Functional, Structural And Catalytic Mechanism Analysis Of Mutagenesis Dextransucrase

We constructed the engineering bacteria of the dextransucrase in Escherichia coli expression system by cloning the dex-YG gene of dextransucrase(EC 2.4.1.5)from Leuconostoc mesenteroides 0326.Dextransucrase can synthesize ?-glucan by hydrolysis of Glucose using sucrose as substrate.In this paper,structure and function of dextransucrase were investigated by truncated,mutated,amino acid insert mutation of the dextransucrase gene dex-YG to expand its application areas.The catalytic functions of dextransucrase were analyzed by different truncation of the fragments of dextransucrase.Mutant enzymes with different catalytic functions were obtained by deleting different fragments.The amino acid of catalytic region were replaced,embedded to investigate different enzymatic properties of mutant enzymes in order to explore the mechanism of product specificity and obtain the dextran products with different properties for expanding the application of the enzyme.1.The gene sequence dex-YG,the secondary structure and the tertiary structure were predicted by the analysis of biological information.The C-terminal sequence was truncated and analyzed to study structure and function of dextransucrase.The region of ?-glucan chain extension,the oligosaccharide synthesis,and the completely conserved region were studied by fragment truncation.Repeated sequence deletion at the end of dextransucrase would greatly affect the ability of its synthesis of dextran.As the length of the truncated fragment increases,and its ability to synthesize the polymer dextran decreases sharply.After a certain length of truncation,the synthesis of dextran ability were completely inactive.The ability of synthesis of oligosaccharides was significantly improved.As the further fragment was truncated until its conserved sequence motif I,the catalytic activity of the oligonucleotides was also significantly decreased,almost completely inactive.2.Dextransucrase belongs to the family of GH70,and the catalyzed mechanism of the family is completely conserved.All of GH70 enzymes can synthesize the?-glucan product with sucrose as the substrate,but the glycosidic bonds of ?-glucan products from different types of GH70 enzyme have obvious difference,including ?(1-2),?(1-3),?(1-4),?(1-6)glycosidic bonds.Based on the analysis of molecular docking and kinetics of dextransucrase,the key amino acids of the receptor and the substrate binding region were replaced by the analysis of its effect on the synthesized product.The mechanism of product specificity was explored by analysis of themolecular modeling structure.More dextran products can expand the application of dextransucrase.By changing the amino acid of the catalytic region,the glycosidic bonds of the dextran 5% ?(1-3)and 95% ?(1-6)bonds of the original dextran was changed to 1-9% ?(1-3)and 90-98% ?(1-6)bond,and some mutations dextran produce additional ?(1-2)and ?(1-4)bonds.Simulation analysis can be found that the size of the side chain of the replacement amino acid,and the nature of the charge hydrophobicity will have a greater impact on the receptor of the most stable conformation,thus affecting the nature of the enzyme and product specificity.3.The amino acid substitution of the catalytic pocket will affect the bond type of the product,but its variation has some limitations.The structure of the product dextran was changed to a greater extent by embedding the amino acid on the critical site of the conserved sequence but not directly with the substrate or receptor.By homologous recombination,663 and 553 site were inserted of amino acids.Through the screening of active strains,as well as synergistic mutations obtained,the highly branched dextran product mutants were obtained.Amino acid embedding method,although it would affect the enzyme activity to a certain extent,but its active mutant its catalytic properties change was more significant.With further synergistic mutantion,the product specific changes more significant.Different properties of dextran products also provide a basis for subsequent research and application.In summary,the structure and function of dextransucrase were studied by molecular means,and the mutant enzymes with different catalytic properties and different mutants were obtained,which laid the foundation for the subsequent research,application and modified dextransucrase.