The noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn 2؉ significantly increased the epimerization rate from D-fructose to D-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn 2؉ . The turnover number (k cat ) and catalytic efficiency (k cat /K m ) of the enzyme for D-psicose were markedly higher than those for D-tagatose, suggesting that the enzyme is not D-tagatose 3-epimerase but D-psicose 3-epimerase. The equilibrium ratio between D-psicose and D-fructose was 32:68 at 30°C. D-Psicose was produced at 230 g/liter from 700-g/liter D-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%.The International Society of Rare Sugars defines rare sugars as monosaccharides and their derivatives that rarely exist in nature. According to this definition, D-psicose is a rare sugar. Rare sugars have recently attracted much attention due to their many uses, including uses as low-calorie sweeteners and bulking agents (11,12,14,17,18), as immunosuppressants in allogeneic orthotopic liver transplantation in rats (6), as potential inhibitors of various glycosidases (20), in ischemiareperfusion injury of the rat liver (5), and in segmented neutrophil production without other detrimental clinical effects (21). The rare sugars can be made by microbial and enzymatic reactions with ketose epimerase, aldose isomerase, aldose reductase, and oxidoreductase. Bioproduction strategies for all rare sugars have been illustrated with ring form structures (4).D-Psicose, a carbon-3 epimer of D-fructose, is present in small quantities in commercial carbohydrate or agricultural products. This rare sugar is absorbed poorly in the digestive tract (19), has zero energy for growth, is a useful sweetener used as an aid for weight reduction (18), represses hepatic lipogenic enzyme activity (16), and is nontoxic (17). Even though few studies have investigated the ability of D-tagatose 3-epimerase to convert D-fructose into D-psicose (7-9, 22), only one enzyme source, D-tagatose 3-epimerase from Pseudomonas cichorii, has been used for D-psicose production (8). The enzyme was purified and characterized (8), and the D-tagatose 3-epimerase gene was then cloned on the basis of the Nterminal amino acid sequence of the purified D-tagatose 3-epimerase. The gene was expressed in Escherichia coli and then characterized (7).In this study, the noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in E. coli. Biochemical properties such as metal ions, pH, temperature, epimerization reactions, and kinetic parameters were investigated. Determination of the equilibrium ratio and bioconversion between D-fructose and D-psicose by the D-psic...
