REVIEW ARTICLE
Enzymes in organic media
Forms, functions and applications
Munishwar N. Gupta and Ipsita Roy
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, India
Enzyme catalysis in low water containing organic solvents is
finding an increasing number of applications in diverse
areas. This review focuses on some aspects which have not
been reviewed elsewhere. Different strategies for obtaining
higher activity and stability in such media are described. In
this context, the damaging role of lyophilization and the
means of overcoming such effects are discussed. Ultrasoni-
cation and microwave assistance are two emerging approa-
ches for enhancing reaction rates in low water media.
Control of water activity and medium engineering are two
crucial approaches in optimization of catalytic behaviour in
nonaqueous enzymology. Organometallics and synthesis/
modification of polymers are two areas where nonaqueous
enzymology can play a greater role in the coming years. The
greater understanding of enzyme behaviour in nonaqueous
media is expected to lead to larger and even more diverse
kinds of applications.
Keywords: antibodies in organic solvents; enzymes in
organic solvents; high activity enzyme formulations;
medium engineering; microwave assisted enzymatic
reactions; synthesis and modification of polymers.
Introduction
The use of enzymes in organic media (with low water
content) has been one of the most exciting facets of
enzymology in recent times. Its importance can be appre-
ciated from the fact that at least four books [1–4] and

been made by many workers. When enzymes are exposed
to such media (stress or denaturing conditions) for a
limited time, and are recovered and checked for biological
activity in water, early studies [16] indicate that full activity
is observed. This implies that no irreversible denaturation
has taken place. On the other hand, enzymes in different
solvents display different k
cat
/K
m
, so operational stability is
different in different organic solvents. A number of solvent
parameters have been described; some have raised the
question of nonpolar solvents not being the same as
hydrophobic solvents [17]. The most acceptable, even if not
totally satisfactory, parameter is log P, where log P is the
partition coefficient of the solvent for the standard octanol/
water two-phase system [18]. Thus, early efforts, and even
some recent ones, have aimed at stabilization of enzymes in
organic media. Chemical modification [19], chemical
crosslinking [20], immobilization [21], protein aggregation
[22,23] and protein engineering [24] have all been tried. Of
these, immobilization has been used most often. In a review
which dispels a lot of vagueness and myths (which are
unfortunately associated with a lot of other papers and
reviews), Halling [25] mentions that the most popular
Correspondence to M. N. Gupta, Department of Chemistry, Indian
Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Fax: + 91 11 2658 1073, Tel.: + 91 11 2659 1503,
E-mail: [email protected]

linked enzyme crystals [22], crosslinked enzyme aggregates
[31] and enzymes coated with ionic liquid [32].
Additives and activation
Lyophilized enzymes are the most common forms of
enzymes used in enzymology. Lyophilization involves
[28,33] (a) initial freezing, (b) primary drying (in which ice
separated from the protein phase is removed by sublimat-
ion) and (c) secondary drying (in which the bound or
trapped water is removed from proteins). Basically, lyo-
philization (or freeze-drying) involves two denaturing
conditions: freezing and drying. Cryoprotectants (very
heterogeneous as far as chemical structures are concerned
(e.g. sugars, amino acids, polyols, salts, etc.) prevent
structural damage due to freeze stress. This protection
may be due to Ôpreferential exclusionÕ [34]. During the drying
stage (dehydration), one needs lyoprotectants. Lyoprotec-
tants substitute water molecules, which are being removed,
by forming H-bonds with the protein structure. Sucrose is a
good example [33]. Evidence (most of it based upon FTIR)
exists, which shows that the absence of lyoprotectant leads
to both the random structure and the a-helical structure
being partially converted to b-sheet structure (in which
peptide bonds are linked to each other via H-bonds). In
conventional enzymology (in aqueous media), rehydration
reverses much of the structural damage during lyophiliza-
tion. In nonaqueous enzymology, this becomes very crucial
and is now considered as largely responsible for the low k
cat
/
K

