Review

Biocatalysis in organic media using enzymes from extremophiles

Gerard A. Sellek*, Julian B. Chaudhuri

Centre for Extremophile Research, Department of Chemical Engineering, University of Bath, Bath BA2 7AY, UK

Received 3 February 1999; received in revised form 10 May 1999; accepted 1 June 1999

Abstract

Enzymes from extremophiles (extremozymes) show activity and stability at extremes of temperature, low water activity, and high

hydrostatic pressure. Aqueous/organic and nonaqueous media allow the modification of reaction equilibria and enzyme specificity, creating
pathways for synthesizing novel compounds. Used in combination with such media, extremozymes show great potential as shown by their
unique properties in aqueous media. This review introduces organic media biocatalysis before addressing the state of the art of recent
fundamental and applied aspects of extremozyme biocatalysis. The aim is to encourage further exploitation of this technology, drawing on
the limited work published in this field and important methods developed using mesophilic enzymes. Enzymes from three classes of
extremophile will be considered: psychrophiles, halophiles, and thermophiles. Low temperature processes using psychrophilic (cold-active)
enzymes may enhance yields of heat-sensitive products and reduce energy consumption. Halophilic enzymes require KCl/NaCl from 1 M
to saturation, i.e. low water activity media, a feature in common with organic solvent systems. Thermophilic enzymes can be active and
stable at up to 130°C and are highly resistant to proteases, detergents, and chaotropic agents. These features may afford resistance to the
effects of organic solvents. Enhancing extremozyme performance via chemical modification, complexation, immobilization, and protein
engineering is also discussed. © 1999 Elsevier Science Inc. All rights reserved.

Keywords: Extremophiles; Organic solvents; Nonaqueous media; Biocatalysis

1. Introduction

Since the discovery of organisms that inhabit environ-

ments of extreme temperatures, extreme pH, high pressure,
and high salinity, there has been a great deal of interest in
the biotechnological potential of their enzymes [1–3].

The potential of enzyme activity observed in the pres-

ence of organic solvents also has received much attention in
the past two decades. Numerous significant advances have
been made in both fields, but they remain to be fully ex-
ploited. Despite the distinct lack of data on the application
of extremozymes in aqueous/organic and nonaqueous media
and comparisons with mesophilic enzymes, it is possible to
see their potential in organic media.

This review concentrates on this emerging field by first

detailing the potential of organic solvent systems in bioca-
talysis. It then considers enzymes from three classes of
extremophile, psychrophiles, halophiles, and thermophiles,
and what these enzymes can give to the already diverse field

of nonaqueous enzymology. Finally, emphasis is placed on
new ways in which extremozymes can be used, drawing on
experience with mesophilic enzymes, and how they can be
produced easily and economically. However, extremozymes
and organic solvent biocatalysis possess so much scope that
this review cannot include all the details. The reader is
recommended to consult the reviews cited in this paper.

2. Organic solvent systems

2.1. Introduction

Aqueous media are the traditional media for biocatalysis.

In recent years, researchers have included organic media
into conventional biocatalysis. Industrial-scale application
has yet to take off. Organic solvents are often regarded as
denaturants (which is an inaccurate view of their effects),
and this plays a part in the lack of uptake of this technology.
Although the interactions between solvents and enzymes are
complex, there are many common phenomena that apply to
biocatalysis in organic media. Table 1 summarizes the ad-

* Corresponding author. Tel.:

⫹44-1225-826-826 ext. 5732; fax: ⫹44-

1225-826-894.

E-mail address: [e-mail] (G. Sellek)

Enzyme and Microbial Technology 25 (1999) 471– 482

0141-0229/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 0 7 5 - 7

vantages and disadvantages common to organic media bio-
catalysis [4 – 8].

2.2. Classification

There are three main types of organic solvent systems:

aqueous/organic, biphasic, and monophasic. A published
system of classification and nomenclature for organic media
was chosen for use in this review [9]. The underlying
principles are introduced below.

Aqueous, organic, vapor, and supercritical phases are

assigned the letters A, O, V, and SC respectively. The
description of the organic solvent system then rests on
listing the phases, by using the aforementioned letters, in
which the biocatalyst, reactant(s), and product(s) reside, in
that order. The authors [9] defined an aqueous phase as that
which contains

⬍20 vol% of a miscible solvent. An organic

phase was defined as being a pure immiscible solvent, or an
aqueous solution of a miscible solvent of

⬎20 vol%.

Subscripts also can be applied to the phase letters to

convey more information on the nature of the biocatalyst,
reactant(s), and product(s): I, immobilized; H, hydrated
(referring to the hydration layer of a biocatalyst); S, solid.
Where the aqueous or organic phases are immiscible or are
separated by a membrane, the phases can also be numbered
and applied as subscripts.

In summary, the four basic types of organic solvent

system can be identified as the following: (1) aqueous so-
lutions with

⬍20 vol% solvent—AAA; (2) aqueous solu-

tions with

⬎20 vol% solvent—OOO; (3) biphasic—AAO,

AOO, and AOA; and (4) monophasic—O

H

OO and SC SC

SC. In this way, the make-up of an organic solvent system
is described by the three-letter classification and requires
little additional explanation. In addition to the reviews al-
ready cited here, additional details can be obtained on or-
ganic media biocatalysis that are beyond the scope of this
review [10 –12].

