Isolation and characterization of Cysteine protease from leguminous cotyledons

Proteolytic enzymes play central role in the biochemical mechanism of germination and intricately involved in many aspects of plant physiology and development. The present study was conducted on the compares studies the Cysteine proteases from four varieties of 62 hours germinated leguminous seeds: lentil, green gram, black gram and pea bean. We elaborated the easy procedure for isolation of protease from leguminous germinated seeds by using (NH4)2SO4 precipitation followed by Gel-filtration and DEAE-cellulose chromatography from the 72h germinated cotyledons of lentil (Lens esculenta), green gram (Vigna radiata), black gram (Vigna mungo) and pea bean (Phaseolus vulgaris). This study revealed that the water-soluble protein concentration of crude extract ranged between 2.03 to 2.36mg/ml in which green gram was highest protein concentration (2.36mg/ml) and lentil accounted was the lowest concentration (2.03mg/ml). Cysteine proteases from all different leguminous seeds show very close monomer with a molecular mass of 29.5–30kDa were determined by SDS-PAGE. The enzyme activities were completely inhibited by pCMB, iodoacetate and DEPC indicating Cysteine and histidine residues at the active site. The enzyme is fairly stable towards pH and temperature. Cysteine protease has broad substrate specificity and stability in pH, temperature, therefore, this protease may turn out an efficient choice for the food, pharmaceutical, medicinal, and biotechnology industries.