Improvement of oligosaccharide synthetic activity of Beta-galactosidase in organic solvents by using cyclodextrins

Abstract

Synthesis of Oligosaccharides from lactose catalyzed by Aspergillus oryzae beta-galactosidase was performed in the presence of aqueous-organic solvent mixtures. By using TLC analysis, an oligosaccharide product (Oligo-I, Rf 0.18) was detected when using citrate buffer containing triethyl phosphate (TEP) or methoxyethyl acetate (MEA), while no product was observed with 1,4-butanediol, 1,5-pentanediol or acetonitrile (all solvents were 50% v/v mixed with citrate buffer). When the amount of oligosaccharide was determined by HPLC, synthesis in TEP solution gave 1.6 and 6.8 times higher product yield than in buffer alone and MEA solution, respectively. However, yield obtained from synthesis by soluble (non-lyophilized) enzyme in TEP solution was only 18.5%. To overcome low enzyme activity under co-solvent condition, co-lyophilization of the enzyme from citrate buffer with the addition of cyclodextrin (CD), a group of macrocyclic excipients,was investigated. Comparing with its hydroxypropyl and methyl derivatives, beta-CD worked best. Beta-galactosidase: beta-CD at1:5 w/w increased oligosaccharide synthesis in 50% TEP by 20.9% compared to synthesis from enzyme lyophilized in the absence of CD or non-lyophilized enzyme. The optimal condition for oligosaccharide synthesis in 50%TEP using the beta-galactosidase-beta-CD co-lyophilizate was 0.3M lactose incubated with 0.025 mg enzyme at pH 4.5 , 40 degree Celsius for 36-72 hours. In addition to the major Oligo-I product, a minor Oligo-II (Rf 0.10) product was also observed. Yield of Oligo-I after optimization was 31.6%, an increase over the non-optimized condition in the absence of beta-CD of 63.6%.Thus beta-CD could significantly enhance oligosaccharide synthesis catalyzed by beta-galactosidase in TEP co solvent system. When oligosaccharide products were analyzed by MS and NMR techniques, Oligo-I is characterized as 0-beta-D-galactopyranosyl-(1-6)-0-beta-D-galactopyranosyl-(1-4)-D-glucopyranose or 6’-galactosyl-lactose,and Oligo-II is a tetrasaccharide mixed with a pentasaccharide.