2.3. AAA and OOO systems

Common solvents in these systems are dimethylsulfox-

ide (DMSO), N-N

⬘-dimethylformamide (DMF), acetoni-

trile, methanol, ethanol, and 1,4-dioxane. In AAA systems,
it is clear that solutions with no solvent fall into this cate-
gory. This is traditional biocatalysis and is not covered by
this review. OOO systems with the aforementioned solvents
are distinct from O

H

OO systems. The catalyst is regarded as

hydrated because of the water-stripping capacity of polar
solvents, and water contents can be higher.

Solvent addition causes the buffer pH to rise because of

the drop in the dielectric constant, but Tris appears to keep
the deviation to

⬍1.5 units from 0 to 90 vol% solvent [13].

However, this paper relies on a pH meter calibrated with
aqueous buffers. The pH so measured does not equal the
actual pH because of changes in the proton activity coeffi-
cient caused by the solvent. Overcoming this using thermo-
dynamic relations [14] is a difficult task and is limited by a
lack of data. A facile method for measuring pH spectropho-
tometrically using p-nitrophenol has been reported [15].
Although its accuracy is not known, it may be preferable to
the use of a pH meter. Despite many papers discussing
enzyme activity in AAA and OOO systems, few studies
have considered stability [16,17] or kinetic effects [18,19].
Moreover, activity and stability data must be considered
together, and under the same conditions, so that the effects
of the solvent can be determined.

With such reliance on enzyme activity data, much work

in AAA and OOO systems has resulted in organic solvents
being regarded as denaturants [20,21] because of their de-
activating effects on enzymes. Deactivation and denatur-
ation give similar observations but occur through different
mechanisms. Considering AAA and OOO systems, we can
place the effects of a solvent on an enzyme in three main
categories: denaturation, stabilization, and inhibition. These
are described below.

Y Denaturation

involves

gross

conformational

changes that tend to unfold the protein and cause
dissociation and can be reversible. Disruption of
the active site reduces the catalytic activity. When
followed by aggregation (due to the entropically
unfavorable exposure of hydrophobic residues),
denaturation becomes irreversible.

Y Stabilization also reduces enzyme activity because

of rigidification of the enzyme, in a reversible
manner. This restricts the large conformational
changes required for catalysis. The drop in water
activity on adding a stabilizing solute removes
water from the enzyme surface, contributing to the
rigidifying effects.

Y Inhibition also can occur when solvent molecules

bind to the active site. Thermolysin, for example,
was shown to be inhibited by alcohols [22]. It is a

Table 1
General effects of organic solvents on biocatalysis

Advantages

Higher solubility of hydrophobic species
Altered substrate-, regio-, and stereo-specificity
Reduced water activity (alters hydrolytic equilibria)
Reduced incidence of the side reactions found in water
Nonpolar solvents cannot desorb enzyme-bound water or

immobilized enzymes

Altered partitioning of substrates/products: aids separations,

improves yields

Reduced microbial contamination
High thermal stability of enzymes in nonpolar media

Disadvantages

Solvents tend to reduce enzyme activity: demands higher

biocatalyst requirement

Polar solvents can act as denaturants
Interfacial inactivation in two-phase systems
Water activity control needed for processes involving

condensation reactions

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G.A. Sellek, J.B. Chaudhuri / Enzyme and Microbial Technology 25 (1999) 471– 482

less common feature of deactivation and will not
be discussed further.

Whether the enzyme is denatured or stabilized relies

solely on the protein–solvent interactions. When the pro-
tein–solvent interactions favor exclusion of the solvent mol-
ecules from the enzyme hydration layer, compaction of the
enzyme occurs because of the unfavorable surface energy
rise, and this stabilizes the enzyme. Preferential binding
leads to denaturation via interaction of the solvent with
nonpolar residues and the concomitant disruption of elec-
trostatic interactions (including H-bonds) [23]. This ap-
proach has been used to explain the effects on proteins of
glycerol [24], acetonitrile [25], and DMSO [26]. DMSO
[26] and glycerol [27] are amphiphilic and have been shown
to exhibit a transition from preferential exclusion to prefer-
ential binding as their concentrations increase. Clearly, sol-
vent–solvent and solvent–water interactions are very impor-
tant

considerations,

in

addition

to

protein–solvent

interactions. The final important considerations involve
those interactions of solvents with substrates and/or prod-
ucts and the effects of the bulk medium on solvation/
desolvation processes.

2.4. AAO, AOO, and AOA systems

In these cases, typical solvents include long-chain alco-

hols and aliphatic/branched hydrocarbons. Reversed micel-
lar media [11] can be included in this category but are not
discussed further here.

The immiscibility of water with nonpolar solvents and

the differing solubilities between phases of the species in-
volved are the features exploited. Storing the substrate in a
different phase to the biocatalyst allows the use of high
concentrations without substrate inhibition. If the product
can be continuously extracted, then the reaction yields will
be improved. The biocatalyst need not be restricted to the
aqueous phase, and there may be a need for simultaneous
aqueous- and organic-phase biocatalysis.

