catalysts

Review
Enzyme Stability and Activity in Non-Aqueous
Reaction Systems: A Mini Review
Shihui Wang 1,† , Xianghe Meng 2,† , Hua Zhou 1 , Yang Liu 1 , Francesco Secundo 3 and Yun Liu 1, *
1 Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of
Chemical Technology, Beijing 100029, China; wangshih@mail.buct.edu.cn (S.W.);
shuka1990@163.com (H.Z.); liuyangtjbd@126.com (Y.L.)
2 Ocean College, Zhejiang University of Technology, Hangzhou 310014, China; mengxh@zjut.edu.cn
3 Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche (CNR),
Via Mario Bianco 9, 20131 Milano, Italy; francesco.secundo@icrm.cnr.it
* Correspondence: liuyun@mail.buct.edu.cn or liuyunprivate@sina.com; Tel.: +86-10-6442-1335;
Fax: +86-10-6441-6428
† These authors contributed equally to this work.

Academic Editor: David D. Boehr

Received: 14 December 2015; Accepted: 21 January 2016; Published: 22 February 2016

Abstract: Enormous interest in biocatalysis in non-aqueous phase has recently been triggered due to
the merits of good enantioselectivity, reverse thermodynamic equilibrium, and no water-dependent
side reactions. It has been demonstrated that enzyme has high activity and stability in non-aqueous
media, and the variation of enzyme activity is attributed to its conformational modifications.
This review comprehensively addresses the stability and activity of the intact enzymes in various
non-aqueous systems, such as organic solvents, ionic liquids, sub-/super-critical fluids and their
combined mixtures. It has been revealed that critical factors such as Log P, functional groups and
the molecular structures of the solvents define the microenvironment surrounding the enzyme
molecule and affect enzyme tertiary and secondary structure, influencing enzyme catalytic properties.
Therefore, it is of high importance for biocatalysis in non-aqueous media to elucidate the links
between the microenvironment surrounding enzyme surface and its stability and activity. In fact, a
better understanding of the correlation between different non-aqueous environments and enzyme
structure, stability and activity can contribute to identifying the most suitable reaction medium for a
given biotransformation.

Keywords: enzyme; conformation; activity; applications; non-aqueous media

1. Introduction
Since the 1980s, biocatalysis in non-aqueous media has undergone a tremendous development and
numerous reactions have been proposed and optimized for synthetic applications. In comparison with
conventional aqueous enzymology, biocatalysis in non-aqueous phase offers unique merits, such as
the possibility of altering enzyme regio- and enantio-selectivity, to reverse thermodynamic equilibrium
toward synthesis (e.g., in the case of reactions catalyzed by hydrolases), to avoid water-dependent
side reactions, and bacterial contamination [1,2]. Organic solvents are the most commonly used
non-aqueous media for biocatalysis. Researchers have investigated that log P of organic solvents
impacts enzyme’s activity [3–5]. In our previous work, we demonstrated that, apart from log P,
functional groups and molecular structure of organic solvents would also exert significant influences
on enzymes activity [6]. Recently, we have also found that some enzymes show high catalytic activity,
enantioselectivity, and stability in ionic liquids (ILs) and sub-/super-critical fluids media, especially

Catalysts 2016, 6, 32; doi:10.3390/catal6020032 www.mdpi.com/journal/catalysts

Catalysts 2016, 6, 32 2 of 16

in their mixture solvents [7,8]. The satisfactory activity of enzymes in non-aqueous media has
allowed many synthetic applications. However, in most cases, enzyme activity in non-aqueous
media is lower than in water (up to several orders of magnitude). Different factors, such as diffusion
limitation, high saturating substrate concentration, restricted protein flexibility, low stabilization of the
enzyme-substrate intermediate, and non-optimal hydration of the biocatalyst, have been suggested to
be responsible for the lower catalytic activity of enzymes in non-aqueous media [9]. It is universally
accepted that conformational changes also play a very important role in the decrease of enzyme activity
in non-aqueous media.
However, Gupta et al. [10] demonstrated that, after incubated in acetonitrile at 70 ˝ C for 3 h, six
enzymes (proteinase K, wheat germ acid phosphatase, α-amylase, β-glucosidase, chymotrypsin and
trypsin) show much higher activity than that of the untreated enzyme. The authors claimed that the
probable reason was attributed to the unchanged stable three-dimensional structure. Taking the above
factors into consideration, it is essential to emphasize that enzyme denaturation is not only due to the
interactions of the enzyme molecules with the components of the non-aqueous media [11], but also
to the (freeze)-drying process used to prepare the enzyme in a suitable form for biocatalysis in these
media [12–14]. So it is crucial to have a deep insight into the conformation variance and activity shift
of enzyme in non-aqueous media, which is helpful for the selection of the suitable reaction medium
for biotransformation. Therefore, this review aims to highlight the mechanisms of enzyme’s activity
variance according to enzyme structure changes in four different common non-aqueous media, i.e.,
organic solvents, ILs, sub-/super-critical fluids, and their combination mixture systems. Furthermore,
some major factors affecting the microenvironment surrounding enzyme in non-aqueous media, such
as log P, solvent type, functional group, molecular structure, cation and anion type, pressure and
temperature, are comprehensively discussed in this review. Besides, some specific applications of
biocatalysis in non-aqueous phases are also addressed in the corresponding section.

