Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61

Abstract

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 Zn2+ 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 Sephracryl 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 acid groups of hydrophobic amino acid residues. Various small polypeptides, such as .alpha.s1-CN(F1-23), .alpha.s1-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 .alpha.s1-CN(f1-54), .alpha.s1-CN(f61-123), .alpha.s1-CN(f136-196), .alpha.s1-casein, .beta.-casein, and .kappa.-casein were not hydrolyzed, LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.