Directed evolution of cholesterol oxidase with improved thermostability using error-prone PCR.
Cholesterol oxidase
Error-prone PCR
Thermostability
Journal
Biotechnology letters
ISSN: 1573-6776
Titre abrégé: Biotechnol Lett
Pays: Netherlands
ID NLM: 8008051
Informations de publication
Date de publication:
Sep 2023
Sep 2023
Historique:
received:
15
06
2022
accepted:
19
05
2023
revised:
14
04
2023
medline:
18
8
2023
pubmed:
8
6
2023
entrez:
8
6
2023
Statut:
ppublish
Résumé
Cholesterol oxidase is industrially important as it is frequently used as a biosensor in food and agriculture industries and measurement of cholesterol. Although, most natural enzymes show low thermostability, which limits their application. Here, we obtained an improved variant of Chromobacterium sp. DS1 cholesterol oxidase (ChOS) with enhanced thermostability by random mutant library applying two forms of error-prone PCR (serial dilution and single step). Wild-type ChOS indicated an optimal temperature and pH of 70 ºC and pH 7.5, respectively. The best mutant ChOS-M acquired three amino acid substitutions (S112T, I240V and A500S) and enhanced thermostability (at 50 °C for 5 h) by 30%. The optimum temperature and pH in the mutant were not changed. In comparison to wild type, circular dichroism disclosed no significant secondary structural alterations in mutants. These findings show that error-prone PCR is an effective method for enhancing enzyme characteristics and offers a platform for the practical use of ChOS as a thermal-resistance enzyme in industrial fields and clinical diagnosis.
Identifiants
pubmed: 37289346
doi: 10.1007/s10529-023-03401-y
pii: 10.1007/s10529-023-03401-y
doi:
Substances chimiques
Cholesterol Oxidase
EC 1.1.3.6
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1159-1167Subventions
Organisme : Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences
ID : 30859
Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer Nature B.V.
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