Purification and Some Properties of an Aminopeptidase from Bifidobacterium breve

Abstract

An aminopeptidase which catalyzes the hydrolysis of L-leucine-ρ-nitroanilide was purified about 1, 000-fold (specific activity 31units/mg protein) from the cell-free extract of Bifidobacterium breve (MT-17) by means of protamine sulphate fractionation, ammonium sulphate fractionation, DEAE-Sepharose CL-6B chromatography, hydroxylapatite chromatography, rechromatography on DEAE-Sepharose CL-6B, rechromatography on hydroxylapatite, and Chromatofocusing and Sephadex G-75 chromatography. The purified enzyme showed a single protein band on polyacrylamide disc gel electrophoresis at pH 6.6, and an isoelectric point pI 3.75 on chromatofocusing. The molecular weight of the enzyme was estimated to be 61, 000 by Sephadex G-75 gel filtration. The enzyme exhibited a pH optimum at 6.6, temperature optimum at 37°C and was stable up to 40°C. It was activated by Mg2+ and inhibited by EDTA, 1, 10-phenanthroline, monoiodoacetic acid and p-chloromercuribenzoate. The enzyme had broad substrates specificity, but was not active on peptides having proline as the N-terminal or C-terminal amino acid, and on carbobenzoxylpeptides. In addition, the enzyme was highly active toward L-leucine-ρ-nitroanilide as compared to L-alanine-ρ-nitroanilide.