Effects Of Signal Peptides On The Nattokinase Secretion In Bacillus Licheniformis

Nattokinase was an alkaline serine protease that secreted from Bacillus subtilis natto,compared to common thrombolytic drugs(urokinase,t-PA and streprokinase),nattokinase showed the advantages of high safety,low cost and long half-life,and nattokinase has an important application value in the prevention and treatment of cardiovascular and cerebrovascular diseases.Heterogenous expression was an important strategy to regulate the yield and property of target protein,and B.licheniformis have been gaining more and more interests as an expression host due to its high secretion ability and biological safety.In this study,B.licheniformis △ 0F-3 has been constructed and nattokinase was expressed secretory successfully.Then,the signal peptides were systematically screened for maximum nattokinase production,and B.licheniformis △0F-3-SacC was obtained for high-level expression of nattokinase,and the researches about nattokinase production in B.licheniformis between signal peptide transformation,signal peptidase and signal peptide peptidase were also been done in this study.The extracellular protease could affect nattokinase secretion,and poly-γ-glutamic acid will affect the post-separation of nattokinase,bprA and aprE were deleted based on B.licheniformis Rf-△0F,and B.licheniformis △0F-3 was gained after pgsC was deleted.Then,based on the expression vector of p HY300 PLK,we have chose the P43 promoter from B.subtilis 168,the aprN coding for nattokinase from B.subtilis natto MBS 04-6 as target production,the terminator of amy L from B.licheniformis WX-02,and the recombinant plasmid was constructed for nattokinase secretion.The recombinant plasmid was electric-transformed to the B.licheniformis △ 0F-3.Through assaying and SDS-PAGE of supernatant after fermentation,the nattokinase has been secreted successfully in B.licheniformis,and the activity of nattokinase was 6.27 FU/m L.Through bioinformatics analysis of extracellular proteins,83 signal peptides from B.licheniformis and B.subtilis were chose for this study.Through constructed the signal peptide screening vector,then the signal peptides of aprN was replaced with the signal peptides of we chose,and 83 vectors containing different signal peptides was obtained.The effects of signal peptides on the secretion expression of nattokinase was done in this study,and the results showed signal peptides have a greater impact on the secretion of nattokinase,and the highest nattokinase produced with the signal peptide of SacC from B.subtilis 168,and the activity was was 32.53 FU/m L,4-fold higher than that of the native signal peptide,and the nattokinase production was 201.18 mg/L.Through this study,we have obtained a B.licheniformis strain with high-level production of nattokinase,and also provided a basis of signal peptide screening for target protein expression in B.licheniformis.Based on the genome annotation of B.licheniformis DW-02,the genes coding for signal peptidases I(sipS,sipT,sipV,sipW)and signal peptide peptidases(sppA,tep A)were identified in B.licheniformis DW-02.In this study,signal peptidases genes and signal peptide peptidases genes were deleted respectively in B.licheniformis △0F-3,and then researches about the signal peptidase and signal peptide peptidase deficiency on the expression of nattokinase were done.The results showed deletion of sipV and sppA affects the secretion of nattokinase obviously in B.licheniformis,and the activity of nattokinase decrease by 43.18% and 42.90 respectively.The results explained that sipV and sppA have an important role in the nattokinase secretion in B.licheniformis.