Recent advances of enzymatic reactions in ionic liquids

Biochemical Engineering Journal 48 (2010) 295–314

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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej

Review

Recent advances of enzymatic reactions in ionic liquids Muhammad Moniruzzaman a , Kazunori Nakashima b , Noriho Kamiya a,c , Masahiro Goto a,c,∗ a

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkoudaicho, Nada-ku, Kobe 657-8501, Japan c Center for Future Chemistry, Kyushu University, Fukuoka 819-0395, Japan b

a r t i c l e

i n f o

Article history: Received 31 July 2009 Received in revised form 2 October 2009 Accepted 5 October 2009 Keywords: Biocatalysis Biotransformation Bioprocesses Enzymes stability Ionic liquids Water-in-ionic liquid microemulsions

a b s t r a c t The tremendous potential of room temperature ionic liquids as an alternative to environmentally harmful ordinary organic solvents is well recognized. Ionic liquids, having no measurable vapor pressure, are an interesting class of tunable and designer solvents, and they have been used extensively in a wide range of applications including enzymatic biotransformation. In fact, ionic liquids can be designed with different cation and anion combinations, which allow the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ionic liquids provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. As compared to those observed in conventional organic solvents, the use of enzymes in ionic liquids has presented many advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. To date, a wide range of pronounced approaches have been taken to further improve the performance of enzymes in ionic liquids. This review presents the recent technological developments in which the advantages of ionic liquids as a medium for enzymes have been gradually realized. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

5. 6.

7.

8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ionic liquids and their properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymes in ionic liquids: a comparison study with organic solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stabilizing enzymes in ionic liquids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Water-in-ionic liquid microemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Immobilization and modification of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Reactions using whole cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recently conducted enzyme-catalyzed reactions in ionic liquids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application to bioprocesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Enzymatic polymerization in ionic liquids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Proteins and enzymes extraction for biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Biofuels production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Combination of ionic liquid and enzymes for biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Ionic liquid membranes for biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Ionic liquid-coated enzymes for biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Ionic liquids and enzymes in sol–gel methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan. Tel.: +81 92 802 2806; fax: +81 92 802 2810. E-mail address: [email protected] (M. Goto). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.10.002

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Fig. 1. Number of articles described the enzymatic reactions in ionic liquids found from the “Scopus”, in the period 2000–2009, 30 September.

1. Introduction Ionic liquids (ILs) that are composed entirely of ions and are liquids at ambient or far below ambient temperature, have been extensively used as a potential alternative to toxic, hazardous, flammable and highly volatile organic solvents [1–4]. Indeed, their many unique and attractive physicochemical properties, including negligible vapor pressure [4], multiple solvation interactions with organic and inorganic compounds [5], excellent chemical and thermal stability [6], high ionic conductivity and large electrochemical window make ILs great candidates for volatile organic compounds replacements. In addition, the physicochemical properties such as the viscosity, hydrophobicity, density, and solubility of ILs can be tuned by simply selecting different combinations of cations and anions as well as attached substituents to customize ILs for many specific demands. This is why ILs have been recognized as “designer solvents.” All these interesting combination of properties opens the road to a wide range of applications, including extraction [7], organic synthesis and catalysis [8–9], inorganic synthesis [10], separation [11], nanomaterial synthesis [12] and enzymatic reactions [13–19]. In particular, the application of environmentally benign ILs1 as media for biotransformation has received tremendous attention in the past few years. The technological utility of enzymes can be enhanced greatly by their use in ILs rather than in conventional organic solvents or in their natural aqueous reaction media due to their unusual solvent characteristics. Studies on enzymatic reactions in ILs over the last 8–9 years have revealed not only that ILs are environmentally friendly1 alternatives to volatile organic solvents, but also that in such a solvent enzymes exhibit excellent selectivity including substrate, regio- and enantioselectivity. Besides these, many enzymes maintained very high thermal and operational stability in ILs. Therefore, ILs are of growing interest as a new and highly efficient reaction medium for biocatalytic reactions (see Fig. 1, researches on biocatalysis in ILs increase linearly). The first successful report on an enzymecatalyzed reaction using IL as a medium was published in 2000 by Russell and co-workers [20]. Since then, a wide number of enzymes have been subjected to ILs to test their catalytic activity; examples are lipases [21–37], alcohol dehydrogenases

1 Since ionic liquids have no detectable vapor pressure, they are often said to be “green” or “environmentally friendly” or “environmentally benign” solvents as compared to traditional volatile organic solvents.

Fig. 2. Structures of typical cations of ionic liquids discussed in the review.

[38,39], proteases [20,40–42], and oxidoreductases [43–50] and so forth. During this time, there have been several excellent reviews that focus on enzyme-catalyzed reactions in ILs [14–19], documenting the advances of the entire fields. Among these, an excellent review published by Yang and Pan has highlighted the solvent properties of ILs, their effects on enzyme performance such as activity, stability and selectivity [17]. Recently, Rantwijk and Sheldon have published a well balanced and comprehensive review that covers the various issues extensively regarding the biocatalysis in ILs [19]. They discussed the effects of ILs on the structure and activity of enzymes as well as on their thermal and operational stability. As shown in Fig. 1, the use of ILs as reaction media is greatly expanding the repertoire of enzyme-catalyzed transformation. Studies over the past 8–9 years have established firmly that many enzymes can work in ILs containing small amount of water or no water [21,22,31]. Furthermore, the performances of enzymes in ILs are improved significantly by immobilization or modification with solid supports [32,42]. These trends are now continuing by discovering new, unique and useful techniques in which enzymes exhibit more pronounced activity, stability as well as selectivity in ILs. Here we will review some of recent technological developments that are proved to be very effective in biocatalysis using ILs as reaction media. In the present work, we put emphasis on the accessible literature published in this field of research from early 2007 to present. This will provide the information of recently developed enzyme-catalyzed reactions studied in ILs or IL-based solvent systems (e.g., water-in-ionic liquid microemulsions and biphasic systems) using any forms of enzymes (e.g., free, modified and immobilized). We also focus on the use of enzymes in ILs for some important bioprocesses applications including biofuels production and polymerization.

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297

Table 1 Ionic liquid anions. Anion −

Cl Br− BF4 − PF6 − (CF3 SO2 )2 N− (FSO2 )2 N− CF3 SO3 − C4 F9 SO3 − CH3 SO3 − CH3 OSO3 − CH3 COO− CF3 COO− CF3 (CF2 )2 COO− C6 H5 COO− HOCH2 COO− CH3 CH(OH)COO− HO2 CCH2 C(CO2 H)–(OH)CH2 COO− NO3 − C8 H17 SO4 − (CN)2 N− (C2 H5 )2 PO4 −

Full name

Abbreviation

Chloride Bromide Tetrafluoroborate Hexafluorophosphate bis((trifluoromethyl)sulfonyl)imide bis(fluorosulfonyl)imide Trifluoromethylsulphonate Perfluorobutylsulfonate Sulphonate Methylsulphate Acetate Trifluoromethylacetate Heptafluorobutanoate Benzyl acetate Glycolate Lactate Citrate Nitrate n-octylsulfate Dicyanamide Diethylphosphate

[Cl] [Br] [BF4 ] [PF6 ] [Tf2 N] [FSI] [TfO] [NfO] [MeSO3 ] [MS] [AcO] [TFA] [HB] [BzO] [glycolate] [lactate] [citrate] [NO3 ] [OcSO4 ] [dca] [(Et)2 PO4 ]

2. Ionic liquids and their properties Typical room temperature ionic liquids (RTILs) are composed of an organic cation (most often an alkyl-substituted imidazolium or a pyridinium or a quaternary ammonium ion) (see Fig. 2) and an inorganic anion (see Table 1). Ionic liquids have low melting points (<100 ◦ C) and remain as liquids within a broad temperature window (<400 ◦ C). The physical, chemical and biological properties of ILs generally depend on the structure of the cation (the symmetry and the length of alkyl substituents, the presence of hydrophobic groups, etc.) as well as on the degree of anion charge delocalization [51]. Ionic liquids are tailorable solvents in which they can be designed to have specific physicochemical properties through structural changes in the cation and anion. Therefore, ILs properties cover a broad range of values and cannot be generalized. A comprehensive database on physical properties of ILs such as melting point, density and viscosity has been presented by Zhang and co-workers [52]. The other solvent properties as regards polarity, hydrophobicity and solvent miscibility behavior of ILs have been described in the literature for specific applications [53–55]. Based on the solvation standpoint, ILs are generally considered to be highly polar solvents. A number of different methods have been used to provide information about their polarity, including solvatochromic dyes [57–60], partition [5,61,62] and fluorescence probe methods [63]. Solvatochromic dyes are compounds with a visible absorption maximum that depends on the polarity of the solvent. In this method, the solvent polarity is determined based on the shift of the charge-transfer absorption band of a solvatochromic probe in the presence of the solvent [64]. Empirical polarity scales, developed using solvatochromic dyes, indicate that the polarity of common ILs based on imidazolium cation such as [bmim][BF4 ] falls in the range of lower alcohols [57,58,60] and formamide [64]. However, the polarity of ILs generally decreases with an increase in the alkyl chain length appended on the imidazolium ring in the cation for a fixed anionic group [54]. Besides, solvatochromic study shows that ILs possessing noncoordinating anion (e.g., PF6 − and Tf2 N− ) are less polar than the lower alcohols [60]. Recently, Schrodle et al. used the dielectric response of ILs to more accurately and reliably obtain information about polarity [65]. The polarity of ILs can affect enzyme stability and selectivity [22,24,25,31]. In general, polar solvents increase the solubility of polar substrates and lead to faster and more selective reactions [27,31]. However, this trend is not true for all cases because some reports showed no relationship between

