Glycosynthase Principle Transformed into Biocatalytic Process Technology: Lacto-N-triose II Production with Engineered exo-Hexosaminidase

Abstract

Glycosynthases are promising enzyme catalysts for glycoside synthesis. Derived from glycoside hydrolases by mechanistic repurposing of their active site, glycosynthases utilize suitably activated glycosyl donors for glycosylation, yet they are unable to hydrolyze the products thus formed. Although primed for synthetic application by their design, glycosynthases have yet to see actual use in carbohydrate production. To challenge limitations on glycosynthase applicability perceived from the process chemistry point of view, here we developed a glycosynthase (D746E variant) from Bifidobacterium bifidum β-N-acetylhexosaminidase that is highly active synthetically (≥100 μmol min–1 mg–1) and fully chemo- and regioselective when using N-acetyl-d-glucosamine 1,2-oxazoline for β-1,3-glycosylation of lactose. We thus established a chemoenzymatic process technology for production of lacto-N-triose II, a core structural unit of human milk oligosaccharides. Using equivalent amounts of oxazoline (prepared chemically in 40% yield from N-acetyl-d-glucosamine) and lactose, we obtained lacto-N-triose II (515 mM; 281 mg mL–1; ∼90% yield; ≤1 h reaction time) immediately recoverable from the reaction in 85% purity. These metrics of process efficiency reveal the prodigious potential of the glycosynthase for trisaccharide production.

β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

“…However, oxazolines are unstable at acidic conditions and less useful, eg, in fungal hexosaminidase reactions having acidic pH optima [64]. Nevertheless, the ability of bacterial hexosaminidases with neutral or alkaline pH optima to utilize Glc-oxa as donor for trans-glycosylation reactions as demonstrated (Table 9) [24,101,105,108,111] together with its comparably good price per mole (1,710,000 €/kg (347,000 €/mol) for Glc-oxa at Carbosynth [112]) may increase the popularity of Glc-oxa as donor molecule for such enzymatic synthesis reactions in the future. The fact that there are no byproducts from trans-glycosylation reactions using Glc-oxa and the option of conducting Glc-oxa synthesis under mild conditions [113,114] with enzymatic trans-glycosylation in a cascade fashion [108] might lead to industrialization of GH20-catalyzed trans-glycosylation with Glc-oxa donors.…”

“…Higher ratios of up to 910:1 have been achieved with non-carbohydrate acceptors like alcohols (Table 10) [71], which are usually highly soluble. However, apart from the auto-condensation reactions, for which the A:D ratio is always considered 1:1 (Table 2), a few examples of GH20 trans-glycosylation reactions using an A:D ratio of 1:1 also exist (Tables 1, 3, 4, 9 and 10) [19,23,24,46,52,53,64,88,90,101,115,116]. Most noteworthy in this context is the recently reported synthesis of LNT II where an isolated yield of 86% (281 g/L) was achieved in a reaction using Lac and Glc-oxa in a ratio of 1:1 at a concentration of 600 mM each (Table 9) [100].…”

“…Four independent studies have reported synthesis of LNT II from pNP-GlcNAc and Lac (Figure 4) using the enzyme BbhI from Bifidobacterium bifidum (Table 5) [101][102][103][104]. Two of the studies used identical conditions for the reaction (pH 5.8, 55 • C, A:D = 20:1, 20% (v/v) DMSO) and reported similar yields: Both, Chen et al [103] and Schmölzer et al [101] thus reported a yield of approximately 45%. In another study, the synthesis of LNT II by BbhI was carried out at neutral pH (pH 7.0) and 37 • C, with an A:D ratio of 4:1 and without DMSO, leading to a yield of 16% (based on HPLC analysis using an internal standard) after 0.6 h [102].…”

“…In another study, the synthesis of LNT II by BbhI was carried out at neutral pH (pH 7.0) and 37 • C, with an A:D ratio of 4:1 and without DMSO, leading to a yield of 16% (based on HPLC analysis using an internal standard) after 0.6 h [102]. In the other two studies, the D746A and D746E mutants of BbhI, which have been used for LNT II synthesis at pH 5.8 and at two different temperatures of 37 and 55 • C [101,104]. For the mutant D746A, the reaction at the lower temperature (37 • C) resulted in a higher yield of 58% [104] compared to 40% in the other study, where the reaction has been performed at 55 • C [101].…”

“…In the other two studies, the D746A and D746E mutants of BbhI, which have been used for LNT II synthesis at pH 5.8 and at two different temperatures of 37 and 55 • C [101,104]. For the mutant D746A, the reaction at the lower temperature (37 • C) resulted in a higher yield of 58% [104] compared to 40% in the other study, where the reaction has been performed at 55 • C [101]. In the case of D746E the opposite trend was observed.…”