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Glycosynthase reaction meets the flow: Continuous synthesis of lacto‐ N ‐triose II by engineered β‐hexosaminidase immobilized on solid support
Author(s) -
Ruzic Lucija,
Bolivar Juan M.,
Nidetzky Bernd
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27293
Subject(s) - chemistry , glycoside hydrolase , flow chemistry , substrate (aquarium) , yield (engineering) , hydrolysis , stereochemistry , combinatorial chemistry , organic chemistry , catalysis , oceanography , materials science , metallurgy , geology
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.