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Chapter 16
Hydrolases in Non-Conventional Media:
Implications for Industrial Biocatalysis
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 Republic
bEnantis, s.r.o., Palackeho trida 1802/129, 61200 Brno, Czech Republic
radka@chemi.muni.cz.
16.1 Introduction
Agrochemical and pharmaceutical industries are urged by environmental regulators to implement sustainable technologies for the
production of enantiomerically pure and value-added compounds.
The use of biocatalysts represents a good solution. The enzymecatalysed reactions often show advantages over the uncatalysed
reactions, including high chemo-, regio- and enantioselectivities
and occurrence under mild reaction conditions. However, the conventional 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,
Industrial Biocatalysis
Edited by Peter Grunwald
Copyright © 2015 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4463-88-1 (Hardcover), 978-981-4463-89-8 (eBook)
www.panstanford.com
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 media 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 identifying 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 biodegradability 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
Solvents
Water
Advantages and disadvantages of various reaction media for
biocatalysis
Advantages
Disadvantages
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
Biocatalysis 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 hydrolysis, suppression of water-induced
side reactions, non-volatile,
thermally stable, enhanced
enzyme enantioselectivity,
designer solvents
Deep
eutectic
solvents
Good solvent for polar substrates High viscosity,
insufficient knowledge
and metal salts, favoured
about their properties
synthesis over hydrolysis,
suppression of water-induced
side reactions, non-volatile,
thermally stable, biodegradable,
easy to prepare, cheap
Supercritical Good solvent for highly
fluids
hydrophobic substrates,
non-toxic, non-flammable,
easy to remove after the
reaction, high diffusion rates,
pressure-tunability of parameters
Fluorous
solvents
Limited data regarding
toxicity, high cost,
energy demanding
synthesis, high viscosity
Good solvent for hydrophobic
substrates, high chemical and
thermal stability, easily recyclable,
temperature-dependent
miscibility with organic
solvents
Use of high pressures
requiring special
reactors, high risk of
enzyme unfolding,
potentially hazardous
Doubts about the
persistence in the
environment, large
quantities of fluorine
and hydrogen fluoride
used in the synthesis
16.2 Biocatalysis in Organic Solvents
The beginning of detailed investigations of enzymes in organic
solvents can be tracked back to the 1980s, when Klibanov and
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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
Biocatalyst
Examples of reactions catalysed by various hydrolases in the
presence of organic solvents
Solvent
Reaction
Effect
Ref.
Turkey pharynx 20–40% (v/v) Esterification of
2-propanol
tributyrin
Higher activity
at lower
temperatures
Cherif and
Gargouri
(2010)
CALB
Enantiomer
excess up to
99% in hexane
and benzene
Raminelli
et al. (2004)
The increase
of enantioselectivity
depending on
the substrate
Ammazzalorso
et al. (2008)
Esterase (EC 3.1.1.1)
Lipase (EC 3.1.1.3)
CALB
THF, diethyl
ether, hexane,
benzene
tert-butanol,
acetone
Candida rugosa 0–80%
(v/v) DMSO,
isopropanol
Rhizomucor
miehei
Esterification of
alcohols
Hydrolysis of
E-value raised
butanoate of 3from 7 to more
chloro-1-(phenyl- than 200
methoxy)-2propanol
Hydrolysis of
β-substituted
aryloxyacetic
esters
5–90% (v/v) Hydrolysis of
DEE, DMF,
esters
DME, 1,4dioxane, DMSO
High
activity with
hydrophobic
substrates
Hansen T. V.
et al. (1995)
Tsuzuki
et al. (2003)
Biocatalyst
Solvent
Reaction
Effect
Ref.
Alcaligenes sp.
DCM, hexane,
decaline,
acetone
cyclohexane,
THF, 1,4dioxane
Kinetic resolution
of sec-alcohols
through transesterification
High enantioWang et al.
