Reference : Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillu...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
Life sciences : Biotechnology
Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans.
Zervosen, Astrid mailto [Research Center Julich (Julich, Germany) > > > >]
Nieder, Veronika [> > > >]
Gallego, Ricardo Gutiérrez [> > > >]
Kamerling, Johannis P [> > > >]
Vliegenthart, Johannes F. G. [> > > >]
Elling, Lothar [> > > >]
Biological Chemistry
Walter de Gruyter
Yes (verified by ORBi)
[en] Bacillus/enzymology ; Biochemistry/methods ; Carbohydrate Sequence ; Hydrogen-Ion Concentration ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Molecular Sequence Data ; Nucleotides/chemistry/metabolism ; Oligosaccharides/chemical synthesis/metabolism ; Stereoisomerism ; Substrate Specificity ; Temperature ; Uridine Diphosphate Sugars/chemical synthesis/chemistry/metabolism ; beta-Galactosidase/chemistry/metabolism
[en] 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.
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