Abstract

Immobilization is a key technology for successful realization of enzyme-based industrial

processes, particularly for production of green and sustainable energy or chemicals from
biomass-derived catalytic conversion. Different methods to immobilize enzymes are critically
reviewed. In principle, enzymes are immobilized via three major routes (i) binding to a support,
(ii) encapsulation or entrapment, or (iii) cross-linking (carrier free). As a result, immobilizing
enzymes on certain supports can enhance storage and operational stability. In addition, recent
breakthroughs in nano and hybrid technology have made various materials more affordable hosts
for enzyme immobilization. This review discusses different approaches to improve enzyme
stability in various materials such as nanoparticles, nanofibers, mesoporous materials, solgel
silica, and alginate-based microspheres. The advantages of stabilized enzyme systems are from
its simple separation and ease recovery for reuse, while maintaining activity and selectivity. This
review also considers the latest studies conducted on different enzymes immobilized on various
support materials with immense potential for biosensor, antibiotic production, food industry,
biodiesel production, and bioremediation, because stabilized enzyme systems are expected to be
environmental friendly, inexpensive, and easy to use for enzyme-based industrial applications.
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Abbreviations
CLEAs
cross-linked enzyme aggregates
CLECs
cross-linked enzyme crystals
GA
glutaraldehyde
MWCNTs
multiwall carbon nanotubes
PVA
polyvinyl alcohol
TMOS
tetramethylorthosilicate
1 Introduction
Enzymes are proteins that catalyze chemical reactions. Like all other catalysts, enzymes work to
reduce the activation energy of a reaction, or the initial energy input necessary for the reaction to
occur; thus, dramatically increasing the reaction rate millions of times [1]. Enzymes have many
benefits in the development of chemical transformation processes, food, and pharmaceutical
production, bioremediation, or biofuel cell production. Furthermore, enzymes can be used for
green and sustainable energy or biomass-derived catalytic conversion technology [2]. Enzymes
usually function under mild conditions, such as physiological ambient temperature and pressure.
Enzyme processes can now be performed in organic solvents and aqueous environments, so not
only nonpolar organic chemicals but water-soluble compounds can be transformed selectively

and efficiently [3]. Thus, industrial chemical synthesis using enzymes has become easier and
more productive.
Biochemists and microbiologists have long seen the potential of enzymes for chemical synthesis,
and during the past decade, it has been shown that there are surprisingly few barriers for use of
enzymes in organic synthesis. As a result, enzymes can be used in either simple or complex
transformations without the need for the inducers or inhibitors that are common in enantio- and
regioselective organic syntheses [2]. Such high selectivity also affords efficient reactions with
few by-products, thereby making enzymes an environmentally favorable alternative to
conventional chemical synthesis, particularly in the food and pharmaceutical industries where
high reaction selectivity on complicated substrates is crucial [4]. Selection of enzymes to
optimize various processes is now becoming a requirement for the chemical industry, and recent
advances in enzymatic catalysis have been extended to the synthesis of specialized chemicals
and polymers [5].
The unique potential of enzymes has still not been fully explored, but some of industrial enzymes
are commercially available [6]. Prices can vary significantly, depending on the degree of
difficulties in isolating the enzyme or availability from recombinant sources. Advances in protein
engineering have made it possible to manipulate enzymes exhibiting desired properties with
regard to substrate specificity, activity, selectivity, stability, and pH optimums using techniques,
such as site-directed mutagenesis and in vitro evolution by gene shuffling [7]. In addition, most
enzymes that are currently used in industry can be produced on a large scale. As a result,
commercialized enzymes have paved the way for widespread industrial applications [8, 9].
Nevertheless, industrial applications are often limited by a lack of long-term operational stability
and difficult recovery and reuse of the enzyme. These obstacles can often be overcome by
stabilizing the enzyme [10]. Once immobilized, enzymes can be stabilized, and, thus, are less
sensitive to their environment. Immobilization can increase dispersion in insoluble hydrophobic
organic media, leading to improved accessibility to the substrate, and avoiding aggregation of the
hydrophilic protein particles. In addition, immobilization ensures that the biocatalyst can be
readily recycled, which reduces the cost of utilization.
In principle, enzymes are immobilized via three major routes (i) binding to a support, (ii)
encapsulation or entrapment, or (iii) cross-linking (carrier-free). Each immobilization method
bears assorted advantages and disadvantages, making a comparison of the methodologies
difficult. Many chemists have tried to increase the performance of immobilized enzymes, such as
specific activity, storage, and recycling stability, and ease of reuse. Meanwhile, materials
scientists have developed more efficient supports to realize better immobilization. Enzyme
stabilization via immobilization has been proposed as a potential strategy. During the last couple
of decades, enzyme immobilization technology has led to successful advances in many applied
disciplines, such as biosensor, antibiotic production, drug metabolism, food industry, biodiesel
production, or bioremediation.
In this review, we will introduce the principal immobilization techniques, such as the use of
binding for support, entrapment methods, and the recent development of cross-linked enzyme
aggregates (CLEAs) along with various carriers such as polymers and inorganic materials. We
will also discuss the role of those nano/micro and hybrid materials in enzyme stabilization,
especially for nanoparticles, nanofibers, mesoporous materials, sol-gel silica, and alginate
microspheres. Last, we will introduce how the immobilized and stabilized enzyme technology is
used in practical applications such as clinical, industrial, and environmental applications.

