Studies on the characteristics of aminopeptidase from barley classified flour.

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The aim of this study is to make clear the caracteristics of aminopeptidase from barley classified flour. Aminopeptidase was obtained from barley classified flour by the investigators described above. The enzyme was purified as follows. 1) cold aceton treatment, 2)extraction with acetic acid buffer(pH 5.5), 3) Sephacryl S-200HR gel filtration, 4)Polyethylene-imine ion exchange chromatography, 5) hyclroxyapatite chromatography, and 6) Sephacryl S-100HR gel filtration chromatography.Optimum pH of enzyme activity was examined using various synthesized substrates. The purifled enzyme showed optimum pH between 7.0 and 8.0. Using leucine-p-nitroanilide as a substrate, optimum pH was 7.0 and Km value 0.22mM.Phenylalanine-p-nitroanilide was hydrolyzed so quickly with the velosity of 2.4 fold compared with that to leucine-p-nitroanilide, whereas the hydrolysis velosities to imino acid and aromatic aminoacis was about 1/3. The enzyme did not hydrolyze p-nitroanilide of acidic amino acid. In the case of phenylalanine- beta-naphtylamide, optimum pH was 8.0 and Km 0.047mM.Aminopeptidase from barley classified had higher affinity to beta-naplitylamide than to p-nitroanilide. The Km values to isoleucine-beta-naphtylamide and serine-beta-naphtylamide were 0.15 and 0.57 mM, respectively. The enzyme was not inhibited by leupeptin, which was an inhibitor to thiol peptidase.It is concludeed that amninopeptidase from barley classified flour is a neutral aminopeptidase.