Hydrolases in Non-Conventional Media: Implications for ... · 584 Hydrolases in Non-Conventional Media ... Table 16.1 Advantages and disadvantages of various reaction media for ...

Chapter 16

Industrial BiocatalysisEdited by Peter GrunwaldCopyright © 2015 Pan Stanford Publishing Pte. Ltd.ISBN 978-981-4463-88-1 (Hardcover), 978-981-4463-89-8 (eBook) www.panstanford.com

Hydrolases in Non-Conventional Media: Implications for Industrial Biocatalysis

16.1 Introduction

Agrochemical and pharmaceutical industries are urged by environ-mental regulators to implement sustainable technologies for the production of enantiomerically pure and value-added compounds. The use of biocatalysts represents a good solution. The enzyme- catalysed reactions often show advantages over the uncatalysed reactions, including high chemo-, regio- and enantioselectivities and occurrence under mild reaction conditions. However, the con-ventional aqueous reaction media for enzymatic reactions can limit the applications of biocatalysts. Only a small number of industrially attractive substrates are sufficiently water-soluble and hydrolysis is favoured over synthetic reactions in aqueous media. Furthermore,

Veronika Stepankova,a,b Jiri Damborsky,a,b and Radka Chaloupkovaa

aLoschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Masaryk University, Kamenice 5/A13, 62500Brno, Czech RepublicbEnantis, s.r.o., Palackeho trida 1802/129, 61200 Brno, Czech Republic

[email protected].

584 Hydrolases in Non-Conventional Media

water represents a challenge for reaction engineering in terms of downstream processing and integration into a process chain due to its high boiling point and high heat of vaporization (Ghanem and Aboul-Enein, 2004). The use of organic solvents as reaction me-dia for biocatalytic reactions has proven to be an extremely useful approach to extend the field of biocatalyst applications. However, exploiting advantages of using aqueous-organic systems or even neat organic solvents is limited by two factors: (i) the risk of enzyme deactivation, and (ii) the environmentally hazardous nature of solvents. Significant progress has been made towards identify-ing the environment-friendly alternatives to the organic solvents as the introduction of green technologies has become a major concern throughout both industry and academia. Such solvents should be associated with low toxicity, low vapour pressure, good biodegrad-ability and easy recycling. Ionic liquids (ILs), deep eutectic solvents (DESs), supercritical fluids (sc-fluids) and fluorous solvents can fill the gap between volatile organic solvents and water.

The aim of this chapter is to describe the scope and limitation of biocatalysis in non-conventional media, including both organic and neoteric solvents (Table 16.1). The most industrial enzymatic transformations are centred on the reactions catalysed by hydrolases. Owing to the broad substrate spectrum, no cofactor requirements and high volume efficiency, hydrolases are preferred industrial biocatalysts, and thus non-conventional media are of special interest for them (Clouthier and Pelletier, 2012). As an example of hydrolytic transformation in non-conventional media, this chapter presents the analysis of the effect of organic co-solvents on structure-function relationships of three representatives of the haloalkane dehalogenase enzyme family.

Table 16.1 Advantages and disadvantages of various reaction media for biocatalysis

Solvents Advantages Disadvantages

Water Good solvent for polar substrates, natural environment for enzymes, non-hazardous, nontoxic, non-flammable, cheap and widely available, easy to handle

Poor solvent for hydrophobic substrates, energy demanding downstream processing

585Biocatalysis in Organic Solvents

Solvents Advantages Disadvantages

Organic solvents

Good solvent for hydrophobic substrates, favoured synthesis over hydrolysis, suppression of water-induced side reactions, potential enhancement of enzyme thermostability and enantioselectivity, favoured product recovery, elimination of microbial contamination

Toxic and volatile, high risk of enzyme inactivation, largest source of wastes in chemical synthesis, laborious and costly preparation of biocatalysts

Ionic liquids Good solvent for polar substrates, favoured synthesis over hydroly-sis, suppression of water-induced side reactions, non-volatile, thermally stable, enhanced enzyme enantioselectivity, designer solvents

Limited data regarding toxicity, high cost, energy demanding synthesis, high viscosity

Deep eutectic solvents

Good solvent for polar substrates and metal salts, favoured synthesis over hydrolysis, suppression of water-induced side reactions, non-volatile, thermally stable, biodegradable, easy to prepare, cheap

High viscosity, insufficient knowledge about their properties

Supercriticalfluids

Good solvent for highly hydrophobic substrates, non-toxic, non-flammable, easy to remove after the reaction, high diffusion rates, pressure-tunability of parameters

Use of high pressures requiring special reactors, high risk of enzyme unfolding, potentially hazardous

Fluoroussolvents

Good solvent for hydrophobic substrates, high chemical and thermal stability, easily recyclable, temperature-dependent miscibility with organic solvents

Doubts about the persistence in the environment, large quantities of fluorine and hydrogen fluoride used in the synthesis

16.2 Biocatalysis in Organic SolventsThe beginning of detailed investigations of enzymes in organic solvents can be tracked back to the 1980s, when Klibanov and


co-workers published several papers denying the received wisdom that enzymes work only in aqueous solutions (Zaks and Klibanov, 1984, 1985, 1988). Their findings immediately attracted attention of researchers from both academia and industry. Shortly it became apparent that enzymes are not only able to work in organic solvents, but also acquire some new properties, such as improved thermal stability, altered regioselectivity or increased enantioselectivity. The possibility of influencing enzyme properties by changing the nature of the solvent in which the reaction is carried out was termed medium engineering (Laane, 1987). Nowadays, medium engineering represents a well-established alternative to the protein engineering and the time-consuming exploration of new catalysts. Many examples of the use of enzymes, mainly hydrolases, in organic solvents have been reported (Table 16.2).

Table 16.2 Examples of reactions catalysed by various hydrolases in the presence of organic solvents

Biocatalyst Solvent Reaction Effect Ref.

Esterase (EC 3.1.1.1)

Turkey pharynx 20–40% (v/v) 2-propanol

Esterification of tributyrin

Higher activity at lower temperatures

Cherif and Gargouri (2010)

Lipase (EC 3.1.1.3)

CALB THF, diethyl ether, hexane, benzene

Esterification of alcohols

Enantiomer excess up to 99% in hexane and benzene

Raminelli et al. (2004)

CALB tert-butanol, acetone

Hydrolysis of butanoate of 3-chloro-1-(phenyl-methoxy)-2-propanol

E-value raised from 7 to more than 200

Hansen T. V. et al. (1995)

Candida rugosa 0–80% (v/v) DMSO, isopropanol

Hydrolysis of β-substituted aryloxyacetic esters

The increase of enantio-selectivity depending on the substrate

Ammaz-zalorso et al. (2008)

Rhizomucor miehei

5–90% (v/v) DEE, DMF, DME, 1,4-dioxane, DMSO

Hydrolysis of esters

High activity with hydrophobic substrates

Tsuzuki et al. (2003)



Alcaligenes sp. DCM, hexane, decaline, acetone cyclohexane, THF, 1,4-dioxane

Kinetic resolution of sec-alcohols through trans-esterification

High enantio-selectivity insmall molecular-sized solvents

Wang et al. (2009)

Bacillus subtilis 10–30% (v/v) THF, acetone, acetonitrile,1,4-dioxane, DMF, DMSO

Hydrolysis of glycidol butyrate

16-fold higher E-value in 18% (v/v) 1,4-dioxane (5°C) than in buffer (25°C)

Li (2008)

Pseudomonas cepacia

35% (v/v) DMSO, DMF, acetone, 1,4-dioxane, THF, 1-propanol

Hydrolysis of 1-chloro-2-acetoxy-3-(1-naphthyloxy)-propane

The highest activity and enantio-selectivity in DMSO and THF

Mohapatra and Hsu (1999)

Carica papaya TCM, hexane, cyclohexane, decane, isooctane

Esterification of 2-(4-chloro-phenoxy)-propionic acid

The highest activity and enantio-selectivity in hexane

Cheng and Tsai (2004)

Wheat germ Toluene, hexane isooctane, isopropanol

Trans-esterification of sec-alcohols

The highest enantio-selectivity in hexane

Xia et al. (2009)

Feruloyl esterase (EC 3.1.1.73)

Aspergillus niger, Neurospora crassa, Talaromyces stipitatus

0–50% (v/v) DMSO, acetone, glycerol, ethanol, methanol, 1,4-dioxane, propanol

Hydrolysis of p-nitrophenyl acetate

Enhanced hydrolytic rate and increased Km in low concentrations of DMSO

Faulds et al. (2011)

β-Galactosidase (EC 3.2.1.23)

Escherichia coli, Kluyveromyces fragilis, Aspergillisoryzae

50% (v/v) acetone, EEE, acetonitrile, MEA, pyridine monoglyme, diglyme, TMP,

Hydrolysis of 2-nitrophenyl- β-D-galacto- pyranoside

20–60% of enzymatic activity retained in the presence of most of organic solvents

Yoon and Mckenzie (2005)

(Continued)



triglyme, DMSO, tetraglyme, 1,4-dioxane, DMF

used, no activity observed in 1,4-dioxane, DMSO, DMF and pyridine

Subtilisin (EC 3.4.21.62)

Bacillus licheniformis, Bacillus amylolique-faciens

TCM, toluene, butyl acetate, THF, acetonitrile, DMF

Trans-esterification of trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoro ethyl ester

The highest activity and enantio-selectivity in acetonitrile

Kawashiro et al. (1997)

Bacillus amylolique-faciens

50% (v/v) DMF

Ester and amide hydrolysis

Improved aminolysis and suppressed hydrolysis in DMF

Kidd et al. (1999)

Pepsin (EC 3.4.23.1)

Porcine pancreas

10–90% (v/v) acetonitrile, 1,4-dioxane, ethanol

Hydrolysis of haemoglobin

The activity preserved up to 60% (v/v) acetonitrile and ethanol

Simon et al. (2007)

Pseudolysin (EC 3.4.24.26)

Pseudomonas aeruginosa

10–90% (v/v) methanol, DMF, DMSO

Synthesis of peptide Cbz-Arg-Leu-NH2

High synthetic rates in organic co-solvents

Ogino et al. (2000)

Thermolysin (EC 3.4.24.27)

Bacillus thermoproteo-lyticus

0–20% (v/v) DMSO, DMF, n-propanol, isopropanol

Hydrolysis of casein

Decreased activity and thermostability

Pazhang et al. (2006)

β-Diketone hydrolase (EC 3.7.1.7)

Rhodococcus sp., Anabaena sp.

