical for true carboxylesterases. In addition of hydrolysing Nitrocefin, Est22 also hydrolysed first generation cephalosporin based derivatives. Detailed selectivity study using different cephalosporin based substrates indicated that Est22 selectively hydrolyse the ester bond of a cephalosporin derivatives leaving the amide bond of the β-lactam ring intact. The selective nature of Est22 makes this enzyme a potential candidate for the use in the synthesis and modification cephalosporin based molecules.
authors.affiliation: CSIR Biosciences, Scientia Campus, P.O. Box 395, Pretoria 0184, South Africa.
authors.forename: Nobalanda
authors.initials: N
authors.lastname: Mokoena
authors.forename: Kgama
authors.initials: K
authors.lastname: Mathiba
authors.forename: Tsepo
authors.initials: T
authors.lastname: Tsekoa
authors.forename: Paul
authors.initials: P
authors.lastname: Steenkamp
authors.forename: Konanani
authors.initials: K
authors.lastname: Rashamuse
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D005798:Genes, Bacterial; D054892:Metagenome / Q000235:genetics*; D008969:Molecular Sequence Data; D011897:Random Allocation; D013379:Substrate Specificity / Q000235:genetics; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D047090:beta-Lactams; D002265:Carboxylic Ester Hydrolases; D001618:beta-Lactamases
keywords: Family VIII carboxylesterase; Functional metagenomics; β-Lactam substrate