Enhancement of hydrolytic activity of thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 through optimization of amino acid residues surrounding the substrate binding site

• AmyL with N-terminal starch binding domain belongs to GH family 13.
• AmyL has high tolerance to high temperature, high pH, chelators, and detergents.
• Y426S/K549M mutation in AmyL increased activity by 340%.
• Y426S/K549M retained high stability at high temperature and pH as the wild type.
• Y426S/K549M was stable in the presence of 1 mg/ml of EDTA and SDS as the wild type.

The hydrolytic activity of a thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 (AmyL) toward soluble starch was enhanced through optimization of amino acid (aa) residues situated near the substrate binding site. Twenty-four selected aa residues were replaced with Ala, and Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of AmyL's aa sequence with related enzymes. Y426A, H431A, I509A, and K549A showed notably higher activity than the wild type at 162–254% of wild-type activity. Tyr426, His431, and Ile509 were predicted to be located near subsite −2, while Lys549 was near subsite +2. Ser, Ala, Ala, and Met were found to be the best aa residues for the positions of Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single mutations at distant positions were effective in enhancing catalytic activity. The double-mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of the selected single mutations, showed 340%, 252%, and 271% of wild type activity, respectively. Triple and quadruple-mutant enzymes of the selected mutations did not show higher activity than the best double-mutant, Y426S/K549M.