20–80% (v/v) EG, acetone, 1,4-dioxane,glycerol, acetonitrile, THF

Hydrolytic cleavage of C-C bond in β-diketones

Excellent activity tolerance towards organic solvents

Siirola et al. (2011)

Table 16.2 (Continued)



Haloalkane dehalogenase (EC 3.8.1.5)

Bradyrhizobium japonicum, Rhodococcus rhodochrous, Sphingobium japonicum

5–75% (v/v) glycerol, PEGs, EG, formamide, DMF, methanol, ethanol, acetone, 1,4-dioxane, isopropanol, THF, DMSO, acetonitrile

Hydrolytic dehalogenation of 1-iodohexane

Glycerol, EG, PEGs, DMSO and methanol well-tolerated by enzymes

Stepankova et al. (2013a)

CALB, Candida antarctica lipase B; DCM, dichloromethane; DEE, diethoxyethane; DME, dimethoxyethane; DMF, dimethylforamide; DMSO, dimethyl sulphoxide; EEE, diethylene glycol diethyl ether; EG, ethylene glycol; MEA, 2-methoxyethyl acetate; PEG, polyethylene glycol; TCM, tetrachloromethane; THF, tetrahydrofuran; TMP, trimethyl phosphate.

16.2.1 Nearly Anhydrous Organic Solvent Systems

The ability of enzymes to work in neat organic solvents was for long time taken with scepticism due to the assumption that enzymes are denatured in organic solvents. This prejudice, however, came from studying enzymes in mixtures of water and organic solvents, not in neat organic solvents containing less than 5% (v/v) of water (Griebenow and Klibanov, 1996). In contrary to aqueous-organic mixtures, enzymes are very rigid in the absence of water. As a consequence of protein rigidity, enzymes are much more stable in organic solvents than in water. The extreme thermostability of enzyme in 99% (v/v) organic medium was reported for the first time by Zaks and Klibanov (1984). The porcine pancreatic lipase was not only able to withstand heating at 100°C for many hours, but also exhibited a high catalytic activity at that temperature.

The organic solvent systems containing little water represent the most widely used non-conventional media for enzymatic reactions. Among the reactions reported in nearly anhydrous organic solvent systems prevail those catalysed by hydrolases, particularly lipases and proteases. Hydrolases are primarily used for resolution processes, where one enantiomer of a racemic mixture is selectively modified to yield a separable derivative. In water, these enzymes catalyse the hydrolysis of esters to the corresponding alcohols and


acids, which obviously cannot occur in nearly anhydrous media. Addition of nucleophiles, such as alcohols, amines and thiols, leads to transesterification, aminolysis and thiotransesterification, respectively. Moreover, a reverse hydrolysis—the synthesis of esters from acids and alcohols—becomes thermodynamically favourable (Zaks and Klibanov, 1985). Several companies are currently using lipase-catalysed reactions in organic solvents for the production of useful intermediates (Table 16.3). For instance, BASF offers a broad range of enzymatically synthesized alcohols for manufacture of enantiopure drugs (Schmid et al., 2001).

Table 16.3 Examples of processes involving lipase-catalysed reactions in organic media developed by several chemical and pharmaceu-tical companies

Company Process Ref.

BASF Synthesis of various enantiomerically pure alcohols, used as intermediates for synthesis of chemicals and pharmaceuticals, by asymmetric (trans)esterification

Schmid et al. (2001)

BASF Synthesis of polyol acrylates, used to prepare pigment dispersions, by reaction of aliphatic polyol with an acrylic acids

Paulus et al. (2003)

Chemie linz Synthesis of enantiomerically pure 2-halopropionic acids, used as intermediates for synthesis of herbicides and pharmaceuticals, by asymmetric esterification

Klibanov and Kirchner (1986)

Schering-Plough Synthesis of antifungal agent involving desymmetrization of 2-substituted-1,3-propanediol

Klibanov (2001)

Bristol-Myers Squibb

Synthesis of enantiomerically pure α-(3-chloropropyl)-4-fluorobenzene-methanol, an intermediate for the synthesis of an antipsychotic agent

Hanson et al. (1994)

Bristol-Myers Squibb

Asymmetric acetylation of (1α,2β,3α)-2-(benzyloxy methyl)-cyclopent-4-ene-1,3-diol to the corresponding monoacetate, a key intermediate for the synthesis of an angiotensin-converting enzyme inhibitor

Patel et al. (2006)


Company Process Ref.

Sepracor Synthesis of enantiomerically pure alkyl 1,4-benzodioxan-2-carboxylates, used as intermediates in the synthesis of pharmaceuticals, such as (S)-doxazosin

Rossi et al. (1996)

Pfizer Synthesis of enantiomerically pure (R)-aminopentanenitrile, an intermediate for manufacture of pharmaceuticals, by selective acylation

Allen et al. (2006)

Despite high stability, enzymes generally exhibit lower activities in neat organic solvents than in aqueous reaction systems. The loss of biocatalytic activity has been ascribed to different reasons, including non-optimal hydration of the biocatalyst, restricted protein flexibility, suboptimal pH, diffusional limitations, unfavourable substrate desolvation, low stabilization of the enzyme–substrate intermediate and changes in the enzyme active site (Carrea and Riva, 2000; Klibanov, 1997; Toth et al., 2010).

The main factor that has to be taken into account when performing biocatalysis in nearly anhydrous organic media is water activity. Even in neat organic solvent media, at least a few water molecules are required to remain bound to the enzyme. It became apparent, that fully dehydrated proteins are inactive. For instance, α-chymotrypsin and subtilisin need about 50 molecules of water per enzyme molecule to be catalytically active (Zaks and Klibanov, 1986). The more hydrophilic the solvent is, the more water has to be added to reach high activity, because hydrophilic solvents have a greater tendency to strip the essential water from the enzyme molecule (Klibanov, 2001). Water, acting as lubricant, allows enzymes to exhibit the conformational mobility required for optimal catalysis. In contrast, organic solvents lack water’s ability to create hydrogen bonds, and also have lower dielectric constants, leading to stronger intra-protein electrostatic interactions. The exception are hydrophilic solvents, such as glycerol, ethylene glycol or formamide, that are capable of forming multiple hydrogen bonds with enzyme molecules, thus partially mimic the water effects (Torres and Castro, 2004; Almarsson and Klibanov, 1996). Addition of small quantities of water or water-mimicking solvent to enzyme in anhydrous solvent can increase the enzyme activity by several


orders of magnitude. Thus, it is very important to control the amount of water in the reaction mixture and keep this parameter close to the optimal value.

Another parameter that affects enzyme activity is pH. In many cases, an enzyme in neat organic solvents keeps the ionization state from the aqueous solution to which it was exposed before removal of water, (Xu and Klibanov, 1996; Klibanov, 1997; Zaks and Klibanov, 1985). This phenomenon is called pH memory. The enzymatic activity can be therefore significantly enhanced if enzymes are lyophilized from solutions of the pH optimal for the catalysis. On the other hand, if the enzymatic reactions involve the formation or consumption of acidic or basic substances, the pH buffering capacity is needed. Triphenylacetic acid and its sodium salts are typical examples of pairs controlling pH in relatively polar solvents, while dendritic polybenzyl ether derivatives have been developed as the alternatives for more hydrophobic media (Dolman et al., 1997; Xu and Klibanov, 1996).

Since enzymes are practically insoluble in most organic solvents, they are usually introduced into neat organic solvents as powders prepared by lyophilisation (Carrea and Riva, 2000; Torres and Castro, 2004). The protein denaturation, which may occur in the process of dehydration, is normally reversible upon rehydration in aqueous media. However, refolding in anhydrous organic solvents is not trivial due to the reduced structural mobility (Mattos and Ringe, 2001; Griebenow and Klibanov, 1995).

In order to minimize this deleterious effect, the lyophilization of enzymes for their application in non-aqueous media should be done in the presence of lyoprotectans, including sugars, inorganic salts, polyethylene glycols and crown ethers (Carrea and Riva, 2000; Klibanov, 1997 and 2001). The approaches used to minimize substrate-diffusion limitations originating from using enzyme powders include (Fig. 16.1): (i) the adsorption or covalent coupling of the enzyme on a solid support, (ii) the encapsulation of the enzyme using polymers, (iii) the entrapment of the enzyme in sol-gel materials or organic polymers, (iv) the solubilisation of the enzyme by formation of complexes with polyethylene glycols, (v) the cross-linking of enzyme crystals or aggregates, and (vi) surfactant-based enzyme preparations (Adlercreutz, 2013; Batistella et al., 2012; Carrea and Riva, 2000; Iyer and Ananthanarayan, 2008).

