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

The hydrolytic activity of a thermophilic alkalophilic alpha-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 wildtype 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. (C) 2014 Elsevier B.V. All rights reserved.