selectivity in
(2009)
small molecularsized solvents
35% (v/v)
DMSO, DMF,
acetone,
1,4-dioxane,
THF,
1-propanol
Hydrolysis of
1-chloro-2acetoxy-3-(1naphthyloxy)propane
Toluene,
hexane
isooctane,
isopropanol
Transesterification of
sec-alcohols
Bacillus subtilis 10–30% (v/v) Hydrolysis of
THF, acetone, glycidol butyrate
acetonitrile,
1,4-dioxane,
DMF, DMSO
16-fold higher
E-value in
18% (v/v) 1,4dioxane (5°C)
than in buffer
(25°C)
Li (2008)
Carica papaya
The highest
activity and
enantioselectivity
in hexane
Cheng and
Tsai (2004)
Pseudomonas
cepacia
Wheat germ
TCM, hexane,
cyclohexane,
decane,
isooctane
Esterification
of 2-(4-chlorophenoxy)propionic acid
Feruloyl esterase (EC 3.1.1.73)
Aspergillus
niger,
Neurospora
crassa,
Talaromyces
stipitatus
The highest
activity and
enantioselectivity in
DMSO and THF
Mohapatra
and Hsu
(1999)
The highest
enantioselectivity
in hexane
Xia et al.
(2009)
Faulds et al.
(2011)
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
50% (v/v)
acetone, EEE,
acetonitrile,
MEA, pyridine
monoglyme,
diglyme, TMP,
Hydrolysis of
2-nitrophenylβ-D-galactopyranoside
20–60% of
Yoon and
enzymatic
Mckenzie
activity retained (2005)
in the presence
of most of
organic solvents
β-Galactosidase (EC 3.2.1.23)
Escherichia coli,
Kluyveromyces
fragilis,
Aspergillis
oryzae
(Continued)
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Table 16.2
Biocatalyst
(Continued)
Solvent
Reaction
Subtilisin (EC 3.4.21.62)
Bacillus
amyloliquefaciens
TCM, toluene,
butyl
acetate, THF,
acetonitrile,
DMF
50% (v/v)
DMF
Pepsin (EC 3.4.23.1)
Porcine
pancreas
Transesterification of
trifluoroacetylDL-phenylalanine
2,2,2-trifluoro
ethyl ester
The highest
activity and
enantioselectivity in
acetonitrile
Kawashiro
et al. (1997)
The activity
preserved up
to 60% (v/v)
acetonitrile
and ethanol
Simon et al.
(2007)
Kidd et al.
Ester and
Improved
(1999)
amide hydrolysis aminolysis
and suppressed
hydrolysis
in DMF
10–90% (v/v) Hydrolysis of
haemoglobin
acetonitrile,
1,4-dioxane,
ethanol
Pseudolysin (EC 3.4.24.26)
Pseudomonas
aeruginosa
10–90% (v/v) Synthesis of
High synthetic
peptide
methanol,
rates in organic
Cbz-Arg-Leu-NH2 co-solvents
DMF, DMSO
Thermolysin (EC 3.4.24.27)
Bacillus
thermoproteolyticus
0–20% (v/v)
DMSO, DMF,
n-propanol,
isopropanol
Ogino et al.
(2000)
Pazhang et
al. (2006)
Hydrolysis of
casein
Decreased
activity and
thermostability
Hydrolytic
cleavage of
C-C bond in
β-diketones
Siirola et al.
Excellent
(2011)
activity
tolerance
towards organic
solvents
β-Diketone hydrolase (EC 3.7.1.7)
Rhodococcus sp., 20–80% (v/v)
Anabaena sp.
EG, acetone,
1,4-dioxane,
glycerol,
acetonitrile,
THF
Ref.
used, no activity
observed in
1,4-dioxane,
DMSO, DMF
and pyridine
triglyme,
DMSO,
tetraglyme,
1,4-dioxane,
DMF
Bacillus
licheniformis,
Bacillus
amyloliquefaciens
Effect
Biocatalyst
Solvent
Reaction
Effect
Ref.
Glycerol, EG,
PEGs, DMSO
and methanol
well-tolerated
by enzymes
Stepankova
et al.
(2013a)
Haloalkane dehalogenase (EC 3.8.1.5)
Bradyrhizobium
japonicum,
Rhodococcus
rhodochrous,
Sphingobium
japonicum
5–75% (v/v) Hydrolytic
glycerol, PEGs, dehalogenation
EG, formamide, of 1-iodohexane
DMF,
methanol,
ethanol,
acetone,
1,4-dioxane,
isopropanol,
THF, DMSO,
acetonitrile
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
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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
Company
Examples of processes involving lipase-catalysed reactions in
organic media developed by several chemical and pharmaceutical companies
BASF
BASF
Chemie linz
Process
Ref.
