Improvement of enzymatic properties of Rhizopus oryzae α-amylase by site-saturation mutagenesis of histidine 286.

Optimal pH and ideal functioning temperature for fungal α-amylase can greatly contribute to improving enzyme efficiency in maltose-forming ability. This work aimed to improve the enzymatic properties of Rhizopus oryzae α-amylase by site-saturation mutagenesis of histidine 286. The biochemical properties of selected mutant enzymes were modified to increase their enzymatic efficiencies compared to their wild-type counterparts. For instance, the optimum temperature of mutants H286 L, H286I, H286S and H286 T was increased from 50 °C to 55 °C, while a similar increase was observed for H286 P from 50 °C to 60 °C. The optimum pH of mutants H286 L, H286I and H286D shifted from 5.5 to 5.0, and the optimum pH of mutant H286E shifted from 5.5 to 4.5. The results obtained showed that the mutant H286I showed a 1.5-fold increase in half-life at 55 °C and the mutant H286E showed a 6.43-fold increase in half-life at a pH of 4.5. Furthermore, the ability to form maltose from soluble starch for mutants H286 L and H286 M was significantly improved under the optimum conditions determined in the study. The catalytic mechanism responsible for improved maltose-forming ability was confirmed through molecular docking simulations with maltotriose among wild-type and mutant enzymes. The mutants with improved enzymatic properties that were attained in this work may help in future computer-aided directed evolution of fungal α-amylase.