To produce N-acetylneuraminic acid (Neu5Ac) efficiently, a coupling reaction of N-acetylglucosamine (GlcNAc)-2-epimerase and sialic acid aldolase was performed in one-pot using GlcNAc and pyruvate as substrates. Using a recombinant human renin binding protein (rhRnBp) showing GlcNAc-2-epimerase activity and Escherichia coli sialic acid aldolase, about 80% conversion yield of Neu5Ac was obtained in the coupling reaction under 10-fold excess of pyruvate to GlcNAc based on the initial concentration of GlcNAc. The equilibrium of GlcNAc-2-epimerase reaction was not affected by temperature, whereas that of sialic acid aldolase reaction was shifted toward Neu5Ac by lowering the reaction temperature. Low temperatures improved the conversion yield of Neu5Ac, but decreased the reaction rate in the coupling reaction. A high reaction rate as well as a high conversion yield could be achieved by shifting the temperature of the coupling reaction during the reaction. (C) 2004 Elsevier Inc. All rights reserved.