2. Structure and Activity of Enzymes in Organic Solvents

2.1. Effect of Log P Value of Organic Solvent

Log P, the partition coefficient, is a measure of hydrophobicity of organic solvent. Generally,
hydrophilic (water miscible) solvents exhibit log P values less than 1.0, while hydrophobic (water
immiscible) solvents exhibit log P values more than 4.0. The higher the log P value, the more
hydrophobic the solvent is. It has been proved that enzyme activity is higher in hydrophobic solvents
than hydrophilic solvents [6,15,16]. The reasonable explanation was that hydrophilic solvents have
a greater tendency to “strip” tightly bound water (which is essential for enzyme catalytic activity)
from the enzyme molecules surface, leading to the decrease of the enzyme activity. Although the log
P value of organic solvent shows no obvious effect on the backbone structure of enzyme, organic
solvent will impact the water content urrounding the enzyme surface and active site region, which
can been observed through the molecular dynamics (MD) simulation. Water stripping is always
accompanied by the penetration of organic solvent molecules into crevices on the enzyme surface,
especially the active site [17,18]. Water activity (aw ) is indicative of water content around enzymes, and
it is defined as the partial vapor pressure of water in a substance divided by the standard state partial
vapor pressure of water. aw is an important consideration for enzyme activity in non-aqueous media
because enzymes require a certain level of water in their structures (bound water) to maintain their
natural conformation, allowing them to deliver their full functionality [19]. Bovara et al. [3] reported
that water activity ranging from <0.1 to 0.53 does not influence the enantioselectivity of enzymes in
organic solvents using lipase PS and lipoprotein lipase as models. Miroliaei and Nemat-Gorgani [20]
reported that the thermophilic alcohol dehydrogenase from Thermoanaerobacter brockii remains approx.
80% of its original activity at 90 ˝ C in n-octane (log P = 4.183).
The Log P value of organic solvents also affects the flexibility of enzyme when biocatalysis is
occurred in the organic solvents media. For instance, Trodler et al. [17] reported that the flexibility of
Catalysts 2016, 6, 32 3 of 16

lipase B from Candida Antarctica (CALB) decreases with increasing log P values of organic solvents.
Fasoli et al. [21] also revealed that the flexibility of subtilisin from Bacillus licheniformis in octane
(log P = 4.183) was lower than in acetonitrile (log P = ´0.334). As discussing the flexibility of the
enzyme, it was worthily noted that enyzme flexibility was usually determined from MD simulations
and it is measured by the relative calculated B-factors [22].
As well known, non-aqueous enzymology is a bell-shaped mechanism in dependence on
hydration [23]. Therefore, the water content and hydrophobicity (Log P) of organic solvent have
a dramatic influence on the properties of enzymes. It was revealed that there was an optimum water
content (~10% w/w) for enzyme properties, at which the enzyme properties are similar to the ones
found in pure water [24]. At lower water content, the enzyme is very rigid; while at higher water
content the enzyme starts to unfold. If the reaction medium is too dry, the enzyme lacks flexibility
resulting in un-efficient catalysis. When the water content increases, the enzyme becomes more flexible
and its activity increases. Beyond the optimum water concentration, the protein starts to unfold and
its activity decreases again.

2.2. Effect of the Functional Groups of Organic Solvent

The functional groups of organic solvents are also critical factors affecting enzyme activity [6].
Alkanes (such as hexane, cyclohexane, octane, and dodecane etc.), just have hydrophobic interactions
with enzyme, so they do not significantly change the global structure and active site of enzyme [25,26].
For the biocatalysis in these organic solvents, the solvent molecules are located near the active site
and/or close to the hydrophobic regions of the enzyme (e.g., CALB, r-chymotrypsin, subtilisin,
cutinase, triosephosphate isomerase, etc.), resulting in the re-orientation of the side chains of some
amino acids, obtained from the MD simulation or X-ray crystal structure analysis [23,27]. Although
these re-orientations of side chains do not necessarily alter the active site of the enzyme, they affect
the enzyme activity by changing the substrate affinity and specificity, as well as the hydration of
enzyme. For example, Pramod et al. [28] reported that although octane almost had no influence on the
secondary and tertiary structures of subtilisin BPN’, the catalytic efficiency kcat /Km of the enzyme in
octane was only 10.6% of that in aqueous solution. Nevertheless, the stability of subtilisin BPN’ in
octane was 645 fold of that in aqueous solution, owing to the absence of autolysis in octane. Therefore,
over its active lifetime the productivity of the soluble enzyme should be higher in octane than in
water. Similarly, Burke et al. [29] also stated that octane had little impacts on the secondary structure
and active site of the α-lytic protease, rather it reduced the activity through the alteration of binding
affinities of substrates. Guinn et al. [30] found that the activity of horse liver alcohol dehydrogenase
(HLAD) dramatically increased from 0% to 370% with the increase of water from 0% to 10% in the
hexane aqueous solution, although the structure of the enzyme was nearly identical to the native
enzyme. These results indicate that the water activity is essential for the activity [30]. Several studies
have corroborated that alkanes (especially hexane and octane) enhance the rigidity and stability of
enzymes, such as Rhizomucor miehei lipase (RML), r-chymotrypsin, subtilisin, cutinase, and horseradish
peroxidase, and the level of increase is positively related to the chain length of the alkane, and thus to
its hydrophobicity [23,26,31].
In many situations, alcohols are used as media for biocatalysis. The introduction of OH group
increases the hydrophilicity of the organic solvent and therefore enhances the interactions between the
solvent and enzyme. MD simulation has been extensively used to investigate the molecular interactions
between the alcohols and enzymes. For instance, it was reported that tert-butanol molecules could
directly bind to the active site of CALB and alter its tertiary structure [32]. Furthermore, ethanol
molecules were found to bind to the surface of the cutinase and strip water molecules from the
hydration layer surrounding the enzyme, which was considered to be an essential factor affecting the
enzyme activity [33]. Using lipase as a model enzyme, Kamal et al. [34] demonstrated that methanol
and isopropanol made lipase structure less rigid and more prone to unfolding, which increased the
instability of the enzyme. The changes of enzyme structures substantially alter enzyme activities. For
Catalysts 2016, 6, 32 4 of 16

