Johan_2022_Int.J.Biol.Macromol__

Carboxylesterases are attractive biocatalysts for various industrial applications, especially hyperthermophilic carboxylesterases, due to their high tolerance towards extreme environments. Such ability confers many advantages, including cost-effectiveness and an increased manufacturing rate. In the current work, we first described the characterization of EstD9, a new carboxylesterase from thermophilic Anoxybacillus geothermalis D9, with a hyper-thermostability characteristic. In this study, the enzyme was expressed, purified and characterised. Additionally, the three-dimensional structure was predicted using Robetta software. Sequence analysis of EstD9 revealed a significant identity (80 %) with thermophilic carboxylesterase Est30 and a typical catalytic triad of carboxylesterase, composed of Ser93-His22-Asp193. As the protein sequence contained a highly conserved pentapeptide (GLSLG), carboxylesterase of A. geothermalis D9 could be proposed as a new member of family XIII. The putative carboxylesterase was recombinantly expressed in E. coli BL21 (DE3) with a molecular mass of 28 kDa and successfully purified via Ni(2+)-Sepharose affinity chromatography with recovery of 88.36 %. Using p-nitrophenyl butyrate (C4), EstD9 presented excellent stability at high temperature range (70 degreesC-100 degreesC) for 5 days of incubation and has broad pH tolerance (pH 6-9), with maximal activity at 80 degreesC and pH 7. Notably, EstD9 activity was stimulated in the presence of 1-propanol and DMSO with 107.8 % and 108.9 % relative activities, respectively. The purified EstD9 maintained 60 % residual activity after 30 min exposure to various surfactants and metal ions. Additionally, the inhibition studies demonstrated that EstD9 was strongly deactivated by phenylmethylsulfonyl fluoride, dithiothreitol, and beta-mercaptoethanol. Circular dichroism (CD) spectra analysis predicted Tm value at 72.12 degreesC. Unlike other carboxylesterases, in silico 3D model of EstD9 disclosed a topological alpha/beta hydrolase fold with a small alpha-helix cap below catalytic region. The enzymatic properties exhibited by EstD9 suggested this enzyme to be a highly suitable catalyst for industrial bioprocesses under harsh conditions.