Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans

The enzymatic access to nucleotide-activated oligosaccharides by a glycosid ase-catalyzed transglycosylation reaction was explored. The nucleotide suga rs UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta -gala ctosidase from Bacillus circulans using lactose as donor substrate. The UDP -disaccharides Gal(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacNAc) and Gal(beta1-4) Glc(alpha1-UDP) (UDP-Lac) and the UDP-trisaccharides Gal(beta1-4)Gal(beta1- 4)GlcNAc(alpha1-UDP and Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) were formed stereo- and regioselectively. Their chemical structures were characterized by H-1 and C-13 NMR spectroscopy and fast atom bombardment mass spectromet ry, The synthesis in frozen solution at -5 degreesC instead of 30 degreesC gave significantly higher product yields with respect to the acceptor subst rates. This was due to a remarkably higher product stability in the small l iquid phase of the frozen reaction mixture. Under optimized conditions, at -5 degreesC and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overal l yield of 8.2% (81.8 mu mol, 62.8 mg with 100% purity) for Gal(beta1-4)Glc NAc(alpha1-UDP) and 3.6% (36.1 mu mol, 35 mg with 96% purity) for Gal(beta1 -4)Gal(beta1-4)GlcNAc(alpha1-UDP) was obtained. UDP-Glc as acceptor gave an overall yield of 5.0% (41.3 mu mol, 32.3 mg with 93% purity) for Gal(beta1 -4)Glc(alpha1-UDPU and 1.6% (13.0 mu mol, 12.2 mg with 95% purity) for Gal( beta1-4)Gal(beta1-4)Glc(beta1-UDP). The analysis of other nucleotide sugars revealed UDP-Gal, UDP-GalNAc, UDP-Xyl and dTDP-, CDP-, ADP- and GDP-Glc as further acceptor substrates for beta -galactosidase from Bacillus circulan s.