instance, the Rhizomucor miehei lipase (ROL) activity decreases from 100% to 0% with the increase of
alcohol concentration, and the decreasing rate increases with the increase of carbon chain length from
methanol to butanol [24]. The activity inhibition of ROL might stemmed from the fact that the OH
group acted as a product inhibitor, competing with that of the substrate in the case of the hydrolytic
reaction [24]. Similarly, the papain showed 60% and 20% of its original activity in 90% and 99% v/v
methanol aqueous solution. Since no global conformational change and minor secondary structure
rearrangements were detected, it was suggested that the active site of the papain was somehow altered
by the methanol molecules [35]. The activities of α-chymotrypsin and trypsin first decreased and then
increased with increasing ethanol concentration from 0% to 100%, in corresponding with the changes
of the secondary structure elements (α-helix and β-sheet) [36]. Moreover, the apparent Km values of
the two enzymes decreasing as the low ethanol concentrations were elevated, but then increased in the
presence of higher ethanol concentrations, indicating the substrate affinity of the two enzymes first
increased and then decreased with increasing ethanol concentrations [36].
Similar phenomena were observed in organic solvents with C=O (e.g., acetone and
N,N-dimethylformamide), C”N (e.g., acetonitrile), and cyclic molecules (e.g., benzene, dioxane and
tetrahydrofuran) as in alcohols [37,38]. Through MD simulation, acetonitrile molecules were found to
penetrate into the active site of lipase, leading to structure variation of the active site and therefore
the drop in the enzymatic activity in acetonitrile aqueous solution [38]; acetone, acetonitrile, and
1,4-dioxane could bind to the active site of subtilis and disturb its structure [21,34]. Gupta et al. [39]
observed that the activity of polyphenol oxidase and trypsin reduced to different extents by 50% of
tetrahydrofuran, dioxane, acetone, and acetonitrile. Liu et al. [6] demonstrated that three commercial
lipases, Novozym 435, lipase PS, and Lipozyme TLIM, showed highest esterification activities when
pretreated (the term “pretreatment” means enzymes were preincubated in organic solvent, and then
the enzymes were filtered and dried to remove the organic solvent. The resulting enzymes were
dissolved in aqueous solution for activity assay) with organic solvents containing C=O and C”N
groups. Instead, the activity was lower if pretreated with alkanes, and even less, with solvents with
OH and aromatic groups.
However, the effects of organic solvents with S=O group (e.g., dimethyl sulfoxide,
DMSO) on enzyme activity and structure are quite different from the above-mentioned ones.
Roy et al. [40], through MD simulation, reported that about 5% (v/v) DMSO could markedly suppress
the flexibility of lysozyme, caused by the preferential solvation of exposed hydrophobic residues by
the methyl groups of DMSO. DMSO with the concentration of 15%–20% (v/v) could partially unfold
lysozyme, accompanied with an increase of both fluctuation and exposure of protein surface area. At
15%–20% (v/v) DMSO, conformational fluctuation and solvent accessible protein surface area suddenly
decrease to form an intermediate collapse state. This structural transformation was attributed to the
cluster of the methyl groups of DMSO on the enzyme surface. When the content of DMSO was higher
than 20% (v/v), the enzyme became denaturation and lost its activity completely.
In summary, hydrophobic functional groups, such as alkaline, could maintain the intact structure
of enzymes so as to dramatically prolong their stability. The enzyme activity usually achieved
maximum in these solvents containing ca. 10% water content, stemmed from the native structure and
water activity. As a result, a great deal of industrial reactions has been successfully applied in alkaline
systems such as hexane, octane, and isooctane [41–43]. Hydrophilic functional groups (e.g., OH, C=O,
C”N, S=O, etc.) changed the enzyme structure to different extents, as well as “stripped” the essential
water from enzymes, and thereby reduced the enzyme activity.

2.3. Effect of Molecular Structure of Organic Solvent

Several experimental studies have shown that the molecular structure of organic solvent has a
dramatic influence on the properties of enzymes. Generally, the organic solvent with its functional
group in the terminal carbon atoms shows higher inhibitory effect on the enzyme activity than
that in the internal carbon atoms. For example, the α-chymotrypsin activity decreased with the
Catalysts 2016, 6, 32 5 of 16

increase of 1-propanol (log P = 0.34) and 2-propanol (log P = ´0.77) concentrations, and the threshold
concentrations (defined as the values at which half inactivation of the enzyme is observed) for
1-propanol and 2-propanol were 27% and 33%, respectively, indicating that 1-propanol had higher
inhibitory effect on the enzyme activity [44]; trypsin showed 97% and 100% of its original activity in
50% 1-propanol and 2-propanol, respectively [39]; lipase PS from Pseudomonas cepacia presented much
higher activity after pretreatment with isopropanol than with n-butanol, although both the solvents
have the same log P of 0.8 [6]; the activity of Candida rugosa lipase in isooctane was much higher than
in octane, although the two solvents are of the same log P (log P = 4.5) [45]. A possible mechanism
might be that organic solvents with functional groups in internal carbon atoms had higher steric effects
than those in terminal carbon atoms. The higher steric effects hinder the effective interactions between
these functional groups and enzyme, the lower inhibitory effect on enzyme activity is caused.

3. Structure and Activity of Enzymes in Ionic Liquids (ILs)

Ionic liquids (ILs), also called molten salts, are organic salts melting below 100 ˝ C. ILs possesses
high thermal stability, negligible vapor pressure, and moderate polarity. Moreover, the physiochemical
properties of ILs (e.g., viscosity, melting point, polarity, and hydrogen bond basicity) can be altered
by simply changing anions or cations. Due to these advantages, ILs has been becoming attractive
alternatives to volatile and unstable organic solvents nowadays [46,47].

