Role of Ionic Liquids in Enzyme Catalysis

1. Introduction
Enzymes are necessary in all living systems, to catalyze all chemical reactions required for their survival and reproduction- rapidly, selectively and efficiently. Isolated enzymes can also carry these reactions. The enzymes can be used as biocatalysts to catalyze chemical reactions on an industrial scale in a sustainable manner. Their application covers the production of desired products for all human material needs (e.g. food, animal feed, pharmaceuticals, fine and bulk chemicals, fibers, hygiene, and environment technology), as well as in a wide range of analytical purposes, especially in diagnostics. Enzyme reactions in nonaqueous environment are gaining importance than in aqueous environment. This is due to higher substrate solubility, the ability to use enzymes synthetically rather than degradatively and the capability to modify native selectivity by simply tailoring the reaction medium rather than the enzyme itself. The use of organic solvents in industrial processes is problematic due to their dangerous properties. The main concerns are the toxicity of the solvents to both the process operators and the environment, and the volatile and flammable nature of these solvents which make them a potential explosion hazard. This has led to strict regulations for their use and containment. To overcome these problems other solvents used include supercritical carbon dioxide and ionic liquids (Buchholz et al., 2005). Ionic liquids are a new attractive reaction medium for biocatalysis. Ionic liquids hold potential as green solvents because of their lack of vapour pressure, and are opening up a new field of nonaqueous enzymology. As compared to those observed in conventional organic solvents, enzymes in ionic liquids have presented enhanced activity, stability and selectivity. Advantages of using ionic liquids over the use of normal organic solvents as reaction medium for biocatalysis also include their high ability of dissolving a wide variety of substrates, especially those highly polar ones, and their widely tunable solvent properties through appropriate modification of the cations and anions (Kaar et al., 2003).

2. Ionic liquids
Ionic liquids are composed entirely of ions. Molten sodium chloride is an ionic liquid but a solution of sodium chloride in water is an ionic solution. The term molten salts, which was previously used to describe such materials, evokes an image of high-temperature, viscous and highly corrosive media. The term ionic liquid, in contrast, implies a material that is fluid at (or close to) ambient temperature, is a colorless, has a low viscosity and is easily handled, i.e. a material with attractive properties for a solvent. Room temperature ionic liquids are generally salts of organic cations, e.g. tetraalkylammonium, tetraalkylphosphonium, N-alkylpyridinium, 1,3-dialkylimidazolium and trialkylsulfonium cations (Sheldon, 2001).

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Role of Ionic Liquids in Enzyme Catalysis Cations:

R1

R1 R2

R2

1,3-Dialkylimidazolium

1,4-Dialkylpyridinium

R2 R3 N N R4 R1 R2 R1

Tetraalkylammonium

1,1-Dialkylpyrrolidinium

Anions: Anion BF4PF6NO3CH3CO2CF3CO2CH3SO4CF3SO3(CF3SO2)2NFull Name tetrafluroborate hexaflurophosphate nitrate acetate trifluroacetate methylsulphate trifluromethylsulfonate bis[(trifluromethyl)sulfonyl]amide Abbreviation [BF4] [PF6] [NO3] [Ac] [TFA] [MeSO4] [TfO] [Tf2N]

In order to be liquid at room temperature, the cation should preferably be unsymmetrical, e.g. R1 and R2 should be different alkyl groups in the dialkylimidazolium cation. The melting point is also influenced by the nature of the anion (Sheldon, 2001). Ethylammonium nitrate, which is liquid at room temperature, was described as first ionic liquid in 1914. The first example of ionic liquids based on dialkylimidazolium cations were reported in the early 1980s by Wilkes et al. They contained chloroaluminate anions (AlCl4- or Al2Cl7-). However, a serious obstacle for widespread use of these ionic liquids is the higher reactivity of chlorobutamine anion towards water. Wilkes et al. reported the first example of new ionic liquids that are currently receiving so much attention as novel media for catalysis, is ethylmethylimidazolium tetrafluroborate. The corresponding hexaflurophosphate salts are synthesized after tetrafluroborate salts. These salts are stable towards hydrolysis. Subsequently, ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 2

Role of Ionic Liquids in Enzyme Catalysis 1,3-dialkylimidazolium salts containing a wide variety of anions, e.g. CF3SO3-, [CF3SO2]2N-, CF3CO2-, CH3CO2-, PhSO3- and many more have been prepared (Sheldon, 2001). 2.1 Properties of ionic liquids Room temperature ionic liquids are usually quaternary ammonium salts, such as tetralkylammonium [R4N]+ or based on cyclic amines, both aromatic (pyridinium, imidazolium) and saturated (piperidinium, pyrrolidinium). Low temperature molten salts based on sulfonium [R3S]+ as well as phosphonium [R4P]+ cations are also known. Cations may be modified by incorporating functionalities to carbon atoms of the ring (Galinski et al., 2006). Traditional solvents are described as molecular liquids whereas ionic liquids are composed of ions. Their unique properties such as nonvolatility, nonflammability, and excellent chemical and thermal stability have made them an environmentally attractive alternative to conventional organic solvents. Ionic liquids have low melting points (less than 100C) and remain liquids within a broad temperature window (less than 300C) (Kaar et al., 2003). 2.1.1 Polarity One of the most special properties for ionic liquids is their high polarity. The polarity of ionic liquids used in biocatalysis is in the range of 0.6-0.7 on the normalized scale, which sets tetramethylsilane at 0.0 and water at 1.0. This puts ionic liquids in the range of lower alcohols or formamide. The effects of alkyl group on the imidazole ring (C4-C8) and the anion (tetrafluoroborate, hexaflurophosphate, bis (trifluromethane sulfonyl) amide) on the polarity seem to be slight. The polarity values of ionic liquids are sometimes sensitive to the temperature and the presence of water. Because of the high polarity, ionic liquids present an ideal reaction media for chemical and biochemical reactions due to their ability to dissolve a wide range of different substances including polar and nonpolar, organic, inorganic, and polymeric compounds (Rantwijk et al., 2003). 2.1.2 Hydrophobicity Despite of their high polarity, most of ionic liquids are hydrophobic and can dissolve up to 1% of water, and the presence of water may affect the physical properties of the ionic liquids. However, the solubility of water in ionic liquids varies unpredictably (Rantwijk et al., 2003). For example [BMIM][BF4] is completely water miscible while [BMIM][PF6] and [BMIM][Tf2N] are slightly soluble in water, although they have same polarity. 2.1.3 Solubility Ionic liquids are generally immiscible with many organic solvents especially when organic solvents are nonpolar such as hexane; whereas some ionic liquids may be miscible with polar solvents such as dichloromethane and tetrahydrofuran (Park and Kazlauskas, 2003). The immiscibility of ionic liquids with either water or organic solvents has made them feasible to be used to form two-phase systems.

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Role of Ionic Liquids in Enzyme Catalysis 2.1.4 Viscosity Compared to typical organic solvents, ionic liquids are much more viscous (35-500 cP viscosity for commonly used ionic liquids versus 0.6 cP for toluene and 0.9 cP for water at 25C) (Park and Kazlauskas, 2003). The viscosity of an ionic liquid represents its tendency to form hydrogen bonding and the strength of its van der Walls interactions, and can be lowered by increasing the temperature or by adding some organic co-solvents. Normally, an ionic liquid with longer alkyl chains on the cation and a larger anion size represents a higher viscosity. 2.1.5 Thermal stability Ionic liquids are thermally stable. This stability is limited by the strength of their heteroatomcarbon and heteroatom-hydrogen bonds respectively. While 150C is considered as maximum working temperature for most quaternary ammonium chloride salts, [EMIM][BF4] (1-ethyl-3methylimidazolium tetrafluoroborate) is stable up to 300C and [EMIM][(CF3SO2)2N] (m.p.3C) is stable to more than 400C (Malhotra and Zhao, 2003). 2.1.6 Designer solvents Ionic liquids are called as designer solvents. One of the advantages of using ionic liquids over the use of normal organic solvents is that the physical and chemical properties of the ionic liquids, including their polarity, hydrophobicity, viscosity and solvent miscibility, can be finely tuned by altering the cation, anion and attached substituents. Due to this, by manipulating the solvent properties one is allowed to design an ionic liquid for specific reaction conditions, such as to increase the solubility, to modify the enzyme selectivity or tailor the reaction rate (Yang and Pan, 2005).