A disadvantage of these systems is that the process

conditions for maximizing interphase mass transfer will
increase the interfacial inactivation of the biocatalyst.
Clearly, enzyme immobilization or retention behind a mem-
brane will be necessary for effective exploitation of this
type of process. Despite low aqueous solubility, dissolved
solvent molecules can be highly disrupting to enzymes.
Two related papers have studied the effects of interfacial
and dissolved solvent inactivation [28,29]. It can now be
seen that immobilized enzyme or membrane-based pro-
cesses, for example, can exploit effectively AAO, AOO,
and AOA systems. The high concentrations/high yields can
allow for smaller processes with a concomitant reduction in
energy consumption, reducing the construction and operat-
ing costs of the process, respectively.

2.5. O

H

OO systems

In these, the most studied of the organic solvent systems,

the medium is either a nonpolar solvent (such as tert-amyl
alcohol, hexane, and toluene) or a supercritical fluid (usu-
ally CO

2

). Supercritical fluid biocatalysis has been reviewed

[11,30] and will not be discussed further here.

Native enzymes are insoluble in nonpolar media, so

application requires lyophilization or immobilization. Ly-
ophilized enzymes are highly thermostable [31,32], but
even when prepared from buffers at optimum pH, they have
very low catalytic activity [33]. Conformational changes
induced by lyophilization, enzyme packing, and diffusion
limitations are factors in this observation. Effects resulting
from the low enzyme hydration are believed to be the most
important. Dramatic activation has been obtained by co-
lyophilization with nonbuffer salts such as KCl, giving
near-aqueous activities with serine proteases [34]. Optimi-
zation of this effect was published recently [35]. An insight
into this effect is given by the finding that in 99 vol%
glycerol with 1 M LiCl [36], lysozyme can be refolded with
the same yield as in buffer [21]. LiCl appears to reduce
aggregation and increases enzyme solubility. It appears that
the presence of salts in an essentially nonaqueous medium
effectively can mimic an aqueous environment.

In processes that produce water, control of water content

(expressed as water activity) is essential. This has been
achieved using salt hydrates [37] and by circulating satu-
rated salt solutions through silicone tubing immersed in the
solvent [38,39]. Use of water activity in monophasic media
is commonplace. It can be measured easily, being simply
the relative humidity of the equilibriated headspace ex-
pressed in decimal form [40].

As well as the aforementioned activation phenomena,

another important advance is the solubilization of modified
enzymes in nonaqueous solvents by polyethylene glycol
(PEG) modification [41], ion-pairing with surfactants [42],
or formation of enzyme-lipid complexes [43]. Solubilities
are of the order of 5 mg enzyme/ml solvent. These prepa-
rations show higher activity than lyophilized enzymes, and
diffusion limitations are removed.

2.6. Characterization of solvents and their effects

Studies aiming to classify organic solvent systems or to

relate solvent properties to the effects on the enzyme require
some way of characterising the medium. Several parameters
have been developed for this, of which the most common
are: log P parameter [44], the Dimroth–Reichardt parameter
E

T

(30) [45], polarity index [46], and water activity (a

w

)

[8,47]. Other parameters developed but yet to be widely
used include denaturation capacity [48], a hydrophobicity
parameter (H) [49], and naphthalene solubility [50].

For characterizing the effects of the solvent on the en-

zyme, structural changes can be monitored by using tryp-
tophan fluorescence [51,52] in AAA and OOO media,

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G.A. Sellek, J.B. Chaudhuri / Enzyme and Microbial Technology 25 (1999) 471– 482

whereas circular dichroism [53] is effective in all media
types. While catalytic activity is most often used to quantify
the condition of the enzyme, it must be remembered that, in
the case of hydrolysis reactions, the increasing favorability
of the reverse reaction with the addition of solvent makes a
contribution to the deactivation effects. Such an observation
has not been incorporated into modeling efforts so far pub-
lished.

3. Extremophiles and their enzymes

3.1. Introduction

3.1.1. Extremophiles

Extremophiles are organisms that inhabit the most ex-

treme of conditions on Earth, compared with the conditions
favored by the organisms with which we are most experi-
enced. The most extreme environments have temperatures
of

⫺50°C and 113°C, hydrostatic pressures of 120 MPa,

water activities of 0.6, and pH values of 0.5 and 12.0.
Acidophiles, alkaliphiles, and barophiles are not considered
further in this paper because there is such limited knowl-
edge on biotechnologically useful enzymes from these or-
ganisms.

Extremophiles, like most organisms that have adapted to

specific conditions, are obligate extremophiles and only
grow in a narrow range of temperatures, pH, etc. One
hyperthermophile, Pyrolobus fumarii, has a maximum
growth temperature of 113°C and is unable to grow below
85°C [54]. Most extremophiles so far isolated have been
classified as members of the proposed third kingdom, the
Archaea [55] (formerly Archaebacteria), on the basis of 16S
rRNA sequences [55] and physiology [56]. A recent phy-
logenetic tree has been published in a comprehensive re-
view of hyperthermophiles [54]. Archaea exist in the form
of psychrophiles [57] and mesophiles [58] (both unculti-
vated) in addition to the isolated halophiles and thermo-
philes. Extremophilic methanogens are also known [59].