3.1. Effect of Hydrophobicity of Ionic Liquid

The activity and stability of enzymes in ILs system can be significantly affected by the
hydrophobicity of ILs [48]. Researchers observed that the enzyme could achieve higher activity
and stability in more hydrophobic ILs. Nakashima et al. [49,50] studied the properties of PEG-modified
lipase and subtilisin in three different ILs, 1-ethyl-3-methylimidazolium bis (trifluoromethanesulfonyl)
imide ([Emim][Tf2 N]), and its ether ([C2 OC1 mim][Tf2 N]) and hydroxyl ([C2 OHmim][Tf2 N]) analogues.
They found that the activities and stabilities of the two enzymes increased with increasing
hydrophobicity of the three ILs (the hydrophobicity decreases in the order of [Emim][Tf2 N] >
[C2 OC1 mim][Tf2 N] > [C2 OHmim][Tf2 N]). Specifically, the transesterification activity of PEG-modified
subtilisin was [Emim][Tf2 N] (65% compared to that in aqueous solution; the same below) >
[C2 OC1 mim][Tf2 N] (55%) > [C2 OHmim][Tf2 N] (28%). This enzyme also showed good stability for a
long period in [Emim][Tf2 N]. Namely, it maintained 80% of its initial activity after 60-h incubation.
For PEG-modified lipase, the initial rate exhibited [Emim][Tf2 N] (29 mmol/h¨ g) > [C2 OC1 mim][Tf2 N]
(25 mmol/h¨ g, which is 86% of [Emim][Tf2 N]) > [C2 OHmim][Tf2 N] (23 mmol/h¨ g, which is 79% of
[Emim][Tf2 N]). PEG-modified lipase was exceedingly stable in [Emim][Tf2 N], maintaining its original
activity for 144 h [50]. Zhang et al. [51] reported that the stability of penicillin acylase was higher
in more hydrophobic IL of 1-butyl-3-methylimidazolim hexafluorophosphate ([Bmim][PF6 ]) than
in 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4 ]) and 1-butyl-3-methylimidazolim
dicyanamide ([Bmim][dca]) after 48-h incubation. Similarly, De Los Ríos et al. [52] observed
that the synthetic activity of CALB was only 0.079 U/mg in IL of 1-butyl-3-methylimidazolium
octylsulfate ([Bmim][OcSO4 ]). However, the activity reached 116.097 U/mg in more hydrophobic
IL of 1-octyl-3-methylimidazolium hexafluorophosphate ([Omim][PF6 ]). Lozano et al. [53] evaluated
the stability of α-chymotrypsin in propanol and four ILs by testing the half-life time (t1/2 ) and
deactivation rate (kd ) at 50 ˝ C. The α-chymotrypsin exhibited an important activity loss in aqueous
solution at temperatures beyond the melting point (43.9 ˝ C). In propanol, the activity falled quickly
with the t1/2 of 0.15 h and the kd of 4.45 h´1 . In ILs, the t1/2 values increased and the kd values
decreased dramatically as compared in propanol, indicating that the enzyme stability was greatly
enhanced. The increase in t1/2 values and decrease in kd values were practically in agreement with
the increase in hydrophobicity of the ionic liquid. Namely, the hydrophobicity of the ILs decreases
in the order of [MTOA][Tf2 N] > [Bmim][BF4 ] > [Bmim][PF6 ] > [Emim][Tf2 N]. Correspondingly, their
t1/2 values decreased in the order of 2.63 h > 1.93 h > 1.58 h > 1.08 h, and the kd values increased in
Catalysts 2016, 6, 32 6 of 16

the order of 0.26 h´1 > 0.36 h´1 > 0.44 h´1 > 0.64 h´1 . The observed correlation between hydrophobicity
of ILs and the enzyme activity and stability might be explained by the fact that the increase in
hydrophobicity of the ILs could increase the preservation of the essential water layer around the
protein molecule, reducing the direct protein-ion interactions and then enhancing the enzyme stability
towards denaturative conditions [53].
In our previous work, biodiesel synthesis and conformation of lipase from Burlkholderia cepacia
(BCL) in 19 different ILs were comprehensively evaluated. Among them, N-octyl-3-pyridine
tetrafluoroborate ([OmPy][BF4 ]) was screened as the best reaction medium for biodiesel synthesis
with the yield of 82.2% ˘ 1.2% (yield = mass of actual yield/mass of theoretical yield ˆ 100%) after
12-h reaction [54]. The high yield of biodiesel achieved by [OmPy][BF4 ] might be explained by the
fact that [OmPy][BF4 ] readily dissolved methanol and byproduct glycerol as a storage phase, which
prevented direct exposure of the lipase to excess methanol and glycerol. Table 1 shows some examples
of biodiesel synthesis by lipases in different IL media.

Table 1. Comparisons of biodiesel yield catalyzed by lipase in ionic liquids (ILs).

ILs Lipase * Biodiesel Yield (%) Refernce

[OmPy][BF4 ] BCL 82.2 ˘ 1.2 [55]
[Bmim][PF6 ] CALB 38 ˘ 1.5 [56]
[Bmim][PF4 ] CALB 2.3 ˘ 0.2 [57]
[Bmim][Tf2 N] PS-D Amano I 24.7 [58]
[Bmim][Tf2 N] PS-C Amano I 73.9 [58]
[Bmim][Tf2 N] PS 21.2 [58]
* CALB: Candida Antarctica lipase B; PS: Pseudomonas cepacia lipase.