3. Enzyme activity in ionic liquids

Enzymes in ionic liquids are active only when they remain suspended as a powder or immobilized by a solid support, like when working in organic solvents. Enzymes if dissolved in ionic liquid are inactive. Many researchers have reported that, a variety of enzymes are capable of performing catalytic activity in ionic liquids and have shown comparable or higher activity than observed in conventional organic solvents (Eckstein et al., 2002; Schofer et al., 2001). When preparing the active enzyme encapsulated cellulose films reconstituted from ionic liquids, Turner et al. found that pre-coating the enzyme (laccase) with a second, hydrophobic ionic liquid prior to dispersion in the cellulose/ionic liquid solution can provide an increase in enzyme activity relative to that of untreated films, and this seemed to suggest that the ionic liquid is capable of providing a stabilizing microenvironment for the enzyme (Yang and Pan, 2005).

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Role of Ionic Liquids in Enzyme Catalysis

Table 1 Examples of using enzymes ionic liquids (Yang and Pan, 2005) Biocatalyst Lipase Reaction Transesterification Alcoholysis, ammoniolysis, perhydrolysis, Kinetic resolution of chiral alcohols Resolution of amino acid ester Esterification of carbohydrates Synthesis of polyesters Enantioselective reduction of 2octanone Synthesis of Z-aspartame Transesterification Transesterification Resolution of amino acid ester Synthesis of N-acetyllactosamine Oxidation of guaiacol Oxidation of anthracene Regeneration of NADH Ionic liquid [BMIM]PF6] [BMIM][PF6], [BMIM][BF4] [BMIM][Tf2N] [EPy][BF4], [EMIM][BF4] [MOEMIM][BF4] [BMIM][PF6] [BMIM][Tf2N] [BMIM][PF6] [EMIM][Tf2N], [MTOA][Tf2N] [BMIM][PF6] [EPy][TFA]H2O (15:85, v/v) [MMIM][MeSO4]H2O [BMIM][PF6] [BMIM][PF6] [MMIM][MeSO4]H2O(25:75, v/v)

Alcohol dehydrogenase Thermolysin -Chymotrypsin Esterase Subtilisin -Galactosidase Peroxidase Laccase Formate dehydrogenase

3.1 Effect of solvent properties of ionic liquids 3.1.1 Hydrophobicity In organic solvents, the most popular solvent descriptor which has been considered as a key determinant of enzyme activity is log P, the logarithm of the partition coefficient of a solvent in an octanol/water mixture. It has been suggested that solvents with a high log P, such as hexane (log P = 3.5), are more hydrophobic and thus more favourable for enzymatic reactions than those with a low log P, such as ethanol (log P = -0.24). So far, ionic liquids have not been treated according to this log P concept. The first determinations of log P for ionic liquids were done based on the fact that the imidazolium ring present in the dialkylimidazolium-based ionic liquids absorbs strongly at approximately 211 nm (Kaar et al., 2003) and their experimentally measured log P values are extremely low (-2.90 to -2.39), which seem to suggest that ionic liquids are highly hydrophilic in nature and hence would likely inactivate enzymes. But inspite of their hydrophilic nature, enzymes are active in ionic liquids (Kaar et al., 2003). Therefore, log P does not seem to be able to act as a useful parameter for determining enzyme activity in ionic liquids. On the other hand, an ionic liquid partitions in the octanol/water mixture as an ion pair, and therefore its log P value may depend not only on the concentration of the cation, but also on that of the anion and the extent of ion pairing in both phases. Unfortunately, it is unclear yet which of these factors would be expected to relate to enzyme activity.

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Role of Ionic Liquids in Enzyme Catalysis 3.1.2 Viscosity Viscosity of the reaction medium may control the enzyme activity by affecting the mass-transfer limitations. Therefore, a lower reaction rate would be expected in an ionic liquid with a higher viscosity. When -chymotrypsin was assayed in two ionic liquids [EMIM][Tf2N] and [MTOA][Tf2N] with a viscosity of 34 and 574 cP, respectively, a reduction in the enzyme activity was corresponding to an increase in the viscosity of the reaction medium (Kaar et al., 2003). 3.1.3 Polarity Park and Kazlauskas, (2001) have studied the rates of the acetylation reaction catalyzed by lipase from Pseudomonas cepacia (PCL) as a function of solvent polarity for 10 ionic liquids with their polarity values ranging from 0.63 to 0.71 (without addition of water). An obvious trend toward higher reaction rates in more polar ionic liquids were observed, which was to the opposite of the trend shown when the reaction was conducted in normal organic solvents. A similar correlation between the polarity of the ionic liquids and reaction rates were also observed by Lozano et al., (2001) who studied a transesterification reaction catalyzed by -chymotrypsin in five ionic liquids. The same research group compared the activity of the free Candida antarctica lipase B in four ionic liquids in the presence of 2 % (v/v) of water and has also found that the synthetic activity of the enzyme increased with the increase in the polarity of the ionic liquid. However, studies of other reactions in ionic liquids showed no relationship between the reaction rate and the polarity of the solvent, e.g. the transesterification catalyzed by Candida rugosa lipase (Kaar et al., 2003), the acetylation of glucose catalyzed by Candida antarctica lipase B (Park and Kazlauskas, 2001), and the transesterification catalyzed by a series of lipases from various sources (Schofer et al., 2001). Polarity of ionic liquid affects the reaction rate differently for different enzymatic reactions. According to the researchers, the enzyme activity may be related more to the viscosity and less to the polarity of the ionic liquids. This is due to the polarity of ionic liquids varies in a relatively narrow range (0.60.7) (Rantwijk et al., 2003; Park and Kazlauskas, 2001) as compared to their viscosity changing over a much broader range (35-500 cP). Also, there seems to be a correlation between polarity and viscosity of ionic liquids. An ionic liquid with shorter alkyl chains on the cation and smaller anion size may have a lower viscosity and also a higher polarity. This suggests that a slight reduction in the polarity of ionic liquids may be corresponding to a huge increase in their viscosity. Therefore, the above observation of higher reaction rates in more polar ionic liquids can be explained by the effect of viscosity (Lozano et al., 2001; Park and Kazlauskas, 2001). Also, the reaction rates have usually been compared in different ionic liquids when the same amount of water was present in the reaction system. Under such conditions, the solvent with a higher polarity would have less water associated with the enzyme and more water remaining in the solvent. This would lead to lower reaction rate and reduction in the viscosity of the ionic liquid and in turn an improvement in the mobility of the protein molecule, thus resulting in an increase in enzyme activity. Depending on which factor plays a more important role on the enzyme, the reaction rate would increase or decrease accordingly. Furthermore, in addition to polarity and viscosity, some other ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 6

