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Immobilization of enzymes on porous silicas – benefits

Cite this: DOI: 10.1039/c3cs60021a
and challenges
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Martin Hartmann* and Xenia Kostrov

Received 23rd January 2013
DOI: 10.1039/c3cs60021a
www.rsc.org/csr
Porous silica materials have extensively been used for the immobilization of enzymes aiming at their
use as biocatalysts or biosensors. This tutorial review will discuss the benefits and challenges of different
immobilization techniques and will provide references to recent papers for further reading. Moreover,
novel trends and unsolved problems will be introduced.

Key learning points

 Using porous silica materials for immobilization of enzymes.
 Selecting a suitable immobilization strategy.
 Factors, which can have influence on enzyme immobilization.
 Current problems in enzyme immobilization.
 Brief overview of current trends in enzyme immobilization on porous supports.

Introduction Immobilization may result in improved stability of the

Enzymes are highly effective and versatile biological catalysts, which
display high chemo-, stereo- and regioselectivity while operating
under ambient conditions (physiological temperature and pH,
atmospheric pressure).1 When enzymes are used as catalysts, no
activation or protection/deprotection of functional groups, which
are typically required in organic synthesis, are necessary. Due to
their operation in water as solvent, less waste is generated and
shorter synthetic routes are achieved in comparison to traditional
organic synthetic routes. Thus, enzyme-catalyzed processes became
increasingly important in large-scale industrial applications for
instance in the pharmaceutical or food industry.2
However, the use of enzymes in their native form is often
hampered by several limitations such as high costs, low operational
stability and difficulties in recovery and reuse. Immobilization of
the enzymes onto porous silica supports provides one of the most
attractive concepts to overcome these drawbacks.3,4 In the broadest
sense, enzyme immobilization can be understood as the attach-
ment of enzyme molecules on different types of supports resulting
in reduced or loss of mobility of the enzyme.5
Erlangen Catalysis Resource Center, Universität Erlangen-Nürnberg, Egerlandstraße 3,
D-91058 Erlangen, Germany. E-mail: Martin.Hartmann@ecrc.uni-erlangen.de,
Xenia.Kostrov@ecrc.uni-erlangen.de; Fax: +49 9131 85 67401;
Tel: +49 9131 85 28792
enzymes towards harsh reaction conditions such as extreme
pH, high temperature or the presence of organic solvents and
enable their easier separation from the reaction media and
their reuse. Moreover, product contamination with enzymes can
be minimized or completely avoided, which is particularly crucial
for applications in the pharmaceutical and food industries.3
For industrial scale applications, immobilization is generally
considered to be favourable since it allows continuous processes,
for example in fixed bed reactors.6,7
A large number of enzymes are nowadays commercially
available. Prices vary significantly, depending on accessibility,
degree of difficulty in isolation and purification of enzymes.
Progress in recombinant DNA technology offers the possibility
to produce the majority of the used enzymes for a commercially
acceptable price.8
The first report on enzyme immobilization dates back to
1916. Nelson and Griffin have shown that invertase physically
adsorbed on charcoal fully retained its catalytic activity.9 Since
then, the immobilization of enzymes has been studied for
nearly 100 years. The first industrially used immobilized
enzyme was aminoacylase, which was physically adsorbed on
polymer DEAE–Sephadex and used for industrial production of
2
L-methionine by Tanabe Seiyaku Co. in 1969.
Nowadays immobilized enzymes are broadly used in several
processes for example in the pharmaceutical industry to prepare

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6-aminopenicillanic acid (APA) from penicillin G by using
immobilized penicillin acylase in an aqueous solution or in
the food industry for the production of high fructose syrup from
starch by using immobilized glucose isomerase (Sweetzyme T,
Novo, Denmark).2
Numerous supports have been investigated with respect to
the immobilization of enzymes, e.g. sol–gels, organic polymers,
porous and non-porous inorganic materials. A good overview of
advantages and disadvantages of using these materials for
enzyme immobilization can be found in several reviews.4,11–14
Mesoporous silicates are promising candidates for enzyme
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immobilization with respect to the requirements of enzyme carriers
such as large surface area, narrow pore size distribution, well-
defined pore geometry, their thermal and mechanical stability
and toxicological safety. Furthermore, the surface of silica supports
can be chemically modified with various functional groups.12,14–16
In this tutorial review, the immobilization of enzymes on
porous silica supports is discussed with respect to the funda-
mental principles governing enzyme immobilization. Selected
examples are highlighted to strengthen important aspects.
Since the first successful immobilization of enzymes on the
mesoporous silica MCM-41 by Diaz and Balkus in 1996,10 mainly
ordered mesoporous silicas have been used in proof-of-principle
studies. However, it is obvious that the mentioned strategies
often also apply to ‘‘normal’’ amorphous silica gels and porous
glasses.
Selecting a suitable carrier for enzyme
immobilization
The properties of the employed carrier are crucial for a successful
immobilization process. The support material has to be thermally,
mechanically and chemically stable and insoluble in the solution
Prof. Dr Martin Hartmann
obtained his PhD in Physical
Chemistry in 1993 from the
Universität Dortmund
(Germany). After postdoctoral
stays with Larry Kevan at the
University of Houston (USA) and
with Jens Weitkamp at the
Universität Stuttgart (Germany),
he finished his Habilitation at TU
Kaiserslautern Chemical
Technology. In 2006 he was
Martin Hartmann appointed Professor of Advanced
Materials Science at the
Universität Augsburg. In 2009, he moved to the Universität
Erlangen-Nürnberg as Professor of Catalysis and Director of the
Erlangen Catalysis Resource Center. He published more than 160
papers and reviews in the area of porous materials and with special
emphasis on their applications in separation and heterogeneous
(bio-)catalysis.
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used for the immobilization process. Moreover, the support
should exhibit a high capacity for enzyme binding and must
not lead to denaturation or deactivation of the enzymes. In
addition for industrial application the support should be
relatively cheap and environmentally benign.3,4,12,14
Various modified/unmodified supports, such as organic poly-
mers, biopolymers, zeolites, magnetic nanoparticles, aluminium
oxide, apatite, controlled pore glasses (CPG), sol–gels, silica gels,
ordered/disordered silica supports, have been investigated with
respect to immobilization of various enzymes.3,4,6,8,12–16 Due to
their small pore size, zeolites (dp o 1.3 nm) are not used as
carriers for adsorption of the large enzyme molecules (molecular
size B3–30 nm).6,8,12–14
Organic supports such as polyacrylamide or polystyrene
polymers either possess suitable functional groups on their
surface (e.g. amino groups), which provide anchoring points for the
interactions with enzymes, or such groups may be introduced
during the polymerization process. However, the low stability
towards organic solvents and low resistance to microbial attack of
certain polymers prevent their large scale industrial applications.4,14
Commercially available silica gels possess moderate to high
surface areas of 300–500 m2 g1. Thermal pretreatment reduces
the number of silanol groups present that provides access to
chemical fixation of enzymes. Often sol–gel methods are
employed for enzyme immobilization which are however
beyond the scope of this review. More information can be
found in ref. 4.
