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PURIFICATION OF AMINOPEPTIDASE FROM THE JAPANESE CLASSIFIED BARLEY FLOUR
Author(s) -
DOI HIROSHI,
KAWAKAMI MISAKO
Publication year - 1995
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1995.tb00544.x
Subject(s) - chromatography , aminopeptidase , chemistry , size exclusion chromatography , high performance liquid chromatography , fractionation , hydroxylapatite , sodium acetate , ion chromatography , column chromatography , enzyme assay , barley flour , enzyme , wheat flour , leucine , biochemistry , amino acid , food science
In a search for biologically active materials from the classified barley flour produced in Japan an aminopeptidase activity was identified. In this paper, the purification of aminopeptidase is described. The activity of the enzyme was monitored using L‐leucine‐p‐nitroanilide as the substrate. After extraction using 20 mM sodium acetate buffer, pH 5.5, from the 95–75% classification flour, ammonium sulfate fractionation was performed between 30 and 50% saturation. The aminopeptidase was then purified about 160‐fold to homogeneity as assessed by HPLC using the following sequential chromatography steps: hydrophobic interaction chromatography, Sephacryl S‐200HR gel chromatography, DEAM‐ion exchange chromatography, hydroxylapatite chromatography, Sephacryl S‐100HR gel chromatography. The molecular weight of this enzyme was estimated as 62 kDα by size exclusion HPLC. The enzyme had a K m value of 0.22 mM and α pH optimum of 7.0.