3.1.2. Extremozymes

Like their host organisms, extremozymes require specific

conditions for activity and stability. Adaptation to extreme
conditions means that homologous enzymes generally have
the same order of magnitude of activity and stability [20]
but exhibit these properties at different temperatures, ionic
strengths, etc. This often has been referred to as the princi-
ple of corresponding states.

All enzymes rely on a library of methods for achieving

stability. Extremozymes rely on some methods more than
others, and differences in their strategies occur not just
between thermophilic enzymes and, for example, halophilic
enzymes, but even between different thermophilic enzymes.
Although many papers have studied thermophilic, halo-
philic, and psychrophilic enzymes in isolation, many re-
views have tackled extremozyme stability in general [60 –

65] with one specifically covering Archaeal extremozymes
[59].

4. Extremozyme sources, structural features, and

application to organic media

4.1. Psychrophiles

Psychrophiles are organisms adapted to life at tempera-

tures below 15°C and are isolated from Antarctica, the
Arctic/Antarctic oceans, and the oceanic abyss. These en-
vironments are estimated to represent

⬇70% of the Earth’s

surface [66]. Isolated psychrophiles consist entirely of bac-
teria and eukarya. An uncultivated Archaeon Crenar-
chaeum symbiosum, a symbiont of a marine sponge, has
been identified from total sponge DNA [57]. Psychrotrophs,
living between 15°C and 30°C, are also of significance.
They have been subjected to more study resulting in part
from food hygiene and preservation issues.

Mesophilic enzymes lose their activity at low tempera-

tures and can slowly unfold because of their greater reliance
on the hydrophobic effect for folding. The adaptation of
psychrophiles to low temperatures has required that their
complement of enzymes overcome the effects of low tem-
peratures on the hydrophobic effect, catalysis, and protein
flexibility. There are many factors that contribute to this
adaptation. Although comprehensive reviews of psychro-
philic enzymes have been published [67–70], the main fea-
tures of cold adaptation are listed below:

1. Increases in ion-pair content overcomes the reduced

contribution to protein folding by the hydrophobic
effect.

2. A reduction in the number of H-bonds and salt-

bridges combined with extended, highly charged sur-
face loops provide the required degree of conforma-
tional flexibility.

3. Greater accessibility to the active site helps overcome

the reduced diffusivity of substrates, and the distribu-
tion of surface charges in some cases may direct
substrate molecules towards the active site.

4. A high K

m

value also has been reported in a cold-

active enzyme [71]. By reducing the enzyme–sub-
strate complex activation energy, catalytic activity is
further enhanced.

At present, there is no published data on the performance

of psychrophilic enzymes in organic media. However, two
psychrotropic lipases have been applied to aqueous/organic
and nonaqueous solvents [72,73]. Pseudomonas strain P38
lipase was used in the synthesis of the flavor compound
butyl caprylate in n-heptane [72], giving a maximum yield
of 75%. Pseudomonas strain B11-1 lipase then was incu-
bated for 1 h at 25°C in aqueous solutions of many solvents
[73], and was activated by methanol, ethanol, DMSO, and
DMF. Acetonitrile was found to be a strong deactivator.

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G.A. Sellek, J.B. Chaudhuri / Enzyme and Microbial Technology 25 (1999) 471– 482

Despite this lack of data, studies on peptide synthesis

with mesophilic enzymes has shown that low temperatures
favor high yields, due to reduced hydrolysis of the acyl-
enzyme intermediate [74]. However, in most other pro-
cesses, that low temperature operation reduces energy con-
sumption will be a big enough advantage.

In addition to the yield enhancement, a PEG-modified

mesophilic lipase dissolved in benzene was found to have a
subzero temperature optimum for activity [75]. It is not
known if this is a property solely of mesophilic enzymes,
resulting, for example, from cold-induced unfolding of the
protein. Certainly, psychrophilic enzymes’ activity at these
temperatures affords a reduction in the biocatalyst loading
compared with mesophilic enzymes, facilitating processes
that are improved by a reduction in operating temperature.
Organic solvent enzymology tends to focus on activity and
stability at high temperatures, as nonpolar solvents for ex-
ample tend to make enzymes highly thermostable. The
existence of cold-active enzymes should now prompt a
change in direction.

4.2. Halophiles

Halophiles inhabit hypersaline environments and can be

isolated from the Dead Sea, Africa, Europe, and the USA
and have even been found in Antarctic lakes [76]. Like
thermophiles, they are mainly made up of archaea, followed
by bacteria, and eukarya. The potential of halophiles, and of
other enzymes isolated from them, has been reviewed [77].
Although halophiles can possess useful activities including
amylases [78], proteases [79,80], glycosidases [81], and
lipases [82], many of these have not been investigated or
applied. So far, the only halophilic enzymes known to be
commercially available are restriction endonucleases [77].

Uniquely, halophilic Archaea accumulate K

⫹

intracellu-

larly for osmoregulation, requiring adaptation of intracellu-
lar and extracellular proteins for activity and stability in 4 M
KCl and

⬍5 M NaCl, respectively [83]. Halophilic eubac-

teria and eukarya tend to accumulate compatible organic
solutes such as sugars, amino acids, and ectoines [77], so
their intracellular enzymes are not halophilic.

