• Korean Journal of Microbiology
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• v.39 no.4
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• pp.230-234
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• 2003

An extracellular protease of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine protease. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. When the enzyme was chemically modified with PMSF, which specifically reacted with serine residue on the enzyme, the activity was eliminated. The endopeptidase activity was inhibited by the modifier which chemically modified carboxyl group of aspartate and glutamate. PLP, which would modify lysine residue, did not affect the endopepetidase activity to a greater extent. This demonstrates that serine and aspartate (or glutamate) residues of enzyme would participate in a important function of the endopeptidase activity.

• Journal of Microbiology
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• v.43 no.3
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• pp.237-243
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• 2005

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, $60^{\circ}C$. The endopeptidase activity was stimulated by $Ca^{++},\;Co^{++},\;Mn^{++},\;Mg^{++},\;and\;Ni^{++}$, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM $Ca^{++}$, and was partially restored by $Co^{++}\;and\;Mn^{++}$, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of $Ca^{++}$ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the $K_m$ value of endopeptidase is 0.315 mM and $V_{max}$ is 0.222 ) is $0.222\;{\mu}mol$ of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.

• Applied Biological Chemistry
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• v.34 no.4
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• pp.334-338
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• 1991

Synthesis of $(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin by solid phase method using a new reaction vessel was carried out. Coupling was performed by dicyclohexylcarbodiimide. After cleavage with dried HBr the peptides were purified by high pressure liquied chromatography. Their purify was assayed by paper and thin layer chromatography, melting point and amino acid analysis. $(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin were incubater in vitro endopeptidase $({\alpha}-chymotrysis)$ and exopeptidase(carboxypeptidase A, leucine aminopeptidase) in order to study the degradation pattern of peptides. $(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin were rapidly degradated by ${\alpha}-chymotrypsin$ and carboxypeptidase A $(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin coution$(D-Phe^7\;-Leu^8)$ bradykinin and bradykinin contain imino peptide bound from proline at N-terminal and therefore they were not attacted by leucine aminopeptidase.

• Journal of Microbiology
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• v.41 no.1
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.