Role of Ionic Liquids in Enzyme Catalysis solvent properties, such as the solubility of substrates and/or products (Park and Kazlauskas, 2001) and especially, the ionic interactions, may also be considered to be responsible for affecting enzyme activity in ionic liquids. Interestingly, regardless of their high polarity, ionic liquids normally do not inactivate enzymes like those polar organic solvents with similar polarity, such as methanol and Nmethylformamide. This important feature enables ionic liquids to create new opportunities for nonaqueous biocatalysis with polar and hydrophilic substrates such as ascorbic acid (Park et al., 2003) and carbohydrates (Park and Kazlauskas, 2001). 3.2 Effect of water In organic media, it is already well-known that the amount of water associated with the enzyme, rather than the total water content in the reaction system, is the key determinant of the enzyme properties (e.g. activity, stability, and specificity) (Zaks and Klibanov, 1988), and thermodynamic water activity (aw) has been generally considered as a parameter to quantify the hydration level on the enzyme. Therefore, it is also necessary to evaluate the role of water and water activity in the biocatalytic ionic liquid systems. Kaar et al., (2003) have reported that water was indeed required by -chymotrypsin when catalyzing both transesterification and hydrolysis reactions in imidazolium-based ionic liquids with a bell-shaped relationship between reaction rates and water contents, similar to the behaviour in normal organic solvents. The fact that the enzyme presented considerably higher activity in [OMIM] [PF6] than in [BMIM][PF6] may result from the former ionic liquid possessing a longer hydrophobic alkyl chain and thus holding less tendency to strip off the essential water from the enzyme. The study on the kinetics of the chymotrypsin catalyzed transesterification and the competing hydrolysis showed that at low water activities (aw = 0.11, 0.33) the enzyme was more active in ionic liquids than in organic solvents (Eckstein et al., 2002). In addition, Berberich et al., (2003) have demonstrated that like in organic solvents, salt hydrate pairs can be used to control water activity for enzyme catalysis in ionic liquids. 3.3 Effect of excipients For biocatalysis in organic solvents, it has been demonstrated that lyophilizing enzymes in the presence of some excipients such as a nonbuffered salt KCl or polyethylene glycol (PEG) can yield dramatically improved enzyme preparations, which may be related to the stabilization of the protein structure (Lee and Dordick, 2002). For example, subtilisin activity in hexane got 4000- fold improvement when the enzyme was pre-lyophilized with 98 % (w/w) KCl, and relyophilization of soybean peroxidase with PEG (MW 10,000) resulted in a 35-fold enhancement in the enzyme activity in acetone. It will be useful if this enzyme activation technique can be extended to the ionic-liquid biocatalytic systems. Maruyama et al. (2002) have investigated the lipase-catalyzed alcoholysis in three ionic liquids and reported that a PEGlipase complex (with a molecular ratio of 10:1) yielded an increased activity of over 14-fold. Similar phenomenon was also observed for -chymotrypsin in [OMIM] [PF6], although the extent of activity stimulation was not so great as compared to that in the organic solvents. Maruyama et al. have further reported that applying PEGlipase complexes in ionic liquids resulted in not only an improved activity with a lower Km, but also a higher enantioselectivity, as compared to the data obtained in common organic solvents such as hexane. ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 7

Role of Ionic Liquids in Enzyme Catalysis 3.4 Effect of pH and impurities As in organic solvents, the activity that the enzyme exhibits depends very much on the pH of the last aqueous solution from which the enzyme is lyophilized, with the optimum preparation conditions coincident with the pH optimum for the enzyme activity in aqueous solution. This apparent pH dependence varies upon changing solvent type and water content present in the reaction medium, both affecting the local polarity of active site of the enzyme. It would be reasonable to expect that the same would happen to the ionic liquid systems, which in fact may be even more complicated due to the ionic and polar nature of the ionic liquids. For example, HTf2N is more acidic in [BMIM][Tf2N] and [BMIM][BF4] than in water. This increased acidity may inactivate the enzyme in ionic liquids. In addition, purification of the ionic liquid, which is essential to enzyme activity (Park and Kazlauskas, 2001), may also result in a pH shift. Unfortunately, not much work has been done related to this pH issue in ionic liquids. Several groups have disagreed on whether or not an enzyme is active in a particular ionic liquid (Table 2). For example, Schofer et al., (2001) reported that CaLB had no activity in [BMIM][BF4] or [BMIM][PF6], but other groups reported good activity for transesterification or ammoniolysis in the same ionic liquids (Kim et al., 2001). Impurities may cause these inconsistencies. Halide ions are a possible impurity. Many procedures for making ionic liquids first prepare the 1-alkyl-3-methylimidazolium chloride or bromide. As mentioned above, enzymes are inactive in these ionic liquids. Next, the halide is exchanged with the desired anion, but an incomplete exchange leaves halide anions behind. Although adding 0.1 % w/v halide salts to purify ionic liquids did not lower the activity of lipase (Park and Kazlauskas, 2001), higher amounts may have an effect. Washing with water followed by vacuum drying can be used to purify water-immiscible ionic liquids. However, the purification of water-miscible ionic liquids, such as [BMIM][BF4], involves filtering through silica gel followed by washing with aqueous sodium carbonate solution. These purified ionic liquids worked reliably, but the purification of ionic liquids still needs further research. Table 2 Contradictory reports of enzyme activity in ionic liquids (Park and Kazlauskas, 2003). Ionic liquid Enzyme Reaction Activity Reference [BMIM][PF6] CaLB Transesterification Yes Itoh et al., 2001 No Schofer et al., 2001 [BMIM][PF6] CRL Transesterification Yes Kaar et al., 2003 No Itoh et al., 2001 [BMIM][PF6] MML Transesterification Yes Husum et al., 2001 No Schofer et al., 2001 CaLB, Candida antarctica lipase B; CRL, Candida rugosa lipase; MML Mucor miehei lipase. 3.5 Enzyme inactivation in ionic liquids Enzymes that show catalytic activities in ionic liquids normally do not dissolve. However, those enzymes that do dissolve in ionic liquids are usually inactive. For example, [BMIM][PF6] can dissolve up to 3.2 mg/ml of thermolysin, the activity of which did not show up until the amount of the enzyme in the reaction system exceeded 3 mg/ml (Erbeldinger et al., 2000). ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 8

Role of Ionic Liquids in Enzyme Catalysis [BMIM][NO3], [BMIM][lactate], and [EMIM][EtSO4] also dissolved and deactivated the Candida antarctica lipase B (Sheldon et al., 2002). The denaturation of the protein structure in an ionic liquid is presumably related to the ionic nature of the ionic liquid; its cation or anion may interact with the charged groups of the enzyme, either in the active site or at its periphery, causing changes in the structure of enzyme. The role of anions may be more crucial in this aspect; enzymes are usually active in ionic liquids containing BF4-, PF6-, and Tf2N- anions, but inactive in those containing anions such as NO3-, CH3CO2-, CF3CO2-, and CF3SO3- (Kaar et al., 2003). There are two possible explanations for this difference. First, the enzyme-compatible anions exhibit lower hydrogen bond basicity, which minimizes interference with the internal hydrogen bonds of an enzyme (Park and Kazlauskas, 2003). Take PF6 anion as an example, it spreads its negative charge over six fluorine atoms. Second, the enzyme-compatible anions exhibit lower nucleophilicity and thus would show lower tendency to change the conformation of enzyme by interacting with the positively charged sites in the enzyme structure (Sheldon et al., 2002).

4. Enzyme stability in ionic liquids

The enzyme stability is usually much higher in organic media, especially at a low water activity, than in aqueous solution, it is expected that ionic liquids should have the same effect on stabilizing enzymes. The activity of thermolysin was well-retained after incubation in [BMIM][PF6] at 37C for 144 h, whereas the same treatment in ethyl acetate resulted in the loss of almost half of the enzyme activity (Erbeldinger et al., 2000). The stability of the esterase from Bacillus stearothermophilus at 40C was also found to be considerably increased in the ionic liquids [BMIM][BF4] and [BMIM][PF6] as compared to that in hexane and MTBE; a half-life of more than 240 h was obtained in [BMIM][PF6], which was more than 30-fold and more than 3-fold higher as compared to that in hexane and MTBE respectively. (Persson and Bornscheuer, 2003). Moreover, the stability of -chymotrypsin was studied by incubation in four different ionic liquids at 2 % (v/v) water content and 50C (Lozano et al., 2001). The half-life of the enzyme (1.08 - 2.63 h) was significantly enhanced, as compared to that of 0.15 h in 1-propanol. The increase in the half-life of enzyme seemed to be in agreement with the increase in hydrophobicity of the ionic liquids. It is reasonable that a hydrophobic reaction medium would allow the preservation of the essential water molecules surrounding the protein structure, thereby reducing the proteinion direct contact and enhancing the enzyme stability toward denaturation (Lozano et al., 2001). The same phenomenon was also observed when Candida antarctica lipase B was used to catalyze the ester synthesis reactions in ionic liquid/supercritical CO2 biphasic systems. By fitting the kinetic data obtained at 50C to a two-step kinetic deactivation model, the authors reported that all the five ionic liquids based on quaternary ammonium cations that were tested acted as stabilizing agents to the enzyme with respect to hexane, producing an increase in the free energy of deactivation and an improvement in the half-life of the enzyme (2000-fold), which agrees with the observed increased hydrophobicity of the cation alkyl side chain. Also reported ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 9

