The transglycosylation ability of wild-type (WT) CfMan2A and variants (collectively "CfMan2A forms") was evaluated with 5 mM p-nitrophenyl-β-D-mannopyranoside (β-D-ManpOpNP) acting as both donor and acceptor substrate. Hence in the transglycosylation reaction a mannosyl unit was transferred from a donor to an acceptor molecule, elongating β-D-ManpOpNP with formation of β-D-Manp-(1→3)-β-D-ManpOpNP and β-D-Manp-(1→4)-β-D-ManpOpNP (both referred to as pNP-Man2) from 5 mM β-D-ManpOpNP as monitored using HPLC (Figs. and). Nuclear magnetic resonance spectroscopy (NMR) and mass-spectrometry (MS) (Fig. , Tables and) were used to determine the chemical structure of the products. Four variants (R360K, N428T, S474A and W612H) showed markedly improved yields for pNP-Man2 synthesis (32-43%) compared to CfMan2A-WT (18%), while W169H, D170N, D386N and H496F were discarded due to low activity (<5 nkat•mg -1 ). It is noteworthy that the best four CfMan2A variants described herein were catalyticallyactive and thus could be applied at loadings that were only moderately greater (2 to 3.5-fold, Fig. ) than that used for CfMan2A-WT, and hundred-fold lower than the ones required with the CfMan2A-E519S glycosynthase . Moreover, compared to WT enzyme, monitoring revealed that the variants catalysed less secondary product hydrolysis and thus ensured high product concentrations even over prolonged reaction times (Fig. ). This result is significant because secondary hydrolysis often impedes enzyme-mediated synthesis of β-D-mannoside conjugates . GH10, β-endo-xylanase RmXyn10A_CM. Compared with most hexopyranoses, the pentopyranose configuration of the D-xylosyl unit confers enhanced flexibility and allows two distinct conformational itineraries that are compatible with GH hydrolysis: . Mechanistically interesting as it may be, the real challenge posed by this enzyme is related to its endo-activity. This implies the use of a substrate that does not possess an exceptionally good leaving group, and thus does not provide kinetic control of the reaction, and also the necessity to monitor multiple products in order to assess transglycosylation efficiency. This is because the reaction allows the transfer of oligosaccharide moieties onto an acceptor in a single catalytic step and provides the means to synthesize higher oligosaccharides displaying degrees of polymerisation (DP) > 10. Endo-1,4β-xylanase is the predominant enzyme class in the GH10 family and the subject of numerous studies . Both negative and positive subsites 23 in GH10 xylanases have been probed to understand their influence on substrate binding and catalysis , including RmXyn10A─CM, the catalytic module of the endo-xylanase from Rhodothermus marinus used here . However, very few reports concerned the transglycosylation reaction, these being limited to mutational studies of the aglycone subsites in a couple of GH10 xylanases .