The transfer of a glycosyl moiety is one of the most important biochemical reactions in living beings. These processes are catalyzed by a wide diversity of glycosidic enzymes, which represent between 1% and 3% of the genes of an organism. Glycosidic bonds are present in a many bioactive glycosides and glycoconjugates. The two main groups of enzymes that act on carbohydrates are glycosidases (GHs, EC 3.2.1) and glycosyltransferases (GTs, EC 2.4). GHs catalyze the hydrolysis of di-, oligo- and polysaccharides, do not require cofactors, and present great availability. GTs transfer glycosyl residues from one activated substrate (sugar-nucleotides for the enzymes of the Leloir route; sugars-phosphate for the glycoside phosphorylases, GPs) to an acceptor. However, a series of GTs called transglycosidases (TGs, e.g. dextransucrases) use “non-activated” carbohydrates (sucrose, starch, etc.) as glycosyl donors. GHs and TGs catalyze both the formation of a glycosidic bond and its hydrolysis, allowing the use of hydrolytic enzymes in synthetic reactions. The following figure illustrates the different alternatives to glucosylate a model carbohydrate (galactose).
We are investigating different applications of carbohydrate-active enzymes, such as the preparation of prebiotic oligosaccharides for incorporation into functional foods or the synthesis of glycoderivatives of polyphenols. As a matter of fact, glycosylation dramatically changes the physico-chemical properties and bioavailability of many vitamins, hormones, flavonoids, antibiotics, etc.
The modification of natural antioxidants in order to increase their miscibility and/or stability towards the action of light and/or oxygen renders a series of “semisynthetic” antioxidants with great impact in the food and pharmaceutical industries. The enzyme-catalysed synthesis of acyl derivatives of antioxidants offers some advantages, such as its high regioselectivity and the moderate reaction conditions. Lipases have been successfully used to catalyse the enzymatic synthesis of antioxidant esters employing saturated and unsaturated free fatty acids, alkyl or vinyl esters as acyl donors.
For enzymatic sugar acylation it is neccesary to find a medium where a polar reagent (the carbohydrate) and the nonpolar acyl donor are soluble and able to react in presence of a biocatalyst. Lipases are readily inactivated by polar solvents capable of dissolving di- and trisaccharides. In this context, we developed a simple process for the lipase-catalysed acylation of sucrose and other sugars based on the pre-solubilization of sucrose in a polar solvent (dimethylsulfoxide -DMSO-) and further addition to a tertiary alcohol. These mixtures of miscible solvents represent a compromise between sugar solubility and enzyme stability.
For the industrial development of the above processes, an effective immobilization method is commonly required to allow the reuse of enzymes or continuous processing. We have employed different strategies to immobilize enzymes, based on adsorption, covalent binding, entrapment and cross-linking.
