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Appl Environ Microbiol. 1987 October; 53(10): 2296-2302
Copyright © 1987, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of a Substrate-Size-Recognizing Metalloendopeptidase from Streptococcus cremoris H61

Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT

During the ripening of Gouda-type cheese, two kinds of endopeptidases were found to participate in the degradation of s1-CN(f1-23), a specific product from s1-casein hydrolyzed by chymosin. One of the endopeptidases, lactic acid bacteria endopeptidase (LEP-II), which can recognize the size of its substrates, has already been purified and characterized (T. R. Yan, N. Azuma, S. Kaminogawa, and K. Yamauchi, Eur. J. Biochem. 163:259-265, 1987). The other endopeptidase, LEP-I, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme appeared to be monomeric, with an apparent molecular weight of 98,000, and its isoelectric point was 5.1. For the hydrolysis of s1-CN(f1-23), the enzyme had an optimum pH and temperature of 7.0 to 7.5 and 40°C, respectively. Its activity was inhibited by such chelating agents as EDTA and 1,10-phenanthrolin, and it could be fully reactivated by Mn²⁺. Inhibitors specific for serine and thiol proteases had no effect on the protease activity. The enzyme showed a high affinity toward the Glu-Asn peptide bond of s1-CN(f1-23) and s1-CN(f91-100) but showed no hydrolysis activity toward s1-CN(f1-52), s1-CN(61-122), s1-CN(136-196), s1-casein, ß-casein, -casein, -lactalbumin, and ß-lactoglobulin. The K_m and V_max of LEP-I for s1-CN(f1-23) were 14.2 pM and 139 U, respectively.

^* Corresponding author.

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