Hydrophobicity of ILs could alter the selectivity of enzymes. It was claimed that enzymes
showed different selectivity in the water-immiscible and water-miscible IL systems, the fact might be
attributed to water activity (aw ) around the enzyme microenvironment was altered in IL media. Shen
et al. [59] reported that Amano lipase PS from Pseudomonas cepacia showed higher enantioselectivity
(eep = 80%) in hydrophobic [Omim][PF6 ] than in hydrophilic 1-hexyl-3-methylimidazolium
tetrafluoroborate ([Hmim][BF4 ]) and 1-ethyl-3-methylimidazolium chloride ([Hmim]Cl) (eep < 5%)
for resolution of racemic cyanohydrins. Lou et al. [60] reported that the enantioselective acylation of
(R,S)-1-trimethylsilylethanol with vinyl acetate catalyzed by Novozym 435 increased with the increase
of hydrophobicity of ILs by the order of [Bmim][PF6 ] (ees = 90.7%) > [Omim][BF4 ] (ees = 86.3%) >
[C7 mim][BF4 ] (ees = 83.7%) > [Hmim][BF4 ] (ees = 76.2%) > [C5 mim][BF4 ] (ees = 70.5%) > [Bmim][BF4 ]
(ees = 62.6%). Hernández-Fernández et al. [61] declaimed that the transesterification activity of CALB
could be reached up to 99.99% in water-immiscible IL systems.

3.2. Effects of Cation and Anion Types of Ionic Liquids

The types of cation and anion of ILs show great influence on the enzyme activity and stability.
Because of a more localized charge and stronger internal polarization of compact anion, the hydrogen
bonding between enzyme and anion is much stronger than the weak van der Waals force between
enzyme and cation [62]. In order to maintain the activity of IL-dissolved enzymes, a balance
of mild hydrogen bond-accepting and donating property is required [63]. Therefore, anions are
universally believed to exert more powerful impact on the catalytic activity and stability of enzyme
than cations. This conclusion can be supported by the study by Liu et al. [54], who comprehensively
investigated the transesterification activity and conformation of BCL in 19 ILs with 6 different
cations and 7 different anions (the 19 ILs were [Omim][Cl], [Emim][TfO], [Bmim][Cl], [Omim][BF4 ],
[Bmim][CH3 SO3 ], [Emim][Cl], [NMP][CH3 SO3 ], [Hmim][TfO], [Hmim][CH3 SO3 ], [Bmim][PF6 ],
[Bmim][OH], [Emim][PF6 ], [Hmim][Cl], [Bmim][Tf2 N], [Bmim][BF4 ], [Omim][PF6], [Emim][BF4 ],
Catalysts 2016, 6, 32 7 of 16

[Hmim][PF6 ], and [OmPy][BF4 ]). By comparing the BCL activity in ILs with same cations or anions, it
was concluded that anions had much greater influence on the BCL activity than cations.
Hofmeister series of cations and anions are also widely used to predict the behaviors of enzyme
in ILs. Generally, kosmotropic anions and chaotropic cations of ILs are deemed as good stabilizers
of enzyme proteins [64]. However, the interactions between ILs and enzyme are complicated in
practice experiments. It was speculated that the shift of enzyme activity in ILs stemmed from the
secondary structure variance of enzyme, especially the alteration of α-helix and β-sheet elements [65].
ILs, in particular anions, which form strong hydrogen bonding may dissociate the hydrogen bonding
that maintains the structural integrity of the α-helices and β-sheets, causing the protein to unfold
wholly or partially [66]. Dabirmanesh et al. [67] demonstrated that imidazolium based ILs could affect
kinetics, structure and stability of the alcohol dehydrogenase from thermophilic Thermoanaerobacter
brockii (TBADH). Ajloo et al. [64] found that ILs could change the tertiary structure of adenosine
deaminase (ADA) after studying the interactions between two ILs (1-allyl 3-methyl-imidazolium
chlorides ([Amim]Cl) and 1-octhyl-3-methyl-imidozolium chlorides ([Omim]Cl)) and ADA. [Amim]Cl
has higher salt properties and then electrostatic interactions dominate, so it denatures ADA by
dissociate the essential hydrogen bonding. While [Omim]Cl has surfactant-like properties and
hydrophobic interaction is dominate. Therefore, the denaturing mechanisms of [Omim]Cl is similar to
that of surfactants.

3.3. Biocatalysis in Mixture Solvents of Organic Solvent and Ionic Liquid

Nowadays, more and more attention has been focussed on the biocatalysis in mixture solvents
of organic solvent and ILs [47,68]. Table 2 shows the typical applications of biotransformation by
enzymes in the mixtures of organic solvent and ILs.

Table 2. Activity of enzyme in mixture solvents of organic solvent and ionic liquids.

Enzyme * Mixture Solvents Applications Reference

[Bmim][PF6 ], [Bmim][Tf2 N],
[Hmim][PF6 ], and Esterification of lauric
BCL [5]
[Bmim][NO3 ]/hexane, benzene, acid with dodecanol
and tert-butanol
Transesterification of
PAL [Bmim][PF6 ]/hexane [69]
1-phenyl ethanol
[Btma][Tf2 N] and Transesterification of
CALB [70]
[Toma][Tf2 N]/hexane rac-1-phenylethanol
[Bmim][PF6 ] and
Aminolysis of
PSL [Bmim][BF4 ]/tert-butanol, [71]
(R,S)-methyl mandelate
and chloroform
Acylation of alaninol,
[Bmim][PF6 ]/tert-amyl 4-amino-1-pentanol and
[72]
alcohol and hexane 6-amino-1-hexanol with
myristic acid
Acylation of
[Bmim][PF6 ]/pyridine 1-β-D-arabinofuranosylcytosine [73]
CALB with vinyl benzoate
[Bmim][SCN], [Bmim]Cl oxidation of
[Bmim][BF4 ], and N-benzyliden-2-ethylhexylamine
[74]
[Bmim][PF6 ]/various to form E- and Z-isomers
organic solvents of oxaziridines
[Hmim][PF6 ], Esterification of ferulic acid to
[75]
[Omim][PF6 ]/isooctane) form oleyl alcohol ester
Catalysts 2016, 6, 32 8 of 16

Table 2. Cont.

