STUDIES ON TRYPSIN INHIBITOR IN LOCAL BEAN (Phaseolus vulgaris L.) CULTIVARS
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Date
2013
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Abstract
ABSTRACT
Trypsin inhibitor from seed flour of Phaseolus vulgaris L. showed highest activity in 0.1
M phosphate buffer (pH 7.5) in 3 hours of extraction time. Among the five local cultivars of
Phaseolus vulgaris L. Baspa cultivar was found to have maximum total and specific inhibitor
activity. The seed flour extracts of all the cultivars inhibited gut protease of Spodoptera littoralis
larvae and highest inhibitory activity was found in Baspa cultivar. The inhibitor activity in Baspa
cultivar was found to decrease during germination period (1-6 days). In developing seeds (8 to 64
DAF) it increased with seed maturation. The extacts of developing seeds inhibited gut protease of
Spodoptera littoralis larvae and maximum inhibition was observed with seed flour extract of
mature seeds (64 DAF). Trypsin inhibitor was partially purified from seed flour extract of Baspa
cultivar to 3.94 fold with 50.37 per cent recovery by ammonium sulfate precipitation and gel
filtration chromatography on Sephadex G-50 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 82.35% activity of control at 77oC. It was found to have pH
optimum of 7.5 and it exhibited non-competitive inhibition pattern. The inhibitor lost its activity
on incubation with increasing concentrations (20-100Mm) of DTT thereby indicating the role of
disulfide linkages in maintaining its stability and three dimensional structures. 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. The larvae survived
after bioassay experiments were shifted to normal leaf discs for further growth and development.
In treatment, delay in pupation was observed, deformed pupa was formed and no adult emerged.
In control, all the larvae formed normal pupa and adults. Pieris brassicae eggs treated with
partially purified inhibitor showed 89.6 percent reduction in hatching as compared to control.
Blood clotting time was found to increase by 11 minutes in treated human blood with partially
(220μg inhibitor/2ml blood) as compared to control.
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Keywords
enzymes, biochemical compounds, sowing, extraction, proteins, animal developmental stages, beans, chromatography, phaseolus vulgaris, inorganic acid salts, Phaseolus vulgaris L,Trypsin inhibitor,seed flour,