Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification

Abstract

In the present study the influence of water content, solvent composition and reaction temperature on the transesterification of 1-phenylpropan-2-ol catalyzed by Candida rugosa lipase was examined. Reactions were carried out in different mixtures of hexane and tetrahydrofurane. The studies showed that an increasing water content of the organic solvent results in an increasing enzyme activity and a decreasing enantiomeric excess. Furthermore, a significant influence of the solvent hydrophilicity both on the enzyme activity and on the enantiomeric excess was found. An increase in solvent hydrophilicity leads to a decrease of enzyme activity and an increase of the enantiomeric excess. This indicates that the enzyme becomes more selective with decreasing flexibility. Similar effects were found by variation of the reaction temperature. Taken together, the decrease in conversion and the increase in selectivity with increasing solvent hydrophilicity are induced by the different water contents on the enzyme surface and not by the solvent itself.

Introduction

The application of organic solvents as reaction medium in enzyme catalysis offers some interesting advantages as for example an increased solubility of substrates, an increased stability of the enzymes as well as the possibility to catalyze reactions which are difficult up to impossible in water. As the biocatalyst is insoluble in organic solvents, it can be easily recovered from the heterogeneous reaction mixture by filtration or centrifugation. On the other hand in organic solvents non-polar substrates can be much better solubilized as in aqueous solutions. Moreover, in organic solvents water-induced side reactions are suppressed and reactions which are not favored in water are possible, e.g., the reversal of hydrolysis reactions (Carrea and Riva, 2000, Gotor-Fernández and Gotor, 2007, Serdakowski and Dordick, 2007).

In organic solvents the enzyme flexibility is reduced due to intramolecular interactions. While in aqueous solutions surface polar and charged amino acid side chains interact with water molecules, in organic solutions these structural elements are oriented towards the protein core developing intramolecular interactions, e.g., like ionic and hydrogen bonds (Hartsough and Merz, 1992). Thus, the enzyme has a higher density in packaging which leads to a reduced solvent-accessible surface area (Tejo et al., 2004). The reduced flexibility of the enzyme induces increased (temperature) stability alongside a reduced activity (Klibanov, 1989, Zaks and Klibanov, 1985).

The enzyme flexibility can be influenced by the solvent hydrophilicity. Hydrophilic organic solvents strip off the essential water from the enzyme surface, causing a rigid and inflexible protein structure (Klibanov, 2001). To minimize this effect, hydrophobic organic solvents are generally preferred compared to hydrophilic ones in biocatalysis. The enzymatic activity in such environments can be enhanced enormously by the addition of a small water amount to the solvent (Zaks and Klibanov, 1988). The enhanced flexibility is based on an enlarged water layer on the enzyme surface. In this case water acts as a “lubricant”, which raises the mobility of the enzyme (Griebenow and Klibanov, 1996).

Besides the influence on enzyme activity, the state of the protein in organic solvents also influences the enantioselectivity. In buffer solutions enantioselectivity can be modified typically by substrate concentration (e.g., Iding et al., 2000), pH (e.g., Liu et al., 1999) and temperature (e.g., Galunsky et al., 1997), respectively. In organic solvents it is assumed that enantioselectivity is primarily a function of water content but also of temperature and solvent nature. Regarding the water content there are contradicting opinions. While in some cases an increase in selectivity with a rising water content was found (Holmberg and Hult, 1990, Kitaguchi et al., 1990, Sinisterra et al., 1994, Stokes and Oehlschlager, 1987), there were also investigations where no correlation between the water content of the solvent and the enantioselectivity could be observed at all (Bovora et al., 1993, Martins et al., 1993, Persson et al., 2002, Secundo et al., 1991, van der Lugt et al., 1992).

The influence of temperature on the enantioselectivity varied profoundly depending on the system under investigation. Considering a hydrolysis reaction, Jin et al. found an increase in enantioselectivity with increasing temperature (Jin et al., 2011), whereas for esterifications and transesterifications a decrease in enantioselectivity with increasing temperature was often observed (Holmberg and Hult, 1991, Watanabe et al., 2001, Yasufuku and Ueji, 1995). Moreover, a so called “racemic temperature” is assumed, i.e. a temperature where the enzyme is unable to discriminate between the two enantiomers (Pham et al., 1989). Recently it was published from Jin and coworkers that some cases of enantiomeric reversal induced by the solvent nature may result from a change in the racemic temperature (Jin et al., 2011).

Another important aspect is the enhanced thermal stability of enzymes in organic solvents compared to aqueous solutions. Thermal denaturation depends on the water amount associated with the enzyme surface (Turner et al., 1995, Zaks and Klibanov, 1984). Due to the high structural flexibility of proteins in water containing environments partial unfolding and heat-induced misfolding of the enzyme molecule could be observed (Kauzmann, 1959, Tanford, 1968). The absence of water stabilizes the enzyme and protects the protein against the formation of covalent bonds within the enzyme structure (Volkin et al., 1991). Despite of the enhanced stability a temperature optimum can still be observed (Ema et al., 2003).

In the present study the influences of water concentration, solvent composition and reaction temperature on the conversion and the enantiomeric excess of the transesterification of 1-phenylpropan-2-ol with vinyl acetate catalyzed by Candida rugosa lipase (CRL) were investigated. The CRL is a widely used effective biocatalyst for the catalysis in organic solvents. In the literature numerous studies of CRL catalyzed transesterifications and esterifications are documented. The focus of several studies is laid on the investigation of the effect of water activity and temperature on enantioselectivity (Csajági et al., 2008, Holmberg and Hult, 1991, Persson et al., 2002, Watanabe et al., 2001, Wehtje et al., 1997, Yasufuku and Ueji, 1995, Xia et al., 2009) as well as on the investigation of the influence of the solvent on enzymatic activity and enantioselectivity, respectively (Overbeeke et al., 2000, Ulbert et al., 2004, Xia et al., 2009). Although a lot of work is done is this field, up to now it is not possible to formulate clear mechanisms and relationships between the parameters mentioned and the conversion and enantiomeric excess of lipase catalyzed reactions in organic solvents.

The aim of this work is to clarify if conversion and enantiomeric excess are primarily influenced by the solvent itself or if the mechanism is mainly triggered by the different water contents of the enzyme surface.