Characterization and Nanoparticle Immobilization of a Thermotogaceae Esterase Active on Aromatic and Aliphatic Esters

Esterases are a class of enzymes that catalyze the hydrolysis of carboxylic ester bonds and are widely utilized in the food, pharmaceutical, and detergent industries. In this study, we report the identification and comprehensive characterization of a novel esterase (TbEst) from the Thermotogaceae family, a group of thermophilic bacteria. The predicted three-dimensional structure of TbEst displays a classical α/β-hydrolase fold with a conserved Ser-His-Asp catalytic triad. TbEst was cloned, heterologously expressed, and purified via a multistep chromatographic protocol. The melting temperature of TbEst at pH 8.0 was 78 °C, confirming its thermostability. Furthermore, TbEst exhibited activity against p-nitrophenyl acetate, confirming its esterase activity, with optimal performance at pH 8.0 and 50 °C. Notably, TbEst exhibited catalytic activity toward broad aromatic and aliphatic esters, reinforcing its versatility for diverse biotechnological applications. Furthermore, the present study describes the successful nanoscale immobilization of TbEst onto Ag/AgCl nanoparticles (TbEst@Ag/AgCl-NPs) via a facile photochemical route. TbEst@Ag/AgCl-NPs were highly stable, with different geometries including spherical and cubic; it was crystalline in nature, and had a mean diameter of 100 nm. Notably, TbEst retained its catalytic activity upon nanoscale immobilization onto Ag/AgCl-NPs. Furthermore, nanoscale immobilization conferred enhanced thermostability and significantly prolonged the active lifetime of the bioconjugate compared to the free enzyme. The data described here should provide a useful basis for future research and biotechnological applications of esterases and nanoimmobilized esterases.