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UDP-glycosyltransferases (UGTs) are a promising class of biocatalysts that offer a sustainable alternative for chemical glycosylation of natural products. In this study, we aimed to characterize plant-derived UGTs from the GT-1 family with an emphasis on their acceptor promiscuity and their potential application in glycosylation processes. Recombinant expression in E. coli provided sufficient amounts of enzyme for the in-depth characterization of the salicylic acid UGT from Capsella rubella (UGT-SACr) and the stevia UGT from Stevia rebaudiana (UGT-76G1Sr). The latter was found to have a remarkably broad specificity with activities on a wide diversity of structures, from aliphatic and branched alcohols, over small phenolics to larger flavonoids, terpenoids and even higher glycoside compounds. As an example for its industrial potential, the glycosylation of curcumin was thoroughly evaluated. Under optimized conditions, 96% of curcumin was converted within 24h into the corresponding curcumin β-glycosides. In addition, the reaction was performed in a coupled system with sucrose synthase from Glycine max, to enable the cost-efficient (re)generation of UDP-Glc from sucrose as abundant and renewable resource., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Sucrose phosphorylase from Bifidobacterium adolescentis was recombinantly expressed in Escherichia coli and purified by use of a His-tag. Kinetic characterization of the enzyme revealed an optimal temperature for phosphorolytic activity of 58°C, which is surprisingly high for an enzyme from a mesophilic source. The temperature optimum could be further increased to 65°C by multipoint covalent immobilization on Sepabeads EC-HFA. The optimal immobilization conditions were determined by surface response design. The highest immobilization yield (72%) was achieved in a phosphate buffer of 0.04 mM at pH 7.2, irrespective of the temperature. The immobilized enzyme was able to retain 65% of its activity after 16 h incubation at 60°C. Furthermore, immobilization of the enzyme in the presence of its substrate sucrose, increased this value to 75%. The obtained biocatalyst should, therefore, be useful for application in carbohydrate conversions at high temperatures, as required by the industry., (Copyright © 2010 Elsevier B.V. All rights reserved.)