Hotspot-based mutation engineering of MAase from Lactobacillus rhamnosus YXY412 for the improvement of hydrolytic activity

The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates. Here, the specific activity of a maltogenic amylase from Lactobacillus rhamnosus YXY412 (LrMA) toward soluble starch was exactly enhanced through hotspot-based research. Based on multiple sequence alignment, three-dimensional structure and existed literature, thirty-eight amino acid residues of LrMA were rationally selected for site-directed mutagenesis. After the screening of the mutants, LrMA^D172A, LrMA^G260A, LrMA^K334A and LrMA^M477A were selected with the activity accounted for 144–209% of that in wild-type. Among all the mutants, LrMA^G260A possessed the highest activity toward soluble starch, reached 133 U/mg, about twice as high as that in the wild-type. Its temperature for optimum activity still maintained at 60 °C, while had no significant loss of thermal stability occurred. In addition, compared with the wild-type in pH stability, the mutant retained over 80% residual activity at a wider pH range of 4.5–8.5. Furthermore, the k_cat/K_m of LrMA^G260A was two times higher than that of the wild-type, indicating that the mutant had a better affinity and a higher conversion efficiency for soluble starch.