Enzyme * Mixture Solvents Applications Reference

[Bmim][PF6 ]/Tween 20 or Esterification of natural fatty acids
[76]
Triton X-100 with various aliphatic alcohols
CRL [Bmim][PF6 ]/AOT and Hydrolysis of 4-nitrophenyl
[77]
Triton X-100 butyrate (p-NPB)
[Bmim][PF6 ]/benzene, toluene,
RML ethylbenzene, hexane, heptane, Esterification of amyl caprylate [78]
octane, and nonane
[MOPMIM][PF6 ]/tBuOMe, Transesterification of various
[79]
THF, CHCl3 bulky secondary alcohols
BCL [Emim][Tf2 N], [Emim][BF4 ], and
[Bmim][PF6 ]/several Acylations of secondary alcohols [79]
organic solvents
[Bmim]Cl and
PSL [Hmim]Cl/methanol Hydrolysis of p-NPB [80]
and 2-propanol
* CALB: Candida Antarctica Lipase B; PAL: Pseudomonas aeruginosa lipase; CRL: Candida rugosa lipase; RML:
Rhizomucor mielei lipase; BCL: Burkholderia cepacia lipase; PSL: Pseudomonas sp. Lipase.

It has been found that the catalytic activity, stability and enantioselectivity of enzymes
are obviously improved in mixture solvents of organic solvent and IL comparing to the
corresponding single organic solvent or ILs. These observations probably stemmed from
the fact that the viscosity of ILs was largely reduced by adding organic solvents, which
largely eliminated the mass transfer limitation of ILs and enhanced the biocatalysis reaction
rate [69]. For example, Singh et al. [81] comprehensively compared the transesterification of
(R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) with vinyl butyrate by lipases in hexane,
[Bmim][PF6 ], [Bmim][BF4 ], and IL/hexane co-solvents systems. Results showed that the maximum
conversion (>49%) and enantiomeric excess (ee > 99.9%) of rac-CDPP were achieved after 6-h incubation
at 30 ˝ C in [Bmim][PF6 ]/hexane co-solvents system, where the tertiary structure of lipase was
supposed to be well stabilized [82]. Ganske and Bornscheuer [83] reported that lipase from Candida
antarctica showed little activity in the synthesis of sugar esters in pure [Bmim][PF6 ] and [Bmim][BF4 ]
media. However, the reaction became feasible in IL/butanol co-solvents system containing 60% of IL
([Bmim][PF6 ] or [Bmim][BF4 ]) and 40% of butanol. Tan et al. [84] applied a mixture of [Bmim][PF6 ]
and pyridine (80:20, v/v) for acylation of 1-β-D-arabinofuranosyl-cytosine using CALB as a biocatalyst,
and the results showed that the conversion was dramatically increased to 99.4% compared with other
solvent systems.
In IL/organic solvent mixture systems, the proportion of organic solvent is an important factor that
affects enzyme activity. For instance, Contesini and Oliveira [85] studied the effect of organic solvent
proportion on the kinetic resolution of (R,S)-Ibuprofen catalyzed by lipases in isooctance/[Bmim][PF6 ]
co-solvents mixture. The enantioselectivity of lipase decreased in the order of 50% [Bmim][PF6 ]
(E-value = 4.6) > 70% [Bmim][PF6 ] (E-value = 4.1) > 30% [Bmim][PF6 ] (E-value = 3.2) > 100%
[Bmim][PF6 ] (E-value = 3.1) > 0% [Bmim][PF6 ] (E-value = 2.1) [85].
However, the inappropriate mix of organic solvent and IL may cause negative influence on
enzyme activity. For instance, enzyme showed higher activity in single ILs benzyltrimethylamine
chloride bis (trifluoromethylsulfonyl)-imide ([Btma][Tf2 N]) and 1-ethyl-3-methylpyridinium
bis(trifluoromethylsulfonyl)-imide ([EMpy][Tf2 N]) or hexane than in their mixture solvents. The
reasons were that [Btma][Tf2 N] and [EMpy][Tf2 N] are not soluble in hexane, so mass-transfer
limitations were introduced in the liquid/liquid biphasic system of their mixture. Moreover, the
authors stated that the homogeneous distribution of the enzyme onto a support with preferential
Catalysts 2016, 6, 32 9 of 16

enzyme-surface interactions and at an optimal hydration level were crucial for the enzyme activity,
indicating that a suitable water content in the enzyme microenvironment was essential for the retaining
of the native structure of the enzyme and therefore its activity [70,71,86].
In summary, ILs hhaveas proven themselves as excellent media for the enzyme catalyzed reactions
in many instances. Enzymes can not only be stabilized in certain ILs, but also is irreversibly activated
once incubated in ILs. Moreover, ILs may retain adequate microenvironment water content stabilizing
the structure of enzyme active site and therefore elevating the activity. The utilization of enzymes in
ILs also has limitations, including the unease of purification, high cost and mass transfer limitations.
However, ILs could provide numerous advantages in biocatalysis reactions due to their great diversity,
and this field surely marks a milestone on the path to future research [87].