2 Enzyme stabilization (types of immobilization)

Immobilization of an enzyme entails the interaction of the enzyme and the carrier. Some of the
parameters to be considered when planning enzyme immobilization are shown in Table 1.
Enzyme immobilization allows the separation of biocatalyst from products, thereby permitting
continuous processing [11]. Immobilization can improve enzyme performance under optimal
reaction conditions (e.g. acidic, alkaline, organic solvents, and elevated temperatures), and allow
applications in industrial chemical synthesis. Due to the long history and obvious advantages and
disadvantages of enzyme immobilization (Table 2), a number of interesting new developments
have been reported in the literature with patent applications [12]. Basically, three traditional
methods of enzyme immobilization have been described, including binding to the supports
(carriers), entrapment or encapsulation with supports, and cross-linking on the supports (Fig. 1).
Table 1. Parameters influencing the activity of immobilized enzymes
Factors related to enzymes

Factors related to the carriers

Specific factors related t

reaction system

Size of the enzyme

Organic or inorganic

Reaction medium

Conformational flexibility
required by the mechanism

Hydrophobic or hydrophilic

Diffusion limitations

Isoelectric point

Surface charges

Enzyme inhibition

Surface functional groups/charge

density

Surface functionalization

Precipitation of produc

Glycosylation

Chemical and mechanical stability

Viscosity of the mixture

Stability under immobilization

conditions

Surface area

Reaction thermodynam

Presence of hydrophobic regions

Porosity

Non-specific solute-sup
interactions

Presence of hydrophilic regions

Particle size

Additives in the enzymatic

preparation
Table 2. Advantages and disadvantages of three types of the immobilization methods: binding to
support, encapsulation, and cross-linking

Immobilization method
Binding to a support(Physical
adsorption)

Advantages
Simple experimental procedure

Disadvantanges

Leaching of enzyme
reaction

No toxic solvents
(Chemical binding)

Entrapment or encapsulation

Enzyme molecules retained

More complicated pr
support preparation

Wide choice of organic linkers

available

Enzyme molecules i
inside carriers

Enzyme molecules retained

Complicated experim
procedure

Enzyme molecules free to move

inside carriers

Reactive species and

solvents may denatu

Decrease diffusion r
reactants and produc
Cross-linking

Enhanced shelf life and

operational stability

Loss of the enzyme's

Easy to recover and reuse

Decrease diffusion r
reactants and produc

Stable towards leaching in

aqueous media
Figure 1.

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Overview of the different enzyme immobilization strategies.
2.1 Binding of enzymes to the supports

Support binding can be performed by physical (such as hydrophobic and van der Waals
interactions), ionic, or covalent binding. Physical bonding is generally known to be too weak to
keep the enzyme fixed to the carriers under industrial conditions. Ionic binding is generally
stronger, and covalent binding of the enzyme is even stronger [13]. Support binding, especially
strong covalent binding, prevents the enzyme leaching from the surface. However, this can also
be a major drawback; if the enzyme is irreversibly deactivated, both the enzyme and support are
unusable. The properties of the immobilized enzyme are governed by the properties of both the
enzyme and the carrier material. The interaction between the two provides specific chemical,
biochemical, mechanical, and kinetic properties. The support (carrier) can be a synthetic
polymer, biopolymer, or an inorganic solid.
So far, various commercialized synthetic organic polymers, such as Eupergit C, beads FP-EP,
and Amberlite XAD-7 (100500 m), were used in enzyme stabilization due to their availability
and cost. But due to their weak mechanical stabilities, chemical, and physical modifications have
been subjected to show better performances on swelling or shrinking over a wide pH ranges from
0 to 14 or even upon drastic pH changes [14]. In this case, however, they are still suffered in
inducing mass transfer limitation and lower enzyme loading, resulted in poor stabilization.
A variety of biopolymers, mainly water-insoluble polysaccharides such as cellulose, starch,
agarose, alginate, and chitosan, or proteins, such as gelatin and albumin have been widely used
as supports for immobilizing enzymes [15]. Enzyme can be immobilized by ionic adsorption and
covalent attachment in a fixed-bed reactor for continuous operation. Enzymes can also be
immobilized in natural or synthetic hydrogels or cryogels. Polyvinyl alcohol (PVA) cryogels
were formed by the freeze-thawing method [16]. Immobilization of free enzymes in PVA
hydrogels can be useful in organic media by preserving the activity of the gel matrix. An
alternative method to increase the size of an enzyme is to form a complex with polyelectrolyte
domains [17]. Covalent attachment to stimulus-responsive or smart polymers is a new
approach to immobilize enzymes. These smart polymers undergo dramatic conformational
changes in response to small changes in their environment, such as temperature, pH, and ionic
strength [18]. The most studied example is the thermo-responsive and biocompatible polymer,
poly-N-isopropylacrylamide. More recently, an alternative thermoresponsive polymer has been
described [19]. It consists of random copolymers derived from 2-(2-methoxyethoxy)
ethylmethacrylate and oligo(ethylene glycol) methacrylate, which combines the positive features
of polyethylene glycol. The properties of these copolymers are similar to poly-Nisopropylacrylamide. Furthermore, the lower critical solution temperature can be controlled by
changing the relative amounts of oligo(ethylene glycol) methacrylate. However, these organic
hydrogels, cryogels, or smart polymer-based supports have drawbacks in making the shape
uniformly due to the arise of rigidity from the hydrophilic interaction [20]. In overcoming this
problem, organicinorganic hybrid technology have been adopted to make fascinating
application potential in enzyme stabilization.
A number of inorganic materials can be used to immobilize enzymes. For example, alumina,
silicas, zeolites, and mesoporous silicas such as MCM-41 and SBA-15 have been reported as
immobilizing carriers [21]. The enzyme needs to be covalently bonded to the silica support to
maintain its integrity in an aqueous environment. Mesoporous silicas have several advantages as
inorganic supports. They have uniform pore diameters (240 nm), very high surface areas (300
1500 m2 g1) and volumes (ca. 1 mL g1), and are inert and stable at elevated temperatures. Their
surface can be easily functionalized for binding moieties, and they can accommodate enzymes in
their pores. Therefore, the enzyme is entrapped inside the pores or immobilized on the outer

