经硫酸铵分级沉淀、热处理、DEAE等方法从掌叶半夏中分离出一种不含糖的具有抗虫作用的胰蛋白酶抑制剂(Pinellia pedatisecta trypsin in hibitor,PPTI),SDS-PAGE电泳与反相液相色谱(RT-HPLC)检测证明分离得到一纯蛋白,质谱(LC-MS)测定其相对分子质量为20596.09.荧光光谱分析研究结果显示:PPTI在pH低于9、温度低于60℃时,PPTI的三级结构相对稳定;抗虫活性结果显示有较好的抗芽虫作用.
To study the thermostability of Nattokinase(subtilisin NAT,NK),three double mutant plasmids(pET-28a-NKG61C/S98C,pET-28a-NKT22C/S87C,pET-28a-NKS24C/S87C)were constructed by site-directed mutagenesis.Target enzymes were detected using SDS-PAGE and disulfide bond formation was detected using Western blotting analysis.Thermostability was tested by rates of inactivation at certain temperature.The results showed that disulfide bond was not formed within two cysteines and the thermostability of three double mutants was not increased compared with the wild-type NK.The thermostability of NK performed in Ca2+was stronger than in ethylenediaminetetraacetic acid(EDTA).But when the temperature reached 62℃,the enzymes rapidly denatured and inactivated even in the presence of Ca2+.Although the thermostability of mutants was not increased,this study shows a tendency of improving thermostability of NK in protein engineering.
