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 screenedfor novel esterases. Of the 110,592 fosmid clones screened, 25 clones demonstrated degradative activ-ity on glyceryl tributyrate (a hit rate of 1:4,423). Four clones displayed ferulic acid esterase activityand were sequenced using 454 Titanium sequencing technology. EstG34, a 410 amino acid protein, wasidentified as having high sequence identity with a number of bacterial -lactamases. EstG34 has theS-X-X-K motif which is conserved in class C -lactamases and family VIII carboxylesterases. Purifiedrecombinant EstG34 had a molecular mass of 42 kDa and displayed hydrolytic activity towards a vari-ety of p-nitrophenyl esters, hydroxycinnamic acid esters and -naphthol acetate. EstG34 represents thefirst family VIII carboxylesterase and -lactamase fold enzyme, able to hydrolyse ferulate and a numberof other hydroxycinnamic acid esters. In addition, EstG34 is the first reported FAE to not adopt the / hydrolase conformation. The sequence similarity and wide substrate utilization capability of this esterasecomplicates its placement within current classification systems, but also draws attention to the enzyme’spotential versatility.