Mesoporous silica materials synthesized from silane precur-
sors via a surfactant templating approach possess an ordered
pore structure, an uniform pore-size distribution, high specific
pore volumes (up to ca. 1.5 cm3 g1), large internal surface area
(up to 1500 m2 g1). Their pore size can be tailored from about
1.5 to 30 nm by choosing an appropriate surfactant and/or
varying the synthesis conditions. These supports offer the possibility
Xenia Kostrov studied chemistry
at the Friedrich-Alexander-
Universität Erlangen-Nürnberg
and received her diploma degree
in the group of Prof. H. Groeger.
In 2011, she joined the group of
Prof. Martin Hartmann at the
Erlangen Catalysis Resource
Center for her doctorate thesis,
which is devoted to the
immobilization of enzymes on
mesoporous materials for
Xenia Kostrov biocatalytic applications.
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of immobilizing large proteins. They are water insoluble,
thermally, mechanically and chemically stable and resistant
towards microbiological attacks. Furthermore, silica supports
exhibit sufficient functional groups on their surface for enzyme
attachment or for further modification. All these features
render them promising candidates for the immobilization of
enzymes.8,12,15 A large number of studies have shown that
immobilization of enzymes onto mesoporous silica materials
may lead to enhanced catalytic activity and stability of the
enzymes.3,11,12,14,15
In the following section we want to give a short introduction
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to mesoporous silica supports along with some current examples
of their use in enzyme immobilization. A more detailed discussion
can be found in other reviews.3,4,6,8,11,13,14,16,17
Mesoporous silica materials
The first report on mesoporous silicas with uniform pore size
distribution is the paper by Kuroda et al. in 1990. The novel
materials were named FSM-n (Folded Sheet Materials, here n
represents the number of carbon atoms in the surfactant alkyl
chain used to synthesize the carrier) and were synthesized by
intercalation of hexadecyltrimethylammonium cations into the
layered polysilicate kanemite in aqueous solution, followed by
removal of the organic template via calcination.18 Inagaki et al.
have reported that the pore diameter of FSM materials can be
controlled by varying the alkyl chain length of the cationic
surfactant.19
Intensive research in the area of mesoporous silica supports
began in 1992, when researchers from Mobil20 introduced a
so-called M41S family of novel highly ordered silicate/alumino-
silicate mesoporous molecular sieves. The initial members of
the M41S family were MCM-41 (Mobil Composition of Matter
No. 41), MCM-48 and MCM-50. Similar to the synthesis of FSM
materials, cationic surfactants with alkyl chains from 8 to 20
carbons were employed as templates during the hydrothermal
synthesis.20 In 1994, Huo et al. reported the synthesis of a novel
mesoporous molecular material, denoted SBA-1 (Santa Barbara
Material No. 1), which exhibited a three-dimensional cubic
structure and was synthesized in highly acidic media.21 The
highly ordered large pore mesoporous silica SBA-15 with pore
sizes ranging from 5 up to 30 nm interconnected by micropores
and with thick pore walls (4–8 nm) was introduced in 1998.22,23
SBA-15, prepared by using the amphiphilic triblock copolymer
(Pluronic P123), showed higher thermal and hydrothermal
stability than previously synthesized mesoporous materials
and can be synthesized in a large variety of shapes (e.g. rods,
fibers, platelets, spheres, etc.) depending on the synthesis
conditions.22,23
The next step in the development of novel mesoporous silica
materials was the synthesis of mesostructured cellular foams
(MCFs) by Stucky and co-workers in 1999.24 These materials
possess a three-dimensional large-pore structure with spherical
cells (24–42 nm) interconnected by uniform windows (9–22 nm).
The synthesis employs triblock copolymers stabilized by oil in
water emulsions. Because of the larger pore size and higher pore
volume, MCFs show improved performance as enzyme carriers
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compared to other mesoporous molecular sieves. Chouyyok
et al. showed that the use of MCF as a support for adsorption
of horseradish peroxidase (HRP) resulted in higher enzyme
loading and activity compared to MCM-41 and SBA-15. More-
over, HRP immobilized in the pores of MCF was also found to
have the highest storage stability.25 Similar observations were
made by Pandya and co-workers for the covalent anchoring of
a-amylase. This biocatalyst exhibits the highest specific activity
in catalytic starch conversion. The authors, thus, concluded
that the smaller pore size of SBA-15 and MCM-41 compared to
MCF might impose diffusional restrictions on substrates and
product molecules.26
Periodic mesoporous organosilicas
Three different research groups independently reported the synth-
esis of periodic mesoporous organosilicas (PMOs), a new class of
organic–inorganic hybrid mesoporous materials, where the organic
fragments are incorporated into the pore walls of the silica
carrier.16 The organic moieties are incorporated into the three-
dimensional network of the silica matrix through two covalent
bonds and, thus, distributed homogeneously in the pore walls.
Compared to their pure mesoporous silica counterparts, the unique
hydrophobic–hydrophilic frameworks of PMOs can be modified
through incorporation of different kinds of organic groups without
pore blocking, thus, maintaining their highly ordered structure,
high specific surface area (up to 1800 m2 g1) and pore volume.