reaction rates and ILs polarity [22,25]. This phenomenon can be explained in terms of viscosity. In fact, the change of the polarity and viscosity of ILs are correlated although the changing rates are different. For example, an IL with shorter alkyl chains on the cation has a lower viscosity and also a higher polarity. The increase of alkyl chain length produces a slight reduction in the polarity of ILs, but a huge increase in their viscosity [66,67]. The reaction rates may be affected by the significant viscosity change. One of the notable properties of ILs is that they are capable of a wider range of intermolecular interactions [5,68], such as dipolar, hydrogen bonding, dispersive and ionic. Hence, many compounds are significantly soluble in ILs. Interestingly, ILs having coordinating anions (e.g., Cl− , NO3 − , CH3 COO− and (MeO)2 PO2 − ) which are strong hydrogen bond acceptors can dissolve many compounds which are insoluble or sparingly soluble in water and most organic solvents. Examples include cellulose [69,70] and some compounds having pharmacological activity [71,72]. The ability of ILs to dissolve such compounds generally depends on the hydrogen bonding ability of anions [5,70]. Based on the solubility of ILs in water, ionic liquids can be divided into two categories: hydrophobic (water immiscible) and hydrophilic (water miscible). This water miscibility generally depends on the anions of ILs. Indeed, water interacts with the anion through the formation of hydrogen bonds [73,74]. However, the miscibility of ionic liquids with water is not well generalized. For example, ILs [bmim][BF4 ], [bmim][PF6 ] and [bmim][Tf2 N] have almost the same polarity [57] and the coordination strength is also comparable [60], but first one is water miscible whereas latter two are not. A recent measurement of the H-bond accepting properties of such ILs revealed that [BF4 ] was better H-bond acceptors (ˇ = 0.61) than [PF6 ] (ˇ = 0.50), which can be considered as a reasonable explanation regarding the difference in water miscibility [75]. Ionic liquids are generally immiscible with many organic solvents such as hexane and ether, whereas some are miscible with polar solvents like lower alcohols, ketones, dichloromethane and tetrahydrofuran [15,53]. The immiscibility of ILs with either water or organic solvents has made them feasible to be used to form a two-phase system. Generally, ionic liquids are not miscible with supercritical carbon dioxide (scCO2 ), but they can absorb a large amount of scCO2 [76]. Ionic liquids are well proved as highly stable solvents and are stable above 100 ◦ C. In particular, dicationic ILs show much higher

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thermal stabilities than monocationic ILs [77,78]. For example, ILs containing bis(2,3-dimethylimidazolium) cation have the excellent thermal stability with only 5% thermal degradation at 440 ◦ C [78]. Consequently, they are very effective as solvents for high temperature reaction systems. The thermal stability of ILs is calculated based on their decomposition temperatures where there is 10% mass loss using thermogravimetric analysis (TGA) [79,80]. The most thermally stable ILs reported were those having the [Tf2 N] anion with various types of cations, including alkylammonium and imidazolium [79]. In contrast, ILs containing a carboxylate anion, particularly formate, may have lower thermal stability due to undergoing a condensation reaction to form amides [81]. On the other hand, the instability of anions BF4 − and PF6 − are well documented [82]. In fact, the hydrolysis of such anions produces the toxic volatiles such as HF, POF3 , which can deactivate the enzymes. The viscosity of ILs is higher compared with that of molecular solvents. Like other solvents, the viscosity of ILs is dependent on the ion-ion interactions, such as van der Waals interactions and hydrogen bonding. Therefore, the value of the viscosity varies significantly with chemical structure, composition, temperature and the presence of solutes of impurities. It has been shown that ionic liquid’s viscosity generally increases with an increase in the alkyl chain length for a fixed anionic group due to the stronger van der Waals interactions [53,54]. Besides, delocalization of the charge on the anion, such as through fluorination, decreases the viscosity by weakening hydrogen bonding [83]. However, the viscosity of ILs was affected more by the change to the anion than to the cation [84]. In the presence of small amount of water or organic solvents or with the increase of temperature, the viscosity of ILs also decreases markedly [53,85]. The solvent viscosity could affect the biocatalytic reactions rate in terms of the mass transfer limitation when the reaction is rapid and the IL is relatively viscous. For example, a higher enzyme activity was observed in [emim][Tf2 N] than in [MTOA][Tf2 N] due to lower viscosity of first one compared to latter one [31]. However, this trend is not true for all biocatalytic reactions performed in ILs [104], particularly when reaction rates are measured in equilibrium instead of kinetics. 3. Enzymes in ionic liquids: a comparison study with organic solvents It is well documented from a large number of articles that an enzyme-catalyzed reaction in ILs provides superior results to those obtained in conventional organic solvents [22]. In addition to enhanced reaction rates and conversions, improved enantioselectivity [22,24] and regioselectivity [24] in ILs have been reported in many cases. Besides the physicochemical properties as well as green aspects of ILs, some important advantages of the RTILs as a medium for enzymes that make them interesting as potential solvents for biocatalysis are the following. • Since ILs are tailorable solvents, it can be designed for particular bioprocesses that are not possible using organic solvents. In fact, as mentioned previously, since ILs are capable of a wider range of intermolecular interactions, they can dissolve many organic and inorganic compounds that are insoluble in organic solvents [20,69,70,86,87]. For example, ILs can be used for carrying out biotransformations with polar or hydrophilic substrates such as amino acids [20,86] and carbohydrates [87], which are sparingly soluble in most organic solvents (e.g., isooctane and hexane). The ionic liquids [bmim][BF4 ] and [bmim][PF6 ] have successfully been applied to increase the solubility of sugars and to maintain the enzymatic activity in lipase-catalyzed synthesis of fatty acid sugar esters [28,88]. • A number of cases, enzymes show excellent stability (both operational and thermal) in ILs. The stability of a wide range of

enzymes/proteins including CALB [27,34,89–91], CRL [92], Cyt C [93], thermolysin [20], lysozyme [66] and ␣-chymotrypsin [31,94,95] was found to increase significantly compared to that in traditional organic solvents. For example, the operational halflife of CALB in a series of ILs [Cn Me3 N][Tf2 N] was up to 2000 times greater than that in hexane [91], and ␣-chymotrypsin at 50 ◦ C has half-lives of several hours in [bmim][BF4 ], [bmim][PF6 ], [emim] [Tf2 N] and [MTOA][Tf2 N] whereas in 1-propanol they deactivate within minutes at that temperature [31]. However, ILs bearing strongly coordinating anions, such as nitrate or acetate caused enzyme deactivation. This was attributed to the strong interactions between enzymes and such anions since this inactivation was not observed in hydrophobic ILs containing noncoordinating anions such as [PF6 ] and [Tf2 N] [26]. The conformational stability of enzymes in ILs was monitored by fluorescence and CD spectroscopy as well as DSC [89,94,95]. All these data indicate that enzymes are highly stable in anhydrous ILs owing to their conformational rigidity in the dehydrate state. Recently, Rantwijk and Sheldon argued that the change of enzymes conformation is slow in high viscous IL solvents, which is effective for maintaining activity and stability of enzymes for long period [19]. Generally, high viscosities of solvents can be translated into good stabilization for enzymes. The excellent stability of enzymes to elevated temperatures in high viscous ILs has opened up the possibilities of biotransformations with several different incubation protocols [19]. • Due to the high thermal stability of enzymes in ILs, bioprocesses can be conducted at high temperature if needed. • Owing to hydrophobic, ionic nature and hydrogen bonding ability, protic ILs (i.e., EAN, ethylammonium nitrate) have been used for protein renaturation and crystallization studies [26,96–99]. In conventional methods, proteins can easily form aggregates during renaturation processing. However, aprotic IL EAN was found to be very useful in preventing such aggregation through a weaker attraction to the hydrophobic part of the protein compared to conventional surfactants [96,97]. In addition, unlike conventional surfactants used to prevent aggregation, EAN does not need to be removed before the protein can refold and the IL can be easily removed at the end of the refolding [97]. Ionic liquid EAN was also used in the sucrose solution to prevent aggregation of lyophilized lysozyme protein [96]. • The most important advantage of ILs as reaction media for biotransformation is the biocatalyst recycling and product recovery schemes that are not feasible with traditional organic solvent systems. As previously mentioned, most of the ionic liquids do not mix organic solvents such as hexane and ether. This unconventional solvent property allowed to extract the product and unconverted reactant simply just washing with diethyl ether and hexane or even supercritical CO2 [99,100]. The biocatalyst remained in the IL phase and can be recycled for further use [23,101,102]. The resulting biocatalyst could be reused at least 4–5 times without loss of their activity. Furthermore, in some cases the products can be simply separated by evaporation due to the lack of vapor pressure of ILs [103].