Synthesis of various enantiomerically pure
alcohols, used as intermediates for synthesis
of chemicals and pharmaceuticals, by
asymmetric (trans)esterification
Schmid
et al. (2001)
Synthesis of polyol acrylates, used to prepare
pigment dispersions, by reaction of aliphatic
polyol with an acrylic acids
Synthesis of enantiomerically pure 2Klibanov
halopropionic acids, used as intermediates for and Kirchner
synthesis of herbicides and pharmaceuticals, (1986)
by asymmetric esterification
Schering-Plough Synthesis of antifungal agent involving
desymmetrization of 2-substituted-1,3propanediol
Bristol-Myers
Squibb
Bristol-Myers
Squibb
Paulus
et al. (2003)
Synthesis of enantiomerically pure
α-(3-chloropropyl)-4-fluorobenzenemethanol, an intermediate for the synthesis
of an antipsychotic agent
Asymmetric acetylation of (1α,2β,3α)-2(benzyloxy methyl)-cyclopent-4-ene-1,3diol to the corresponding monoacetate, a
key intermediate for the synthesis of an
angiotensin-converting enzyme inhibitor
Klibanov
(2001)
Hanson
et al. (1994)
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
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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 crosslinking 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).
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-hydroxy2-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
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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
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 cosolvent-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 watersoluble 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
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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 enantioselectivity. 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 subtilisincatalysed 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
Biocatalysis in Ionic Liquids
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 secondgeneration 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.
597
1-Ethyl-3-methylimidazolium
1-Decyl-3-methylimidazolium
Tetrafluoroborate
1-Butyl-3-methylimidazolium
Yes
[Bmim]I
Yes
Yes
Yes
[Emim][CH3COO]
[Emim]Cl
Acetate
Chloride
No
[Emim][Tf2N]
Yes
[Emim][BF4]
[Dmim]Cl
Yes
No
[Bmim][OctSO4]
[Bmim][Tf2N]
Yes
Yes
No
Bis[(trifluoromethyl)sulphonyl]imide
Tetrafluoroborate
Chloride
Octylsulphate
[Bmim]Cl
Iodide
Chloride
[Bmim][PF6]
Yes
[Bmim][BF4]
[Bzmim]Cl
Yes
Yes
Water miscibility
[Bzmim][BF4]
[Amim]Cl
Notation
Hexafluorophosphate
Chloride
Tetrafluoroborate
Chloride
1-Allyl-3-methylimidazolium
1-Benzyl-3-methylimidazolium
Anion
Examples of the second-generation ILs mentioned in this chapter
Cation
Table 16.4
598
Bis[(trifluoromethyl)-sulphonyl]imide
Triethylammonium
Methyltrioctylammonium
TEAP
TEAA
Phosphate
Acetate
[MTOA][Tf2N]
[CPMA][MS]
[BTMA][Tf2N]
[MEBu3P][Tf2N]
Yes
Yes
Yes
No
Yes
No
No
Yes
[EtPy][CF3COO]
Cocosalkyl pentaethoxy methylammonium Methosulphate
Butyltrimethylammonium
Trifluoroacetate
1-Ethylpyridinium
[Mmim][DMP]
Dimethylphosphate
Yes
No
[Mmim][MeSO4]
[Omim][Tf2N]
Methylsulphate
2-Methoxyethyl(tri-n-butyl)phosphonium
No
No
[Omim][PF6]
[MOEmim][PF6]
Part.
Yes
Yes
Yes
[MOEmim][BF4]
[Hmim]Cl
[Emiml[DEP]
[Emiml[DMP]
Hexafluorophosphate
Hexafluorophosphate
Tetrafluoroborate
Chloride
1,3-Dimethylimidazolium
1-Octyl3-methylimidazolium
1-Methoxyethyl3-methylimidazolium
1-Hexyl3-methylimidazolium
Diethylphosphate
Dimethylphosphate
599
Transesterification of methyl methacrylate
Burkholderia
cepacia
Pseudomonas sp.
Candida rugosa
Transesterification of secondary alcohols
Kinetic resolution of 1-phenylethanol
[MEBu3P][Tf2N]
[Bmim][Tf2N]
Hydrolysis of methyl ester of naproxen
Water-saturated
[Bmim][PF6]
Synthesis of butyl propionate
[Bmim][PF6]
Candida rugosa
Thermomyces
lanuginosus
[Bmim][PF6],
[Omim][PF6]
Resolution of tetrahydro-4-methyl-3-oxo1H-1,4-benzo diazepine-2-acetic acid
methyl ester
85–97% (v/v)
[Bmim][PF6]
Xin et al. (2005)
Kaar et al. (2003)
De Diego et al.