Out of all the halophilic enzymes isolated, malate dehy-

drogenase from Haloarcula marismortui has been studied in
the most detail by Eisenberg, Mevarech, and coworkers.
Their findings have been reviewed, in addition to other work
on the halophilic adaptations of proteins [56,59,83]. The
main structural features of halophilic enzymes are described
briefly below:

1. The high content (up to 20% of all residues) of as-

partic and glutamic acid residues found in clusters on
the enzyme surface. These attract hydrated counter-
ions to the enzyme surface, reducing the surface ten-
sion at the protein–solvent interface, and hence pre-
venting precipitation of the protein.

2. Extensive ion-pair networks have been observed.

These increase in effectiveness with increasing salt
concentrations. They result in part from the aforemen-
tioned increase in acidic residue content. They are
also believed to responsible for the stability observed
at high temperatures with halophilic malate dehydro-
genase [84] and dihydrolipoamide dehydrogenase
[59].

3. Fewer strongly hydrophobic residues (tyrosine, tryp-

tophan, phenylalanine) are found to compensate for
the highly salting-out nature of the medium.

As a result of this adaptation, halophilic enzymes rapidly

denature and dissociate at NaCl/KCl concentrations below
⬇1 M and can be irreversible. This feature, rather than
stability at high salt concentrations, has been proposed as
the defining feature of halophilicity [85]. This distinguishes
true halophilic enzymes from enzymes such as thermolysin,
which despite being soluble and activated by

⬎1 M salt, is

still active and stable in the absence of salt [86].

Because salt has the effect of reducing water activity,

enzymes from the halophilic Archaea are thought to be
important biocatalysts in aqueous/organic and nonaqueous
media [1]. The a

w

of saturated NaCl is 0.75, corresponding

to a DMF concentration of 60 vol% (calculated from a
published model) [47]. Although enzyme activity can be
observed in such media, it is generally low, and the high
specific activity of halophilic enzymes at such an a

w

is an

important property. Unpublished work by J. S. Dordick has
been reported in which a lyophilized halophilic enzyme in
an O

H

OO system medium retained almost all of its aqueous

activity [87]. If this can be observed with other halophilic
enzymes, they may become the enzymes of choice for
biocatalysis in organic media.

Two previous papers have reported the effects of sol-

vents on halophilic enzymes [88,89]. One of these reports
the activity of Halobacterium cutirubrum catalase in the
presence of different concentrations of solvents [88]. Glyc-
erol, ethylene glycol, and DMSO resulted in activation,
which peaked at

⬇2.5 M solvent. All of these solvents

deactivated bovine liver catalase. Despite little overall
knowledge on these enzymes at the time, the inferences
made by the authors appear to be consistent with what is
now known. Haloferax mediterranei protease was studied
briefly in AAA media, giving greater activity in solvents of
increasing polarity [90]. These form the only published
work that quantifies the activity of halophilic enzymes in
any kind of organic media.

The most significant and useful work published to date

with halophilic enzymes concerns the application of an
extracellular protease from Halobacterium halobium [80,
91,92] in AAA/OOO systems. While k

cat

/K

m

for substrate

hydrolysis reduced by three orders of magnitude from 0 to
33 vol%—and 42 vol%—DMF [80] (similar to that seen in
earlier work) [92], a peak was observed in the ratio of
esterase : amidase k

cat

/K

m

values at 32 vol% DMF. At this

point, the protease’s peptide synthetic capacity is at a max-

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G.A. Sellek, J.B. Chaudhuri / Enzyme and Microbial Technology 25 (1999) 471– 482

imum. When this experiment was repeated with subtilisin
Carlsberg, changes in this ratio were minimal, showing the
halophilic protease’s greater potential for peptide synthesis.
Further work showed the enzyme possessed broad substrate
specificity.

DMSO was shown to stabilize the halophilic protease at

all salt concentrations, whereas tetrahydrofuran and 1,4-
dioxane caused destabilization [91]. Protection was af-
forded by DMSO at low salt concentrations, and the salting
out capacity of DMSO was proposed to have a similar effect
to salt. Subtilisin Carlsberg was stabilized by DMSO, tetra-
hydrofuran, and dioxane and is apparently less sensitive to
the salting-out nature of the solvent. Although combining
solvent and high salt can be seen to enhance stability, the
solubility of the salt limits the solvent concentration as
found by Dordick and coworkers. Immobilization, however,
reduced the salt requirement of H. mediterranei aldolase
[93] and is important in ensuring that the use of halophilic
enzymes with polar solvents is not to be limited by the salt
solubility.

4.3. Thermophiles

Thermophiles, extreme thermophiles, and hyperthermo-

philes have adapted to environments with temperatures
from 50°C to the known upper limit of 113°C [94]. Despite
the differing classifications according to the extent of ther-
mophily, the term “thermophiles” is often sufficient for
general purposes. The environments colonized by thermo-
philes include hot springs, fumaroles, submarine hydrother-
mal vents, oil reservoirs, and even man-made coal refuse
piles. The deep terrestrial subsurface is emerging as a new
source of thermophiles [95]. In many cases, these environ-
ments may possess a low pH or high hydrostatic pressure in
addition to high temperature.

