| Levan has extensive and important applications in many fields,such as pharmaceuticals,food industry,cosmetics,nano-materials,and so on.In order to obtain large amounts of levan,two methods are usually used,including microbiological fermentation and enzymatic synthesis.However,the yield and conversion rate of levan produced by microbiological fermentation are relatively low,and other polymers in the fermentation broth are not conducive to the large-scale purification of levan.Therefore,enzymatic synthesis has become the main method for in industrial producti of levan.The ability of levansucrase from different sources and structures to synthesize levan is different,and correspondingly,their products have different properties.Therefore,the key to enzymatically synthesize levan is how to develop new levansucrase and improve its utilization efficiency.Zymomonas mobilis invertase SacC(Zm-SacC)belongs to glycoside hydrolase family 68,and it has a high sequence identity with levansucrase SacB,however,there are few studies on its function to synthesis levan.In this dissertation,the molecular evolution of sacC gene of Z.mobilis,the catalytic activity of SacC,the catalytic molecular mechanism and the production of levan by yeast cell surface system,were studied to provide the basis for revealing the molecular mechanism of SacC catalysis and further producing levan.The main results are as follows:(1)It was found that Zm-SacC and Zm-SacB has a common ancestor,and SacC retained the typical structure of levansucrase.At the same time,SacC fusion protein was obtained by prokaryotic expression.Based on the evolutionary tree analysis,it was found that sacB and sacC genes may originate from levansucrase(AAT81165.1)with a common ancestor of Firmicutes,Leuconostoc mesenteroides,and then replicated in Proteobacteria to form sacB and sacC homologies.Then the pET-32a(+)-sacC recombinant plasmid was constructed and transformed into Escherichia coli BL21(DE3)to achieve efficient heterologous expression of SacC recombinant protein.High purity SacC protein was obtained through the optimization of expression condition and protein purification.(2)Zm-SacC has levansucrase activity and can synthesize high molecular weight levan.The optimal hydrolysis and transfer conditions of SacC are quite different.Polysaccharides were prepared with sucrose as substrate.Through the analysis and identification(through.TLC,HPLC,GPC)of its reaction products,it was confirmed that SacC has the levansucrase activity and can synthesize macromolecular levan.The results showed that the optimal hydrolysis conditions of SacC were pH 6.0 and 40℃,while the optimal transfer conditions were pH 5.5 and 25℃.When the temperature is higher than 30℃,or the pH is higher than 7.0,it will cause to a large loss of its transferase activity.In addition,its transferase activity has poor temperature tolerance.The results showed that the highest yield of levan was 4.5 g/L at 25℃ and 40%sucrose concentration.At low sucrose concentration(20%),the molecular weight(Mw)of the product was low(10 kDa),while at high sucrose concentration(50%),the product mostyly has high Mw levan(3,800 kDa),indicating that SacC is well-tolerated to high sucrose concentration.Futhermore,the synthesized product has high Mw levan(2,300 kDa)with a small polydispersity(PD,2.06)under low temperature,while it has low Mw levan but much more diverse(Mw=506 kDa,PD=86.93)under high temperature.(3)The catalytic mechanism of Zm-SacC was clarified,which laid the foundation for the subsequent rational design.The catalytic mechanism of SacC was studied through homology modeling and molecular docking.The results showed that the mechanism of glycoside hydrolysis catalyzed by SacC is a retention mechanism.The catalytic process was as follows:In the first step,sucrose is bound to the active site,and the fructosyl residue is stabilized by the transition state stabilizer Asp 192.The acid-base catalyst,Glu276,acts as a general acid,to protonate the glycosidic oxygen of sucrose.Glucose is release,and oxocabenium ion of the fructosyl residue is formed.Then,a nucleophile Asp44 attacks C2 of the oxocabenium ion,and the covalent fructosyl-enzyme intermediate is formed.In the second step,sucrose binds to the receptor-binding site.Glu276 acts as a general base removing a proton from O6 at the end of the receptor.Then,this O6 attacks the fructosyl C2 of the covalent fructosyl-enzyme intermediate,creating the β-(2,6)linkage to extend the levan chain.Finally,the bond between the fructosyl and Asp44 is broken,and the product is released.At the same time,bioinformatics tools were used to analyze the structure of SacC and explore the function of key amino acid residues.The results showed that there are a large number of salt bridges(23)in the protein,among them,Glu276 forms salt bridges with Arg191 and His294,and interacted with many amino acid residues around,which plays an important role in the stability of the protein.Many loops of SacC have high B-factor and RMSD values;it was found that His294 played an auxiliary role in localization by interacting with the glucose group of sucrose.(4)The mutants with increased enzyme activity and stability were obtained through molecular modification.Seven sites were selected through molecular docking and kinetic simulation,and seven SacC mutants were constructed through whole plasmid site-directed mutagenesis.It was found that H294R significantly enhanced the transfer activity of SacC,and the introduction of disulfide bonds can significantly improve the stability of SacC.By unifying these two mutants,a multi-site mutant was obtained,which can significantly improve the enzyme’s thermostability(3 times),transfer activity(35 U/mg→138 U/mg),yield(4.5 g/L→12.4 g/L),Mw(2,500 kDa→4,990 kDa)and homogeneity(PD,254→31.15).(5)The effects of different surface display systems were explored to improve the utilization efficiency of SacC.The multi-site mutant was immobilized on the cell wall of Saccharomyces cerevisiae by surface display through N-terminal fusion(a-agglutinin)and C-terminal fusion(Flolp).The results showed that the whole-cell display technique not only improves the stability of SacC,but also realizes the repeated use of SacC.In addition,the yield of levan synthesized by the a-agglutinin system and the Flolp system were 13.4 g/L and 15.2 g/L,respectively,while it was 11.6 g/L by the free enzyme.Moreover,it was found that the surface display system is conducive to synthesize low Mw levan.The average molecular weights produced by the two surface display systems were 480 kDa(Flolp)and 640 kDa(a-agglutinin),respectively,which is significantly lower than that of the free enzyme product(5,200 kDa).Based on this work,the surface display technology was used to produce levan with waste molasses,and the applications of several levans with different molecular weights were further explored.The results showed that levl(8.7 kDa)has a good prebiotic effect,while lev3(5,200 kDa)can be used as a cell protective agent alone.In summary,SacC has been proved to have the activity of levansucrase,and its catalytic mechanism has been preliminarily elucidated in our work.Through the rational design of its molecular modification,the mutants with significantly improved stability of transferase activity was obtained.Finally,yeast surface display was carried out to expand the application range of SacC,which provided a new option for the production of levan. |
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