(2009)
Roberts et al.
(2004)
Kim et al. (2001)
De Diego
et al. (2009)
Madeira Lau
et al. (2000)
Ref.
Slightly faster reaction than in
diisopropyl ether
Abe et al. (2008)
Higher enantioselectivity than in MTBE Eckstein et al.
(2002b)
Enantioselectivity 6-fold higher than in
isooctane
Reaction rate 1.5-fold higher than in
hexane
High activity
Higher solubility of the substrate and
ability to operate at high temperatures
Higher enantioselectivity than in THF
and toluene
Transesterification of secondary alcohols
[Emim][BF4],
[Bmim][PF6]
CALB
CALB
[CPMA][MS]
Transesterification of ethyl butanoate and
Reaction rates comparable or better
octanoate, ammoniolysis of ethyl octanoate, than those observed in organic media
epoxidation of cylohexene
Increased activity and thermostability
Effect
[Bmim][PF6],
[Bmim][BF4]
Synthesis of butyl propionate
Reaction
CALB
CALB
Solvent
Examples of reactions catalysed by various hydrolases in the presence of ILs
Lipase (EC 3.1.1.3)
Biocatalyst
Table 16.5
600
15% (v/v)
[EtPy][CF3COO]
20% (v/v)
[Mmim][MeSO4]
Bacillus circulans
β-Galactosidase (EC 3.2.1.23)
5–20% (v/v)
[Emim][DMP],
[Mmim] [DMP]
20% (v/v)
[Amim]Cl,
[Bmim]Cl,
[Emim]Cl
Hydrolysis of α-cellulose
Hydrolysis of starch
Hydrolysis of various esters of amino acids
Synthesis of
N-acetyllactosamine by transglycosylation
Hydrolysis of p-nitrophenyl β-Dxylopyranoside
10% (v/v) [Mmim] Hydrolysis of α-cellulose
[DMP], [Bmim]Cl,
[Amim]Cl,
[Emim]
[CH3COO]
Anoxybacillus sp.
Xylanase (EC 3.2.1.8)
Halorhabdus
utahensis
Trichoderma
reesei
Cellulase (EC 3.2.1.4)
Bacillus amylolique- 5–40% (v/v)
faciens, Bacillus
[Bmim]Cl,
lichiniformis
[Hmim]Cl
α-Amylase (EC 3.2.1.1)
Porcine pancreatic
The suppression of the secondary
hydrolysis
Slight increase of activity
Activity unchanged or slightly
stimulated
Decrease of activity
Decrease of activity and stability
High enantio-selectivity
(Continued)
Kaftzik et al.
(2002)
Thomas et al.
(2011)
Zhang et al.
(2011)
Engel et al.
(2010)
Dabirmanesh
et al. (2011)
Malhotra and
Zhao (2005)
601
Solvent
(Continued)
[Emim]
[Tf2N]
Hydrolysis of p-nitrophenyl butyrate
Higher activity than in toluene
Nakashima et al.
(2006)
Liu et al. (2005)
Lou et al. 2006b)
CALB, Candida antarctica lipase B; MTBE, methyl tert-butyl ether; TEAA, triethylammonium acetate; TEAP, triethylammonium phosphate; TBPBr,
tetrabutylphosphonium bromide; THF, tetrahydrofuran.
Bacillus
licheniformis
Increase of substrate solubility and
enantioselectivity
Higher activity than in ethyl acetate and Eckstein et al.
MTBE
(2002a)
Transesterification of N-acetyl-L-phenylalanine ethyl ester
80% (v/v) [Bmim] Hydrolysis of hydroxyphenylglycine
[BF4]
methyl ester
[Bmim]
[Tf2N]
Carica papaya
Papain (3.4.22.2)
Bovine
Attri et al. (2011)
Yang et al. (2012)
Thomas et al.
(2011)
Ref.
