Ž . Journal of Molecular Catalysis B: Enzymatic 11 2001 817–824 www.elsevier.comrlocatermolcatb Preparation of new lipases derivatives with high activity–stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine J.M. Guisan a, ) , P. Sabuquillo a , R. Fernandez-Lafuente a , G. Fernandez-Lorente a , C. Mateo a , P.J. Halling b , D. Kennedy b , E. Miyata c , D. Re c a Department of Biocatalysis, Instituto de Catalisis, CSIC, Campus UAM - Cantoblanco, 28049 Madrid, Spain ´ b Department of Pure and Applied Chemistry, UniÕersity of Stracthclyde, Glasgow, UK c ( ) Resindion Srl Mitsubishi Chemical Corp. , Milan, Italy Abstract A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports Ž . having large internal surfaces covered by hydrophobic groups e.g. polyacrylic resins covered by C18 moieties . Then, Ž . immobilized lipases are incubated in the presence of polyethyleneimine PEI at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media. Ž . Ž . Lipases from Rhizomucor miehie RML and Candida rugosa CRL were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in Ž anhydrous media. For example, RML derivatives exhibited a very high synthetic activity more than 1000 Unitsrg . immobilized biocatalyst even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar Ž . conditions less than 10 Unitsrg of immobilized biocatalyst . Moreover, these new RML derivatives preserve 100% activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90% of activity in less than 10 min. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Lipases; Interfacial adsorption; Enzymes in organic medium; Artificial enzyme microenvironment; Protein surface hydrophiliza- tion; Sepabeads ) Corresponding author. Instituto de Catalisis, CSIC, Campus ´ UAM - Cantoblanco, 28049 Madrid, Spain. Tel.: q 34-91-585-48- 00; fax: q 34-91-585-47-60. Ž . E-mail address: jmguisan@icp.csic.es J.M. Guisan . 1. Introduction 1.1. Immobilized lipases in organic solÕents The impressive possibilities of lipases as catalysts of biotransformations in organic solvents have been 1381-1177r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. Ž . PII: S1381-1177 00 00011-4