STUDIES ON CHARACTERIZATION AND PURIFICATION OF α-AMYLASE INHIBITOR FROM BEAN (Phaseolus vulgaris L .) CULTIVAR
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Date
2013
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ABSTRACT
Seven bean (Phaseolus vulgaris L.) cultivars of Himalayan region were analyzed for α-amylase inhibitor activity. α-Amylase inhibitor from seed flour of Phaseolus vulgaris L. showed highest activity in 0.1 M phosphate buffer (pH 7.6) contaning 0.15 NaCl in 1 hours of extraction time. Among the seven cultivars of Phaseolus vulgaris L. Triloki cultivar was found to have maximum total and specific inhibitor activity. The α–amylase inhibitor from seeds of Triloki cultivar was partially purified to 15.25 fold. The specific inhibitor activity increased from 0.19 in crude extract to 3.01 in Gel filtration (Sephadex G-100) chromatography. A single band of the purified inhibitor was obtained by Native–PAGE. SDS-PAGE revealed the purified inhibitor to be a monomer with molecular weight of 25,000 daltons. The nature of inhibition was found to be of non-competitive type as determined Dixon’s plot. The inhibitor was found to be heat labile was stable up to 30°C and retained 55.51 percent activity at 70°C temperature. Inhibitor was found to have two pH optima of 5 and 7.6. The purified inhibitor was found to have inhibitory activity against α–amylase extracted from larvae of Corcyra cephalonica. 100 percent larval mortality of Corcyra cephalonica was observed after 11 days when they were fed on wheat flour mixed with 252 µg of partially purified α- amylase inhibitor. Partially purified α–amylase inhibitor was also found to inhibit the activity of α-amylases of Corcyra cephalonica larvae, Aspergillus oryzae, Human salivary and Bacillus spp. Molecular characterization of bean cultivar Triloki was done by amplification of its genomic DNA using primer OPS-02. The number of bands amplified was 2.