Purification of a novel α-amylase inhibitor from local Himalayan bean (Phaseolus vulgaris) seeds with activity towards bruchid pests and human salivary amylase

Abstract

Six bean (Phaseolus vulgaris L.) cultivars of Himalayan region were analysed for α- amylase inhibitor activity. The α-amylase inhibitor from seeds of screened bean cultivar KR-9, showing maximum inhibitory activity was purified using ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-100) and ion exchange chromatography (DEAE-Sephadex). The inhibitor was purified to homogeneity as judged by native-PAGE with 14.22 fold purification and 71.66% recovery. Purified inhibitor consisted of three subunits of molecular weight 15,488, 18,620 and 26,302 daltons, respectively as determined by SDS-PAGE. It was found to be heat stable up to 30 °C-40 °C and had two pH optima of 5.0 and 6.9. Nature of inhibition was found to be of non-competitive type. The purified inhibitor was found to be effective against α-amylases extracted from larvae of Callosobruchus chinensis, Tribolium castaneum and gut enzyme of Spodoptera littoralis. Larvae of Tribolium castaneum fed on flour mixed with purified inhibitor for 5 days showed 100% larval mortality. Purified α-amylase inhibitor was also found to inhibit human salivary α-amylase, suggesting its potential in prevention and therapy of obesity and use as drug design targets for treatment of diabetes. The gene encoding the inhibitor may be used to develop transgenic plants resistant against insect pests.

Keywords: Callosobruchus chinensis; Phaseolus vulgaris; Spodoptera littoralis and Human salivary amylase; Tribolium castaneum; α-Amylase inhibitor.

Fig. 1

Elution profile of alpha amylase inhibitor on (a) Sephadex G-100 column and (b) DEAE-Sephadex (A50) column

Fig. 2

Effect of temperature (a) and pH (b) using acetate, phosphate and tris buffers on alpha-amylase inhibitor activity n = 6 Each observation is a mean of three replicates with duplicates

Fig. 3

Lineweaver burk plot (a) of alpha amylase activity in the absence and presence of two fixed concentrations of inhibitors and Dixons plot (b) of alpha amylase activity in presence of different concentrations of inhibitor at two fixed concentrations of substrate