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

Area of Science:

• Biotechnology
• Enzyme Engineering

Background:

• Cholesterol oxidase (ChOS) is crucial for biosensors in food, agriculture, and diagnostics.
• Limited thermostability of natural enzymes restricts their industrial application.
• Enzyme engineering is vital for developing robust biocatalysts.

Purpose of the Study:

• To enhance the thermostability of Chromobacterium sp. DS1 cholesterol oxidase (ChOS) using random mutagenesis.
• To identify mutations responsible for improved thermal resistance.
• To evaluate the potential of engineered ChOS for industrial applications.

Main Methods:

• Construction of a random mutant library of ChOS using two forms of error-prone PCR.
• Screening for mutants with enhanced thermostability.
• Characterization of the best mutant (ChOS-M) including amino acid substitutions and stability assays.
• Analysis of secondary structural changes using circular dichroism.

Main Results:

• An improved ChOS variant (ChOS-M) with enhanced thermostability was obtained.
• ChOS-M exhibited a 30% increase in thermostability at 50°C for 5 hours.
• The mutant possessed three key amino acid substitutions: S112T, I240V, and A500S.
• Optimal temperature and pH remained unchanged, and no significant secondary structural alterations were observed.

Conclusions:

• Error-prone PCR is an effective strategy for improving enzyme thermostability.
• The engineered ChOS-M variant offers a promising platform for thermal-resistant enzyme applications.
• This work facilitates the practical use of ChOS in industrial fields and clinical diagnostics.