Thermal stabilization of immobilized lipase B from Candida antarctica on different supports: Effect of water activity on enzymatic activity in organic media Miguel Arroyo, Jose ´ Marı ´a Sa ´nchez-Montero, and Jose ´ Vicente Sinisterra Department of Organic & Pharmaceutical Chemistry, Faculty of Pharmacy, Universidad Complutense, Madrid, Spain Covalent immobilization of C. antarctica lipase B (CALB) on sepharose, alumina, and silica was undertaken. The thermal stability of these covalently immobilized catalysts were studied and compared to adsorbed derivatives from Novo Nordisk at 50°C under wet conditions. Native enzyme and Novozym 435 follow a deactivation model E 3 E 1 whereas covalently immobilized derivatives and SP435A follow the model E 3 E 1 3 E 2 . This different behavior is related to the nature of the support and the immobilization methodology. Water absorption isotherms of dry solid biocatalysts in air or isooctane were used to predict the optimum preequilibrium a w value to obtain the highest rate in the esterification of (r,s)-ibuprofen. © 1998 Elsevier Science Inc. Keywords: Lipase; immobilization; stability; ibuprofen; water activity Introduction Lipase B from Candida antarctica (CALB) 1 is an interest- ing lipase with potential application in a number of indus- trial processes such as the synthesis of triglycerides, 2 esterification of terpenic alcohols, 3 etc. Adsorbed CALB on different supports has also proven to be very regioselective in the esterification of sugars, 4 nucleosides, 5 and steroids, 6 and very enantioselective in the resolution of secondary alcohols via hydrolysis 7 or esterification in organic sol- vents. 8 One of these derivatives, called SP435A, has been employed successfully in the preparation of pure S( +)-2- arylpropionic acids with antiinflammatory effect. 9 In the current paper, several immobilization methods and supports have been tested for the covalent bonding of pure lipase B from C. antarctica. We have also compared the thermal stability of our covalent immobilized derivatives with those obtained by Novo Nordisk by absorption of the same lipase on different polymers. All the biocatalysts were tested in the hydrolysis of triacetin due to the low activity of this lipase in the hydrolysis of triglycerides with long-chain fatty acids. 10 Afterwards and at different initial water activity ( a w ), the best immobilized derivatives catalyzed the stereospecific esterification of racemic ibuprofen in isooctane in order to find a method to predict the best pre-equilibrium a w of the whole system to perform the reaction. Materials and methods Materials Native lipase B from C. antarctica (SP525) and the same lipase immobilized on Lewatit OC 1600 (SP435A) and Lewatit E (Novozym 435) were kindly supplied by Novo Nordisk Bioindus- trias (Madrid, Spain). Tresylated sepharose 4B and epoxy-acti- vated sepharose 6B were purchased from Pharmacia (Uppsala, Sweden). Silica (Kiesegel 60, size 0.015– 0.040 nm, average pore Address reprint requests to Dr. J. M. Sanchez-Montero, Universidad Complutense de Madrid, Facultad de Farmacia, Dept. Organic & Pharma- ceut. Chemist., 28040 Madrid, Spain Received 22 January 1998; revised 7 April 1998; accepted 28 April 1998 Enzyme and Microbial Technology 24:3–12, 1999 © 1998 Elsevier Science Inc. All rights reserved. 0141-0229/99/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0141-0229(98)00067-2