Another advantage is the tunable surface properties imparted by
the organic components in the framework.15
Controlled pore glasses
Another promising candidate for enzyme immobilisation is so
called Controlled Pore Glass (CPG). CPG can be understood as
the leaching product of phase-separated alkali borosilicate
glasses. CPGs are amorphous materials with a SiO2 content of
around 96 wt%.27 CPGs exhibit a broad pore size ranging
between 3 and 1000 nm, high thermal, mechanical and
chemical stability and high resistance towards aggressive chemicals
e.g. acids. Thus, CPGs can be regenerated many times by heating or
boiling in acid to remove all organic residues adsorbed and to
create more silanol groups on the support surface for further
functionalization.27,28 The surface properties of CPGs are mainly
determined by the silanol groups. Thus, porous glasses can be
easily modified by a variety of functional groups in a similar
manner than mesoporous silica molecular sieves. Furthermore,
CPGs can be prepared in diverse geometrical forms, i.e. rods, beads,
(hollow) fibers or plates. These properties render them attractive
supports for enzyme immobilization.27,29
Recently, Coniglio and Kreis have reported the use of porous
glasses as supports for the immobilization of flavonol 3-O-
heterodisaccharidase (FHG 1) from the dried herb of Fagopyrum
esculentum. The results demonstrated that the immobilized FHG
1 is more stable than the free enzyme. About 40% of the activity
was lost after three weeks storage at 25 1C for the immobilized
enzyme, while the native enzyme is inactive after several days.30
Kahraman et al. used controlled pore glass as a carrier for
covalent immobilization of a-amylase (from porcine pancreas).
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Table 1 Structural characteristics of porous silica materials employed for the immobilization of enzymes

Porous support Pore structure/symmetry Typical template used Pore size [nm] BET surface [m2 g1]
FSM-16 Folded-sheet/p6mm CTAB, CTMAa 3–9 500–900
MCM-41 2D hex. channels/p6mm CTAB, CTMA 2–10 300–1200
MCM-48 Cubic/Ia3% d CTAB, CTMA 2–10
SBA-15 2D hex. channels/p6mm P123b 5–30 500–1400
SBA-16 Spherical cages/Im3% m F127c 3–15 600–1200
MCF Mesocellular foam P123 25–40 500–1000
PMOs 2D or 3D CTMA, P123, F127 2–50 Up to 1800
FDU-12 3D-cages/Fm3% m F127, TMB,d KCl 10–15 200–700
CPG Monomodal or hierarchical Alkali borosilicate glass 1 to >1000 20–500
a
CTMA: CnH2n+1(CH3)N+, n = 8–18. b
P123: EO20PO70EO20. c F127: EO106PO70EO106. d
TMB: tetramethylbenzene.
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Prior to the immobilization step, the porous glasses were
functionalized with 3-aminopropyltriethoxysilane and then
treated with phthaloyl chloride, thereby the acid chloride
moieties are used for covalent attachment of enzyme via reaction
with NH2-groups. It is reported that the immobilized amylase is
more thermostable than the native one (about 40% of the
original activity is retained at 90 1C).31
In the same year, Karakus and Pekyardimci have demon-
strated that immobilization of apricot pectinesterase from
Prunus armeniaca L. on 3-aminopropyltriethoxysilane-coated
porous glass beads leads to improved thermal stability of the
immobilized enzyme compared to the free enzyme.32 The
structural characteristics of the mainly discussed porous silicas
in this article are summarized in Table 1. For further details about
the preparation and characterization of porous silica materials,
organic–inorganic hybrid materials and controlled pore glasses
the readers are referred to recent extensive reviews.12,15,16
Immobilization
Immobilization is a major route for the creation of a stable
biocatalyst with high enzyme loading, retained or even
improved catalytic activity, which can be easily recovered and
reused. Another important parameter to be considered is the
productivity (kg of the product per kg of enzyme) of the
prepared biocatalyst. For the development of a successful
immobilizing process, several factors such as immobilization
conditions, properties and characteristics of both enzymes and
carriers must be considered. Table 2 summarizes some general
factors to be taken into account when optimizing an enzyme
immobilization protocol.
Immobilization methods
Since the first publication of invertase immobilization on
charcoal in 1916, research in this area has developed rapidly
and numerous reports have appeared in the open literature.
The progress in this area is summarized in several comprehensive
reviews.3,5,6,8,11,13,14 Different methods have been proposed for
immobilization of enzyme molecules on porous silica supports.
Three main strategies are typically used: physical adsorption,
covalent attachment onto the pore walls and lately cross-linking
of enzyme molecules.3–6,11,13,14 It should be pointed out that the
classification of immobilization techniques presented here is
somewhat arbitrary and in the literature many other divisions
can be found.
Physical adsorption
The most popular approach of immobilizing enzymes onto any
kind of (porous) silica support is physical adsorption. This
method is characterized by its simplicity, because previous
functionalization of the carrier surface is typically not required.
Denaturation and/or deactivation of the enzyme can be avoided

Table 2 Critical parameters for the development of the optimized immobilization protocol

Parameters to be
considered
Carrier Chemical parameters
Physical parameters
Examples
Composition, functional groups, stability against solvents, swelling behavior, hydrophobicity.
Morphology, particle size, porosity (pore structure, pore size distribution) active surface area,
surface charge, abrasion (for stirred tanks), flow resistance (for a fixed-bed reactor).

Enzyme Biochemical properties Availability, storage stability, toxicology, purity, conformational flexibility, molecular weight,
size, active site, morphology, functional groups on the surface, surface charge (pI).
Kinetic parameters Specific activity, pH and temperature profiles, stability towards reaction conditions
(pH, solvents, temperature), stabilizing or activating additives.

Immobilization Immobilization strategy Physical adsorption, encapsulation, covalent binding, cross-linking.

process Immobilization PH, temperature, solvents, stabilizing agents.
conditions
Diffusion/mass transfer Buffer effect, viscosity of reaction media, pore diffusion.
Performance Space-yield time, recycling, reusability, product removal, toxicity, enzyme inhibition, kinetics.
Process cost Costs of enzyme and support, waste disposal, energy.

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and, thus, retention of catalytic activity of the immobilized
enzyme is very high.4
Physisorption of enzymes onto silica surfaces is governed by
the interaction of functional moieties of the enzyme chains
with the solid surface. The principal driving forces are hydro-
phobic interactions, hydrogen bonding, van der Waals and
electrostatic (attraction and repulsion) forces. Since the enzyme
molecules are only physically adsorbed on the silica surface,
leaching from the carrier may be triggered by even minor
changes in reaction conditions, such as temperature/pH altera-
tions or variation in substrate concentration.4 To solve this
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problem, different strategies were proposed for the improve-
ment of enzyme binding to the support.
One possibility to avoid or minimize enzyme leaching from
the support is enhancing enzyme–silica surface interactions
through straightforward variation in immobilizing conditions.
