Abstract: | An intracellular endopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by anion exchange chromatography on DEAE-Sepharose, hydroxyapatite chromatography, second anion exchange chromatography on Mono-Q, and metal-chelating affinity chromatography. The endopeptidase was a monomer with a molecular mass of approximately 70 kDa determined by SDS-PAGE and gel filtration. Various oligopeptides (e.g. Met-enkephalin, bradykinin) were hydrolysed by the endopeptidase. Exopeptidase activity and cleavage of dipeptides or tripeptides was not observed. The KM value for the cleavage of Metenkephalin was 1.2 mM. Temperature and pH optima were 47 °C and pH 7.7, respectively. The endopeptidase was inhibited by the classical agents for metal-dependent (EDTA) and serine (DFP) enzymes. Activity was increased by Co2+ and Mg2+, no effect was observed with Ca2+. After inhibition with EDTA, enzyme activity could be restored fully by Co2+. Activity was inhibited by Zn2+, Mn2+, Fe2+, Cu2+, Cd2+ and Hg2+. The N-terminal sequence of the endopeptidase was determined as: H2N-Val-Arg-Gly-Gly-Ser-Gly-Asp-Thr-Thr-Val-0H. |