Purification and characterization of black gram (Vigna mungo) husk peroxidase

Black gram is one of the important pulse crops in India and a variety of food products are made with its dhal (splits). During milling of black gram into dhal about 25% is a by-product. Currently, it is used as cattle feed or wasted and, therefore, it does not have any commercial value. In the present study, the milled by-product was fractionated into husk, germ, plumule and aleurone layer rich husk fractions. Few oxidative and hydrolytic enzyme activities such as peroxidase, polyphenol oxidase, protease, amylase and xylanase were determined in these fractions. All the fractions exhibited more peroxidase activity compared to other enzyme activities and aleurone layer rich husk fraction showed maximum peroxidase activity (1155 × 103 units/g husk; 275 × 102 μmol/min/mg protein). Peroxidase is an important enzyme having a variety of applications in analytical chemistry, biochemistry and food processing. Peroxidase from aleurone layer rich husk fraction (BGHP) was purified using two steps, ion-exchange chromatography followed by gel filtration. The purity was determined from the high specific activity, purification fold (44), by the single peak obtained by HPLC and capillary electrophoresis, and a single band in native PAGE and SDS-PAGE. The molecular weight of the enzyme was found to be around 35 kDa. o-Dianisidine was found to be the good substrate for the enzyme compared to other hydrogen donor substrates. Dithiothretol and sodium azide were found to be non-competitive inhibitors for the enzyme. The Km value of the enzyme for hydrogen peroxide and o-dianisidine was found to be 43.5 mM and 4.7 mM, respectively. Optimum pH of the enzyme was 5.5. The half-life of the enzyme at 50 °C was found to be 5 h and 15 min. Black gram husk, by-product of milling industry, is a rich source for a peroxidase.