DOI: 10.1002/adsc.

200700082
Enzyme Immobilization: The Quest for Optimum Performance
Roger A. Sheldon
a,
*
a
Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
Fax: (+31)-15-278-1415; e-mail : r.a.sheldon@tudelft.nl
Received: February 5, 2007
Abstract: Immobilization is often the key to optimiz-
ing the operational performance of an enzyme in in-
dustrial processes, particularly for use in non-aque-
ous media. Different methods for the immobilization
of enzymes are critically reviewed. The methods are
divided into three main categories, viz. (i) binding to
a prefabricated support (carrier), (ii) entrapment in
organic or inorganic polymer matrices, and (iii)
cross-linking of enzyme molecules. Emphasis is
placed on relatively recent developments, such as the
use of novel supports, e.g., mesoporous silicas, hydro-
gels, and smart polymers, novel entrapment methods
and cross-linked enzyme aggregates (CLEAs).
1 Introduction
2 Types of Immobilization
3 Immobilization on Supports: Carrier-Bound En-
zymes
3.1 Synthetic Organic Polymers
3.2 Biopolymers
3.3 Hydrogels
3.4 Inorganic Supports
3.5 Smart Polymers
4 Entrapment
5 Carrier-Free Immobilization by Cross-Linking
5.1 Cross-Linked Enzyme Crystals (CLECs)
5.2 Cross-Linked Enzyme Aggregates (CLEAs

)
6 Combi-CLEAs and Catalytic Cascade Processes
7 Enzyme-Immobilized Microchannel Reactors for
Process Intensification
8 Conclusions and Prospects
Keywords: biotransformations; cross-linked enzyme
aggregates; entrapment; enzymes; immobilization;
support binding
1 Introduction
In the drive towards green, sustainable methodologies
for chemicals manufacture
[1]
biocatalysis has much to
offer:
[2]
mild reaction conditions (physiological pH
and temperature), a biodegradable catalyst and envi-
ronmentally acceptable solvent (usually water), as
well as high activities and chemo-, regio- and stereo-
selectivities. Furthermore, the use of enzymes general-
ly obviates the need for functional group protection
and/or activation, affording synthetic routes which are
shorter, generate less waste and, hence, are both envi-
ronmentally and economically more attractive than
traditional organic syntheses.
Modern developments in biotechnology have paved
the way for the widespread application of biocatalysis
in industrial organic synthesis.
[3–14]
Thanks to recombi-
nant DNA techniques
[15]
it is, in principle, possible to
produce most enzymes for a commercially acceptable
price. Advances in protein engineering have made it
possible, using techniques such as site-directed muta-
genesis and in vitro evolution via gene shuffling,
[16–19]
to manipulate enzymes such that they exhibit the de-
sired properties with regard to, inter alia, substrate
specificity, activity, selectivity, stability and pH opti-
mum. Nonetheless, industrial application is often
hampered by a lack of long-term operational stability
and difficult recovery and re-use of the enzyme.
These drawbacks can often be overcome by immobili-
zation of the enzyme.
[20–25]
There are several reasons for using an enzyme in
an immobilized form. In addition to more convenient
handling of the enzyme, it provides for its facile sepa-
ration from the product, thereby minimizing or elimi-
nating protein contamination of the product. Immobi-
lization also facilitates the efficient recovery and re-
use of costly enzymes, in many applications a conditio
sine qua non for economic viability, and enables their
use in continuous, fixed-bed operation. A further ben-
efit is often enhanced stability,
[26]
under both storage
and operational conditions, e.g., towards denaturation
by heat or organic solvents or by autolysis. Improved
enzyme performance via enhanced stability and re-
peated re-use is reflected in higher catalyst productiv-
ities (kg product/kg enzyme) which, in turn, deter-
mine the enzyme costs per kg product. As a rule of
thumb the enzyme costs should not amount to more
than a few percent of the total production costs. In
Adv. Synth. Catal. 2007, 349, 1289 – 1307 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 1289
REVIEWS
the production of 6-aminopenicillanic acid (6-APA)
by penicillin G amidase (penicillin amidohydrolase,
E.C. 3.5.1.11)-catalyzed hydrolysis of penicillin G, for
example, 600 kg of 6-APA are produced per kg of im-
mobilized enzyme and the production of the com-
modity, fructose, by glucose isomerase-catalyzed iso-
merization of glucose has a productivity of 11,000.
[22]
Indeed, the development of an effective method for
its immobilization was crucial for the application of
penicillin amidase in the industrial synthesis of b-
lactam antibiotics.
[27]
Immobilization is generally necessary for optimum
performance in non-aqueous media. In the traditional
method of using enzymes as lyophilized (freeze-dried)
powders, many of the enzyme molecules are not read-
ily accessible to substrate molecules. Furthermore,
lyophilization can cause pronounced structural pertur-
bations often leading to deactivation. In contrast, dis-
persion of the enzyme molecules by immobilization
generally provides for a better accessibility and/or an
extra stabilization of the enzymes towards denatura-
tion by the organic medium.
Another benefit of immobilization is that it enables
the use of enzymes in multienzyme and chemoenzy-
matic cascade processes (see later).
[28–31]
A major
problem encountered in the design of catalytic cas-
cade processes is the incompatibility of the different
catalysts and a possible solution is compartmentaliza-
tion (i.e. , immobilization) of the different catalysts
thus circumventing their mutual interaction which
could result in inhibition and or deactivation.
In this review we shall focus on recent develop-
ments in novel immobilization techniques, such as the
use of novel supports, e.g., smart polymers, novel en-
trapment methods, and the recent development of
cross-linked enzyme aggregates (CLEAs), in the
quest for optimum performance in biotransforma-
tions. Another approach to facilitating the recovery
and re-use of enzymes is to perform the reactions,
with the free enzyme, in a membrane bioreactor con-
sisting of an ultrafiltration membrane which retains
the enzyme on the basis of its size but allows sub-
strates and products to pass through. This combines
ease of recovery and recycling with the high activity
of the free enzyme. The methodology is applied com-
mercially, for example, by Degussa in the commercial
synthesis of enantiomerically pure amino acids using
hydrolases or dehydrogenases.
[32]
However, since it in-
volves the free enzyme it falls outside the scope of
this review.
2 Types of Immobilization
Basically, three traditional methods of enzyme immo-
bilization can be distinguished, binding to a support
(carrier), entrapment (encapsulation) and cross-link-
ing.
(i) Support binding can be physical (such as hydro-
phobic and van der Waals interactions), ionic, or co-
valent in nature. However, physical bonding is gener-
ally too weak to keep the enzyme fixed to the carrier
under industrial conditions of high reactant and prod-
uct concentrations and high ionic strength. Ionic bind-
ing is generally stronger and covalent binding of the
enzyme to the support even more so, which has the
advantage that the enzyme cannot be leached from
the surface. However, this also has a disadvantage: if
the enzyme is irreversibly deactivated both the
enzyme and the (often costly) support are rendered
unusable. The support can be a synthetic resin, a bio-
polymer or an inorganic polymer such as (mesopo-
rous) silica or a zeolite.
(ii) Entrapment via inclusion of an enzyme in a po-
lymer network (gel lattice) such as an organic poly-
mer or a silica sol-gel, or a membrane device such as
a hollow fiber or a microcapsule. The physical re-
straints generally are too weak, however, to prevent
enzyme leakage entirely. Hence, additional covalent
attachment is often required. The difference between
entrapment and support binding is often not clear.
For the purpose of this review we define support
binding as the binding of an enzyme to a prefabricated
support (carrier) irrespective of whether the enzyme
is situated on the external or internal surface. Entrap-
ment requires the synthesis of the polymeric network
in the presence of the enzyme. For example, when an
enzyme is immobilized in a prefabricated mesoporous
Roger Sheldon (1942)
is Professor of Bioca-
talysis and Organic
Chemistry at Delft
University of Technol-
ogy (Netherlands). He
received a PhD in or-
ganic chemistry from
the University of Lei-
cester (UK) in 1967.
This was followed by
post-doctoral studies
with Prof. Jay Kochi in
the USA. From 1969 to 1980 he was with Shell Re-
search in Amsterdam and from 1980 to 1990 he was
R&D Director of DSM Andeno. His primary re-
search interests are in the application of catalytic
methodologies – homogeneous, heterogeneous and
enzymatic – in organic synthesis, particularly in rela-
tion to fine chemicals production. He developed the
concepts of E factors and atom utilization for assess-
ing the environmental impact of chemical processes.
1290 asc.wiley-vch.de 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Adv. Synth. Catal. 2007, 349, 1289 – 1307
REVIEWS
Roger A. Sheldon
silica the enzyme may be situated largely in the meso-
pores but this would not be entrapment. On the other
hand when the enzyme is present during the synthesis
of a silica sol-gel the enzyme is entrapped.
(iii) Cross-linking of enzyme aggregates or crystals,
using a bifunctional reagent, to prepare carrierless
macroparticles.
The use of a carrier inevitably leads to dilution of
activity, owing to the introduction of a large portion
of non-catalytic ballast, ranging from 90% to >99%,
which results in lower space-time yields and produc-
tivities.
[22]
Moreover, immobilization of an enzyme on
a carrier often leads to the loss of more than 50%
native activity,
[33]
especially at high enzyme load-
ings.
[34]
The design of carrier-bound immobilized en-
zymes also relies largely on laborious and time-con-
suming trial and error experiments, because of the
lack of guidelines that link the nature of a selected
carrier to the performance expected for a given appli-
cation.
[35]
Consequently, there is an increasing interest
in carrier-free immobilized enzymes, such as cross-
linked enzyme crystals (CLECs),
[36]
and cross-linked
enzyme aggregates (CLEAs).
[37]
This approach offers
clear advantages: highly concentrated enzyme activity
in the catalyst, high stability and low production costs
owing to the exclusion of an additional (expensive)
carrier.
It should be pointed out that, from the literally
thousands of papers on enzyme immobilization, it is
difficult to make comparisons of the different meth-
odologies as most authors compare the performance
of the immobilized enzyme, prepared using a particu-
lar technique, with that of the free enzyme but do not
compare different methods of immobilization. In ad-
dition, details of the immobilization of industrial bio-
catalysts are often not disclosed.
3 Immobilization on Supports:
Carrier-Bound Enzymes
The properties of supported enzyme preparations are
governed by the properties of both the enzyme and
the carrier material. The interaction between the two
provides an immobilized enzyme with specific chemi-
cal, biochemical, mechanical and kinetic properties.
The support (carrier) can be a synthetic organic poly-
mer, a biopolymer or an inorganic solid.
3.1 Synthetic Organic Polymers
Acrylic resins such as Eupergit

C are widely used as
supports. Eupergit

C is a macroporous copolymer of
N,N’-methylene-bi-(methacrylamide), glycidyl meth-
acrylate, allyl glycidyl ether and methacrylamide with
average particle size of 170 mm and a pore diameter
of 25 nm.
[38]
It is highly hydrophilic and stable, both
chemically and mechanically, over a pH range from 0
to 14, and does not swell or shrink even upon drastic
pH changes in this range. It binds proteins via reac-
tion of its oxirane moieties, at neutral or alkaline pH,
with the free amino groups of the enzyme to form co-
valent bonds which have long-term stability within a
pH range of pH 1 to 12 (see Figure 1). The remaining
epoxy groups can be rendered inactive by capping
using a variety of reagents (mercaptoethanol, ethanol-
amine, glycine, etc.) to prevent any undesired sup-
port-protein reaction. Due to the high density of oxir-
ane groups on the surface of the beads enzymes are
immobilized at various sites of their structure. This
“multi-point-attachment” is largely responsible for
the high operational stability of enzymes bound to
Eupergit