surface [22]. Another type of immobilization on inorganic supports comprises the so-called
protein-coated microcrystals. Nevertheless, due to the lack of specific interactions with enzyme
molecules, inorganic materials suffer from leaching of the immobilized enzyme during the
reaction process. Therefore, being decorated with functional organic groups for specific
interactions with enzymes makes them attractive to stabilize enzymes for successful catalytic
applications.
2.2 Entrapment or encapsulation of enzymes
Entrapment or encapsulation is to entrap the enzyme in a polymer network (gel lattice), such as
an organic polymer, a silica sol-gel, or a membrane device such as a hollow fiber or a
microcapsule. Additional covalent attachment is required. Entrapment requires the synthesis of a
polymeric network in the presence of the enzyme. Typically, alginic acid-based
microencapsulation forms an insoluble polymeric matrix for enzyme entrapment. In fact, alginate
microbeads or microspheres have been used in the enzymatic reactor process. But, due to their
mechanical stability, their utilization is limited to only a few applications [23]. In addition, a
novel polymer-incarceration methodology for immobilizing enzymes has been reported recently.
Polystyrene with pendant hydrophilic tetraethylene glycol and glycidol moieties and
dichloromethane can cause coacervation with an enzyme after adding 1-hexane, leading to a
precipitate containing the enzyme in the polymer phase. In water, organic chemicals are not
uniformly dispersed but may be separated out into layers or droplets. If the droplets formed
contain a colloid, rich in organic compounds and surrounded by a tight skin of water molecules,
then they are known as coacervates [24]. Of course, entrapment of enzyme in polymer network
can protect enzymes by preventing direct contact with the environment, thereby minimizing the
effects of gas bubbles, mechanical sheer, and hydrophobic solvents, but has the drawback of
mass transfer limitations and low enzyme loading.
As an inorganic network, solgel matrices have been used for enzyme immobilization through
entrapment formed by hydrolytic polymerization of metal alkoxides or tetraethoxysilane that was
pioneered by Avnir et al., and a wide variety of enzymes has been used for solgel
immobilization [19, 25]. It should be pointed out that silica solgel morphology depends on the
drying methods [26]. Drying by evaporation affords so-called xerogels, in which capillary stress
causes the shrinkage of the nano cages and pores. By adding porous supports, such as Celite, to
bind the enzyme the solgel process can lead to enzyme-containing gels. This double
immobilization creates materials with higher thermal stability and activity [27]. Reetz et al.
used higher alkyl groups in a RSi(OMe)3 precursor, and this second generation solgel
immobilization lead to high enzyme loadings, high activity, and recyclable uses [27, 28]. In
addition, additives such as polyethylene glycol, polyvinyl alcohol, and albumin have a stabilizing
effect on solgel entrapped enzymes [29]. Silicone elastomers and polydimethylsiloxane
membranes are used to entrap enzymes [30]. In contrast, a new concept for entrapping enzymes
using a biosilification process has been reported by Naik et al. [29]. In nature, diatoms are able to
synthesize silica nanoparticles by polymerization of silicic acid, catalyzed by enzymes known as
silicateins. When this process is performed in the presence of an enzyme it results in the
entrapment of silica [31]. Meanwhile, nanoporous silica spheres with a surface area of 630
m2g1 and mesopores with pore sizes up to 40 nm, and a subsequently assembled nanocomposite
shells, composed of three layers of poly-dimethyldiallylammonium chloride and 21-nm silica
nanoparticles, has been reported for enzyme immobilization [32]. These biomimetic silica

nanoparticles and nanoporous silica spheres can generally provide a large surface area and low
mass-transfer resistance arisen from nanostructure, for useful enzyme stabilization.
2.3 Cross-linked enzymes
Cross-linking of enzyme aggregates or crystals with a bifunctional reagent is used to prepare
carrier-less macroparticles. The use of a carrier inevitably leads to dilution of activity, but the
cross-linking strategy results in lower efficiency and productivity [33]. In 1964, Quiocho and
Richards discovered that dissolved enzymes via reaction of surface NH2 groups with a
bifunctional chemical cross-linker, such as glutaraldehyde (GA), afforded insoluble cross-linked
enzymes (CLEs) that retained their catalytic activity. However, CLEs have several drawbacks,
such as low retention of activity, poor reproducibility, low mechanical stability, and difficulties in
handling the gelatinous CLEs. The cross-linking of a crystalline enzyme using GA was first
described in 1964 [34]. The crosslinked enzyme crystals (CLECs) are more stable against
denaturation by heat, organic solvents, and proteolysis than that of soluble enzyme or lyophilized
(freeze-dried) powder.
In contrast, adding of salts, water-miscible organic solvents, or nonionic polymers to enzymes
results in precipitation as physical aggregates without denaturation [35]. Subsequent crosslinking of these physical aggregates renders them permanently insoluble while maintaining their
superstructure, and, hence, their catalytic activity. This lead to the development of CLEAs
(Fig. 2). The CLEAs methodology essentially combines both purification and immobilization
into a single step operation that does not require highly pure enzymes. Remarkably, the
productivity of CLEAs is even higher than that of the free enzyme and CLECs. These results
clearly demonstrate the tremendous potential of CLEAs as an enzyme immobilization method.
While the mass transfer limitations and filterability are concerned in CLEAs, enzyme, and GA
concentrations, a very popular commercially available inexpensive cross-linking agent, are
important factors when determining the particle size of CLEAs [36].
Figure 2.

