棉铃虫中肠类胰蛋白酶的部分纯化和性质测定及其对苏云金杆菌δ-内毒素的降解作用

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

将棉铃虫Helicoverpaarmigera中肠液经SephadexG 75、DEAE SephadexA 50阴离子交换和CM SephadexC 50阳离子交换柱层析进行分离纯化 ,得到部分纯化的 2 4 5kD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 ,2 4 5kD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。SephadexG 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Bacillusthuringiensisvar.kurstakiHD 1原毒素降解为 60 5kD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 5kD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 SephadexC 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 。

A 24 5 kD protein was obtained from the midgut extracts of Helicoverpa armigera larvae by the purification with Sephadex G 75, DEAE Sephadex A 50 and CM Sephadex C 50 columns. Enzyme activity assay showed that this protein could hydrolyze BApNA and TAME, the specific substrates of trypsin, but couldn't hydrolyze BTEE, the specific substrate of chymotrypsin, and the BApNA hydrolytic activity was significantly inhibited by TLCK, PMSF, STI and SBBI, though no inhibition was caused by TPCK. So this 24 5 kD protein is a trypsin like enzyme in H. armigera larval midgut. Using BApNA as substrate, the optimum pH of trypsin like enzyme activity was between 10 5～11 0. SDS PAGE results showed that the δ endotoxin of Bacillus thuringiensis var. kurstaki (HD 1) could be degraded into 60 5 kD active fragments by all the fractions eluted from the Sephadex G 75 column, although no obvious proteolytic activity on BApNA and TAME could be measured with peak Ⅳ. Peak Ⅱ possessed the highest trypsin like enzyme activity, and degraded the δ endotoxin and 60 5 kD protein completely. When the concentration of peak Ⅱ was reduced to 1/3, the 60 5 kD protein appeared again. The results suggest that multiple proteases take part in the degradation of δ endotoxin in the H. armigera midgut, and the trypsin like enzyme is most important among them. Trypsin like enzyme further purified from CM Sephadex C 50 could degrade the protoxin of Btk HD 1, and the degradation was inhibited by TLCK, PMSF, STI, and SBBI, but no obvious inhibition occurred with TPCK.