C.
Immobilization by covalent attachment to Eupergit
C has been successfully applied to a variety of en-
zymes for industrial application.
[27,38]
Penicillin ami-
dase on Eupergit C, for example, maintained 60% of
its initial activity over >800 cycles.
[38]
A major draw-
back of Eupergit C is diffusion limitations, the effects
of which, as would be expected, are more pronounced
in kinetically controlled processes. Sepa beads FP-EP
(Resindion, Milan, Italy) consist of a polymethacry-
late-based resin functionalized with oxirane groups
and exhibit characteristics similar to Eupergit C.
[39]
Similarly, various porous acrylic resins, such as Am-
berlite XAD-7, are used to immobilize enzymes via
simple adsorption without covalent attachment. For
example, the widely used enzyme C. antarctica lipase
B (CaLB),
[40]
is commercially available in immobilized
form as Novozym 435 which consists of the enzyme
adsorbed on a macroporous acrylic resin. A disad-
vantage of immobilization in this way is that, because
Figure 1. Immobilization of enzymes on Eupergit C.
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Enzyme Immobilization: The Quest for Optimum Performance
it is not covalently bound, the enzyme can be leached
from the support in an aqueous medium. In a compar-
ison of the immobilization of lipases of Humicola la-
nuginosa, Candida antarctica and Rhizomucor miehei
on supports with varying hydrophobicity with Novo-
zym 435 (see below), in the esterification of oleic acid
with 1-butanol in isooctane, the highest activity was
observed with sepa beads (see above) containing octa-
decyl chains.
[41]
This was attributed to the hydropho-
bic nature of the support facilitating opening of the
hydrophobic lid of the lipase.
3.2 Biopolymers
A variety of biopolymers, mainly water-insoluble
polysaccharides such as cellulose, starch, agarose and
chitosan
[42]
and proteins such as gelatin and albumin
have been widely used as supports for immobilizing
enzymes. Indeed, the first industrial application of an
immobilized enzyme in a biotransformation is the
Tanabe process,
[43]
first commercialized more than 40
years ago, for the production of l-amino acids by res-
olution of racemic acylamino acids using an aminoa-
cylase from Aspergillus oryzae (Figure 2). The
enzyme was immobilized by ionic adsorption on
DEAE-Sephadex which consists of cellulose modified
with diethylaminoethyl functionalities (Figure 2) and
the process was performed in continuous operation in
a fixed-bed reactor.
This method is still widely used, e.g., in the immo-
bilization of a recombinant epoxide hydrolase from
Aspergillus niger.
[44]
An activity retention of 70% was
observed in the resolution of p-chlorostyrene oxide
under biphasic conditions. The immobilized biocata-
lyst was active at high substrate concentrations (306 g/
L) and could be recycled 7 times but it exhibited a
slightly lower enantioselectivity compared to the free
enzyme (E=76 vs. E=90).
3.3 Hydrogels
In non-aqueous media enzymes can also be immobi-
lized in natural or synthetic hydrogels or cryogels.
Polyvinyl alcohol (PVA) cryogels formed by the
freeze-thawing method,
[45]
for example, have been
widely used for immobilization of whole cells.
[46]
A
mild and highly efficient method for preparing PVA
hydrogels by partial drying at room temperature af-
forded lens-shaped hydrogels (Lentikats) exhibiting
good mechanical stability, easy separation (diameter
3–5 mm and thickness 200–400 mm) and stability to-
wards degradation.
[47]
Lentikats have been successful-
ly used for the entrapment of whole cell biocata-
lysts,
[48,49]
for example, in the immobilization of whole
cells of Rhodococcus equi A4, which contains nitrile
hydratase and amidase activities.
[49]
However, free en-
zymes, owing to their smaller size, can diffuse out of
the gel matrix and are, consequently, leached in an
aqueous medium. In order to entrap free enzymes the
size of the enzyme must be increased, e.g., by cross-
linking (see later). In contrast, immobilization of free
enzymes in PVA hydrogels can be useful in organic
media, where the enzyme is not leached from the gel
matrix. For example, Ansorge-Schumacher and co-
workers reported the co-immobilization of an alcohol
dehydrogenase (EC 1.1.1.1) from Lactobacillus kefir
together with its co-factor, NADP, in PVA beads.
[50]
The resulting immobilisate was used for the enantio-
selective reduction of a broad range of hydrophobic
prochiral ketones to the corresponding (R)-secondary
alcohols (Figure 3) in n-hexane as solvent. For exam-
ple, acetophenone afforded (R)-1-phenylethanol in
>98% ee. Co-factor regeneration was achieved
within the gel matrix using isopropyl alcohol as the
co-substrate (total turnover number of the co-factor
10
2
–10
3
). In addition to stability towards the organic
solvent, the immobilized biocatalyst showed improved
thermal stability and long-term stability under the re-
action conditions.
The same group reported the enantioselective ben-
zoin condensation of aromatic and heteroaromatic al-
dehydes, in n-hexane as solvent, using a recombinant
benzaldehyde lyase (BAL) from Pseudomonas fluo-
rescens immobilized in a PVA hydrogel.
[51]
As noted
above, in order to retain the enzyme in a PVA hydro- Figure 2. Tanabe aminoacylase process.
1292 asc.wiley-vch.de 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Adv. Synth. Catal. 2007, 349, 1289 – 1307
REVIEWS
Roger A. Sheldon
gel, in the presence of water, the molecular weight
must be increased. Grçger and co-workers
[52]
achieved
this by cross-linking an (R)-oxynitrilase using a mix-
ture of glutaraldehyde and chitosan (Figure 4). The
cross-linked enzyme was subsequently entrapped in a
Lentikat PVA hydrogel. The resulting immobilized
biocatalyst had a well-defined particle size of 3–5 mm
and showed no leaching in the enantioselective hydro-
cyanation of benzaldehyde in a biphasic aqueous
buffer/organic solvent system. It could be recycled 20
times without loss of yield or enantioselectivity.
An alternative method to increase the size of the
enzyme is to form a complex with a polyelectrolyte.
Owing to their ampholytic character, proteins exist as
polycations or polyanions, depending on the pH of
the medium. Hence, they can form complexes with
oppositely charged polyelectrolytes. This principle
was used by Dautzenberg and co-workers
[53]
to immo-
bilize amyloglucosidase (EC 3.2.1.3) by coupling to an
excess of a complex of sodium polystyrenesulfonate
(PSS) or poly-diallyldimethylammonium chloride
(PDM). The immobilized biocatalyst retained 45% of
its activity and lost no activity over five cycles.
In an interesting recent development, directed evo-
lution of the formate dehydrogenase (EC 1.2.1.2)
from Candida boidinii, in two rounds of error-prone
PCR, was used to create variants that were more
suited for immobilization in a polyacrylamide gel.
[54]
The best mutant had a 4.4-fold higher activity com-
pared to the free enzyme immobilized in the gel. The
stabilization resulted from a substitution of lysine,
glutamic acid and cysteine residues remote from the
active site.
Bruns and Tiller have reported
[55]
a novel immobili-
zation of enzymes in a prefabricated, nanophase sepa-
rated, amphiphilic network (see Figure 5), whereby
the enzyme is situated in its hydrophilic domains
which consist of poly(2-hydroxyethyl acrylate)
(PHEA). The substrate that diffuses into the hydro-
phobic polydimethylsiloxane (PDMS) phase can
access the enzyme via the large interface, owing to its
nanophase separation and peculiar swelling proper-
ties. The amphiphilic network was synthesized by UV-
initiated radical copolymerization of a silylated 2-hy-
droxyethyl acrylate monomer and a bifunctional a,w-
methacroyloxypropyl-poly(dimethylsiloxane) (see Fig-
ure 5).The hydrophilic domain swells in the presence
of water and the polymer is loaded with the enzyme
by immersing it in an aqueous solution of the biocata-
lyst. Upon drying the phase shrinks, trapping the
enzyme in an enzyme-friendly environment. When
the polymer network is immersed in a non-polar or-
ganic solvent the hydrophobic PDMS domain swells
allowing access of a dissolved substrate which can ap-
proach the enzyme via the interface between the two
domains. Immobilization of horseradish peroxidase
(HRP; EC 1.11.1.7) and chloroperoxidase (CPO; EC
1.11.1.10) in this way afforded immobilisates showing
substantially enhanced activities and operational sta-
bilities in heptane, compared to the free enzymes (see
Figure 5). In contrast, no enhancement of activity was
observed in isopropyl alcohol as solvent, which was
Figure 3. Alcohol dehydrogenase in Lentikat.
Figure 4. Cross-linked (R)-oxynitrilase in Lentikat hydrogel.
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Enzyme Immobilization: The Quest for Optimum Performance
attributed to swelling of the PHEA phase rather than
the PDMS phase in this polar solvent.
3.4 Inorganic Supports
A variety of inorganic solids can be used for the im-
mobilization of enzymes, e.g., alumina, silica,
[56]
zeo-
lites
[57,58]
and mesoporous silicas
[59–62]
such as MCM-
41, and SBA-15. One of the simplest and most inex-
pensive methods to immobilize an enzyme is by silica
granulation.
[40]
It is used, for example, to formulate
enzymes for detergent powders which release the
enzyme into the washing liquid during washing. Gran-
ulation technology was used to immobilize CaLB
lipase on silica granules, by first adsorbing the lipase
on silica powder followed by agglomeration.
[40]
Owing
to the composition of the granulates, they are intend-
ed for use only in organic media. In an aqueous
medium the lipase is desorbed and the particle slowly
disintegrates. However, the CaLB silica granules can
be used in a direct ester synthesis if the water is re-
moved by, e.g., evaporation under vacuum. Applying
the granules in packed-bed reactors also minimizes
the contact time with high water concentrations. The
CaLB silica granules exhibited a similar activity to
Novozym 435 in the direct synthesis of the skin emol-
lient, isopropyl myristate.
In order to maintain its integrity in an aqueous en-
vironment the enzyme needs to be covalently bonded
to the silica support. For example, immobilization of
epoxide hydrolase from Aspergillus niger by covalent
attachment to functionalized silica resulted in 90%
activity retention, which was retained over a period of
months, in the enantioselective (E=85) hydrolysis of
p-nitrostryrene oxide.
[56]
Furthermore, an enhanced
stability towards 20% DMSO as solvent was observed
and no leaching occurred in a filtration test.
Mesoporous silicas, which are nowadays often refer-
red to as nanosilicas, have several advantages as sup-
ports: they have uniform pore diameters (2–40 nm),
very high surface areas (300–1500 m
2
g
À1
) and volumes
(ca. 1 mLg
À1
), and are inert and stable at elevated
temperatures. Moreover, the surface can be easily
functionalized. Because of the large pore sizes of
these materials they can accommodate relatively
small enzymes in the pores. Whether the enzyme is
situated inside the pores or on the outer surface can
be determined by comparing immobilization on cal-
cined and non-calcined material (i.e. , the latter still
contains the template). If these values are roughly the
same then most of the enzyme is on the outer surface.
On the other hand, when the calcined material ad-
sorbs much more enzyme this indicates that most of
the enzyme is situated in the pores.
[59]
Covalent binding of a-chymotrypsin (EC 3.4.21.2)
to a mesoporous sol-gel glass, which had been modi-
fied by reaction of surface hydroxy groups with 3,3,3-
trimethoxypropanal, afforded an immobilized catalyst
with a half-life one thousand times that of the free
enzyme.
[61]
Immobilization of chloroperoxidase (CPO)
from Caldariomyces fumago on the same material re-
sulted in increased stability towards organic sol-
vents.
[62]
The immobilized preparation was used to-
gether with free glucose oxidase which generated hy-
drogen peroxide in situ,by aerobic oxidation of glu-
cose.
[62]
Similarly, immobilization of Mucor javanicus
lipase (EC 3.1.1.3) on functionalized silica nanoparti-
cles resulted in enhanced thermal stability and a high
retention of activity over a wider pH range
(Figure 6).
[63]
In a variation on this theme, Wang and Caruso
[64]
immobilized catalase (EC 1.11.1.6) on nanoporous
silica spheres, having a surface area of 630 m
2
g
À1
and
mesopores with a pore size of up to 40 nm, and subse-
quently assembled a nano-composite shell coating
composed of three layers of poly-dimethyldiallylam-
monium chloride (PDM) and 21 nm silica nanoparti-
cles. The resulting immobilisate displayed an activity
Figure 5. Amphiphilic polymer network for enzyme immobilization.
1294 asc.wiley-vch.de 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Adv. Synth. Catal. 2007, 349, 1289 – 1307
REVIEWS
Roger A. Sheldon
75 times that of catalase immobilized on mesoporous
silica spheres.
A novel type of immobilization on inorganic sup-
ports comprises the so-called protein-coated micro-
