Research lines

The transfer of a glycosyl moiety is one of the most important biochemical reactions in living beings. The processing of glycosidic molecules involves a wide diversity of enzymes, which accounts for 1-3% of an organism’s genes.Glycosidic bonds are present in a wide variety of bioactive glycosides and glycoconjugates, in which the glycone part often exerts a great influence in their activity.

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, there exist some GTs called transglycosidases (TGs, e.g. dextransucrases, cyclodextrin glucosyltransferases, amylosucroses, inulosucrases or levansucrases) that use “non-activated” carbohydrates (sucrose, starch, etc.) as glycosyl donors. GHs and some GTs are capable of catalyzing both the formation of a glycosidic bond and its hydrolysis, and this duality permits the use of hydrolytic enzymes in synthetic reactions. Fig. 1 illustrates the different alternatives we are exploring to glucosylate a model carbohydrate (galactose).
 Fig. 1. Strategies to glucosylate a model sugar (galactose) employing carbohydrate-active enzymes: Glycosidases (GH) can act via thermodynamic or kinetic control; Transglycosidases (TGs) may use sucrose as glucosyl donor.

We are investigating different applications of enzymes active on carbohydrates, such as the preparation of prebiotic oligosaccharides for incorporation into functional foods, the production of second-generation biofuels (bioethanol), the preparation of lactose-free dairy products, or the synthesis of glycoconjugates with biomedical use. In fact, glycosylation dramatically changes the physico-chemical properties and bioavailability of many vitamins, hormones, flavonoids, antibiotics, etc. For example, a-glucosylation of the flavonoid rutin (vitamin P) increases its solubility more than 30,000 times.

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 a 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 its further mixing with a tertiary alcohol (2-methyl-2-butanol -2M2B-), being the final DMSO content close to 20% v/v. 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, granulation and entrapment in polymers and cross-linking of enzyme crystals or protein aggregates.