Domain structure and multiplicity of raw-starch-digesting amylase from Bacillus circulans: extensive proteolysis with proteinase K, endopeptidase Glu-C and thermolysin.
Raw-starch-digesting amylase (RSDA) is a key extracellular enzyme of mesophilic Bacillus circulans F-2 which uses raw starch granules as a carbon source. Previous work has demonstrated that there are two domains of the enzyme during digestion with subtilisin, and that RSDA activity is selectively inactivated by limited proteolysis with subtilisin, which cleaves the enzyme between these hydrolytic and adsorption domains (Kim, C.-H., Kwon, S.-T., Taniguchi, H. and Lee, D.-S. (1992) Biochim. Biophys. Acta 1122, 243-250). In this work we show that a similar phenomenon is observed during limited proteinase K, thermolysin and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63 and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the proteinase-sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive towards anti-RSDA were detected through whole bacterial cultivation. 93, 75, 63, 55, 38 and 31-kDa proteins were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results suggest that enzyme heterogeneity of the raw-starch hydrolysis system might arise from the endogenous proteolytic activity of the bacterium.
Kim
C H
CH
Genetic Resources Center, Genetic Engineering Research Institute, K.I.S.T., Taejon, South Korea.
Kho
Y H
YH
D000681:Amylases / Q000737:chemistry* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D019286:Endopeptidase K; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D012697:Serine Endopeptidases; D013213:Starch / Q000378:metabolism; D013820:Thermolysin
D010446:Peptide Fragments; D013213:Starch; D000681:Amylases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D019286:Endopeptidase K; D013820:Thermolysin