Isolation and purification of a papain inhibitor from Egyptian genotypes of barley seeds and its in vitro and in vivo effects on the cowpea bruchid, Callosobruchus maculatus (F.).

The cysteine inhibitors that are known as cystatin have been identified and characterized from several plant species. In the current study, 44 barley (Hordeum vulgare) genotypes including 3 varieties and 41 promising lines were screened for their potential as protease inhibitors. The barley genotypes showed low inhibitory activity against trypsin and chymotrypsin enzymes with a mean of 4.15 TIU/mg protein and 4.40 CIU/mg protein. The barley variety, Giza 123, showed strong papain inhibitory activity of 97.09 PIU/mg proteins and was subjected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Barley purified proteins showed two bands on SDS-PAGE corresponding to a molecular mass of 12.4-54.8 kDa. The purified barley PI was found to be stable at a temperature below 80 °C and at a wide range of pH from 2 to 12. Barley PI was found to have higher potential inhibitory activity against papain enzyme compared to the standard papain inhibitor, E-64 with an IC50 value of 21.04 µg/ml and 25.62 µg/ml for barley PI and E-64, respectively. The kinetic analysis revealed a non-competitive type of inhibition with a Ki value of 1.95 × 10(-3 )µM. The antimetabolic effect of barley PI was evaluated against C. maculatus by incorporating the F30-60 protein of the purified inhibitor into the artificial diet using artificial seeds. Barley PI significantly prolonged the development of C. maculatus in proportion to PI concentration. Barley PI significantly increased the mortality of C. maculatus and caused a significant reduction in its fecundity. On the other hand, barley PI seemed to have non-significant effects on the adult longevity and the adult dry weight. The in vitro and in vivo results proved the efficiency of the papain inhibitory protein isolated from barley as a tool for managing the cowpea bruchid, C. maculatus.