Discovery and characterization of an enantioselective family VIII esterase from effluent treatment plant sludge metagenome.

This study reports on the biochemical characterization of a novel esterase (EstN3) belonging to class C β-lactamases of family VIII esterases from the functional screening of an Effluent Treatment Plant (ETP) sludge metagenome library. The enzyme is 410 amino acids long and does not contain a signal peptide. It showed maximum amino acid sequence similarity with uncharacterized serine hydrolases from Phenylobacterium and Caulobacter species, suggesting that it is a member of family VIII esterase. EstN3’s primary structure contains SxxKs, YSx, KTG, PLGMxDTxF, LxxxPGxxW, and GGxG motifs observed in class C β-lactamases, peptidases, and carboxylesterases of family VIII. This supports its designation as a class C β-lactamase. EstN3 favored shorter-chain p-nitrophenyl esters (C2-C6) based on substrate specificity profiling with p-nitrophenyl esters (C2-18). EstN3 exhibited excellent stereoselectivity in the production of S-mandelic acid under aqueous hydrolytic conditions. We have discovered that EstN3, a family VIII esterase, shows enantioselectivity towards methyl mandelate. The novel esterase was identified from the ETP sludge, indicating that unexplored environments serve as rich reservoirs for the discovery of novel enzymes with unique properties, offering valuable opportunities for advancing biocatalysis and industrial biotechnology.