@article {1436, title = {Glycosynthase-based synthesis of xylo-oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi}, journal = {Organic \& Biomolecular Chemistry}, volume = {4}, number = {10}, year = {2006}, note = {ISI Document Delivery No.: 041NHTimes Cited: 20Cited Reference Count: 39}, pages = {2025-2032}, type = {Article}, abstract = {Glycosynthases are synthetic enzymes derived from retaining glycosidases in which the catalytic nucleophile has been replaced. The mutation allows irreversible glycosylation of sugar acceptors using glycosyl fluoride donors to afford oligosaccharides without any enzymatic hydrolysis. Glycosynthase technology has proven fruitful for the facile synthesis of useful oligosaccharides, therefore the expansion of the glycosynthase repertoire is of the utmost importance. Herein, we describe for the first time a glycosynthase, derived from a retaining xylanase, that synthesizes a range of xylo-oligosaccharides. The catalytic domain of the retaining endo-1,4-beta-xylanase from Cellulomonas fimi (CFXcd) was successfully converted to the corresponding glycosynthase by mutation of the catalytic nucleophile to a glycine residue. The mutant enzyme (CFXcd-E235G) was found to catalyze the transfer of a xylobiosyl moiety from alpha-xylobiosyl fluoride to either p-nitrophenyl beta-xylobioside or benzylthio beta-xylobioside to afford oligosaccharides ranging in length from tetra- to dodecasaccharides. These products were purified by high performance liquid chromatography in greater than 60\% combined yield. H-1 and C-13 NMR spectroscopic analyses of the isolated p-nitrophenyl xylotetraoside and p-nitrophenyl xylohexaoside revealed that CFXcd-E235G catalyzes both the regio- and stereo-selective synthesis of xylo-oligosaccharides containing, exclusively, beta-(1 {\textendash}> 4) linkages.}, keywords = {4-GLYCANASE, BETA-1, DIRECTED EVOLUTION, EFFICIENT SYNTHESIS, ENZYMATIC-SYNTHESIS, GLYCOSIDASES, MASS-SPECTROMETRY, SPECIFICITY, SUBSTRATE, TRANSGLYCOSYLATION, XYLOOLIGOSACCHARIDES}, isbn = {1477-0520}, url = {://000237462400022}, author = {Kim, Y. W. and Fox, D. T. and Hekmat, O. and Kantner, T. and McIntosh, L. P. and Warren, R. A. J. and Withers, S. G.} }