For instance, the hydrophilic interactions between carriers and
enzymes can be improved by the use of glycosylated enzymes,
which results in the formation of hydrogen bonds between
enzymes and carriers. This strategy is suitable for various silica
supports such as porous glasses, silica gel and Celite.4,6,12–14
Another way to prevent enzyme leaching is the enhancement
of electrostatic interactions between the enzyme and the silica
surface. Numerous studies4,6,8,11,14 reported that electrostatic
interactions between the surface of the silica carrier and the
enzyme molecules were stronger, when the adsorption was
performed at a pH, where the enzyme possessed a positive
charge while the silica support was negatively charged. Silica
supports are typically negatively charged at pH values above
three.15 The enzyme is positively charged when the adsorption
is performed at a pH value below the isoelectric point (pI) of the
enzyme. The pI is the point at which the overall charge of the
enzyme is zero and is determined by the distribution of surface
functional groups (e.g. –NH2, –COOH, –OH).6,15,16 The larger
the positive charge of enzyme, the stronger the electrostatic
interactions between the enzyme and the surface will be.
However, it should not be forgotten that the repulsion forces
between positively charged enzyme residues adsorbed on the
surface are also intensified. At pH above the pI, the enzyme will
be negatively charged, which will lead to increased repulsive
interactions between the enzyme and the negatively charged
carrier. The loading of enzymes (proteins) is found to vary with
pH of adsorption medium following a bell-shaped curve, where
the maximum loading is observed near the pI of the enzyme
(Fig. 1).15
Thus proper adjustment of the pH value of solution allows
improvement of electrostatic interactions between silica supports
and enzyme molecules. The disadvantage of this method is that
the pH value should be kept constant during the application e.g.
the catalytic reaction, and be close to the pH of the preparation
otherwise significant leaching may occur. In some cases, an
inversion of the surface charge is required, e.g. through functio-
nalization of porous silica supports with different organic groups
or metals.11,13,14
Vinu et al. described in 2004 that the amount of cytochrome
c adsorbed on MCM-41 and SBA-15 varies significantly with the
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Fig. 1 Dependence of the maximum of enzyme adsorption as function of the
pH of the solution (the shaped area represents the range of isoelectric points
reported in the literature).15
solution pH.33 The maximum loading of the enzyme was
achieved near its isoelectric point (pI = 9.8). Similar observations
were reported for the adsorption of myoglobin and bovine serum
albumin on SBA-15.34
One further possibility to avoid or minimize leaching from
the support is enzyme encapsulation inside the pore after
physical adsorption by introducing bulky functional groups to
decrease the pore size entrance. Several routes have been
explored including silanation and coating with microporous
silicas or organic layers. First, the protein is adsorbed from the
solution and then the opening of mesopores of the silica
support is reduced in size through silanation in order to
prevent subsequent leaching of the enzymes during e.g. a
catalytic reaction.13
For instance, porcine pancreatic lipase (PPL) was physically
adsorbed in the mesopores of SBA-15 followed by simple
silanation of the pore entrance with 3-(trimethoxysilyl)propyl
methacrylate, which effectively reduced the pore opening and
suppressed the leaching of lipase from the support. An alternative
approach for reduction of the pore opening is silanation with vinyl
groups followed by in situ polymerization with free 3-(trimethoxy-
silyl)propyl methacrylate. Activity assays confirmed that the second
strategy was more effective in suppressing enzyme leaching from
the pores. The authors assumed that the enhanced stability was
due to reducing the pore opening to a size smaller than the size of
the lipase.35
An interesting strategy to encapsulate the adsorbed enzymes
inside the pores of the support was presented by Wang and
Caruso. The authors used bimodal mesoporous silica spheres
(BMS), with different pore sizes and a bimodal pore system
(dp = 2–3 nm and dp = 10–40 nm), for the immobilization of
catalase, cytochrome c, lysozyme, peroxidase and protease. All
enzymes were physically adsorbed on the mesoporous silica
spheres followed by layer-by-layer (LbL) coating with nanoscale
multilayer shells to encapsulate the enzymes. It has been
demonstrated that BSM particles show a faster immobilization
rate and exhibit higher enzyme immobilization capacity than
similar mesoporous silica spheres with smaller pores (2–3 nm).36
When enzymes are entrapped inside the pores by using one
of the above described immobilization strategies, they possess
higher conformational freedom than covalently anchored enzymes,
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and, thus, their environment is much closer to that of free
enzymes. This results in the most cases in higher enzymatic
activity of physically adsorbed enzymes. Moreover, reduction of
the pore entrance size using silanation methods effectively
prevents leaching. However, modification of the support after
adsorption of enzymes may result in reduced enzyme activity
due to denaturation or deactivation of the enzyme through the
chemicals used for this purpose. Moreover, the diffusion rate of
the substrates to the active site of the enzyme inside the pores
may also be affected.3,13,16
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Covalent binding
Often physical interactions between enzymes and silica supports
are not strong enough to keep the enzyme molecules inside the
pores. Enzyme leaching is a critical limitation for the use of silica
carriers in their ‘‘pure’’ form.
Another promising method for prevention of leaching of the
enzyme from the porous (silica) carrier is covalent binding of
enzyme to the support surface. Most covalent immobilization
methods require chemical modification of the support surface.
Thereby, silanol groups on the surface of silica supports are
condensed with organosilane species, for example alkoxysilanes,
chlorosilanes or methallylsilanes to generate covalently bound
organic moieties.3,4,15,16
The modification of silica surfaces is a well-established and
widely used technology in enzyme immobilization and the literature
offers numerous synthetic protocols.4,11,13–15 The advantage of this
method is that enzyme molecules are tightly fixed, thus, enzyme
desorption from the silica supports is minimized and e.g. enzyme
contamination of the product can be avoided.3
Xu et al. compared the immobilization of porcine pancreatic
lipase (PPL) on SBA-15 functionalized with (3-aminopropyl)-
triethoxysilane with an unfunctionalized surface of the same sup-
port and found that chemically anchored PPL showed higher
loading and catalytic activity compared to physically adsorbed
PPL. Furthermore, covalently immobilized PPL showed improved
stability towards temperature and pH changes of the reaction
medium and exhibited better reusability.37
There are two general pathways to introduce organic groups
onto the silica support, namely in situ modification during
Fig. 2 Co-condensation method (left) and grafting (right) for organic modification of mesoporous silica supports (R = organic functional groups). Reproduced with
permission from ref. 16.