(w/w) of KCl was tentatively explained by either rigid salt
structure protecting the enzyme in organic solvents or
polar environment of salt helping in maintaining native
structure of the enzyme. Soon after, Bedell et al. [39]
provided evidence that it is not a result of reduced
diffusional limitation. Subsequently, it was shown [40,41]
that salt activation is the result of kosmotropicity and this
stabilizing effect (during lyophilization) operates via prefer-
ential hydration. Also, a combination of kosmotropic salt
sodium acetate and sodium carbonate buffer had a cumu-
lative effect. Hsu & Dordick [42] found that salt activation
enhances enantioselectivity of subtilisin because the
favoured reaction is more sensitive to the structural integrity
of the enzyme. On the other hand, Altreuter et al. [43] found
that salt activation is accompanied by expanded and
unnatural regioselectivity of subtilisin. The latter feature
is very valuable in the use of enzymes for developing
combinatorial biocatalysis [10]. Recently, Lindsay et al. [44]
reported the salt activation of a nonprotease, penicillin
amidase and found that salt activation was dependent upon
the water content in the solvent medium. However, the
preparation lyophilized in the presence of trehalose behaved
differently. The conclusion was that salt activation is
mechanistically distinct from lyoprotection. On the other
hand, Morgan & Clark [45] believe that the Ôpresence of salt
protected enzymes from irreversible activationÕ in several
cases.
To sum up, the picture is still far from clear. That is not
surprising as our appreciation of the role of lyophilization,
while obtaining enzyme preparations for nonaqueous enz-

K
m
values may lead to lower reaction rates [25]. The nature
of the solvent media affects both the enantio- and regio-
selectivity of the enzymes. Various hypotheses have been
given to rationalize these important effects. No consensus
seems to have emerged so far [7]. Finally, one must
mention Ôsolvent-freeÕ media wherein one or more of the
substrate(s) form the medium and no other solvent is
required. In some cases, this results in more efficient
conversion rates. A good example is the production of
biodiesel with lipases [14]. Biodiesel consists of monoalkyl
esters of long chain fatty acids. There is no petroleum or
other fossil fuel in biodiesel; the diesel part of its name is
based upon the fact that it can be substituted in place of
petroleum diesel fuel. It is produced from vegetable oils or
fats by lipase catalyzed transesterification with methanol or
ethanol [48].
Importance of water activity in nearly
anhydrous media
It is now recognized that less than a monolayer of water is
needed for an enzyme molecule to start showing biological
activity. Beyond this, addition of more water molecules
increases biological activity. However, in the case of
hydrolases, beyond a threshold limit of water concentration,
hydrolytic activity starts effectively competing with trans-
ferase or synthetic activity [49].
The first clue that water content may not be the best
parameter to look at was the work of Zaks & Klibanov [46].
Working with alcohol dehydrogenase in a variety of

levels were listed [55].
One area which needs more work is the effect of water
activity on the stereoselectivity of the reaction. There are
already a few reports which show that both rate and
enantiomeric ratio change simultaneously when water
activity is increased [56].
pH tuning and pH memory
This is one dramatic observation [46] for which clear
rationalization is now available [3]. The correct protonation
state of side chains of amino acid residues of enzymes is
important in nonaqueous media as well. Hence pH tuning
(placing the enzyme in water at optimum pH of the enzyme,
and lyophilizing) results in higher rates in organic solvents.
However, this is valid only if the reaction does not change
the acid/base concentration. Such changes have been
mentioned by the same authors [57] who have also described
useful protocols for pH tuning by the use of special buffers.
Microwave-assisted reactions in nonaqueous
enzymology
The microwave region (0.3–300 GHz) falls between the
infrared and radiofrequency regions of the electromagnetic
spectrum. Chemical synthesis using microwave irradiation
has been extensively reported. In this approach, microwave
irradiation replaces conventional forms of heating [58].
Upon irradiation with microwaves, a polar molecule
continually aligns itself with the fluctuating field. This
converts electromagnetic energy into heat energy. It is not
very clear whether these rate enhancements are purely due
to thermal effects or whether some nonthermal effects are
involved [59,60]. For example, Whittaker & Mingos [61]

