α-Amylase inhibitor in local Himalyan collections of Colocasia: Isolation, purification, characterization and selectivity towards α-amylases from various sources

The alpha-amylase inhibitor from corms of Colocasia collected from Bhota village of Hamirpur district, Himachal Pradesh was purified to 17.21 folds with 61.61% recovery using ammonium sulfate precipitation, gel filtration chromatography (sephadex G-200) and ion exchange chromatography (DEAE-sephadex). 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 13,900 daltons. The nature of inhibition was found to be of non-competitive type as determined by Lineweaver-Burk plot and a K-i value of 0.54 nmole was obtained by Dixon's plot. The inhibitor was found to be heat stable and retained 81.50% activity at 70 degrees C temperature. Inhibitor was found to have pH optima of 6.9. The purified inhibitor was found to have inhibitory activity against alpha-amylases extracted from the larvae of Callosobruchus chinensis, Tribolium castaneum, Corcyra cephalonica and midgut alpha-amylase of Spodoptera littoralis. 100% larval mortality of C cephalonica was observed when fed on wheat flour mixed with 0.0036% (w/w) of purified inhibitor. Purified alpha-amylase inhibitor was found to inhibit the activity of human salivary alpha-amylase. It also had inhibitory activity against potato alpha-amylases and reduced sugar content in treated potato slices. The purified inhibitor was found to be a glycoprotein. In the present study, the ability of the inhibitor to inhibit insect amylases highlights its possible role in pest resistance and post harvest decay of crop plants. Inhibitory activity of co-amylase inhibitor against mammalian amylases could suggest its potential in treatment of diabetes and cure of nutritional problems, which result in obesity. (C) 2012 Elsevier Inc. All rights reserved.