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synthesis, so called co-condensation, or postmodification,
also known as grafting.15,16 Co-condensation allows surface
modification of mesoporous silicas in a single step (one-pot
synthesis) by copolymerization of tetraalkoxysilanes [(RO)4]Si
(typically, tetraethylorthosilicate (TEOS) or tetramethylortho-
silicate (TMOS)) with terminal trialkoxyorganosilanes of the type
(R0 O)3SiR in the presence of one surfactant (Fig. 2, left).15,16 This
modification technique provides a higher and more homo-
geneous loading of the surface with organosilane groups com-
pared to materials functionalized via grafting. Nevertheless, the
co-condensation method has several shortcomings: the amount
of trialkoxyorganosilanes incorporated into the silica materials is
generally lower than one would expect from their initial concen-
tration in the reaction medium. Increasing concentration of
trialkoxyorganosilanes in the reaction medium may result in
the formation of products with highly disordered structure. The
consequence is a reduced surface area and a lower degree of
loading with organic moieties.15,16
Furthermore, functionalized alkoxysilane must hydrolyze at
a similar rate to the unfunctionalized alkoxysilane, otherwise a
heterogeneous gel will be formed and, thus, the functional
groups will not be evenly distributed.15
Grafting is achieved by covalent linking of organotrialkoxy-
silanes or other reactive silane species such as trichloro-
organosilanes or silylamines with the free surface silanol
groups on the (ordered or disordered) silica support (Fig. 2,
right). By using the appropriate organosilane species, various
functional groups such as chloride, alkyl, thiol, cyano/isocyano,
vinyl/allyl, organophosphine, phenyl, alkoxy or amino groups
can be attached to the surface of silica carriers.12,15,16
Although due to the attachment of a layer of functional
moieties, the pore size will slightly be reduced, the structure of
the starting material is usually preserved.
In contrast to co-condensation, grafting is more time-
consuming, because two steps are required for the formation
of modified silica supports. Furthermore, the control of loading
and position of organosilanes is difficult and diffusion problems
may occur, if the organosilanes react preferentially with silanol
groups at the pore entrance during the initial phase of
the reaction. Consequently, the content of organic groups in
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Fig. 3 Post-synthesis (grafting) functionalization of mesoporous silica materials with different functional groups.
the modified silica support will be low and their distribution
within the pores of the silica support will be inhomogeneous.
In extreme cases, this may result in pore blocking.15,16 Since
grafting is easy feasible for a broad range of functional groups
and does not interfere with the formation of the silica frame-
work, post-synthesis modification is the commonly used
method for surface functionalization with respect to use for
enzyme immobilization (Fig. 3).15
Enzymes are highly complex molecules and possess several
different functional groups on their surface, which may react
with a functionalized silica support. The most commonly used
functional moieties of the enzymes are the amino groups of
arginine, lysine and histidine residues, the carboxylic groups of
glutaric acid or the sulfhydryl groups of cysteine residues.1 The
most popular route for covalent immobilization of enzyme on
porous silica supports is the functionalization of the material
surface with amino groups (mostly via (3-aminopropyl)triethoxy-
silane, APTES) followed by reaction with glutaraldehyde (GA) to
provide a suitable linker (Fig. 4). The aldehyde groups on the
Fig. 4 Covalent enzyme immobilization, APTES-modified mesoporous silica reacts with GA and N-terminus of the enzyme.
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surface of the silica support subsequently react with the amino-
groups of the enzyme under formation of imine bindings. In some
immobilizing protocols, the imine bindings were further reduced
by using NaBH4.7 Other bifunctional spacers such as glutaric
anhydride, succinimido-3-maleimidopropanoate, cyanuric or bulky
polyaldehydes, chlorides can be also used as cross-linking agents.11
Other popular and commercially available organosilanes
used in surface modification are 3-(glycidoxypropyl)-trimethoxy-
silane39 and 3-(mercaptopropyl)-triethoxysilane,40 which will be
used for functionalization of the surface with epoxy groups or
thiol groups, respectively. 3-(Mercaptopropyl)-triethoxysilane
functionalized silicas can further react with thiol groups of
cysteine residues of the enzyme resulting in the formation of
disulfide bonds.11
A major advantage of covalent binding of enzymes to silica
carriers is that the resulting biocatalyst can also be used in
polar solvents such as water, while physically adsorbed
enzymes may only be used in organic solvents or with pure
hydrophobic reactants in order to avoid leaching.6
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The main disadvantage of the covalent immobilization of
enzymes is the lower specific activity of the resulting biocatalysts
compared to the free enzymes in solution. The operational
stability, however, is in most cases improved, especially, when
multipoint attachment on the supports is achieved.3,4
Nevertheless, in some cases, random or poor immobilization
resulting in bad mass transport, undesirable enzyme active site
orientation or covalent agent induced denaturation of the
enzyme are observed, which often leads to a significant decrease
or even to a complete loss of biocatalytic activity.11
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Cross-linked enzyme aggregates (CLEAs)
Cross-linking originally is a carrier-free approach based on the
formation of cross-linked enzyme aggregates (CLEAs).41 CLEAs
can be prepared easily by a two-step process. In the first
step, precipitation of enzyme aggregates is performed through
addition of precipitants (salts, organic solvents or polymers)
under mild reaction conditions (typically: pH = 7.0, T = 4 1C).
Thereafter, enzyme aggregates are stabilized by addition of
cross-linking agents, such as glutaraldehyde, which react with
amino groups of the enzyme in a base-catalyzed reaction
forming CLEAs (Fig. 5).15
The advantage of this method is that cross-linked enzymes
often retain their catalytic activity even under harsh reaction
conditions such as extreme pH values and high temperature or
by application of organic solvents. The cross-linking of enzymes
as a immobilizing strategy was reported by several research
groups. A good overview can be found in ref. 6, 8, 11, and 12.
Based on preliminary work, by Sheldon et al.,38 a new
approach was developed. Thereby the enzymes are physically
adsorbed on (meso)porous materials, aggregated and subsequently
cross-linked with GA, resulting in CLEAs entrapped in the pores.
The carrier-free CLEAs can grow up to a size of 100 mm.8
Thus, a large fraction of active enzyme is located in the center
of the CLEAs and does not take part in the reaction, resulting in
a low effective activity of the enzyme. Due to the formation of
CLEAs inside the channels of (meso)porous silicas, the size of
Fig. 5 Formation of CLEAs for enzyme immobilization (left) in solution and (right) in the pores of a suitable support.
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the CLEAs is restricted by the size of pores and channels of the
carrier. Another advantage of the carrier-bound CLEAs in
comparison to the carrier-free counterparts is that no addition
of (aggressive) precipitants is necessary since the enzyme
molecules naturally aggregate in the pores of the porous solids2
by adsorption. Kim and co-workers employed mesocellular
mesoporous silica materials (HMMS) and SBA-15 for the immo-
bilization of a-chymotrypsin and lipase from Mucor javanicus.
