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.

Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions

“…The similar thermostability of the single-substitution mutant enzymes, on the one hand, and the higher stability of the double mutant-mutated enzyme, on the other, suggest that each substitution contributed independently to the overall stability of the enzyme as was previously observed for stabilizing mutations in protein structures (Ben Messaoud et al, 2010;Declerck et al, 1997).…”

The optimized production, purification, characterization, and application in the bread making industry of three acid‐stable alpha‐amylases isoforms from a new isolated Bacillus subtilis strain US586

“…Bacterial amylases were chosen over other origins because of their specific properties (Goesaert et al, ) including an optimum acidic pH and an intermediate thermostability (Sharma & Satyanarayana, ). B. subtilis strains, were considered as an important cell factory producing α‐amylase (Messaoud, Mabrouk, Jemli, & Bejar, ; Villaverde & Miralles, ) and are endotoxin‐free (Jan & Michael, ; Liu et al, ). The microbial amylase production level could be greatly enhanced by the selection of proper media components and appropriate cultural parameters.…”

“…B. subtilis strains, were considered as an important cell factory producing α-amylase (Messaoud, Mabrouk, Jemli, & Bejar, 2010;Villaverde & Miralles, 2013) and are endotoxin-free (Jan & Michael, 2013;Liu et al, 2013). The microbial amylase production level could be greatly enhanced by the selection of proper media components and appropriate cultural parameters.…”