title: Processing of an NH2-terminally extended thermostable alpha-amylase by Bacillus subtilis alkaline protease.
abstract: To analyze the processing of extracellular enzymes of Bacillus subtilis, an NH2-terminally extended hybrid alpha-amylase [pTUBE638-alpha-amylase (E24)] was purified from the periplasm of E. coli(pTUBE638) as the substrate for the in vitro processing reaction, in which a 21-amino-acid extra-peptide was added at the NH2-terminus of the mature thermostable alpha-amylase. The extended peptide in pTUBE638-alpha-amylase (E24) was completely processed by the extracellular alkaline protease of B. subtilis alone at pH 7.5 to 10.0. The processing was inhibited by 2 mM PMSF. In contrast, the neutral protease did not process the extended peptide. The processing activity of the purified alkaline protease was fully active in 100 mM phosphate and glycine-NaCl-NaOH buffer while it was partially active in 100 mM Tris-HCl or MOPS buffer. The optimum pH of the activity ranged from 8.0 to 9.0, although the optimum pH of the alkaline protease activity toward casein and Azocoll was 10.5. The NH2-terminal amino acid sequences of the enzymes processed in vitro coincided with those of the mature extracellular thermostable alpha-amylases in the culture medium of B. subtilis (pTUBE638). The appearance of the processing activity of alkaline protease was correlated with the changes of the pH in the culture medium.
authors: Institute of Biological Sciences, University of Tsukuba, Ibaraki.
authors: Y
authors: Y
authors: Itoh
authors: M
authors: M
authors: Sumi
authors: K
authors: K
authors: Nakamura
authors: K
authors: K
authors: Yamane
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology*; D010450:Endopeptidases / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D000516:alpha-Amylases / Q000378:metabolism*
chemical_list: D011480:Protease Inhibitors; D000516:alpha-Amylases; D010450:Endopeptidases