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

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 degrees 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.

Automated summary

An improved variant of cholesterol oxidase with enhanced thermostability was obtained through error-prone PCR. This mutant showed 30% increased thermostability at 50 degrees C for 5 h compared to the wild type, without changing the optimum temperature and pH. Circular dichroism analysis revealed no significant structural changes in the mutant enzymes. These findings indicate that error-prone PCR is an effective method for enhancing enzyme characteristics and provide a platform for the practical use of cholesterol oxidase in industrial and clinical settings.