Abstract

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^oC. 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 mmol of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.

• Keywords: Bacillus cereus; leucine endopeptidase; Ca^+^+-activated protease