title: Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans.
abstract: The enzymatic access to nucleotide-activated oligosaccharides by a glycosidase-catalyzed transglycosylation reaction was explored. The nucleotide sugars UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta-galactosidase 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 1H and 13C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 degrees C instead of 30 degrees C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at -5 degrees C and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overall yield of 8.2% (81.8 micromol, 62.8 mg with 100% purity) for Gal(beta1-4)GlcNAc(alpha1-UDP) and 3.6% (36.1 micromol, 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 micromol, 32.3 mg with 93% purity) for Gal(beta1-4)Glc(alpha1-UDP) and 1.6% (13.0 micromol, 12.2 mg with 95% purity) for Gal(beta1-4)Gal(beta1-4)Glc(alpha1-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 circulans.
authors: Institute of Enzyme Technology, Heinrich-Heine-University Düsseldorf, Research Center Jülich, Jülich, Germany.
authors: A
authors: A
authors: Zervosen
authors: V
authors: V
authors: Nieder
authors: R G
authors: RG
authors: Gallego
authors: J P
authors: JP
authors: Kamerling
authors: J F
authors: JF
authors: Vliegenthart
authors: L
authors: L
authors: Elling
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D001671:Biochemistry / Q000379:methods; D002240:Carbohydrate Sequence; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D014539:Uridine Diphosphate Sugars / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D009844:Oligosaccharides; C433831:UDP-lactose; D014539:Uridine Diphosphate Sugars; C083732:uridine diphosphate N-acetyllactosamine; D001616:beta-Galactosidase