Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio-and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.hydroxynitrile lyases catalyzed the enzymatic hydrolysis of the glycoside amygdalin [3]. Moving almost two centuries forward, the largest volumetric biocatalytic industrial process is the application of glucose isomerase for the production of high fructose syrup for food and drink applications, producing fructose from glucose at 10 7 tons per year [4]. The secret of the success of enzymes in the production or treatment of carbohydrates and glycosides is their exquisite stereo-and regioselectivity. The excellent selectivity of enzymes is required due to the diversity of structural features of carbohydrates [5], comprising d-and l-epimers, ring size, anomeric configuration, linkages, branching, and oxidation state(s). Since drug targets often exhibit specificity for all of these structural features, the production process should not contain any side-products to prevent undesired side-effects [6].The challenge in the synthesis of carbohydrates is their wide variety of functionalities and stereochemistry ( Figure 1). (Poly)hydroxyaldehydes containing a terminal aldehyde are referred to as aldoses and (poly)hydroxyketones are defined as ketoses. In aqueous solutions, monosaccharides form equilibrium mixtures of linear open-chain and ring-closed 5-or 6-membered furanoses or pyranoses, respectively. For aldoses, the asymmetric ring forms at C-1. For ketoses, it closes at C-2 as an axial (α) or equatorial (β) hemiacetal or hemiketal, respectively (commonly defined as the anomeric center). A glycosidic linkage is a covalent O-, S-, N-, or C-bond connecting a monosaccharide to another residue resulting in a glycoside, while glucoside is specific for a glucose moiety. The equatorial or axial position of the glycosidic bond is referred to as α-(axial) or β-linkage (equatorial). The number of carbohydrates linked via glycosidic bonds can be subdivided into oligosaccharides with two to ten linked carbohydrates, while polysaccharides (glycans) contain more than ten glycosidic bonds. A glycan either con...

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Biocatalysis that produces economically interesting compounds can be carried out by using free enzymes or microbial cells. However, often the cell metabolism does not allow the overproduction or secretion of activated sugars and thus downstream processing of these sugars is complicated. Here enzyme immobilization comes into focus in order to stabilize the enzyme as well as to make the overall process economically feasible. Besides a robust immobilization method, a highly active and stable enzyme is needed to efficiently produce the product of choice. Herein, we report on the identification, gene expression, biochemical characterization as well as immobilization of the uridine-5′-diphosphate-glucose (UDP-glucose) pyrophosphorylase originating from the thermostable soil actinobacterium Thermocrispum agreste DSM 44070 ( Ta GalU). The enzyme immobilization was performed on organically modified mesostructured cellular foams (MCF) via epoxy and amino group to provide a stable and active biocatalyst. The soluble and highly active Ta GalU revealed a V max of 1698 U mg –1 (uridine-5′-triphosphate, UTP) and a K m of 0.15 mM (UTP). The optimum reaction temperature was determined to be 50°C. Ta GalU was stable at this temperature for up to 30 min with a maximum loss of activity of 65%. Interestingly, immobilized Ta GalU was stable at 50°C for at least 120 min without a significant loss of activity, which makes this enzyme an interesting biocatalyst for the production of UDP-glucose.

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Production of specialty chemicals increasingly makes use of enzyme catalysts, and Novozym 435 (N435) is among most often applied. However, its polymeric skeleton is unstable in many solvents. In this context, we report results of a systematic study of the biocatalysts, fabricated using highly porous siliceous pellets/enzyme (MH), grafted with octyl (-O), amino (-A) and octyl and amino (-OA) groups, deployed in a rotating bed reactor and tested in hydrolysis and esterification reactions. N435 appeared the most active in both reactions, when activity was related to the catalyst's mass, mainly owing to very large enzyme load. But its structure degraded in many typical solvents, whereas no such effect was detected in MH-O-and MH-OA-catalysts. MH-O showed the highest specific activity, however, a significant enzyme leaching was observed in a hydrolytic reaction, in contrast to MH-OA. In esterification reaction the MH-O-bound lipase was not only most active but also quite stable.