A novel cold-adapted esterase from Enterobacter cloacae: Characterization and improvement of its activity and thermostability via the site of Tyr193Cys

Abstract Background In industries lipolytic reactions occur in insensitive conditions such as high temperature thus novel stout esterases with unique properties are attracts to the industrial application. Protein engineering is the tool to obtain desirable characters of enzymes. A novel esterase gene was isolated from South China Sea and subjected to a random mutagenesis and site directed mutagenesis for higher activity and thermo-stability compared to wild type. Results A novel esterase showed the highest hydrolytic activity against p-nitrophenyl acetate (pNPA, C2) and the optimal activity at 40 °C and pH 8.5. It was a cold-adapted enzyme and retained approximately 40% of its maximum activity at 0 °C. A mutant, with higher activity and thermo-stability was obtained by random mutagenesis. Kinetic analysis indicated that the mutant Val29Ala/Tyr193Cys shown 43.5% decrease in K m, 2.6-fold increase in K cat, and 4.7-fold increase in K cat /K m relative to the wild type. Single mutants V29A and Y193C were constructed and their kinetic parameters were measured. The results showed that the values of K m, K cat, and K cat /K m of V29A were similar to those of the wild type while Y193C showed 52.7% decrease in K m, 2.7-fold increase in K cat, and 5.6-fold increase in K cat /K m compared with the wild type. The 3-D structure and docking analysis revealed that the replacement of Tyr by Cys could enlarge the binding pocket. Moreover Y193C also showed a better thermo-stability for the reason its higher hydrophobicity and retained 67% relative activity after incubation for 3 h at 50 °C. Conclusions The superior quality of modified esterase suggested it has great potential application in extreme conditions and the mutational work recommended that important information for the study of esterase structure and function.

Abstract licence: CC BY-NC-ND

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.