Enzymatic Synthesis Of D-tagatose From Lactose With The Combination Ofβ-Galactosidase And L-arabinose Isomerase

D-tagatose, a rare natural hexoketose, is one of the rare sugars. D-tagatose is a C-4fructose epimer, with92%of the sweetness of sucrose. It can be considered as a possible alternative bulking sweetener. The studies have show that D-tagatose can be used in food and cosmetics safely.The processe for the conversion from lactose to D-tagatose wasresearched in this paper. The recombinant strain containing both β-galactosidase and L-arabinose tandem recombinant plasmidwas constructed. When the resuspended cells were used as enzyme source,the lactose can be degraded into D-Glucose and D-galactose, then the later was converted into D-tagatose.The gene encoding β-Galactosidase(lacZ) from Escherichia coli K-12was cloned into expression vector pET-28a(+), then the recombinant plasmid was transformed into the strain E. coli BL1(DE3). Therefore, the recombinant strain(E-lacZ) expressing β-Galactosidase was successfully constructed. The resuspended cells were used as enzyme source and lactose as substrate, the degradation rate of lactose could reach to90%or more at40～50℃and pH7.0. The addition of Mn2+ions could increase lactose activity.The genes encoding L-arabinose isomerase and一-Galactosidase from Escherichia coli K-12were cloned into expression vector pET-28a(+) in tandem, and the rbs site was inserted in the upstream of the lacZ genes to ensure these two genes could be expressed in host bacteria simultaneously. Then the tandem recombinant plasmid was transformed into the strain E.coli BL1(DE3). The recombinant strain(E-araA-lacZ) simultaneously expressing一-Galactosidaseand L-arabinose isomerase was successfully constructed.(3-Galactosidaseand L-arabinose could be highly expressed simultaneously in soluble form after induction with IPTG. To investigate the impact of the concentration of substrate, temperature, pH and divalent metallic ion on the conversion of lactose to D-tagatose, the resuspended cells were used as enzyme source and lactose as substrate, the results showed that the optimal temperature for the D-galactose isomerization reaction was50℃. The rate of production could reach21%or more at pH7.0. The addition of Mn2+enhanced the conversion of lactose to D-tagatose.