593

Figure 16.1 Schematic presentation of enzyme preparations suitable for use in anhydrous organic media.

Dramatic changes in enzyme stereoselectivity upon switching from one organic solvent to another have been documented by several studies (Table 16.2). For instance, enantioselectivity of α-chymotrypsin in the transesterification of methyl 3-hydroxy-2-phenylpropionate with propanol has been found by Wescott et al. (1996) to span a 20-fold range simply by switching between different solvents. The completely inverted enantiopreference upon changing the organic solvent was observed by Ke et al. (1996). The dominant products of the chymotrypsin-catalysed acetylation of prochiral 2-substituted 1,3-propanediols in diisopropyl ether or cyclohexane were S-enantiomers, whereas R-enantiomers were formed preferentially in acetonitrile or methyl acetate. Hypotheses formulated in order to rationalize the observed solvent effects on enzyme enantioselectivity can be grouped into three different classes: (i) the solvent modifies the enzyme conformation, leading to the alteration of the enzyme-substrate recognition process, (ii) the solvent influences the substrate desolvation, and (iii) the solvent binds into the enzyme active site, interfering with the association of one enantiomer more than the other one (Carrea and Riva, 2008). However, all of them lack reliable predictive value and are not sufficient to explain every case. Recently, the relationship between enantioselectivity and water content on the enzymatic surface was reported by Herbst et al. (2012), who investigated the enantioselectivity of Candida rugosa lipase in different binary mixtures of hexane and tetrahydrofuran. They revealed a decrease in conversion but increase in selectivity with increasing solvent

Biocatalysis in Organic Solvents


hydrophilicity. The observation was ascribed to the extraction of water molecules from the enzyme surface, resulting in enzyme rigidification, which is in agreement with previous studies by Fitzpatrick and Klibanov (1991), and Gubicza and Kelemen-Horvàth (1993). On the other hand, contradictory studies showing an increasing enantiomeric excess with increasing enzyme flexibility have been published by Nakamura et al. (1991), Persson et al. (2002), and Carrea et al. (1995).

16.2.2 Biphasic Systems

The concept of biphasic systems includes the use of two immiscible liquids phases, where one of the phases is aqueous and provides a protective environment for biocatalyst, whereas the second phase is a water-immiscible organic solvent and provides a substrate/product pool. Owing to the high substrate concentrations that can be achieved in this way, high volumetric productivities can be envisaged in comparison with aqueous systems. The hydrophobic products pass to the organic phase, thus can be relatively simply isolated. Furthermore, the potential inhibitory effect of substrate/product towards the biocatalyst is minimized. On the other hand, disadvantage of biphasic systems is the interfacial inactivation of the biocatalyst (Sellek and Chaudhuri, 1999). Moreover, dissolved solvent molecules present in the aqueous phase with the biocatalyst may interact with nonpolar groups in the protein and disrupt its hydrophobic core (Ross et al., 2000). Although the majority of the work on bioconversions has been done with enzymes, the use of whole cells as biocatalysts in biphasic systems is becoming a very promising field especially for certain bioconversions, such as oxidations, which usually involve cofactor addition (Leon et al., 1998). Whole cell biocatalysts require more water than isolated enzymes, thus two-phase systems are more suitable for them than neat hydrophobic solvents.

16.2.3 Organic Co-Solvent Systems

Organic co-solvent systems are produced when water-miscible solvents are added to the aqueous medium to improve the solubility of compounds sparingly soluble in water, to modify the enzyme

595

enantioselectivity or to favour synthesis over hydrolysis (Doukyu and Ogino, 2010). Surprisingly, enzymes are much more tolerant to pure organic solvents than to water-solvent mixtures (Griebenow and Klibanov, 1996). This is due to the interplay of two effects. On the one hand, as the water content declines, the protein conformational mobility is diminished. On the other hand, as the organic solvent concentration is raised the tendency of protein to denature is increased. Thus, an increase of organic co-solvent concentration in aqueous media generally decreases the enzyme activity. Most enzymes become almost totally inactive at an organic co-solvent concentration of 60–70% (v/v). Conformational changes are the most common reason for enzyme deactivation in the presence of organic co-solvents (Graber et al., 2007; Mozhaev et al., 1989; Stepankova et al., 2013b). Miscible solvents are known to turn the hydrophobic core of the protein from buried to more exposed position (Yoon and Mckenzie, 2005). The strategies employed to enhance the enzyme structure stability in the presence of organic co-solvents include: (i) protein engineering, (ii) chemical modification of enzymes with amphipathic compounds and lipids, (iii) immobilization of enzymes, (iv) reverse micelle formation, and (v) addition of salts. Nevertheless, several naturally occurring enzymes exhibiting high tolerance towards organic co-solvents have been reported (Table 16.2). A good example of organic co-solvent-tolerant enzymes represent PST-01 proteases secreted by Pseudomonas aeruginosa PST-01 discovered by Ogino et al. (1999). Stability of these proteases in the solutions containing water-soluble organic solvents was even higher than that in the absence of organic solvents. Recently, the excellent tolerance towards organic co-solvents showed β-diketone hydrolases, representatives of the crotonase superfamily. These enzymes retained their activity even in the presence of 80% (v/v) 1,4-dioxane or tetrahydrofuran, in which only highly stable lipases have previously been shown to retain the activity (Siirola et al., 2011). Organic solvent-stable lipases discovered till now, originate mainly from Pseudomonas and Bacillus genera (Chakravorty et al., 2012).

Organic co-solvents can be helpful also for improvement of enzyme enantioselectivity (Table 16.2). For instance, by addition of water miscible organic co-solvents, such as tert-butanol and acetone, the E-value for Candida antarctica lipase B-catalysed hydrolysis of

Biocatalysis in Organic Solvents


butanoate of 3-chloro-1-(phenylmethoxy)-2-propanol raised from 7 to more than 200 (Hansen et al., 1995). Similarly high enhancement of enantioselectivity was observed by Watanabe and Ueji (2001) for Candida rugosa and Pseudomonas cepacia lipase-catalysed hydrolysis of 2-(4-substituted phenoxy)-propionates in the presence of DMSO. They found out that the improvement of enantioselectivity proceeds via different mechanisms. For Pseudomonas cepacia lipase, the high DMSO-induced enantios-electivity was caused by the acceleration of the initial rate for the preferred R-enantiomer, while the enantioselectivity enhancement for Candida rugosa lipase was attributed to the almost complete inactivation of the conversion of incorrectly bound S-enantiomer.

The addition of hydrophilic co-solvent to anhydrous hydrophobic solvent was also found to be beneficial for enzyme enantio-selectivity. If the target compounds are not the natural substrates for the enzyme, enantioselectivity in neat organic solvents is not always high enough to obtain optically pure compounds. These non-natural substrates require sufficient flexibility of the enzyme for proper binding to the active site. This could be achieved by the addition of a small amount of denaturing co-solvent. For example, Watanabe et al. (2004) significantly enhanced the enantioselectivity for subtilisin-catalysed reaction in dry isooctane by the addition of 0.3% (v/v) DMSO.

16.3 Biocatalysis in Ionic Liquids

Ionic liquids (ILs) are organic salts composed of bulky asymmetric cations and weakly coordinating anions, with melting points below 100°C (Wasserscheid and Keim, 2000). In contrast to organic solvents, ILs are non-flammable and have extremely low volatility, which introduces the possibility of products removal by distillation without further contamination by solvents (Weingartner, 2008). Thus, ILs can be classified as “green solvents”, whose application results in significantly reduced environmental impact. Visser et al. (2002) estimated that 1018 different ILs are theoretically possible. The physico-chemical properties of ILs, such as solubility characteristics, viscosity, density, polarity and melting point can

597

be finely tuned by altering their ionic components, opening up the possibility to optimize the ionic reaction medium to meet the criteria of specific applications (Freemantle, 1998; Wasserscheid and Keim, 2000).

The pioneer report of biocatalysis using ILs as reaction media was published by Magnuson et al. (1984), who used an aqueous mixture of ethylammonium nitrate as a solvent for alkaline phosphatase. Unfortunately, ILs did not attract significant attention until 2000 when several examples of using enzymes in ILs appeared (Cull et al., 2000; Erbeldinger et al., 2000; Madeira Lau et al., 2000). These first studies were remarkably successful, showing the enhancement in the solubility of substrates or products without inactivation of the enzymes. ILs with dialkylimidazolium and alkylpyridinium cations (Table 16.4), the so-called second-generation ILs, are generally recognized as the most suitable for biocatalytic reactions (Park and Kazlauskas, 2003). The representative structures of the second-generation ILs are given in Fig. 16.2. These ILs exhibit interesting properties, such as low melting point, high thermostability, low viscosity and different solubility. Their solubility in water is influenced by the ability of anions to form hydrogen bonds. Thus, ILs with [PF6]– or [Tf2N]– are water immiscible, whereas those with [BF4]– or Br– are water miscible (Gorke et al., 2007).

Figure 16.2 Representative structures of cations and anions comprising the second-generation ILs commonly used in biocatalysis.

