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.
D000596:Amino Acids / Q000032:analysis; D000681:Amylases / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology*; D002845:Chromatography; D005944:Glucosamine / Q000378:metabolism; D006824:Hybridization, Genetic; D008239:Lysine / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000378:metabolism; D011817:Rabbits / Q000276:immunology; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D011506:Proteins; D001120:Arginine; D000681:Amylases; D014357:Trypsin; D008239:Lysine; D005944:Glucosamine