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Principal of enzyme aggregation and cross-linking to prepare cross-linked enzyme aggregates
(CLEAs).
The cross-linking of enzymes involves the reaction of amino groups of lysine residues on the
external surface of the enzyme. Bulky polyaldehydes, obtained by periodate oxidation of
dextrans, are occasionally used as cross-linkers, followed by reduction of Schiff base bonding
with sodium borohydride to form irreversible amine linkages [37]. The cross-linking of enzymes
is expected to be less effective for electronegative enzymes, due to a shortage of lysine residues
on the surface. One way of compensating for this lack of surface amino groups is to coprecipitate
the enzyme with a polymer containing numerous free amino groups, such as poly-lysine or
polyethylene imine [38, 39]. Adding bovine serum albumin as a proteic feeder during the
preparation of CLEAs facilitates CLEA formation [40]. CLEAs not only improve enzyme
stability under the reaction conditions but also reduce enzyme cost. Another benefit of the
CLEAs technology is that it can stabilize the quaternary structures of multimeric enzymes, a
structural feature often encountered with redox metalloenzymes [33]. Furthermore, CLEAs
technlogy can be adopted in so called catalytic cascade processes. Catalytic cascade processes

have numerous potential benefits, including fewer unit operations, lower reactor volume, higher
volumetric, and space-time yields, shorter cycle times, and less waste generation [41]. In
addition, an unfavorable equilibrium can be driven toward product formation by coupling steps
together. In principle, this can be achieved by coprecipitation and cross-linking of two or more
enzymes in combi CLEAs. Inside the combi CLEAs, the close proximity of the two enzymes
is more favorable for the transfer of the product during the first step and to the active site of the
enzyme for the second step [42]. In addition, multienzyme stabilization may be really important
tool to mimic metabolic synthetic pathway. Therefore, the combi-CLEAs can be helpful to
multienzyme stabilization. When the CLEAs method using specific nano/microsized hybrid
supports is applied for immobilization, the immobilized enzyme can form a multilayer-coated
structure on the support. Then it results in the formation of enzyme-support complexes, which
show the long-term storage stability. In addition, their reusability and the convenient usages of
enzymes-support complex in the reaction are favorable features for stable enzyme systems or
carriers desirable in terms of cost effectiveness and its successful application. Furthermore, new
supports that hold functional moieties, high surface areas, and magnetic separabilities come into
the positive effect on enhanced enzyme loading, specific activity, and easy separation.
3 Advances in enzyme stabilization
Advancements in nanotechnology have lead to the rapid growth of nanobiotechnology. As a
result, various nanostructured materials have received attention as enzyme immobilizing carriers
due to their intrinsic large surface areas. This large surface area results in improved enzyme
loading, which increases enzyme activity per unit mass or volume, compared to that of
conventional materials [43]. One of the advantages of nanostructured materials is to be able to
control the size at the nanometer scale, such as the pore size in nanopores, thickness of
nanofibers or nanotubes, and nanoparticle size (Fig. 3). Various nanomaterials and nanostructures
generally provide a large surface area and low mass-transfer resistance, which enables better
interaction with the enzyme, increases immobilization efficiency, and enhances the long-term
storage and recycling stability of the enzyme [44].
Figure 3.

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Enzyme immobilization using various nano/microsized materials.
A new class of materials based on combined organic and inorganic species has recently received
more attention [45]. These so called organicinorganic hybrid materials provide novel features
that enhance mechanical properties such as strength, elasticity, plasticity, and chemical bonding
in an appropriate microenvironment. The hybrid materials have both the advantages of organic
materials, such as lightweight, flexibility, and good moldability, and those of inorganic materials,
such as high strength, heat stability, and chemical resistance. Inorganic silica, for example, is
built up on the support as a shield, called a frustule, which not only protects the enzyme from
denaturation due to the environment but also provides ample space for conjugation.
3.1 Nanoparticles
Recently, nanoparticles have been used as enzyme immobilization carriers [46]. Effective
enzyme loading on nanoparticles has been achieved for up to 10 wt% due to a large surface area

