Lucija RuzicBolívar Bolívar, Juan ManuelBernd Nidetzky2025-01-152025-01-152020-02-130006-35921097-029010.1002/bit.27293https://hdl.handle.net/20.500.14352/114451The D746E variant of Bifidobacterium bifidum β-N-acetyl-hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective β-1,3-glycosylation of lactose from a d-glucosamine 1,2-oxazoline donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145–200 mM) at quantitative yield from the donor substrate. The wild-type β-N-acetyl-hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d-glucosamine. By enabling short residence times (2 min) that are challenging for mixed-vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80–100 mM/min) from the slower side reactions (decomposition of donor substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/journal articlehttps://doi.org/10.1002/bit.27293https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/full/10.1002/bit.27293open access577.11,2-oxazoline-activated donor substrateFlow chemistryGlycosynthaseHuman milk oligosaccharidesβ-glycosaminidaseBioquímica (Química)Ingeniería químicaQuímica industrial2302 Bioquímica3303 Ingeniería y Tecnología Químicas3303 Ingeniería y Tecnología Químicas