The Production Of D-tagatose From Lactose Using ?-galactosidase And L-arabinose Isomerase

D-Tagatose,as a new type of low calorie functional sweetener,has many important physiological functions such as anti caries and reduced the blood sugar.The sweetness of D-tagatose is 92%that of sucrose.the taste is similar with sucrose.the energy is much lower than the traditional sweetener.and has a good prospect in application.L-arabinose isomerase(LAI)is the most effective enzyme in the production of D-tagatose by biological method.In order to decrease the production cost,D-tagatose was produced using the low cost lactose by using the combinant enzymes of ?-Galactosidase and L-arabinose isomerase in this study.The optimal preparation method of aminated silica gel for ?-galactosidase immobilization was obtained by comparing different amino-silica gel preparation methods.Then,the immobilization method of ?-galactosidase using the amino-silica gel prepared above was optimized.The crosslinking agent concentration of glutaraldehyde was 2%,the cross-linking time was 3 h,the immobilization pH was 6,the immobilization temperature was 30 ? overnight,and the ratio of carrier to enzyme was 1:1000(g:U).Under the optimal conditions,the immobilization efficiency of the immobilized enzyme reached 60%.The optimum reaction conditions for the immobilized ?-galactosidase were 55 ?and pH 5.0.Under the optimal conditions,the activity of the immobilized enzyme reached 560±10 U/g.After 10 rounds of batch reaction experiments,the activity of the immobilized enzyme decreased slightly,and showed good reutilization ability,which was suitable for industrial production and utilization.After the hydrolysis of 2 h,the conversion rate of lactose reached more than 95%,which prepared the substrate for production of D-tagatose in the next step.The L-arabinose isomerase(LAI)(GenBank:HM150718.1)gene from Lactobacillus.fermentum CGMCC 2921 were synthesized and cloned,the plasmid pET-22b-araA was successfully constructed and transformed into Escherichia coli BL21-GroEL-GroES(DE3).The recombinant E.coli BL21(DE3)(pET-22b-araA)was induced and expressed,and the relative molecular mass of the recombinant enzyme was about 55 KDa.The concentration of IPTG for induction was 1 mM,and the induction temperature was 25 ? overnight.The optimal reaction conditions for the free enzyme are as follows:the temperature 60-65 ? and pH 8.0.The enzyme has good heat resistance,and alkali resistance up to pH 9.0.The activation effect of Mn2+ on the enzyme activity of the free enzyme was most obvious at 1 mM.The matrix for immobilization of L-arabinose isomerase was tested with different resins and the results showed that amino resin of SQ-100EA is the best in immobilization of the crude enzyme solution.The optimal immobilization conditions were optimized with the concentration of glutaraldehyde of 1%,25 ?,pH 7.0,the ratio of the carrier to the crude enzyme of 1:130(g:U),and immobilization for 3 h.Addition of 1 mM Mn2+ in the immobilization process can not only increasse the activity,but also the heat resistance of the immobilized enzyme.The optimum immobilization conditions were 60 ?,pH 8.0.Under the optimal conditions,the activity of the immobilized enzyme reached 49±2 U/g.The heat and pH resistance of the immobilized enzyme were improved compared with the free enzyme.In convertion of lactose into D-tagatose by using the immobilized ?-Galactosidase and L-arabinose isomerase,the conversion rate of D-tagatose reached 12%in 2 h and 23%in 48 h.