Both enzymes were physically adsorbed inside the pores of
HMMS followed by cross-linking with glutaraldehyde. CLEAs of
a-chymotrypsin showed besides the high enzyme loading also
an increased enzyme stability towards shaking and trypsin
digestion in comparison to free and physically adsorbed
a-chymotrypsin on the same support.43,44
Jung et al. described the successful use of the CLEA
strategy for the immobilization of chloroperoxidase (CPO) from
Caldariomyces fumago and glucose oxides (GOx) from Aspergillus
niger in the pores of mesocellular foams (MCF). Optimization of
immobilization conditions by adjustment of pH during the
preparation of CLEAs increased the specific activity of the
biocatalyst by a factor of five compared to physisorbed CPO
on MCF. A similar increase in specific activity by adjustment of
pH was observed for GOx. Furthermore, it was shown that the
resulting heterogeneous biocatalyst is significantly more stable
against leaching than the conventional catalyst prepared by
physical adsorption of CPO on the same support. Finally,
the location of CLEAs in the cages of MCF was confirmed by
small-angle neutron scattering (SANS) after perfluoropentane
adsorption.45
The advantages and disadvantages of the different immobi-
lization methods discussed in this review are compared in
Table 3.
Each immobilization technique has its own advantages and
disadvantages and a ‘‘perfect’’ universally applicable method of
enzyme immobilization is not available. Typically a trial and
error strategy is employed until a biocatalyst with the desired
target properties is found. Independent of the method selected
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Table 3 Advantages and disadvantages of the different immobilization methods
Immobilization
method Advantages
Physical  Cheap, simple and rapid experimental procedure
adsorption  (Mostly) no functionalization of support is required
 No toxic solvents are required
 No conformational changes of the enzyme
 No destruction of the active site of the enzyme
Chemical  No leaching of enzymes from the support
(covalent)  Tight binding of enzyme to the support
binding  Wide choice of organic linkers is available
 Established methods of functionalization/
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modification of supports
Entrapped  Stabilization of multimeric enzymes
CLEAs/  Stabilization towards harsh reaction conditions
encapsulation (e.g. extreme pH)
 High purity of enzyme is not required
 No leaching from the support
 Different enzymes can be co-immobilized
(tandem-system)
 No or minimal conformational changes of
the enzyme
 Size of the CLEAs is restricted by the cage size
for enzyme immobilization, the main goals are to achieve high
loading of enzyme with high retention of activity and enhanced
operational stability and durability. With respect to industrial
scale application the immobilization process should be straight-
forward as well as energy and cost efficient.
Factors influencing enzyme immobilization
As discussed in the preceding chapters, the successful immobili-
zation of enzymes on porous silica supports and the properties of
the resulted biocatalyst are largely determined by several factors.
These include the experimental conditions such as the pH and
temperature of the buffer solution, and material properties such
as size of enzyme and physicochemical properties of the porous
hosts viz. morphology, particle size, composition and pore size.4
For the enzyme, the presence of polar groups (e.g. amino
groups of lysine or/and acid groups of glutamic acid), apolar
surface regions or sugar residues largely influences enzyme
immobilization.4,11
Pore size
Numerous studies indicated that enzyme loading and activity
are clearly dependent on pore size of the silica support. There-
fore, not all porous silica materials are applicable for enzyme
immobilization. Carriers with pore diameters too small to
accommodate enzyme molecules such as microporous zeolites
do not provide a stable support, because in this case enzyme
molecules will be adsorbed on the external surface and will no
longer be protected towards their environments.3,4,13,14 If the
pores are much bigger than the enzyme molecules, the latter
cannot be protected and retained as well as in smaller pores. In
the case of the physical adsorption a leaching of enzyme
molecules from the pores is possible. Porous materials are often
associated with large specific surface area, which is, however, not
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Disadvantages
 Leaching of enzymes from the support during the catalytic reaction
due to changes in reaction conditions (e.g. temperature, pH) or
through mechanical shear forces
 Most complicated and expensive immobilization method
 Functionalization/modification of support surface is necessary
 Use of toxic chemicals (e.g. glutaraldehyde)
 Reduction or even loss of catalytic activity resulting from
conformational changes of the enzyme
 Complicated experimental process (more than two steps are
necessary)
 Use of toxic chemicals (e.g. glutaraldehyde)
 Decreased diffusion rate of substrates/products due to reduced
pore volume
a guarantee for obtaining high enzyme loadings. For example,
although MCM-41 materials possess a very large surface area
(B1000 m2 g1), often only low enzyme loading and slow
immobilization rates have been reported for these materials.
Since the pore size of MCM-41 is typically around 4 nm, they are
restricted to immobilization of enzymes with relatively small
sizes.36 To reach high enzyme loading and high activity reten-
tion, a compromise between the pore size of the support and the
site of enzyme has to be found.
Very recently, Hisamatsu et al. studied the adsorption of
a-amylase from Bacillus licheniformis on FSM, KIT-6 and two-
dimensional SBA-15 with different pore sizes. The amount of
a-amylase (dimensions 4.5  5.1  8.5 nm) encapsulated increased
with increasing pore size of the silica support in the following
order: SBA-15 (dp = 11 nm) o KIT-6 (dp = 11 nm) o FSM (dp =
9 nm). Due to its morphological characteristics, viz. the presence of
larger pores with disordered arrangements and smaller particles
size, FSM exhibited the highest adsorption capacity in comparison
to KIT-6 and SBA-15 with somewhat similar pore size. Moreover,
a-amylase immobilized on FSM-type materials exhibits higher ther-
mal stability than the free enzyme.46 Although the ordered arrange-
ment of the pores is beneficial for characterization of the material, a
clear advantage for any application has not been reported.
