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

Abstract

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. K E Y W O R D S 1,2-oxazoline-activated donor substrate, flow chemistry, glycosynthase, human milk oligosaccharides, β-glycosaminidase

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

“…In Bb H1 with GlcNAc-oxazoline as activated donor and lactose acceptor, LNT-II was produced in 86% yield with mutant D746E [ 30 ]. The yield was further improved with an immobilized version of the enzyme packed into a fixed bed for continuous production of LNT II [ 31 ]. The high yields with the Bb H1 enzyme mutants both with GlcNAc- p NP and GlcNAc-oxazoline donors do not translate to LnbB.…”

Production of Lacto-N-triose II and Lacto-N-neotetraose from Chitin by a Novel β-N-Acetylhexosaminidase Expressed in Pichia pastoris

“…7 Therefore, protein engineer- ing, especially HaHex74 modifications, such as directed evolution and rational design, should be performed. 6 Furthermore, enzyme engineering, such as enzyme immobilization and multienzymatic cascade reaction construction, 47,48 also represents an efficient method to improve the conversion ratios of LNT2 and LNnT.…”

The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives

“…105 Further work of the group includes among others process intensification for O 2 -dependent enzymatic transformations in continuous single-phase pressurized flow, 106 and the continuous synthesis of lacto-N-triose II by engineered β-hexosaminidase immobilized on solid support. 107…”