2 3 Enzyme immobilization: an update

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Journal of Chemical Biology ISSN 1864-6158Volume 6Number 4 J Chem Biol (2013) 6:185-205DOI 10.1007/s12154-013-0102-9

Enzyme immobilization: an update

Ahmad Abolpour Homaei, ReyhanehSariri, Fabio Vianello & RobertoStevanato

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REVIEW

Enzyme immobilization: an update

Ahmad Abolpour Homaei & Reyhaneh Sariri &Fabio Vianello & Roberto Stevanato

Received: 5 June 2013 /Accepted: 31 July 2013 /Published online: 29 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Compared to free enzymes in solution, immobilizedenzymes are more robust and more resistant to environmentalchanges. More importantly, the heterogeneity of the immo-bilized enzyme systems allows an easy recovery of both en-zymes and products, multiple re-use of enzymes, continuousoperation of enzymatic processes, rapid termination of reac-tions, and greater variety of bioreactor designs. This paper is areview of the recent literatures on enzyme immobilization byvarious techniques, the need for immobilization and differentapplications in industry, covering the last two decades. Themost recent papers, patents, and reviews on immobilizationstrategies and application are reviewed.

Keywords Enzyme immobilization . Biocatalyst .

Enzyme reuse

AbbreviationsALG AlginateAuNPs Gold nanoparticlesCLEAs Cross-linked enzyme aggregatesCLECs Cross-linked enzyme crystalsCLIO Cross-linked iron oxideCS ChitosanDEAE DimethylaminoethylEDC 1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimideFDA Food and Drug AssociationHPLC High-performance liquid chromatographyIMAC Immobilized metal affinity chromatographyKd Dissociation constantKM Michaelis constantLC–MS Liquid chromatography–mass spectrometryLCST Low critical solution temperatureMIONs Monocrystalline iron oxide nanoparticlesNHS N -HydroxysuccinimideNi-NTA Nickel nitrilotriacetic acidPLA Poly(lactic acid)PLGA Poly(lactic-co-glycolic acid)polyNIPAM Poly-N -isopropylacrylamidePVA Polyvinyl alcoholTm Transition temperatureUSPIO Ultrasmall superparamagnetic iron oxide

A. A. Homaei (*)Department of Biology, Faculty of Science, University ofHormozgan, Bandarabbas, Irane-mail: [email protected]

R. Sariri (*)Reyhaneh Sariri, Department of Microbiology, Lahijan Branch,Islamic Azad University, Lahijan, Irane-mail: [email protected]

F. VianelloDepartment of Comparative Biomedicine and Food Science,University of Padua, Padua, Italy

R. Stevanato (*)Department of Molecular Sciences and Nanosystems, University ofVenice, Venice, Italye-mail: [email protected]

J Chem Biol (2013) 6:185–205DOI 10.1007/s12154-013-0102-9

Author's personal copy

Introduction

The relationship between humans and enzymes hasevolved over time. Even during historical times, wherethere was no concept of enzymes, ancient Egypt peopleproduced beer and wine by enzymatic fermentation.After several thousand years, enzymatic studies havesignificantly progressed. Enzymes are proteins that ac-celerate many biochemical and chemical reactions. Theyare natural catalysts and are ubiquitous, in plants, ani-mals, and microorganisms, where they catalyze process-es that are vital to living organisms. The growing knowledgeand technique improvement about protein extraction and pu-rification lead to the production of many enzymes at ananalytical grade purity for research and biotechnological ap-plications. Enzymes are intimately involved in a wide varietyof traditional food processes, such as cheese making, beerbrewing, and wine industry. Recent advances in biotechnolo-gy, particularly in protein engineering, have provided the basisfor the efficient development of enzymes with improved prop-erties. This has led to establishment of novel, tailor-madeenzymes for completely new applications, where enzymeswere not previously used.

Application of enzymes in different industries is continu-ously increasing, especially during the last two decades.