[66] have provided a protocol for using microwave assist-
ance for protease catalyzed peptide synthesis. Unfortu-
nately, most of these protocols do not take account of
temperature control during microwave irradiations.
Recently, esterification by a-chymotrypsin and transeste-
rification by subtilisin Carlsberg were accelerated (in the
range of 2.1–4.7 times) in six solvents of differing polarities
and at different water activities [60]. Interestingly, micro-
wave irradiation could be used in conjunction with pH
tuning and salt activation. For example, at the same level of
water activity (0.3%, v/v) in n-octane, untuned subtilisin
showed a transesterification rate of 0.5 mmolÆh
)1
at 25 °C.
The salt-activated and pH tuned subtilisin showed about 20
times increase in reaction rates when microwave irradiations
were used at 25 °C. It was also observed that the choice of
the reaction medium was a factor which dominated the
microwave effect.
Maugard et al. [67] have exploited microwave assistance
for a somewhat different kind of application, i.e. synthesis of
galactooligosaccharides from lactose by using Kluyvero-
myces lactis b-galactosidase. Low water activity, high
lactose concentration and cosolvents with higher log P
value all favoured oligosaccharide synthesis. By optimizing
conditions and using microwave irradiations, the synthesis
of galactooligosaccharides could be increased 217 times. It is
hoped that when more experience with different systems
becomes available, microwave assistance will become a
powerful approach in nonaqueous enzymology.

temperature as well. It is also difficult for others to
reproduce results obtained with such ill-defined condi-
tions. In other cases, temperature has been controlled
either by use of a water-cooled reactor or interrupting
ultrasonication so that the temperature does not rise. In
the latter cases, cycles of ultrasonication and cooling have
been used.
Vulfson et al. [73] carried out subtilisin-catalyzed inter-
esterification and reported (a) that pretreatment of
subtilisin suspensions by ultrasound in alcohols led to
an increase in enzyme activity. This effect was more
pronounced with long-chain alcohols, being 6–8 times
more in octanol as compared to the effect observed in
short chain alcohols. The effect was dependent both on
sonication power and water content of the medium,
(b) the enhancement of reaction rates was, however,
much higher if the reaction was carried out in the
presence of ultrasound. The authors speculated on the
mechanism of these effects; the reduction in mass transfer
constraint because of reduction in enzyme particle size
and increased fluid velocity seemed to be the most
probable factors. Sinisterra [74], in his review on the
application of ultrasound to biotechnology, has men-
tioned data on ultrasonication increasing the percent yield
in protease-catalyzed peptide synthesis in organic solvents.
In hydrocarbons, the more hydrophobic the solvent
(greater log P value), the larger was the sonication effect.
In the case of organic halides, the relative increases under
ultrasonication were CCl
4

Transmission electron microscopy by Bracey et al. [76]
found that ultrasonication changed the open honeycomb
structure of freeze-dried subtilisin into a plate-like struc-
ture. Thus, size reduction did not result in significant
increase in surface area. However, the hydration state of
the enzyme may be critical. According to Rozicwski &
Russell [77], solvation of a subtilisin particle in a
hydrophobic solvent swells it, increasing the diffusion
path for the substrate. If these pores have water
molecules, reactant diffusion is inhibited. In such cases,
2578 M. N. Gupta and I. Roy (Eur. J. Biochem. 271) Ó FEBS 2004
increased fluid velocity by sonication would overcome this
inhibition. Bracey et al. [76] believe that their enzyme was
drier than those of other workers and hence they failed to
observe any increase in reaction rate by ultrasonication. It
is also worth noting that it is the hydration level before
transferring the enzyme to the organic media, which was
found to be critical. Thus, this mechanism does not
explain the correlation between water content of the
medium and the effect of ultrasonication reported earlier
[73]. We need more structural work before ultrasonication
can be used in nonaqueous enzymology in a predictable
manner.
Synthesis and modification of polymers
Dordick et al. [78] have used about 80–85% organic
solvents as media for peroxidase-catalyzed polymerization
of phenols. The use of predominantly nonaqueous media
has two advantages: the substrate phenols can be solubilized
easily, and unlike in water where dimers and trimers
precipitate out of solutions thereby terminating chain

