Role of Val289 Residue in the $\alpha$-Amylase of Bacillus amyloliquefaciens MTCC 610: An Analysis by Site Directed Mutagenesis

The Val289 residue in the α-amylase of Bacillus amyloliquefaciens, which is equivalent to the Ala289 and Val286 residues in the α-amylases of B. stearothermophilus and B. licheniformis, respectively, was studied by site-directed mutagenesis. This residue was substituted with 10 different amino acids by random substitution of the Val codon. In these mutant α-amylases, Val289 was substituted with Ile, Tyr, Phe, Leu, Gly, Pro, Ser, Arg, Glu, and Asp. Compared with the wild-type α-amylase, the mutant α-amylase Val289Ile showed 20% more hydrolytic activity, whereas Val289Phe and Val289Leu showed 50% lesser activity. On the other hand, the mutant α-amylases Val289Gly, Val289Tyr, Val289Ser, and Val289Pro showed less than 15% activity. The substitution of Val289 with Arg, Asp, or Glu resulted in complete loss of the α-amylase activity. Interestingly, the mutant α-amylase Val289Tyr had acquired a transglycosylation activity, which resulted in the change of product profile of the reaction, giving a longer oligosaccharide.