Biochemical characterization of α-amylases from gut and hemolymph of Rhynchophorus ferrugineus Olivieri (Col.: Curculionidae) and their inhibition by extracts from the legumes Vigna radiata L. and Phaseolus vulgaris L.

α-amylase inhibitors represent an important tool in engineering crop plants against insect pests. For achieving this goal, it is necessary to find the nature of α-amylases and their properties for possible use in a pest management procedure. Because Rhynchophorus ferrugineus Olivieri is a devastating pest of palm trees in the southeast of Iran, we attempted to characterize α-amylases from larval gut and hemolymph, and to study their interaction with inhibitors extracted from the common bean and the green mung bean. The optimal pHs for gut and hemolymph α-amylases were 4 - 5 and 5 - 6, respectively. Also, high gut amylolytic activity was found at temperatures of 40 – 50 °C. The highest and lowest specific α-amylase activities were detected in the guts of last instar and adult males, and in the hemolymph of last instar, respectively. As calculated from Lineweaver-Burk plots, the Km values for gut and hemolymph α-amylases of the last instar were 0.54 and 2.15 %, respectively, when glycogen was used as the substrate. Also, when starch was used as the substrate, the Km values for gut and hemolymph α-amylases were 1.37 and 0.15 %, respectively. Zymogram pattern in the native gel revealed that R. ferrugineus gut and hemolymph α-amylases had two isoforms. α-amylase inhibitors partially purified from Vigna radiata L. and Phaseolus vulgaris L. by ionic exchange DEAE cellulose column, inhibited the R. ferrugineus gut α-amylase activity by 19 ± 0.64 % and 11.56 ± 0.69 %, respectively

Protease Inhibitor from the Crude Extract of Plant Seeds Affects the Digestive Proteases in Hyphantria Cunea (Lep.: Arctiidae)

Proteases are one of the most important digestive enzymes in the midgut of Hyphantria cunea Drury. Proteases are responsible for protein digestion. In the present study, we evaluated the efficiency of some plant inhibitors on proteases in the gut of the H. cunea. Last instar larvae were collected from mulberry trees. The digestive system of the larvae was used as an enzyme source. The total proteolytic and trypsin activity were assessed by the hemoglobin and BApNA, respectively, as the substrate. The evaluation of the total proteolytic and trypsin activities in various pHs showed the highest relative activity at a pH of 11. Also, the inhibitory effect of inhibitors extracted from Alhagi maurorum Medik., Lathyrus sativus L., Vicia faba L., Prosopis farcta (Banks & Sol.) Eig., and Panicum miliaceum L. on the digestive protease of the fall webworm was measured. Protease inhibitors extracted from A. maurorum, P. farcta and P. miliaceum showed negligible inhibition but L. sativus was able to inhibit 34.72% and 100% of the total activity of proteolytic and trypsin, respectively. Also, the total proteolytic and trypsin activities were inhibited by the inhibitor from V. faba, at 22.27% and 100%, respectively. The zymogram pattern of trypsin with nitro-cellulose membranes showed 2 isoforms in the gut of H. cunea. The inhibitor from L. sativus completely inhibited both isoforms. Gel electrophoresis of proteolitytic activity revealed at least 6 isoforms the inhibitor extracted from L. sativus; completely inhibiting some of them. The inhibitor from L. sativus was purified by ammonium sulfate precipitation and gel-filtration. The molecular mass of the inhibitor was determined as 45 kDa. The highest inhibition of trypsin activity by the inhibitor from L. sativus occurred at a pH of 10. The stability of the inhibitor from L. sativus was evaluated at different pHs and temperatures. The results showed that the inhibitor from L. sativus was stable at a pH of 11.0, and showed 45% inhibition on trypsin activity at a pH of 11. Also, this inhibitor revealed stability up to 50°C