Construction Of A Recombinant Bacillus Subtilis Strain Producing Nattokinase And Optimization Of Maltose Induction Conditions

Bacillus subtilis has been regarded as safe by United States Food and Drug Administration(FDA).And it can be used for the development and production of health products,food and drugs.Nattokinase is used for the prevention and treatment of thrombosis diseases,which can be highly expressed by Bacillus subtilis recombinant.So building a B.subtilis expression system is of great significance for realizing Nattokinase industrialization.In this research,a maltose-induced expression vector and two recombinants of nattokinase were constructed,and the maltose induced conditions and fermentation conditions were optimized.The main results are as follows:(1)Construction of maltose inducible expression vectorPCR-based two-step DNA synthesis(PTDS)method was used for expression cassette synthesis,including the maltose-inducible promoter(Pglv),the ribosome binding site(RBS),signal peptide(bpr),multiple cloning site(MCS),his tag,and terminator(gnt).Then the cassette was cloned into the Escherichia coli-Bacillus subtilis shuttle vector pWB402,resulting pGWB402.In order to alleviate repression of glucose,CG of Cre site was mutated into AT,resulting the mutated vector pMB402.(2)Construction of recombinant strains of nattokinaseThe nattokinase gene aprN,derived from B.subtilis natto,was cloned into the vectors pGWB402 and pMB402,then the recombinant aprN-pGWB402-B.subtilis 168 and aprN-pMB402-B.subtili1s 168 were constructed respectively.The enzyme activity of the two strains were 1144 IU/mL and 1047 IU/mL respectively,as 3.81 times and 3.49 times as B.subtilis natto.And the mutant transformant was selected for the subsequent research.(3)Optimization of the maltose-induced conditions of the recombinant strain aprN-pMB402-B.subtilis 168The optimal results were as follows:maltose concentration was 3.5%,induction time was OD600 1.2,and the harvest time was 144 h.Under the best conditions,NK activity reached 9180 IU/mL,as 7.38 times as that without maltose induction.The study of the optimal reaction conditions showed the optimal temperature was 45 ? and the pH was 8.(4)Optimization of the fermentation methodIn order to shorten the fermentation time,precipitates in LB without maltose was collected into new LB with 3.5%maltose,in a ratio of 1:1 and 2:1 respectively.The results showed that the enzyme activity of the enrichment culture were 9300 IU/mL and 8400 IU/mL respectively,which were 81.06%and 81.06%of the control.So induction after enrichment can not shorten the fermentation time and influenced the enzyme production.(5)The selection of industrialisation production mediumSix kinds of medium were studied.And the optimal medium was soybean maltose medium:maltose 20 g/L,soybean powder 10 g/L,yeast extract 5 g/L,KH2PO4 3 g/L,NaCl 5 g/L,MnS04 0.02 g/L,CaC03 0.3 g/L.(5)High cell density fermentationIn the high cell density fermentation of 50 L fermentor,enzyme activity reached the maximum 9774 IU/mL at 36 h,which is 0.69 times higher than flask fermentation.At the same time the fermentation time was much shorter than 144 h.