4. Structure and Activity of Enzymes in Sub-/Super-Critical Fluids

4.1. Effects of Pressure and Temperature on the Structure and Activity of Enzyme
Supercritical fluids are materials above their critical temperature and critical pressure. Sub-critical
fluids refer to liquid at temperatures between their atmospheric boiling point and critical temperature.
The physical properties of sub-/super-critical (SC) fluids, such as density, polarity, diffusivities and
viscosities, are sensitive to the pressure and temperature. Since these properties of solvents exert great
impacts on the structure, stability, enantioselectivity and mass transfer rate of enzyme, biocatalyzed
reactions with specific requirements (especially high enzyme activity and enantioselectivity) can be
achieved by tuning the temperature and pressure of the SC fluids.
In SC fluids, enzyme activity usually firstly increases with increasing temperature, and then
decreases with the further increase of temperature due to thermal deactivation. For instance,
Knez et al. [88] studied the activity of lipase in SC-CO2 in the temperature range of 40–80 ˝ C and
pressure range of 80–450 bar. They found that, at various pressures, the lipase activity showed maximal
activity within 50–60 ˝ C. Similarly, the subtilisin and Aspergillus proteases had highest activity at
50 ˝ C in supercritical fluids [89]. Kamat et al. [89] studied the effect of pressure on the lipase activity in
SC-fluoroform, and found that the activity reached maximum value near the critical point of fluoroform,
and then gradually approached zero as pressure increased. In our previous work, we evaluated
the effects of SC-CO2 pretreatment, including pressure (6 and 10 MPa), exposure time (20, 30, and
150 min) and temperature (35 and 40 ˝ C), on the conformation (e.g., secondary and tertiary structures)
and catalytic properties (e.g., residual activity, kinetics constants (Km and V max ), activation energies
(Eα ), thermo-stability, and organic solvent tolerance) of two commercial enzymes CALB and lipase
PS in their solution forms. Results showed that the catalytic activities and kinetic constants of both
lipases were markedly altered by SC-CO2 pretreatment due to the changes of α-helix content in the
secondary structure as well as tertiary structure of the enzymes [8]. In particular, for the biocatalysis in
SC-CO2 , pressure variance could significantly alter the interactions between CO2 and enzyme through
the formation of carbamates by CO2 and the free amine groups of the enzyme. These interactions
might gradually change the conformation and activity of the enzyme in response to pressure [8,90–92].
The stability of enzyme is usually assessed by measuring residual activity after incubation with
sub-/super-critical fluids. Hu et al. [93] reported that the residual activity of tyrosinase showed a
significant reduction of about 25%–30% after the pretreatment of SC-CO2 under the condition of
8–12 MPa, 35 ˝ C, and 20 min pretreatment time. At 8 MPa and 55 ˝ C, the residual activity decreased
by 40% after 20-min pretreatment of SC-CO2 . However, Liu et al. [94] observed that after high pressure
SC-CO2 pretreatment (100 MPa and 25 ˝ C), the activity of mushroom polyphenoloxidase enhanced
by 11% compared with the native enzyme (0.1 MPa and 25 ˝ C). Kamat et al. [89] reported that the
lipase stability increased with increasing temperature in SC-CO2 since high temperature could inhibit
carbamate formation.
Natalia et al. [95] studied the selectivity of benzaldehyde lyase (BAL) in four supercritical
fluids (carbon dioxide, fluoroform, ethane, and sulphur hexafluoride), and found that the enzyme
Catalysts 2016, 6, 32 10 of 16

enantioselectivity was almost racemic with the highest enantiomeric excess for fluoroform (40%).
However, when excess water was added to the supercritical fluids, the enantiomeric excess increased
up to more than 90% for fluoroform, ethane, and sulphur hexafluoride, indicating that water activity
was a main factor in the selectivity. Ottosson et al. [96] demonstrated that there was a correlation
between enzyme enantioselectivity and the molecular volume of the solvent when CALB was used
as a catalyst for the transesterification of sec-alcohol in eight liquid organic solvents and SC-CO2 .
The correlation was explained by the fact that a solvent with large molecular volume would lose
translational entropy of fewer solvent molecules than that with smaller molecular volume when
restricted in the active site, resulting in higher enantioselectivity.

4.2. Biocatalysis in Mixture Solvents of Organic Solvent and Supercritical Fluid

Randolph et al. [97] investigated for the first time the catalytic performance of cholesterol oxidase
from G. Cirysocreas in SC-CO2 mixed with six organic solvents. Results indicated that reaction rate
slightly decreased with increasing methanol content, while progressively increased with increasing
contents (v/v, 0%–2.5%) of acetone, n-butanol, ethanol, isobutanol, and tert-butanol. The authors
asserted that this phenomenon could not be explained by the solubility data of substrate cholesterol
in these solvents. However, it was attributed to the conformational change of the enzyme and the
aggregation degree of cholesterol in organic solvent. The greater the aggregation of cholesterol caused
by organic solvent, the larger increase in the rate of enzymatic oxidation in the mixture solvent.
Liu et al. [8] reported that two commercial lipases, CALB and lipase PS, showed high tolerance
towards the five tested organic solvents with the relative activity of more than 85% following
SC-CO2 pretreatment in the condition of subcritical CO2 (6 MPa, 35 ˝ C, 30 min) and supercritical
CO2 (10 MPa, 40 ˝ C , 30 min). CALB showed higher hydrolysis activity in hexane (log P = 3.5)
and isooctane (log P = 4.5), in which CALB performed more than 80 U/g, whereas lipase PS from
Pseudomonas cepacia showed higher activity in isopropanol (log P = 0.28) and acetonitrile (log P = ´0.33).
The residue activity of lipase PS in isopropanol and acetonitrile was 140 and 110 U/g, respectively. It
was postulated that log P was an important factor that characterizes the catalytic activity of lipases in
an organic solvent. This was probably due to a variation of water retained in the microenvironment
of the catalytic active site, which is essential for the maintenance of the dynamical properties of the
enzyme. However, conformation analysis of enzyme has not been reported in the mixture solvents of
organic solvent and supercritical fluid so far.

