Converting hydrolytic GHs into potent glycosynthetic tools is an attractive approach to extend the synthetic chemist's toolbox, while introducing catalysts that obey green chemistry principles (e.g. use of non-toxic catalysts, aqueous solvents). However, so far, the success of this approach has been hampered by the time and effort necessary to develop appropriate biocatalysts for each target reaction. The strategy presented herein goes a long way to surmounting this obstacle, making it much simpler to obtain tailored biocatalysts that can then be used to operate straightforward, relatively inexpensive synthesis reactions that do not require difficult to obtain sugar donors, lengthy protection/deprotection cycles or exorbitant quantities of enzyme. Moreover, the transferability implies that the reported mutations can be readily transferred to other GHs from the five described families, expanding the portfolio of available evolved transglycosylases and synthetic oligosaccharides. pellets resuspended, lysed, and centrifuged. The enzymes were purified from the supernatant by Ni 2+ (or Co 2+ )-affinity IMAC chromatography. Eluates were analysed by SDS-PAGE, and protein concentration was determined spectrophotometrically using UV280 absorption and molar extinction coefficients calculated in ExPASy (www.expasy.org). GH Family-specific procedures GH2, CfMan2A. Activity of the CfMan2A and its variants were determined by measuring the release of p-nitrophenol (pNP) from p-nitrophenyl-β-D-mannopyranoside (β-D-ManpOpNP), using a version of the method described by Zechel et al adapted for microplate assay. Briefly, reactions mixtures containing 1 or 5 mM β-D-ManpOpNP, 35 mM sodium phosphate pH 7.0 and appropriately diluted enzyme were incubated at 35℃ for 10 min. The reaction was stopped by adding 1 M Na2CO3 and released pNP was measured on an Epoch Microplate spectrophotometer (BioTek Instruments, USA) at 405 nm. All assays and reactions (below) were in duplicates. To evaluate the transglycosylation ability of the CfMan2A forms, reactions containing 5 mM β-D-ManpOpNP and 3-34 µg•mL -1 enzyme in 35 mM sodium phosphate pH 7.0 were incubated up to 6 h at 35℃. Aliquots were collected at appropriate time intervals throughout the reaction, heat denatured (95°C, 10 min) and filtered (0.22 µm PTFE). Aliquots were separated at 40℃ on a Luna Omega SUGAR HPLC-column (Phenomenex, USA) using an Ulti-Mate 3000 HPLC system (Thermo Fisher Scientific, USA) with 40:60 water:acetonitrile eluent (v/v) at 1 mL•min -1 . A VWD-3400RS detector was used (Thermo Fisher Scientific, USA), measuring absorbance at 300 nm. The presence of transglycosylation products were determined with mass spectrometry and analysed with NMR spectroscopy as described in Supplementary Information, section 1.2. Progress curves of pNP-Man2 production for CfMan2A forms were generated from HPLC analysis as above for reaction times up to 6 h (7-10 sampling points, see Fig. ). pNP-Man2 was quantified using pNP-cellobioside as a standard. The yield of pNP-Man2 was calculated as the amount of β-D-ManpOpNP used as either acceptor or donor in the production of pNP-Man2 divided by the amount of loaded β-D-ManpOpNP. GH10, RmXyn10A_CM. GH10 transglycosylation activity was characterized by incubating 20 mM X4 with RmXyn10A_CM forms. Reaction mixtures containing 10-43 µg•mL -1 GH10 in 20 mM sodium phosphate pH 7.0 were incubated at 65℃ for 4 h. Aliquots (15 µL) were withdrawn at different time points, diluted in 0.5 mM NaOH, filtered, and analysed on high performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) using an ICS-5000 (Dionex) monitored by the software Chromeleon. Separation was carried out at 30℃ on a CarboPac PA-200 using 100 mM NaOH at 0.5 mL•min -1 and a linear gradient of 0-120 mM sodium acetate. The reaction mixtures for MALDI-TOF-MS analysis contained 10-43 µg•mL -1 GH10 in 20 mM sodium phosphate pH 7.0 and were incubated at 60℃ for 4 h. MALDI-TOF-MS spectra were obtained on a Bruker Daltonics Autoflex Speed MALDI-TOF (/TOF) spectrometer in positive ion reflector