Using D-glucurono-6,3-lactone (GlcL) and sucrose (Suc) as raw materials, we synthesized sucuronic acid (SucA), in which the D-glucose (Glc) residue of Suc was replaced with D-glucuronic acid, by a three-step chemoenzymatic method. In the 1st chemical step, methyl D-glucuronate (GlcAM) was synthesized by treating GlcL with a strong base anion exchange resin, Amberlite IRA402BL OH AG, in anhydrous methanol. In the 2nd step, which included an enzyme reaction, methyl sucuronate (SucAM) was synthesized from GlcAM and fructose by exploiting the transfructosylation activity of the Microbacterium saccharophilum K-1 beta-fructofuranosidase, a reaction that is suppressed in the presence of high-concentration Glc. In this reaction, the addition of a Suc-non-assimilating yeast, Saccharomyces bisporus NBRC1131, to the reaction mixture increased the amount of SucAM generated, because Glc was removed from the mixture by this yeast. In the 3rd chemical step for producing sodium sucuronate (SucANa), SucAM was treated with Amberlite IRA402BL OH AG in water to hydrolyze SucAM's ester bond, and product was then treated with NaOH. The molar yield of SucA.Na from GlcL was 34.2%. SucA was stable at 37 degrees C in buffer solutions at pH 3, 5, 7, or 9. However, at temperatures exceeding 75 degrees C, the glycosidic bond of this disaccharide was hydrolyzed not only in acidic buffers (pH 3 and 5) but also in alkaline buffer (pH 9). SucA was not a suitable substrate for the beta-fructofuranosidases of M. saccharopilum K-1 and Saccharomyces cerevisiae.