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The 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.

Glycosynthase reaction meets the flow: Continuous synthesis of lacto‐ N ‐triose II by engineered β‐hexosaminidase immobilized on solid support