Applied Biocatalysis Group

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Directed evolution

Directed Molecular Evolution is a new tool in protein engineering that is meaning a revolution in biotechnology. This methodology has been employed to design enzymatic functions never required in natural environments.

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Enzymatic synthesis of oligosaccharides (prebiotics)

Our objective is to apply glycosyltransferases enzymes (Transglycosydases) using agro-products (sucrose and starch) as substrates for the synthesis of valuable non-digestible oligosaccharides (esp. with prebiotic properties) of interest for food, feed and dermocosmetic application. Non-digestible oligosaccharides are widely used as soluble fibers in Japan and their market is rapidly growing in Europe. The key advantage of using such natural and simple carbohydrate oligomers is to improve the ecological equilibrium of the intestinal bacterial flora by promoting the growth of beneficial bacteria (bifidobacteria, for example) thus preventing the growth of potentially detrimental microorganisms. At the level of human or animal nutrition, these products can play the role of essential food or feed ingredients in the future. 

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Enzymatic modification of antioxidants

Antioxidants protect cells against the effects of harmful free radicals and play an important role in preventing many human diseases (e.g. cancer, atherosclerosis, neurodegeneration, inflammatory disorders, etc.) and aging itself. In addition, antioxidant molecules are employed to prevent unsaturated oil products from becoming rancid during storage, thus extending oil life. 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 feed industries. L-Ascorbic acid (vitamin C), the major water-soluble natural antioxidant, acts as a free radical scavenger and plays an important role in regenerating vitamin E. However, due to the low miscibility of ascorbic acid with a-tocopherol, it is necessary to use ascorbyl fatty acid derivatives. The enzymatic synthesis of acyl L-ascorbates offers some advantages compared with the current chemical process, such as its high regioselectivity and the moderate reaction conditions. Vitamin E enhances the oxidative stability of polyunsaturated fatty acids from peroxidation acting as a free radical scavenger and is generally administered in the form of all-rac-α-tocopheryl acetate to increase its stability. Several approaches have been described for the enzyme-catalysed synthesis of vitamin E acetate, based on the transesterification of vitamin E with vinyl acetate, or the regioselective hydrolysis of alpha-isophorone followed by reaction with isophytol.

Bioremediation with enzymes

In the last few years, enzymatic bioremediation has risen as an attractive alternative to further support the bio-treatment techniques currently available, since enzymes are more simple systems than a whole organism. Most xenobiotics can be submitted to enzymatic bioremediation, e.g. polycyclic aromatic hydrocarbons (PAHs) , polynitrated aromatic compounds, pesticides such as organochlorine insecticides, bleach-plant effluents, synthetic dyes, polymers and wood preservatives (creosote, pentachlorophenol). Laccases (belonging to the multicopper blue family) have also been extensively investigated for new and challenging decontamination programs because they affect the oxidation of many aromatic compounds towards more benign and less toxic products. Laccases (EC 1.10.3.2) are blue multi-copper-containing enzymes that catalyze the oxidation of a variety of organic substances coupled to the reduction of molecular oxygen to water . Because of their broad specificity for the reducing substrates, laccases from white-rot fungi are receiving increasing attention as potential industrial enzymes in various applications, such as pulp delignification, wood fiber modification, dye or stain bleaching, chemical or medicinal synthesis, and contaminated water or soil remediation.

Enzymatic synthesis of carbohydrate esters

Enzymatic synthesis of fatty acid esters of di- and trisaccharides is limited by the fact that most biological catalysts are inactivated by the polar solvents (e.g. dimethylsulfoxide, dimethylformamide) where these carbohydrates are soluble. We have explored different methodologies to overcome this limitation, namely those involving control over the reaction medium, the enzyme and the support, as well as the fatty acid donor. We have obtained successful results using mixtures of miscible solvents (e.g. dimethylsulfoxide and 2-methyl-2-butanol) as a general strategy for di- and trisaccharides acylation. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesize different positional isomers, whose properties could vary considerably. In addition, the support textural properties affect significantly the reaction rate and/or the selectivity of the process. As a result, careful control over all these parameters is necessary for effective enzymatic sugar acylation strategies.