Applications of enzymes in food industries include baking[1], dairy products [2, 3], starch conversion [4] and beverageprocessing (beer, wine, fruit and vegetable juices). In textilesindustries, enzymes have found a special place due to theireffect on end products [5]. In industries such as pulp and papermaking [6] and detergents [7], the use of enzymes has becomean inevitable processing strategywhen a perfect end product isdesired. Application of enzymes in more modern industriesincluding biosensors is improving rapidly due to the specific-ity of enzymes which is of prime importance in biosensor [8].Many other important industries including health care andpharmaceuticals [9] and chemical [10] manufacture are in-creasingly taking advantages of these natures amazing cata-lysts. During the last few years, enzymes have widely beenused in biofuels, such as biodiesel and ethanol [11].One of thebest use of enzymes in the modern life is their application intreatment of wastes in general [12] and especially for solidwastes treatment [13] and waste water purification [14].

In some cases, industrial applications of enzymes in organ-ic solvents are also developed [15]. Moreover, enzymes areproduced from renewable raw materials and are completelybiodegradable. In addition, the mild operating conditions ofenzymatic processes mean that they can be operated in rela-tively simple and totally controlled equipment. In short, theyreduce environmental drawbacks of manufacturing by

Structural abstract. A summary of enzyme immobilization techniques investigated in this review work showing their advantage,disadvantages, and various applications.

Immobilizationprinciple

Advantage Disadvantage Application Reference

Deposition on solid Retaining almost allactivity

Low enzyme loading Inversion of carbohydrates [1]

Adsorption on mesoporoussilicates

Support is chemically andmechanically stableand resistant to microbialattack

Variable pore size preparationin harsh conditions causingdenaturation of enzyme

Scaffold for mesoporouscarbon materials

[2, 3]

Immobilization on polyketoneby hydrogen bonds

Easy immobilization, highbinding capacity,extraordinary stableimmobilization

Only small increase inKM value

Applicable for largeenzymes, peroxidase,and amine oxidases

[46]

Classical covalentimmobilization

Relatively stable to hydrolysisat neutral pH

Esters are unstable in aqueousconditions

Immobilization of antibodies,proteases, and oxidases

[4, 5]

Physical entrapment Avoid negative influence onenzyme surface, thermallyand mechanically verystable

Diffusion of substrate to theenzyme is restricted

Applicable for most enzymesand antibodies,development of biosensors

[6]

Immobilization usingaffinity tag

Possibility of in situ immobilization Relatively low selectivity Capture of proteins duringpurification in affinitychromatography

[7]

Encapsulation with lipidvesicles (liposomes)

High degree of reproducibility Enzyme inactivation byshear force

Medical, biomedical fields,enzyme-replacementtherapy, ripening process

[8]

Immobilization onbiodegradable polymers

Longer circulation in theblood stream

Low entrapment efficiencies,burst release, instability ofencapsulated enzyme

Control release for enzymereplacement therapy

[9]

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reducing the consumption of energy and chemicals and con-comitant generation of wastes.

However, all these desirable characteristics of enzymes andtheir widespread industrial applications are often hampered bytheir lack of long-term operational stability and shelf-storagelife and by their cumbersome recovery and re-use. Thesedrawbacks can generally be overcome by immobilization ofenzymes. In fact, a major challenge in industrial bio-catalysisis the development of stable, robust, and preferably insolublebiocatalysts.

Historical background

The first scientific observation that led to the discovery ofimmobilized enzymes was made in 1916 [1]. It was demon-strated that invertase exhibited the same activity when absorbedon a solid, such as charcoal or aluminum hydroxide, at thebottom of the reaction vessel as when uniformly distributedthroughout the solution. This discovery was later developed tothe currently available enzyme immobilization techniques.

Early immobilization techniques provided very low enzymeloadings, relative to available surface areas. During 1950s and1960s, different covalent methods of enzyme immobilizationwere developed. Continuing from 1960s, to date more than5,000 publications and patents have been published on enzymeimmobilization techniques. Several hundred enzymes havebeen immobilized in different forms and more than a dozenimmobilized enzymes; for example, penicillin G acylase, in-vertase, lipases, proteases, etc. have been used as catalysts invarious large-scale processes. While enzyme immobilizationhas been studied for a number of years, the appearance ofrecent published research and review papers indicates a con-tinued interest in this area [16–18]. According to PubMeddatabase only in the first 6 months of 2010, many hundredspapers on enzyme immobilization have been published.

