Purification Of β-galactosidase From Bovine Liver And Regioselective Glycosylation Of Nucleosides By Using This Enzyme

Nucleoside analogs constitute a vital class of anticancer and antiviral agents. However, these nucleoside agents generally suffer from various severe side effects and poor oral bioavailability in the clinical application. It was reported that glycosylated derivatives of nucleoside drugs could mask their toxicity or/and improve their pharmacokinetic properties. Because the conventional chemical methods are characterized by low regioselectivity, the requirement of protection/deprotection, and being environmentally unfriendly, an efficient and selective enzymatic approach to disaccharide nucleosides with bovine liver β-galactosidase as the biocatalyst was established in this dissertation. Firstly, we purified and characterized β-galactosidase from bovine liver; then, with the fucosylation of floxuridine (FUdR) and the galactosylation of acyclovir mediated by this β-galactosidase as model reactions, the effects of key parameters on the enzymatic reaction were examined, respectively; finally, enzyme substrate recognition in the glycosylation of nucleosides was explored and a group of β-fucosyl and β-galactosyl-containing disaccharide nucleosides were synthesized.β-Galactosidase from bovine liver was purified62-fold to homogeneity by successive ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography and gel filtration chromatography, with a activity yield of24%. Its molecular mass was estimated to be54KDa by SDS-PAGE, and to be60KDa by gel permeation chromatography, and to be57KDa by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) tandam mass spectrum. With p-nitrophenyl-β-D-galactopyranoside (pNPGal) as the substrate, it displayed the optimum activity at pH7.0and40℃, and was stable at temperatures of＜40℃and in a pH range of4.5-8.0. Its activity was completely inhibited by5mM Hg2+and Ag+. The substrate specificity study revealed that this enzyme displayed the highest catalytic efficiency toward pNPGal (Vmax/Km value,0.0173min-1), and lower toward p-nitrophenyl-β-D-fucopyranoside (pNPF,0.0156min-1) and p-nitrophenyl-β-D-glucopyranoside (pNPGlu,0.0126min-1).With the enzymatic fucosylation of floxuridine (FUdR) as a model reaction, the effects of four key conditions on the enzymatic reaction were studied. The optimal enzyme dosage, buffer pH, temperature and substrate molar ratio (FUdR/pNPF) were0.104U/mL,7.0,40℃and3:1, respectively. Under the optimum conditions, the initial reaction rate, the maximum yield and the5’-regioselectivity were0.30mM/h,52%and97%, respectively. And the enzymatic synthesis of a group of β-fucosyl-containing disaccharide nucleosides using the purified β-galactosidase was conducted. The desirable5’-O-β-D-fucosyl derivatives of pyrimidine nucleosides were obtained with44-60%yields. Besides, the5’-regioselectivities decreased with increasing bulk of5-substituents present in the base moiety of nucleosides. In addition, this enzyme could accept acyclic nucleoside analogs (acyclovir and ganciclovir) as the substrates and catalyze fucosylation of these nucleosides, furnishing the glycosylated products with the yields of36%and32%, respectively.With the enzymatic galactosylation of acyclovir as a model reaction, four key reaction conditions were optimized. The optimal enzyme dosage, buffer pH, temperature and acyclovir/oNPGal were0.225U/mL,7.0,40℃and2.5:1, respectively. Under the optimum conditions, the initial reaction rate and the maximum yield were0.40mM/h and26%, respectively. And four β-galactosyl-containing disaccharide nucleosides using this β-galactosidase were synthesized with the yields of25-71%.This study not only enriches fundamental enzymology knowledge of β-galactosidase from bovine liver, but also provides a novel and efficient route to regioselective glycosylation of nucleosides. In addition, several novel glycosylated derivatives of nucleosides have been successfully synthesized.