关键词: 臭腹腺蝗, α-淀粉酶, 纤维素酶, 消化酶, 纯化, 蛋白类抑制剂

Abstract: 【Aim】 This study aims to gain information on carbohydrate digestion in the variegated grasshopper Zonocerus variegatus. 【Methods】 Crude enzyme extracts of the midgut with specific activity of 283±1.72 U/mg for α-amylase and 17±0.83 U/mg for cellulase, were subsequently purified by a combination of cation-exchange on CM-Sepharose CL-6B, anion-exchange on QAE Sephadex A-50 and gel filtration on Sephadex G-100. The co-existing enzymes were separated into alpha amylase and cellulase by the final step of chromatography. 【Results】 Both enzymes are monomers with molecular weights of 38 kDa for αamylase and 21 kDa for cellulase. Alpha amylase has a K_m of 13.8±1.29 mg/mL for starch and a V_max of 3 883±52.25 U/mg protein, whereas the values of 5.03±0.57 mg/mL and 433.00±5.24 U/mg protein were respectively obtained for cellulase with carboxymethyl cellulose (CMC) as substrate. Z. variegatus α-amylase could hydrolyse raw starches with varying efficiency while the cellulase could also hydrolyze various cellulosic materials to varying degrees. Hydrolytic activity was maximal for α-amylase at pH 6 and 40℃ and cellulase at pH 9 and 45℃. 【Conclusion】 The proteinaceous inhibitors were isolated from Carica papaya seeds and found to be effective inhibitors to both enzymes, suggesting that these protein inhibitors (CpAI and CpCI) may be useful in the pest control if overexpressed in crop plants that the pests feed on. Though both cellulosic and starchy materials may contribute significantly to the pests’ energy requirements, kinetic analysis suggests that Z. variegatus prefer starchy foods.

Key words: Zonocerus variegatus, α-amylase, cellulase, digestive enzymes, purification, proteinaceous inhibitors