opening/polymerization of 12-dodecanolide and e-capro-
lactone [84,85]. Enzyme-catalyzed polytransesterification of
alkanedioates and butane-1,4-diol in organic solvents has
also been successfully carried out [86].
Use of subtilisin (solubilized by formation of protein-
surfactant ion-pair method) in iso-octane for transesterifica-
tion of a thin layer of amylose (deposited onto zinc-selenide
plates) has been described [80]. The modification was
followed by FTIR and thermogravimetric analysis of
amylose.
1
H-NMR showed that all available primary amino
groups were acylated. Further work in this area will
definitely be useful in obtaining novel carbohydrate mate-
rials for a variety of applications.
Enzyme modification by directed evolution
This approach originated in the realization that an enzyme
is designed or evolved for a particular function and works
in vivo as a part of a complex metabolic network. In many
situations, these two features are constraints for indus-
trial biocatalysts, especially if they are to be used in
unnatural environments such as organic media. The
approach consists of:
Random mutations [87–89]
At the outset, it is clear that one has to limit the number of
mutations to only one or two residues at a time, otherwise
the number of sequences generated will be unmanageably
large. In the next step, it is possible to recombine and
accumulate such beneficial mutations. This approach has
been successfully used by Moore & Arnold [88] for directing

may not have been screened out.
Arnold & her coworkers have combined the two
approaches (of random mutagenesis and in vitro recombi-
nation) to obtain an esterase for hydrolyzing p-nitrobenzyl
ester in the presence of polar organic solvents [88,93]. The
application was to selectively remove p-nitrobenzyl groups
during the synthesis of cephalosporin type antibiotics. The
starting enzyme was from Bacillus subtilis with low p-nitro-
benzyl esterase activity, especially in the polar organic
solvents required to solubilize the esters. The clone obtained
Ó FEBS 2004 Enzymes in organic media (Eur. J. Biochem. 271) 2579
by directed evolution showed more than 150-fold greater
total activity than the wild type clone. It was also more
stable in 15% (v/v) dimethyl formamide.
Unlike site-directed mutagenesis, directed evolution can
be applied without much structural and mechanistic infor-
mation about an enzyme.
Using antibodies in organic solvents
Using antibodies in organic media provides a good oppor-
tunity to carry out immunoassays of water insoluble
antigens [94]. One major application of this is in environ-
mental control and analysis [95]. Russell et al. [96] showed
that antigen–antibody interaction is possible in low water
containing organic solvents. Stocklein et al. [97] studied
binding of triazine herbicides to antibodies in nearly
anhydrous media. The work of Matsuura et al. [98] on
screening monoclonals for okadaic acid in the context of
shellfish poisoning is worth mentioning. Okahata &
Yamaguchi [99] described a lipid-coated catalytic antibody
which was soluble in organic cosolvents and had higher k

nonaqueous enzymology can be clearly seen. The first trend
is that although lipases and proteases continue to dominate
vis-a
`
-vis applications in the nonaqueous media, other
enzymes like oxidoreductases (alcohol dehydrogenase,
catalase, peroxidase) [3], penicillin amidase [106], b-glucosi-
dase [2], b-galactosidase [68], catalytic antibodies [2] and
whole cells [2] are also beginning to be used in organic
media. Given the large number of lipases and proteases
available with a wide range of specificities, this picture is
unlikely to change significantly. The second trend is that
greater understanding (at the molecular level) of the way
enzymes behave in such media is emerging. The focus right
now is on creating formulations/designs with greater
activity. The control of water activity during the reaction
is crucial. Microwave assistance and ultrasonoenzymology
in such media have not yet been extensively studied,
therefore one is likely to see more work in this area in the
coming years. The iterative process of application fi basic
understanding fi more applications is likely to continue
for a while in this area.
Acknowledgements
The financial assistance from Department of Science and Technology,
Department of Biotechnology, Council for Scientific and Industrial
Research (both Extramural Division and Technology Mission on
Oilseeds, Pulses and Maize) and National Agricultural Technology
Project (Indian Council for Agricultural Research), all of which are
Government of India organizations, is gratefully acknowledged.
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