Improving the enzyme property of ornithine transcarbamylase from Lactobacillus brevis through site-directed mutation

Abstract Ornithine transcarbamylase (OTC) is an enzyme that participates in the degradation of arginine by catalyzing the formation of ornithine and carbamoyl phosphate from citrulline, which is a major precursor of ethyl carbamate (EC), a potential carcinogen in fermented foods and beverages. Prior research has not focused on the study of OTC, although it has been previously found to have a negative relationship with EC formation. In this study, nine mutants (H140A, H140W, Q143A, Q143F, Q143W, Q143Y, D236A, D236W, and D236R) were constructed by site-directed mutagenesis using the Discovery Studio software to increase the catalytic properties or thermal stability of the enzyme. Compared with the wild-type enzyme, the catalytic activity of Q143W and H140A mutants increased 2–3 times at the most. The optimal temperature decreased to 35 °C, and the optimal pH decreased to 8.5. Meanwhile Q143W and H140A also exhibited excellent ethanol tolerance, which indicated new possibilities for further application in wine fermentation. The thermostability of D236R was successfully enhanced with a 1.4-fold improvement of t1/2 and T5010 higher by 9.9 °C. It is expected that OTC will be transformed by site-directed mutation to achieve an increase in enzyme activity while also enhancing the thermal stability.