Isolation, purification and characterization of proteinaceous inhibitor(s) from Echinochloa crus-galli seeds
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Date
2008-07
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
The production of cereals can be increased by increasing the resistance in cereals against pathogens. Several studies demonstrated the efficiency of proteinaceous inhibitors isolated from millets, acting against digestive enzymes of the insects, acting as biocontrol. In the present study characterization of the bifunctional inhibitor from barnyard millet was conducted. From three varieties of barnyard millet one variety VL-29 was selected on the basis of biological control efficacy against storage pest, bacterial strains and fungus and qualitative tests conducted on them. Crude protein extracted from VL-29, was purified to isolate the protein(s) responsible for showing amylase/trypsin inhibitory activity by first undergoing ammonium sulphate fractionation and then followed by gel filtration on a Sephadex G-75 column. All the fractions were qualitatively assayed by the DNS method for the amylase inhibition to check their inhibitory activity. The fractions showing maximum activity were pooled and lyophilized. Finally protein profiling was conducted on 10% SDS-PAGE. In the purified sample 2 bands were observed which corresponded to 18 kDa and 59 kDa. The presence of the 2 bands suggested the inhibitor protein(s) consisting either of 2 subunits- 18 kDa and 59 kDa or a single protein present in multimeric form.
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