Role of Ionic Liquids in Enzyme Catalysis results state that at elevated temperatures, Candida rugosa lipase in ionic liquids kept its activity and enantioselectivity much better than in traditional organic solvents (Yang and Pan, 2005). The stabilization effect of the ionic liquids on enzymes can be improved by incubating the enzyme in the presence of the substrate, in this way -chymotrypsin obtained a 10- fold increase in its half-life (Lozano et al., 2001). The reuse of free lipase (Candida antarctica lipase B) in [BMIM][PF6] in continuous operation cycles showed a half-life of 2300 times greater than that observed when the enzyme was incubated in the absence of substrate(Lozano et al., 2001). The association of the substrate to the enzyme may cause a conformational change that activates the enzyme, and this active conformation is well-retained during the presence of the substrate. Studies on enzymes after incubation in ionic liquids by Sheldon et al., (2002) have also revealed that enzymes are not only strongly tolerant against ionic liquids, but they can even be activated. The thermostability of Candida antarctica lipase B, either free (Novozyme SP525) or adsorbed on a macroporous acrylic support (Novozyme 435), was investigated by incubating the enzyme preparation in anhydrous [BMIM][PF6] at 80C for a specific period and then after dilution with water, measuring the residual activity in triacetin hydrolysis (Sheldon et al., 2002). Both enzyme forms showed a significant increase in their activities; 120 % increase for the free enzyme after incubation for 20100 h, and 350 % increase for Novozym 435 after 40 h incubation. It is necessary to mention the two recent spectroscopic studies of protein thermostability in ionic liquids. By conducting the fluorescence studies of a single tryptophan protein, monellin, (Kaar et al., 2003) reported the first detailed protein spectroscopy in ionic liquids, which indicated that the significant thermodynamic stabilization effect offered by the ionic liquid may result from alteration in the protein hydration level and the structural compaction; the authors also suggested that some other factors such as free volume contributions, ionic interactions and confinement effects may also contribute to the protein stabilization by ionic liquids. More recently, a research group reported a more detailed spectroscopic study on protein thermostability. They studied the stability of -chymotrypsin in the ionic liquid [EMIM][Tf2N] at elevated temperatures and compared that to the stability in other liquid media such as water, 3M sorbitol, and 1-propanol. Their results have revealed that the enzyme stabilization by ionic liquids seemed to be related to the associated structural changes of the protein: differential scanning calorimetry (DSC) revealed that both the melting temperature and heat capacity of the enzyme was enhanced by the ionic liquid as compared to the other media; the fluorescence spectra clearly showed the ability of the ionic liquid to compact the native structural conformation of the enzyme, preventing the usual thermal unfolding which occurs in other media; the circular dichroism (CD) spectra have also demonstrated the changes in the secondary structure of the protein in the presence of the ionic liquid, reflecting its stabilization power (Kaar et al., 2003). Not all ionic liquids are suitable for biocatalysis. Enzymes are usually active in ionic liquids containing BF4-, PF6-, and NTf2- anions, but not in ionic liquids containing Cl-, NO3-, CF3SO3-, trifluroacetate or acetate anions (Kaar et al., 2003). A possible reason for this difference is the lower hydrogen-bond basicity of the enzyme-compatible anions. The BF4 anion spreads its negative charge over four fluorine atoms, the PF6 anion over six fluorine atoms and the NTf2 anion over five atoms. The lower hydrogen-bond basicity minimizes interference with the ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 10

Role of Ionic Liquids in Enzyme Catalysis internal hydrogen bonds of an enzyme. Enzymes are inactive in [BMIM][Cl] which has high hydrogen bond basicity. Ionic liquids with anions having high hydrogen-bond basicity cannot be used for the enzyme catalysis (Anderson et al., 2002). 4.1 Thermal stability of enzymes in ionic liquids Sheldon et al., (2002) studied the thermal stability of different CaLB preparations by suspending them in an organic solvent or [BMIM][PF6], allowing the mixture to stand at 80C and taking aliquots at various time intervals and measuring the residual activity, after dilution with water, in triacetin hydrolysis. Incubation of free enzyme (SP 525) in [BMIM][PF6] at 80C resulted in an increase in activity. After an incubation time of 20 h, the maximum activity found was 120 % of the activity of the native untreated enzyme. This activity value did not decrease up to an incubation time of 100 h, while in tert-BuOH a nearly linear deactivation in time was observed. Incubation of Novozym 435 in [BMIM][PF6] at 80C showed a maximum activity of 350 % compared to the activity of the untreated SP 435 after an incubation time of 40 h. After 5 days this preparation still exhibited 210 % of the initial activity. Here again, incubation in tert-BuOH showed a linear deactivation in time. Recently, Lozano et al., (2001) have similarly reported a stabilizing effect of ionic liquids on CaLB. Interestingly, they found that the half life of the enzyme was three orders of magnitude greater when it was incubated in the presence of the substrate. In their study, experiments were performed in the presence of 2 % (v/v) water. In contrast to the results obtained with free CaLB and Novozyme 435, no stabilizing effect was observed when a CLEC or CLEA from CaLB was incubated in [BMIM][PF6] at 80C. A decrease in activity was observed in both the ionic liquid and tert-butyl alcohol which was comparable to that found for CaLB and Novozyme 435 in tert-butyl alcohol. The stabilizing effect of the ionic liquid on the free and supported enzyme possibly results from a protecting action of a coating of ionic liquid on the microenvironment (hydration layer) of the enzyme. The reason for the lack of a stabilizing effect on the CLEA and CLEC preparations is not clear and forms the subject of further investigations (Sheldon et al., 2002).

5. Enzyme selectivity in ionic liquids

One of the novel properties exhibited by enzymes in organic solvents, as compared to in aqueous solution, is their altered specificity, such as enhanced enantioselectivity and regioselectivity. In ionic liquids, some enzymes are even more enantioselective and regioselective. 5.1 Enantioselectivity Several research groups have reported that lipases showed higher enantioselectivity when used for kinetic resolution of chiral alcohols in ionic liquids. Sometimes, this enantioselectivity is remarkably dependent on the nature of the reaction medium. Take the lipase-mediated acetylation of racemic P-chiral hydroxymethanephosphinates and hydroxymethylphosphine oxides as an example; the enantioselectivity in [BMIM][[PF6] was up to six times higher than in common organic solvents, but was negligible in [BMIM][BF4]. A higher enantioselectivity for ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 11