Biocatalysis in Ionic Liquids


Tabl

e 16

.4

Exam

ples

of t

he se

cond

-gen

erat

ion

ILs m

entio

ned

in th

is ch

apte

r

Cati

onA

nio

nN

otat

ion

Wat

er m

isci

bil

ity

1-Al

lyl-3

-met

hylim

idaz

oliu

mCh

lori

de[A

mim

]Cl

Yes

1-Be

nzyl

-3-m

ethy

limid

azol

ium

Tetr

aflu

orob

orat

e[B

zmim

][BF

4]Ye

s

Chlo

ride

[Bzm

im]C

lYe

s

1-Bu

tyl-3

-met

hylim

idaz

oliu

mTe

traf

luor

obor

ate

[Bm

im][

BF4]

Yes

Hex

aflu

orop

hosp

hate

[Bm

im][

PF6]

No

Chlo

ride

[Bm

im]C

lYe

s

Iodi

de[B

mim

]IYe

s

Bis[

(tri

fluor

omet

hyl)s

ulph

onyl

]imid

e[B

mim

][Tf

2N]

No

Octy

lsul

phat

e[B

mim

][Oc

tSO 4

]Ye

s

1-De

cyl-3

-met

hylim

idaz

oliu

mCh

lori

de[D

mim

]Cl

Yes

1-Et

hyl-3

-met

hylim

idaz

oliu

mTe

traf

luor

obor

ate

[Em

im][

BF4]

Yes

Bis[

(tri

fluor

omet

hyl)s

ulph

onyl

]imid

e[E

mim

][Tf

2N]

No

Chlo

ride

[Em

im]C

lYe

s

Acet

ate

[Em

im][

CH3C

OO]

Yes

599

Dim

ethy

lpho

spha

te[E

mim

l[DM

P]Ye

s

Diet

hylp

hosp

hate

[Em

iml[D

EP]

Yes

1-H

exyl

-3-

met

hylim

idaz

oliu

mCh

lori

de[H

mim

]Cl

Yes

1-M

etho

xyet

hyl-

3-m

ethy

limid

azol

ium

Tetr

aflu

orob

orat

e[M

OEm

im][

BF4]

Part

.

Hex

aflu

orop

hosp

hate

[MOE

mim

][PF

6]N

o

1-Oc

tyl-

3-m

ethy

limid

azol

ium

Hex

aflu

orop

hosp

hate

[Om

im][

PF6]

No

Bis[

(tri

fluor

omet

hyl)s

ulph

onyl

]imid

e[O

mim

][Tf

2N]

No

1,3-

Dim

ethy

limid

azol

ium

Met

hyls

ulph

ate

[Mm

im][

MeS

O 4]

Yes

Dim

ethy

lpho

spha

te[M

mim

][DM

P]Ye

s

1-Et

hylp

yrid

iniu

mTr

ifluo

roac

etat

e[E

tPy]

[CF 3

COO]

Yes

2-M

etho

xyet

hyl(t

ri-n

-but

yl)p

hosp

honi

umBi

s[(t

riflu

orom

ethy

l)sul

phon

yl]im

ide

[MEB

u 3P]

[Tf 2

N]

No

Buty

ltrim

ethy

lam

mon

ium

Bis[

(tri

fluor

omet

hyl)-

sulp

hony

l]im

ide

[BTM

A][T

f 2N

]N

o

Coco

salk

yl p

enta

etho

xy m

ethy

lam

mon

ium

Met

hosu

lpha

te[C

PMA]

[MS]

Yes

Met

hyltr

ioct

ylam

mon

ium

Bis[

(tri

fluor

omet

hyl)s

ulph

onyl

]imid

e[M

TOA]

[Tf 2

N]

No

Trie

thyl

amm

oniu

mAc

etat

eTE

AAYe

s

Phos

phat

eTE

APYe

s



Tabl

e 16

.5

Exam

ples

of r

eact

ions

cata

lyse

d by

var

ious

hyd

rola

ses i

n th

e pr

esen

ce o

f ILs

Bio

cata

lyst

Solv

ent

Rea

ctio

nEf

fect

Ref

.

Lipa

se (

EC 3

.1.1

.3)

CALB

[Bm

im][

PF6]

,[B

mim

][BF

4]Tr

anse

ster

ifica

tion

of e

thyl

but

anoa

te a

nd

octa

noat

e, a

mm

onio

lysi

s of e

thyl

oct

anoa

te,

epox

idat

ion

of cy

lohe

xene

Reac

tion

rate

s com

para

ble

or b

ette

r th

an th

ose

obse

rved

in o

rgan

ic m

edia

Mad

eira

Lau

et a

l. (2

000)

CALB

[CPM

A][M

S]Sy

nthe

sis o

f but

yl p

ropi

onat

eIn

crea

sed

activ

ity a

nd th

erm

osta

bilit

yDe

Die

go

et a

l. (2

009)

CALB

[Em

im][

BF4]

, [B

mim

][PF

6]Tr

anse

ster

ifica

tion

of se

cond

ary

alco

hols

H

ighe

r ena

ntio

sele

ctiv

ity th

an in

TH

F an

d to

luen

eKi

m e

t al.

(200

1)

CALB

85–9

7% (v

/v)

[Bm

im][

PF6]

Reso

lutio

n of

tetr

ahyd

ro-4

-met

hyl-3

-oxo

-1H

-1,4

-ben

zo d

iaze

pine

-2-a

cetic

aci

d m

ethy

l est

er

Hig

her s

olub

ility

of t

he su

bstr

ate

and

abili

ty to

ope

rate

at h

igh

tem

pera

ture

sRo

bert

s et a

l. (2

004)

Ther

mom

yces

la

nugi

nosu

s[B

mim

][PF

6],

[Om

im][

PF6]

Synt

hesi

s of b

utyl

pro

pion

ate

Hig

h ac

tivity

De D

iego

et a

l. (2

009)

Cand

ida

rugo

sa[B

mim

][PF

6]Tr

anse

ster

ifica

tion

of m

ethy

l met

hacr

ylat

eRe

actio

n ra

te 1

.5-fo

ld h

ighe

r tha

n in

he

xane

Kaar

et a

l. (2

003)

Cand

ida

rugo

saW

ater

-sat

urat

ed[B

mim

][PF

6]H

ydro

lysi

s of m

ethy

l est

er o

f nap

roxe

nEn

antio

sele

ctiv

ity 6

-fold

hig

her t

han

in

isoo

ctan

eXi

n et

al.

(200

5)

Pseu

dom

onas

sp.

[Bm

im][

Tf2N

]Ki

netic

reso

lutio

n of

1-p

heny

leth

anol

Hig

her e

nant

iose

lect

ivity

than

in M

TBE

Ecks

tein

et a

l. (2

002b

)

Bur

khol

deri

a ce

paci

a[M

EBu 3

P][T

f 2N

]Tr

anse

ster

ifica

tion

of se

cond

ary

alco

hols

Slig

htly

fast

er re

actio

n th

an in

di

isop

ropy

l eth

erAb

e et

al.

(200

8)

601

Porc

ine

panc

reat

ic15

% (v

/v)

[EtP

y][C

F 3CO

O]H

ydro

lysi

s of v

ario

us e

ster

s of a

min

o ac

ids

Hig

h en

antio

-sel

ectiv

ityM

alho

tra

and

Zhao

(200

5)

α-Am

ylas

e (E

C 3.

2.1.

1)

Bac

illus

am

ylol

ique

-fa

cien

s, B

acill

us

lichi

nifo

rmis

5–40

% (v

/v)

[Bm

im]C

l, [H

mim

]Cl

Hyd

roly

sis o

f sta

rch

Decr

ease

of a

ctiv

ity a

nd st

abili

tyDa

birm

anes

het

al.

(201

1)

Cellu

lase

(EC

3.2

.1.4

)

Tric

hode

rma

rees

ei10

% (v

/v) [

Mm

im]

[DM

P], [

Bmim

]Cl,

[Am

im]C

l, [E

mim

] [C

H3C

OO]

Hyd

roly

sis o

f α-c

ellu

lose

Decr

ease

of a

ctiv

ity

Enge

l et a

l. (2

010)

Hal

orha

bdus

ut

ahen

sis

20%

(v/v

) [A

mim

]Cl,

[Bm

im]C

l, [E

mim

]Cl

Hyd

roly

sis o

f α-c

ellu

lose

Activ

ity u

ncha

nged

or s

light

ly

stim

ulat

edZh

ang

et a

l. (2

011)

Xyla

nase

(EC

3.2

.1.8

)

Anox

ybac

illus

sp.

5–20

% (v

/v)

[Em

im][

DMP]

,[M

mim

] [DM

P]

Hyd

roly

sis o

f p-n

itrop

heny

l β-D

-xy

lopy

rano

side

Slig

ht in

crea

se o

f act

ivity

Thom

as e

t al.

(201

1)

β-Ga

lact

osid

ase

(EC

3.2.

1.23

)

Bac

illus

cir

cula

ns20

% (v

/v)

[Mm

im][

MeS

O 4]

Synt

hesi

s of

N-a

cety

llact

osam

ine

by tr

ansg

lyco

syla

tion

The

supp

ress

ion

of th

e se

cond

ary

hydr

olys

isKa

ftzik

et a

l. (2

002)


(Con

tinu

ed)


Bio

cata

lyst

Solv

ent

Rea

ctio

nEf

fect

Ref

.

