Abstract
A Bacillus subtilis strain, 207-SV1, producing thermostable α-amylase was constructed by transformation as reported previously using a nonhomologous DNA prepared from an unidentified and Gram negative thermophilic bacterium, Thermophile V2. In the study here, enzymatic characterization of an extracellular thermostable α-amylase was conducted in comparison with B. subtilis α-amylase. Both α-amylases were found to be equally insensitive to 5mM EDTA but differed from each other in thermostability, optimum temperature, electrophoretic mobility on polyacrylamide gel, and immunological specificity. These results suggest that the structure of the thermostable α-amylase was different from that of the B. subtilis α-amylase. The structural gene for the thermostable α-amylase was named amyV2+. PBS1 phage mediated transduction and DNA mediated transformation studies with 207-SV1 and its derivative strains suggested that the chromosomal order around amyV2+ was recA-pyrA-cysC-amyV2-metC. Since the amyV2+ locus differs from that of amyE+, DNA from B. subtilis strain B7, which has a tunicamycin-resistant mutation (tmr7) located close to amyE, was transferred into strain 207-SV1, and transformants producing both thermostable α-amylase and B. subtilis α-amylase were isolated.
