STUDIES ON PROTEASE INHIBITOR IN GERMINATED HORSEGRAM (Dolichos biflorus L.) SEEDS
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Date
2015
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ABSTRACT
Protease inhibitors present in seeds of leguminous plants possess strong inhibitory activity against trypsin and confer resistance against pests and microbes. Trypsin inhibitor from cotyledons of 24 hours soaked seeds of Dolichos biflorus L. cultivar HPK5 showed highest activity in distilled water (pH 7.0) in 3 hours of extraction time. The inhibitor activity was found to decrease gradually with increase in germination period. Decrease in the intensity and number of bands on native PAGE at different stages of seed germination indicated the reduction of soluble protein content with increasing germination period. Partially purified inhibitor protein from Sephadex G-50 was characterized. Linear increase in per cent inhibition with increase in inhibitor concentration was observed till 72 %. The inhibitor protein was found to be thermostable and retained 98 % activity of control at 100 оC. It had pH optima of 7.5. The inhibitor lost its activity on incubation with varying concentrations of DTT thereby indicating the role of disulfide linkages in maintaining its stability and three dimensional structures. Trypsin inhibitor was further partially purified to 3.83 fold with 78.5 per cent recovery by ammonium sulfate precipitation and gel filtration chromatography on Sephadex G-100 column. It exhibited competitive inhibition pattern and showed stability on incubation with urea even at high temperature (97 оC). However, it lost its stability on incubation with 0.6 N HCl at 37 оC and 97 оC. Partially purified inhibitor protein (6.17mg) inhibited the growth of fungus Alternaria spp. to 70 %. It also inhibited the growth of bacterium Ralstonia solanacerum. This study signifies the antimicrobial potential of Dolichos biflorus L. cultivar HPK5 which might be exploited for crop protection programmes.