per unit mass of nanoparticles [44]. Overall, nanoparticles are considered to be an ideal support
for enzyme immobilization due to their minimized diffusional limitations, maximum surface area
per unit mass, and high enzyme loading capability. Theoretical and experimental studies have
demonstrated that particle mobility, which is governed by particle size and solution viscosity, can
impact the intrinsic activity of the particle-attached enzymes [46]. As discussed above, most of
the studies using nanoparticles have shown improved enzyme activity and loading, but not
improved enzyme stability. A recent report using magnetic nanoparticles for enzyme
immobilization demonstrated good enzyme stabilization. Especially, the silica-coated single
magnetic nanoparticles or magnetic nanoparticle clusters embedded in silica nanoparticles
seemed to be attractive since its magnetic core composed of Fe3O4 enables magnetic capturing,
while the silica surface allows a great variety of chemical modifications that make them
adaptable to enzyme conjugation [47-49]. In one report by Kim and Hyeon et al., the silica shell
and magnetic core could be controlled with various combination. As shown in Fig. 4, 25 nm of
silica shell dimension with 5 nm of magnetic core particles were used in stabilization of
chymotrypsin and lipase [48]. Contrastingly, an another report by Lee et al. showed the magnetic
nanoparticles ranged from 5 to 12 nm, with an average diameter of 7 nm, resulted in the
formation of clusters in the range of 70 to 120 nm, with an average size of 100 nm and an
average thickness of the silica shell of 20 nm. This magnetic/silica nanoparticles with a core of
magnetic clusters were applied to stabilization of His-taggedBacillus stearothermopilus L1 lipase
[49].
Figure 4.

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Some examples of nanoparticles and silica-coated nanoparticles used for enzyme immobilization
(A) silica-coated single magnetic nanoparticle and (B) magnetic/silica nanoparticles with a core
of magnetic clusters for enzyme stabilization.
Immobilized enzymes on magnetic nanoparticles exhibit high stability and can be easily
separated from the reaction medium using a magnetic field. As a result, immobilization of
various enzymes on magnetic particles has become an important area of research. Several
magnetic particles, microspheres containing magnetic particles encapsulated, and copolymers
with magnetic particles have been used with good results [50].
3.2 Electospun nanofibers
One-dimensional nanostructured materials such as fibers, wires, rods, belts, tubes, spirals, and
rings have attracted much attention because of their unique properties and interesting
applications. Among them, electrospun nanofibers seems to be the simplest, by which fiber that
are exceptionally long, uniform in diameter and diversified in composition can be fabricated
[43]. These unique features of electrospun nanofibers ensure their potential applications in
biocatalysis. Electrospun nanofibers show distinct and superior characteristics. Briefly,
nanofibers are excellent supports, because (i) a variety of polymers can be electrospun and meet
different requirements as supports, (ii) the high porosity and the interconnectivity of electrospun
fiber supports endows them with a low hindrance for mass transfer, and (iii) the nanofiber
surfaces can be modified to benefit enzyme activity [51]. Although each nanofiber provides high
surface area for hosting enzymes, the collection of randomly arrayed nanofibers usually forms a

nonwoven mesh (or membrane) that can be reused [52, 53]. Electrospun nanofiber mats are
durable and easily separable, and can also be processed in a highly porous form to relieve masstransfer of the substrate through the mats. Therefore, these nanofibers as hosts for enzyme
immobilization on the surface show highly stable and enhanced activity. For example, the
nanofiber-enzyme composites improved activity by more than three orders of magnitude than
that of native enzyme suspended in organic solvents [54].
Xia's group reported on encapsulated enzymes in nanofibers by direct coelectrospinning [55]. In
many cases, the polymers to be coelectrospun with enzymes form homogeneous enzymeencapsulated nanofibers that are highly bioactive [55]. As an alternative, a cross-linking reaction
could damage enzyme activity; however, the extremely high enzyme loading led to high overall
enzymatic activity [54, 56]. In addition, it is reasonable to choose pristine nanofibers as carriers
for enzyme immobilization [57]. Depending on the types of polymers and their surfaces, the
behavior and loading of enzyme can be optimized. Therefore, the specific activity and stability of
immobilized enzymes can be improved by modifying the nanofibers or changing immobilizing
methods. Several surface modification modes have been reported, such as surface induced
undesired conformational change, increased mobility by the flexible spacer, reduced nonspecific
interactions by the biomimetic layer, and fastened electron transfer by electrical conductivity of
the support [51].
Various organic or inorganic nanomaterials such as multiwall carbon nanotubes (MWCNTs),
magnetic nanoparticles, silica nanoparticles, and quantum dots have been used to make hybrid
composite nanofibers with additional physical properties and mechanical stability [57-59]. The
outstanding electrical conductivity of MWCNTs or quantum dots improves the composite
nanofibers and has led to potential applications for redox enzyme immobilization with enhanced
activity [57]. MWCNTs increase the mechanical stability of nanofibers, and makes them more
durable under operating conditions. The distance between the nanofibers can be several dozen to
hundreds nanometers, because its formation in elecrospinning is random and cannot control the
regular deposition. However, as shown in Fig. 5B, hybrid nanofibers embedded with quantum
dots-nanofibers observed much better uniform compactness, showing consistent deposition.
Practically, it has been utilized to obtain unique features for enzyme-immobilized enzyme
applications inducing enhanced stability of immobilized enzyme [57]. These unique features
derived from hybrid technology ensure potential applications of electrospun nanofibers,
including membranes with biocatalytic and separation functions, biosensors, biofuel cells, and
enzymatic membrane bioreactors [44].
Figure 5.

