Roberto Fernández-Lafuente

Modulation of the enantioselectivity of lipases via controlled immobilization and medium engineering: hydrolytic resolution of mandelic acid esters

Abstract Lipase from Candida rugosa (CRL) has been purified and immobilized by using different immobilization protocols: interfacial adsorption on hydrophobic supports, ionic adsorption on PEI-coated supports, and covalent immobilization (on glutaraldehyde supports). This gave enzyme immobilized with different orientations and microenvironments. The catalytic properties (activity, specificity, and enantioselectivity) of the different derivatives have been found to be dramatically different. Very significant changes on activity with different substrates were found. For example, interfacially adsorbed derivative was the most active using simple substrates (ethyl butyrate) while PEI derivative was the most active hydrolysing ionic substrates (2-phenyl-2-butyroylacetic acid at pH 7) or methyl mandelate. The E value also depends strongly on the derivative and the conditions employed. Thus, the interfacially absorbed enzyme varied its enanatioselectivity (toward S isomer) from 1.6 to 85 in the hydrolysis of ( R , S )-2-phenyl-2-butyroylacetic acid when the pH value varied from 7 to 5. However, the glutaraldehyde derivative presented a high enantioselectivity ( E =400) toward R isomer (the inverse E value compared to the previous derivative) at both pH conditions. Polyethyleneimine (PEI) derivative presented a slight enantiopreference toward the S isomer. Thus, using different derivatives, it has been possible to obtain both pure enantiomers from the ester or the product. Similar changes in the E values were obtained in the hydrolysis of methyl mandelate though here always there was a enantiopreference for the S isomer. Using this substrate, the best derivative was the PEI derivative at pH 5 ( E =300), while the glutaraldehyde one presented an E value of only 10.

Synthesis of antibiotics (cephaloglycin) catalyzed by penicillin G acylase: Evaluation and optimization of different synthetic approaches

Abstract Two different approaches have been utilized to synthesize cephaloglycin using immobilized-stabilized penicillin G acylase derivatives. These are thermodynamically and kinetically controlled strategies. The thermodynamically controlled strategy could be employed to synthesize cephalotin or penicillin G, but this approach in the synthesis of cephaloglycin presented serious difficulties because of the absence of conditions where the thermodynamics of the process and the enzyme activity/stability properties were good enough. The kinetically controlled strategy has given much better results. The systematic study of the different parameters that defined the maximum yields of this strategy has permitted the identification of its main problem as the hydrolysis of the antibiotic. Because of the rapid enzymatic hydrolysis of cephaloglycin that has been previously synthetized, the yields were poor and they decreased very rapidly after reaching the maximum yield. Three different strategies have been used to decrease this amidase activity (an excess of acyl donor, selection of acidic pH, and distortion of the enzyme molecule by methanol). Simultaneous utilization of these strategies has significantly improved this synthetic process with very high yields (around 95%), reaction rates, and enzyme stability.

Selective oxidation: stabilisation by multipoint attachment of ferredoxin NADP+ reductase, an interesting cofactor recycling enzyme

Ferredoxin-NADP+ reductase (FNR, EC 1.18.1.2) is an enzyme that is able to catalyse the oxidation of NADPH + H+. A strategy to prepare industrial derivatives of this enzyme for use as an ‘NADP’ regenerating enzyme in oxidizing reactions is presented. The strategy is based on a strictly controlled process of multipoint covalent attachment between the enzyme, via its amino groups, and a pre-existing solid activated with a monolayer of simple aldehyde groups linked by a space-arm of moderate length to the surface of the support. Controlling the variables which may have an influence in the multi-interaction process, we have prepared a number of enzyme derivatives with very different activity/stability properties. n nThe ‘optimum derivative’ was found to be much more stable than its corresponding soluble enzyme under all the denaturation conditions assayed (high temperatures, extreme pH, organic solvents, etc.). Because of the excellent properties of this enzyme derivative, we can regenerate NADP+ by using molecular oxygen directly as the oxidizing agent under a wide range of conditions. Coupling this oxidative system to other NADP-dependent redox enzymes, we should be able to develop a very specific and selective oxidative procedure under very mild oxidizing conditions.

Engineering of Enzymes via Immobili-zation and Post-Immobilization Techniques: Preparation of Enzyme Derivatives with Improved Stability in Organic Media

The high potential of enzyme biotransformations in nonaqueous media has been adequately emphasised throughout the present volume. However, enzymes also have important limitations for working in non-aqueous media. For example, enzymes are usually inactivated in the presence of high concentrations of organic cosolvents or in the presence of hydrophobic interfaces of non-miscible solvents and so on[1].Such limitations may not be very relevant when working at laboratory scale (e.g. by performing a short unique biotransformation) but they can become critical when trying to scale up such exciting biotransformations to an industrial scale (e.g. trying to perform a number of long reaction cycles)