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

Autor: Fang J; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China., An L; Medical and Health Analysis Center, Peking University, Beijing 100191, China., Yu J; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China., Ma J; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China., Zhou R; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China., Wang B; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases and Key Laboratory of Biomedicine in Gene Diseases and Health of Anhui Higher Education Institutes, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China; Auhui Provincial Engineering Research Centre for Molecular Detection and Diagnostics, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China. Electronic address: wangbaojuan@ahnu.edu.cn.
Jazyk: angličtina
Zdroj: Journal of bioscience and bioengineering [J Biosci Bioeng] 2024 Sep; Vol. 138 (3), pp. 181-187. Date of Electronic Publication: 2024 Jun 12.
DOI: 10.1016/j.jbiosc.2024.05.001
Abstrakt: 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 °C, pH 9.0 and excellent catalytic performances (K m  = 0.263 mM, k cat /K m  = 562.3 s -1  mM -1 for p-nitrophenyl acetate (pNPA 2 ) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstF P144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstF P144G displayed a 5.10-fold increase of half-life at 100 °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 EstF P144G 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 enzymes.
(Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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