AbstractImmobilization 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.Enhanced Article FeedbackAbbreviationsCLEAscross-linked enzyme aggregatesCLECscross-linked enzyme crystalsGAglutaraldehydeMWCNTsmultiwall carbon nanotubesPVApolyvinyl alcoholTMOStetramethylorthosilicate1IntroductionEnzymes 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 andin vitroevolution 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.2Enzyme 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 Table1. 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 (Table2), 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).Table1.Parameters influencing the activity of immobilized enzymesFactors related to enzymesFactors related to the carriersSpecific factors related to the reaction system

Size of the enzymeOrganic or inorganicReaction medium

Conformational flexibility required by the mechanismHydrophobic or hydrophilicDiffusion limitations

Isoelectric pointSurface chargesEnzyme inhibition

Surface functional groups/charge densitySurface functionalizationPrecipitation of products

GlycosylationChemical and mechanical stabilityViscosity of the mixture

Stability under immobilization conditionsSurface areaReaction thermodynamics

Presence of hydrophobic regionsPorosityNon-specific solute-support interactions

Presence of hydrophilic regionsParticle size

Additives in the enzymatic preparation

Table2.Advantages and disadvantages of three types of the immobilization methods: binding to support, encapsulation, and cross-linkingImmobilization methodAdvantagesDisadvantanges

Binding to a support(Physical adsorption)Simple experimental procedureLeaching of enzymes during reaction

No toxic solvents

(Chemical binding)Enzyme molecules retainedMore complicated procedure in support preparation

Wide choice of organic linkers availableEnzyme molecules immobile inside carriers

Entrapment or encapsulationEnzyme molecules retainedComplicated experimental procedure

Enzyme molecules free to move inside carriersReactive species and toxic solvents may denature enzymes

Decrease diffusion rate of reactants and products

Cross-linkingEnhanced shelf life and operational stabilityLoss of the enzyme's flexibility

Easy to recover and reuseDecrease diffusion rate of reactants and products

Stable towards leaching in aqueous media

Figure1. Open in figure viewer Download Powerpoint slideOverview of the different enzyme immobilization strategies.2.1Binding of enzymes to the supportsSupport 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-n-isopropylacrylamide. 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 (3001500 m2g1) 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.2.2Entrapment or encapsulation of enzymesEntrapment 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 etal., 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 etal. used higher alkyl groups in a RSi(OMe)3precursor, 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 etal. [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 m2g1and 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.2.3Cross-linked enzymesCross-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 NH2groups 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 cross-linking 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].Figure2. Open in figure viewer Download Powerpoint slidePrincipal 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.3Advances in enzyme stabilizationAdvancements 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].Figure3. Open in figure viewer Download Powerpoint slideEnzyme 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.3.1NanoparticlesRecently, 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 Fe3O4enables 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 etal., 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 etal. 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 stearothermopilusL1 lipase [49].Figure4. Open in figure viewer Download Powerpoint slideSome 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].3.2Electospun nanofibersOne-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 mass-transfer 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 enzyme-encapsulated 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].Figure5. Open in figure viewer Download Powerpoint slideTEM image of (A) electrospun polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers and (B) hybrid nanofibers embedded with quantum dots for enzyme stabilization.3.3Mesoporous materialIn 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 etal. [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 (Table3). 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.Table3.Some typical cases of mesoporous materials to stabilize enzymesPorous materialDerivationPore size (nm)BET surface (m2g1)

MCM-41Mobil Composition of Matter No. 412.5123001200

FSM-16Folded-sheet mesoporous material39500900

SBA-15Santa Barbara Amorphous Material No. 155205001400

SBA-16Santa Barbara Amorphous Material No. 163156001200

FDU-121015200700

MCFMesocellular foam25405001000

SMSSponge-like mesoporous silica3.34.7430800

BMSBimodal mesoporous silica spheres1040600

SibunitMesoporous carbon550

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 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 Fe3O4magnetic 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 etal. [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].3.4Sol-gel silicaEnzyme 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 nano-capsule 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.3.5Alginate-based microspheresOne 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 etal. [78]. As a result, a large number of colloidal silica particles were dispersed well in the alginate gel. The use of TMOS Si(OCH3)4as 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-aminopropyl-trimethoxysilane 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.4Practical applications for stabilized enzymesImmobilized 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. Table4describes the typical application of stabilized enzymes using nano/microsized hybird materials.Table4.Examples of some typical application of stabilized enzymes using nano/microsized hybrid materialsApplicationsEnzymesStabilization support

BiosensorGlucoes oxidaseMesocellular foam silica

Alcohol dehydrogenaseSilica-coated alginate gel beads

AcetylcholinesteraseBiomimetically synthesized silica-carbon nanofibers

Horseradish peroxidasePANCAA/MWCNT nanofibers

Antibiotic productionPenicillin G acylaseEpoxy-activated magnetic cellulose microspheres

Penicillin acylaseHollow silica nanotube

D-amino-acid oxidaseSilica coated magnetic nanoparticles

Food industryGlucose isomeraseAlginate/carbon composite beads

-GlucosidasePS-PSMA nanofibers with entrapped magnetic nanoparticles

AmylaseMagnetic chitosan beads

ProteaseMagnetically-separable mesoporous silica

TrypsinHybrid silica monolith

Biodiesel productionLipaseMeso-structured onion-like silica

Magnetic silica nanotubes

Chitosan-alginate hydrogel

Polysiloxanepolyvinyl alcohol hybrid matrix

BioremidationPeroxidaseSilicatefructosePVA nanofibers

LaccaseMagnetic biomodal mesoporous carbon

Polyphenol oxidaseAlginate-SiO2hybrid 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 enzyme-linked 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 fromE. colias 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.5ConclusionRecent 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.AcknowledgmentsThis 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.