Modification of cis-epoxysuccinic acid hydrolase by random mutation combined with semi-rational design

Abstract:

[Background] D(−)-tartaric acid is a non-natural organic acid, which has great application potential in health care products, food, and tumor drug synthesis. At present, D(-)-tartaric acid is produced from cis-epoxysuccinic acid (ESH) hydrolyzed by cis-epoxysuccinic acid hydrolase[CESH(D)] through biotransformation. This method is simple and mild but has bottleneck problems such as low enzyme conversion efficiency of CESH(D). [Objective] To improve the enzyme activity, temperature, and pH stability of CESH(D) through genetic engineering. [Methods]Directional evolution and semi-rational design were used to modify CESH(D) in vitro to screen out forward mutants with high throughput. Then the enzymatic properties were studied, including the enzyme activity, the influence of temperature and pH on the catalytic efficiency of the enzyme, the temperature and pH stability of the enzyme, and enzymatic kinetics analysis. Finally, the primary mechanism of mutation sites affecting catalytic activity was analyzed using molecular docking. [Results] Four positive mutants L231P/N226S, V77I, D183E, and T223S were screened out. The specific enzyme activity of the four mutants was increased by 2.2, 1.6, 1.5, and 1.4 folds of the wild-type enzyme, respectively. The temperature stability and pH stability of L231P/N226S mutant were significantly higher as compared with the wild-type CESH(D), and its catalytic activity was 1.6 folds at 55℃, and 1.2 folds at pH 6.0 as compared with the wild-type enzyme, respectively. Kinetic analysis showed that the affinity of L231P/N226S mutant and T223S mutant to ESH substrates was significantly increased, with K_m values of 20 mmol/L and 21 mmol/L, respectively, which were 18% and 16% lower than that of the wild-type enzyme, respectively. Finally, the results of molecular docking showed that the mutated site promoted the interaction between enzyme and substrate mainly by changing the substrate binding pocket, thus affecting enzyme activity. [Conclusion] Through experiments, CESH(D) mutants with good temperature and pH stability and significantly improved catalytic activity were obtained. The mechanism of mutation site affecting enzyme activity is preliminarily analyzed in this research, which lays a foundation to study the relationship between the structure and function of CESH(D) and its further improvement.