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%.