Today, in many cases immobilized enzymes have revealedhighly efficient for commercial uses. They offer many advan-tages over enzymes in solution, including economic conve-nience, higher stability, and the possibility to be easily re-moved from the reaction mixture leading to pure productisolation. An immobilized enzyme is, therefore, attached toan inert, organic, or inorganic or insoluble material, such ascalcium alginate or silica. Furthermore, the attachment of anenzyme to a solid support can increase its resistance to variousenvironmental changes such as pH or temperature [19].

Immobilization strategies

Despite of many advantages in the use of enzymes comparedto traditional catalysts, there are few practical problems asso-ciated with their utilization in industrial applications. Enzymes

are generally expensive, which means that the cost of theirisolation and purification is many times higher than that ofordinary catalysts. Being protein in structure, they are alsohighly sensitive to various denaturing conditions when isolat-ed from their natural environments. Their sensitivity to pro-cess conditions, such as temperature, pH, and substances attrace levels, can act as inhibitors which add to their costs. Onthe other hand, unlike conventional heterogeneous chemicalcatalysts, most enzymes operate dissolved in water in homo-geneous catalysis systems, leading to product contaminationand ruling out their recovery, for reuse, in the active form frommost of the reaction mixtures.

One of the most successful methods proposed to overcomethese limitations is the use of an immobilization strategy (vande Velde 2002). Immobilization is a technical process inwhich enzymes are fixed to or within solid supports, creatinga heterogeneous immobilized enzyme system. Immobilizedform of enzymes mimic their natural mode in living cells,where most of them are attached to cellular cytoskeleton,membrane, and organelle structures. The solid support sys-tems generally stabilize the structure of the enzymes and, as aconsequence, maintain their activities. Thus, as compared tofree enzymes in solution, immobilized enzymes are morerobust and more resistant to environmental changes. In addi-tion, heterogeneous immobilized enzymes systems allow theeasy recovery of both enzymes and products, multiple reuse ofenzymes, continuous operation of enzymatic processes, rapidtermination of reactions, and greater variety of bioreactordesigns. On the other hand, compared with free enzymes,most commonly immobilized enzymes show lower activityand, generally, higher apparentMichaelis constants because ofa relative difficulty in accessing the substrate [20].

In recent years, a wide range of interest and high attentionhas been directed toward exploring the potential ofimmobilized enzymes [21]. Compared to their free forms,immobilized enzymes are generally more stable and eas-ier to handle. In addition, the reaction products are notcontaminated with the enzyme (especially useful in thefood and pharmaceutical industries), and in the case ofproteases, the rate of the autolysis process can be dra-matically reduced upon immobilization [22].

These alterations result from structural changes introducedinto the enzyme molecule by the applied immobilizationprocedure and from the creation of a microenvironment inwhich the enzyme works, different from the bulk solution.The result would be a pure product not contaminated withother environmental ingredients and easy to be isolated fromthe solution. The attached enzyme is then ready for the sub-sequent reactions without the need for repeated, time-consuming, and costly extraction and purification procedures.Enzymes may be immobilized by a variety of methods, whichmay be broadly classified as physical, where weak interac-tions between support and enzyme exist, and chemical, where

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covalent bonds are formed between the support and theenzyme.

In particular, the development and applications of site-selective protein immobilization have undergone significantadvances in recent years. Notably, advances in organic chem-istry and molecular biology have resulted in the developmentof some very powerful, efficient, site-specific, and importantapplications of anchoring proteins onto supports. These havebeen followed by the development of functional protein mi-croarrays, biosensors, and continuous flow reactor systems.The advent of high-density array printing technology andimproved methods for high-throughput production and puri-fication of large numbers of proteins have, in recent years,allowed the preparation and exploitation of protein microar-rays. However, many of the applications reported have reliedon protein attachment methods that result in non-specificimmobilization, either covalently through amine, aldehyde,and epoxy-derivatized surfaces or through adsorption onnitrocellulose-, hydrogel-, or polylysine-coated slides. A num-ber of more selective approaches have been demonstratedemploying affinity reagents that bind specific epitopes or tagson proteins and expose them in a correctly orientated manner,such as nickel nitrilotriacetic acid (Ni-NTA)-coated slides,which are used to bind histidine-tagged proteins andstreptavidin (or avidin). Figure 1 schematically explains themechanism by which a protein is first tagged by two mole-cules of histidine followed by interaction with a Ni-NTAnanogold. It can be seen in this figure that this type ofinteraction can only provide non-covalent attachment [3].

