HYPEREXPRESSION OF THE GENE FOR A BACILLUS ALPHA-AMYLASE IN BACILLUS-SUBTILIS CELLS - ENZYMATIC-PROPERTIES AND CRYSTALLIZATION OF THE RECOMBINANT ENZYME

We have constructed a new excretion vector, pHSP64, to develop a hyper excretion system for Bacillus subtilis [Sumitomo et al., Biosci, Biote ch, Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel l iquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus s p, KSM-1378 was amplified by PCR, It was cloned into a Sal I-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 1 0(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physicochemical an d catalytic properties were observed between the recombinant enzyme an d the native enzyme produced by Bacillus sp. KSM-1378, The enzymatic p roperties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffere d solution of 10% (w/v) ammonium sulfate (pH 6.5).