Role of Ionic Liquids in Enzyme Catalysis lipase in [BMIM][PF6] than in [EMIM][BF4] was also observed (Kim et al., 2001). The enzymemediated kinetic resolution in ionic liquids has shown its advantages when conducted at elevated temperatures due to the high enzyme thermostability in ionic liquids. The kinetic resolution of (R,S)-1-phenylethanol catalyzed by a lipase from Pseudomonas sp. in [BMIM][Tf2N] remained highly enantioselective, with only a minimal decrease in the E-value from 200 to 150 when the temperature was raised from 25 to 90C; while in MTBE, the E-value dropped dramatically to 4 at 55C (Eckstein et al., 2002). Subtilisin has been used in the enzymatic resolution of amino acid esters in the ionic liquid [EPy][TFA] (Zhao and Malhotra, 2002). The hydrolysis of N-acylamino acid esters into the corresponding (S)-amino acids became more enantioselective when it was carried out in [EPy][TFA]H2O (15:85) instead of acetonitrileH2O (15:85). 5.2 Regioselectivity The lipase-catalyzed esterification of carbohydrates in ionic liquids can be more advantageous as compared to that occurring in organic solvents because of the enhanced regioselectivity (Park and Kazlauskas, 2001). In organic solvents, acetylation of insoluble glucose by lipase yielded a mixture in the product, because the first monoacylated product (6-o-acetyl glucose) was more soluble than the substrate glucose in the reaction medium, therefore tending to undergo further acetylation into a diester (3,6-o-diacetyl glucose). In ionic liquids, however, the reaction was highly selective in the best ionic liquid [MOEMIM][BF4] which dissolved 5 mg/ml glucose at 55C, 100 times more than acetone or THF, the reaction at 55C for 36 h reached 99 % yield, of which 93 % was the desired monoacylated product. By contrast, in the ionic liquid [BMIM][PF6], in which glucose has a low solubility of less than 1 mg/ml at 55C, the reaction showed both a slow rate (29 % conversion) and a low selectivity (29 % monoacetyl). This experiment has thus demonstrated that for the regioselective acetylation of glucose in ionic liquids, the ability of the ionic liquid to dissolve the substrate is an important factor, and this ability has enabled the extension of using ionic liquids for other substrates which are difficult in organic solvents, such as maltose, ascorbic acid, and even cellulose (Park and Kazlauskas, 2003). 5.3 Suppression of side reactions Kaftzik et al., (2002) have studied galactosylation with -galactosidase for the synthesis of Nacetyllactosamine from lactose and N-acetylglucosamine. This reaction is problematic when performed in aqueous solution because the yield is limited by the competing further hydrolysis of the product. However, this secondary hydrolysis can be effectively suppressed by addition of 25 % (v/v) [MMIM][MeSO4] as a water-miscible co-solvent, resulting in an increase in the product yield from less than 30 % in aqueous buffer to 58 % in aqueous ionic liquid (Yang and Pan, 2005).

6. Effect of ionic liquids on enzyme activity and the Hofmeister series

Most biocatalysis in ionic liquids involved no or little water as co-solvent (Song, 2004). Most ionic liquids used in biocatalysis are hydrophobic types of PF6-and (CF3SO2)2N-salts (water ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 12

Role of Ionic Liquids in Enzyme Catalysis immiscible or partially water-miscible). Hydrophobic solvents could be superior to hydrophilic ones (water-miscible) because the latter might remove internally bound water (essential water) from the enzyme (Zaks and Klibanov, 1988). Currently, there are limited results of enzymatic reactions in hydrophilic ionic liquids containing a considerable amount of water. The effect of cations and anions of ionic liquids on the enzyme activity and stability is as shown in the Table 3 (Zhao, 2005). When the biocatalysis was conducted in hydrophobic or anhydrous hydrophilic ionic liquids, the enzyme activity does not seem to follow the Hofmeister series (Lau et al., 2004) even though NO3- (B-coefficient = -0.043), BF4- (-0.093) and PF6- (-0.21) are all chaotropic anions. The same conclusion applies to the enantioselectivity of enzymes. The hydrophobicity of ionic liquids may be described by the log P, a concept derived from the partition coefficient of ionic liquids between water and octanol. Generally, enzymes are more stable in solvents with a larger log P (>3) (such as hexane has a log P of 3.9) than lower log P (such as ethanol has a log P of 0.24). Free lipase (Candida rugosa) was found only active in [BMIM][PF6] (log P = -2.39) for the transesterification of methyl methacrylate, but inactive in other ionic liquids including [BMIM][CH3COO-] (log P = -2.77) [BMIM][NO3], (log P = -2.90) and [BMIM][CF3COO-] (Karr et al.,2003). This example illustrated that the high hydrophobicity (large log P) of ionic liquids could be beneficial to the enzyme activity. It was possible that the enzyme was destabilized by the latter three ionic liquids because they are hydrophilic and could strip off the essential water from the enzyme at high salt concentrations (Zaks and Klibanov, 1988). Fig. 1 revealed the relationship of ionic liquids hydrophobicity and the enzyme hydration. If the ionic liquid is more hydrophobic (less negative of the log P value), then less water is required for maintaining optimal enzyme activity and enantioselectivity. When the ionic liquid is hydrophilic ([BMIM][BF4]), more water presence is needed because high concentrations (high ionic strength) of hydrophilic ionic liquids tend to remove the essential water from the enzyme causing the deactivation (Zhao, 2005).

Fig.1 The relationship of log P values of three ionic liquids and the optimal hydration of Candida rugosa lipase {cw (activity) is the water concentration for optimal hydration of the lipase activity; cw (enantioselectivity) is the water concentration for optimal hydration of lipase enantioselectivity; ONIM is 1- octyl-3-nonylimidazolium}. ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 13

Role of Ionic Liquids in Enzyme Catalysis Example of the importance of water activity on biocatalysis in hydrophobic ionic liquids is of chymotrypsin. A number of studies illustrated that water was required by -chymotrypsin to maintain its enzymatic activity (Lozano et al., 2001; Eckstein et al., 2002) and the bell-shaped relationship between reaction rates and water contents is similar to that in organic solvents. Table 3 also demonstrated that higher enzyme activity might be observed in ionic liquids with larger cations (such as [OMIM]+) than those with smaller ones (such as [BMIM]+). The longer hydrophobic alkyl chain in the cation has fewer tendencies to take away the essential water from the enzyme (Yang and Pan, 2005). The water-stripping ability of hydrophobic [BMIM][(CF3SO2)2N] was found comparable to that in polar organic solvents (Eckstein et al., 2002).

Fig.2 The rate of enzyme-catalyzed reaction with low and high water content as a function of total water content (Buchholz et al., 2005). Table 3 Effect of ionic liquids (ILs) on the enzyme activity and stability. Nature of Effect Hydrophobic or anhydrous ILs Stability of Novozym 435 in ILs at 30C Order of effectiveness Anions: CH3COO- >PF6- >NO3([BMIM]+ or [MMEP]+ based ILs) Reference Kaar et al., 2003

Initial reaction rates of PEGlipase catalyzed alcoholysis in ILs Enantioselective acylation of 1phenylethanol by lipase CaLB

Cations: [MMEP]+ > [BMIM]+ (CH3COO-, PF6-, or NO3- salts) Cations: [OMIM]+ > [HMIM]+ > Maruyama et al., 2002 [BMIM]+ (PF6- based ILs) Anions: [(CF3SO2)2N]-, [CF3SO3]- >BF4- >PF6([BMIM]+ based ILs) Cations: [OMIM]+ > [HMIM]+ > [BMIM]+ (BF4-based ILs) Schofer et al., 2001

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Role of Ionic Liquids in Enzyme Catalysis Nature of Effect Enantioselectivity of the acetylation of 1-phenylethanol with vinyl acetate by lipase from Pseudomonas cepacia in ILs (purification method B, no additive) Enantioselectivity of Candida rugosa lipase in the esterification of 2-substituted-propanoic acids and 1-butanol in ILs Activity of Candida antarctica lipase B in transesterification of ethyl butanoate and 1-butanol Hydrophilic ILs containing water Specific activity of esterase from Anions: [(CF3SO2)2N] >BF4 >PF6 ([BMIM]+ based ILs) Bacillus stearothermophilus in the kinetic resolution of 1-phenylethanol (10 mM) with vinyl acetate (200 mM) at 40C, aw = 0.11 Stability of -chymotrypsin in ILs (2% water, v/v) at 50C Stability of Candida antarctica lipase B in ILs (2% water, v/v) exhibited by incubation without substrates Activity of cellulase from Trichoderma reesei in salt solutions containing 20100% water Anions: PF6 >BF4 ([BMIM]+ based ILs) Anions: BF4 >PF6 > [(CF3SO2)2N] ([EMIM]+ and [BMIM]+ based ILs) Cations: [MMIM]+ > [BMIM]+ ([(CF3SO2)2N] based ILs) Sodium citrate buffer > sodium dodecylsulfate > NaCl > [BMIM]Cl Persson and Bornscheuer, 2003 Order of effectiveness Anions: BF4 >PF6 ([BMIM]+ based ILs) Cations: [EMIM]+ > [PrMIM]+, [BuPy]+ > [PrPy]+ > [BMIM]+ (BF4- based ILs) Anions: PF6 >BF4 ([BMIM]+ based ILs) Cations: [BMIM]+ > [OMIM]+ (PF6 based ILs) Anions: BF4 >PF6 > [lactate] >NO3 ([BMIM]+ based ILs) Reference Park and Kazlauskas, 2001

