清酒麹中の酸性カルボキシペプチダーゼ活性の新規測定法

Abstract

A simple assay for acid carboxypeptidase activity in Sake koji with carbobenzoxy-L-tyrosyl-Lalanine (Cbz-Tyr-Ala) as a substrate was developed.In the proposed method, L-Ala released from the substrate by the action of acid carboxypeptidase was oxidized by alanine dehydrogenase (ADH) in the presence of NAJD+ at pH 9.0 to pyruvic acid and NADH.NADH readily reduced 2-(2-methoxy- 4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfopheny1)-2H-tetrazoliulm (WST) to WST formazan dyes in the presence of the coupled electron carrier 1-methoxy-5-methylphenazinium methosulfate (1- methoxy PMS).The increment of the formazan dyes, measured at 460nm, was directly proportional to the activity of acid carboxypeptidase.The optimum activity of acid carboxypeptidase from Aspergillus oryzae was found to be approx.pH 4.0 in this study.The coefficients of variation (CVs) were within 1% in both within-day and day-to-day experiments.This method was not affected by the various amino acids present in Sake koji extracts.The values of the acid carboxypeptidase activities obtained by the proposed method correlated well with those obtained by a standard method (National Tax Administration Agency).This new assay would fulfill the need for an accurate, specific and simple assay for acid carboxypeptidase.