STUDIES ON CHARACTERIZATION OF TRYPSIN INHIBITOR FROM Dolichos biflorus
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Date
2014
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Abstract
ABSTRACT Protease inhibitors present in seeds of leguminous plants possess strong inhibitory activity against trypsin and show resistance against pests. Trypsin inhibitor from seed flour of Dolichos biflorus L. showed highest activity in distilled water (pH 7.0) in 4 hours of extraction time. Among the three cultivars of Dolichos biflorus L. HPK-4 cultivar was found to have maximum total and specific inhibitor activity. In developing seeds (7 to 70 DAF) inhibitor activity increased with seed maturation. After six months of storage at room temperature there was significant decline in trypsin inhibitor activity and seeds were infested with beetles but seeds stored at 4oC showed no decline in inhibitor activity. The inhibitor activity in HPK-4 cultivar was found to decrease during germination period (1-11 days). Trypsin inhibitor was partially purified from seed flour extract of HPK-4 cultivar to 4.78 fold with 72.89 per cent recovery by ammonium sulfate precipitation and gel filtration chromatography on Sephadex G-10 and G-100 column. Linear increase in percent inhibition with increase in inhibitor concentration was observed till 71%.The partially purified inhibitor was found to be heat stable and retained 98.98% activity of control at 100oC. It was found to have pH optimum of 7.0 and it exhibited non-competitive inhibition pattern. The inhibitor lost its activity on incubation with varying concentrations of DTT there by indicating the role of disulfide linkages in maintaining its stability and three dimensional structures. Pieris brassicae eggs treated with partially purified inhibitor showed 93.4 percent reduction in hatching as compared to control. Feeding bioassay of Pieris brassicae larvae on leaf discs of cabbage coated with partially purified inhibitor showed decline in per cent survival of larvae with increase in inhibitor concentration. Blood clotting time was found to increase by 33 minutes in human blood treated with partially purified inhibitor (200µg) as compared to control. Partially purified protein (120 µg) showed inhibition of fungus Aspergillus versicolor growth in PDA (potato dextrose agar) medium.
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enzymes, biochemical compounds, sowing, proteins, extraction, beans, chromatography, animal developmental stages, electrophoresis, flours, Protease inhibitors,pests,Trypsin inhibitor, enzymes, biochemical compounds, sowing, proteins, extraction, beans, chromatography, animal developmental stages, electrophoresis, flours