Particle size and morphology
Early research has already shown that the morphology and
particle size of the silica carrier have a significant influence on
immobilization ability. Furthermore, with respect to large scale
industrial applications, these carrier properties often influence
the selection of the reaction system and the reactor configu-
ration. For example, for a fixed-bed reactor large size particles
are required to reduce the pressure drop.4
Lei et al. studied the influence of support morphology on
immobilization behaviour of lysozyme. For that purpose four
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silica carriers with different morphologies were synthesized
and evaluated with respect to enzyme adsorption. Rod-like
SBA-15 (B1–2 mm in length) exhibits in addition to higher
achievable loadings a faster adsorption rate than the conven-
tional SBA-15 (B20 mm in length). Furthermore, it was shown
that the number of pore entrances increases with decreasing
particle size of mesoporous silicas used in this study, leading to
obvious improvement of enzyme uptake.47 Zhou and co-work-
ers investigated the influence of particle morphology on the
immobilization of Candida rugosa lipase (CRL). They showed
that immobilization on vesicle-like silica results in higher
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thermal stability and reusability compared to CRL immobilized
on a rod-like silica. The catalytic activity of lipase adsorbed
on the vesicle-like silica in the hydrolysis of tributyrin in
phosphate buffer solution was also obvious higher compared
to rod-like silica adsorbed CRL.48
Very recently, three kinds of mesoporous silica supports
with different particle sizes (40 nm, 300 nm and 1000 nm),
but the same pore size of 9 nm, respectively, were used by
Gustafsson et al. for the immobilization of lipases from Mucor
miehei (MML) and Rhizopus oryzae (ROL).49 The used lipases
have the same molecular size, but differ largely in their iso-
electric points (pI (MML) = 3.8; pI (ROL) = 7.6). Although, the
enzyme loading was similar for the samples used, it was shown
that both lipases were most active when immobilized onto the
300 nm particles.
Surface chemistry
The surface chemistry of the chosen carrier also plays an
important role in binding enzymes as this will influence the
strength of the interactions between the biomolecules and the
surface of the supports.
The hydrophobicity or hydrophilicity of the support may
have a strong influence on immobilization behaviour and
catalytic activity of immobilized enzymes. Most enzymes have
hydrophilic character and thus adsorb rather to hydrophilic
silica supports with a polar surface.4,11 The immobilization of
other enzymes e.g. lipases is dominated by hydrophobic inter-
actions. Sörensen and co-workers immobilized lipase from
Thermomyces lanuginosus (TLL) on hydrophilic and hydrophobic
support surfaces. The specific activity of lipase immobilized on
the hydrophilic support was similar to the specific activity of
native lipase in solution. On the other hand, the specific activity
of the enzyme immobilized onto the hydrophobic support was
improved in comparison to lipase immobilized on the hydro-
philic support and native lipase. This was attributed to inter-
facial activation of the lipase when it is attached to a
hydrophobic surface and a reduced denaturation. Subsequently,
it could be shown that the rate and the amount of enzyme
leached from the hydrophobic support were reduced.52 Similar
observations were made also by Zhou et al. for the immobiliza-
tion of TLL on PMOs with different organic groups, viz. ethylene,
ethenylene and benzene. They found that lipase immobilized on
these materials showed better storage stability than lipase
immobilized on pure silica materials. Moreover, the catalytic
activity of lipase immobilized on organosilica supports for
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hydrolysis of 4-nitrophenyl palmitate was even higher as for
native lipase.55
In some immobilization protocols were reported that different
methods of binding enzymes to the porous silica support resulted
in varying degrees of stability. Changing one type of binding
functionality (organic linker) to another can also influence the
performance of the catalytic activity of immobilized enzyme.23,24
Jung et al. functionalized a mesoporous SBA-15 support with two
different organic linkers, 3-aminopropyltrimethoxysilane (further
functionalization with GA) and 3-glycidooxypropyltrimethoxy-
silane (GTS) and investigated the influence of different functional
moieties on the surface on the adsorption capacity and catalytic
activity of CPO and GOx. The GTS-functionalized SBA-15 adsorbed
both enzymes, but only glucose oxidase catalytic activity stayed
after immobilization. Since the catalytic activity of CPO immobi-
lized on GTS–SBA-15 was nearly zero, the authors supposed that
the epoxy moiety is too reactive and probably binds several other
functional groups of enzyme like hydroxyl or thiol moieties, which
leads to disruption of the quaternary structure of enzyme and its
deactivation. Glutaraldehyde-functionalized APTES–SBA-15 in
contrast adsorbed both enzymes without losing any activity. The
catalytic activity of immobilized enzymes was tested in a fix-bed
reactor under continuous operating conditions. It was also shown
that enzymes, covalently immobilized on functionalized meso-
porous hosts, can be stored for weeks without losing their catalytic
activity in contrast to the physically adsorbed enzymes, which
show a decrease of activity after a certain period of time.39
The influence of metal-ions viz. Al, Ti, Co, Cr, V which were
incorporated onto the pores of mesoporous silicates, on
enzyme immobilization has also been investigated.15,42 Initially
aluminium-substituted mesoporous silicates have also been
used to adsorb different enzymes. It has been found that the
amount of cyt c adsorbed on the aluminium-substituted SBA-15
was higher in comparison to the pure silica analogues. The
authors suggested that the increase in adsorption can be
explained as a result of the strong electrostatic interactions
between the positively charged amino acid residues on the
surface of enzyme and the negative charges on the aluminium
sites.50 Very recently, Secundo et al. investigated the effect of
the incorporation of Al into the SBA-15 framework on the
adsorption behaviour of a-chymotrypsin and catalytic activity
of the resulted biocatalyst in organic solvents. The amount of
adsorbed a-chymotrypsin was found to be dependent on the
Si/Al ratio of the support and the pH of the adsorption solution.
The catalytic activity of the biocatalyst was also strongly dependent
on the nature of the supports.51
Challenges in enzyme immobilization
All immobilization strategies discussed in this review refer
to co-factor free enzymes or to enzymes with tightly bound
co-factors. Many important enzymes that are employed in the
field of chemical processing, e.g. oxidases or reductases,
require a co-factor for their catalytic cycle. A co-factor, such as
nicotinamide adenine dinucleotide (NAD(H)) or nicotinamide
adenine dinucleotide phosphate (NADP(H)), is a non-protein
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Fig. 6 Model for a co-factor recycling system.
chemical compound that acts as a stoichiometric agent in
biotransformation reactions.1 The cofactors undergo transfor-
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mation during (bio)chemical reactions with the result that
the cofactor-dependent enzymes are incapable of catalyzing
further reactions, until the cofactor is replaced. The majority
of cofactors are expensive and often relatively sensitive toward
reaction conditions to be used in stoichiometric amounts for
biocatalysis. Co-factors can be regenerated1 by using e.g.
a second enzyme, typically alcohol dehydrogenase (ADH) or
formate dehydrogenase (FDH), which catalyzes the reverse
reaction (Fig. 6).