Ulbert et al., 2004

Lau et al., 2004

Lozano et al., 2001 Lozano et al., 2003

Turner et al., 2003

Note: BMIM: 1-butyl-3-methylimidazolium; EMIM: 1-ethyl-3-methylimidazolium; MMIM: 1,3dimethylimidazolium; HMIM: 1-n-hexyl-3-methylimidazolium; OMIM: 1-n-octyl-3methylimidazolium; MMEP: 1-methyl-1-(2-methoxyethyl) pyrrolidinium; PrMIM: 1-propyl-3methylimidazolium; BPy: 1-n-butylpyridinium; PrPy: 1-propylpyridinium; EPy: 1ethylpyridinium. ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 15

Role of Ionic Liquids in Enzyme Catalysis From the molecular level, the enzyme stabilization by water immiscible ionic liquids (such as [(CF3SO2)2N] types) was explained as a more compact enzyme confirmation formed from the evolution of -helix to -sheet secondary structure of the enzyme. In conclusion, hydrophobic ionic liquids (1) may strip off the essential water from the enzyme as organic solvents do; (2) may interact with the enzyme through electrostatic interactions. However, the inhibition mechanisms of enzymes by ions could not be explained entirely by the electrostatic interactions; (3) may interact with substrates or products as organic solvents do (Zhao, 2005). On the other hand, with the presence of water in hydrophilic ionic liquids, the effect of ions on the enzyme activity is related to the Hofmeister series (Table 3) because these ionic liquids dissociate into individual ions in water. Lau et al., (2000) reported a high conversion in pure [EMIM][BF4] for the transesterification of ethyl butanoate with 1-butanol catalyzed by Novozym 435, but a much lower conversion in the same ionic liquid containing 10 % (v/v) water. When water was involved, the chaotropic BF4- and kosmotropic [BMIM]+ could deactivate the enzyme. Zhao,(2005) indicated that the protease stability in low concentrations of ionic liquid solutions could be related to the Hofmeister series (Table 3): kosmotropic anions (such as CH3COO- and CF3COO-) stabilize the enzyme while chaotropic ones (such as BF4-) destabilize it. In the presence of water, the cations in ionic liquids impose a considerable effect on the enzyme activity. Most data in the second part of Table 3 seem to support that enzymes exhibited higher activity and stability in ionic liquids with smaller cations. The reason is because organic cations experience hydrophobic hydration, and smaller organic cations are chaotropic while larger ones are kosmotropic.s Turner et al., (2003) noticed that the high Cl- ion concentration did not seem to be the only reason for the inhibition of cellulase in [BMIM]Cl solution. Maybe the cation was relevant to the enzyme activity. Besides regularly seen quaternary alkyl-substituted ammonium, phosphonium, imidazolium and pyridinium ionic liquids, it is noticeable that new ionic liquids based on pyrrolidinium cations (as well as triazolium, pyrazinium, pyridazinium, pyrimidinium, pyrazolium, piperazinium, thiazolium, oxazolium, oxazolidinium and morpholinium cations) have recently been prepared, and their physical properties have been characterized. The Zhao, (2005) reported higher stabilities of Novozym 435 in pyrrolidinium ([MMEP]+) ionic liquids than in [BMIM]+ based ionic liquids. Another example of using pyrrolidinium Ionic liquids for biocatalysis is the lipasecatalyzed synthesis of polyesters. Although the B-coefficients and other hydration information of pyrrolidinium cations are not yet available, these new aliphatic ions are less toxic than aromatic species and thus are worthy of a further investigation as greener media for biocatalysis.

7. Applications of using enzymes in ionic liquids

Applying enzymes in ionic liquids has a very short history (Yang and Pan, 2005). More recently, ionic liquids have been employed to the whole-cell in situ fermentation and showed its great potential in whole-cell biocatalytic processing due to their low toxicity to microorganisms (Howarth et al., 2001).

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Role of Ionic Liquids in Enzyme Catalysis Proteases The first successful biotransformation in ionic liquids was reported by Erbeldinger et al., (2000) who used the protease thermolysin in the ionic liquid [BMIM][PF6] containing 5 % (v/v) water for the synthesis of the dipeptide Z-aspartame. A yield of 95 % was obtained with enzyme activity comparable to that observed in conventional organic solvents such as ethyl acetate, and excellent enzyme stability was also achieved as compared to that in the organic solvent. They found that the effect of enzyme concentration on rate of reaction in ionic liquids has some interesting features. At low thermolysin concentration (<3 mg/ml), they were not able to observe synthesis of Z-aspartame in [BMIM][PF6] because below 3.2 mg/ml, thermolysin dissolves in [BMIM][PF6] and its dissolved form is completely inactive. By using Bradford protein assay, they measured thermolysin saturation in ionic liquid at 3.2 mg/ml and they hypothesized that thermolysin needs to be in suspension in order to maintain its activity in [BMIM][PF6].
O OMe NH2
L-Phe Thermolysin

O HO
+

OH O NHZ
Z-L-Asp [bmim][PF6 ]/H2O 95:5v/v, 37oC, 50h

NHZ H N

O OH

O OMe

95% yield, Aspartame Precursor

Lipases The work from Sheldon et al., (2002) was the second report to demonstrate the potential use of ionic liquids for biocatalysis and the first one to show the use of ionic liquids with lipases. Their experiments have shown the broad use of lipases in ionic liquids, namely catalyzing alcoholysis, ammoniolysis, and perhydrolysis reactions with reaction rates generally comparable with, or better than, those observed in organic solvents. However, the types of ionic liquids and enzymes used so far are quite restricted; the ionic liquids that are commonly used are the imidazolium salts such as [BMIM][PF6], and the majority of enzymes reported to be active and frequently used in ionic liquids are hydrolases, or more specifically, lipases. ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 17

Role of Ionic Liquids in Enzyme Catalysis Sheldon et al., (2002) showed that Candida antarctica lipase B (CaLB), either as the free enzyme (SP 525) or in an immobilised form (Novozym 435), is able to catalyze a variety of transformations in [BMIM][BF4] or [BMIM][PF6] in the absence of added water. For example, transesterification (Scheme 1) proceeded with rates comparable to those observed in tert-butyl alcohol, a commonly employed solvent for such transformations. The immobilised enzyme (Novozym 435) gave higher rates than the free enzyme (SP 525) suspended in the ionic liquid. This is consistent with the generally observed higher reactivity of the immobilised lipase, compared to the free enzyme, in organic media.
CaLB [bmim][PF6] or [bmim][BF4] 40oC

R1CO2Et

+ R2OH

R1CO2R2

EtOH

(Scheme 1) Sheldon et al., (2002) showed that CaLB-catalysed ammoniolysis of carboxylic esters to the corresponding amides occurs smoothly under mild conditions. Primary fatty acid amides are important commodities that are conventionally prepared by reaction of the ester with ammonia under forcing conditions of temperature and pressure. Sheldon et al., (2002) found that the reaction of octanoic acid with ammonia (Scheme 2) in the presence of Novozym 435 at 40C in [BMIM][BF4], proceeded to complete conversion in 4 days, compared to 90100 % conversion in 17 days using ammonium carbamate in methylisobutylketone.
OH

NH3

O CaLB [bmim][BF4]

NH2 O

(Scheme 2)

