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.
