Secretory Expression Of Heterologous Levansucrase In B.subtilis And Its Immobilization

Levan exists in a variety of organisms.Due to its numerous biological functions,levan shows great application potential in fields of food and medicine.The content of levan in microorganisms and plants is low and its extraction is difficult.While levan is synthesized easily by the levansucrase and the purification method is relatively simple,too.At present,levansucrase produced by microbial fermentation is difficult to be separated from the fermentation products,which is not conducive to subsequent research and industrial application.Bacillus subtilis is a food grade engineered bacteria,which has advantages of simple operation,fast growth and no endotoxin production.B.subtilis is an ideal engineering bacterium to study the secretion and expression of heterogenous protein due to its strong ability to secrete protein.In this study,the gene of levansucrase(sacB)derived from B.amyloliquefaciens BH072 was inserted into the shuttle plasmid pHT43 harboring the signal peptide Amy Q.The constructed plasmid was then introduced into B.subtilis 168for expression and secretion,so as to study the enzymatic properties of the recombinant enzyme.Finally,the recombinant enzyme was immobilized on the magnetic nanomaterials by covalent binding to obtain a kind of highly stable and reusable immobilized enzyme.The main results are as follows:(1)Firstly,the active sites of levansucrase from B.amyloliquefaciens BH072 were predicted by the amino acid sequence homology comparison.The gene sacB was amplified using the genomic of B.amyloliquefaciens BH072 as a template and then connected to the inducible plasmid pHT43,which contained the signal peptide Amy Q and the promoter P_lac.The constructed plasmid was confirmed in Escherichia coli and then injected into B.subtilis 168.In order to increase the production of recombinant enzyme,the expression conditions of the recombinant strain were optimized.We found that the optimal concentration of IPTG was 40?m and the optimal induction time was 16 h.In addition,the introduction of heterogenous gene had no effect on the growth of B.subtilis168.(2)Then,the enzymatic properties of the purified recombinant enzyme Ba-Sac B were investigated.The maximum specific activity of the enzyme was 283.77±0.3 U/mg in the reaction buffer containing 6 m M Ca Cl₂(pH 6.0)and the optimal reaction temperature was 40?.Ba-Sac B should be stored at low temperature and pH 7.0.The K_m and V_mvalues for Ba-Sac B were calculated to be 0.71 m M sucrose and 0.49?mol·(min·mg)^-1.The optimum conditions for the synthesis of levan catalyzed by Ba-Sac B are as follows:150?g/m L enzyme solution,200 g/L sucrose,20 m M Ca Cl₂,0.05 M Na₂HPO₄/Na H₂PO₄buffer(pH 7.0).The reaction temperature and time were 35?and 9 h,respectively.Under the above conditions,the sucrose conversion rate was 65.21%and the yield of levan was 39.17%.(3)Magnetic nanoparticles(Fe₃O₄MNPs)were prepared by hydrothermal method.After being modified by EDC-NHS,Fe₃O₄MNPs have good magnetic properties and can covalently combine with Ba-Sac B to form the immobilized enzyme Ba-Sac B@Fe₃O₄MNPs.Compared with the free enzyme,the immobilized enzyme showed better stability,higher tolerance to temperature and pH and could be reused.The best conditions for Ba-Sac B@Fe₃O₄MNPs to be applied to levan synthesis are as follows:150?g/m L Ba-Sac B@Fe₃O₄MNPs solution,200 g/L sucrose,5 m M Ca Cl₂,0.05 M Na₂HPO₄/Na H₂PO₄ buffer(pH 7.0).The reaction temperature and time were 45?and 9h.The sucrose conversion rate and yield of levan of the immobilized enzyme were 63.42%and 37.81%,respectively.At the same time,the characteristic peaks of Levan were also characterized by infrared method.