Nan_2019_Mol.Biotechnol_61_892

A novel carboxylesterase gene, named dlfae4, was discovered and sequenced from a soil metagenomic library. The dlfae4 gene was composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2 kDa. DLFae4 exhibited strong hydrolytic activity towards methyl ferulate under optimum pH and temperature conditions (pH 8.6, 50 degrees C) and displayed remarkable thermostability, with residual activity as high as 50% after incubation for 3 h at 60 degrees C. A family VIII esterase DLFae4 was found to contain a typical serine residue within the S-X-X-K motif, which serves as a catalytic nucleophile in class C beta-lactamases and family VIII esterases. As a consequence of its high sequence similarity with beta-lactamases, DLFae4 exhibited significant hydrolytic activity towards ampicillin. In addition, DLFae4 was found to be the first known member of family VIII carboxylesterases with phthalate-degrading ability. Site-directed mutagenesis studies revealed that Ser11, Lys14, and Tyr121 residues play an essential catalytic role in DLFae4. These new findings, which are of great importance for further in-depth research and engineering development of carboxylesterases, should advance the implementation of biotechnological applications.