crystals (PCMCs).
[65,66]
The stabilization of lyophilized
enzyme powders through the addition of lyoprotec-
tants
[67]
and inorganic salts
[68]
is well documented.
PCMCs are prepared by mixing an aqueous solution
of the enzyme with a concentrated solution of a salt
such as potassium sulfate (a sugar or an amino acid
can also be used). The resulting solution is added
dropwise with vigorous mixing to a water-miscible sol-
vent such as isopropyl alcohol, whereupon micron-
sized crystals, containing the enzyme on the surface,
are formed. A major advantage of the technique is
that the enzyme molecules are dehydrated by a mech-
anism that leaves the majority of the enzymes in an
active conformation and minimizes denaturation. The
PCMCs can be separated and stored or used as a sus-
pension in an organic solvent. Obviously in an aque-
ous medium they dissolve to liberate the free enzyme.
In a transesterification of N-acetyltyrosine ethyl ester
with isopropyl alcohol (Figure 7) PCMCs of subtilisin
Carlsberg (EC 3.4.21.62) exhibited an activity three
Figure 6. Immobilization of a lipase on silica nanoparticles.
Figure 7. Resolutions with protein coated micro crystals (PCMCs).
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Enzyme Immobilization: The Quest for Optimum Performance
orders of magnitude higher than that of the lyophi-
lized powder.
[65]
Similarly, PCMCs of lipases showed a
substantial rate enhancement in most cases, compared
to the corresponding lyophilized powders, in the ki-
netic resolution (Figure 7) of 1-phenylethanol by
transesterification.
[65]
A further elaboration of the
technique employs solid state buffers as the support
to be coated.
[66]
The technique was also successfully
applied to oxidoreductases – an alcohol dehydrogen-
ase, catalase, soybean peroxidase and horse radish
peroxidase – affording PCMCs with enhanced activi-
ties in organic media.
[66]
3.5 Smart Polymers
A novel approach to immobilization of enzymes is via
covalent attachment to stimulus-responsive or smart
polymers which undergo dramatic conformational
changes in response to small changes in their environ-
ment, e.g., temperature, pH and ionic strength.
[69–71]
The most studied example is the thermo-responsive
and biocompatible polymer, poly-N-isopropylacryla-
mide (polyNIPAM). Aqueous solutions of polyNI-
PAM exhibit a critical solution temperature (LCST)
around 328C, below which the polymer readily dis-
solves in water while, above the LCST it becomes in-
soluble owing to expulsion of water molecules from
the polymer network. Hence, the biotransformation
can be performed under conditions where the enzyme
is soluble, thereby minimizing diffusional limitations
and loss of activity owing to protein conformational
changes on the surface of a support. Subsequently,
raising the temperature above the LCST leads to pre-
cipitation of the immobilized enzyme, thus facilitating
its recovery and reuse. An additional advantage of
using such thermo-responsive immobilized enzymes is
that runaway conditions are avoided because when
the reaction temperature exceeds the LCST the cata-
lyst precipitates and the reaction shuts down.
Two methods are generally used to prepare the
enzyme-polyNIPAM conjugates: (i) introduction of
polymerizable vinyl groups into the enzyme followed
by copolymerization with NIPAM or (ii) reaction of
NH
2
groups on the surface of the enzyme with a co-
polymer of NIPAM containing reactive ester groups
or the homopolymer containing an N-succinimide
ester function as the end group (Figure 8).
For example, penicillin G amidase (PA) was immo-
bilized by condensation with a copolymer of NIPAM
containing active ester groups.
[72]
The resulting
enzyme-polymer conjugate exhibited hydrolytic activ-
ity close to that of the free enzyme and was roughly
as effective as the free PA in the synthesis of cepha-
Figure 8. Thermoresponsive polymers for enzyme immobilization.
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Roger A. Sheldon
lexin by reaction of d-phenylglycine amide with 7-
ADCA (Figure 9).
More recently, an alternative thermo-responsive po-
lymer has been described.
[73]
It consists of random co-
polymers derived from 2-(2-methoxyethoxy)ethyl
methacrylate and oligo(ethylene glycol) methacrylate
(OEGMA) (see Figure 8) and combines the positive
features of poly(ethylene glycol), non-toxicity and
anti-immunogenicity, with thermo-responsive proper-
ties similar to polyNIPAM. The LCST could be
varied between 26 and 908C depending on the rela-
tive amounts of OEGMA used. These properties
make this a potentially interesting support for biocat-
alysts.
4 Entrapment
Enzymes can be immobilized by entrapment in sol-gel
matrices formed by hydrolytic polymerization of
metal alkoxides. Immobilization in silica sol gels pre-
pared by hydrolytic polymerization of tetraethoxysi-
lane, in the presence of the enzyme, was pioneered by
Avnir and co-workers
[74]
and has been used for the
immobilization of a wide variety of enzymes.
[75]
It
should be pointed out that the morphologies of the
silica sol-gels depend on the method of drying.
[76]
Drying by evaporation affords so-called xerogels in
which capillary stress causes a shrinkage of the nano
cages and pores. When alkylsiloxanes, RSi(OR)
3
are
used together with Si(OR)
4
the surface of the sol-gel
is more densely populated by the hydrophobic alkyl
groups and the capillary stresses which operate during
evaporation are largely attenuated, affording a so-
called ambigel in which there is no contraction of the
nano cages. Alternatively, drying with supercritical
carbon dioxide affords a so-called aerogel in which
the delicate pore structure is maintained. Silica aero-
gels have a phenomenal porosity; the Guinness Book
of Records refers to a silica aerogel with a density of
0.001 as the worlds lightest solid.
[76]
Reetz and co-workers found that when lipases were
entrapped in sol-gels produced from Si (OEt)
4
the re-
sulting immobilisates exhibited disappointingly low
activities in the esterification of lauric acid by 1-octa-
nol.
[77]
Reasoning that the microenvironment in the
sol-gel may be too hydrophilic they entrapped the
lipase in a sol-gel prepared from a mixture of Si-
(OMe)
4
and RSi (OMe)
3
containing non-hydrolyzable
alkyl moieties, on the premise that the more hydro-
phobic matrix would facilitate interfacial activation of
the lipase. This proved to be the case; they observed
rate enhancements of 2–8-fold compared with the tra-
ditional lyophilized lipase powder. This method has
been widely used for the immobilization of en-
zymes.
[78]
An interesting elaboration involves the ad-
dition of porous supports such as Celite during the
sol-gel process to bind the lipase-containing gels. This
“double immobilization” afforded materials with
higher thermal stability and activity.
[79]
More recently,
Reetz and co-workers
[80]
have reported a further im-
provement of the methodology, involving the use of
higher alkyl groups in the RSi (OMe)
3
precursor and/
or the use of additives such as isopropyl alcohol,
crown ethers, surfactants and KCl, with or without
the addition of Celite. These second generation sol-
gel immobilisates contained high lipase loadings, and
were highly active, robust and recyclable.
Pierre and co-workers have reported seminal stud-
ies on the entrapment of enzymes in silica aero-
gels.
[76,81,82]
For example, entrapment of Burkholderia
cepacia lipase in a hydrophobic silica sol-gel resulted
in an increase of all the kinetic constants, e.g., V
max
by
a factor of 10, in the esterification of lauric acid with
1-octanol.
[83]
More recently, lipases from Burkholderia
cepacia and Candida antarctica were entrapped in
silica aerogels, prepared from mixtures of Si (OMe)
4
and MeSi (OMe)
3
and reinforced with silica quartz
fiber felt to improve their mechanical properties.
[84]
The resulting biocatalysts showed activities similar to
that of Novozym 435 in the synthesis of biodiesel by
interesterification of sunflower seed oil with methyl
acetate in iso-octane. However, at high substrate con-
centrations, in the absence of solvent, they were less
effective than Novozym 435, presumably owing to dif-
fusion limitations.
Additives such as polyethylene glycol (PEG), poly-
vinyl alcohol and albumin, can have a stabilizing
effect on sol-gel entrapped enzymes. For example,
Zanin and co-workers
[85]
compared three different
methods – physical binding, covalent attachment and
gel entrapment, in the presence and absence of PEG
Figure 9. Cephalexin synthesis with pen G amidase/polyNIPAM conjugate.
Adv. Synth. Catal. 2007, 349, 1289 – 1307 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim asc.wiley-vch.de 1297
REVIEWS
Enzyme Immobilization: The Quest for Optimum Performance
1450 – for the immobilization of Candida rugosa
lipase. Their activities were determined in the hydrol-
ysis of olive oil. Immobilization yields varied from 3
to 32%, the most active biocatalyst resulting from the
encapsulation in the presence of PEG.
In an interesting variation on this theme, Naik and
co-workers
[86]
entrapped catalase and horseradish per-
oxidase (HRP) using a biosilification process. In
nature, diatoms are able to synthesize silica nanopar-
ticles by polymerization of silicic acid, catalyzed by
enzymes known as silicateins. A peptide subunit of
the silicatein from Cylindrotheca fusiformis has been
shown
[87]
to catalyze the formation of silica in vitro.
When this process was performed in the presence of
catalase or HRP this resulted in their entrapment.
Enzymes can also be entrapped in silicone elasto-
mers
[88]
and polydimethylsiloxane membranes.
[89]
Ko-
bayashi and co-workers
[90]
have recently described a
novel polymer-incarceration methodology for immo-
bilizing enzymes. The immobilization procedure is de-
picted in Figure 10. It involves dissolving polystyrene
containing hydrophilic tetraethylene glycol and glyci-
dol moieties as pendant groups in dichloromethane
and then adding a solution of CaLB. This is followed
by the addition of 1-hexane which causes coacerva-
tion to occur, affording a precipitate containing the
lipase in the polymer phase. After decantation of the
supernatant the polymeric matrix was cross-linked by
reaction of the pendant glycidol groups with a tria-
mine at 608C in hexane (see Figure 10) to afford a
polymer incarcerated lipase. The immobilisate was
used in the kinetic resolution of chiral secondary alco-
hols and could be reused 5 times without any loss of
activity.
5 Carrier-Free Immobilization by
Cross-Linking
In the early 1960s, studies of solid phase protein
chemistry led to the discovery that cross-linking of
dissolved enzymes via reaction of surface NH
2
groups
with a bifunctional chemical cross-linker, such as glu-
taraldehyde, afforded insoluble cross-linked enzymes
(CLEs) with retention of catalytic activity.
[91]
Howev-
er, this method of producing cross-linked enzymes
(CLEs) had several drawbacks, such as low activity
retention, poor reproducibility, low mechanical stabili-
ty, and difficulties in handling the gelatinous CLEs.
Mechanical stability and ease of handling could be
improved by cross-linking the enzyme in a gel matrix
or on a carrier but this led to the disadvantageous di-
lution of activity mentioned above. Consequently, in
the late 1960s, emphasis switched to carrier-bound en-
zymes, which became the most widely used industrial
methodology for enzyme immobilization for the next
three decades.
5.1 Cross-Linked Enzyme Crystals (CLECs)
The cross-linking of a crystalline enzyme by glutaral-
dehyde was first described by Quiocho and Richards
Figure 10. Polymer-incarcerated (PI) Candida antarctica lipase.
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Roger A. Sheldon
in 1964.
[92]
Their main objective was to stabilize
enzyme crystals for X-ray diffraction studies but they
also showed that catalytic activity was retained. The
use of cross-linked enzyme crystals (CLECs) as indus-
trial biocatalysts was pioneered by scientists at Vertex
Pharmaceuticals in the early 1990s
[93]
and subsequent-
ly commercialized by Altus Biologics.
[36]
The initial
studies were performed with CLECs of thermolysin
(EC 3.4.24.4), of interest in the manufacture of aspar-
tame, but the method was subsequently shown to be
applicable to a broad range of enzymes.
[36]
CLECs
proved significantly more stable to denaturation by
heat, organic solvents and proteolysis than the corre-
sponding soluble enzyme or lyophilized (freeze-dried)
powder. CLECs are robust, highly active immobilized
enzymes of controllable particle size, varying from 1
to 100 mm. Their operational stability and ease of re-
cycling, coupled with their high catalyst and volumet-
ric productivities, renders them ideally suited for in-
dustrial biotransformations. In a more recent exam-
ple, a CLEC of chloroperoxidase (CPO) from Caldar-
iomyces fumago exhibited a higher thermal stability
and tolerance to organic solvents than the free
CPO.
[94]
5.2 Cross-Linked Enzyme Aggregates (CLEAs

