The Research On The Expression Of Bacillus Subtilis LNUB236 Nattokinase And Site-directed Mutagenesis To Modify Thermostability

Natto was as a kind of traditional food in Japan for thousands of years.Nattokinase(Nattokinase,abbreviated as NK)was a kind of serine protease produced in natto fermentation process by Bacillus subtilis.Studies have shown that NK has a strong ability to dissolve thrombus.nattokinase has a lot of advantages such as more safer,easier to be absorbed by gastrointestinal tract,more direct and persistent ability of degrading fibrin in thrombi.Therefore,NK as a new type of dissolved drug has a great development potential.PCR amplified the protogene of nattokinase including mature peptide,leader peptide and mature peptide,full length gene.Constructed pET-26 b prokaryotic recombinant expression vector and pGAPZ?A eukaryotic recombinant expression vector respectively,transfered into E.coli BL21 strain and Pythagorean yeast X-33 strain to induce expression,and analyzed the result by SDS-PAGE.Fibrin plate method is used to detect the fibrinolytic activity of expression.Used FoldX software to predict the thermostability amino acid of nattokinase by bioinformatics method.Site-directed mutagenesis was used to mutate the 103 glutamine,226 histidine and 232 alanine which predicted by overlap extension PCR method.Constructed the protogene of nattokinase including mature peptide,leader peptide and mature peptide,full length gene prokaryotic expression vector.Specific protein band about 38 KD was shown in SDS-PAGE electrophoresis diagram.Only the supernatant of the prokaryotic expression vector which including leader peptide and mature peptide of nattokinase showed the fibrinolytic activity by fiber plate method.But the eukaryotic expression protein showed no fibrinolytic activity.After mutation of the three amino acids predicted by bioinformatics method,the enzyme activity test results showed that only 226 mutation has enzyme activity,but enzyme activity was lower compared with original.The thermostability test result showed that compared with original nattokinase the activity of the mutant decreased gently,calculated the inactivity rate of enzyme activity after different temperature treat with the nattokinase,the result showed that the inactivity rate of enzyme activity of mutation nattokinase was 56.82% when temperature rose from 35? to 60? and the original nattokinase was 88.65%,nattokinase maximum heat resistant temperature result showed that the mutation nattokinase was 64? and the original nattokinase was 62?.The result showed that the thermostability of mutant was improved.