4. Stabilizing enzymes in ionic liquids Although enzymes in ILs have presented enhanced activity, stability, and selectivity, the practical obstacle of using ILs is that many enzymes do not dissolve readily in most ILs, which has ruled out many potential biotechnological applications. In fact, enzymes that show catalytic activities in ILs normally do not dissolve in ILs. When enzymes become active in ILs, they remain suspended as a powder. Although some ILs can dissolve enzymes through the weak hydrogen bonding interactions, they often induce enzyme confor-

[118] rt, W0 = 18 Ethanol → acetaldehyde

4.1. Water-in-ionic liquid microemulsions The insolubility limitation could be overcome by the formation of nano/micrometer-sized water domains in an IL continuous phase (noted as w/IL microemulsions) stabilized by a suitable surfactant or mixture of surfactants. It is well recognized that enzymes can be solubilized in organic solvents by the use of surfactants (generally called w/o microemulsions) without the loss of their catalytic activity [105–107]. In this microheterogeneous medium, enzyme molecules are entrapped in tiny water domains and thus become protected against unfavorable contact with the surrounding organic solvent by a layer of water and surfactant molecules, thereby exhibiting good stability and activity. Indeed, w/o microemulsions are highly versatile reaction media, which are currently used for many applications. The insolubility of enzymes in compressed or supercritical carbon dioxide (scCO2 ) was also solved by the formation of water domains in CO2 (w/c microemulsions), which is now being used for various purposes including enzymatic reactions [108,109]. However, the formation of water domains in an ionic liquid continuous phase is generally hindered by the insolubility of most conventional surfactants, particularly anionic surfactants including AOT (sodium bis(2-ethyl-1-hexyl) sulfosuccinate) which has been used extensively to form microemulsions in organic solvents [105].

TX-100 ADH (yeast) 6

299

mational changes resulting in inactivation [26,34]. For example, EAN can dissolve CALB through the strongly coordinating nitrate anion resulting in denaturation [26]. To improve the enzyme solubility as well as activity in ILs various attempts have been made by modifying the form of enzymes in which it is used, including immobilized enzymes, the use of microemulsions, and the use of whole cells.

bmimPF6

51 ␮M L−1 min−1

a. 100% (conv.) b. 14.2% 30 ◦ C, 24 h, W0 = 5 Lipase (T. lanu-ginosa) 5

a. Tween-20 b. TX-100

bmimPF6

Lauric acid + 2-propanol → propyl laurate

[116] 33 ␮M L−1 min−1 30 ◦ C, W0 = 18, pH = 4.2 2,6-Dimethoxyphenol + H2 O2 → 2,3-di–amino phenazine Laccase (Trametes versicolor) 4

TX-100

bmimPF6

4.5 ␮M L−1 min−1 30 ◦ C, W0 = 10, pH = 3.2 o-Phenylenediamine + H2 O2 → 2,3-di-amino phenazine Lip (P. chry-sosporium) 3

TX-100

kcat,app = 16.4 s−1 35 ◦ C, W0 = 5 Pyrogallol + H2 O2 → purpurogallin HRP 2

AOT

omimTf2 N + 1hexanol (10 vol.%) bmimPF6

[116]

[113]

First example of enzyme reaction in w/IL microemulsion Better activity and stability obtained compared to oil ME Obtained improved activity compared to pure/water saturated IL Apparent viscosity decreased than that in pure/water saturated IL Lipase recycled 10 times without loss of its activity No activity found in pure ILs Vmax = 5.3 ␮M min−1 Km,app = 7.5 mM 35 ◦ C, W0 = 4 p-Nitrophenyl butyrate + water → p-nitrophenol + butyric acid Lipase (P. cepacia) 1

AOT

omimTf2 N + 1hexanol (10 vol.%)

[115]

Ref. Remarks Yield (%) or initial rate Reaction conditions Substrates/products Solvent Surfactant Enzymes Entry

Table 2 Enzymatic reactions studied in water-in-ionic liquid (w/IL) microemulsions (MEs).

[117]

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

Fig. 3. Structures of the surfactants used to solubilize enzymes in ionic liquids.

300

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

Fig. 4. Fluorescent emission of lipase PS in various systems, reproduced from our previous article [113].

Presently, there are some reports in the literature dealing the development of water-in-ionic liquid microemulsions using nonionic and anionic surfactants (see Fig. 3) [110–112]. Although nonionic surfactants (e.g., Tween-20 and TX-100) are readily soluble in hydrophobic ionic liquids, anionic surfactant AOT is only dissolved in the presence of 1-hexanol as a cosurfactant or a cosolvent. Such w/IL MEs were successfully used for enzymatic reactions (see Table 2). The fluorescence study of lipase PS encapsulated in the AOT/water/1-hexanol/IL system showed that the lipase experiences an environment similar to that of bulk buffered water which is in contrast with the AOT reverse micelles formed in a nonpolar solvents (see Fig. 4) [113]. This notable result can be explained by the large size of water domains formed in the IL MEs [110] because Venables et al. [114] provided the evidence for bulk-like character for water in large microemulsions. We reported the first example of an enzymatic reaction carried out in w/IL MEs comprised AOT surfactant [113]. In this study, we described the successful lipase-catalyzed hydrolysis of p-nitrophenyl butyrate, and it was found that the catalytic activity of lipase PS dissolved in the water domains of ILs became much higher than that in microemulsions of AOT in isooctane. Later, we tested the activity of HRP in the same system using pyrogallol as a substrate [115]. It was shown that the HRP intrinsic activity in the new ionic liquid microemulsions was 5 times higher than those in microemulsions of AOT in isooctane. This enhanced catalytic activity of enzymes in w/IL microemulsions is due to the following: firstly, the partition of the substrate, products or other molecules involved in the reaction between the aqueous pseudophase and the IL continuous phase; secondly, a change in the enzyme microenvironment, and thirdly, the presence of 1-hexanol as a cosurfactant affected the reaction rate. For example, solubility studies showed

that both the substrate (pyrogallol) and the product (purpurogallin) in the HRP-catalyzed reaction are significantly soluble in IL [omim][Tf2 N] whereas they are poorly soluble in isooctane [115]. Therefore, the product concentration in the aqueous pseudophase was reduced significantly due to the favorable partitioning to the ionic liquid continuous phase. Consequently, the product inhibitory effect may be less effective in the IL systems than that in the organic solvents systems. The stability study showed that HRP was found to retain almost 70% of its initial activity after 30 h in the w/IL system, whereas the half-life of HRP in AOT/water/isooctane microemulsions was 33 h [115]. We hope that this ME may be suitable for bioprocesses to be deigned with multi-components (e.g., more than one enzymes and substrates) due to the large size of water domain. Zhou et al. investigated the activity of lignin peroxidase and laccase in w/IL MEs comprised Triton X-100 as the surfactant in hydrophobic ionic liquid [bmim][PF6 ] [116]. The authors reported that the catalytic activity of such enzymes is greatly enhanced in [bmim][PF6 ] based MEs compared to that in pure or water saturated IL[bmim][PF6 ]. The apparent viscosity of the medium was also decreased by forming the microemulsion systems. The enzyme ADH from yeast also found to be active in the above mentioned w/IL ME, whereas no activity was observed in a pure IL [118]. More recently, w/IL MEs formulated with nonionic surfactants Tween-20 and TX-100 in [bmim][PF6 ] were successfully used as media for lipase-catalyzed esterification reactions using lipases from Candida rugosa, Chromobacterium viscosum and Thermomyces lanuginosa as biocatalysts [117]. The lipases exhibited higher catalytic performance and operational stability, particularly at higher incubation temperature (50 ◦ C) (see Table 3) as compared to that found in nonpolar solvents based MEs. Fourier transform-infrared (FTIR) and circular dichroism (CD) studies suggested that lipases entrapped in IL-based microemulsions retain their native structure or adapt more rigid structure compared to other MEs, which is accounted as the main reason for excellent stability in the MEs. Lipase from Thermomyces lanuginose retained 90% of its original activity after 10 reaction recycle in w/IL MEs formed with Tween-20 at 30 ◦ C. In conclusion, it is to be expected to design more suitable ILphilic surfactants for the interface between water and ionic liquids that will offer new opportunities in biotransformation in “green” solvents ionic liquids1 . 4.2. Immobilization and modification of enzymes Due to the insolubility as well as the relatively low activity of crude preparations of enzymes in ILs, a wide range of enzymes immobilized on solid supports that are commercially available such as Novozyme 435 (CALB), Lipozyme (RML), Chirazymes [Candida antarctica lipase A (CALA), CALB, Psedumonas cepacia lipase (PCL),

Table 3 Half-life constants (t1/2 , h) for three lipases in various systemsa . Enzyme

T (◦ C)

Lipase (C. rugosa)

30 40 50

Lipase (C. viscosum)

Lipase (T. lanuginosa)

a b c d

w/IL MEb

w/IL MEc

w/o MEd

2.5 1.3 0.7

23.5 8.3 6.3

30.1 13.3 9.9

0.2 0.1 <0.1

30 40 50

6.5 2.2 0.9

>100 48.0 41.3

>100 22.7 4.3

>100 90 4.9

30 40 50

75.0 53.4 6.4

>100 47.7 31.2

>100 67.9 38.9

0.9 0.8 0.6

Aqueous

For further information and more data, see Ref. [117]. Microemulsion compositions % (w/w); IL/Tween-20/water = 60/35/5. Microemulsion compositions % (w/w); IL/Tween-20/water = 45/50/5. Microemulsion compositions: AOT = 100 mM, W0 = 6, hexane was used as oil phase.

Table 4 Lipase-catalyzed reactions involving fatty acids and esters in ionic liquids. Entry

Enzyme

Preparation

Solvent

Substrate + product

Reaction conditions

Yield (%) or initial rate

Remarks

Ref.

1

CALB

NZ435

bmim TfO

Glucose + vinyl laurate → 6-Olauroyl-d-glucose

40 ◦ C, 12 h

>90% (conv.)