4.3. Biocatalysis in Combined Mixture Solvents of Ionic Liquid and Supercritical Fluid
Reports have easily been available on the biocatalysis reactions in the mixture solvents
of IL and SC-CO2 fluid [97–100]. Bogel-Łukasik et al. [101] applied a ternary system of
[Omim][PF6 ]/SC-CO2 /products for the acylation of (R,S)-2-octanol with succinic anhydride catalyzed
by lipase. They stated that the recovery of >99.99 mol % was obtained at optimized conditions
of 35 ˝ C and 11 MPa. Lozano et al. [102] described the utilization of [Emim][Tf2 N]/SC-CO2
and [Bmim][Tf2 N]/SC-CO2 systems for the transesterification of vinyl butyratewith 1-butanol and
the kinetic resolution of rac-1-phenylethanol with vinyl propionate by CALB. In both systems,
the enantiomeric excess of the recovered product fraction (eep ) was above 99.9% for continuous
(R)-1-phenylethyl propionate synthesis at 100 ˝ C and 15 MPa, and the enzyme showed excellent
activity and stability.
Through assaying the property of CALB in five different SC-CO2 /IL systems based on quaternary
ammonium cations and Tf2 N anion, it was observed that all of the five ILs acted as enzyme stabilizing
agents with respect to hexane, leading to increasing the free energy of deactivation (to 25 kJ/mol
protein) and an improvement in the half-life time of the enzyme (2000-fold) [103]. Monhemi et al. [100]
confirmed it through all-atom MD simulation. It was showed that enzyme and IL molecules formed a
supramolecular-like structure in SC-CO2 , where IL molecules function as a coating layer and protect
enzyme from denaturing condition in SC-CO2 . The data of root mean square deviation implied that
Catalysts 2016, 6, 32 11 of 16

the enzyme had more native and stable conformation in SC-CO2 /IL system than in SC-CO2 . Moreover,
based on the radius of gyration values, it was found that enzyme had a more compact and active
conformation in SC-CO2 /IL system than in SC-CO2 .
On the other hand, the combination of SC-CO2 and IL could also achieve higher reaction rate than
IL alone by decreasing the viscosity of IL and enhancing the mass transfer [104]. Therefore, enzymes
showed higher activity and stability in the mixture solvents of IL and SC-CO2 than the corresponding
single medium. Interestingly, a homogeneous enzymatic reaction in SC-CO2 /IL system could be
achieved by elevating pressure; and a subsequent phase separation would be attained by lowering the
pressure, where free or immobilized enzyme dissolved or suspended in the ionic liquid phase (catalytic
phase), while substrates and/or products resided largely in the supercritical phase (extractive phase).
In summary, more attention should be focused on developing the bioreactor integrated with high
efficiency reaction and easy product separation in SC-CO2 /IL systems in the coming years.

5. Remarks and Prospects

Enzymes in non-aqueous solutions have been largely studied and employed in the areas of
food, synthesis, pharmaceuticals, and analysis. The utilization of non-aqueous solutions as reaction
media could enable high enzyme activity and stability, alter enzyme selectivity, and facilitate the
transformation of substrates that are unstable or poorly soluble in water. To take full advantage
of the opportunities afforded by non-aqueous enzymology and develop more feasible industrial
processes, this article comprehensively reviewed the structure-activity relationship of enzymes in
organic solvents, ILs, sub-/super-critical fluids, and their combined mixtures systems. As for organic
solvents and ILs media, molecular interactions between the enzyme and the solvent dramatically
affect the advanced structure of the enzyme and therefore its activity and stability. Generally, solvent
molecules or functional groups that interact with the enzyme through weak interactions could hold
the essential bound water on the enzyme surface, stabilizing the native structure and retaining the
activity of the enzyme. Unlike organic solvents, ILs also interacts with the enzyme through electrostatic
interaction due to the charged cation and anion. Kosmotropic anions and chaotropic cations of ILs,
according to the Hofmeister series, usually act as good stabilizers of enzymes, though anions exert
much greater influence on the enzyme properties owing to the strong hydrogen bonding between them.
However, due to the complex interactions involved in them, it is difficult to provide a general basis for
assessing the impacts of ILs on enzyme conformation and activity. In case of sub-/super-critical fluids
medium, the main advantage for enzyme-catalyzed reactions is the tunability of solvent properties.
Therefore, the enzyme activity and product separation efficiency are dependent on temperature and
pressure. It is commonly suggested that the deactivation of enzyme in sub-/super-critical fluids is
caused by carbamate formation or acidification of reaction media. Interestingly, using binary mixture
media of organic solvents, ILs, or sub-/super-critical fluid could effectively eliminate the demerits of
the single solvent whilst preserving the merits [105].
Despite the advance of mechanisms and applications on non-aqueous enzymology, there is still a
great deal of space to be improved on the way of reserach. First of all, more efforts should be made
to understand the causes of reduced enzyme activity in non-aqueous media and how to prevent it.
The synergies of solvent engineering and protein engineering could be a potential strategy to enhance
enzyme catalytic properties in non-aqueous media. Second, it is urgent to test more enzymes in
non-aqueous media, especially complex enzymes. Finally, it is required to illustrate the mechanisms
in deeply and to screen more solvent-tolerant bacteria and fungal strains producing enymes. The
advances in the understanding of biocatalysis in non-aqueous systems will open a new pathway to
elucidate the mechanism between structure and activity of enzymes, which will facilitate the screening
of a suitable reaction medium for biotransformation.

Acknowledgments: This work was financially supported by the Natural Science Foundation of China (NSFC)
(31070709, 31270858, and 21476016).
Catalysts 2016, 6, 32 12 of 16

Author Contributions: Y.L. (Yun Liu) conceived and designed the review article; S.W. wrote the paper; X.M.
collected the documents and wrote the section of Biocatalysis in Combined Mixture Solvents of Ionic Liquid and
Supercritical Fluid; H.Z. and Y.L. (Yang Liu) collected and analyzed the documents; F.S. revise the paper and
correct the final vesion of this paper.
Conflicts of Interest: The authors declare no conflict of interest.

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