Sharifloo A, Zibaee A, Jalali Sendi J, Talebi Jahroumi K
Department of Plant Protection,Faculty of Agricultural Sciences,University of Guilan,Rasht 416351314,Iran.
Department of Plant Protection,College of Agriculture and Natural Resources,University of Tehran,Karaj,Iran.
Bull Entomol Res. 2018 Aug;108(4):501-509. doi: 10.1017/S0007485317001067. Epub 2017 Nov 7.
A comprehensive study on digestive trypsin was undertaken in the larval midgut of Pieris brassicae L. Results of enzymatic compartmentalization showed a significantly higher activity of crude trypsin in the anterior larval midgut rather than posterior-midgut. Using Diethylaminoethyl cellulose fast flow column chromatography a purified trypsin was obtained by specific activity of 21 U mg-1 protein, recovery of 22%, purification fold of 28-fold and molecular weight of 25 kDa. This purified enzyme showed the highest activity at pH 8 and the corresponding temperature of 40°C. However, the specific inhibitors used including 4-(2-Aminoethyl) benzenesulfonyl fluroride hydrochloride, N-p-Tosyl-L-lysine methyl ester hydrochloride and Soybean Trypsin Inhibitor significantly lowered the activity of the purified enzyme in vitro. Moreover, the activity of trypsin and likewise the nutritional indices were significantly altered in the larval midgut feeding upon the leaves treated by 1 mM concentration of each inhibitor in comparison with control. Determination of enzymatic characteristics of insect trypsins is crucial in paving the path for controlling pests by potential natural compounds via transgenic plants.
对粉纹夜蛾幼虫中肠的消化性胰蛋白酶进行了全面研究。酶区室化结果显示，幼虫中肠前部的粗胰蛋白酶活性显著高于中肠后部。使用二乙氨基乙基纤维素快速流动柱色谱法，获得了一种纯化的胰蛋白酶，其比活性为21 U mg-1蛋白质，回收率为22%，纯化倍数为28倍，分子量为25 kDa。这种纯化的酶在pH 8和40°C的相应温度下表现出最高活性。然而，所使用的特异性抑制剂，包括4-(2-氨基乙基)苯磺酰氟盐酸盐、N-对甲苯磺酰-L-赖氨酸甲酯盐酸盐和大豆胰蛋白酶抑制剂，在体外显著降低了纯化酶的活性。此外，与对照相比，用1 mM浓度的每种抑制剂处理叶片后，幼虫中肠中胰蛋白酶的活性以及营养指标均发生了显著变化。确定昆虫胰蛋白酶的酶学特性对于通过转基因植物利用潜在天然化合物控制害虫至关重要。