[35]

2

CALB

NZ435

a. bmimTfO b. bmimTf2 N c. bmimTfO + bmimTf2 N

Glucose + vinyl laurate → 6-Olauroyl-d-glucose

40 ◦ C

a. 4.1 b. 3.75 c. 0.77 ((M min−1 g−1 )

3

CALB

NZ435

a. PCL b. CALB

a. 40% b. 26% (conv.) a. 30% b. 61%

5

CALB

a. Immobilized on ceramic particle b. NZ435 NZ435

6

PSL

Free

7

CALB

NZ435

Esculin + plamitic acid → esculin ester 1,6-anhydroglucopyranose + VA → 4-O-acetyl -1-6-anhydroglucopyranose Benzyl acetate + benzyl -amine → acetamide Ethyl 3-phenylpropanoate + butanol → 3-phenylpropanoic acid esters Acetic acid + geraniol → geranyl acetate

40 ◦ C, 96 h

4

a. MebPy.dca b. MebPyO.dca MOemim.dca

Activity improved using supersaturated glucose solution in IL Optimal activity and stability obtained in the mixture (1/1,v/v) of two ILs Hydrophobic IL gave better bioconversion Various ILs tested

8

CALB

Chirazyme® L-2

9

CALB

NZ435

10

CALB

NZ435

11

PCL

Free

CALB

NZ435

13

a. CALA b. CALB c. TLL d. RML a. CALA b. CALB c. TLL d. RML a. CALB b. RML c. CRL CALB

NZ868L NZ525L NZ871L NZ388L NZ868L NZ525L NZ871L NZ388L NZ435 Lipozyme IM Free NZ435

14

15

16

a. bmimBF4 b. bmimPF6 c. bmimTf2 N bmimPF6

60 ◦ C, 24 h

94%

50 ◦ C, 48 h

a. 19% b. 95% c. 96% 30–35%

30 ◦ C, 72 h

Oleic acid + ascorbic acid → ascorbyl oleate

60 ◦ C

Ascorbic acid + CLA → CLA ascorbyl ester

70 ◦ C, 24 h

CLA + l-carnitine → conjugated-linoleoylL-carnitine 2-Phenylethanol + VA → phenylethyl acetate Acetic acid + isoamyl alcohol → isoamyl acetate

60 ◦ C

CPMA.MS

Vinyl propionate + 1butanol → butyl propionate

40 ◦ C, 2% (v/v) Water

bmimPF6

Vinyl propionate + 1-butanol → butyl propionate

40 ◦ C

emimPF6

Benzyl alcohol + VA → benzyl acetate

bmimPF6

Vinyl butyrate + 1-butanol → butyl butyrate

a. 40 ◦ C b. 40 ◦ C c. rt 30 ◦ C, 2h 0.4% (v/v) TEA

a. bmimBF4 b. emimTOS c. emimOcSO4 d. bmimMDEGSO4 e. Acetone a. t-OMA.Tf2 N b. bmim.CF3 SO3 c. t-butanol d. t-OMA.Tf2 N + t-butanol bmimPF6

a. bmimPF6 b. i-bmimPF6 bmimPF6

rt, 48 h ◦

50 C, 72 h

a. 0.18 b. 0.08 c. 0.12 d. 0.10 e. 0.02 (mM h−1 mg−1 ) a. 44.44% b. 31.32% c. 16.25% d. 71.01% (conv.) 98.27%

a. 65% b. 55% >95%

a. 15.1 b. 655.5 c. 12.1 d. 2.4 (U mg protein−1 ) a. 5.1 10 b. 24.1 c. 217.6 d. 50.9 a. 4.08 b. 0.13 c. 0.15 (mM min−1 g−1 ) 14.02 U mg−1

[147] [56]

Various esters and amines investigated Enzymes recycled 5 times without loss its activity Better conversion but slower reaction rate compared to org. solvents Continuous reaction

[148]

Higher yields obtained in IL/alcohol mix. than those in single solvent

[153]

2.13 times higher conv. obtained in IL than that of acetonitrile activity and stability improved using isomeric IL IL and enzyme reused together in biphasic system A wide range of ILs used as solvents

[154]

[149]

[36]

[150]

[156] [157]

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

12

bmimPF6

50 ◦ C

[159]

[37]

Increased temperature facilitate the ester synthesis

[37]

Chloride impurity influenced lipase activity IL pretreatment with aqu. Na2 CO3 enhanced the enzyme activity

[158]

[152]

301

[155]

[113]

a. 24.3 b. 134.1 c. 30.8 d. 33.5 (U mg protein−1 ) 30 ◦ C, 2 h 2% (v/v) water CALB 19

Free

omimPF6

Vinyl butanoate+ a. Methanol b. 1-Butanol c.1-Hexanol d.1-Octanol → alkyl butanoate

Better results found in w/IL MEs than that of oil ME; first example of biocatalysis in w/IL ME Activity and selectivity improved in ILs compared to hexane Vmax = 5.3 ␮M min−1 35 ◦ C p-nitrophenyl butyrate + water → p-nitrophenol + butyric acid PCL 18

w/IL ME

omimTf2 N + 1-hexanol

Higher yields of mono-ester obtained in all ILs than those found in acetone a. 86.8% b. 98.1% c. 68.3% d. 23.6% 60 ◦ C, 72 h Vinyl butyrate+ a. Naringin b. Esculin c. Helicon d. Salicin → mono-ester CALB 17

NZ435

bmimBF4

Remarks Yield (%) or initial rate Reaction conditions Substrate + product Solvent Preparation Enzyme Entry

Table 4(Continued)

[151]

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

Ref.

302

etc.], Lipolases [Aspergillus oryzae lipase (AOL), and Humicola lanuginose lipase (HLL)]. All these show varying degrees of biocatalytic activity in ILs, just like biocatalysis in organic solvents [119]. In particular, CALB can catalyze a wide range of biotransformations in ILs, such as transesterification, ammonolysis and polymerization [21,23–25,34,43,120–123]. In addition to this, many research groups have modified the enzymes through the immobilization on celite [86,124], carbon nanotubes [125,126], PEG-modification [32,42,127–131], and immobilization through multipoint attachment in polyurethane foam, have been investigated to improve the enzyme solubility as well as activity in ILs. All these strategies provide more robust, more efficient, and more enantioselective biocatalyst suitable for biotransformation with ILs. Since the phenomenon of using immobilized enzymes in ILs is well established, it is not focused again in this review.

4.3. Reactions using whole cells The whole cells biotransformation is of great interest in the chemical industry to produce fine chemicals, since it is found to be very potential from the economical point of view with established chemical processes [132–135]. To overcome the poor solubility of substrates and products in aqueous media as well as inhibitory effects of the reactants on the biocatalyst, biphasic biocatalytic processes with water immiscible organic solvents are mainly used to enhance the process efficiency. However, this approach has some problems because organic solvents are found to damage bacterial cell membranes and promote enzyme denaturation [135]. Considering these points, RTILs could be a good candidate for the replacement of such toxic organic liquids. It was therefore interesting to see that, following from pioneering work of Cull et al. on the use of ILs for whole cell biocatalysis [13], a number of groups have successfully used water immiscible ILs in a range of whole cell bioconversions [103,136–143,171,177]. In this process, RTILs are employed as a substrate reservoir and in situ extracting agent for whole cell biocatalytic reactions. In most examples studied to date, reaction rates and yields are comparable to, or greater than, those obtained in organic systems. Significant enhancements in biocatalyst stability have also been observed. Although various ILs have been used, hydrophobic ILs particularly bearing PF6− and Tf2N− anions are reported to be less toxic to the cell membranes than the organic solvents [136]. Examples of cells used for whole cell biocatalysis include Rhodococcus R312 [13,177], baker’s yeast [144], Escherichia coli [142], Lactobacillus kefir [137], Saccharomyces cerevisiae [142], Pichia pastoris, Bacillus cereus and Geotrchum candidum [142]. Very recently, Wang et al. [171] reported the biocatalytic asymmetric reduction of 4 -methoxyacetophenone (MOAP) to enantiopure (S)1-(4-methoxy-phenyl) ethanol ((S)-MOPE) using Rhodotorula sp. AS22241 cells (Fig. 5). With the faster reaction rates, the operational stability of cells was improved markedly; the cells remained above 90% of their original activity in the [bmim][PF6]-based biphasic system, which was much higher than that in the monophasic buffer system (about 25% of their original activity), after being repeatedly used for 8 batches (50 h per batch). It was initially supposed that toxicity of ILs (still now debatable) causes cell membranes damage and results in cell death in a similar manner of organic solvents. This idea was, however, proved to be wrong by experimental evidences. In addition, ILs can be designed to be nontoxic using nontoxic, biodegradable, and pharmaceutical tolerable organic cations and inorganic anions. Some of ILs have already been synthesized with such organic and inorganic compounds [145,146]. This finding may further accelerate the use of ILs in whole cells biocatalysis for practical applications.

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

303

Fig. 5. Whole cell reaction in ionic liquid: asymmetric reduction of 4 -methoxyacetophenone [171].

