Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate.
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 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 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.
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D010450:Endopeptidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases; D008970:Molecular Weight; D013291:Streptococcus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D010450:Endopeptidases; D008666:Metalloendopeptidases