Volv

arie

lla v

olva

cea

5–20

% (v

/v)

[Em

im][

CH3C

OO],

[Em

im][

DEP]

, [E

mim

][DM

P],

[Mm

im][

DMP]

Hyd

roly

sis o

f p-n

itrop

heny

l β-D

-glu

copy

- ra

nosi

deSh

arp

decr

ease

of a

ctiv

ity a

t co

ncen

trat

ions

abo

ve 1

0% (v

/v)

Thom

as e

t al.

(201

1)

Prun

e se

ed m

eal

Mix

ture

of [

Bmim

]I,

ethy

lene

gly

col a

nd

diac

etat

e

Synt

hesi

s of a

ryla

lkyl

β-D

-glu

copy

rano

side

s vi

a re

vers

e hy

drol

ysis

Yiel

ds e

nhan

ced

betw

een

0.2-

fold

and

0.

5-fo

ldYa

ng e

t al.

(201

2)

α-Ch

ymot

ryps

in (

EC 3

.4.2

1.1)

Bovi

ne50

% (v

/v) T

EAA,

TE

AP, T

BPBr

, [B

zmim

]Cl,

[Bzm

im][

BF4]

Hyd

roly

sis o

f Suc

–Ala

–Ala

–Pro

–Phe

–p-

nitr

oani

lide

The

stro

nges

t sta

biliz

atio

n in

trie

thyl

am

mon

ium

salts

At

tri e

t al.

(201

1)

Bovi

ne[B

mim

] [T

f 2N

]Tr

anse

ster

ifica

tion

of N

-ace

tyl-L

-phe

nyl-

alan

ine

ethy

l est

er

Hig

her a

ctiv

ity th

an in

eth

yl a

ceta

te a

nd

MTB

EEc

kste

in e

t al.

(200

2a)

Papa

in (

3.4.

22.2

)

Cari

ca p

apay

a 80

% (v

/v) [

Bmim

] [B

F 4]

Hyd

roly

sis o

f hyd

roxy

phen

ylgl

ycin

e m

ethy

l est

erIn

crea

se o

f sub

stra

te so

lubi

lity

and

enan

tiose

lect

ivity

Liu

et a

l. (2

005)

Lo

u et

al.

2006

b)

Subt

ilisi

n (E

C 3.

4.21

.62)

Bac

illus

lic

heni

form

is

[Em

im]

[Tf 2

N]

Hyd

roly

sis o

f p-n

itrop

heny

l but

yrat

eH

ighe

r act

ivity

than

in to

luen

eN

akas

him

a et

al.

(200

6)

CALB

, Can

dida

ant

arct

ica

lipas

e B;

MTB

E, m

ethy

l ter

t-bu

tyl e

ther

; TEA

A, tr

ieth

ylam

mon

ium

ace

tate

; TEA

P, tr

ieth

ylam

mon

ium

pho

spha

te; T

BPBr

, te

trab

utyl

phos

phon

ium

bro

mid

e; T

HF,

tetr

ahyd

rofu

ran.

Tabl

e 16

.5

(Con

tinu

ed)

603

Biocatalysis in ILs is rapidly expanding, and a great number of reactions in ILs have been published. The selected examples are given in Table 16.5. On the other hand, literature search for large- scale industrial applications with biocatalysis in ILs provided no result. Following challenges must be solved before catalytic processes in ILs will be broadly used: (i) the high cost, (ii) the presence of impurities, such as water and unreacted halides, (iii) the antibacterial activity and toxicity of some imidazolium and pyridinium ILs, (iv) the decomposition of [PF6]– and [BF4]– in water, yielding hydrofluoric acid, and (v) the high viscosity (Docherty and Kulpa, 2005; Marsh et al., 2004). Despite current obstacles, it is believed that the use of ILs will open up a new field in biocatalysis as the use of enzymes in organic solvents did in 1980s.

16.3.1 Nearly Anhydrous IL Systems

Typical non-aqueous reaction conditions, used to make the condensation reaction thermodynamically favourable, are non-polar organic solvent such as toluene or hexane. Polar organic solvents cannot be used because they usually denature enzymes (Serdakowski and Dordick, 2008). In contrast, polar ILs are well tolerated by many enzymes, suggesting the use of ILs for non-aqueous synthetic applications with highly polar substrates, for example, carbohydrates, which are only sparingly soluble in most organic solvents. The polarity of the second-generation ILs is in the range of lower alcohols and formamide (Park and Kazlauskas, 2003). Some ILs are polar but hydrophobic, which is an exceptional property not known for other solvents (Fischer et al., 2011).

Most of the reactions in pure ILs are carried out with lipases (Table 16.5). On the contrary, a majority of other hydrolases lose the activity in such media. In general, the factors influencing enzyme activities in non-aqueous organic solvents are, in most cases, also relevant for enzymes in non-aqueous ILs. Controlling the water content is of the highest importance to achieve a high conversion (Berberich et al., 2003). Previous studies showed that the higher catalytic activities under non-aqueous conditions are obtained in hydrophobic ILs, whereas hydrophilic ILs usually have a deleterious impact on enzyme stability and activity, since they might remove internally bound water from the enzyme (Ventura et al., 2012).



For hydrophobic ILs, Kurata et al. (2010), Attri et al. (2011) and Ha et al. (2012) showed that an increase in alkyl chain length attached to the imidazolium ring decreased the catalytic efficiency of α-chymotrypsin and Candida antarctica lipase B. Ventura et al. (2012) explained this effect by the increase of the cation hydrophobicity, leading to the increase in the van der Waals interactions with the non-polar domains of the enzyme. However, this trend is not generally accepted since De Diego et al. (2009) and De Los Ríos et al. (2007) reported an opposite effect, leaving this issue open for the future studies.

Several reports describe the effect of ILs on enzyme enantioselectivity. Lipases were shown to be active in pure 1-butyl-3-methylimidazolium-based ILs with enantioselectivities up to 25-times higher than in conventional organic solvents (Kim et al., 2001; Schofer et al., 2001; Ulbert et al., 2004; Xin et al., 2005). More recently, Abe et al. (2008) reported that [MEBu3P][Tf2N] is a good solvent for lipase-catalysed resolution of alcohols, because it lacks acidic protons and the reaction is faster than that with imidazolium salts.

16.3.2 IL-Based Biphasic Systems

Recently, the development of biotechnological processes using biphasic systems based on ILs attracted a lot of attention. Here, hydrophobic ILs replace water-immiscible organic solvents in two-phase system with water. Jiang et al. (2007) developed a process for the hydrolysis of penicillin G using two different ILs and phosphate buffer. Commonly used reaction medium for penicillin acylase is a two-phase system based on aqueous buffer and butyl acetate. However, the main problem with this system is its low pH, which decreases activity of the enzyme. To overcome this disadvantage, butyl acetate was replaced with two ILs, [Bmim][BF4] and [Bmim][PF6]. This new reaction system had a pH value of 5, which is beneficial for the activity and stability of the penicillin acylase. Likewise organic solvents, ILs are usually toxic to microorganisms. Nevertheless, IL-based biphasic systems can be applied in whole- cell biocatalysis as shown by Pfruender et al. (2004) and Weuster-Botz (2007). The membrane integrity of the Escherichia coli, Lactobacillus kefir and Saccharomyces cerevisiae as well as the

605

reaction yield were significantly higher in biphasic systems of [Bmim][PF6], [Bmim][Tf2N] and [MTOA][Tf2N] than in organic solvent-based systems.

16.3.3 IL Co-Solvent Systems

The water-miscible ILs are used as co-solvents to increase the solubility of substrates or to suppress unwanted non-enzymatic hydrolysis of reactants. Although the majority of enzymes reported to work in non-aqueous ILs are lipases, reaction involving these enzymes usually do not use ILs as co-solvents. On the contrary, many other hydrolases have been investigated in aqueous ILs mixtures (Table 16.5). It was established that the enzyme activity, stability and enantioselectivity generally follow the Hofmeister series when the aqueous solutions of hydrophilic ILs are used as reaction media (Zhao, 2005). Although Yang et al. (2008 and 2009) and Zhao et al. (2006) demonstrated that the enzymes maintain high level of activity and enantioselectivity in water-mimicking ILs composed of chaotropic cations and kosmotropic anions, the connection between the Hofmeister series and the enzymatic behaviour cannot be taken as an universal rule. For instance, Lou et al. (2006a) showed that the lipase activity was increased three-times in the co-solvent systems with 20% (v/v) [Bmim][BF4] comprising kosmotropic cation and chaotropic anion.

Kamiya et al. (2008) and Lee et al. (2009) address pretreatment of cellulosic biomass by ILs as co-solvents, which is of a great importance in industry. It has been shown that cellulose after IL pretreatment has reduced crystallinity, and thus is more acceptable for cellulolytic enzymes. Unfortunately, commercially available fungal cellulases are usually inhibited even by trace amounts of ILs (10–15% v/v) left after cellulose pretreatment (Engel et al., 2010; Zhang et al., 2011). Therefore, the identification of IL-resistant cellulases or development of enzyme-compatible ILs is very important for the enzymatic hydrolysis of cellulose in industrial applications.

The activity of various lipases has been investigated also in co-solvent mixtures of organic solvents and ILs. Interestingly, Wallert et al. (2005), Singh et al. (2009), Pan et al. (2010) and Lou et al. (2005) found out, that in some cases, the enzyme activity is



higher in mixture of organic solvent and IL than in corresponding pure organic solvent or IL. Pan et al. (2010) assigned this effect to the diminished viscosity of ILs in the presence of organic solvent, which eliminates the mass transfer limitations.