5. Recently conducted enzyme-catalyzed reactions in ionic liquids It is well documented that lipases are the most choice for enzymatic reactions conducted in ILs, which is analogue to organic solvents. To date, a wide range of reactions catalyzed by lipases have been carried out extensively in ILs. Examples include esterification, transesterification, alcoholysis, aminolysis, hydrolysis and polymerizations. Therefore, firstly, we will summarize the recently studied lipase-catalyzed reactions in ILs. Table 4 is focused on reactions involved acids and esters. All examples included here indicate that reaction rates and yields are comparable to, or greater than, those obtained in organic systems. Table 5 summarizes some important reactions related to the oils and fats, whereas stereospecific reactions catalyzed by lipases are listed in Table 6. Lipase-catalyzed polymerization is listed in Table 9, in which polymerization in ILs conducted by other enzymes is also included.Compared to lipases, the use of other enzymes in ILs is limited. Tables 7 and 8 summarize some important biocatalytic reactions in ionic liquids catalyzed by other enzymes such as HRP, ADH, SC and cellulase. 6. Application to bioprocesses In this section, we focus on the use of RTILs in place of organic solvents for some important multiphase bioprocessing operations. 6.1. Enzymatic polymerization in ionic liquids Enzymatic polymerization has been receiving great interest as one of the new methodologies that provide biodegradable polymer synthesis without the use of toxic catalysts [183,184]. For this polymerization, a significant amount of volatile organic solvent has to be used to overcome the viscosity problem happened during the bulk polymerization [183]. However, organic solvents suffer from several drawbacks. For example, many polymers are poorly soluble in water and organic solvents, which causes premature precipitation of the polymer leading to low molecular weights. Therefore, in comparison with the metal-catalyzed polymerization, it was much more difficult to obtain high molecular weight polymers by the enzymatic polymerization [185]. Although highly polar aprotic solvent such as DMF and DMSO are able to solubilize many higher molecular weight fractions of polymers [186,187], enzyme activ-

ity in such polar solvents is often low [187,188]. To solve such problems, ILs have been used as an efficient reaction medium for enzyme-catalyzed polymerization (see Table 9). Due to the unconventional properties of ILs, they offer the possibility of enzymatic polymerization and the size of composition of the polymers generated. To date, a wide range of biocatalytic polymers were synthesized using ILs as a reaction medium [122,185,189–192]. The first experiment that showed the enzymatic synthesis of polyesters in ILs was conducted by Uyama and co-workers [122]. In this study, a ring-opening polymerization (ROP) of ␧-caprolactone was performed using lipase from C. antarctica as a catalyst in two ILs, [bmim][BF4 ] and [bmim][PF6 ] at 60 ◦ C. Polyesters were obtained within a week. It was demonstrated that polymerization of lactone in ILs was suitable for the production of higher molecular weight polymer. Later, Gorke et al. [189] reported synthesis of poly(hydroxyalkanotes) using the same lipase as the catalyst in water miscible and water immiscible ILs. Reactions in the water immiscible ILs like [bmim][PF6 ] or [bmim][Tf2 N] generally gave higher conversions than those in water miscible ILs like [bmim][dca]. This is due to the deactivation of lipase by highly polar ILs [33], which is consistent with other biocatalytic reactions in such highly polar ILs [24]. Fujita et al. reported the lipase (CALB)-catalyzed ROP of l-lactide in ILs at room temperature [185]. They found that higher molecular weight polymer could be produced in ILs compared to conventional bulk and toluene methods. However, the conversion was lower in ILs. The authors suggested that the high solubility of generated polymers in ILs may be the possible reason for such a lower polymer yields. Very recently, Dordick and co-workers [191] have shown that enzyme Soybean peroxidase can be used effectively to catalyze phenolic polymerization in ILs/aqueous solutions at 60 ◦ C. Here, they employed two water miscible ILs, [bmim][BF4 ] and [bmpy][BF4 ]. Gel permeation chromatography (GPC) and MALDI-TOF analysis indicated that the polymer size could be controlled by varying the IL concentrations, and polymer with higher molecular weight could be synthesized in the presence of higher ILs concentrations. Thermal analysis showed that phenolic polymers generated in this way were highly thermostable and functioned as thermosets. Rumbau et al. have shown that HRP immobilized in hydrophobic IL [bmim][PF6 ] acts as an efficient biocatalyst for biocatalytic synthesis of polyaniline [43]. The HRP/IL phase was added to the aqueous solution containing aniline, the template dodecylbenzenesulfonic acid and hydrogen peroxide. This method is found to be

304

Table 5 Lipase-catalyzed reactions involving oils in ionic liquids. Enzyme

Preparation

Solvent

Substrate + product

Reaction conditions

Yield (%) or initial rate

Remarks

Ref.

1

a. PCL b. PCL c. PCL d. PFL e. CRL f. CALB

a. PS-C Amano I b. PS-D Amano I c. PS-Amano d. crude e. crude f. NZ435

bmimTf2 N

Soybean oil + methanol → biodiesel

rt

a. 74.6 b. 48.8 c. 39.1 d. 27.0 e. 0 f. 38.6

[160]

2

CALB

NZ435

Sunflower oil + methanol → FAME

60 ◦ C 4h

3

CALB

NZ435

a. emimPF6 b. bmimPF6 c. bmimBF4 a. emimTfO b. emimMS

Soybean oil + methanol → FAME

50 ◦ C 12 h

a. 98% b. 97% c. 0% a. >80% (conv.) b. <20%

4

CALB

NZ435

a. TOMA.Tf2 N b. ammoeng 102 c. TOMA Tf2 N + ammoeng 102 (1:3, v/v)

Triolein + glycerol → diglycerides

60 ◦ C 24 h

a. 56.6% b. 37.9% c. 91.4%

5

CALB

NZ435

CPMA.MS

70 ◦ C 24 h

a. 89.09 b. 88.61 c. 90.7 d. 88.2

6

a. CALB b. RML

ammoeng 102

60 ◦ C 24 h

a. 90.31 b. 31.8

7

CALB

NZ435 Lipozyme RMIM NZ435

Glycerol+ a. Sunflower oil b. Rapeseed oil c. Palm stearin d. Fish oil → monoglyceride Triolein + glycerol → monoglycerides

Recovered IL/lipase catalytic system reused 4 times without loss its activity; biodisel separated by decatation Hydrophilic ILs are poor solvents for this methanolysis Yield 8 times higher than those of solvent free system One IL used for improved DG production whereas other one increased oil conversion Higher yield obtained; improved operational stability of enzyme

Triolein + glycerol → fatty acids + monoglycerides

60 ◦ C 12 h

a. 90.45% (MG) 2.86% (FA) b. 92.50% (MG) 2.42% (FA)

a. ammoeng 100 b. ammoeng 102

IL anions influenced the bioconversions Improved triolein solubility and reaction rate obtained for hydrophobic ILs

[161]

[162]

[163]

[164]

[165]

[166]

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

Entry

Table 6 Lipase-catalyzed stereospecific reactions in ionic liquids. Enzyme

Preparation

Solvent

Substrate + product

Reaction conditions

Yield (%) or initial rate

Stereoselectivity (eep unless otherwise stated)

Ref.

1

CALB

NZ435

bmimPF6

40 ◦ C, 3 h

50% (conv.)

>99%

[167]

2

PAL

Free

bmimPF6 + hexane (1/1, v/v)

30 ◦ C

48%

>99%

[168]

3

PPL

Free

bmimPF6

rt

49.9% (alcohol) 49.8% (acetate)

>98% >96%

[172]

4

CALB

NZ435

bmimPF6

rac-1-phenylethanol + VA → (R)-1-phenylethyl acetate rac-1-chloro-3 (3,4-difluoro phenoxy)-2-propanol + VB → (S) butyric acid cis-(±)-4-O-TBS-2-cyclo pentene-1-ol + VA → cis(−)-4-O-TBS-2-cyclopentene1-ol + cis-(+) 4-O-TBS-2cyclopentenyl acetate Ethan-1,2diol + ethyacetate → monoacetate + diacetate

5

a. CALA b. CALB c. PCL d. MML e. AL

Free NZ435 Free Free Free

bmimTf2 N

rac-1-phenylethanol + VA → (R)-1-phenyl ethyl acetate

a. 40 ◦ C b. 50 ◦ C c. 60 ◦ C 24 ◦ C, 3 d

a. 85.3% (mono acetate) b. 84.5% c. 85.3% a. 98% (conv.) b. 50% c. 47% d. 40% e. 89%

0% >98% >98% >98% 15%

[22]

6

CALB

NZ435

CALB

NZ435

a. 25 ◦ C, 3.5 h b. 25 ◦ C, 5 h 24 ◦ C aw = 0.2

a. 44% b. 45% a. 16.9% (conv.) b. 88.5%

a. >99% b. >99% a. >99% b. >99%

8

CALB

NZ435

bmimBF4 H2 O (20% of IL)

30 ◦ C

53.1%

ees 93% E = 34

[169]

9

CALB

NZ435

a. bmimPF6 b. bmimBF4 c. bmimTf2 N d. bmim.dca e. aliq.dca

rac-5-phenyl-1-penten-3-ol + VA → (S)-5-phenyl pentyl acetate 1-␤-d-Arabinofuranosyl cytosine +vinyl benzoate → 5 -O-benzoyl 1-␤-darabinofuranosylcytosine d,l-Phenylglycine-methyl ester + H2 O → d–phenylglycine l-phenylglycine (±)-cis-benzyl N(1-hydro-xyindan-2-yl) carbamate + VA → corresponding acetate

[23]

7

a. bmimBF4 b. bmimPF6 a. bmimPF6 b. bmimBF4 + pyridine (20%)

35 ◦ C, aw = 0.2, 72 h

a. 19% b.12% c. 8% d. 19% e. 56%

a. 85% (eeester ) b. 99% c. 96% d. 3% e. 69%

[210]

[173]

[170]

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

Entry

305

306

Table 7 Esterification, glycosylations, hydrolysis reactions catalyzed by other (nonlipases) enzymes in ionic liquids. Enzyme

Preparation

Solvent

Substrate + product

Reaction conditions

Yield (%) or initial rate

Remarks

Ref.

