Open AccessPurification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61
Author(s) -
YAN TsongRong,
AZUMA Norihiro,
KAMINOGAWA Shuichi,
YAMAUCHI Kunio
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb10796.x
Subject(s) - chemistry , casein , biochemistry , hydrolysis , enzyme , size exclusion chromatography , endopeptidase , protease , peptide , molecular mass , peptide sequence , chromatography , amino acid , lactococcus lactis , subtilisin , biology , bacteria , lactic acid , gene , genetics
An endopeptidase (LEP‐II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10‐phenanthroline; the metal‐depleted enzyme could be fully reactivated by micromolar levels of Zn 2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S‐300 gel filtration and high‐performance liquid chromatography with a TSK‐G3000SW column. The enzyme consisted of two identical subunits and the N‐terminal sequence of LEP‐II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as αs1‐CN(f1–23), αs1‐CN(f91–100). oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as αs1‐CN(f1–54), αs1‐CN(f61–123), αs1‐CN(f136–196), αs1‐casein, β‐casein, and K ‐casein were not hydrolzyed. LEP‐II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.