Characterization of calcium-activated bifunctional peptidase of the psychrotrophic Bacillus cereus.
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 degrees 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 Km value of endopeptidase is 0.315 mM and Vmax 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.
Kim
Jong-Il
JI
Department of Food and Microbial Technology Seoul Women's University 126 Kongnung 2-dong, Nowon-Gu, Seoul 139-774, Republic of Korea. jikim@swu.ac.kr
Lee
Sun-Min
SM
Jung
Hyun-Joo
HJ
D001409:Bacillus cereus / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D003080:Cold Temperature; D010450:Endopeptidases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D004790:Enzyme Induction; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine; D013379:Substrate Specificity
D010450:Endopeptidases; D007930:Leucine; D002118:Calcium