1

SC (B. licheniformis)

bmimPF6

APEE + 1-propanol → APPE + ethanol

40 ◦ C

a. 41.6 b. 0.01 (mM h−1 mg−1 )

emimTfO

N-Ac-L-Phe-OH + ethanol → APEE

30 ◦ C, 0.2% H2 O

a. 9.2 b. 11.0 (␮M min−1 mg−1 )

3

a. SC (B. licheniformis) b. CMT (bovine pancreas) Tannase (A. niger)

SC treated with PEG, trehalose, TPP tested; EPRP of SC superior to others Activity varied with water content in IL

[178]

2

a. EPRP b. Crude (lyophilized from pH 7.8) Free

bmim.MEESO4

3.5% (-EC conv.)

Various ILs tested

[175]

FaeA (A. niger)

Gallic acid + (−)epicatechin → epicatechin gallate Glycerol + sinapic → glycerol cinapate

40 ◦ C, 24 h, 20% H2 O

4

Immobilized on Eupergit C Immobilized (CLEAs)

40 ◦ C, 24 h, 15% H2 O

8%

[176]

5

SC (B. licheniformis)

Modified with PEG

emimTf2 N

ZGGLpNA + ethanol → p-nitroaniline

40 ◦ C, 1 M EtOH

kcat = 7.3 (min−1 )

6

Cellulase

emimDEP pretreatment

Buffer

50 ◦ C

>50%

7

Cellulase (Trichoderma reesei)

Free

emim(Et)2 PO4 / buffer (1/4,v/v)

Wheat straw + water → glucose + cellbiose Cellulose + water → glucose + cellbiose

The first example of esterification of glycerol with SA by AnFaeA in ILs Various 1-alcohol tested; benzyl alcohol unsuitable IL recycled for further use

50 ◦ C, pH 5.0, 24 h

70% (conv.)

8

CMT (bovine pancreas)

Free

a. emimFSI b. emimBF4 c. emimTf

N-AC-Trp-OEt + Gly-Gly -NH2 → N-Ac-Trp-Gly -Gly-NH2 + N-Ac-Trp-OH

25 ◦ C, 5% H2 O

a. 280 (peptide) b. 0.031 c. 0 (␮M min−1 mg−1 )

EOHmimPF6

Batch; first example on cellulose saccharification in water/IL mixture Superior peptide synthesis in emimFSI compared to organic solvents

[174]

[130]

[181]

[182]

[243]

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

Entry

Table 8 Oxidations and carboxylation reactions catalyzed by other (nonlipases) enzymes in ionic liquids. Preparation

Solvent

Substrate + product

Reaction conditions

Yield (%) or initial rate

Remarks

Ref.

1

HLDH

Free

a. bmimCl b. bmimTf2 N

Ethanol → acetaldehyde

30 ◦ C, [IL] 50 mg ml−1

Activation energy reduced in the presence of IL

[180]

2

ADH (Rhodococcus Erythropolis)

Isolated

bmpyTf2 N + water

30 ◦ C, IL 10% (v/v)

ADH (yeast)

w/IL ME

bmimPF6

rt; W0 = 18

51 ␮M L−1 min−1

4

HRP

w/IL ME

omimTf2 N + 1-hexanol

Pyrogallol + H2 O2 → purpurogallin

35 ◦ C, W0 = 5

kcat,app = 16.4 s−1

5

a. Lip (P. chrysosporium) b. Laccase (T. versicolor)

w/IL ME

bmimPF6

o-Phenylenediamine + H2 O2 → 2,3-di-amino phenazine

30 ◦ C, W0 =a. 8 b. 20

a. 18 b. 4.5 (␮M L−1 min−1 )

6

HRP

Lyophilization with PBS

T2HEA.TfO + H2 O

Guaiacol + H2 O2 → tetraguaiacol

rt, 5% H2 O

200–250 (␮M min−1 )

7

HRP

Free

bmimBF4

4-Phenylphenol + H2 O2 2,2 -bi-4-phenylphenol

rt

–

8

Rhodotorula sp.

AS2.2241 cells

a. emimTf2 N + buffer b. bmimPF6 + buffer

MOAP + glucose → (S)-MOPE

rt

a. 1.04 ␮M h−1 eep : >99% b. 1.19 ␮M h−1 eep : >99%

9

ADH (G. candidum)

Immobilized cells

a. bmimPF6 b. bmimBF4 c. bmimTf2 N d. bmimTfO

o-Fluoroacetophenone + 2-propanol → (S)-1-(ofluorophenyl)-ethanol + propanone

rt, 16 h

a. 92%, eep >99% b. 82%, eep >99% c. 49%, eep >99% d. 1%

Cofactor NADH generated by GDH103 mediated oxidation of glucose Surfactant TX-100; no activity observed in pure IL Surfactant AOT; better activity and stability obtained in IL MEs than that in oil MEs Surfactant TX-100; both enzyme showed increased activity compared to pure or water saturated IL Activity increased with increase the water in IL/water mixture Activity decreased with the increased IL content; pH higher than 9 suitable for this reaction Faster reaction rate, enhanced stability in IL/water biphasic system than monophasic aqueous system No activity observed in hydrophilic ILs; substrate specificity investigated

[179]

3

4 -Br-2,2,2-trifluoro acetophenone → (R)-4 -Br-2,2,2trifluoro acetophenyl alcohol Ethanol → acetaldehyde

a. 2.16 b. 6.14 Vmax /Km (h−1 ) 100% (conv.)

[118] [115]

[116]

[45]

[46]

[171]

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

Entry Enzyme

[142]

307

308

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Table 9 Enzymes-catalyzed polymerization and poly condensation in ionic liquids. Entry

Enzyme

Preparation

Solvent

Substrate/products

Reaction temperature

Yield%/Mw

Ref.

1

CALB

NZ435

l-Lactide → poly-l-lactide

120 ◦ C

CALA

Chirazyme L-5

90 ◦ C

3

CALB

NZ435

bmimTf2 N

4

CALB

NZ435

5

CALB

NZ435

a. bmimBF4 b. bmimPF6 bmimBF4

6

CALB

NZ435

bmimTf2 N

7

CALB

NZ435

8

HRP

Immobilized in bmimTf2 N

a. bmimBF4 b. bmimTf2 N c. bmimPF6 IL + water

␧-caprolactone → poly (6-hydroxyalkanoates) a. ␤-propiolactone → poly (3-hydroxypropionate) b. ␧-caprolactone → poly (6-hydroxyalkanoates) c. ␦-valerolactone → poly (5-hydroxyvalerate) ␧-caprolactone → polyester Diethyl adipate + 1,4butanediol → polyester ␧-caprolactone → polyester Dimethyl adipate + 1,4butanediol → polyester

a. 35.2%/50300 b. 10.5%/50100 80–90%/8100

[185]

2

a. bmimBF4 b. bmimTf2 N bmimTf2 N

9

SBP

Free

10

SBP

Free

a. bmimBF4 + water b. bmpyBF4 + water bmpyBF4 (70%) + water (30%)

Aniline + dodecylbenzene sulfonic acid + H2 O2 → polyaniline p-Phenylphenol + H2 O2 → poly(p-phenylphenol) a. Phenol+H2 O2 → poly (phenol) b. 1-naphthol+H2 O2 → poly (1-naphthol) c. 2-naphthol+H2 O2 → poly (2-naphthol)

faster and easier than the classical immobilization of HRP into solid supports. In conclusion, although enzyme-catalyzed polymerization in ILs of various monomers has demonstrated the production of higher molecular weight polymers, the monomer conversion was low compared to the bulk and solvent polymerizations. Therefore, further technical improvements are needed to achieve high polymer yields. In this approach, the optimization of IL structures and extraction method may be one of the ways for better yields. 6.2. Proteins and enzymes extraction for biocatalysis In recent years, the extraction of biomolecules such as proteins, enzymes and amino acids using ILs as a medium is gaining increasing attention due to their unconventional solvent properties [193–197]. In 2006, our group have reported that the Lys-rich protein Cyt-c could be extracted to the hydroxyl group containing hydrophobic ILs (e.g., [C2 OHmim][Tf2 N]) with crown ethers via supramolecular complexation [193,194]. Crown ether dicyclohexano-18-crown-6 (DCH18C6) was used as the extractant. The interactions between DCH18C6 and the amino groups (NH3 + ) in the lysine residues on the protein surface were accounted as the main driving force for the Cyt-c extraction into ILs. In fact, the cavity of DCH18C6 has a strong affinity for the NH3 + group in the lysine residues on the protein surface owing to the tripodal hydrogen bonding interaction, the ion-dipole interaction and the inclusion effect. Dicyclohexano-18-crown-6 recognizes the NH3 + group in the lysine residues on the protein surface, which promotes the partitioning of Lys-rich proteins into IL. The Cyt-c-DCH18C6 complex in IL provides remarkably high peroxidase activity compared with native Cyt-c, because of enhancement of the affinity for H2 O2 [194]. Later, the functionalized IL having crown ether moiety on the imidazolium cation [DCH18C6mim][PF6 ] was used as the extractant in the place of ordinary DCH18C6 [198]. Although

[189]

60 ◦ C

a. 11900 b. 9700 c. 2200

[189]

60 ◦ C

[122]

