Glycosynthase activity of Geobacillus stearothermophilus GH52 β-xylosidase: Efficient synthesis of xylooligosaccharides from α-D-xylopyranosyl fluoride through a conjugated reaction

Alon Ben-David, Tsafrir Bravman, Yael S. Balazs, Mirjam Czjzek, Dietmar Schomburg, Gil Shoham, Yuval Shoham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

54 Scopus citations

Abstract

Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 β-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using α-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction of α-D-xylopyranosyl fluoride, providing mainly α-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis-Menten behavior, with kinetic constants of 1.3 s-1 and 2.2 mM for kcat and KM(acceptor) respectively, and a kcat/ KM(acceptor) value of 0.59 s-1 mM-1. When the β-xylqsldase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable α-D-xylopyranosyl fluoride as the sole substrate.

Original languageAmerican English
Pages (from-to)2145-2151
Number of pages7
JournalChemBioChem
Volume8
Issue number17
DOIs
StatePublished - 23 Nov 2007

Keywords

  • Enzymes
  • Glycoside hydrolases
  • Glycosynthases
  • Green chemistry
  • Xylo oligosaccharides

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