Hybrid alpha-amylases produced by the transformants of Bacillus subtilis. III. A possible mechanism of formation of hybird alpha-amylases.

Alpha-Amylases (NA64 and NA20) produced by the representative transformants Bacillus subtilis NA64 and NA20 were hybrid enzymes between the two parental alpha-amylases (NAT and MAR) produced by the DNA donor strain of Bacillus natto IAM 1212 and the DNA recipient strain of B. subtilis 6160, a derivative of B. subtilis 168. In order to elucidate a possible mechanism of formation of the hybrid alpha-amylases, 14C-labeled alpha-amylase (SAC) produced by B. subtilis var. amylosarcchariticus, [3H]lysine- and [3H]arginine-labeled alpha-amylases (MAR, NA64, NA20, NAT and SAC), [3H]lysine-labeled alpha-amylase (SAC) and [3H]glucosamine-labeled alpha-amylase (NA64) were purified through ammonium sulfate precipitation, carboxy-methylcellulose and DEAE-Sephadex A-50 column chromatography and immunoprecipitation with rabbit antiserum against alpha-amylase (SAC). Peptide compositions of the tryptic digests from the labeled alpha-amylases were analyzed by double-label AG 50W-X2 column chromatography. On the other hand, amino- and carboxy-terminal amino acid residues of unlabeled alpha-amylases (MAR, NA64, NA20 and NAT) were analyzed. Based on these results, the possibility of DNA recombination events in the alpha-amylase structure gene and on the previous results, we attempted to estimate possible peptide arrangements for the four alpha-amylases (MAR, NA64, NA20 and NAT) and possible recombination regions to form the hybrid enzymes introduced by the DNA-mediated transformation of B. subtilis 6160.