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Role of Ionic Liquids in Enzyme Catalysis Enantioselectivity Recently, several groups have investigated lipase-catalyzed transesterification of chiral substrates in ionic liquids. A primary aim of these studies was to ascertain the effect of ionic liquid media on the enantioselectivities of these transformations. For example, Schofer et al., (2001) investigated the kinetic resolution of 1-phenylethanol with nine different lipases in ten different ionic liquids.
HO Lipase/24oC/3 days HO AcO

OAc

CH3CHO

Lipase mediated enantioselective acylation of 1-phenylethanol

(Scheme 3) Enantioselective esterification of ()-menthol was studied using Candida rugosa lipase (CRL) in ionic liquids (1-butyl-3-methylimidazolium hexafluorophosphate) and 1-butyl-3-methylimidazolium tetraflouroborate) and organic solvents of different hydrophobicities. Propionic anhydride was employed as an acylating agent. Comparison of the activity, stability and enantioselectivity of CRL was carried out by examining the effects of the mole ratio of substrates, temperature, incubation time and enzyme recycling. It was found that temperature control was more crucial in the ionic liquid than in hexane to reach high conversion and enantioselectivity. The ionic liquid system showed an advantage of using less acid anhydride to achieve higher ()-menthol conversion yield and better enantioselectivity. Moreover, during an incubation of 460 days in the ionic liquid, CRL activity was 2.5 times higher than its initial value, while that in hexane decreased to less than 60 % in 2 days. In addition, the enzyme showed potentiality of recycled use in the ionic liquid. These advantages of the ionic liquid suggest that it would be used as a green alternative to organic solvents for the enantioselective esterification of ()-menthol (Yuan et al., 2006). Kim et al., (2001) studied the transesterification of several chiral alcohols catalyzed by CaLB and PCL in [EMIM][BF4] and [BMIM][PF6]. Markedly enhanced enantioselectivities were observed compared with the same reactions performed in toluene or THF. Ionic liquid anchored substrate for enzyme catalyzed kinetic resolution A hydroxyl group appended task specific ionic liquid was designed and synthesized by researchers. The ionic liquid was used as a vehicle for the substrate (ibuprofen anchored ionic liquid) for lipase catalyzed kinetic resolution. The ionic liquid anchored ibuprofen underwent Candida antarctica lipase catalyzed hydrolysis yielding the S-enantiomer. The strategy facilitated easy post-resolution isolation of the enantiomer and also carries the prospect of ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 19

Role of Ionic Liquids in Enzyme Catalysis recyclability. The ibuprofen was anchored on the ionic liquid via ester linkage so that it can be hydrolyzed enantioselectively by lipases (Naik et al., 2007).

PF6-

Fig.3: Ibuprofen anchored ionic liquid for the lipase catalyzed kinetic resolution. Oxidoreductases Much less attention has been paid to oxidoreductases in ionic liquids. Recently, the immobilized bakers yeast reduction of ketones in a 10:1 [BMIM] [PF6]/water was reported (Howarth et al., 2001). Sheldon et al., (2003) showed that formate dehydrogenase from Candida boidinii exhibited 98% residual activity (compared to aqueous buffer) in a 3:1(v/v) [MMIM] [MeSO4]/H2O mixture. The carbonyl reductase from Candida parapsilosis (CPCR) showed no activity in 1:3 [MMIM][MeSO4]/H2O in the reduction of acetophenone. Similarly, yeast alcohol dehydrogenase displayed only a very low activity in ethanol oxidation in ionic liquid/water mixture. Glycosidases Sheldon et al., (2003) have reported the successful use of ionic liquid/water mixtures as reaction media for the -galactosidase catalyzed synthesis of N-acetyllactosamine. The addition of 25% (v/v) [MMIM][MeSO4] as a water miscible ionic liquid suppressed secondary hydrolysis of the N-acetyllactosamine product, resulting in an increase in yield from 30-58 %. They further showed that the enzyme could be recycled several times, after ultrafiltration of the reaction mixture, without loss of activity. Horseradish peroxidase The peroxidase catalyzed reaction of phenolic compounds in organic solvents yielded interesting products, often characterized by the presence of more homogeneous polymeric products with respect to the same reaction conducted in aqueous buffers. However, most co-solvents used endowed with intrinsic toxicity and low biodegradability; thus rendering the catalytic process less convenient for large scale synthesis.

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Role of Ionic Liquids in Enzyme Catalysis The reactivity of horseradish peroxidase with water insoluble compounds has been studied in [BMIM][BF4]/water mixtures. The enzyme retained some catalytic activity up to 90 % ionic liquid in water at 25C only at pH values higher than 9.0. Activity steadily decreased towards neutral and acidic conditions. The quenching effect of [BMIM][BF4] on the peroxidase turnover is interpretated according to three different mechanisms that are the binding of F- anion to the peroxidase active site at neutral and acidic pH values, the dielectric constant induced lowering of the heme iron redox potential and a solvent shielding effect that impairs the productive encounters of radical species. As a result of these effects, products distribution is inhibited thus favouring the formation of discrete dimmers. Thus, although the narrow operational window determined by the interplay between pH (higher than 9.0) and [BMIM][BF4] concentration (50 75%, v/v) may appear limiting for most traditional peroxidase chemistry applications, the high product homogeneity and the ease of raw product recovery (precipitation after water dilution) pave the way to novel possible synthetic strategies based on the use of peroxidases for selective C-C bond (Simona et al., 2007). Laccase Oxidative enzymes, laccase C from Trametes sp. and horseradish and soybean peroxidases, catalyzed oxidation reactions in systems with ionic liquids whose content varied from several volume percent to almost total non-aqueous ionic liquids. For the oxidation of anthracene, catalyzed by laccase C and assisted by a number of mediators, addition of 4-methyl-Nbutylpyridinium tetrafluoroborate, instead of tert-butanol, increased the yield of the oxidation product several-fold (Hinckley et al., 2002). Oxidation of syringaldazine catalyzed by laccase does not require the presence of mediators. The results of catalytic activity measurements of laccase in this reaction show that the enzyme tolerates moderate concentrations of a water-miscible ionic liquid [4-MBP][BF4]. Increasing concentrations of this solvent resulted in a decrease of the laccase activity, causing enzyme precipitation at concentrations of [4-MBP][BF4] above 50% (v/v). A detailed analysis of kinetic data revealed that the decrease in the laccase activity was a result of a simultaneous decrease in Vmax and increase in Km (Table 4) (Hinckley et al., 2002). Enzymes have been shown to retain catalytic activity in ionic liquids at very low water contents (Sheldon, 2001). In order to explore the feasibility of using this type of reaction systems as a medium for laccase catalyzed reactions, they measured the catalytic activity of laccase C suspended in a water-immiscible ionic liquid [BMIM][PF6]. While the enzyme did not show a measurable activity in anhydrous [BMIM][PF6], the oxidation of syringaldazine was observed in the ionic liquid saturated with aqueous buffer. The values of kinetic parameters Vmax and Km determined for this reaction are given in Table 4, showing that the observed catalytic activity of laccase in [BMIM][PF6] is several orders of magnitude lower than the aqueous activity of the enzyme, which is typical for catalysis by enzymes in nonaqueous solvents with low water content (Klibanov, 2001).

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Role of Ionic Liquids in Enzyme Catalysis Table 4 Kinetic parameters for syringaldazine oxidation catalyzed by laccase C in reaction systems with different ionic liquids at 25C Kinetic parameter Vmax (nmol min1 mg) Km (M)
a b

Aqueous tert-Butanol (4-MBP)BF4a (BMIM)PF6b buffer buffer 20% (v/v) 25% (v/v) 510 0.9 420 5.0 42 21 5.7 103 40

4-methyl-N-butylpyridinium tetrafluroborate. 1-butyl-3-methylimidazolium hexaflurophosphate.