The strongest stabilization in triethyl
ammonium salts
Yields enhanced between 0.2-fold and
0.5-fold
Sharp decrease of activity at
concentrations above 10% (v/v)
Effect
Hydrolysis of Suc–Ala–Ala–Pro–Phe–pnitroanilide
50% (v/v) TEAA,
TEAP, TBPBr,
[Bzmim]Cl,
[Bzmim][BF4]
Bovine
α-Chymotrypsin (EC 3.4.21.1)
Mixture of [Bmim]I, Synthesis of arylalkyl β-D-glucopyranosides
ethylene glycol and via reverse hydrolysis
diacetate
Hydrolysis of p-nitrophenyl β-D-glucopyranoside
Reaction
Prune seed meal
Volvariella volvacea 5–20% (v/v)
[Emim][CH3COO],
[Emim][DEP],
[Emim][DMP],
[Mmim][DMP]
Biocatalyst
Table 16.5
602
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 largescale 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 nonpolar 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 nonaqueous 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).
603
604
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 αchymotrypsin 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-butyl3-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 wholecell biocatalysis as shown by Pfruender et al. (2004) and WeusterBotz (2007). The membrane integrity of the Escherichia coli,
Lactobacillus kefir and Saccharomyces cerevisiae as well as the
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
605
606
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).
Biocatalysis in Deep Eutectic Solvents
Table 16.6
Examples of the commercially available DESs
Component 1
Component 2
Urea
Choline
chloride
&+
+2
1
&+
+1
1
+
+1
+1
2
&
&
2
&
&
2
Ethylene ++2
+2
glycol
+2
&+
–
&O
Glycerol
Malonic
acid
Tf , Freezing point; Ruß and Konig (2012).
DES
2
2
+2
+2
+2
+2
2
2
&22
+2 &
+2 &&
++
22
1+
1+ Reline
1+
1+
2+
2+
2+
2+
Ratio Tf (°C)
1:2
Ethaline 1:2
2+
2+
Glyceline
2+ 2+
2+
2+
2+
2+
Maline
2
2
&22
& 2+
&
& 2+
2+
2+
12
–20
1:2
–40
1:1
10
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.
607
608
(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
Biocatalyst
Examples of reactions catalysed by various hydrolases in the
presence of DESs
Solvent
Reaction
Effect
Ref.
Hydrolysis of
p-nitrophenyl
Moderately
increased
reaction rates
Gorke
et al.
(2008)
Esterase (EC 3.1.1.1)
Pseudomonas
fluorescens,
Rhizopus
oryzae,
Pig liver
10% (v/v)
ChCl:Gly (1:2)
Biocatalysis in Deep Eutectic Solvents
Biocatalyst
Solvent
Reaction
Effect
Ref.
ChCl:Acet (1:2)
Transesterification
ChCl:Gly (1:2)
of ethyl valerate
ChCl:U (1:2) EACl: with 1-butanol
Acet (1:1.5) EACl:
Gly (1:1.5)
Gorke
et al.
(2008)
ChCl:Gly (1:2)
ChAc:Gly (1:1.5)
ChCl:U (1:2)
Transesterification
of miglyol
Conversions
similar or
higher than
those in
toluene or ILs
70% (v/v)
ChCl:Gly (1:2)
in methanol
Transesterification
of soybean oil
88%
triglyceride
conversion
in 24 h
Zhao
et al.
(2013)
Lipase (EC 3.1.1.3)
CALA, CALB,
Burkholderia
cepacia
CALB
CALB
Rhizopus
oryzae
ChCl:U (1:2)
Epoxide hydrolase (EC 3.3.2.10)
Agrobacterium 25% (v/v)
radiobacter
ChCl:Gly (1:2)
Potato
20–60% (v/v)
ChCl:Gly (1:2)
ChCl:EG (1:2)
ChCl:U (1:2)
α-chymotrypsin (EC 3.4.21.1)
Bovine
97% (v/v)
ChCl:Gly (1:2)
ChAc:Gly (1:1.5)
Bacillus
licheniformis
97% (v/v)
ChCl:Gly (1:2)
ChAc:Gly (1:1.5)
Synthesis
of dihydropyrimidines
Conversion of
styrene oxide to
styrene glycol
Hydrolysis of
methylstyrene
oxide
The best
Zhao
combination
et al.
of high activity (2011a)
and selectivity
in ChAc:Gly
High efficiency Borse
and selectivity et al.
(2012)
Activity 20-fold Gorke
higher than in et al.
buffer alone
(2008)
The least
Lindberg
influenced
et al.
catalysis in
(2010)
ChCl:Gly, but
slightly altered
regioselectivity
Transesterification Lower activity Zhao
of N-acetyl-L-phenyl
et al.
alanine ethyl ester
(2011b)
with 1-propanol
Transesterification High activity
Zhao
of N-acetyl-L-phenyl and selectivity et al.
alanine ethyl ester
(2011b)
with 1-propanol
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.
