Biochemical characterization of an acetylesterase from and its application for 7-aminocephalosporanic acid deacetylation.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA), which could be converted from 7-aminocephalosporanic acid (7-ACA), is a crucial starting material that is used for synthesizing industrial semisynthetic β-lactam antibiotics. Enzymes involved in the conversion from 7-ACA to D-7-ACA present critical resources in the pharmaceutical industry. In the present study, a putative acetylesterase, EstSJ, identified from KATMIRA1933, was first heterologously expressed in BL21(DE3) cells and biochemically characterized. EstSJ belongs to carbohydrate esterase family 12 and is active on short-chain acyl esters from -NPC to -NPC. Multiple sequence alignments showed that EstSJ was also an SGNH family esterase with a typical GDS(X) motif at its N-terminal end and a catalytic triad composed of Ser-Asp-His. The purified EstSJ displayed the highest specific activity of 1,783.52 U mg at 30°C and pH 8.0, and was stable within the pH range of 5.0-11.0. EstSJ can deacetylate the C3' acetyl group of 7-ACA to generate D-7-ACA, and the deacetylation activity was 4.50 U mg. Based on the structural and molecular docking with 7-ACA, the catalytic active sites (Ser-Asp-His) together with four substrate-binding residues (Asn, Arg, Thr, and Leu) of EstSJ are revealed. This study provided a promising 7-ACA deacetylase candidate that could be applied to produce D-7-ACA from 7-ACA in the pharmaceutical industry.