Methods for Improving Enzymatic Trans-glycosylation for Synthesis of Human Milk Oligosaccharide Biomimetics

Abstract

Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is a novel field of research in advanced food ingredient chemistry, involving the use of rare enzymes, which have until now mainly been studied for their biochemical significance, not for targeted biosynthesis applications. For the enzymatic synthesis of biofunctional glycans reaction parameter optimization to promote "reverse" catalysis with glycosidases is currently preferred over the use of glycosyl transferases. Numerous methods exist for minimizing the undesirable glycosidase-catalyzed hydrolysis and for improving the trans-glycosylation yields. This review provides an overview of the approaches and data available concerning optimization of enzymatic trans-glycosylation for novel synthesis of complex bioactive carbohydrates using sialidases, α-l-fucosidases, and β-galactosidases as examples. The use of an adequately high acceptor/donor ratio, reaction time control, continuous product removal, enzyme recycling, and/or the use of cosolvents may significantly improve trans-glycosylation and biocatalytic productivity of the enzymatic reactions. Protein engineering is also a promising technique for obtaining high trans-glycosylation yields, and proof-of-concept for reversing sialidase activity to trans-sialidase action has been established. However, the protein engineering route currently requires significant research efforts in each case because the structure-function relationship of the enzymes is presently poorly understood.

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Glycosidases, on the other hand, do not require an input of energy, use cheap donor substrates, and has relaxed substrate specificity for acceptors and are wide spread enzymes. Although glycosidases suffer from limitations such as product hydrolysis and low transglycosylation activity [143,144]. In this context, some β-glucosidase can catalyze transglycosylation under high substrate or product concentration [31].…”

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

“…The effects of lactose concentration (50, 100, 200, 300, 400, and 500 mM) on HexNAc-Lac synthesis were measured using 25 mM glycosyl donor at pH 7.0 and 37°C for 7.5 h (pNP-␤-GalNAc) or 2.5 h (pNP-␤-GlcNAc). The effects of the concentration of glycosyl donor (5,10,15,20,25, and 30 mM) were measured using 400 mM lactose at pH 7.0 and 37°C for 7.5 h (pNP-␤-GalNAc) or 2.5 h (pNP-␤-GlcNAc). The effects of pH were examined at 19 different pH values (acetate buffer at 3.6, 4.0, 4.6, 5.0, 5.4, and 5.8; phosphate buffer at 5.8, 6.0, 6.5, 7.0, 7.5, and 8.0; Tris-HCl buffer at 8.0, 8.5, and 9.0; glycine buffer at 9.0, 9.5, 10.0, and 10.7; Na 2 HPO 4 -NaOH buffer at 10.7, 11.0, 11.5, and 12.0) at 37°C by using 400 mM lactose in the presence of 10 mM pNP-␤-GalNAc for 7.5 h or 20 mM pNP-␤-GlcNAc for 2.5 h. The effects of temperature and reaction time were investigated using 400 mM lactose and 10 mM pNP-␤-GalNAc or 20 mM pNP-␤-GlcNAc at pH 5.8 at six different temperatures (35°C, 40°C, 45°C, 50°C, 55°C, and 60°C), followed by interval sampling within 14 h. All reactions were stopped by heating at 100°C for 10 min, and the remaining donor and resulting products were detected by HPLC.…”

“…Moreover, addition of organic solvents can increase the solubility of a substrate and reduce water activity, promoting transglycosylation over hydrolysis. Other conditions, such as reaction time, temperature, and pH, can also affect product yield (5). In this work, the final concentration of 20% (vol/vol) DMSO was used for the synthesis reaction by BbhI.…”

“…The regioselectivity of glycosidases mainly depends on two main factors: the sources of the enzymes and the kinds of substrates (5,54,55). The ␤-Nacetylhexosaminidase from A. oryzae synthesizes GlcNAc␤1-3/1-6Gal␤1-4Glc via reverse hydrolysis by using GlcNAc and lactose (44).…”

“…Glycosyltransferases, the natural enzymes for specific synthesis of oligosaccharides and polysaccharides, require expensive nucleoside sugars as glycosyl donors and generally exhibit strict acceptor specificity. In contrast, glycosidases, which are enzymes that normally hydrolyze carbohydrates, can use lowcost, simple glycosides as glycosyl donors and show a broad acceptor specificity for glycosylation or transglycosylation reactions in vitro; thus, the use of glycosidases is a more effective practical approach for large-scale synthesis of oligosaccharides (4,5).…”