An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range

Abstract

A thermophilic compost metagenomic library constructed in Escherichia coli was functionally screened for novel esterases. Of the 110,592 fosmid clones screened, 25 clones demonstrated degradative activity on glyceryl tributyrate (a hit rate of 1:4,423). Four clones displayed ferulic acid esterase activity and were sequenced using 454 Titanium sequencing technology. EstG34, a 410 amino acid protein, was identified as having high sequence identity with a number of bacterial β-lactamases. EstG34 has the S-X-X-K motif which is conserved in class C β-lactamases and family VIII carboxylesterases. Purified recombinant EstG34 had a molecular mass of 42kDa and displayed hydrolytic activity towards a variety of p-nitrophenyl esters, hydroxycinnamic acid esters and α-naphthol acetate. EstG34 represents the first family VIII carboxylesterase and β-lactamase fold enzyme, able to hydrolyse ferulate and a number of other hydroxycinnamic acid esters. In addition, EstG34 is the first reported FAE to not adopt the α/β hydrolase conformation. The sequence similarity and wide substrate utilization capability of this esterase complicates its placement within current classification systems, but also draws attention to the enzyme's potential versatility.