Study On Prokaryotic Expression And Enzymatic Properties Of ?-GalActosidase From GeobaciLLus Thermodenitrificans

?-GalActosidase,alias melibiase,It is widespread in nature.?-GalActosidase an exoglycosidase and it can specifically catalyze polysaccharides,glycoproteins,glycolipids containing?-1.6 galactosidic linkages at the ends of sugar chains,it can Catalyzes substrate such as verbascose,raffinose,stachyose,melibiose and some heteropolysaccharides containing?-galactosidase.At present,the isolated?-galactosidase is mostly normal temperature enzyme,normal temperature enzyme greatly limits the use of this enzyme in high temperature industries.Thermophilic alpha-galactosidase sources limited,mainly isolated from naturally occurring thermophile strains,however,the separation process is cumbersome and the yield is extremely low and cannot be satisfied with production applications.Therefore,This study uses molecular biology,microbiology,and bioinformatics technologies and synthesis of thermophilic thermobacterlus?-galactosidase gene encoding thermophilic bacteria Geobacillus thermodenitrificans,Construction of?-galactosidase Engineered Bacteria Using Prokaryotic Expression System.The enzymatic properties and active sites were studied,It provides a theoretical basis for the subsequent molecular modification of the enzyme.The main experimental contents and results of this study are as follows:1.This experiment was based on the thermophilic Geobacillus thermodenitrificans?-galactosidase amino acid sequence provided by the Gen Bank database?Login ID:WP?011887668?.We design Eco R I and Bam H I sites,gene company synthesizes the coding gene.Recombining the gene of interest into a expression vector p GEX-4T-3,andp GEX-4T-GalA transformed into competent E.coil BL21?DE3?,thus Obtained recombinant engineering bacteria after verification and sequencing.2.Through bioinformatics methods,the enzyme was found to belong to the 36th family of glycoside hydrolases,physicochemical properties show,Molecular formula is C₃₇₆₇H₅₇₅₇N₁₀₄₁O₁₀₈₃S₂₂,p I is 6.29,molecular weight is 83.66KDa.No transmembrane region was identified in the transmembrane prediction.The protein is not a membrane protein,use Network Server PSIPRED to Predict Protein Secondary Structure,It was found that the protein was composed of a large number of committed-Sheel and garden-helix,in 3D structure prediction,discovering a suspected active site in a three-dimensional structure.3.The recombinant plasmid E.coil BL21-p GEX-4T-GalA was Induced Expression and Purification of Recombinant Proteins.The results indicate that 28??0.08mmo L/L IPTG,induce 5 hours is Optimum.At this point the protein of interest exists in a soluble form,Purification using affinity chromatography,The concentration of purified protein was determined,The results is 14.443?g/m L,enzyme activity is 82.385U/mg.4.pNPG as substrate,Study the enzymatic properties of the enzyme,results show The optimal temperature of the enzyme is 70?,the optimal p H was p H 7.0,The enzyme has good thermal stability and p H stability,The impact of metal ions on?-galactosidase activity displayed that?-galactosidase activity was activated by Fe³⁺?Ca²⁺?Mn²⁺?Zn²⁺,Zn²⁺has the best activation effect on?-galactosidase,Cu²⁺,Ag⁺,and Hg⁺can completely inhibit enzyme activity.Determine the kinetic constants of the enzyme,result that Km is 10.04mmo L/L,Vmax is 18.25?mo L/min.5.Molecular docking by using Auto Dock and the best molecular conformation was selected for molecular dynamics simulation.The binding energy between small-molecule raffinose and?-galactosidase was calculated using MM-PBSA method,The results show that van der Waals interactions,electrostatic interactions and non-polar solvation free energies are the main driving forces for the formation of complexes.Through study on the contribution of each amino acid residue,ASN-334?TRP-336?GLU-337?TYR-340?ASP-367?TRP-411?ASP-548?SER-576?ALA-577?PRO-579 and ASP-606 that is an amino acid residue that plays a key role in the binding of small molecule cottonseed sugar and prun-galactosidase.