16.3.4 Ionic Liquids as Coating Agents

Besides acting as a reaction media, ILs can be used as coating agents for the enzymes. In recent years, the coating of enzymes by ILs during lyophilization has emerged as an efficient method for the preparation of highly stable biocatalysts, showing better catalytic activities and enantioselectivities under harsh reaction conditions required for industrial applications (Abdul Rahman et al., 2012; Moniruzzaman et al., 2010). An emerging biotransformation technique employs IL-coated enzyme beads in organic solvents. For example, lipase coated by dodecyl imidazolium salt during the lyophilization was 660-fold more active and exhibited higher enantioselectivity in anhydrous toluene, when compared to the free enzyme (Lee and Kim, 2011).

16.4 Biocatalysis in Deep Eutectic Solvents

Deep eutectic solvents are physical mixtures of salts and hydrogen bond donors that melt at low temperatures due to the charge delocalisation, Abbott et al. (2003). At the eutectic ratio, typically 1–4 molecules of hydrogen bond donor per molecule of salt, the mixtures form a liquid at room temperature. Although DESs are often called as advanced ILs, they contain uncharged components, and therefore they are not entirely ionic. Deep eutectic solvents were established by Abbott et al. (2003), who reported low melting mixture of (2-hydroxyethyl) trimethyl-ammonium (choline) chloride (ChCl), so called vitamin B4, and urea. Consequently, different hydrogen bond donors, such as alcohols, carboxylic acids and urea derivatives, were used in combination with ChCl or ethylammonium chloride (EACl) (Abbott et al., 2004; Ruß and Konig, 2012). Some of DESs are now available commercially (Table 16.6).

607

Table 16.6 Examples of the commercially available DESs

Component 1 Component 2 DES Ratio Tf (°C)

Choline chloride

–

Urea Reline 1:2 12

Ethylene glycol

Ethaline 1:2 –20

Glycerol Glyceline 1:2 –40

Malonic acid

Maline 1:1 10

Tf , Freezing point; Ruß and Konig (2012).

Despite the fact that the physico-chemical properties of DESs have not yet been investigated into detail, it is believed that many properties of ILs could be extrapolated to DESs. They are thermally stable, polar and have low vapour pressures (Abbott et al., 2006). Nevertheless, in contrast to ILs, they are nontoxic, biodegradable and easy to prepare at the low cost. Moreover, DESs can dissolve several compounds like metal salts, organic acids, and various polyols, which are problematic for conventional aprotic ILs (Abbott et al., 2004, 2007; Morrison et al., 2009; Zhao et al., 2011b). These properties make DESs applicable as green solvents in several industrial processes. One potential drawback of DESs stems from their relatively high viscosities, leading to the mass transfer limitations and requirements for the agitation. For example, ChCl:urea (1:2) mixture has a viscosity of around 1200 m Pa.s at room temperature and 170 m Pa.s at 40°C (Abbott et al., 2006). Recently, new species of eutectic mixtures have been developed. The combination of choline acetate (ChAc) with glycerol (1:1.5) led to a lower viscosity (Zhao et al., 2011a).

The similarity with ILs was the main rationale behind investigation of DESs as the reaction media for biotransformations. Even though the use of DESs in biocatalysis is still in the early stage of development, the number of articles has been growing nearly exponentially since the first publication in 2008. Gorke et al.

Biocatalysis in Deep Eutectic Solvents


(2008) showed that several hydrolases exhibited comparably or even better activities in DESs than in conventional organic solvents (Table 16.7). For instance, the addition of 25% (v/v) ChCl:glycerol (1:2) increased the rate of conversion of styrene oxide to styrene glycol by epoxide hydrolase from Agrobacterium radiobacter by 20-fold compared to buffer alone, while adding 25% (v/v) DMSO or acetonitrile decreased activity 2–6-fold. This was a groundbreaking study, because strong hydrogen-bond donors, for instance urea, are expected to denature proteins and alcohols can interfere with hydrolase-catalysed reactions. Surprisingly, the components of DESs were found to be significantly less reactive with enzymes than expected. For example, ethylene glycol and glycerol were found to be 9-fold and 600-fold, respectively, less reactive in lipase-catalysed transesterification when they were present as components of DES. Thus, it seems that the hydrogen bond network in DESs lowers the chemical potential of the individual components and makes them suitable for a much wider range of reactions. Nevertheless, in order to obtain DES minimally destructive to protein, it is critical to mix both components in a proper molar ratio. The effect of various molar ratios was illustrated by Zhao et al. (2011a), who observed the highest lipase-catalysed conversion of miglyol in 1:1.5 ChAc:glycerol, while the 1:1 and 1:2 mixtures showed lower total conversions.

It is believed, that DESs may serve as a key solvents for the future industrial production. The transfer of knowledge from ILs to DESs represents a logical step in the area of medium engineering for biocatalysis. Deep eutectic solvents might contribute to the economically feasible, sustainable and efficient biocatalytic processes.

Table 16.7 Examples of reactions catalysed by various hydrolases in the presence of DESs


Esterase (EC 3.1.1.1)

Pseudomonas fluorescens, Rhizopus oryzae, Pig liver

10% (v/v) ChCl:Gly (1:2)

Hydrolysis of p-nitrophenyl

Moderately increased reaction rates

Gorke et al. (2008)

609


Lipase (EC 3.1.1.3)

CALA, CALB, Burkholderia cepacia

ChCl:Acet (1:2)ChCl:Gly (1:2) ChCl:U (1:2) EACl:Acet (1:1.5) EACl:Gly (1:1.5)

Transesterification of ethyl valerate with 1-butanol

Conversions similar or higher than those in toluene or ILs

Gorke et al. (2008)

CALB ChCl:Gly (1:2)ChAc:Gly (1:1.5) ChCl:U (1:2)

Transesterification of miglyol

The best combination of high activity and selectivity in ChAc:Gly

Zhao et al. (2011a)

CALB 70% (v/v) ChCl:Gly (1:2) in methanol

Transesterification of soybean oil

88% triglyceride conversion in 24 h

Zhao et al. (2013)

Rhizopus oryzae

ChCl:U (1:2) Synthesis of dihydro-pyrimidines

High efficiency and selectivity

Borse et al. (2012)

Epoxide hydrolase (EC 3.3.2.10)

Agrobacterium radiobacter

25% (v/v) ChCl:Gly (1:2)

Conversion of styrene oxide to styrene glycol

Activity 20-fold higher than in buffer alone

Gorke et al. (2008)

Potato 20–60% (v/v) ChCl:Gly (1:2)ChCl:EG (1:2)ChCl:U (1:2)

Hydrolysis of methylstyrene oxide

The least influenced catalysis in ChCl:Gly, but slightly altered regioselectivity

Lindberg et al. (2010)

α-chymotrypsin (EC 3.4.21.1)

Bovine 97% (v/v)ChCl:Gly (1:2)ChAc:Gly (1:1.5)

Transesterification of N-acetyl-L-phenyl alanine ethyl ester with 1-propanol

Lower activity Zhao et al. (2011b)

Subtilisin (EC 3.4.21.62)

Bacillus licheniformis

97% (v/v)ChCl:Gly (1:2)ChAc:Gly (1:1.5)

Transesterification of N-acetyl-L-phenyl alanine ethyl ester with 1-propanol

High activity and selectivity

Zhao et al. (2011b)

CALA, Candida antarctica lipase A; CALB, Candida antarctica lipase B; ChCl, choline chloride; ChAc, choline acetate; EACl, ethylammonium chloride; Acet, acetamide; EG, ethylene glycol; Gly, glycerol; U, urea.

Biocatalysis in Deep Eutectic Solvents


16.5 Supercritical Fluids

A supercritical fluid (sc-fluid) is defined as the state of a compound or an element above its critical temperature and critical pressure, but below the pressure required to condense it into a solid state. In the supercritical region, the densities of fluids are comparable to those of liquids, while the viscosities are comparable to those of gases (Hobbs and Thomas, 2007). Liquid-like densities and dissolving power let the sc-fluid to work as an effective reaction solvent. Diffusion is typically faster in sc-fluids than in water, which can speed up the diffusion-limited reactions. Another key feature of sc-fluids is pressure-tunability of parameters such as dielectric constant, partition coefficient and solubility, allowing their rational control (Cantone et al., 2007).

Since 1980’s, the use of sc-fluids as non-aqueous reaction media for enzymatic processes has been an area of active research (Table 16.8). The advantages of using sc-fluids in the enzymatic reactions include non-toxicity, non-flammability, easy removal of the solvents by post-reactional depressurisation, the ability to dissolve hydrophobic compounds and good control of enzyme selectivities by simply changing the pressure and the temperature (Housaindokht et al., 2012; Matsuda et al., 2003). Sc-fluids are also attractive for their swelling capability on cellulose pretreatment, as reported for example, by Nishino et al. (2011) and Gremos et al. (2012). The use of enzymes in combination with sc-fluid as an impregnation factor and reaction medium make the esterification of cellulose a green procedure. The range of sc-fluids used as a solvent for enzyme-catalysed reactions is relatively small due to the tendency of proteins to unfold at the elevated temperatures. The vast majority of reactions employed sc-CO2, which is cheap, chemically inert and non-toxic with relatively low critical parameters (Tc = 31°C, p = 74 bar). On the other hand, Cantone et al. (2007) reported sc-CO2 as a solvent with potentially strong deactivating effect on enzymes. Therefore, current research has shifted to other sc-fluids better suited to act as a reaction medium for biocatalytic reactions, such as sc-ethane, sc-propane or sc-butane. For instance, the transesterification activity of immobilized cutinase was found out by Garcia et al. (2004) to be one order of magnitude higher in sc-ethane than in sc-CO2.