Lyases The cleavage of mandelonitrile catalysed by hydroxynitrile lyases (HNL) from Prunus mygdalus (PaHNL) and Manihot esculenta (MeHNL) proceeded more rapidly in monophasic aqueous media containing 1-propyl-3-methylimidazolium tetrafluoroborate [PrMIM][BF4] than in media containing acetonitrile or THF. Both HNLs were much more thermostable in [PrMIM][BF4] than in acetonitrile or THF. The addition of each of the four ionic liquids 1-butyl-, 1-pentyl- and 1hexyl-3-methylimidazolium tetrafluoroborates at 26% (v/v in the aqueous phase) increased both the enzyme activity and the product e.e. in the PaHNL-catalysed transcyanation in an aqueous/DIPE biphasic system. However, MeHNL was inactivated by the ionic liquids, as indicated by the decreased reaction rate, substrate conversion and product yield. The effect of ionic liquids on enzymatic reactions varies widely with the enzyme used. To see whether HNLs could work well in such novel media, they examined the HNL catalysis in ionic liquids. In solvent mixtures with low water content, a slow initial reaction rate was observed. Enzymes exhibited higher activity in the aqueous solution of [PrMIM][BF4] than in acetonitrile or THF-containing system, under the same conditions. This suggested that the denaturation of HNLs by [PrMIM][BF4] was less serious compared to the organic solvent-containing systems (Lou et al., 2005). Enzymatic condensation reactions in ionic liquids In an aqueous environment glycosidases and peptide amidases usually hydrolyse glycosidic bonds or amides, respectively. The reaction can be reversed by incubating the enzyme at lower water activity in the presence of ionic liquids, resulting in higher yield of disaccharide or peptide amide. -galactosidases from Bacillus circulans can be applied in nearly anhydrous ionic liquids for reverse hydrolysis with yields of lactose of up to 17 %. Peptide amidase from Stenotrophomonas maltophilia is used for the direct C-terminal peptide amidation. Kaftzik et al., (2003) studied the reverse hydrolytic activity of -galactosidases from Bacillus circulans in both aqueous media containing different amounts of ionic liquid and the pure ionic liquid 1,3-dimethylimidazole methylsulphate. To favour the formation of disaccharides, high concentration of monosaccharides is necessary. Ionic liquids offer the advantage of dissolving ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 22

Role of Ionic Liquids in Enzyme Catalysis carbohydrates very well allowing higher concentrations than in water especially mixtures of water and organic solvents as cosolvents. Kaftzik et al., (2003) also studied the synthetic activity of peptide amidase. The substrate used was H-Ala-Phe-OH. The ammonium bicarbonate was used as ammonium source. They used a series of different ionic liquids from total water miscible to water insoluble ionic liquids. Solubility properties and amidation rates were compared with results obtained in organic solvents like acetonitrile, glycerin or ethyleneglycol. The best results were obtained in [BMIM][MeSO4].

8. Ionic liquids allow unconventional reaction techniques

The unique properties of ionic liquids allow the use of unconventional reaction techniques.

Catalyst recycling Ionic liquids, such as [BMIM][PF6], do not mix with ether. This unconventional behaviour was advantageously used by extracting the products and the unconverted reactant from the transesterification mixture of 5-phenyl-1-pentene-3-ol with ether (Sheldon et al., 2002). The lipase biocatalyst remained suspended in the ionic liquid phase and could be recycled.

Product evaporation Because ionic liquids lack a vapour pressure, products can be removed by evaporation, as was shown in the reduction of prochiral ketones by bakers yeast in [BMIM][PF6].Evaporation of the alcohol side-product in lipase-catalyzed transesterification reactions can be used to drive the equilibrium towards complete conversion. (Rantwijk, 2003) Two-phase system with supercritical CO2 The above-mentioned approach of retaining the biocatalyst in the ionic liquid reaction medium has been further developed into a biphasic reaction system. The enzyme is retained in an ionic liquid working phase and the reactants and products largely reside in a scCO2 extractive phase (Reetz et al., 2002). The high operational stability of CaLB, which contrasts with the generally ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 23

Role of Ionic Liquids in Enzyme Catalysis rapid deactivation in pure scCO2, is one of the attractive aspects of this approach. The reaction rate was approximately eight times better than that in pure scCO2 under otherwise identical conditions (Lozano et al., 2002). Two-phase aqueous systems Two-phase aqueous reaction systems, consisting of an aqueous working phase and an organic extractive phase, are widely used in biotransformations of hydrophobic compounds. They are useful with biocatalysts that require an aqueous phase for activity, in particular when water does not interfere with the desired reaction, as is the case, for example, with non-hydrolase biocatalysts. The replacement of organic solvents as the extractive phase by the hydrophobic ionic liquid [BMIM][PF6] has, until now, only been demonstrated with whole-cell nitrile hydratase-amidase (Cull et al., 2000) and redox biocatalysts (Howarth et al., 2001), as well as in the recovery of butanol from acetonebutanol-ethanol fermentations(Rantwijk et al., 2003). The technique could also prove to be useful with isolated enzymes, which are prone to deactivation at aqueousorganic interfaces by proper design of the ionic liquid (Rantwijk et al., 2003). Two phase system with organic solvent Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liquid (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF4] or 1-butyl-3methyl imidazolium hexafluorophosphate [BMIM][PF6]) and tert-butanol as organic solvent was investigated by Ganske and Bornscheuer, (2005). The best enzyme commercially available was lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginose (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temperature, reaction time, percentage of co-solvent) conversions up to 60 % could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF6] in the presence of 40 % tert-BuOH with CAL-B at 60C (Ganske and Bornscheuer, 2005).

9. Conclusion
The results reported until now on enzyme catalyzed reactions in ionic liquids have clearly shown that the potential of using ionic liquids as solvents for biocatalysis. Enzymes in ionic liquids have shown enhanced activity, stability and selectivity, than observed in conventional organic solvents. Due to high polarity of ionic liquids they can be used as reaction medium for biotransformation of wide range of substrates, especially highly polar ones such as carbohydrates and amino acids, which are sparingly soluble in organic solvents. Another important feature of ionic liquids is that their solvent properties can be finely tuned by selecting appropriate combinations of cations and anions, thus allowing the ionic liquids to be specially designed for different reaction conditions, hence ionic liquids are called as designer solvents. The research on use of ionic liquids as reaction media for enzyme catalyzed reaction is at its infant stage. The further investigation needs to be done to understand the fundamentals of biocatalysis in ionic liquids in more details. Also the studies are necessary to find out the reason ___________________________________________________________________________ Institute of Chemical Technology, Mumbai. 24

Role of Ionic Liquids in Enzyme Catalysis behind the observed advantages, such as better activity, stability, selectivity and suppression of side reactions. Ionic liquids are more expensive than organic solvents (800 times more). A key to their industrial use will be their efficient recovery, product isolation and reuse. A suitable method of isolation needs to be developed for the products that are less volatile or non-volatile.

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Role of Ionic Liquids in Enzyme Catalysis

10. References
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Role of Ionic Liquids in Enzyme Catalysis Ulbert O., Frater T., Belafi-Bako L. and Gubicza (2004) Enhanced enantioselectivity of Candida rugosa lipase in ionic liquids as compared to organic solvents Journal of Molecular Catalysis B: Enzymatic31:39-45. Yang Z. and Pan W., (2005) Ionic Liquids: Green solvents for nonaqueous biocatalysis Enzyme and Microbial technology 37: 19-28. Yuan Y., Shu B. and Sun Y. (2006) Comparison of lipase-catalyzed enantioselective esterification of ()-menthol in ionic liquids and organic solvents Food Chemistry 97: 324-330. Zaks A. and Klibanov A. M. (1988) The Effect of Water on Enzyme Action in Organic Media The Journal of Biological Chemistry 263: 8017-8021. Zhao H. (2005) Effect of ions and other compatible solutes on enzyme activity, and its implication for biocatalysis using ionic liquids Journal of Molecular Catalysis B: Enzymatic 37: 16-25. Zhao, H. and Malhotra, S.V. (2002), Enzymatic resolution of amino acid esters using ionic liquid N-ethyl pyridinium trifluoroacetate Biotechnology Letters 24: 1257.

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