beta-Galactosidase of A. oryzae was used for oligosaccharide synthesis in aqueous-organic co-solvent mixtures. In a 50% (v/v) solvent system (water-miscible solvent mixed with citrate buffer), an oligosaccharide product (Oligo-I) was observed only in triethyl phosphate (TEP) or methoxyethyl acetate (MEA) co-solvents, while no product was observed with 1,4-butanediol, 1,5-pentanediol or acetonitrile mixtures. Determination of the amount of oligosaccharide by HPLC revealed that synthesis in TEP gave a 1.8 and 8.3 times higher product yield than in buffer alone and MEA solution, respectively. However, the yield obtained from synthesis by soluble (non-lyophilized) enzyme in TEP solution was only 22 +/- 0.5%. To overcome the low enzyme activity under co-solvent conditions, co-lyophilization of the enzyme from citrate buffer with the addition of cyclodextrin (CD), a group of macrocyclic excipients, was investigated. The enhancement of oligosaccharide synthesis by beta-CD was more than that of the hydroxypropyl or methyl derivative. A beta-galactosidase:beta-CD ratio of 1:5 (w/w) increased oligosaccharide synthesis in 50% (v/v) TEP by 28% compared to the synthesis attained by either non-lyophilized or lyophilized enzyme in the absence of CD. The optimal conditions for oligosaccharide synthesis in 50% (v/v)TEP using the beta-galactosidase-beta-CD co-lyophilizate were: 0.3 M lactose substrate incubated with 0.025 mg enzyme at pH 4.5, 40 degrees C for 36-72 h. In addition to the major Oligo-I product, a minor Oligo-II product was also observed. Yield of Oligo-I after optimization was 37 +/- 2%, an average increase over the non-optimized condition in the absence of beta-CD of 65%. Thus beta-CD can significantly enhance the oligosaccharide synthesis catalyzed by beta-galactosidase in a TEP co-solvent system. The product Oligo-I was characterized using MS and NMR techniques and was found to be the trisaccharide, O-beta-D-galactopyranosyl-(1-6)-O-beta-D-galactopyranosyl-(1-4)-D-glucop yranose or 6'-galactosyl-lactose. (C) 2008 Elsevier Inc. All rights reserved.