Thermophiles are mainly Archaea with the remainder

consisting of eubacteria, although a eukaryotic thermophile
also has been recorded [96]. Comprehensive reviews of
isolated hyperthermophiles, their ecology, and biotechno-
logical potential have been published [54,97]. They have
many nutritional requirements, with anaerobic thermophiles
gaining energy from sulfur reduction and heterotrophs ca-
pable of degrading polymers such as starch, protein, and
saccharides. Therefore they are host to many useful en-
zymes [98], with different degrees of thermophilicity. How-
ever, it is possible for mesophiles to express enzymes with
thermophilic properties, as has been found with eubacterial/
eukaryotic dihydrolipoamide dehydrogenase [99]. Extracel-
lular enzymes are also more thermostable than expected
from their host’s growth temperature [20].

Despite the large differences in operating temperatures

between mesophilic and thermophilic enzymes, the required
change in

⌬G

N

3 U

is small enough to be provided by only

a few interactions. The major stabilizing interactions in-
clude:

1. Increased salt-bridge content,
2. reductions in cavity size,
3. increased content of hydrophobic inter-subunit inter-

actions,

4. reduced content of thermolabile residues (it is be-

lieved that steric effects protect the remaining labile
residues).

Reduced conformational flexibility often is stated as im-

portant to thermostability. However, the higher content of
long-range interactions has been proposed as a mechanism
for maintaining conformation at high temperatures [100].
This appears to be a more realistic proposal because homol-
ogous thermophilic and mesophilic enzymes tend to have
similar specific activities as mentioned earlier in the review.

One enzyme that highlights the remarkable stability that

thermophilic enzymes are capable of showing is

␣-amylase

from Pyrococcus furiosus. It has a half-life of 2 h in aqueous
buffer at 120°C and is fully inactivated after 1 h at 130°C
[101]. In addition to such stability at high temperatures,
thermophilic enzymes also possess great resistance to pro-
teolysis [102] and the effects of detergents [103,104] and
chaotropic agents [104]. These properties have encouraged
their use in organic solvents, with the belief that they will be
more stable.

So far, the largest application of thermophilic enzymes

has been in the polymerase chain reaction [105], and future
large-scale applications may include the production of hy-
drogen from biomass [106,107]. The potential of thermo-
philic enzymes in organic media is equally promising.

The denaturation of thermophilic enzymes in AAO,

AOO, and AOA systems has been reviewed [20] but gives
little insight into the advantages of these biocatalysts in such
systems. This review also showed changes in the enantio-
selectivity of sec-alcohol dehydrogenase induced by sol-
vents including DMSO, DMF, and acetonitrile. Increasing
the solvent concentration tended to increase the selectivity
for the R(

⫹) isomer. However, thermophilic enzymes have

been studied mainly in AAA and OOO systems. Car-
boxypeptidase [104] and malate dehydrogenase [108] from
Sulfolobus solfataricus have been studied, and the latter
enzyme showed higher activities in alcohols of increasing
polarity. Glyceraldehyde-3-phosphate dehydrogenase from
Thermus thermophilus HB8 was activated by acetone and a
number of alcohols including ethanol, with solvents of in-
creasing hydrophobicity giving lower levels of activation
[109]. An opposite trend was found with glutamate dehy-
drogenase from Pyrobaculum islandicum, which appeared
to perform better in less polar solvents [110]. No specific
relationship was observed with S. solfataricus alcohol de-
hydrogenase [102]. Activation was seen with solvents
across a range of polarities, i.e. methanol, ethanol, propan-
2-ol, ethyl acetate, and acetonitrile, in most cases the acti-
vation remaining after incubation for 72 h at 50°C.

There appears to be no clear trend between solvent po-

larity and enzyme performance with the thermophilic en-

476

G.A. Sellek, J.B. Chaudhuri / Enzyme and Microbial Technology 25 (1999) 471– 482

zymes so far tested. Yet these studies show that thermo-
philic enzymes can be highly solvent tolerant and have great
potential when in organic media. However, without publish-
ing data for a homologous mesophilic enzyme used under
the same conditions, it is difficult to see if there are any
advantages (other than operation at high temperature) over
mesophilic enzymes. If more information on protein–sol-
vent interactions were known, even if only for mesophilic
enzymes, it may be possible to predict how thermophilic
enzymes behave in organic media and how to use them to
their best advantage.

In summary, Table 2 gives an overview of the use of

extremozymes in organic media, using the examples cited
previously in this section. Extremozymes have great poten-
tial when applied to organic media, as shown by the avail-
able data. However, the available data are scant, and where
extremozymes have been used in organic media, there have
been very few comparisons between them and their meso-
philic equivalents. These two factors limit the depth to
which extremozyme biocatalysis can be analysed at present.

