Fluorescence and CD Spectroscopic Analysis of the A-Chymotrypsin Stabilization by the Ionic Liquid, 1-Ethyl-3-methylimidazolium Bis[(trifluoromethyl)sulfonyl]amide Teresa De Diego, 1 Pedro Lozano, 1 Said Gmouh, 2 Michel Vaultier, 2 Jose ´ L. Iborra 1 1 Departamento de Bioquı ´mica y Biologı ´a Molecular B e Inmunologı ´a, Facultad de Quı ´mica, Universidad de Murcia, P.O. Box 4021, E-30100 Murcia, Spain; telephone: (34) 968367398; fax: (34) 968364148; e-mail: jliborra @um.es. 2 Universite ´ de Rennes-1, Institut de Chimie, UMR-CNRS 6510, Campus de Beaulieu. Av. Ge ´ne ´ral Leclerc, 35042, Rennes cedex, France Received 7 June 2004; accepted 26 August 2004 Published online 28 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20330 Abstract: The stability of a-chymotrypsin in the ionic liq- uid, 1-ethyl-3-methyl-imidizolium bis[(trifluoromethyl)sul- fonyl]amide ([emim][NTf 2 ]), was studied at 30 and 50jC and compared with the stability in other liquid media, such as water, 3M sorbitol, and 1-propanol. The kinetic analysis of the enzyme stability pointed to the clear denaturative effect of 1-propanol, while both 3M sorbitol and [emim] [NTf 2 ] displayed a strong stabilizing power. For the first time, it is shown that enzyme stabilization by ionic liquids seems to be related to the associated structural changes of the protein that can be observed by differential scanning calorimetry (DSC) and fluorescence and circular dichroism (CD). The [emim][NTf 2 ] enhanced both the melting tem- perature and heat capacity of the enzyme compared to the other media assayed. The fluorescence spectra clear- ly showed the ability of [emim][NTf 2 ] to compact the native structural conformation of a-chymotrypsin, prevent- ing the usual thermal unfolding which occurs in other me- dia. Changes in the secondary structure of this h/h protein, as quantified by the CD spectra, pointed to the great en- hancement (up 40% with respect to that in water) of h- strands in the presence of the ionic liquid, which reflects its stabilization power. B 2004 Wiley Periodicals, Inc. Keywords: ionic liquids; enzyme stability; fluorescence; circular dichroism; a-chymotrypsin INTRODUCTION Applied biocatalysis in nonaqueous environments has gained increasing interest during recent years because of its potential to foment enzymatic-chemical transformations of interest for the fine chemical industries (Klibanov, 2001). In this context, the use of neoteric solvents (e.g., ILs, scCO 2 , etc.) as novel media for enzyme-catalyzed trans- formations has recently attracted considerable attention, and could be considered an important axis for development of green chemistry in the near future (van Rantwijk et al., 2003). Indeed, some ionic liquids (ILs) have been shown to be excellent nonaqueous media for enzyme-catalyzed re- actions, not only because the enzymes display a high level of activity and stereoselectivity for many different chemi- cal transformations, e.g., aliphatic ester synthesis (Lozano et al., 2003), the kinetic resolution of racemic alcohols (Scho ¨fer et al., 2001; Lozano et al., 2004; Noe ¨l et al., 2004), carbohydrate ester synthesis (Park and Kazlauskas, 2001), and polymer synthesis (Nara et al., 2003), but also because of their important stabilizing effect on enzymes (up to 2,300-fold increased half-life time with respect to classical organic solvents) (Lozano et al., 2001a,b; Persson and Bornscheuer, 2003). Understanding the molecular mechanism of enzyme sta- bilization by ionic liquids is an untreated question in ap- plied biocatalysis. The serine protease, a-chymotrypsin, is one of the most widely analyzed enzymes with regard to deactivation due to the large number of potential appli- cations for ester and peptide synthesis in nonconventional media. Different strategies to increase a-chymotrypsin sta- bility have been assayed, such as adding polyols (Lozano et al., 1994; Simon et al., 2002) or hydrophilization of the external surface by chemical modification (Kudryashova et al., 1997; Levitsky et al., 1999), thus improving enzyme stability by preserving the water shell around the protein molecule. In aqueous media, a-chymotrypsin stability has been analyzed mechanistically using a two-step enzyme deactivation kinetic model, and excellent agreement has been obtained between both experimental and theoretical data (Lozano et al., 1997). Complementary spectroscopy B 2004 Wiley Periodicals, Inc. Correspondence to: Prof. Jose ´ L. Iborra Contract grant sponsors: CICYT; SENECA Foundation Contract grant numbers: PPQ2002-03549; PB/75/FS/02