)
An inherent disadvantage of CLECs is the need to
crystallize the enzyme, which is often a laborious pro-
cedure requiring enzyme of high purity. On the other
hand, it is well-known
[95]
that the addition of salts, or
water-miscible organic solvents or non-ionic polymers,
to aqueous solutions of proteins leads to their precipi-
tation as physical aggregates of protein molecules,
held together by non-covalent bonding without per-
turbation of their tertiary structure, that is without de-
naturation. It was reasoned that subsequent cross-
linking of these physical aggregates would render
them permanently insoluble while maintaining their
pre-organized superstructure, and, hence their catalyt-
ic activity. This indeed proved to be the case and led
to the development of a new family of immobilized
enzymes: cross-linked enzyme aggregates (CLEA

)
(Figure 11). Since precipitation from an aqueous
medium, by addition of ammonium sulfate or polyeth-
ylene glycol, is often used to purify enzymes, the
CLEA methodology essentially combines purification
and immobilization into a single unit operation that
does not require a highly pure enzyme. It could be
Figure 11. Preparation of a CLEA.
Figure 12. Ampicillin synthesis.
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Enzyme Immobilization: The Quest for Optimum Performance
used, for example, for the direct isolation of an
enzyme, in a purified and immobilized form suitable
for performing biotransformations, from a crude fer-
mentation broth.
The first examples of CLEAs were derived from
penicillin G amidase, an industrially important
enzyme used in the synthesis of semi-synthetic peni-
cillin and cephalosporin antibiotics (see earlier). The
free enzyme exhibits limited thermal stability and a
low tolerance to organic solvents, making it an ideal
candidate for stabilization by immobilization. Indeed,
penicillin G amidase CLEAs, prepared by precipita-
tion with, for example, ammonium sulfate or tert-
butyl alcohol, proved to be effective catalysts for the
synthesis of ampicillin (Figure 12).
[96]
The CLEA exhibited a synthesis/hydrolysis ratio
(S/H) comparable with that of the commercial cata-
lyst, PGA-450 (penicillin G amidase immobilized on
polyacrylamide), and substantially higher than that of
the penicillin G amidase CLEC suggesting that diffu-
sional limitations are more severe in the CLEC. Re-
markably, the productivity of the CLEA was higher
even than that of the free enzyme that it was made
from and substantially higher than that of the CLEC.
In stark contrast, the commercial catalyst mainly con-
sists of non-catalytic ballast in the form of the poly-
acrylamide carrier which was reflected in its much
lower productivity. Analogous to the corresponding
CLECs, the penicillin G amidase CLEAs also main-
tained their high activity in organic solvents.
[97,98]
CLEAs were subsequently prepared from seven
commercially available lipases (EC 3.1.1.3) and the
effects of various parameters, such as the precipitant
and the addition of additives such as surfactants and
crown ethers, on their activities were investigated.
[99]
The activation of lipases by additives, such as surfac-
tants and crown ethers, is well-documented and is
generally attributed to the lipase being induced to
adopt a more active conformation.
[100]
Co-precipita-
tion of such additives with the enzyme followed by
cross-linking of the enzyme aggregates, can lock the
enzyme in this more favourable conformation. Fur-
thermore, since the additive is not covalently bonded
to the enzyme, it can subsequently be washed from
the CLEA using, for example, an appropriate organic
solvent to leave the immobilized enzyme locked in
the favourable confirmation. Using this procedure, a
variety of hyperactive lipase CLEAs exhibiting activi-
ties up to twelve times that of free enzyme were pre-
pared (Figure 13).
[99]
The experimental procedure was
further simplified by combining precipitation, in the
presence or absence of additives, with cross-linking
into a single operation.
[99]
These results clearly demonstrate the tremendous
potential of the CLEAs as immobilized enzymes, with
high catalyst and volumetric productivities, prepared
in a simple procedure from relatively impure enzyme
preparations. Indeed, the simplicity of the operation
lends itself to automation, e.g., using 96-well
plates.
[101]
Initial studies of CLEAs derived from the popular
Candida antarctica lipase B (CaLB) revealed that the
excellent performance observed in water, compared
to that of the standard immobilized form, Novozym
435 (CaLB immobilized on a macroporous acrylic
resin), could not be directly translated to organic
media. Consequently the preparation was modified to
produce a more lipophilic CLEA which could better
accommodate organic solvents. This afforded a dra-
matic improvement in the activity of CaLB CLEA in
the enantioselective acylation of 1-phenethylamine in
diisopropyl ether as solvent (Figure 14).
[102]
Clearly
the optimized CaLB CLEAs have activities surpass-
ing those of Nov 435 in both aqueous and organic
media.
Figure 13. Hyperactivation of lipase CLEAs.
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REVIEWS
Roger A. Sheldon
CaLB CLEAs also exhibit excellent activities in su-
percritical carbon dioxide
[103]
and ionic liquids.
[104]
Thus, CaLB CLEA displayed superior activity to that
of Novozym 435 in the kinetic resolution of 1-phenyl-
ethanol and 1-tetralol, by acylation with vinyl acetate,
in scCO
2
. The enzymatic resolution could also be
combined with the production of the 1-phenylethanol
substrate, by palladium-catalyzed hydrogenation of
acetophenone, using two separate columns in series
(one containing the Pd catalyst and the other the
CaLB CLEA), without the need for depressurization
(Figure 15).
[103]
In the kinetic resolution of 1-phenylethanol and 1-
phenylethylamine in the ionic liquids, [bmim] [NO
3
]
and [bmim] [N(CN)
2
] the best results were observed
with CaLB adsorbed and cross-linked on a polypropy-
lene carrier (Accurel EP100). In contrast, the free
CaLB or immobilized as Novozym 435 dissolves in
these ionic liquids with complete loss of activity.
An important property of CLEAs from the point of
view of large-scale applications is their particle size
which obviously has a direct effect on mass transfer
limitations and filterability. The enzyme and glutaral-
dehyde concentrations are, inter alia, important fac-
tors in determining the particle size of CLEAs as was
reported for Candida rugosa lipase.
[105]
Optimum ac-
tivity was observed with particles of 40–50 nm.
Glutaraldehyde is generally the cross-linking agent
of choice as it is inexpensive and readily available in
commercial quantities. However, with some enzymes,
e.g., nitrilases, low or no retention of activity was
sometimes observed using glutaraldehyde as the
cross-linker. A possible cause of deactivation is reac-
tion of the cross-linker with amino acid residues
which are crucial for the activity of the enzyme.
Owing to its high reactivity and small size, which
allows it to penetrate the interior of the protein, this
will be particularly severe with glutaraldehyde.
Hence, this could be avoided by using bulky polyalde-
hydes, obtained by periodate oxidation of dextrans, as
the cross-linkers,
[106]
followed by reduction of the
Schiffs base moieties with sodium borohydride to
form irreversible amine linkages. The activity reten-
tion of these CLEAs was generally much higher than
that observed with CLEAs prepared using glutaralde-
hyde. Dramatic results were obtained, for example,
Figure 14. Comparison of Novozym 435 with CaLB-CLEAs.
Figure 15. Sequential hydrogenation and resolution in scCO
2
.
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Enzyme Immobilization: The Quest for Optimum Performance
with a nitrilase (EC 3.5.5.13) from P. fluorescens and
with a nitrilase from the company Biocatalytics.
Cross-linking with glutaraldehyde produced a com-
pletely inactive CLEA while with dextran polyalde-
hyde 50–60% activity retention (not optimized) was
observed (see Table 1). Similarly, better activity reten-
tions were observed when penicillin amidase CLEAs
were prepared with dextran polyaldehyde compared
to glutaraldehyde.
[106]
Since cross-linking largely involves reaction of the
amino groups of lysine residues on the external sur-
face of the enzyme, every enzyme can be expected to
be, and is, different. For electronegative enzymes, that
contain a paucity of lysine residues on the surface,
cross-linking is expected to be less effective. One way
of compensating for this lack of surface amino groups
is to co-precipitate the enzyme with a polymer con-
taining numerous free amino groups, e.g., poly-l-
lysine
[107]
or polyethylene imine.
[108,109]
In a variation
on this theme, Gupta and co-workers
[110]
reported that
addition of bovine serum albumin (BSA) as a “prote-
ic feeder” in the preparation of CLEAs from solu-
tions containing low concentrations of enzymes facili-
tated the formation of the CLEA.
Other examples of hydrolases that have been suc-
cessfully cleated include pig liver esterase (EC
3.1.1.1), aminoacylase (EC 3.5.1.14), proteases, and
glycosidases (EC 3.2.1).
[111]
For example, the alkaline
protease from Bacillus licheniformis (alcalase, EC
3.4.21.62, also known as subtilisin Carlsberg), an inex-
pensive enzyme used in laundry detergents, has been
widely used in organic synthesis, e.g., in the resolution
of (amino acid) esters
[112]
and amines
[113]
and peptide
synthesis.
[114]
An alcalase CLEA showed excellent ac-
tivities and enantioselectivities in amino acid ester hy-
drolyses.
[115]
Interestingly, a CLEA of aminoacylase prepared
from a crude extract from Aspergillus sp. fermenta-
tion lacked the esterase activity observed with the
crude enzyme.
[116]
This strongly suggests that the ester-
olytic activity is derived from a protein impurity in
the crude aminoacylase preparation and illustrates the
potential of the CLEA methodology for performing
purification and immobilization in a single operation.
Similarly, CLEAs were successfully prepared from
the glycosidase, b-galactosidase (EC 3.2.1.23) from
Aspergillus oryzae and phytase (EC 3.1.3.26) from As-
pergillus niger.
[101]
The former enzyme catalyzes the
hydrolysis of lactose in dairy products and is adminis-
tered as tolerase to people suffering from lactose in-
tolerance and the latter is an acid phosphatase which
is added to animal feed in order to hydrolyse phytate
(inositol hexaphosphate).
Recyclable CLEAs were also prepared
[101]
from a
variety of oxidoreductases, e.g., glucose oxidase
(EC1.1.3.4), galactose oxidase (EC 1.1.3.9) and lac-
case (EC 1.10.3.2). Laccase, in particular, has many
potential applications, e.g., for bleaching in the pulp
and paper or textile industries, aqueous effluent treat-
ment and, in combination with the stable radical
TEMPO (2,2,6,6-tetramethyl-1-piperidinoxyl), for the
catalytic aerobic oxidation of starch to carboxystarch
(Figure 16). The latter can be made by TEMPO cata-
lyzed hypochlorite oxidation of starch but greener,
aerobic oxidation is more attractive. Immobilization
as a CLEA improves the stability of the laccase under
the reaction conditions, thereby reducing the enzyme
cost contribution.
Another benefit of the CLEA technology is that it
can stabilize the quaternary structures of multimeric
enzymes, a structural feature often encountered with
redox metalloenzymes. For example, the stability of
CLEAs from two tetrameric catalases (EC 1.11.1.6)
which, for the soluble enzymes is dependent on con-
centration, became independent of this parameter in
the CLEA, which allowed for the use of low concen-
trations of catalase.
[117]
Similarly, CLEAs have been
prepared from an alcohol dehydrogenase (EC 1.1.1.1)
from Rhodococcus erythropolis and a formate dehy-
drogenase (EC 1.2.1.2) from Candida boidinii.
[101]
Table 1. Effect of cross-linker on recovered activity of CLEA.
[106]
Enzyme Recovered activity [%]
[a]
Glutaraldehyde Dextran polyaldehyde
Nirilase (Ps. fluorescens) 0 50
Nitrilase (Biocatalytics 1004) 0 60
Penicillin G amidase 48 85–90
[a]
Relative to the free enzyme.
Figure 16. Laccase-catalyzed aerobic oxidation of starch.
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REVIEWS
Roger A. Sheldon
The methodology has also been successfully ap-
plied
[102]
to various C
À
C bond forming lyases, notably
the R- and S-specific oxynitrilases (hydroxynitrile
lyases, EC 4.1.2.10) which catalyze the hydrocyana-
tion of a wide range of aldehydes. For example, a
CLEA prepared from the (R)-specific oxynitrilase
from almonds, Prunus amygdalis (PaHnL) was highly
effective in the hydrocyanation of aldehydes under
microaqueous conditions and could be recycled ten
times without loss of activity.
[118]
CLEAs were similar-
ly prepared from the (S)-specific oxynitrilases from
Manihot esculenta and Hevea brasiliensis.
[119,120]
These
oxynitrilase CLEAs perform exceptionally well in or-
ganic solvents, affording higher enantioselectivities
than observed with the free enzymes owing to the es-
sentially complete suppression of competing non-en-
zymatic hydrocyanation.
A further elaboration of the CLEA methodology
involves the immobilization of lipase CLEAs by inclu-
sion in hydrophobic polytetrafluoroethylene mem-
branes for use in membrane bioreactors.
[121]
This
method has advantages compared with physical ad-
sorption or covalent binding. Similarly, inclusion of
glucose oxidase CLEAs in magnetic mesocellular
carbon foam was used to construct a magnetically
switchable bioelectrocatalytic system.
[122]
The prepara-
tion of CLEAs from CO
2
expanded micellar solutions
afforded dendritic CLEAs with tunable nanometer di-
mensions (7–38 nm).
[123]
6 Combi-CLEAs and Catalytic Cascade
Processes
The ultimate in environmental and economic efficien-
cy is to combine atom-efficient, catalytic, steps into a
one-pot, catalytic cascade process without the need
for separation of intermediates.
[28]
Catalytic cascade
processes have numerous potential benefits: fewer
unit operations, less reactor volume, and higher volu-
metric and space-time yields, shorter cycle times and
less waste generation. Furthermore, by coupling steps
together unfavourable equilibria can be driven to-
wards product. In principle, this can be achieved by
co-precipitation and cross-linking of two or more en-
zymes in combi CLEAs. For example, combi CLEAs
have been prepared from catalase in combination
with glucose oxidase or galactose oxidase. The cata-
lase serves to catalyze the rapid degradation of the
hydrogen peroxide formed in the aerobic oxidation of
glucose and galactose, respectively, catalyzed by these
enzymes.
A combi CLEA containing an S-selective oxynitri-
lase from Manihot esculenta and an aselective nitrilase
from Pseudomonas fluorescens, catalyzed the smooth,
one-pot conversion of benzaldehyde to S-mandelic
acid (Figure 17)
[124]
in diisopropyl ether/water (9:1) at
pH 5.5. The enantioselectivity is provided by the oxy-
nitrilase and in situ conversion by the nitrilase serves
to drive the equilibrium of the first step towards prod-
uct. In principle, this could also be achieved by using
an S-selective nitrilase in combination with non-enzy-
matic hydrocyanation but, unfortunately, there are no
nitrilases that exhibit S-selectivity with mandeloni-
triles. Interestingly, the combi CLEA was more effec-
tive than a mixture of the two separate CLEAs. A
possible explanation is that the close proximity of the
two enzymes inside the combi CLEA is more favour-
able, compared to the case with two separate CLEAs,
for transfer of the product of the first step to the
active site of the enzyme for the second step.
7 Enzyme-Immobilized Microchannel Reactors for
Process Intensification
Microreactor technology is an interdisciplinary field
that has attracted much attention recently.
[125]
Process
intensification through the use of microchannel reac-
tors (microfluidic devices) has many advantages com-
pared with traditional batch process technologies,
such as rapid mass and heat transfer and large surface
area to volume ratios. These are attractive features
for conducting catalytic reactions in microreactors
containing the enzyme immobilized on their inner
walls. Maeda and co-workers
[107,126,127]
developed a mi-
croreactor in which enzymes are immobilized as an
enzyme-polymer membrane on the inner walls of the
microchannels.
[127]
Thus, a solution of the enzyme
(e.g., a-chymotrypsin) in aqueous buffer was mixed
with glutaraldehyde and formaldehyde as cross-link-
ers in commercially available polytetrafluoroethylene
(PTFE) tubing (inner diameter 500 mm). This results
in the formation of a CLEA membrane on the inner
walls of the tubing. This technique has been used to
prepare CLEA-based enzyme microreactors (CEMs)
Figure 17. One-pot conversion of benzaldehyde to (S)-man-
delic acid.
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from a wide variety of enzymes.
[107]
With electronega-
tive enzymes (such as aminoacylase) coprecipitation
of the enzyme in the presence of poly-l-lysine (see
above) was used to realize fast and efficient CLEA
formation.
[107]
The use of such enzyme-immobilized
microchannel reactors clearly has considerable poten-
tial for the design of green and sustainable biotrans-
formations.
In the context of incorporating the immobilized
biocatalyst in the reactor configuration, the develop-
ment of a monolithic stirrer reactor by Lathouder and
co-workers deserves a special mention.
[128]
According
to this novel concept, the enzyme is immobilized on a
honeycomb, ceramic monolith, analogous to those
used in catalytic exhaust systems in automobiles. Cor-
dierite monoliths were functionalized, by coating with
polyethylene imine or different types of carbon, in
order to create adsorption sites for the enzyme
(CaLB). These were then employed in a monolithic
stirrer reactor in which the monolithic structure is in-
corporated in the stirrer blades. This concept was
tested in the CaLB-catalyzed transesterification of 1-
butanol with vinyl acetate.
[128]
Carbon nanofiber-coated monoliths performed the
best with regard to catalyst productivity and did not
exhibit any leaching under the reaction conditions.
The activity was lower (by a factor of 2–4) than Novo-
zym 435 or free CaLB but the latter preparations de-
activated much faster. In contrast, the monolithic cat-
alysts were operationally stable for several weeks,
without significant loss of activity.
8 Conclusions and Prospects
Hopefully, it is clear from this review that the subject
of enzyme immobilization continues to attract consid-
erable attention from researchers in both industry
and academia. Novel concepts continue to appear, a
recent example being single enzyme nanoparticles
(SENs) in nanoporous silica.
[129]
However, many of
these innovations involve the use of rather exotic sup-
ports which are substantially more expensive than the
enzyme to be immobilized. Consequently, they are
unlikely to be applied in industrial biotransformations
but could be interesting for applications in biosensors
or other devices where the enzyme cost contribution
is less of an issue. They can also provide important in-
sights into the effects of enzyme immobilization on
activity and stability.
For application in industrial biotransformations the
cost contribution of the (immobilized) enzyme is an
important issue. Clearly, the immobilization method-
ology, in addition to providing an active and stable
biocatalyst, should be a relatively simple operation
that does not require a highly pure enzyme prepara-
tion or an expensive support that may not be com-
mercially available. Immobilization as silica granu-
lates, for example, meets all these criteria but the
methodology is not applicable to aqueous environ-
ments (see earlier). Cross-linked enzyme aggregates
(CLEAs) would appear to have considerable industri-
al potential based on their high activity retention and
stability coupled with ease of preparation from crude
enzyme samples and no requirement for a support.
Because they are close to 100% active catalyst they
also display high catalyst productivities and space-
time yields. However, properties such as mechanical
strength and filterability still have to be demonstrated
on an industrially relevant scale.
It is also clear that every enzyme is different and,
consequently, there is no all-encompassing, one size
fits all solution to the problem of enzyme immobili-
zation. Based on the increasing importance of en-
zymes in a plethora of industrial applications, interest
in improving their operational performance will cer-
tainly continue unabated. The quest for optimum per-
formance continues.
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REVIEWS
Enzyme Immobilization: The Quest for Optimum Performance