5. Biotechnological implications

5.1. Enzyme production

Before extremozymes can be exploited on a large scale,

there must be economical and effective means of produc-
tion. Here, there are two options. One is to express the
enzyme’s gene in a native extremophilic host, e.g. dihydro-
lipoamide dehydrogenase (DHLipDH) from Haloferax vol-
canii has been overexpressed using H. volcanii as host
[111]. However, cultivation of extremophiles is not an easy
process. The low yields, slow growth, and the specialized
apparatus required result in a high-cost product that may
limit the production of extremozymes with this method.
Furthermore, there is a lack of suitable gene transfer sys-
tems for extremophiles, especially the Archaea. Thus, there
has been considerable interest in heterologous gene expres-
sion using a more common host, such as Escherichia coli.

A large number of thermophilic enzymes have been

produced successfully by heterologous expression in E. coli,
e.g. citrate synthase from P. furiosus [112] and glucose
dehydrogenase from Thermoplasma acidophilum [113]. It
appears that expression of themophilic enzymes in E. coli is
a straightforward process, and an advantage is gained during
purification by heat treating the supernatant [114]. At the
other end of the scale, only a few psychrophilic enzymes
have been successfully expressed in E. coli, e.g. citrate
synthase from an Antarctic bacterium [71] and

␣-amylase

from Alteromonas haloplanctis [115]. In both cases, the
yield of active enzyme was increased by gene expression at
low temperatures (25–27°C).

Heterologous expression of halophile genes in E. coli has

resulted in two outcomes. The expression of 3-hydroxy-3-
methylglutaryl-coenzyme A reductase from H. volcanii
[116] and malate dehydrogenase from Haloarcula maris-
mortui [117] resulted in a soluble product that was inactive
and required reactivation by the addition of salt. This has the
added advantage of precipitating much of the contaminating
E. coli protein. However, the expression of dihydrofolate
reductase [118] and dihydrolipoamide dehydrogenase [119]
from H. volcanii resulted in the formation of inclusion
bodies. In both cases, inclusion body solubilization and
refolding were required to obtain an active enzyme. The
unfolding and refolding of a halophilic enzyme has been
studied in detail [120].

Recombinant DNA technology is crucial to the produc-

tion of enzymes from uncultivated or unculturable organ-
isms, which represent at least 99% of the biosphere [98].
The potential for obtaining enzymes with useful activities
and stability is clear. The identification and production of a
DNA polymerase from the uncultivated psychrophilic Ar-
chaeon Crenarchaeum symbiosum using a method pub-
lished previously [57] has been reported [121]. Addition-
ally, enzymes also can be identified from the complete
genome sequences of several extremophiles (e.g. Methano-
coccus jannaschii [122] and Archaeoglobus fulgidus [123]),
the gene cloned, then expressed.

5.2. Enzyme purification

For thermophilic and psychrophilic enzymes, normal pu-

rification processes can be used without any modification.
Halophilic enzymes provide an extra complication, with
their requirement of high salt concentrations for stability.
Whereas glycerol and other solutes can afford stability in
the absence of salt, the increase in viscosity in often unac-
ceptable. However, ammonium sulfate-mediated chroma-
tography [124] relies on high salt conditions and has been
used to purify halophilic enzyme proteins on a large scale
[125]. Immobilized metal-ion affinity chromatography
[126] is another effective technique and is compatible with
NaCl/KCl concentrations of the order of 4 M. Both methods
are facile and have the added advantage of offering very
high protein yields.

Table 2
Application of extremozymes in organic solvent systems

System

Enzymes

AAA, OOO

Psychrotropic: lipase [73]
Halophilic: catalase [88], lactate dehydrogenase [89],

protease [80,90–92]

Thermophilic: alcohol dehydrogenase [102]

carboxypeptidase [104], malate dehydrogenase [108],
glyceraldehyde-3-phosphate dehydrogenase [109],
glutamate dehydrogenase [110]

AAO, AOO,

AOA

Thermophilic: crude extracts and sec-alcohol

dehydrogenase [63]

O

H

OO

Psychrotropic: lipase [72]

477

G.A. Sellek, J.B. Chaudhuri / Enzyme and Microbial Technology 25 (1999) 471– 482

5.2. Enzyme application: biotransformations

Biocatalysis in the presence of organic solvents already

has proved to be capable of synthesizing novel compounds
and opening up new synthetic pathways. There are many
papers concerning biotransformations in organic media. Ta-
ble 3 summarizes some novel transformations and important
applications of established reactions such as acylations and
peptide synthesis. New ways of exploiting these pathways
are equally significant, such as combinatorial biocatalysis
[138].

As has been a common theme throughout this review,

there is a lack of data concerning extremozymes in biotrans-
formations in organic media. One example of the synthetic
potential of, for example, thermophilic enzymes is in the
synthesis of lactosamine under kinetic control [139]. Al-
though not performed in organic media, a substantially
higher yield was obtained when the mesophilic glycosidases
were replaced with enzymes from a commercial thermo-
philic glycosidase library. However, we can look at the new
methods of application of mesophilic enzymes for inspira-
tion, with every chance that extremozymes will enhance the
processes in some way.

5.4. Enhancement of enzyme performance

Although extremozymes have an inherent stability that

may be of use in the desired catalytic reaction, it is possible
that they may be denatured over a period of time or that
there is scope to improve their activity/stability. A number
of techniques have been developed for the enhancement of
the performance of mesophilic enzymes. Few if any of these
methods have been applied to extremozymes. They are
discussed in the following three subsections.