mode and recorded in the mass range from 200 to 4000 or 5000 Da. Samples were diluted in MilliQ H2O to a total salt concentration <10 mM, and 1 μL of this dilution was mixed with 0.5 μL aqueous 10% dihydroxybenzoic acid (DHB) matrix solution on a stainless steel target and left to dry at room temperature. Sample irradiation was done at 55% laser power by targeting the laser pulses at amorphous crystal regions, regularly shifting to remove heterogeneity in the sample. Calibration was done internally by addition of xylo-oligosaccharides X1-X6. The hydrolysis assay was performed in 0.1 mL reactions with 2 mM β-D-xylotriosideOpNP in 20 mM sodium phosphate pH 7.0, at 70℃ for 5 min and the reaction was stopped using one volume of 0.1 M NaOH. The absorbance was measured at 400 nm using a Multiskan spectrophotometer from Thermo Scientific. The extinction coefficient (18250 M•cm⁻ 1 ) of released p-nitrophenol (pNP) was determined using a standard of pNP (Sigma). GH20, BbHI. GH20 transglycosylation was monitored by HPAEC-PAD. Samples with 0.5-5 µM GH20, 10 mM β-D-GlcpNAcOpNP and 40 mM lactose in 50 mM sodium phosphate, 0.1% BSA pH 7.0 were incubated (2 h, 37°C), then heat denatured (2 min, 98°C), centrifuged, and the obtained supernatant was diluted five-fold in milliQ H2O and filtered. Separation was carried out at 30°C on a CarboPac PA-1 (Dionex) using an ICS-5000 (Dionex) monitored by the software Chromeleon (Dionex). NaOH 0.1 M was used as eluent in 20 min separations at 250 µL•min -1 . GH29, AlfB and AlfC. GH29 transfucosylation was assessed from NMR spectra recorded on an 800 MHz Bruker Avance III (799.75 MHz for 1 H) equipped with a 5 mm TCI cryoprobe using 1 H with presaturation. AlfB-catalysed reactions were carried out with 20 mM α-L-FucpOCNP, 20 mM D-GlcNAc, and 20-750 μg•mL -1 (0.4-16 nM) GH29 in 600 μL 40 mM sodium phosphate pD 7.0 (pD=pHmeter reading+0.4, 6.6 on readings) . Time course experiments were obtained using pseudo-2D kinetics experiments, with 1 H NMR spectra recorded every 3 min. Integration of anomeric protons is inaccurate due to the closeness of the presaturated HOD peak, therefore chemical shifts at 4.26 (H-2 of D-GlcNAc in α-L-Fucp-(1→3)-D-GlcNAc) and 4.05 ppm (α-L-FucpOCNP) were used for integration (Fig. ). AlfC-catalysed reactions were identical but using 8 mM donor and acceptor, and the use of the fucose methyl group protons to assess concentrations (Figs. and). GH51, TxAbf. The activities of TxAbf and mutants thereof were determined using a discontinuous enzyme assay . Reactions were performed in triplicate at 45°C in 50 mM sodium phosphate pH 7.0 containing 1 mg•mL -1 BSA, using 5 mM α-L-ArafOpNP without/with 10 mM xylotriose as acceptor in hydrolysis and transglycosylation modes, respectively. The amount of pNP released, which was measured at 401 nm, was calculated using an appropriate standard curve of pNP. Negative controls containing all of the reactants except the enzyme were used to correct for spontaneous hydrolysis of the donor substrate. To monitor the transglycosylation products profiles from 1 H NMR spectra at 500 MHz on a Bruker Avance II spectrometer equipped with a TCI probe, reactions were performed at 45°C in NMR tubes containing 600 µL 5 mM α-L-ArafOpNP, 10 mM xylotriose and TxAbf enzymes (3.5-12 800 nM) in 10 mM sodium phosphate pH 7.0/D2O: 9/1, v/v, with 1 mg•mL -1 BSA. The quantity of enzyme (Fig. ) was adjusted to suit the 13-19 h reaction time frame. Time course of NMR monitoring was obtained using pseudo-2D kinetics experiments based on a phase sensitive NOESY sequence with presaturation, with spectra collected every approximatively 9 min (i.e. twice 32 scans). The transglycosylation yields were determined by relative integration of anomeric proton signals from the α-L-Araf unit of each AXOS (Table ) . Concentration of α-L-ArafOpNP donor was quantified by integrating its relevant anomeric proton signals at 5.86 ppm. Molar balances, based on initial donor signal as internal reference, were used to convert the transglycosylation product signal integral into concentration.