609
610
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.
Hungerhoff et
al. (2001)
Romero et al.
(2005)
Garcia et al.
(2004)
Reetz et al.
(2002)
Ref.
(Continued)
Alcoholysis between cinnamate and Higher activity of the PEG-lipase Maruyama et al.
benzyl alcohol
complex in fluorous solvents
(2004)
than in organic solvents
Alcaligence sp.
Perfluorooctane/
isooctane and
perfluorohexane/
isooctane biphasic
systems
Acetylation of 1-(p-chlorophenyl)2,2,2-trifluoroethanol with vinyl
acetate
Higher yield and stereospecific- Matsuda et al.
(2003)
ity at low pressure and low
temperature than at high
pressure and high temperature
Facile separation of products
Continuous operation for one
month
High activity detected in both
sc-fluids
Enzyme/IL mixture reused
many times without loss of
enzymatic activity
Effect
Sc-CO2
CALB, Pseudomonas
aeruginosa,
Pseudomonas cepacia, Candida
rugosa, Rizomucor miehei
CALB
Synthesis of isoamyl acetate
Transesterification of 2-phenyl-1propanol by vinyl butyrate
Acylation of octan-1-ol by vinyl
acetate
Reaction
Perfluorohexane/
Esterification of 1-phenylethanol
methanol biphasic system with highly fluorinated acyl donor
Sc-CO2
CALB
CALB
[BMIM][Tf2N] and sc-CO2
as the mobile phase
CALB
Sc-ethane, sc-CO2
Reaction medium
Examples of reactions catalysed by various hydrolases in the presence of sc-fluids and fluorous solvents
Lipase (EC 3.1.1.3)
Biocatalyst
Table 16.8
Supercritical Fluids
611
(Continued)
Sc-CO2
Valencia orange
Sc-ethane, sc-CO2
PFMC, perfluoro(methylcyclohexane).
Fusarium solanipisi
Cutinase (EC 3.1.1.74)
Pectin methylesterase (EC 3.1.1.11)
PFMC/hexane biphasic
system
Burkholderia cepacia
Perfluorohexane/hexane
biphasic system
Transesterification of 2-phenyl-1propanol by vinyl butyrate
Demethylation of pectin
Esterification of 1-phenylethanol
and vinyl acetate
Esterification between sterols and
fatty acids
Lower activity determined in
sc-CO2 than sc-ethane
Decrease in enzyme activity
with increasing pressure
High stability, stereospecificity and multiple recovery of
lipase-Krytox complex
Continuous high-yield (99%)
production
Esterification of 2-methylpentanoic Facile separation of products
acid with fluorinated alcohol
Sc-CO2
Candida rugosa
Burkholderia cepacia
High yield with immobilized
enzyme
Synthesis of citronellol ester by
transesterification
Garcia et al.
(2004)
Zhou et al.
(2009)
Shipovskov
(2008)
King et al.
(2001)
Beier and
O’Hagan (2002)
Dhake et al.
(2011)
Ref.
In batch reaction under optimal Lee et al.
conditions, the yield was 99.9% (2011)
at 2 h
Effect
Reaction
Sc-CO2/water (10% v/v)/ Biodiesel production
supply of methanol
(90 mmol per 0.75 h)
Sc-CO2
Reaction medium
Mixture of Candida rugosa,
Rhizopus oryzae
Rhizopus oryzae
Biocatalyst
Table 16.8
612
Supercritical Fluids
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.
613
614
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 scCO2 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
Case Study
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 scfluids 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 structurefunction relationships of haloalkane dehalogenases (HLDs, EC 3.8.1.5)
demonstrate how different reaction media influence catalytic behaviour 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-
615
616
clohaloalkanes haloalkenes, haloalcohols, halohydrins, haloethers,
haloesters, haloamides and haloacetonitriles (Damborsky et al.,
2001; Westerbeek et al., 2011). The broad substrate specificity together with relatively high robustness makes the members of HLD
family attractive for both academic research and practical applications (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 Bradyrhizobium japonicum USDA110, DhaA from Rhodococcus rhodochrous 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.
Case Study
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).
617
618
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 cosolvent 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 steadystate 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 cosolvent 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 cosolvent 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
Case Study
molecules. These algorithms can be used to analyse trajectories
from molecular dynamics simulations and study the effect of waterorganic 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).
619
620
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 activesite 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
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