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TEM image of (A) electrospun polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA)
nanofibers and (B) hybrid nanofibers embedded with quantum dots for enzyme stabilization.
3.3 Mesoporous material
In 1992, a new class of mesoporous material (MCM-41), which possesses high surface area, high
pore volume, and a well-ordered pore structure was developed [60]. Mesoporous materials
received considerable attention as excellent supports for catalysts, organometallic compounds,
and enzymes [21]. Enzyme immobilization on MCM-41 was first reported in 1996 by Balkus

et al. [61], discovering that enzyme immobilization was dependent on the molecular size of the
enzyme. The unique advantages of various ordered mesoporous solids that make them suitable
for enzyme immobilization are their narrow pore size distribution, their well-defined pore
geometry and connectivity, their mechanical stability, and the ease of synthesis (Table 3).
Physical adsorption is the simplest method of enzyme immobilization on mesoporous silica.
Under physical adsorption conditions, the active site of the enzyme is often unaffected, and
nearly full activity is retained [62]. Even though the enzyme adsorption approach is simple and
easy, the critical problem is that the adsorbed enzymes are continuously leached out from the
mesoporous material, resulting in poor operational stability.
Table 3. Some typical cases of mesoporous materials to stabilize enzymes
Porous material

Derivation

Pore size (nm)

BET sur

MCM-41

Mobil Composition of Matter No.

41

2.512

30012

FSM-16

Folded-sheet mesoporous material

39

50090

SBA-15

Santa Barbara Amorphous Material

No. 15

520

50014

SBA-16

Santa Barbara Amorphous Material

No. 16

315

60012

1015

20070

FDU-12
MCF

Mesocellular foam

2540

50010

SMS

Sponge-like mesoporous silica

3.34.7

43080

BMS

Bimodal mesoporous silica spheres

1040

600

Sibunit

Mesoporous carbon

A common method to reduce leaching is functionalizing the mesoporous silica support. Easy
access to organicinorganic hybrid materials based on SBA-15 is provided by surface
modifications [63, 64]. In this method, a variety of functional groups, including aliphatic
hydrocarbons, thiol groups, vinyl groups, phenyl groups, amine groups, and perfluoro groups
have been incorporated into mesoporous materials. In particular, the amine group (NH2) is
useful for covalent coupling of the enzyme to the surface of silica materials via linkers such as
GA [62]. This approach usually results in stabilized enzyme activity by preventing enzyme
leaching. Another strategy for enzyme immobilization that suppresses leaching is enzyme

550

encapsulation in the pores of a suitable support. Bimodal mesoporous silica with an

organic/inorganic composite shell and modified hydrophobic silica matrices prevents enzyme
leaching [65]. Furthermore, a magnetic particle is applied to mesoporous silica as a hybrid
composite to recycle immobilized enzyme. Amino-functionalized magnetite-containing
mesoporous silica spheres (Fe3O4@MSS) and composites of Fe3O4 magnetic nanocrystals with
mesoporous silica nanospheres are hosts for enzyme immobilization [66].
Recently, to overcome the enzyme leaching, and to show promising novel approach for enzyme
immobilization, a simple ship-in-a-bottle approach has been employed as an effective means to
immobilize enzymes in mesoporous materials and prevent leaching. This approach is referred to
as nanoscale enzyme reactors (NERs) by Kim et al. [43, 44]. The NER approach is based on a
simple two-step process; the first step is adsorption of the enzyme onto mesoporous material and
the second step is GA treatment, which cross-links the absorbed enzyme within the mesopores.
This approach not only improves enzyme loading and activity, but also prevents enzyme
denaturation and leaching. -Chymotrypsin, trypsin, glucose oxidase, and lipase have been
successfully immobilized and stabilized via the NER approach [67-69]. Furthermore, entrapped
magnetic nanoparticles among cross-linked enzymes or directly incorporated magnetic
nanoparticles into mesoporous materials led to the development of magnetically separable and
stabilized enzyme systems for easy reuse in organic synthesis [70]. Immobilized enzymes in
mesoporous materials have found applications in biosensors, peptide synthesis, and pulp
biobleaching. It is anticipated that additional diversified applications will be reported in the near
future [71, 72].
3.4 Sol-gel silica
Enzyme encapsulation via the solgel approach has also been a method, since the first report
showing that encapsulated enzymes into solgel matrices maintained activity [73]. In a typical
synthetic protocol, tetramethylorthosilicate (TMOS), or tetraorthosilicate is hydrolyzed into
sol, and adding an enzyme solution to the sol initiates a condensation reaction leading to the
gel, where enzymes are encapsulated in silicate matrices. During this process, the enzyme acts
as a template, so that its nano-capsule size is usually much larger than most of the pores in the
gel walls, in particular in xerogels. Various pores and channels are formed in the final silicate
matrices. The pore size of solgel silica particle can be varied according to the synthesis
methods. In the middle of synthesis, the gelation process is the main factor influencing the pore
diameters and porosity. The gelation altered in certain conditions (such as pH, temperature, and
pressure) induces the change of their properties. The pore size of 0.1500 nm in solgel silica
can be fabricated according to the condition specifically used [74]. The solgel process requires
synthesis conditions, such as optimized pH, which are not always favorable for enzyme
encapsulation. Of course, gels can be used to immobilize enzymes by adsorption or surface
covalent binding on presynthesized gel beads. However, gels are a most interesting technique for
encapsulation, entrapment, or embedding of an enzyme due to the porous gel network. Gel
encapsulation consists of knitting a porous wall by chemical condensation of the silica gel
network around the enzyme. Thus, each enzyme molecule is captured inside a nano-cage that has
enough room to change its conformation as required for a full catalytic cycle [74]. Thus,
adsorption or other ionic or covalent bonds to the gel walls are not needed, although such
interactions may naturally occur and might interfere with enzyme efficiency. Additionally,
external substrates and transformed products must remain free to diffuse in and out of the nanocapsule walls. Once enzyme leaching is prevented, the solgel approach leads to a highly stable

