Improvement of thermostability of cholesterol oxidase from streptomyces Sp. SA-COO by random mutagenesis.

Autor: Fazaeli A; Department of Clinical Biochemistry, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran., Fana SE; Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Student Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran., Golestani A; Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran., Aminian M; Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Electronic address: amminian@tums.ac.ir.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2022 Mar; Vol. 191, pp. 106028. Date of Electronic Publication: 2021 Dec 01.
DOI: 10.1016/j.pep.2021.106028
Abstrakt: To enhance the thermal stability of Streptomyces Sp. SA-COO cholesterol oxidase, random mutagenesis was used. A random mutant library was generated using two types of error-prone PCR (single step and serial dilution) and two mutants (ChOA-M1 and ChOA-M2) with improved thermostability were obtained. The best mutant ChOA-M1 acquired three amino acid substitutions (G49T, W52K, and F62V) and improved thermostability (at 50 °C for 5 h) by 40% and increased the kcat/Km value by 23%. The optimum pH was desirably changed to encompass a broad range from alkali to acid and circular dichroism revealed no significant secondary structure changes in mutants against wild type. These findings indicated that random mutagenesis was an effective technique for optimizing cholesterol oxidase properties and make a foundation for practical applications of Cholesterol oxidase in clinical diagnosis and industrial fields.
(Copyright © 2021. Published by Elsevier Inc.)
Databáze: MEDLINE
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