Construction Of Food-grade Expression Platform Of ?-Dextranase And Analysis Of Its Enzymatic Properties

During cane harvest,strorage and juice extraction,the accumulation of dextran produced by microorganisms would result in sucrose losses and energy over-consumption,and thus bring great challenge to sugar making industry.The application of dextranase is considered to be the most effective way to remove dextran in sugar cane juice.However,most of the ?-Dextranase-producing strains found so far were not have food safety grade.Chaetomium gracile could produce ?-Dextranase from ?-Dextran,and general recognized as safe(GRAS).Nevertheless,Chaetomium gracilis has many defects,such as longer fermentation time and lower yield,which affected its industrial application.Bacillus subtilis is recognized as a food safety host,and has been widely used in the microbial produciton of food enzyme.In this dissertation,the expression of ?-Dextranase derived from Chaetomium gracile in Bacillus subtilis was investigated.The main results are described as follows:1.The codon-optimized a-DEX gene was sub-cloned into vector pSTOP1622 and transformed into B.subtilis WB800,which derived from C.gracile,got a recombinant strain.Under the induction of 15 g/L xylose,the a-DEX yield reaches to 3.46 U/mL.Then the expression cassette double-p43 promoter was constructed to enhance the DEX yield,which up to 14.22 U/mL,310.98%higher than control.In addition,the fermentation condition of the double-p43 strain was optimized.The optimal carbon source was starch,and a-DEX yield reached to 13.38 U/mL;Also,metal ions Fe2+and Mg2+ increased the a-DEX yield by 32%and 15%,respectively;2.The codon-optimized a-DEX gene was integrant expressed in B.subtilis WB800 by replace the native amyE gene,and the knockout cassate was constructed,after transform we got a recombinant strain B.subtilis WB800-p43-DEX-(lox71-spc-lox66).To further delete the resistance marker gene spc,a temperature-sensitive plasmid pTSC,which secreted Cre recombinase,was transformed into B.subtilis WB800-p43-DEX-(lox71-spc-lox66).Then non-antibiotic integrated strain B.subtilis WB800-p43-DEX was screened.The strain B.subtilis WB800-p43-DEX was fermented in the medium,the OD600 reached the maximum of 1.766 at 14 h,and the enzyme activity was 6.21 U/mL;3.By analyzing the characterization of a-DEX from B.subtilis WB800-p43-DEX,the optimal pH was 5.0,the optimal temperature was 55?,?-DEX still maintain 87.97%of the enzyme activity when placed 45? in 1 h,and which still stay 98.69%of the enzyme activity while placed 4? in 28 h.the optimal reaction concentrations was 20 g/L,and the optimum substrate was dextran T-2000,the activation effect of Co2+ reached 21.25 U/mL,but Ag+ had significant inhibitory effects on a-DEX activity,which decreased 44%.