CLONING AND SEQUENCING OF THE α-AMYLASE GENE FROMBACILLUS SUBTILISUS116 STRAIN ENCODING AN ENZYME CLOSELY IDENTICAL TO THAT FROMBACILLUS AMYLOLIQUEFACIENSBUT DISTINCT IN THERMAL STABILITY

Abstract

The gene encoding for the α-amylase AMYUS116 was cloned and sequenced. The amino acid sequence of AMYUS116 exhibited an almost perfect homology with the α-amylase BACAAM, excluding the residues N205 and N217 of AMYUS116 that were changed to H205 and I217 into BACAAM. Three mutant derivatives from AMYUS116 (N205H, N217I and N205H/N217I) were created by site-directed mutagenesis and their physicochemical and kinetic properties were compared with those of the wild-type enzymes. Therefore, the undertaken amylases mainly generated maltohexaose from starch and had radically the same kinetic parameters and optimum pH and temperature. They, however, were significantly distinct in thermal stability; AMYUS116 was more thermosensible as its half-life time at 80C was 13 min, while those of BACAAM and the double mutant were likewise 38 min. The single-mutant amylases exhibited an identically intermediate thermal stability as their half-life times at 80C were roughly 22 min. PRACTICAL APPLICATIONS Of particular interest to the current search is that the different thermal stability between AMYUS116 and BACAAM can lead to novel findings pertaining to protein stability, which can bring about new strategies for protein engineering. Basically, the comparative study of closely related amylases and the protein engineering of already existing ones are certainly important because they offer opportunities to understand the structure–function relationships of these biocatalysts.