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Partial purification and characterization of trypsin-like enzyme from Helicoverpa armigera larval midgut and its effect on the degradation of δ-endotoxin from Bacillus thuringiensis
Author(s): 
Pages: 282-289
Year: Issue:  3
Journal: ACTA ENTOMOLOGICA SINICA

Keyword:  棉铃虫类胰蛋白酶苏云金杆菌降解;
Abstract: 将棉铃虫Helicoverpa armigera中肠液经Sephadex G-75、 DEAE-Sephadex A-50阴离子交换和CM-Sephadex C-50阳离子交换柱层析进行分离纯化,得到部分纯化的24.5 kD蛋白.该蛋白可以水解胰蛋白酶的专性底物BApNA和TAME,不能水解胰凝乳蛋白酶的专性底物BTEE.蛋白酶抑制剂的抑制试验显示,TLCK、PMSF、STI及SBBI均可显著抑制该蛋白对BApNA的水解作用,而TPCK无抑制作用.由此推断,24.5 kD蛋白是棉铃虫的类胰蛋白酶.以BApNA为底物,类胰蛋白酶的最适pH为10.5~11.0.Sephadex G-75分离得到了4个洗脱峰,电泳结果显示,各洗脱峰均能使苏云金杆菌库斯塔克变种Bacillus thuringiensis var. kurstaki HD-1原毒素降解为60.5 kD的活力片段.峰Ⅳ虽然不能水解BApNA和TAME,但仍可对原毒素进行降解.而类胰蛋白酶活力最高的峰Ⅱ已将60.5kD蛋白完全降解,当减小用量至1/3时,才出现60.5 kD蛋白.因此,苏云金杆菌δ-内毒素在棉铃虫中肠内的降解有多种蛋白酶参与,类胰蛋白酶在其中起重要作用.CM-Sephadex C-50进一步分离得到的类胰蛋白酶可对原毒素进行降解,其降解作用受到TLCK、PMSF、STI及SBBI的显著抑制,不受TPCK的影响.
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