Biochemical characterization of an esterase from Thermobifida fusca YX with acetyl xylan esterase activity

Abstract

Background Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fne industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. Methods and results In this study, a gene encoding an esterase from Thermobifda fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purifed using afnity chromatography. The TfEst kinetic assay revealed catalytic efciencies of 0.58 s −1 mM−1, 1.09 s−1 mM−1, and 0.062 s−1 mM−1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. Conclusions The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with signifcant implications for the advancement of biotechnology and biofuels industries.