Characterization of a novel carboxylesterase from Streptomyces lividans TK24 and site-directed mutagenesis for its thermostability

As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 degrees C, pH 9.0 and excellent catalytic performances (Km = 0.263 mM, kcat/Km = 562.3 sL1 mML1 for p-nitrophenyl acetate (pNPA2) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 degrees C and 2.86 h at 100 degrees C. Furthermore, the EstF was modified to obtain EstFP144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstFP144G displayed a 5.10-fold increase of half-life at 100 degrees C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstFP144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other (c) 2024, The Society for Biotechnology, Japan. All rights are reserved, including those for text and data mining, AI training, and similar technologies.