Effect of Inhibitors from Plant Seeds on Digestive Proteolytic Activities in Larvae of the Date Palm Fruit Stalk Borer, Oryctes elegans Prell (Coleoptera: Scarabaeidae)

Abstract

The date palm fruit stalk borer is one of the most important pests of date palm in the world. Biochemical properties of digestive proteases in Oryctes elegans Prell larvae were investigated in this research and optimal total proteolytic and trypsin activities were obtained at pH 9.0 and 11.0, respectively. Activity staining of protease on SDS-PAGE showed one isoform. Also, zymogram pattern of trypsin using nitro-cellulose membrane revealed two isoforms. The inhibitory effect of PMSF, TLCK, TPCK, EDTA, iodoacetate and iodoacetamide were determined on O. elegans proteolytic activity. The iodoacetamide showed the highest inhibition on total proteolytic activity. Therefore, cysteine protease accounted for the major proteases in the gut of O. elegans. Total proteolytic activity was inhibited 22.3 and .15% by inhibitors extracted from Vicia faba and Lathyrus sativus, respectively. However, the inhibitors extracted from seeds of Prosopis farcta, Panecum miliaceum, and Alhagi maurorum showed negligible inhibitory effects on proteolytic activities. Trypsin activity was inhibited 91.5 and 82.3% by inhibitors extracted from V. faba and L. sativus, respectively. Electrophoretic analysis showed that inhibitors extracted from V. faba reduced the intensity of total proteolytic and trypsin activities. The inhibitor from V. faba was purified by ammonium sulfate precipitation and gel-filtration, also the molecular mass of inhibitor was determined 35 kDa. This purified inhibitor was able to inhibit trypsin activity by 72.7%. In addition, the highest inhibition of trypsin activity by inhibitor from V. faba occurred at pH 11.0. Also, the stability of inhibitor from V. faba was evaluated at different pHs and temperatures. This inhibitor was stable at pH 11.0 and 30 °C. Keywords: Digestive protease type, Plant protease inhibitors, Trypsin. The date palm fruit stalk borer is one of the most important pests of date palm in the world. Biochemical properties of digestive proteases in Oryctes elegans Prell larvae were investigated in this research and optimal total proteolytic and trypsin activities were obtained at pH 9.0 and 11.0, respectively. Activity staining of protease on SDS-PAGE showed one isoform. Also, zymogram pattern of trypsin using nitro-cellulose membrane revealed two isoforms. The inhibitory effect of PMSF, TLCK, TPCK, EDTA, iodoacetate and iodoacetamide were determined on O. elegans proteolytic activity. The iodoacetamide showed the highest inhibition on total proteolytic activity. Therefore, cysteine protease accounted for the major proteases in the gut of O. elegans. Total proteolytic activity was inhibited 22.3 and .15% by inhibitors extracted from Vicia faba and Lathyrus sativus, respectively. However, the inhibitors extracted from seeds of Prosopis farcta, Panecum miliaceum, and Alhagi maurorum showed negligible inhibitory effects on proteolytic activities. Trypsin activity was inhibited 91.5 and 82.3% by inhibitors extracted from V. faba and L. sativus, respectively. Electrophoretic analysis showed that inhibitors extracted from V. faba reduced the intensity of total proteolytic and trypsin activities. The inhibitor from V. faba was purified by ammonium sulfate precipitation and gel-filtration, also the molecular mass of inhibitor was determined 35 kDa. This purified inhibitor was able to inhibit trypsin activity by 72.7%. In addition, the highest inhibition of trypsin activity by inhibitor from V. faba occurred at pH 11.0. Also, the stability of inhibitor from V. faba was evaluated at different pHs and temperatures. This inhibitor was stable at pH 11.0 and 30 °C. Keywords: Digestive protease type, Plant protease inhibitors, Trypsin.