611

Tabl

e 16

.8

Exam

ples

of r

eact

ions

cata

lyse

d by

var

ious

hyd

rola

ses i

n th

e pr

esen

ce o

f sc-

fluid

s and

fluo

rous

solv

ents

Bio

cata

lyst

R

eact

ion

med

ium

Rea

ctio

nEf

fect

Ref

.

Lipa

se (

EC 3

.1.1

.3)

CALB

[BM

IM][

Tf2N

] and

sc-C

O 2

as th

e m

obile

pha

seAc

ylat

ion

of o

ctan

-1-o

l by

viny

l ac

etat

eEn

zym

e/IL

mix

ture

reus

ed

man

y tim

es w

ithou

t los

s of

enzy

mat

ic a

ctiv

ity

Reet

z et

al.

(200

2)

CALB

Sc-e

than

e, sc

-CO 2

Tran

sest

erifi

catio

n of

2-p

heny

l-1-

prop

anol

by

viny

l but

yrat

eH

igh

activ

ity d

etec

ted

in b

oth

sc-fl

uids

Garc

ia e

t al.

(200

4)

CALB

Sc-C

O 2Sy

nthe

sis o

f iso

amyl

ace

tate

Cont

inuo

us o

pera

tion

for o

ne

mon

th

Rom

ero

et a

l. (2

005)

CALB

Perf

luor

ohex

ane/

met

hano

l bip

hasi

c sys

tem

Este

rific

atio

n of

1-p

heny

leth

anol

w

ith h

ighl

y flu

orin

ated

acy

l don

orFa

cile

sepa

ratio

n of

pro

duct

sH

unge

rhoff

et

al. (

2001

)

CALB

, Pse

udom

onas

ae

rugi

nosa

,Ps

eudo

mon

as c

epac

ia, C

andi

da

rugo

sa, R

izom

ucor

mie

hei

Sc-C

O 2Ac

etyl

atio

n of

1-(

p-ch

loro

phen

yl)-

2,2,

2-tr

ifluo

roet

hano

l with

vin

yl

acet

ate

Hig

her y

ield

and

ster

eosp

ecifi

c-ity

at l

ow p

ress

ure

and

low

te

mpe

ratu

re th

an a

t hig

h pr

essu

re a

nd h

igh

tem

pera

ture

Mat

suda

et a

l. (2

003)

Alca

ligen

ce sp

. Pe

rflu

oroo

ctan

e/is

ooct

ane

and

perf

luor

ohex

ane/

isoo

ctan

e bi

phas

ic

syst

ems

Alco

holy

sis b

etw

een

cinn

amat

e an

d be

nzyl

alc

ohol

Hig

her a

ctiv

ity o

f the

PEG

-lipa

se

com

plex

in fl

uoro

us so

lven

ts

than

in o

rgan

ic so

lven

ts

Mar

uyam

a et

al.

(200

4)

Supercritical Fluids

(Con

tinu

ed)


Bio

cata

lyst

R

eact

ion

med

ium

Rea

ctio

nEf

fect

Ref

.

Rhiz

opus

ory

zae

Sc-C

O 2Sy

nthe

sis o

f citr

onel

lol e

ster

by

tran

sest

erifi

catio

nH

igh

yiel

d w

ith im

mob

ilize

d en

zym

eDh

ake

et a

l. (2

011)

Mix

ture

of C

andi

da r

ugos

a,

Rhiz

opus

ory

zae

Sc-C

O 2/w

ater

(10%

v/v

)/su

pply

of m

etha

nol

(90

mm

ol p

er 0

.75

h)

Biod

iese

l pro

duct

ion

In b

atch

reac

tion

unde

r opt

imal

co

nditi

ons,

the

yiel

d w

as 9

9.9%

at

2 h

Lee

et a

l. (2

011)

Cand

ida

rugo

saPe

rflu

oroh

exan

e/he

xane

bi

phas

ic sy

stem

Este

rific

atio

n of

2-m

ethy

lpen

tano

ic

acid

with

fluo

rina

ted

alco

hol

Faci

le se

para

tion

of p

rodu

cts

Beie

r and

O’

Hag

an (2

002)

Bur

khol

deri

a ce

paci

aSc

-CO 2

Este

rific

atio

n be

twee

n st

erol

s and

fa

tty

acid

sCo

ntin

uous

hig

h-yi

eld

(99%

) pr

oduc

tion

King

et a

l. (2

001)

Bur

khol

deri

a ce

paci

a PF

MC/

hexa

ne b

ipha

sic

syst

emEs

teri

ficat

ion

of 1

-phe

nyle

than

ol

and

viny

l ace

tate

Hig

h st

abili

ty, s

tere

ospe

cifi-

city

and

mul

tiple

reco

very

of

lipas

e-Kr

ytox

com

plex

Ship

ovsk

ov

(200

8)

Pect

in m

ethy

lest

eras

e (E

C 3.

1.1.

11)

Vale

ncia

ora

nge

Sc-C

O 2De

met

hyla

tion

of p

ectin

Decr

ease

in e

nzym

e ac

tivity

w

ith in

crea

sing

pre

ssur

eZh

ou e

t al.

(200

9)

Cuti

nase

(EC

3.1

.1.7

4)

Fusa

rium

sol

anip

isi

Sc-e

than

e, sc

-CO 2

Tr

anse

ster

ifica

tion

of 2

-phe

nyl-1

-pr

opan

ol b

y vi

nyl b

utyr

ate

Low

er a

ctiv

ity d

eter

min

ed in

sc

-CO 2

than

sc-e

than

eGa

rcia

et a

l. (2

004)

PFM

C, p

erflu

oro(

met

hylc

yclo

hexa

ne).

Tabl

e 16

.8

(Con

tinu

ed)

613

Reactor design is an important feature of the processes employing the sc-fluids. Continuous flow processes are preferred because of practical and technical advantages, such as the improvement of mass and heat transfer, possibility to perform virtually solventless reactions, or easier scale-up of the supercritical processes.

The use of sc-CO2 flow reactor (Fig. 16.3) for large-scale kinetic resolution of alcohols by lipase improved the yield of the optically active compounds over 400-fold compared to the corresponding batch reaction using sc-CO2 (Matsuda et al., 2004). The biocatalyst maintained its performance in terms of the reactivity and selectivity under supercritical conditions (13 MPa at 42°C) for 3 days.

Figure 16.3 Reaction apparatus for continuous lipase-catalysed kinetic resolution of alcohols using scCO2-flow reactor. The scheme was reproduced from Matsuda (2013).

Besides acting as reaction media, sc-fluids can also find use as the extraction agents. Combination of kinetic resolution in ILs and selective extraction with sc-fluids provides a new approach to asymmetric synthesis (Fan and Qian, 2010). Using this approach, sc-CO2 can serve both to transport the substrate to IL phase containing the biocatalyst and to extract the products form IL. CO2 readily dissolves in the liquid phase of the vast majority of ILs, reducing their viscosity, while IL remains insoluble in the CO2 vapour phase (Fan and Qian, 2010). This improves the mass transfer of the IL/sc-CO2 system. Another variation of the supercritical extraction is the use of sc-CO2 to separate IL and an organic solvent.

Supercritical Fluids


For instance, MeOH and [BMIM][PF6] that are completely miscible at ambient conditions, form three phases in the presence of sc-CO2 (Blanchard and Brennecke, 2001). This behaviour can be exploited in all biocatalytic processes leading to an alcohol as a by-product.

Study of biocatalysis in the sc-fluids has just begun due to the strong concern about the natural environment. Promising examples of the large-scale asymmetric synthesis by hydrolytic enzymes in sc-CO2 or IL/sc-CO2 have been already described (Matsuda et al., 2004; Fan and Qian, 2010). On the other hand, the intrinsic limitation is the cost of the equipment for sc-fluids production and manipulation. Therefore, the development of continuous-flow catalytic systems in which fluids can be recycled is clearly needed for their commercial applications.

16.6 Fluorous Solvents

Fluorous solvents are another class of low polarity recyclable green solvents with a wide range of industrial applications. Their wide spread use is particularly due to a high chemical and thermal stability, originating from the strength and low polarizability of C–F bonds. Fluorous solvents form at low temperatures biphasic systems with most organic solvents and become completely miscible at a certain temperature (Hildebrand and Cochran, 1949; Lozano, 2010). The catalytic reaction can then occur in homogenous system and at the end of the reaction the fluorous-soluble compounds can be separated from organic ones by simple recooling of the reaction mixture.