[27] Immobilization is generally necessary for optimum performance in non-aqueous media. the development of an effective method for its immobilization was crucial for the application of penicillin amidase in the industrial synthesis of blactam antibiotics. which has the advantage that the enzyme cannot be leached from the surface. Catal. many of the enzyme molecules are not readily accessible to substrate molecules. for example. by glucose isomerase-catalyzed isomerization of glucose has a productivity of 11. Jay Kochi in the USA. However. a biopolymer or an inorganic polymer such as (mesoporous) silica or a zeolite. Hence. novel entrapment methods. by Degussa in the commercial synthesis of enantiomerically pure amino acids using hydrolases or dehydrogenases. e. Ionic binding is generally stronger and covalent binding of the enzyme to the support even more so. In the traditional method of using enzymes as lyophilized (freeze-dried) powders. Weinheim .. KGaA. to prevent enzyme leakage entirely.wiley-vch.de  2007 Wiley-VCH Verlag GmbH & Co.C. binding to a support (carrier). or a membrane device such as a hollow fiber or a microcapsule. additional covalent attachment is often required. This was followed by post-doctoral studies with Prof. such as the use of novel supports. fructose.[22] Indeed. Furthermore. The difference between entrapment and support binding is often not clear. three traditional methods of enzyme immobilization can be distinguished. Another approach to facilitating the recovery and re-use of enzymes is to perform the reactions. From 1969 to 1980 he was with Shell Research in Amsterdam and from 1980 to 1990 he was R&D Director of DSM Andeno. for example.e. with the free enzyme. entrapment (encapsulation) and cross-linking.. since it involves the free enzyme it falls outside the scope of this review. immobilization) of the different catalysts thus circumventing their mutual interaction which could result in inhibition and or deactivation. Another benefit of immobilization is that it enables the use of enzymes in multienzyme and chemoenzymatic cascade processes (see later).REVIEWS Roger Sheldon (1942) is Professor of Biocatalysis and Organic Chemistry at Delft University of Technology (Netherlands).11)-catalyzed hydrolysis of penicillin G. 600 kg of 6-APA are produced per kg of immobilized enzyme and the production of the commodity. smart polymers. E. He received a PhD in organic chemistry from the University of Leicester (UK) in 1967. when an enzyme is immobilized in a prefabricated mesoporous Adv. dispersion of the enzyme molecules by immobilization generally provides for a better accessibility and/or an extra stabilization of the enzymes towards denaturation by the organic medium.[28–31] A major problem encountered in the design of catalytic cascade processes is the incompatibility of the different catalysts and a possible solution is compartmentalization (i. For example. or covalent in nature. Synth. 1290 asc. 349. this also has a disadvantage: if the enzyme is irreversibly deactivated both the enzyme and the (often costly) support are rendered unusable. particularly in relation to fine chemicals production. The support can be a synthetic resin. The methodology is applied commercially. (ii) Entrapment via inclusion of an enzyme in a polymer network (gel lattice) such as an organic polymer or a silica sol-gel. For the purpose of this review we define support binding as the binding of an enzyme to a prefabricated support (carrier) irrespective of whether the enzyme is situated on the external or internal surface. 3. Sheldon In this review we shall focus on recent developments in novel immobilization techniques. and the recent development of cross-linked enzyme aggregates (CLEAs).5. Entrapment requires the synthesis of the polymeric network in the presence of the enzyme. lyophilization can cause pronounced structural perturbations often leading to deactivation. However. however. in a membrane bioreactor consisting of an ultrafiltration membrane which retains the enzyme on the basis of its size but allows substrates and products to pass through. His primary research interests are in the application of catalytic methodologies – homogeneous. 2007.1. Roger A. (i) Support binding can be physical (such as hydrophobic and van der Waals interactions).[32] However. 1289 – 1307 the production of 6-aminopenicillanic acid (6-APA) by penicillin G amidase (penicillin amidohydrolase. ionic.g. In contrast. physical bonding is generally too weak to keep the enzyme fixed to the carrier under industrial conditions of high reactant and product concentrations and high ionic strength. He developed the concepts of E factors and atom utilization for assessing the environmental impact of chemical processes.000. in the quest for optimum performance in biotransformations. The physical restraints generally are too weak. heterogeneous and enzymatic – in organic synthesis. 2 Types of Immobilization Basically. This combines ease of recovery and recycling with the high activity of the free enzyme.

[38] A major drawback of Eupergit C is diffusion limitations. 349. ranging from 90 % to > 99 %. from the literally thousands of papers on enzyme immobilization. as would be expected. and does not swell or shrink even upon drastic pH changes in this range. Catal. at neutral or alkaline pH. details of the immobilization of industrial biocatalysts are often not disclosed. glycidyl methacrylate. 3. various porous acrylic resins.[35] Consequently. Italy) consist of a polymethacrylate-based resin functionalized with oxirane groups and exhibit characteristics similar to Eupergit C. It binds proteins via reaction of its oxirane moieties.[34] The design of carrier-bound immobilized enzymes also relies largely on laborious and time-consuming trial and error experiments. The use of a carrier inevitably leads to dilution of activity.wiley-vch. The remaining silica the enzyme may be situated largely in the mesopores but this would not be entrapment. to prepare carrierless macroparticles.[33] especially at high enzyme loadings.[36] and cross-linked enzyme aggregates (CLEAs).de 1291  2007 Wiley-VCH Verlag GmbH & Co. there is an increasing interest in carrier-free immobilized enzymes. Synth. it is difficult to make comparisons of the different methodologies as most authors compare the performance of the immobilized enzyme. allyl glycidyl ether and methacrylamide with Adv. using a bifunctional reagent.[27. such as crosslinked enzyme crystals (CLECs).[37] This approach offers clear advantages: highly concentrated enzyme activity in the catalyst. The interaction between the two provides an immobilized enzyme with specific chemical. such as Amberlite XAD-7. 2007. a biopolymer or an inorganic solid.) to prevent any undesired support-protein reaction. owing to the introduction of a large portion of non-catalytic ballast. are more pronounced in kinetically controlled processes. high stability and low production costs owing to the exclusion of an additional (expensive) carrier. 1289 – 1307 epoxy groups can be rendered inactive by capping using a variety of reagents (mercaptoethanol. antarctica lipase B (CaLB). For example.Enzyme Immobilization: The Quest for Optimum Performance REVIEWS average particle size of 170 mm and a pore diameter of 25 nm. Immobilization of enzymes on Eupergit C. biochemical. 3 Immobilization on Supports: Carrier-Bound Enzymes The properties of supported enzyme preparations are governed by the properties of both the enzyme and the carrier material. Figure 1. immobilization of an enzyme on a carrier often leads to the loss of more than 50 % native activity. ethanolamine. mechanical and kinetic properties. because of the lack of guidelines that link the nature of a selected carrier to the performance expected for a given application.[39] Similarly.[22] Moreover. KGaA. prepared using a particular technique. because asc. for example. A disadvantage of immobilization in this way is that. maintained 60 % of its initial activity over > 800 cycles. etc. It should be pointed out that. This “multi-point-attachment” is largely responsible for the high operational stability of enzymes bound to Eupergit C. The support (carrier) can be a synthetic organic polymer. Sepa beads FP-EP (Resindion.[38] It is highly hydrophilic and stable. over a pH range from 0 to 14. Weinheim . Eupergit C is a macroporous copolymer of N. Due to the high density of oxirane groups on the surface of the beads enzymes are immobilized at various sites of their structure. the effects of which. glycine. Milan. both chemically and mechanically. which results in lower space-time yields and productivities.N’-methylene-bi-(methacrylamide).1 Synthetic Organic Polymers Acrylic resins such as Eupergit C are widely used as supports. On the other hand when the enzyme is present during the synthesis of a silica sol-gel the enzyme is entrapped.38] Penicillin amidase on Eupergit C. Immobilization by covalent attachment to Eupergit C has been successfully applied to a variety of enzymes for industrial application. In addition. with that of the free enzyme but do not compare different methods of immobilization. the widely used enzyme C.[40] is commercially available in immobilized form as Novozym 435 which consists of the enzyme adsorbed on a macroporous acrylic resin. with the free amino groups of the enzyme to form covalent bonds which have long-term stability within a pH range of pH 1 to 12 (see Figure 1). are used to immobilize enzymes via simple adsorption without covalent attachment. ACHTUNGRE(iii) Cross-linking of enzyme aggregates or crystals.

[50] The resulting immobilisate was used for the enantioselective reduction of a broad range of hydrophobic prochiral ketones to the corresponding (R)-secondary alcohols (Figure 3) in n-hexane as solvent. The enzyme was immobilized by ionic adsorption on DEAE-Sephadex which consists of cellulose modified with diethylaminoethyl functionalities (Figure 2) and the process was performed in continuous operation in a fixed-bed reactor. consequently. the highest activity was observed with sepa beads (see above) containing octadecyl chains.2 Biopolymers A variety of biopolymers. 3.g. in the immobilization of whole cells of Rhodococcus equi A4. agarose and chitosan[42] and proteins such as gelatin and albumin have been widely used as supports for immobilizing enzymes. KGaA. For example. Polyvinyl alcohol (PVA) cryogels formed by the freeze-thawing method. using a recombinant benzaldehyde lyase (BAL) from Pseudomonas fluorescens immobilized in a PVA hydrogel.[43] first commercialized more than 40 years ago. Synth. the immobilized biocatalyst showed improved thermal stability and long-term stability under the reaction conditions.[44] An activity retention of 70 % was observed in the resolution of p-chlorostyrene oxide under biphasic conditions. In addition to stability towards the organic solvent. The immobilized biocatalyst was active at high substrate concentrations (306 g/ L) and could be recycled 7 times but it exhibited a slightly lower enantioselectivity compared to the free enzyme (E = 76 vs. In non-aqueous media enzymes can also be immobilized in natural or synthetic hydrogels or cryogels. can diffuse out of the gel matrix and are. by crosslinking (see later). Candida antarctica and Rhizomucor miehei on supports with varying hydrophobicity with Novozym 435 (see below). in the esterification of oleic acid with 1-butanol in isooctane. where the enzyme is not leached from the gel matrix. immobilization of free enzymes in PVA hydrogels can be useful in organic media.3 Hydrogels 3.[49] However. Roger A. the enzyme can be leached from the support in an aqueous medium. 349.de  2007 Wiley-VCH Verlag GmbH & Co.wiley-vch. Co-factor regeneration was achieved within the gel matrix using isopropyl alcohol as the co-substrate (total turnover number of the co-factor 102–103). have been widely used for immobilization of whole cells. 2007. 1292 asc. leached in an aqueous medium.[41] This was attributed to the hydrophobic nature of the support facilitating opening of the hydrophobic lid of the lipase. Catal. in the immobilization of a recombinant epoxide hydrolase from Aspergillus niger. owing to their smaller size. easy separation (diameter 3–5 mm and thickness 200–400 mm) and stability towards degradation. Indeed. which contains nitrile hydratase and amidase activities. Sheldon This method is still widely used.[46] A mild and highly efficient method for preparing PVA hydrogels by partial drying at room temperature afforded lens-shaped hydrogels (Lentikats) exhibiting good mechanical stability. 1289 – 1307 Figure 2. in n-hexane as solvent. acetophenone afforded (R)-1-phenylethanol in > 98 % ee.[47] Lentikats have been successfully used for the entrapment of whole cell biocatalysts. In order to entrap free enzymes the size of the enzyme must be increased. the first industrial application of an immobilized enzyme in a biotransformation is the Tanabe process.[45] for example. e. The same group reported the enantioselective benzoin condensation of aromatic and heteroaromatic aldehydes.. in order to retain the enzyme in a PVA hydroAdv. For example. Tanabe aminoacylase process. In contrast. free enzymes. Ansorge-Schumacher and coworkers reported the co-immobilization of an alcohol dehydrogenase (EC 1. E = 90).1) from Lactobacillus kefir together with its co-factor. for the production of l-amino acids by resolution of racemic acylamino acids using an aminoacylase from Aspergillus oryzae (Figure 2).g.REVIEWS it is not covalently bound. e.[51] As noted above. Weinheim .49] for example. In a comparison of the immobilization of lipases of Humicola lanuginosa.. starch. mainly water-insoluble polysaccharides such as cellulose.1.1.[48. in PVA beads. NADP.