immobilized enzyme, as the close fit of the enzyme molecule within the solgel pore probably
protects unfolding and denaturation of encapsulated enzymes [75]. In the past decade, the
encapsulation of enzymes inside inorganic solgel matrices has become a universal method to
prepare biocatalysts that are easy to recycle. Therefore, the solgel processes are useful for
enzyme encapsulation, mostly solgel silica.
3.5 Alginate-based microspheres
One of the microencapsulation techniques is to immobilize enzyme behind a semipermeable
membrane [76]. This semipermeable membrane, which allows diffusion of small substrates and
products, is crucially important for the efficiency of enzyme immobilization. Numerous
biopolymers and polymers useful for microencapsulation exhibit gelation properties and
chemical functionalities. Among them, alginate-based microcapsules are well designed for
enzyme encapsulation. Alginate beads are formed by gelation of a droplet in alginate solution
using divalent cations (Ca2+), leading to the formation of an outer membrane [50]. Thus, alginate
gels have become one of the most studied matrices for enzyme encapsulation. But alginate beads
have severe problems; the Ca2+ in the alginate beads is exchanged with Na+ ions in a phosphate
buffer solution, resulting in swelling, followed by enzyme leakage, and a subsequent inhibition
on recycling of enzyme in the reaction media. Various strategies to enhance the mechanical
strength of alginate microcapsules have been attempted. In recent years, there have been attempts
in fabricating organicinorganic silica-based hybrid materials using the solgel process, because
the functional versatility of organic materials can be integrated with inorganic substrates and
create thermal stability. This attempt has lead to the suggestion of applying alginate
microcapsules with silica [77]. This approach appears very promising, as it allows the association
of a soft biocompatible component, alginate, with a tough, thermostable, nonswelling
component, silica. This allowed hybrid composites to be manipulated, permitting enzyme
encapsulation in mineral hosts. The first attempt to associate silica and alginate to prepare
microcapsules was reported in 1995 by Chang et al. [78]. As a result, a large number of colloidal
silica particles were dispersed well in the alginate gel. The use of TMOS Si(OCH3)4 as the silica
source has also been reported. Wet calcium alginate beads were suspended in a solution of
TMOS in hexane. Partial hydrolysis of the alkoxide led to the formation of water-soluble silicon
species that permeated the alginate gel and polymerized within the capsule [79]. To prevent
electrostatic repulsion between alginate and silicon species, the surface is modified with a
positive charge. As a result, a smooth mineral layer composed of charged polymer was observed
at the capsule surface. The use of colloidal silica has also been investigated; however, the
resulting mineral coating was more fragile as nanoparticles were only deposited on the capsule
surface and did not form strong SiOSi bonds, in contrast to the silicate polymerization process
[80, 81]. Alternatively, a very promising approach using a mixture of TMOS and 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane to form a silica layer on Ca2+-alginate beads
has been reported. The methoxy groups of the TMOS molecules were hydrolyzed to form a silica
gel, whereas the positively charged amino groups interacted with the alginate surface to anchor
the mineral deposit. Another alginate layer could also be added, providing a biocompatible outer
surface [77]. The presence of silica enhances capsule stability while controlling membrane
diffusion properties and allows efficient enzyme immobilization.
4 Practical applications for stabilized enzymes

Immobilized and stabilized enzyme technology has advanced to multidisciplinary fields such as
clinical, industrial, and environmental applications. Therefore, immobilized enzymes can be
utilized in biosnesor, antibiotic production, drug metabolism, the food industry, biodiesel
production, and bioremediation. Table 4describes the typical application of stabilized enzymes
using nano/microsized hybird materials.
Table 4. Examples of some typical application of stabilized enzymes using nano/microsized
hybrid materials
Applications
Biosensor

Antibiotic production

Food industry

Biodiesel production

Enzymes

Stabilization support

Glucoes oxidase

Mesocellular foam silica

Alcohol dehydrogenase

Silica-coated alginate gel b

Acetylcholinesterase

Biomimetically synthesize
carbon nanofibers

Horseradish peroxidase

PANCAA/MWCNT nanof

Penicillin G acylase

Epoxy-activated magnetic
microspheres

Penicillin acylase

Hollow silica nanotube

D-amino-acid oxidase

Silica coated magnetic nan

Glucose isomerase

Alginate/carbon composite

-Glucosidase

PS-PSMA nanofibers with

magnetic nanoparticles

Amylase

Magnetic chitosan beads

Protease

Magnetically-separable m
silica

Trypsin

Hybrid silica monolith

Lipase

Meso-structured onion-lik

Applications

Enzymes

Stabilization support

Magnetic silica nanotubes

Chitosan-alginate hydroge

Polysiloxanepolyvinyl al
hybrid matrix
Bioremidation

Peroxidase

SilicatefructosePVA nan

Laccase

Magnetic biomodal mesop

carbon

Polyphenol oxidase

Alginate-SiO2 hybrid gel

Enzyme-based electrodes are a representative application of immobilized enzymes for diagnosis