5.4.1. Immobilization

Immobilization is a well-established technique. How-

ever, it is the new approaches to this area that are to be

considered here, especially those designed for organic me-
dia. Modification of an immobilized enzyme and the sup-
port has been achieved such that a hydrophilized prepara-
tion can be produced [140]. There is the possibility of
mimicking the enzymes’ natural environment. Of the wealth
of work on immobilization, the most important (with re-
spect to organic media) has been the incorporation of en-
zymes into artificial polymers. Sugar-based biocatalytic hy-
drogels [135] and enzymes co-polymerized with methyl
methacrylate and styrene monomers result in highly active
and stable preparations for catalysis in organic media [131].
Other similar work has been reported [141–143].

The applications of biocatalytic plastics were discussed

recently [144]. The use of protein monolayers also has been
proposed [145]. This has given mesophilic enzymes melting
temperatures of up to 200°C, and application to AAA/OOO
was suggested.

Stability advantages aside, immobilization also affords

protection against aggregation and ensures that solvent-
denatured enzyme can be reactivated. This has been per-
formed with several cycles of denaturation-reactivation
[146]. In the absence of thermal degradation of the enzyme,
reactivation can be total. Such recycling is important to
large-scale processes and smaller-scale processes that use
more expensive biocatalyst.

5.4.2. Complexation

Another significant advance in enhancing enzyme per-

formance has been in the formation of noncovalent compl-
exation of enzymes with poly-ionic species. When

␣-chy-

motrypsin (CT) was complexed with the poly-cationic
polybrene (hexadimethrine bromide) [147], the activation
normally seen with native CT in 20 vol% ethanol and 10
vol% DMF was increased more than 2-fold. The solvent
concentration required for full inactivation also increased
nearly 2-fold [121]. When more negative surface charges
were introduced into the enzyme surface through chemical
modification, CT was soluble and active in 95 vol% ethanol,
and the activation at 20 vol% ethanol was increased 3-fold
over native CT [148]. Adding solvents also enhances the
poly-ion-enzyme interactions, due to the reduction in the
dielectric constant. The authors propose that this method
could be used as with halophilic enzymes, with their high
surface negative charge. This could enable them to be less
dependent on salt for activity and stability.

5.4.3. Protein engineering

Much research is concerned with modifying enzyme

structures to give enhanced properties. Some research in-
vestigating the stability mechanisms of thermophilic en-
zymes aims to develop protein engineering methodologies
to confer thermophilicity upon mesophilic enzymes. Al-
though this has not been successful, it has been reported that
a thermophilic protein was given hyperthermophilic stabil-
ity (including a higher resistance to denaturants and pro-
teases) without loss of activity [149]. Despite this, it should

Table 3
Enzymatic syntheses in organic media

Peptide synthesis

One-spot synthesis of a pentapeptide [127]
Lipase-catalyzed synthesis of peptides containing

D

-amino acids [128]

Carbohydrate reactions

Alkyl-

␤-

D

-glycoside synthesis [129]

Regioselective synthesis of ethoxylated glycoside esters [130]
Sucrose and thymidine acylation in tetrahydrofuran by subtilisin

incorporated into polymer [131]

Galactosylation of chloramphenicol and chlorphenisin [132]
Galactosylation of serine methyl ester [133]

Miscellaneous

Enhancement of aqueous solubility of Taxol (Paclitaxel) by enzymatic

acylation [134]

Transesterification of N-Ac-Phe-OEt with propan-1-ol by

CT-hydrogel [135]

Transesterification and acylation by lipases [136]
Lipase-catalyzed ester synthesis [137]

478

G.A. Sellek, J.B. Chaudhuri / Enzyme and Microbial Technology 25 (1999) 471– 482

be noted that using a thermophilic enzyme initially pre-
cludes the need to engineer thermostability.

Protein engineering of enzymes for biocatalytic func-

tions can be achieved through either rational protein design
or random mutagenesis, including directed evolutionary ap-
proaches. The former requires an enzyme structure from
which to make the rational changes. On the other hand,
directed evolution of the enzyme (through sequential rounds
of mutagenesis followed by selection or screening) only
requires the enzyme gene. Recent results that illustrate the
potential of the evolutionary approach include: increasing
the temperature stability of Bacillus subtilis p-nitrobenzyl
esterase [150]; increasing 50-fold the half-life of subtilisin E
at 65°C [151]; and improving its activity in DMF [152].

6. Conclusions

Extremozymes only recently have become the focus of

attention. Most work to date has been particularly focused
on unravelling the structural features of these enzymes that
confer upon them their remarkable properties. Although the
exploitation of extremozymes lies solely in the field of
molecular biology, this provides a clear example of the
contribution these enzymes can make to the development of
new technologies. Given that

⬍1% of microbial biodiver-

sity has been explored, the potential for the discovery and
use of new enzymes in biocatalysis is immense. The wealth
of information that exists for the use of mesophilic enzymes
in organic solvents will be required to facilitate the devel-
opment of new extremozyme-based biocatalytic processes.

Acknowledgments

The authors thank the Engineering and Physical Sciences

Research Council (EPSRC) for financial support.

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