Enzyme and Microbial Technology 31 (2002) 775–783 Modulation of the enantioselectivity of lipases via controlled immobilization and medium engineering: hydrolytic resolution of mandelic acid esters Jose M. Palomo, Gloria Fernandez-Lorente, Cesar Mateo, Claudia Ortiz, Roberto Fernandez-Lafuente ∗ , Jose M. Guisan 1 Laboratorio de Tecnolgia Enzimatica, Departamento de Biocatalisis, Instituto de Catalisis—CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain Received 18 April 2002; received in revised form 22 May 2002; accepted 14 June 2002 Abstract Lipase from Candida rugosa (CRL) has been purified and immobilized by using different immobilization protocols: interfacial adsorp- tion 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 enantios- electivity) 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. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Conformational engineering; Lipase enantioselectivity; Interfacial adsorption; Methyl mandelate; 2-Phenyl-2-butyroylacetic acid 1. Introduction Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are perhaps the most popular enzymes in biocatalysis [1–9] be- cause they couple a wide specificity to a high regio and enantioselectivity and specificity, therefore, may be used in many different reactions. However, lipase catalysis implies dramatic conforma- tional changes of the enzyme molecule. Lipases may be in two different structural forms. One of them, where the ∗ Corresponding author. Tel.: +34-91-585-48-09; fax: +34-91-585-47-60. E-mail addresses: rfl@icp.csic.es (R. Fernandez-Lafuente), jmguisan@icp.csic.es (J.M. Guisan). 1 Co-corresponding author. active center of the lipase is secluded from the reaction medium by a polypeptide chain called “lid”, is considered an inactive (closed) form. The other one, which presented the lid displaced and the active center exposed to the re- action medium, is considered as the lipase in an active (open) form. The lipase molecule is in equilibrium be- tween the open-active and the closed-inactive structures of the immobilized lipases. This equilibrium shifts towards the open form in the presence of hydrophobic interfaces by the adsorption of the open form [10–16]. It is very likely that if this equilibrium or the exact shape of the enzyme is altered in any way, the catalytic properties of the enzyme may be dramatically altered. This could be achieved via immobilization techniques involving different areas of the enzyme, giving different rigidity to the enzyme structure or even generating a certain special 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0141-0229(02)00169-2