Upon drying the phase shrinks. EC 1. In contrast. the molecular weight must be increased. nanophase sepaAdv. Synth. which was asc. in two rounds of error-prone PCR. Immobilization of horseradish peroxidase (HRP.2.2.1. In an interesting recent development. Hence. EC 1. directed evolution of the formate dehydrogenase (EC 1. When the polymer network is immersed in a non-polar organic solvent the hydrophobic PDMS domain swells allowing access of a dissolved substrate which can approach the enzyme via the interface between the two domains.1. Alcohol dehydrogenase in Lentikat. KGaA. gel. 1289 – 1307 Figure 4. whereby the enzyme is situated in its hydrophilic domains which consist of poly(2-hydroxyethyl acrylate) (PHEA). rated.wmethacroyloxypropyl-poly(dimethylsiloxane) (see Figure 5). Catal. The amphiphilic network was synthesized by UVinitiated radical copolymerization of a silylated 2-hydroxyethyl acrylate monomer and a bifunctional a. The cross-linked enzyme was subsequently entrapped in a Lentikat PVA hydrogel. 2007. compared to the free enzymes (see Figure 5). The resulting immobilized biocatalyst had a well-defined particle size of 3–5 mm and showed no leaching in the enantioselective hydrocyanation of benzaldehyde in a biphasic aqueous buffer/organic solvent system.1. Owing to their ampholytic character. glutamic acid and cysteine residues remote from the active site.1. was used to create variants that were more suited for immobilization in a polyacrylamide gel. Weinheim . 349.10) in this way afforded immobilisates showing substantially enhanced activities and operational stabilities in heptane. Grçger and co-workers[52] achieved this by cross-linking an (R)-oxynitrilase using a mixture of glutaraldehyde and chitosan (Figure 4). no enhancement of activity was observed in isopropyl alcohol as solvent. The stabilization resulted from a substitution of lysine. amphiphilic network (see Figure 5). trapping the enzyme in an enzyme-friendly environment.[54] The best mutant had a 4. depending on the pH of the medium. they can form complexes with oppositely charged polyelectrolytes. in the presence of water.4-fold higher activity compared to the free enzyme immobilized in the gel.The hydrophilic domain swells in the presence of water and the polymer is loaded with the enzyme by immersing it in an aqueous solution of the biocatalyst. proteins exist as polycations or polyanions. Cross-linked (R)-oxynitrilase in Lentikat hydrogel. Bruns and Tiller have reported[55] a novel immobilization of enzymes in a prefabricated.7) and chloroperoxidase (CPO. An alternative method to increase the size of the enzyme is to form a complex with a polyelectrolyte.2) from Candida boidinii. The immobilized biocatalyst retained 45 % of its activity and lost no activity over five cycles. It could be recycled 20 times without loss of yield or enantioselectivity. The substrate that diffuses into the hydrophobic polydimethylsiloxane (PDMS) phase can access the enzyme via the large interface.Enzyme Immobilization: The Quest for Optimum Performance REVIEWS Figure 3. This principle was used by Dautzenberg and co-workers[53] to immobilize amyloglucosidase (EC 3.11. owing to its nanophase separation and peculiar swelling properties.3) by coupling to an excess of a complex of sodium polystyrenesulfonate (PSS) or poly-diallyldimethylammonium chloride (PDM).de 1293  2007 Wiley-VCH Verlag GmbH & Co.11.wiley-vch.

3. The CaLB silica granules exhibited a similar activity to Novozym 435 in the direct synthesis of the skin emollient. 349. attributed to swelling of the PHEA phase rather than the PDMS phase in this polar solvent. e.de  2007 Wiley-VCH Verlag GmbH & Co.g. KGaA.[59] Covalent binding of a-chymotrypsin (EC 3. The resulting immobilisate displayed an activity asc. which had been modified by reaction of surface hydroxy groups with 3.[56] zeolites[57. Mesoporous silicas. the CaLB silica granules can be used in a direct ester synthesis if the water is removed by. by first adsorbing the lipase on silica powder followed by agglomeration. Because of the large pore sizes of these materials they can accommodate relatively small enzymes in the pores. isopropyl myristate. Granulation technology was used to immobilize CaLB lipase on silica granules. and SBA-15. evaporation under vacuum.4 Inorganic Supports A variety of inorganic solids can be used for the immobilization of enzymes. One of the simplest and most inexpensive methods to immobilize an enzyme is by silica granulation. 1289 – 1307 . Synth.[40] Owing to the composition of the granulates. In an aqueous medium the lipase is desorbed and the particle slowly disintegrates. to formulate enzymes for detergent powders which release the enzyme into the washing liquid during washing. However. which are nowadays often referred to as nanosilicas.[56] Furthermore..6) on nanoporous silica spheres.. In order to maintain its integrity in an aqueous environment the enzyme needs to be covalently bonded to the silica support. Wang and Caruso[64] immobilized catalase (EC 1. silica. very high surface areas (300–1500 m2 gÀ1) and volumes (ca.[62] Similarly. and are inert and stable at elevated temperatures. the surface can be easily functionalized. Sheldon Figure 5. alumina. e. Applying the granules in packed-bed reactors also minimizes the contact time with high water concentrations. have several advantages as sup1294 ports: they have uniform pore diameters (2–40 nm).e.4. in the enantioselective (E = 85) hydrolysis of p-nitrostryrene oxide. afforded an immobilized catalyst with a half-life one thousand times that of the free enzyme. Weinheim Adv.1.wiley-vch. Amphiphilic polymer network for enzyme immobilization.2) to a mesoporous sol-gel glass. On the other hand.11. and subsequently assembled a nano-composite shell coating composed of three layers of poly-dimethyldiallylammonium chloride (PDM) and 21 nm silica nanoparticles.21.[62] The immobilized preparation was used together with free glucose oxidase which generated hydrogen peroxide in situ. for example. 3.3) on functionalized silica nanoparticles resulted in enhanced thermal stability and a high retention of activity over a wider pH range (Figure 6). 1 mL gÀ1).g.[40] It is used. Moreover.[61] Immobilization of chloroperoxidase (CPO) from Caldariomyces fumago on the same material resulted in increased stability towards organic solvents.3trimethoxypropanal. when the calcined material adsorbs much more enzyme this indicates that most of the enzyme is situated in the pores. they are intended for use only in organic media. the latter still contains the template). an enhanced stability towards 20 % DMSO as solvent was observed and no leaching occurred in a filtration test.REVIEWS Roger A. which was retained over a period of months.1. immobilization of epoxide hydrolase from Aspergillus niger by covalent attachment to functionalized silica resulted in 90 % activity retention.[63] In a variation on this theme. For example. If these values are roughly the same then most of the enzyme is on the outer surface. 2007.1. Whether the enzyme is situated inside the pores or on the outer surface can be determined by comparing immobilization on calcined and non-calcined material (i.by aerobic oxidation of glucose.. immobilization of Mucor javanicus lipase (EC 3. Catal.58] and mesoporous silicas[59–62] such as MCM41. having a surface area of 630 m2 g À1and mesopores with a pore size of up to 40 nm.

KGaA. The PCMCs can be separated and stored or used as a suspension in an organic solvent. PCMCs are prepared by mixing an aqueous solution of the enzyme with a concentrated solution of a salt such as potassium sulfate (a sugar or an amino acid can also be used). 1289 – 1307  2007 Wiley-VCH Verlag GmbH & Co.de 1295 .4.66] The stabilization of lyophilized enzyme powders through the addition of lyoprotectants[67] and inorganic salts[68] is well documented. In a transesterification of N-acetyltyrosine ethyl ester with isopropyl alcohol (Figure 7) PCMCs of subtilisin Carlsberg (EC 3. 75 times that of catalase immobilized on mesoporous silica spheres. The resulting solution is added dropwise with vigorous mixing to a water-miscible sol- vent such as isopropyl alcohol. Weinheim asc. A major advantage of the technique is that the enzyme molecules are dehydrated by a mechanism that leaves the majority of the enzymes in an active conformation and minimizes denaturation. are formed. containing the enzyme on the surface. Adv. 349.wiley-vch. Obviously in an aqueous medium they dissolve to liberate the free enzyme.Enzyme Immobilization: The Quest for Optimum Performance REVIEWS Figure 6. 2007.62) exhibited an activity three Figure 7. Immobilization of a lipase on silica nanoparticles. Synth.21. A novel type of immobilization on inorganic supports comprises the so-called protein-coated microcrystals (PCMCs).[65. Resolutions with protein coated micro crystals (PCMCs). whereupon micronsized crystals. Catal.

thereby minimizing diffusional limitations and loss of activity owing to protein conformational changes on the surface of a support. the biotransformation can be performed under conditions where the enzyme is soluble. poly-N-isopropylacrylamide (polyNIPAM). Catal. Aqueous solutions of polyNIPAM exhibit a critical solution temperature (LCST) around 32 8C.wiley-vch. temperature.[65] A further elaboration of the technique employs solid state buffers as the support to be coated. soybean peroxidase and horse radish peroxidase – affording PCMCs with enhanced activities in organic media. 349. Thermoresponsive polymers for enzyme immobilization. Weinheim Adv. KGaA. penicillin G amidase (PA) was immobilized by condensation with a copolymer of NIPAM containing active ester groups. Subsequently.5 Smart Polymers A novel approach to immobilization of enzymes is via covalent attachment to stimulus-responsive or smart polymers which undergo dramatic conformational changes in response to small changes in their environment. Hence. below which the polymer readily dissolves in water while.[72] The resulting enzyme-polymer conjugate exhibited hydrolytic activity close to that of the free enzyme and was roughly as effective as the free PA in the synthesis of cepha- Figure 8. Synth. 2007. raising the temperature above the LCST leads to precipitation of the immobilized enzyme.REVIEWS orders of magnitude higher than that of the lyophilized powder. pH and ionic strength.de  2007 Wiley-VCH Verlag GmbH & Co. Sheldon 3. e.[65] Similarly.[66] The technique was also successfully applied to oxidoreductases – an alcohol dehydrogenase. above the LCST it becomes in- soluble owing to expulsion of water molecules from the polymer network. PCMCs of lipases showed a substantial rate enhancement in most cases.[69–71] The most studied example is the thermo-responsive and biocompatible polymer. 1289 – 1307 .[66] Roger A. in the kinetic resolution (Figure 7) of 1-phenylethanol by transesterification. catalase. 1296 asc.g. compared to the corresponding lyophilized powders. Two methods are generally used to prepare the enzyme-polyNIPAM conjugates: (i) introduction of polymerizable vinyl groups into the enzyme followed by copolymerization with NIPAM or (ii) reaction of NH2 groups on the surface of the enzyme with a copolymer of NIPAM containing reactive ester groups or the homopolymer containing an N-succinimide ester function as the end group (Figure 8). thus facilitating its recovery and reuse. For example. An additional advantage of using such thermo-responsive immobilized enzymes is that runaway conditions are avoided because when the reaction temperature exceeds the LCST the catalyst precipitates and the reaction shuts down..

affording a socalled ambigel in which there is no contraction of the nano cages. 4 Entrapment Enzymes can be immobilized by entrapment in sol-gel matrices formed by hydrolytic polymerization of metal alkoxides. at high substrate concentrations. surfactants and KCl. This proved to be the case. lexin by reaction of d-phenylglycine amide with 7ADCA (Figure 9). in the presence and absence of PEG asc. 1289 – 1307 sol-gel may be too hydrophilic they entrapped the lipase in a sol-gel prepared from a mixture of SiACHTUNGRE(OMe)4 and RSiACHTUNGRE(OMe)3 containing non-hydrolyzable alkyl moieties. crown ethers. Additives such as polyethylene glycol (PEG).wiley-vch. Reetz and co-workers[80] have reported a further improvement of the methodology. More recently.[83] More recently. an alternative thermo-responsive polymer has been described.[78] An interesting elaboration involves the addition of porous supports such as Celite during the sol-gel process to bind the lipase-containing gels. they were less effective than Novozym 435. in the absence of solvent. non-toxicity and anti-immunogenicity.Enzyme Immobilization: The Quest for Optimum Performance REVIEWS Figure 9. the Guinness Book of Records refers to a silica aerogel with a density of 0.001 as the worlds lightest solid. and were highly active. drying with supercritical carbon dioxide affords a so-called aerogel in which the delicate pore structure is maintained. The LCST could be varied between 26 and 90 8C depending on the relative amounts of OEGMA used. presumably owing to diffusion limitations. Weinheim . Pierre and co-workers have reported seminal studies on the entrapment of enzymes in silica aerogels.. However. When alkylsiloxanes. on the premise that the more hydrophobic matrix would facilitate interfacial activation of the lipase. This “double immobilization” afforded materials with higher thermal stability and activity. Synth.g. These properties make this a potentially interesting support for biocatalysts. Vmax by a factor of 10. These second generation solgel immobilisates contained high lipase loadings. prepared from mixtures of SiACHTUNGRE(OMe)4 and MeSiACHTUNGRE(OMe)3 and reinforced with silica quartz fiber felt to improve their mechanical properties. Catal.82] For example. they observed rate enhancements of 2–8-fold compared with the traditional lyophilized lipase powder. Zanin and co-workers[85] compared three different methods – physical binding.[79] More recently. Cephalexin synthesis with pen G amidase/polyNIPAM conjugate.[76. with thermo-responsive properties similar to polyNIPAM. lipases from Burkholderia cepacia and Candida antarctica were entrapped in silica aerogels. in the presence of the enzyme. Silica aerogels have a phenomenal porosity.[76] Drying by evaporation affords so-called xerogels in which capillary stress causes a shrinkage of the nano cages and pores.[73] It consists of random copolymers derived from 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate (OEGMA) (see Figure 8) and combines the positive features of poly(ethylene glycol). Alternatively. For example. robust and recyclable.[84] The resulting biocatalysts showed activities similar to that of Novozym 435 in the synthesis of biodiesel by interesterification of sunflower seed oil with methyl acetate in iso-octane. in the esterification of lauric acid with 1-octanol. RSi(OR)3 are used together with Si(OR)4 the surface of the sol-gel is more densely populated by the hydrophobic alkyl groups and the capillary stresses which operate during evaporation are largely attenuated. 2007. entrapment of Burkholderia cepacia lipase in a hydrophobic silica sol-gel resulted in an increase of all the kinetic constants. Immobilization in silica sol gels prepared by hydrolytic polymerization of tetraethoxysilane. with or without the addition of Celite. can have a stabilizing effect on sol-gel entrapped enzymes.[75] It should be pointed out that the morphologies of the silica sol-gels depend on the method of drying. polyvinyl alcohol and albumin. 349.[76] Reetz and co-workers found that when lipases were entrapped in sol-gels produced from SiACHTUNGRE(OEt)4 the resulting immobilisates exhibited disappointingly low activities in the esterification of lauric acid by 1-octanol. KGaA. covalent attachment and gel entrapment. e.[77] Reasoning that the microenvironment in the Adv. was pioneered by Avnir and co-workers[74] and has been used for the immobilization of a wide variety of enzymes. involving the use of higher alkyl groups in the RSiACHTUNGRE(OMe)3 precursor and/ or the use of additives such as isopropyl alcohol.de 1297  2007 Wiley-VCH Verlag GmbH & Co. This method has been widely used for the immobilization of enzymes.81.