Like enzymes, co-factors can also be immobilized on differ-
ent supports. A co-factor regenerating system composed of two
enzymes and a co-factor, immobilized together or separately on
the same support would be interesting for academia and
industry. Such a system was recently proposed by Zheng
et al.56 Two enzymes, glutamate dehydrogenase and glucose
dehydrogenase, and cofactor NADH were immobilized both
separately from each other (E–C–E) and together (co-immobilized,
ECE) on silica-coated magnetic nanoparticles. Both systems were
tested with respect to their catalytic activity and reusability. It was
demonstrated that in the case of separately immobilized enzymes
and cofactors, only 20% of initial activity were lost after 10 cycles of
reuse. For the co-immobilized system, the retention of residual
activity was even higher with over 90% after 10 reuse-cycles. More-
over, due to using magnetic nanoparticles as a support, the
immobilized species were easily separated from the solution by
applying a magnetic field.
Another problem is to present unambiguous evidence for
the presence of the enzymes inside the pores of the silica
supports. Reducing the pore diameter or pore volume after
enzyme immobilization does not give evidence for enzyme
uptake into the pore. Another possible explanation for the
observed reduction of specific pore volume or pore diameter
is blocking of the pore entrances by enzyme molecules that
have not fully entered the pore. However, constant pore volume,
before and after the immobilization process, suggests that the
adsorption of enzyme occurred only on the external surface of
the silica materials.14,33,50
One promising possibility to detect whether and how the
enzyme molecules are distributed inside the pores of the
support is the so called confocal laser scanning microscopy
(CLSM). It is possible to image individual particles in the bulk
of a concentrated dispersion by CSLM, due to a combination of
powerful depth discrimination (optical sectioning) and an
increased resolution compared to conventional light. Sörensen
et al. used this technique to determine the spatial distribution
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of lipase from Thermomyces lanuginosus, labeled with Alexa
Fluors 488 and immobilized on commercial fumed silica.52
Piras and co-workers proved the presence of human lysozyme
inside the pores of mesoporous SBA-15 with the aid of the
immunogold staining (IGS) method. For this purpose enzyme
loaded SBA-15 was first mixed with unmarked primary antibody,
which was specific to human lysozyme protein. At the following
step a secondary antibody, conjugated with colloidal gold particles
was added. Subsequently transmission electron microscopy (TEM)
was used for visualization of enzymes inside the pores.53
Very recently Mayoral et al. reported that the presence of
enzyme inside the pores can be visualized by using electron
energy loss spectroscopy (EELS).54
Novel trends in enzyme immobilization
In this paragraph we want to highlight novel developments in
enzyme immobilization with probably high potential for
chemical synthesis and industrial applications.
Novel surface functionalization strategies
Several publications devoted to ionic liquids as functionalizing
agents for mesoporous silica supports appeared in recent years.
Very recently, Bian et al. used a so-called room temperature
ionic liquid (RTIL), denoted [Simim+][Cl] for modification of
silica SBA-15 (RTIL–SBA-15) and applied the resulting materials
for adsorption of papain. It was shown that the papain loading
on RTIL–SBA-15 (261 mg g1) was higher than on unfunctiona-
lized SBA-15 (185 mg g1), when the adsorption was conducted
at pH = 9, probably as a consequence of electrostatic interactions
between the cation [Simim+] and the negatively charged papain14
(pI = 8.75). Subsequently, the resulting biocatalyst was tested in
the hydrolysis of casein. It was shown that the catalytic activity of
the papain immobilized on RTIL–SBA-15 was also improved in
comparison to that of SBA-15.57
Another promising surface modification method was
reported in 2010 by Gaffney and co-workers. The authors
functionalized the surface of SBA-15 with Ni-cyclam and used
the resulted hybrid material for immobilization of the His6-
tagged protease inhibitor (Spi). It was demonstrated that
the protein uptake in SBA-15–Ni(II)-cylam was relatively high
(6.7 mmol g1, 85% of protein). It was also shown that the
protein uptake without His6-tag was only 0.43 mmol g1 (B7%).58
Tandem-systems
A large advantage of enzymes as catalysts is the rather narrow
range of reaction conditions. Thus the combination of different
enzymes in one-pot reaction is possible.
Jung et al. investigated the catalytic behaviour of immobi-
lized chloroperoxidase for oxidation of indole. It was reported
that external addition of hydrogen peroxide to the batch reactor
results in deactivation of the immobilized CPO due to high
local H2O2 concentrations. Deactivation was largely suppressed
by in situ hydrogen peroxide generation. Thereby H2O2 was
produced by oxidation of glucose with glucose oxidase immobilized
on SBA. Furthermore, the tandem catalyst can be recycled several
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Fig. 7 Tandem reaction with chloroperoxidase and glucose oxidase immobi-
lized on ordered porous silica.
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times without significant loss of catalytic activity of enzymes
(Fig. 7).59
The surface of some silica supports such as porous glasses is not
usable for attachment of enzymes without suitable derivatization.
An interesting method of enzyme immobilization, combined
enzyme engineering and the use of silica carriers was developed
recently by Bolivar and co-workers. They immobilized successful two
enzymes of industrial interest, D-amino acid oxidase and sucrose
phosphorylase, on the underivatized porous glass surface under
physiological pH conditions via so called SBM (silica binding
module). As SBM contains a strongly positively charged mini-protein
called Zbasic 2, an engineered mini-protein of 7 kDa size, which
anchors the enzymes under studies to the unfunctionalized glass
surface. It has been shown that immobilized enzymes retained their
full biological activity after the immobilization process. The authors
suggested that binding of enzymes to the glass surface occurred in a
preferred orientation via the Zbasic 2 module.60
Conclusions
Porous silicas are suitable supports for the immobilization of
enzymes. Depending on the enzyme and the targeted application a
suitable immobilization strategy has to be chosen. Physical adsorp-
tion, encapsulation, covalent binding and recently cross-linking are
the predominantly employed routes that possess certain advantages
and drawbacks. The use of tailor-made silica supports with opti-
mized particle size and morphology, pore diameter and surface
properties will result in biocatalysts with increased activity, higher
stability and reusability. Novel surface functionalization strategies
including the use of ionic liquids for modification of the support
properties are constantly being developed. The immobilization of
two or more enzymes on the same support is possible and should be
explored further with respect to one-pot reactions. However, until
now, industrial applications exploring the specific features of porous
silica supports have not been disclosed. In our opinion, the prepara-
tion of CLEAs in the pores of a suitable support is most promising.
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