Screening Of Dextranase Producing Strains,Enzymetic Characteristics And Activity Modification

Dextran is the main impurity present in the sugar production process.The presence of dextran reduces the efficiency of sugar production,affects product quality,and damages equipment,therefore,causes economic losses.In addition,dextran will adhere to the tooth surface and gradually forms caries,causing oral diseases.Therefore,how to remove dextran in the process of making sugar and in the oral cavity is one of the hot spots of research at home and abroad.Dextranase（EC 3.2.1.11）can specifically hydrolyzeα-1,6 glycosidic bonds in dextran.Enzymes degradation method is currently the most effective method for hydrolyzing glucan,and is widely used in the sugar industry and stomatology.In this study,A strain that producing dextranase was screened from enrich sugar soil by the selective medium.After 16S r RNA sequence analysis and comparison,it was found that the similarity between the strain and Paenibacillus sp.,and it was named P.sp GXD-1.The gene of P.sp GXD-1 dextranase（dex）was obtained by homologous cloning.The size of dex is 2145 bp.DEX belongs to the exoenzyme type of glycoside hydrolase 66 family.Based on the expression vector p SE380,the soluble expression of DEX in E.coli BL21 was achieved.DEX was purified by Ni-NTA column affinity chromatography and determined by DNS method with dextran as substrate.The results showed that the optimum temperature of DEX was 40℃,the optimum p H was6.5,DEX to the substrate DEX-T2000 k_m=2.5926±0.09672 mg/m L,V_max=0.9578±0.05675μmol·min^-1mg^-1.The specific activity of DEX was11.3±0.529 U/mg.In p H 6-8 buffer solution for 12 hours,the enzyme activity remained at 80%.Metal ions Fe³⁺,Co²⁺,K⁺,Li⁺,Cr³⁺,Cu²⁺,Ni²⁺,Ca²⁺inhibited the enzyme activity of DEX in varying degrees,but the inhibition was not strong,and the enzyme activity was between60%-80%.PCR technology was used to cut out the CBM segment contained in dex,and the deletion plasmid p SE-dexΔcbm was constructed to determine the enzyme properties.The optimum temperature and p H value of the delectase DEXΔCBM were 40℃and 7.0.The K_mof DEXΔCBM to the substrate DEX-T2000 was 2.7495±0.06217 mg/ml,V_maxwas 1.8818±0.06217μmol·min^-1mg^-1,the specific activity was 22.3±1.09 U/mg,96%higher than that of the wild enzyme.After 30 minutes of holding at different temperatures,It was kept at 40℃for 30 minutes,The relative enzyme activity was 110%,100%at 30℃,at 90℃for 30 min enzyme activity still have 20%,and the wild enzyme was completely inactivated at 90℃.Mg^2+,Co²⁺,K⁺have certain activation effect,and the relative enzyme activity reaches 110%;Cu²⁺has the strongest inhibition effect on DEXΔCBM,only 50%of the relative enzyme activity is left,and Li⁺and Fe³⁺have no effect on the enzyme activity.Through sequence alignment and homology modeling,the 262amino acid residue（D）of wild enzyme was selected for site mutation,and the mutant D262G was obtained.Its enzymatic properties were studied.The results showed that the amino acid residue was predicted the key amino acid residue related to enzyme activity.The breeding of high-yield strains,the efficient expression of enzymes and the optimization of enzymatic properties have become urgent problems.The development and in-depth study of dextranase have great significance for the development of large-scale industrial production and the field of medicine.