It involves dissolving polystyrene containing hydrophilic tetraethylene glycol and glycidol moieties as pendant groups in dichloromethane and then adding a solution of CaLB. When this process was performed in the presence of catalase or HRP this resulted in their entrapment. Immobilization yields varied from 3 to 32 %. After decantation of the supernatant the polymeric matrix was cross-linked by reaction of the pendant glycidol groups with a triamine at 60 8C in hexane (see Figure 10) to afford a polymer incarcerated lipase. 5. Synth. Mechanical stability and ease of handling could be improved by cross-linking the enzyme in a gel matrix or on a carrier but this led to the disadvantageous dilution of activity mentioned above. the most active biocatalyst resulting from the encapsulation in the presence of PEG. Their activities were determined in the hydrolysis of olive oil.REVIEWS 1450 – for the immobilization of Candida rugosa lipase. this method of producing cross-linked enzymes (CLEs) had several drawbacks. 2007. afforded insoluble cross-linked enzymes (CLEs) with retention of catalytic activity. such as glutaraldehyde. poor reproducibility. and difficulties in handling the gelatinous CLEs. In an interesting variation on this theme. such as low activity retention. 1298 asc. in the late 1960s. 5 Carrier-Free Immobilization by Cross-Linking In the early 1960s.de  2007 Wiley-VCH Verlag GmbH & Co. This is followed by the addition of 1-hexane which causes coacervation to occur. 349. Consequently. Catal. KGaA. The immobilisate was used in the kinetic resolution of chiral secondary alco- Roger A. catalyzed by enzymes known as silicateins. emphasis switched to carrier-bound enzymes.[91] However. Naik and co-workers[86] entrapped catalase and horseradish peroxidase (HRP) using a biosilification process.[89] Kobayashi and co-workers[90] have recently described a novel polymer-incarceration methodology for immobilizing enzymes. A peptide subunit of the silicatein from Cylindrotheca fusiformis has been shown[87] to catalyze the formation of silica in vitro. 1289 – 1307 .wiley-vch. diatoms are able to synthesize silica nanoparticles by polymerization of silicic acid. which became the most widely used industrial methodology for enzyme immobilization for the next three decades. Sheldon hols and could be reused 5 times without any loss of activity. low mechanical stability.1 Cross-Linked Enzyme Crystals (CLECs) The cross-linking of a crystalline enzyme by glutaraldehyde was first described by Quiocho and Richards Figure 10. The immobilization procedure is depicted in Figure 10. In nature. Enzymes can also be entrapped in silicone elastomers[88] and polydimethylsiloxane membranes. Polymer-incarcerated (PI) Candida antarctica lipase. affording a precipitate containing the lipase in the polymer phase. studies of solid phase protein chemistry led to the discovery that cross-linking of dissolved enzymes via reaction of surface NH2 groups with a bifunctional chemical cross-linker. Weinheim Adv.

This indeed proved to be the case and led to the development of a new family of immobilized enzymes: cross-linked enzyme aggregates (CLEA) (Figure 11).[94] Figure 11. The use of cross-linked enzyme crystals (CLECs) as industrial biocatalysts was pioneered by scientists at Vertex Pharmaceuticals in the early 1990s[93] and subsequently commercialized by Altus Biologics. a CLEC of chloroperoxidase (CPO) from Caldariomyces fumago exhibited a higher thermal stability and tolerance to organic solvents than the free CPO.[92] Their main objective was to stabilize enzyme crystals for X-ray diffraction studies but they also showed that catalytic activity was retained. the CLEA methodology essentially combines purification and immobilization into a single unit operation that does not require a highly pure enzyme. Synth. On the other hand. which is often a laborious procedure requiring enzyme of high purity. Ampicillin synthesis. coupled with their high catalyst and volumetric productivities. It was reasoned that subsequent crosslinking of these physical aggregates would render them permanently insoluble while maintaining their pre-organized superstructure. is often used to purify enzymes. it is well-known[95] that the addition of salts. Adv. varying from 1 to 100 mm.[36] CLECs proved significantly more stable to denaturation by heat. It could be in 1964. or water-miscible organic solvents or non-ionic polymers. Their operational stability and ease of recycling. held together by non-covalent bonding without perturbation of their tertiary structure. 349. organic solvents and proteolysis than the corresponding soluble enzyme or lyophilized (freeze-dried) powder. CLECs are robust. hence their catalytic activity. 1289 – 1307  2007 Wiley-VCH Verlag GmbH & Co.Enzyme Immobilization: The Quest for Optimum Performance REVIEWS 5. Catal.[36] The initial studies were performed with CLECs of thermolysin (EC 3. to aqueous solutions of proteins leads to their precipitation as physical aggregates of protein molecules.24.4. Preparation of a CLEA. In a more recent example.4).de 1299 . by addition of ammonium sulfate or polyethylene glycol. Figure 12. of interest in the manufacture of aspartame. KGaA.wiley-vch.2 Cross-Linked Enzyme AggregatesACHTUNGRE(CLEAs) An inherent disadvantage of CLECs is the need to crystallize the enzyme. Since precipitation from an aqueous medium. Weinheim asc. and. 2007. that is without denaturation. but the method was subsequently shown to be applicable to a broad range of enzymes. highly active immobilized enzymes of controllable particle size. renders them ideally suited for industrial biotransformations.

proved to be effective catalysts for the synthesis of ampicillin (Figure 12). an appropriate organic solvent to leave the immobilized enzyme locked in the favourable confirmation.[101] Initial studies of CLEAs derived from the popular Candida antarctica lipase B (CaLB) revealed that the excellent performance observed in water. The first examples of CLEAs were derived from penicillin G amidase. for example.[102] Clearly the optimized CaLB CLEAs have activities surpassing those of Nov 435 in both aqueous and organic media. 1300 asc. Weinheim Adv. the simplicity of the operation lends itself to automation. Indeed. and substantially higher than that of the penicillin G amidase CLEC suggesting that diffusional limitations are more severe in the CLEC. KGaA. prepared in a simple procedure from relatively impure enzyme preparations. compared to that of the standard immobilized form. for the direct isolation of an enzyme. it can subsequently be washed from the CLEA using. Novozym 435 (CaLB immobilized on a macroporous acrylic resin). 349. a variety of hyperactive lipase CLEAs exhibiting activities up to twelve times that of free enzyme were prepared (Figure 13). 2007. The free enzyme exhibits limited thermal stability and a low tolerance to organic solvents. with high catalyst and volumetric productivities. an industrially important enzyme used in the synthesis of semi-synthetic penicillin and cephalosporin antibiotics (see earlier). the penicillin G amidase CLEAs also maintained their high activity in organic solvents. Figure 13. Analogous to the corresponding CLECs.REVIEWS used. with cross-linking into a single operation. using 96-well plates.[99] The activation of lipases by additives. Remarkably.3) and the effects of various parameters. the productivity of the CLEA was higher even than that of the free enzyme that it was made from and substantially higher than that of the CLEC. penicillin G amidase CLEAs. This afforded a dramatic improvement in the activity of CaLB CLEA in the enantioselective acylation of 1-phenethylamine in diisopropyl ether as solvent (Figure 14).[99] The experimental procedure was further simplified by combining precipitation.[96] The CLEA exhibited a synthesis/hydrolysis ratio (S/H) comparable with that of the commercial catalyst. such as the precipitant and the addition of additives such as surfactants and crown ethers. Indeed. Furthermore. the commercial catalyst mainly consists of non-catalytic ballast in the form of the polyacrylamide carrier which was reflected in its much lower productivity. can lock the enzyme in this more favourable conformation.de  2007 Wiley-VCH Verlag GmbH & Co. could not be directly translated to organic media. from a crude fermentation broth. Sheldon adopt a more active conformation..1. Synth.98] CLEAs were subsequently prepared from seven commercially available lipases (EC 3. 1289 – 1307 . since the additive is not covalently bonded to the enzyme. PGA-450 (penicillin G amidase immobilized on polyacrylamide). Catal. In stark contrast. Consequently the preparation was modified to produce a more lipophilic CLEA which could better accommodate organic solvents. in the presence or absence of additives. prepared by precipitation with.g. on their activities were investigated.[97.1.[99] These results clearly demonstrate the tremendous potential of the CLEAs as immobilized enzymes. making it an ideal candidate for stabilization by immobilization. in a purified and immobilized form suitable for performing biotransformations. ammonium sulfate or tertbutyl alcohol.wiley-vch. such as surfactants and crown ethers. for example. Hyperactivation of lipase CLEAs. for example.[100] Co-precipitation of such additives with the enzyme followed by cross-linking of the enzyme aggregates. e. Using this procedure. is well-documented and is generally attributed to the lipase being induced to Roger A.

CaLB CLEA displayed superior activity to that of Novozym 435 in the kinetic resolution of 1-phenylethanol and 1-tetralol. Sequential hydrogenation and resolution in scCO2. Hence. in scCO2. low or no retention of activity was sometimes observed using glutaraldehyde as the cross-linker.[103] In the kinetic resolution of 1-phenylethanol and 1phenylethylamine in the ionic liquids. An important property of CLEAs from the point of view of large-scale applications is their particle size which obviously has a direct effect on mass transfer limitations and filterability. obtained by periodate oxidation of dextrans. as the cross-linkers. for example.Enzyme Immobilization: The Quest for Optimum Performance REVIEWS Figure 14. using two separate columns in series (one containing the Pd catalyst and the other the CaLB CLEA). Glutaraldehyde is generally the cross-linking agent of choice as it is inexpensive and readily available in commercial quantities.[105] Optimum activity was observed with particles of 40–50 nm. inter alia. Weinheim .[106] followed by reduction of the Schiffs base moieties with sodium borohydride to form irreversible amine linkages. CaLB CLEAs also exhibit excellent activities in supercritical carbon dioxide[103] and ionic liquids. KGaA. e. Dramatic results were obtained. this will be particularly severe with glutaraldehyde.[104] Thus. with some enzymes. nitrilases. this could be avoided by using bulky polyaldehydes. without the need for depressurization (Figure 15).de 1301  2007 Wiley-VCH Verlag GmbH & Co. 1289 – 1307 dehyde concentrations are. important factors in determining the particle size of CLEAs as was reported for Candida rugosa lipase.wiley-vch. However.g. A possible cause of deactivation is reaction of the cross-linker with amino acid residues which are crucial for the activity of the enzyme. Comparison of Novozym 435 with CaLB-CLEAs. Catal. which allows it to penetrate the interior of the protein. asc. Figure 15. In contrast.. Owing to its high reactivity and small size. 2007. 349. Synth. The enzymatic resolution could also be combined with the production of the 1-phenylethanol substrate. [bmim] [NO3] and [bmim] [N(CN)2] the best results were observed with CaLB adsorbed and cross-linked on a polypropylene carrier (Accurel EP100). by palladium-catalyzed hydrogenation of acetophenone. the free CaLB or immobilized as Novozym 435 dissolves in these ionic liquids with complete loss of activity. The activity retention of these CLEAs was generally much higher than that observed with CLEAs prepared using glutaraldehyde. by acylation with vinyl acetate. The enzyme and glutaralAdv.

