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Directed evolution of cholesterol oxidase with improved thermostability using error-prone PCR

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Abstract

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.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Funding

This study was funded by Tehran University of Medical Sciences (Grant Number 30859).

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Author notes

  1. Saeed Ebrahimi Fana and Aliakbar Fazaeli have contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, P.O. Box: 14155–6447, Tehran, Iran

    Saeed Ebrahimi Fana & Mahdi Aminian

  2. Student Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran

    Saeed Ebrahimi Fana

  3. Department of Clinical Biochemistry, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran

    Aliakbar Fazaeli

Authors
  1. Saeed Ebrahimi Fana
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  2. Aliakbar Fazaeli
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  3. Mahdi Aminian
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Contributions

Study conception and design performed by MA. Investigation, material preparation, data collection and analysis were performed by AF, MA and SEF. The original draft of the manuscript was written by SEF and all authors commented on previous versions of the manuscript. All authors read, edited and approved the final manuscript.

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Correspondence to Mahdi Aminian.

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The authors have no relevant financial or non-financial interests to disclose.

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This study was reviewed and approved by the Ethics Committee of the Tehran University of Medical Sciences (IR.TUMS.REC.1395.2376).

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Ebrahimi Fana, S., Fazaeli, A. & Aminian, M. Directed evolution of cholesterol oxidase with improved thermostability using error-prone PCR. Biotechnol Lett 45, 1159–1167 (2023). https://doi.org/10.1007/s10529-023-03401-y

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  • DOI: https://doi.org/10.1007/s10529-023-03401-y

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