Thermostability of Subtilisin Nattokinase Obtained by Site-Directed Mutagenesis  

作　　者：WENG Meizhi DENG Xiongwei WU Jieyuan ZOU Guolin 

机构地区：[1]Jiangxi Province Blood Center [2]College of Life Sciences,Wuhan University [3]Hongdu Chinese Medical Hospital

出　　处：《Wuhan University Journal of Natural Sciences》2014年第3期229-234,共6页武汉大学学报（自然科学英文版）

基　　金：Supported by the National Natural Science Foundation of China(30670464,20873092,30800190);Science and Technology Project of Wuhan(200960323115)

摘　　要：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.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.

关 键 词：nattokinase subtilisin thermostability 

分 类 号：Q816[生物学—生物工程]