Purification of aminopeptidase from Australian classified barley flour

Barley is one of the major crops cultivated in the world. A new milling system, which removes successive layers of the grain has been developed, the preparation being called a classified flour. We have detected aminopeptidase activity in the flour obtained from the near surface zone. Barley was the Weeah species cultivated in Australia. Our aim in this paper is to describe the complete purification of aminopeptidase from barley classified flour. Aminopeptidase activity was determined using L-leucine-p-nitroanilide in 10 mM sodium phosphate buffer, pH 7.0. Aminopeptidase was extracted in 20 mM sodium acetate buffer, pH 5.5. Fractions (HP-1 and HP-2) were obtained upon chromatography using an HI-propyl hydrophobic interaction column in 20 mM acetate buffer, pH 5.5, with a linear gradient of ammonium sulfate from 15 to 0% saturation after the ammonium sulfate fractionation (from 40 to 60% saturation) of the extract. The final purified preparation A-1-I-G was obtained from HP-1 by gel filtration, hydroxylapatite chromatography, diethylaminomethyl-ion exchange chromatography, and re-chromatography on a Sephacryl S-100HR gel column. It was pure as assessed by native and sodium dodecylsulfate-polyacrylamide gel electrophoreses. The specific activity of A-1-I-G was 64.8 fold that of the crude extract. A K(m) value of 0.138 mM when using L-leucine-p-nitroanilide was calculated. A-1-I-G is active over a wide pH range and has a strong affinity for its substrate. The classification process is effective as a step of enzyme purification. Also, the results with metal ions suggest that the enzyme is a metalloenzyme. It was concluded that complete purification of an aminopeptidase from barley was achieved.