60 ◦ C

a. 4200 b. 540 1500

60 ◦ C

44%/10500

[190]

70 ◦ C

a. 35%/5900 b. 21%/5100 c. 24%/4900

[190]

20 ◦ C

60 ◦ C 60 ◦ C

[43]

a. 96.2%/1820 b. 95%/1860 a. 50%/1100 b. 69%/2010 c. 75%/170

[191] [191]

the extraction ability was found to be lower than that of DCH18C6, the functionalized IL system gave a better stripping performance compared to the ordinary crown ether system. Functionalized IL [bmim][CB] synthesized from Cibacron Blue 3GA (CB) dye was also effectively used as an extractant for the extraction of lysozyme from aqueous phase to IL phase [202]. An extraction higher than 90% was observed at pH 4, and the extraction efficiency of the IL phase remained essentially the same after eight cycles of extraction. Besides, IL-based ATPS (ionic liquid-based aqueous two-phase systems) were found to be very effective for the extraction of proteins and enzymes [195–197]. The first work regarding the use of ILs to produce ATPS ([bmim][Cl]/K3 PO4 ) was reported by Gutowski et al. in 2003 [199], in which above critical concentrations of such two compounds in aqueous solution, phase separation takes place resulting in the formation of an IL-enriched upper and a salt-enriched lower phase. Traditional liquid extraction procedures with organic solvents cause contamination of the obtained proteins, which might also pose critical problems for subsequent biological investigations, as such organic solvents might be toxic to bioprocesses [200]. In contrast, IL-based ATPS provides a biocompatible environment for the gentle extraction and purification of biological materials, including proteins, enzymes and nucleic acids. Ionic liquid-based ATPS was exploited for the first time to extract proteins from human body fluids by employing a [bmim][Cl]/K2 HPO4 system by Du et al. in 2007 [195]. In this approach, quantitative extraction of proteins into the [bmim][Cl] rich upper phase from human urine was facilitated in the presence of appropriate amounts of [bmim][Cl] and sufficient quantities of K2 HPO4 , while metal species and some other concomitants were separated, remaining in the K2 HPO4 -rich lower phase. The extraction of BSA, trypsin, Cyt-c and ␥-globulins by using aqueous two-phase systems of [bmim][Br]/K2 HPO4 , [hmim]Br/K2 HPO4 and [omim]Br/K2 HPO4 was also conducted [201].

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

Fig. 6. Structure of ionic liquid found to be suitable for the formation of ionic liquidbased aqueous two-phase systems with K2 HPO4 /KH2 PO4 [196,197].

In the similar approaches, Dreyer and Kragl reported the formation of IL-based ATPS using IL AmmoengTM 110 having cations with oligoethyleneglycol units (see Fig. 6) and K2 HPO4 /KH2 PO4 [196,197]. They studied the stabilization of two different alcohol dehydrogenases (from Lactobacillus brevis and thermophilic bacterium) and the performance of enzyme-catalyzed reactions in such ATPS. Improved conversion and yield were observed using both enzymes, especially when reactions are conducted using hydrophobic substrates [196]. The electrostatic interaction between the charged amino acid residues at a protein’s surface and the positively charged IL-cation mainly acts as the driving force of the extraction process [197]. 6.3. Biofuels production In the last few years, the application of RTILs as (co)solvents and/or reagents in the enzymatic catalysis for the production of biofuels is an emerging research area [160,161,182,205–207] (for example see Table 5). Recently, the production of biodiesel (also known as mono alcohol fatty acids esters) in ILs through lipasecatalyzed alcoholysis of vegetable oils was reported by several research groups [160–162]. The production yield was improved markedly using CALB as a biocatalyst compared to organic solvents or a solvent free system. Among the various types of ILs used, hydrophobic ILs especially having Tf2 N− anion were found to be the most effective in production of biodiesel [160]. This trend can be explained considering the terms of methanol-induced enzyme deactivation [203,204]. In fact, hydrophobic ILs are protecting lipase from such deactivation because lipase is entrapped in IL matrix, whereas hydrophilic ILs cannot [161,222]. More recently, Gamba et al. [160] used IL-supported PCL for the production of biodiesel from the soybean oil. This transesterification reaction can be performed at room temperature, in the presence of water and without the use of organic solvents. Interestingly, the use of water improved the oil hydrolysis rate yielding the fatty acid that is converted into respective ester faster than the transesterification pathway (Fig. 7). The most notable advantages of the use of ILs in this bioconversions is that the biodiesel can be separated by simple decantation and the recovered ionic liquid/enzyme catalytic system can be re-used several times without loss of catalytic activity and selectivity [160,162]. Furthermore, ILs provide the ideal medium for the removal of byproduct glycerol that is also accounted for increasing biodiesel yield. On the other hand, ILs have been used effectively as a cosolvent and/or pretreatment agent for the production of bioethanol from cellulosic biomass [182,205–207]. Cellulose, the most abundant renewable biomaterials in the world, has long been recognized as a potential sustainable source of mixed sugars for fermentation to liquid biofuels and other biomaterials [208,209,235]. Among the various technologies used to date, enzymatic hydrolysis has been found as an effective way to break down pure cellulose into fermentable reducing-sugars. However, this approach is challenged

309

Fig. 7. Hydrolysis, transesterification and esterification reaction catalyzed by lipase from PS in ionic liquids [160].

by the poor enzymatic hydrolysis of cellulose biomass, mainly due to its structural heterogeneity and complexity of cell-wall microfibrils. In fact, the highly ordered structured cellulose is not soluble in water and most of the organic solvents due to the extensive network of inter- and intramolecular hydrogen bonds and van der Waals interactions between cellulose fibrils, and such strong networks make crystalline cellulose resistant to enzymatic hydrolysis [209]. In this context, ILs possessing coordinating anions (e.g., Cl− , NO3 − , CH3 COO− , (MeO)2 PO2 − ) that are strong hydrogen bond acceptors have been found to be capable of dissolving cellulose in mild conditions by forming strong hydrogen-bonds with cellulose and other carbohydrates at elevated temperatures [70,234,236,237,238]. However, the activity of cellulase decreased significantly in the presence of cellulose dissolving ILs [239], which is consistent with what is found for other enzymatic reactions [25,34]. To overcome the negative effect of ILs during enzymatic hydrolysis, many research groups regenerated cellulose from ILs prior to enzymatic saccharification and observed faster hydrolysis of IL-regenerated cellulose compared to untreated cellulose [206,207,240]. Ionic liquid treated cellulose was found to be essentially amorphous and porous than native cellulose, which are effective for enhancing enzyme action. Although a considerable research is needed to further enhance the enzyme performance using this process, this generally appears easy to operate and more environmentally friendly than other pretreatment processes such as thermochemical and inorganic acid pretreatment. More recently, our group reported the enzymatic in situ saccharification of cellulose dissolved in IL [emim][(EtO)2 PO2 ], in which the recovery of regenerated cellulose was not required (see Fig. 8) [182]. However, cellulase activity was highly dependent on the IL content in IL-aqueous media, and it was found that 20% (v) IL in the media gave the best results. At this condition, glucose formation was found to be approximately 2-fold higher than that of the aqueous solution. In conclusion, it seems that although hydrophilic ILs are very effective in dissolution of cellulose, they are not suitable for enzymes. Certainly, future researches should focus on the development of ILs that would be compatible with cellulose solubility as well as cellulase activity. 7. Combination of ionic liquid and enzymes for biotransformation Besides the use of ILs as a reaction medium for enzymes, the application of this green solvent as cosolvents and/or regents for biotransformation is well recognized. Examples include ioniccoated enzymes, the use of ILs as a protective agent for enzymes in sol–gel methods, the use of ILs based liquid membrane for enzyme-facilitated selective separation of organic compounds and the combination of enzymes and RTILs with electrochemistry.

310

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Fig. 8. A new route for enzymatic in situ saccharification in water-ionic liquid mixture [182].

7.1. Ionic liquid membranes for biotransformation Supported liquid membranes (SLMs), a porous supports whose pores are filled with a liquid, have been used as a potential carrier for separation of optically active compounds [211–213]. Like other potential applications of ILs, during the recent years, the use of SLMs based on ILs was found to be very effective in selective transport of organic compounds such as alcohols, esters, acids, ketones and amines [214–220]. In ionic liquid supported membranes, mainly hydrophobic ILs are immobilized inside the porous structure of a polymeric or ceramic membrane and act as a separative phase of two additional phases, named feed phase and receiving phase. In many cases, ionic liquids are confined between two membranes. For example, lipase-facilitated transport of (S)-ibuprofen through an ionic liquid-based SLM is shown in Fig. 9 [216]. In this system, the feed phase contains (rac)-ibuprofen and lipase from C. rugosa and the enantioselective esterification takes places at the interface 1. Afterwards, esters selectively pass through the SLM to the receiving phase where they are further hydrolyzed by PPL. Very recently, the integrated reaction/separation process for the kinetic resolution of rac-2pentanol and rac-1-phenylethanol was carried out using ILs based SLM [219,220]. 7.2. Ionic liquid-coated enzymes for biocatalysis Organic solvents (e.g., hexane, acetone and toluene) are often used to pretreatment of enzymes to improve their activity [119,221]. This approach stimulated the use of RTILs as pretreatment agents to enhance the activity and stability of enzymes [30,222–225]. The treatment strategy depends on the nature of ILs. Lee and Kim have used hydrophobic IL [ppmim][PF6 ] (melts at 53 ◦ C) for coating a lipase PS. In this process, firstly, ILs are heated and melted. Then aggregated enzymes are dispersed carefully in the melted liquid [222]. After cooling the mixture, the samples cut into small species, which is termed as ionic liquidcoated enzymes. The coated enzyme can be reused with several times with excellent enantioselectivity in nonaqueous media [222]. Later, to the similar approach, Itoh et al. [223,224] used the lipases from two different sources with the coating agent [bdim][cetyl-PEG-10-sulphate]. For this case, a freeze drying was performed after mixing the ionic liquid with the enzyme solution. MALDI-TOF mass spectrometric analysis indicated that IL binds to the enzymes and provides a favorable environment for the reaction [224]. Besides, RTILs were used as agents for immobilization of enzymes, which can be used in nonaqueous [225] and aqueous