Biocatalysis in the presence of fluorous solvents is an active area of research, thus only few examples have been described to date (Table 16.8). In one of the pioneering studies, Beier and O’Hagan (2002) reported lipase-catalysed kinetic resolution of 2-methylpentanoic acid with highly fluorinated decanol in biphasic perfluorohexane-hexane system. The acid substrate was dissolved in hexane, while fluorinated alcohol was dissolved in the fluorous phase. The reaction was initiated by warming to 40°C leading to the one-phase formation, followed by the enzyme addition. At the end of the reaction, the biocatalyst was separated by filtration and the reaction mixture was recooled to 25°C. An important problem of this strategy is the necessity to use an enzyme soluble

615

in the fluorous solvent; otherwise the reuse of the biocatalyst cannot be carried out. The enhanced solubilisation of enzymes in perfluoromethylcyclohexane (PFMC) by using the anionic surfactant perfluoropolyether carboxylate (Krytox) has been reported by Shipovskov (2008). Krytox interacts by hydrophobic ion pairing with basic amino acid residues on the protein surface, resulting in a complex that can easily be extracted into PFMC. The formation of a non-covalent complex between Burkholderia cepacia lipase and the surfactant Krytox was shown to increase the solubilisation of the enzyme in PFMC, and thus promote its operation in the PFMC/hexane biphasic system.

The application of enzymes in fluorous solvents, although being a recent topic with only a handful of examples published, is an attractive field and further research will certainly be of great interest. Particularly, the combination of fluorous phases with sc-fluids or ILs is currently being explored. Nevertheless, doubts over the persistence of flourinated solvents in the environment are still a topic of debate. The negative aspects that prevent fluorochemicals to be considered as environment-friendly solvents are the following: (i) synthesis involving large quantities of fluorine and hydrogen fluoride, (ii) high persistence in the environment, (iii) the accumulation in the atmosphere, and (iv) bioaccumulation (Marques et al., 2012). Moreover, industrial interest in fluorous solvents is limited due to their high cost. The possible solution is the replacement of conventional perfluorinated compounds by less toxic and more cost-effective hydrofluoroethers.

16.7 Case Study: Haloalkane Dehalogenases in the Presence of Organic Co-Solvents

Recent investigations of the effect of organic solvents on structure-function relationships of haloalkane dehalogenases (HLDs, EC 3.8.1.5) demonstrate how different reaction media influence catalytic be-haviour of these hydrolytic enzymes. Haloalkane dehalogenases convert halogenated compounds to the corresponding alcohols, halides and protons. The hydrolytic cleavage of a carbon-halogen bond proceeds by the SN2 mechanism, followed by the addition of water, which is the only co-factor required for catalysis. The set of substrates converted by HLDs consists of haloalkanes, cy-

Case Study


clohaloalkanes haloalkenes, haloalcohols, halohydrins, haloethers, haloesters, haloamides and haloacetonitriles (Damborsky et al., 2001; Westerbeek et al., 2011). The broad substrate specificity to-gether with relatively high robustness makes the members of HLD family attractive for both academic research and practical applica-tions (Koudelakova et al., 2013). Biodegradation is one of the most promising fields for application of HLDs. The enzymes have already been successfully used for bioremediation of 1,2-dichloroethane and hexachlorocyclohexane and to neutralize sulphur mustard (Lal et al., 2010; Prokop et al., 2006; Stucki and Thueer, 1995). Besides, both the substrates, e.g., haloalkanes or haloamides, and the products, e.g., haloalcohols, alcohols, diols or hydroxyamides, of HLDs are valuable building blocks in organic and pharmaceutical synthesis, making this group of enzymes attractive for biocatalysis (Mozga et al., 2009; Patel, 2001).

The broader use of haloalkane dehalogenases is limited by poor solubility of their substrates in water, evoking the necessity of introduction of organic co-solvents to the reaction media. In an effort to choose the most compatible organic co-solvent for HLDs, the effects of 14 co-solvents on activity, stability and enantioselectivity of three model enzymes—DbjA from Bradyr-hizobium japonicum USDA110, DhaA from Rhodococcus rhodo-chrous NCIMB13064, and LinB from Sphingobium japonicum UT26—were systematically evaluated by Stepankova et al. (2013a). All co-solvents caused at high concentration loss of activity and conformational changes. The highest inactivation was induced by tetrahydrofuran, while more hydrophilic co-solvents, such as ethylene glycol, methanol and dimethyl sulphoxide, were more tolerated (Fig. 16.4). Surprisingly, the effects of co-solvents at low concentration were different for each enzyme-solvent pair. The increase in DbjA activity was induced by the majority of organic co-solvents tested, while activities of DhaA and LinB decreased at comparable concentrations of the same co-solvent. Moreover, significant increase of DbjA enantioselectivity was observed. Ethylene glycol and 1,4-dioxane were shown to have the most positive impact on the enantioselectivity. The E-value increased from 132 in pure buffer to more than 200. The favourable influence of the co-solvents on both activity and enantioselectivity makes DbjA the most suitable for biocatalytic applications.

617

Figure 16.4 The relative activities of DbjA (green), DhaA (blue) and LinB (yellow) in the presence of different concentrations of organic co-solvents. The relative activities were measured at 37°C and are expressed as a percentage of the specific activity in glycine buffer (100 mM, pH 8.6). The specific activities (in µmol s–1 mg–1 of enzyme) of DbjA, DhaA and LinB in glycine buffer were 0.0213, 0.0355 and 0.0510, respectively. DMF, dimethylformamide; DMSO, dimethyl sulphoxide; PEG, polyethylene glycol; THF, tetrahydrofuran. Reprinted from Stepankova et al. (2013a).

Case Study


Even though the loss of HLD activity at high co-solvent concentrations was attributed to the alterations in enzyme secondary structure, the spectroscopic data could not explain observed changes in enzyme activity under non-denaturing co-solvent concentrations. To gain an insight into the mechanisms, the behaviour of the same haloalkane dehalogenases in the presence of three representative organic co-solvents—acetone, formamide and isopropanol—was investigated by employing molecular dynamics simulations, time-resolved fluorescence spectroscopy and steady-state kinetic measurements (Stepankova et al., 2013b). A generally applicable computational method, involving molecular dynamics simulations and quantitative analysis of co-solvent occupancies inside the access tunnels and active sites, was developed to aid the selection process for an appropriate organic co-solvent. It was revealed that the inhibition of the enzymes correlates with the expansion of the active-site pockets and their occupancy by co-solvent molecules. Two different mechanisms of co-solvent induced inhibition were identified. First, enzyme inactivation correlated with increased substrate inhibition, which was not sufficiently compensated by an increase in substrate binding to the free enzyme. Molecular dynamics simulations revealed that this mechanism is coupled to the most extensive occupancy of the active site by the organic solvent, accompanied by significant dehydration of the protein cavity (Fig. 16.5).

Second, diminishing enzyme activity was due to a reduction in the catalytic constant. Stepankova et al. (2013b) observed this mechanism for cases where organic solvent molecules predominantly occupied the access tunnel, connecting the active site cavity with the surrounding environment, causing either substrate entry or product release to be impaired. Moreover, organic co-solvent molecules affect the volume and geometry of the active site pockets to different extents (Fig. 16.6). The big changes in the volume and geometry of the pocket were observed for LinB, followed by DhaA. On the contrary, no enlargement of the pocket was observed for DbjA, which is a possible explanation why DbjA exhibited the highest co-solvents resistance in the experiments. The newly developed algorithms enable calculation of the volumes of active-site pockets and their occupancy by the co-solvent

619

molecules. These algorithms can be used to analyse trajectories from molecular dynamics simulations and study the effect of water- organic co-solvent mixtures on enzyme catalytic performance.

Figure 16.5 Relative solvation of DbjA (green), DhaA (blue) and LinB (yellow) is determined as the ratio of the volume of the access tunnel or active site occupied by co-solvent molecules to the total volume of the access tunnel or active site. Reprinted from Stepankova et al. (2013b).

Case Study


Figure 16.6 Representative geometries of DbjA (green), DhaA (blue), and LinB (yellow) active-site pockets obtained from 35 ns molecular dynamics simulations in water or organic co-solvents. Only the protein surface and the active-site pockets are shown for clarity. The values given are the average volumes of the active-site pockets calculated over 4000 snapshots. Reprinted from Stepankova et al. (2013b).

16.8 Concluding Remarks

The application of hydrolases in non-conventional reaction media at laboratory and industrial scale represent an area of active research and development. Two obvious reasons underlying the need for non-aqueous media are the low water solubility of many substrates and demanding downstream processing. Moreover, using existing enzymes in non-conventional reaction media may uncover unexploited biocatalytic activities, and thus extend the repertoire of enzyme-catalysed transformations. Organic solvents represent the most commonly used non-conventional media for biocatalysis. However, their possible drawback is a negative effect on enzyme activity. This is not surprising, since natural enzymes have evolved over millions of years to work in a water environment of living cells. Therefore, they often sustain lower concentrations

621

of an organic solvent than is required for industrial processes. Presented case study with the haloalkane dehalogenases de-monstrates that the effect of organic solvents on enzyme structure and function can be mechanistically explained at the molecular level and mathematically modelled. Compatible enzyme-solvent pairs can be rationally selected based on molecular dynamics simulations using the newly developed algorithms analysing the protein-solvent interactions.

Another aspect that needs to be taken into account for the organic solvents is their explosive and environmentally hazardous nature. Growing environmental concerns favour the replacement of organic solvents by more benign solvents: ILs, DESs, sc-fluids and fluorous solvents. Their unique properties, such as non-flammability, thermal stability, easy recycling and dissolving power, together with the possibility to further tailor these properties by selection of appropriate components, pave the way to many new biotransformation processes that previously required the use of traditional organic solvents. Several enzymatic reactions have already been reported in these systems and many others may be on the way. We expect that these environmentally more benign solvents are likely to replace traditional solvents in many industrial applications in the next few decades. The speed at which these new solvents will be put into practice may depend on the results of research focused on compatibility with enzymes, ecological footprint and economical viability.

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