[114] An alcalase CLEA showed excellent activities and enantioselectivities in amino acid ester hydrolyses. cross-linking is expected to be less effective.2. a structural feature often encountered with redox metalloenzymes. which allowed for the use of low concentrations of catalase.1. in the resolution of (amino acid) esters[112] and amines[113] and peptide synthesis. e. glucose oxidase (EC1. The latter can be made by TEMPO cata- Figure 16. Similarly. and glycosidases (EC 3. fermentation lacked the esterase activity observed with the crude enzyme.6-tetramethyl-1-piperidinoxyl). galactose oxidase (EC 1. also known as subtilisin Carlsberg)..1.1. aqueous effluent treatment and. became independent of this parameter in the CLEA.[116] This strongly suggests that the esterolytic activity is derived from a protein impurity in the crude aminoacylase preparation and illustrates the potential of the CLEA methodology for performing purification and immobilization in a single operation.26) from As1302 pergillus niger.10.1). Gupta and co-workers[110] reported that addition of bovine serum albumin (BSA) as a “proteic feeder” in the preparation of CLEAs from solutions containing low concentrations of enzymes facilitated the formation of the CLEA. thereby reducing the enzyme cost contribution. Effect of cross-linker on recovered activity of CLEA. EC 3.2.2). b-galactosidase (EC 3.g.109] In a variation on this theme.21.5.23) from Aspergillus oryzae and phytase (EC 3. Laccase-catalyzed aerobic oxidation of starch..3. has many potential applications.2.g. Sheldon Recovered activity [%][a] Glutaraldehyde 0 0 48 Dextran polyaldehyde 50 60 85–90 Relative to the free enzyme. KGaA. For electronegative enzymes.[101] Adv. CLEAs have been prepared from an alcohol dehydrogenase (EC 1.3. Immobilization as a CLEA improves the stability of the laccase under the reaction conditions. fluorescens) Nitrilase (Biocatalytics 1004) Penicillin G amidase [a] Roger A.g.g.6) which. Weinheim . Other examples of hydrolases that have been successfully cleated include pig liver esterase (EC 3. 349. the alkaline protease from Bacillus licheniformis (alcalase. 1289 – 1307 asc. for bleaching in the pulp and paper or textile industries. different.6.[101] The former enzyme catalyzes the hydrolysis of lactose in dairy products and is administered as tolerase to people suffering from lactose intolerance and the latter is an acid phosphatase which is added to animal feed in order to hydrolyse phytate (inositol hexaphosphate). and is. aminoacylase (EC 3. in combination with the stable radical TEMPO (2.4.2) from Candida boidinii. proteases. in particular. Laccase. with a nitrilase (EC 3. For example. Cross-linking with glutaraldehyde produced a completely inactive CLEA while with dextran polyaldehyde 50–60 % activity retention (not optimized) was observed (see Table 1).1. Similarly. 2007. for the catalytic aerobic oxidation of starch to carboxystarch (Figure 16). a CLEA of aminoacylase prepared from a crude extract from Aspergillus sp.13) from P. Synth.[111] For example.3.5.5. aerobic oxidation is more attractive.REVIEWS Table 1.[115] Interestingly. e.1). Another benefit of the CLEA technology is that it can stabilize the quaternary structures of multimeric enzymes.1. Catal. has been widely used in organic synthesis.14). that contain a paucity of lysine residues on the surface.4). the stability of CLEAs from two tetrameric catalases (EC 1. lyzed hypochlorite oxidation of starch but greener.de  2007 Wiley-VCH Verlag GmbH & Co.[117] Similarly.11.62.[106] Enzyme Nirilase (Ps.1.1.. e.1. every enzyme can be expected to be. e. One way of compensating for this lack of surface amino groups is to co-precipitate the enzyme with a polymer containing numerous free amino groups. better activity retentions were observed when penicillin amidase CLEAs were prepared with dextran polyaldehyde compared to glutaraldehyde.3. for the soluble enzymes is dependent on concentration.. poly-llysine[107] or polyethylene imine. Recyclable CLEAs were also prepared[101] from a variety of oxidoreductases.2.1.wiley-vch. CLEAs were successfully prepared from the glycosidase.9) and laccase (EC 1.1.1. fluorescens and with a nitrilase from the company Biocatalytics.[108.1) from Rhodococcus erythropolis and a formate dehydrogenase (EC 1.[106] Since cross-linking largely involves reaction of the amino groups of lysine residues on the external surface of the enzyme. an inexpensive enzyme used in laundry detergents.

2007. for transfer of the product of the first step to the active site of the enzyme for the second step.[121] This method has advantages compared with physical adsorption or covalent binding. A possible explanation is that the close proximity of the two enzymes inside the combi CLEA is more favourable.120] These oxynitrilase CLEAs perform exceptionally well in organic solvents. this can be achieved by co-precipitation and cross-linking of two or more enzymes in combi CLEAs.2. catalyzed the smooth. respectively.[122] The preparation of CLEAs from CO2 expanded micellar solutions afforded dendritic CLEAs with tunable nanometer dimensions (7–38 nm). such as rapid mass and heat transfer and large surface area to volume ratios. Prunus amygdalis (PaHnL) was highly effective in the hydrocyanation of aldehydes under microaqueous conditions and could be recycled ten times without loss of activity.127] developed a microreactor in which enzymes are immobilized as an enzyme-polymer membrane on the inner walls of the microchannels.g. inclusion of glucose oxidase CLEAs in magnetic mesocellular carbon foam was used to construct a magnetically switchable bioelectrocatalytic system.1.and S-specific oxynitrilases (hydroxynitrile lyases.[127] Thus. Catal. unfortunately. Interestingly. catalytic cascade process without the need for separation of intermediates. affording higher enantioselectivities than observed with the free enzymes owing to the essentially complete suppression of competing non-enzymatic hydrocyanation. One-pot conversion of benzaldehyde to (S)-mandelic acid. a-chymotrypsin) in aqueous buffer was mixed with glutaraldehyde and formaldehyde as cross-linkers in commercially available polytetrafluoroethylene (PTFE) tubing (inner diameter 500 mm). catalyzed by these enzymes. notably the R. combi CLEAs have been prepared from catalase in combination with glucose oxidase or galactose oxidase. A further elaboration of the CLEA methodology involves the immobilization of lipase CLEAs by inclusion in hydrophobic polytetrafluoroethylene membranes for use in membrane bioreactors. Furthermore. 6 Combi-CLEAs and Catalytic Cascade Processes The ultimate in environmental and economic efficiency is to combine atom-efficient. this could also be achieved by using an S-selective nitrilase in combination with non-enzymatic hydrocyanation but.. For example. there are no nitrilases that exhibit S-selectivity with mandelonitriles. steps into a one-pot. catalytic. The catalase serves to catalyze the rapid degradation of the hydrogen peroxide formed in the aerobic oxidation of glucose and galactose. Synth. less reactor volume. compared to the case with two separate CLEAs. one-pot conversion of benzaldehyde to S-mandelic Adv.Enzyme Immobilization: The Quest for Optimum Performance REVIEWS The methodology has also been successfully applied[102] to various CÀC bond forming lyases. A combi CLEA containing an S-selective oxynitrilase from Manihot esculenta and an aselective nitrilase from Pseudomonas fluorescens. Similarly. These are attractive features for conducting catalytic reactions in microreactors containing the enzyme immobilized on their inner walls. the combi CLEA was more effective than a mixture of the two separate CLEAs. In principle. shorter cycle times and less waste generation. a CLEA prepared from the (R)-specific oxynitrilase from almonds. This results in the formation of a CLEA membrane on the inner walls of the tubing. 7 Enzyme-Immobilized Microchannel Reactors for Process Intensification Microreactor technology is an interdisciplinary field that has attracted much attention recently. a solution of the enzyme (e.de 1303  2007 Wiley-VCH Verlag GmbH & Co.126. and higher volumetric and space-time yields.[118] CLEAs were similarly prepared from the (S)-specific oxynitrilases from Manihot esculenta and Hevea brasiliensis. The enantioselectivity is provided by the oxynitrilase and in situ conversion by the nitrilase serves to drive the equilibrium of the first step towards product.[119.10) which catalyze the hydrocyanation of a wide range of aldehydes. For example.wiley-vch. Maeda and co-workers[107. 349. Weinheim . In principle.5.[125] Process intensification through the use of microchannel reactors (microfluidic devices) has many advantages compared with traditional batch process technologies. 1289 – 1307 acid (Figure 17)[124] in diisopropyl ether/water (9:1) at pH 5. This technique has been used to prepare CLEA-based enzyme microreactors (CEMs) asc.[123] Figure 17. by coupling steps together unfavourable equilibria can be driven towards product. KGaA. EC 4.[28] Catalytic cascade processes have numerous potential benefits: fewer unit operations.

A. 281 – 289. In contrast. M. [15] J. Curr. 359. B. Liese. Dordick. Patel. Opin. many of these innovations involve the use of rather exotic supports which are substantially more expensive than the enzyme to be immobilized. Stemmer. Panke. Hauer. Schmid. del Cardayre. [5] R. 595. W. Biotechnol. J. Novel concepts continue to appear. there is no all-encompassing. Powell. Huisman. they are unlikely to be applied in industrial biotransformations but could be interesting for applications in biosensors or other devices where the enzyme cost contribution is less of an issue. 130. 2000. Curr. M. N. However. Witholt. H. Patel. [12] S. T. 2002. Opin. Adv. Catal. K. 2001. 317. Kiener. 1999. G. Biotechnol. M. 2004. Technol. [10] A. analogous to those used in catalytic exhaust systems in automobiles. Reetz. Microb. M. Angew. Catal. Opin. 13. Opin. O. interest in improving their operational performance will certainly continue unabated. Chem. They can also provide important insights into the effects of enzyme immobilization on activity and stability. Wubbolts. 72. Opin. ceramic monolith. 587. 409. 425. 13. Based on the increasing importance of enzymes in a plethora of industrial applications. 120. B. Villela Filho. S. Stewart. S.[107] The use of such enzyme-immobilized microchannel reactors clearly has considerable potential for the design of green and sustainable biotransformations. Catal. Opin. Immobilization as silica granulates. V. [7] K. [13] S. 2002. Schoemaker. Thomas. 13. 111. Zaks. Ghisalba. 284. 5. should be a relatively simple operation that does not require a highly pure enzyme preparation or an expensive support that may not be com1304 asc. Pure Appl. Roger A. Cordierite monoliths were functionalized. M. [4] A. B. Cross-linked enzyme aggregates (CLEAs) would appear to have considerable industrial potential based on their high activity retention and stability coupled with ease of preparation from crude enzyme samples and no requirement for a support. 57. Chem. the immobilization methodology. P. [17] M. Sheldon. 10. This concept was tested in the CaLB-catalyzed transesterification of 1butanol with vinyl acetate. for example. D. J. S. Tibtech 2002. N. M. B. 548. DiCosimo. P. R. These were then employed in a monolithic stirrer reactor in which the monolithic structure is incorporated in the stirrer blades. the monolithic catalysts were operationally stable for several weeks. [16] K. 2002. J. A. Panke. Biol. in addition to providing an active and stable biocatalyst. Curr. 2000. 1694. Chem. Jaeger. Longchamp. J. E. Synth.wiley-vch. 19–20. Hollmann.[129] However. 2001. Chem. 299. 5. without significant loss of activity. 2001. van Rantwijk. F. [6] A. Straathof.[107] With electronegative enzymes (such as aminoacylase) coprecipitation of the enzyme in the presence of poly-l-lysine (see above) was used to realize fast and efficient CLEA formation.de  2007 Wiley-VCH Verlag GmbH & Co. Mol. 20. Gray. 2003. Laumen. [19] O. G. the enzyme is immobilized on a honeycomb. [2] R. 55. 2002. R. Curr. Biotechnol. It is also clear that every enzyme is different and. F. meets all these criteria but the methodology is not applicable to aqueous environments (see earlier). 2007. J. Weinheim . B: Enzymatic 2002. A. P. Because they are close to 100 % active catalyst they also display high catalyst productivities and spacetime yields. Biol. Schmid. T. G. Chem. W. 407. consequently. Biotechnol. M. Mink . Curr. Biol. A. 31. 18. References [1] R. Nature 2001. Nagarajan.[128] Carbon nanofiber-coated monoliths performed the best with regard to catalyst productivity and did not exhibit any leaching under the reaction conditions. Biotechnol.-E. Shaw. the development of a monolithic stirrer reactor by Lathouder and co-workers deserves a special mention. T. 3. 3948. C. Consequently. Minshull. K. Curr. [8] N.[128] According to this novel concept. [14] G. [11] A. 804. Tobin. May. Robins. properties such as mechanical strength and filterability still have to be demonstrated on an industrially relevant scale. 6. Wubbolts. Biotechnol. S. 349. J. Opin. Nat. Kiener. Kittelmann. [3] H. 1999. Bühler. Schmid. it is clear from this review that the subject of enzyme immobilization continues to attract considerable attention from researchers in both industry and academia. The activity was lower (by a factor of 2–4) than Novozym 435 or free CaLB but the latter preparations deactivated much faster. Synth. Clearly. F. W. The quest for optimum performance continues. Huisman. Opin. 1289 – 1307 8 Conclusions and Prospects Hopefully. Enz. Adv. Arnold. [18] J. Eur. For application in industrial biotransformations the cost contribution of the (immobilized) enzyme is an important issue. C. KGaA. [9] A. Biotechnol. Curr. in order to create adsorption sites for the enzyme (CaLB). In the context of incorporating the immobilized biocatalyst in the reactor configuration. 238. 2001. Opin. Curr. M. A. 12.REVIEWS from a wide variety of enzymes. W. P. a recent example being single enzyme nanoparticles (SENs) in nanoporous silica. J. 13. one size fits all solution to the problem of enzyme immobilization. Nguyen. 2002. Sheldon. 2000. by coating with polyethylene imine or different types of carbon. D. 258. Park. F. Stemmer. B. Chem. 40. Sheldon mercially available. Curr. 345. Science 2003. B. A. 1233. Ramer. W. Aust. Wubbolts. D. 352.

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