Bioconjugation is an important aspect of the biologicalsciences and critical to the applications discussed above; as aresult, many methods have been described through recentyears. Of these, a small number have become widely applieddue to their ease of use, flexibility, and reagent commercialavailability. In general, these methods rely on physicochemi-cal adsorption phenomena or on functional groups that arenaturally present in proteins. They are, therefore, extremelystraightforward to employ and applicable to all proteins, bothnative and modified.

Modes of immobilization

Traditionally, four methods are used for enzyme immobiliza-tion, namely (1) non-covalent adsorption and deposition, (2)physical entrapment, (3) covalent attachment, and (4) bio-conjugation (Fig. 2). Support binding can be physical orchemical, involving weak or covalent bonds. In general, phys-ical bonding is comparatively weak and is hardly able to keepthe enzyme fixed to the carrier under industrial conditions.The support can be a synthetic resin, an inorganic polymersuch as zeolite or silica, or a biopolymer. Entrapment involvesinclusion of an enzyme in a polymer network (gel lattice) suchas an organic polymer or a silica sol–gel, or a membranedevice such as a hollow fiber or a microcapsule. Entrapmentrequires the synthesis of the polymeric network in the pres-ence of the enzyme. The last category involves cross-linkingof enzyme aggregates or crystals, using a bifunctional reagent,to prepare carrier-free macroparticles [23].

Many other methods, which are either original combina-tions of the ones listed, sometimes specific for a given supportor enzyme have been developed. However, no single methodand support is the best for all enzymes and their variousapplications. This is because of the widely different chemicalcharacteristics and composition of enzymes, the differentproperties of substrates and products, and the various uses ofthe product. However, all of the methods may present anumber of advantages and drawbacks. Adsorption is simple,cheap, and effective but frequently reversible; covalent attach-ment and cross-linking are effective and durable, but expen-sive and easily worsening the enzyme performance; diffusion-al problems are inherent in membrane reactor-confinement,entrapment, and micro-encapsulations.

Protein adsorption

Non-covalent methods of protein immobilization are widelyemployed and involve either passive adsorption onto hydro-phobic surfaces or electrostatic interactions with charged sur-faces. The use of nitrocellulose membranes or polylysine-

Fig. 1 Selective approachesemploying affinity reagents

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coated slides for electrostatic binding is perhaps the mostwidely familiar. As noted above, the major advantage of thiskind of immobilization is that neither additional couplingreagents nor modification of the protein of interest is required.However, non-covalent immobilization typically involves rel-atively weak and reversible interactions. As a result, proteinscan leach out from the support, which in turn results in loss ofactivity with time and contamination of the surrounding me-dia. This has implications in the overall robustness and recy-clability of systems, particularly when used in analytical as-says and sensor devices. It is also well-known that absorptionof protein onto surfaces often results in conformationalchanges and denaturation of proteins that can lead to massivelosses of protein activity. Furthermore, since there is no con-trol over the packing density of the immobilized proteins, theiractivity may be further reduced by excessive crowding.

Protein immobilization by absorption on mesoporous silicates

In the past decade, much work has been undertaken in thesynthesis of mesoporous silicates and in the immobilization ofenzymes onto these supports. In 1992, the Mobil researchgroup [24] discovered a family of mesoporous silicates whichhad a narrow pore size distribution, amorphous silica surfaces,and pore sizes in the range of 20–300 Å. These new regularrepeating mesoporous structures offered the possibility ofadsorbing or entrapping large molecules within their pores.Since their development, these materials have promised animprovement about catalytic research of immobilized en-zymes. It was anticipated that mesoporous silicates wouldprovide a sheltered protected environment in which reactionswith selected substrates could proceed. Following their initialdiscovery, mesoporous structures have been synthesized using

cationic, neutral, and block copolymer surfactants. Thesecopolymers contain organic functional groups and metalslocated within their framework or grafted onto their surfaceand have been used as a scaffold to develop mesoporouscarbon materials [2].

Mesoporous silicates possess a number of additional attri-butes which make them attractive candidates for the immobi-lization of proteins. It is possible to chemically modify theirsurfaces with various functional groups, enabling electrostaticattraction or repulsion between the mesoporous silicate sup-port and the biological molecule of interest. As a result of theirsilicate inorganic framework, mesoporous silicates are chem-ically and mechanically stable and are resistant to microbialattack.