and treatment of various diseases. The high specificity and reactivity of immobilized enzymes
are being exploited in the biosensing field. These studies have resulting in replacing existing
diagnostic tools such as glucose test strips, chromatography, mass spectroscopy, and enzymelinked immunosorbent assays with faster and cost effective diagnostic devices [67]. These
devices can be used to provide an early signal of metabolic imbalances and assist in preventing
and curing diabetes and obesity [82].
Fine chemical synthesis processes for antibiotics such as -lactam is a major challenge for
industrial implementation. Significant advancement has also been made in the resolution of
racemic mixtures by means of stereo-selective acylation/hydrolysis using -lactam acylases [83].
Enzymatic production of cephalexin using immobilized penicillin G acylase has also been
studied in detail [83]. Conversion of 7-amino-3-deacetoxy cephalosporanic acid to cephalexin by
immobilized peincillin G acylase has been investigated with an 85% conversion yield under
optimized conditions [84]. Furthermore, stabilized peincillin G acylase can be reused for about
10 cycles. Production of cefazolin by stabilized cefazolin synthetase from E. coli as a biocatalyst
is possible. Physico-chemical studies have made it possible to design a highly efficient
technological process to produce cefazolin [8]. Synthesis of various antibiotics by different
enzymes immobilized on different supports has been achieved [85, 86].
Stabilized enzymes are of great value in the processing and analysis of food samples. The extent
of lactose hydrolysis for skimmed milk production has been greatly enhanced using respective
enzymes in immobilized forms [87]. High fructose corn syrup has been produced with the use of
immobilized glucose isomerase. Similarly, processing efficiency for amino acids through the use
of immobilized amino acid acylase has been increased [88]. A relatively new concept is the use
of a single matrix for immobilizing more than two enzymes to enhance food processing.
Immobilized multienzyme systems offer many attractive advantages. Immobilizing other
enzymes such as -glucosidase, amylase, trypsin, protease, and flavor modifying enzymes has

received some attention recently for food processing [87]. Various food substrates are catalyzed
by stabilized enzymes.
A typical synthetic product produced via enzyme-catalyzed transesterification is biodiesel fuel,
which refers to fatty acid alkyl esters. Biodiesels have attracted attention due to concerns about
depleting oil reserves, as an environmentally friendly alternative fuel for diesel engines [89].
Recently, lipase-catalyzed transesterification has offered considerable advantages, including
reducing process operations for production and easy separation of the glycerol byproduct without
any complex operation steps [90, 91]. Biodiesel can be produced from vegetable oils, animal,
fats, microalgal oils, and vegetable oil waste products. Stabilized enzymes could be employed in
biodiesel production with the aim of reducing production costs by reusing the enzyme [92].
Different carriers such as ceramics, polymer, silica, zeolite, and their hybrid materials have been
used to immobilize lipase [93, 94].
More than 100000 commercially available dyes have been produced and used extensively in the
textile, dyeing, and printing industries, resulting in the formation and accumulation of colorless
aromatic amines that are highly toxic and carcinogenic [95, 96]. Recent studies indicate that an
enzymatic approach has attracted much interest to remove phenolic pollutants from aqueous
solution as an alternative strategy [97]. Stabilized peroxidases on some cheaper supports have
been found to be highly effective for decolorizing reactive textile dyes. Furthermore, various
immobilized laccases have been used to decolorize or degrade various textile and nontextile dyes
as well as phenolic compounds. Peroxidase, laccases, and polyphenol oxidase in various
immobilized forms have been utilized to remove phenolic compounds and decolorize or degrade
dyes [98-100].
As the structure and mechanism of action of enzymes become available, more controlled
immobilization methods will be developed. The use of additional immobilized and stabilized
enzymes in clinical, biotechnological, pharmacological, and other industrial fields has great
promise among future technologies.
5 Conclusion
Recent advances in the design of immobilization support have enabled more precise control of
enzyme immobilization. The development of new support to stabilize enzymes has been of
interest for many years, and new opportunities for stabilizing enzymes with improved intrinsic
and operational stabilities have been developed. Each of the immobilization methods such as
binding to a support, encapsulation, and cross-linking using supports bear assorted advantages
and disadvantages, making a comparison of the different methodologies difficult. Many chemists
have tried to increase immobilized enzyme performance such as specific activity, storage, and
recycling stability, and ease of reuse, whereas materials scientists have developed more efficient
supports to realize their objectives. This review has illustrated advanced enzyme immobilization
and stabilization with nano/micro and hybrid materials, from nanoparticles, nanofibers,
mesoporous materials, solgel silica, and alginate-based microspheres. Stabilized enzyme
technology has led to successful advances in various disciplines in the last few decades. As these
approaches are successfully applied to a wider range of enzymes, they will result in new and
expanded uses of enzymes in practical applications such as medicine, antibiotic production, drug
metabolism, food industry, biodiesel production, and bioremediation.
Acknowledgments

This work was supported by the National Research Foundation of Korea Grant funded by the
Korean Government (MEST) (NRF-C1ABA001-2010-0020501) and a Korea University Grant
(2012). We appreciate Mr Jinyang Chung and Mr Haemin Gang in Korea University for his help
in graphic work.
The authors have declared no conflict of interest.