Glycosynthases in Biocatalysis

Abstract

Glycosynthases, engineered glycoside hydrolases that are able to synthesize glycans in quantitative yields without hydrolyzing them, are among the most interesting tools for the chemoenzymatic synthesis of carbohydrates that have been made available so far. From their invention in 1998, these enzymes have been developed enormously and demonstrated to be convenient alternatives to the more expensive glycosyltransferase. Glycosynthases have been proved to be efficient catalysts for the synthesis of oligosaccharides, polysaccharides, and glycoconjugates of various lengths containing different monosaccharides bound through a-and b-linkages, which have interesting applications. In this review we describe the impact that glycosynthases have had on the biocatalysis and biosynthesis of carbohydrates through the description of the novel activities produced and of the development of the glycosynthasebased process. In addition, we summarize the approaches that, according to the current literature, can be adopted to produce novel glycosynthase activities and to improve the existing process.

Structural basis for the flexible recognition of α‐glucan substrates by Bacteroides thetaiotaomicron SusG

“…The observation of this apparent transglycosylation product was particularly unexpected as catalytic nucleophile mutants of retaining glycosidases typically possess <10 4 -fold residual activity of the wild-type. 10 After ruling-out the potential of buffercatalyzed rescue of activity leading to transglycosylation 11 (data not shown), we suspected that trace contamination of the wild type enzyme in the mutant enzyme preparation was responsible for this apparent activity. Such contamination may result from sidechain deamidation or translational misincorporation, which has precedent in other glycoside hydrolases.…”

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

“…3 While high yields and selectivities are key features of the glycosynthase reaction, mutagenesis can also play an important role in the improvement and alteration of the catalytic activities of glycosynthases. [4][5][6][7][8][9] Exoglycosynthases, glycosynthases derived from exoglycosidases, have moderate substrate specificity and regioselectivity and can synthesize short chain oligosaccharides (di-, tri-, and tetra-oligosaccharides) that have various glycosidic linkages. [10][11][12] Endoglycosynthases, which are derived from endoglycosidases, generally have high regioselectivity and can catalyze the synthesis of specific glycosidic linkages.…”

Characterization of a Galactosynthase Derived from Bacillus circulans β‐Galactosidase: Facile Synthesis of D‐Lacto‐ and D‐Galacto‐N‐bioside

“…[2] To understand the cellular functions of GNB-and LNB-containing oligosaccharides and glycoconjugates, tools are required for the efficient synthesis of Gal-b-(1,3)-HexNAc (GlcNAc or GalNAc) disaccharides. [14] Although glycosynthases offer a versatile strategy for the synthesis of oligosaccharides, there is no report of a successful GH35-derived glycosynthase that is able to form Gal-b-(1,3)-HexNAc linkages. [3] Although naturally occurring galactosylations are performed by galactosyl transferases with activated nucleotide galactoside as the sugar donor, galactosidase-catalyzed galactosylations have also been reported.…”

“…[6] They also exhibit transglycosylation activity in the synthesis of galactosyl b-1,3-linked transfer products, but with modest yields because of the significant rehydrolysis of the transfer products by the enzyme. [14] Although glycosynthases offer a versatile strategy for the synthesis of oligosaccharides, there is no report of a successful GH35-derived glycosynthase that is able to form Gal-b-(1,3)-HexNAc linkages. [9] In addition, coupled enzyme systems that used d-galactosyl-b-(1,3)-N-acetyl-d-hexosamine phosphorylase with enzymes that supply galactosyl-1-phosphate as the donor produced LNB and GNB with yields above 90 %.…”

“…[7,8] Several successful attempts, with ionic liquid as the co-solvent to improve the yields of the reactions catalyzed by wild-type (WT) BgaC, resulted in accumulation of transfer products in the reaction mixtures, with high yields because of the low hydrolysis of the products. [14] Although glycosynthases offer a versatile strategy for the synthesis of oligosaccharides, there is no report of a successful GH35-derived glycosynthase that is able to form Gal-b-(1,3)-HexNAc linkages. [10] Despite the high efficiency of these enzymatic methods, there are still limitations, such as the high cost of the ionic liquids, the requirement for an expensive cofactor (adenosine triphosphate) for the kinase reaction, and different optimal conditions for each of the individual enzymes used in the coupled-enzyme system.…”