media [30]. In this strategy, enzymes simply suspended into the ILs, and then such a mixture could be used for biocatalytic reactions. It was shown that lipase from C. antarctica immobilized with IL was active at very high temperature (95 ◦ C) in hexane and solvent free conditions [225]. Recently, Koo and co-workers [30] investigated the activity and stability of Mucor javanicus lipase pretreated with hydrophobic and hydrophilic ILs in an aqueous solution. The activities of treated lipase were well maintained even after 7 days of incubation at 60 ◦ C, while untreated lipase in phosphate buffer was fully inactivated only after 12 h of incubation at the same temperature. This excellent stabilization of IL-supported immobilized ILs may lead to develop many practical applications. 7.3. Ionic liquids and enzymes in sol–gel methods Sol–gel derived silica glasses are effectively used for the immobilization of biomolecules due to their porosity, transparency, chemical stability and convenient preparation, and such immobilized enzymes usually exhibit better activity and stability than free enzymes [226,227]. However, one important limitation of this strategy is that the enzymes are deactivated by the shrinking of gel during condensation and drying processes as well as the released alcohols during the hydrolysis of silicon alkoxide [228]. Interestingly, such problems were overcome by employing ILs as a protective agent in sol–gel methods [226,229,230]. The first example on enzymes encapsulated into an IL-based silica gel matrix was reported by Liu et al. [226], where they used HRP as a model biomolecule and IL [bmim][BF4 ] as a template solvent. It was demonstrated that HRP immobilized in ionic liquid-based silica sol–gel matrix showed enhanced activity and excellent thermal stability as compared to immobilized HRP prepared by conventional sol–gel methods (without ILs). Later, Koo and coworkers [230] used various ILs as additives in the sol–gel immobilization of lipase and found the improved activity and stability. The reason considered here for such dramatically enhanced activity and stability of immobilized enzymes is that ILs in the sol–gel process can act as a template during gelation and behave as a stabilizer to protect the enzyme from the inactivation by released alcohol or heat. This strategy for enzyme coimmobilized with ILs is very useful for the reaction in organic solvents, and ILs can be tuned for this strategy to lead many practical applications. Another useful approach is the combination of enzymes and ionic liquids with electrochemistry as ILs show a relative wide potential window of electrochemical stability and conductivity [229,231–233]. For example, the ionic liquid sol–gel enzyme electrodes retained the high activity of HRP and provided long-term stability of HRP in storage [229].

M. Moniruzzaman et al. / Biochemical Engineering Journal 48 (2010) 295–314

311

Fig. 9. Schematic diagram of lipase-facilitated membrane support [216].

8. Conclusions and remarks

Abbreviations

In conclusion, RTILs are emerged as promising solvents in the field of biocatalysis under “green” conditions that have been well proved over the past 9–10 years. In fact, ILs provide a new and powerful platform for enzymes, in which they can catalyze many important reactions impossible in commonly used organic solvents. The activities of biocatalyst are generally comparable with or higher than those observed in molecular organic solvents. Of particular importance has been found that enzymatic selectivity including substrate, regio- and enantioselectivity. The enzymes also show the outstanding thermal and operational stability in ILs. On the whole, there are an incredible number of opportunities to improve further enzymatic biotransformation in ILs. However, to take the full advantage of the opportunities afforded by ILs in biotransformation, there are still a number of challenges ahead on their potential industrial applications. Before the large-scale utilization of RTILs in biocatalysis, several urgent issues need to be addressed. A much more emphasis should be put on the exploitation of new and effective routes to synthesis of RTILs, especially new “clean” synthesis methods. Furthermore, it is expected to develop techniques together with corresponding facilities for large-scale production of ILs because it is clear that the number of ILs presently on the market is still limited. Hopefully, researches into these issues are going on and therefore new generations of ILs are being produced. From economical view point, it is needed to find more innovative methodologies in which the reuse and recycle of ILs as well as biocatalysts can be conducted in effective way. In this context, the combinations of ILs and scCO2 [100,241,242] as well as the unconventional ILs properties could be used to discover a suitable way. The research in this direction is just starting. Although lipases have been used extensively for biocatalysis in ILs, the applications of other enzymes or complex enzymes are very limited. The use of complex enzymes in ILs, particularly which requires a cofactor may be used for industrial biotransformation due to the tailoable properties of ILs. The last issue is perhaps the most important on the significant uncertainty regarding the toxicity and potential impact of RTILs on the environment, in which researches are going on. Fortunately, some reports show that nontoxic ILs can be produced by selecting biocompatible cations and anions [145,146]. We believe that green and biocompatible ILs will be available in near future, which stimulate the use of ILs in industrial biotransformation.

ADH alcohol dehydrogenase AL Alcaligenes AOT sodium bis(2-ethyl-1-hexyl) sulfosuccinate APEE N-acetyl-l-phenylalanine ethyl ester APPE N-acetyl-l-phenylalanine propyl ester ATPS aqueous two-phase system water activity aw BSA bovine serum albumin CALA Candida antarctica lipase A CALB Candida antarctica lipase B CLA conjugated linoleic acid CMT ␣-chymotrypsin CRL Candida rugosa lipase Cyt-c cytochrome C DCH18C6 dicyclohexano-18-crown-6 DMF dimethyl formamide DMSO dimethyl sulfoxide E enantiomeric ratio ee enantiomeric excess EPRP enzyme precipitated and rinsed with propanol FA fatty acid FAME fatty acid methyl ester FaeA feruloyl esterase GDH 103 glucose dehydrogenase 103 Gly-Gly-NH2 glycyl glycinamide HLADH horse liver alcohol dehydrogenase HRP horseradish peroxidase ILs ionic liquids kcat intrinsic activity apparent Michaelis constant Km,app Lip lignin peroxidase ME microemulsion MML mucor miehei lipase MOAP 4 -methoxyacetophenone MOPE 1-(4-methoxyphenyl) ethanol Mw molecular weight N-Ac-Trp-OEt N-acetyl-l-tryptophan ethyl ester N-Ac-L-Phe-OH N-acetyl-l-phenylalanine N-Ac-Trp-OH N-acetyl-l-tryptophan N-Ac-Trp-Gly-Gly-NH2 N-acetyl-l-tryptophan glycyl glycinamide NAD nicotinamide adenine dinucleotide NL lanuginosa lipase NZ 435 Novozyme 435

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PAL Pseudomonas aeruginosa lipase PCL Pseudomonas cepacia lipase PEG poly(ethylene glycol) PFL Pseudomonas fluorescens lipase PPL porcine pancreas lipase Lipase PS Pseudomonas sp. lipase RML Rhizomucor miehei lipase ROP ring-opening polymerization RTILs room temperature ionic liquids SC subtilisin Carlsberg sc supercritical SLM supported liquid membrane TPP three phase partitioning TLL Thermomyces lanuginosus lipase TX-100 Triton X-100 U unit enzyme activity VA vinyl acetate Vmax maximum velocity v/v volume per volume w/c water-in-CO2 w/IL water-in-ionic liquid w/o water-in-oil w/w weight per weight ZGGLpNA benzyloxycarbonyl-glycyl-glycyl-l-leucine p-nitroanilide W0 water contents expressed as molar ratio of water to surfactant molecules Ionic liquids Ammoeng 100 ethyloctadecanoyl oligoethyleneglycol ammonium methylsulfate Ammoeng 102 ethyloctadecanoyl oligoethyleneglycol ammonium ethylsulfate bdim 1-butyl-2,3-dimethylimidazolium bmim 1-butyl-3-methylimidazolium CPMA.MS cocosalkyl pentaethoxi methyl ammonium methosulfate EAN ethylammonium nitrate emim 1-ethyl-3-methylimidazolium hmim 1-hexyl-3-methylimidazolium MTOA methyl trioctylammonium MebPy 1-butyl-3-methylpyridinium MEESO4 2-(2-methoxyethoxy)-ethylsulfate MDEGSO4 diethyleneglycol monomethylethersulfate OcSO4 n-octylsulfate omim 1-octyl-3-methylimidazolium ppmim 1-(3 -phenylpropyl)-3-methylimidazolium T2HEA tetrakis (2-hydroxyethyl) ammonium TOMA trioctylmethylammonium TOS tosylate Acknowledgements The present work is supported by a Grant in-Aid for the Global COE Program, “Science for Future Molecular Systems” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] P. Wasserscheid, T. Welton (Eds.), Ionic Liquids in Synthesis, Wiley-VCH, Weinheim, 2003. [2] R.D. Rogers, K.R. Seddon (Eds.), Ionic Liquids; Industrial Applications for Green Chemistry. ACS Symposium Series 818, American Chemical Society, Washington, DC, 2002. [3] T. Welton, Chem. Rev. 99 (1999) 2071–2084.

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