Materials, such as silica sol–gels, display similar stability tomesoporous silicates and have been used to encapsulate pro-teins for the development of biosensors. However, sol–gelssuffer from the disadvantage of possessing a highly variablepore size distribution (10–400 Å). More importantly, theirpreparation can involve the use of harsh conditions or re-agents, which can cause denaturation of proteins and aredetrimental to enzyme activity. Using mesoporous silicates,protein encapsulation occurs after the synthesis of the support,avoiding this difficulty [25].

Protein immobilization on polyketone polymer by hydrogenbonds

An innovative immobilization process has been recently pro-posed [46] which utilizes polyketone polymer, a completelynew support. Polyketone polymer –[–CO-CH2CH2–]n–,obtained by copolymerization of ethane and carbon monox-ide, has been utilized for immobilization of three different

Fig. 2 Some methods used forenzyme immobilization

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enzymes, one peroxidase from horseradish and two amineoxidases from bovine serum and lentil seedlings. The easyimmobilization procedure was carried out in diluted aqueousbuffer, gently mixing the proteins with the polymer. No bi-functional agents or spacer arms are required for the immobi-lization, which occurs exclusively via a large number ofhydrogen bonds between the carbonyl groups of the polymerand the –NH groups of the polypeptidic chain (Fig. 3).

Experiments demonstrate a high binding capacity and ex-traordinary stable immobilization, at least for amine oxidases.Moreover, activity measurements demonstrate that immo-bilized enzymes retain their fully catalytic characteristics,where only a small increase of KM value is observed. Theperoxidase activated polymer was used as active packed bedof an enzymatic reactor for continuous flow conversion andflow injection analysis of hydrogen peroxide containingsolutions.

Classical covalent immobilization methods

For more stable attachment, the formation of covalent bonds isrequired, and these are generally formed through reaction withfunctional groups present on the protein surface. In commonwith non-covalent adsorption, these methods can be used onunmodified proteins since they rely only on naturally presentfunctional groups. For example, the exposed amine groups oflysine residues readily react with supports bearing activeesters, with the most common being N -hydroxysuccinimide(NHS) esters, to form stable amide bonds. However, onedisadvantage of using NHS esters is that they are unstable inaqueous conditions, and thus, the attachment of proteins inaqueous buffers will compete with ester hydrolysis, possiblyresulting in a modest immobilization yield. As an alternative,aldehyde groups can be coupled with exposed amino groupsfollowed by reduction using sodium cyanoborohydride, orother reagent, to form a stable secondary amine linkage.

The nucleophilicity of the amine group also allows reactionwith epoxide-functionalized materials (diglycidyl ethers).These epoxides have the advantage of being relatively stable

to hydrolysis at neutral pH, which allows easy handling of thematerials but can result in slow or incomplete coupling. More-over, it is necessary to have supports with the highest degreeof epoxy groups, in conditions where they may be very stableto permit long enzyme–support reactions and having theshortest possible spacer arms (mainly in the third generationof epoxy supports) to effectively freeze the enzyme structure.New heterofunctional supports or new uses of the above-described supports may speed up the implementation of manyindustrial bioprocesses, at present hampered by the lack ofsuitable biocatalysts [4, 5].

Cysteine residues, bearing the thiol group, are alsooften employed for protein immobilization and readilyundergo conjugate addition with unsaturated carbonyls(e.g., maleimides) to form stable thioether bonds. It hasbeen shown that maleimide groups strongly favor conju-gate addition with thiols at physiological pH (6.5–7.5)since under these conditions amines are predominantlyprotonated and un-reactive (Fig. 4). As proteins generallyhave very few surface-exposed cysteine residues, it ispossible to achieve site-selective immobilization, espe-cially if the protein of interest can be engineered to removeall but one surface cysteine residue or to insert a singlecysteine on the surface where none previously existed. Thenucleophilicity of the thiol group also means that it can reactwith epoxides and NHS esters, although in practical terms thislatter reaction is relatively slow and the resultant thioestermoiety is susceptible to hydrolysis.

As regard aspartic and glutamic acid residues, the genericmethod in which they can be used for immobilization is byconversion to their corresponding active esters in situ with acarbodiimide coupling agent and an auxiliary nucleophile.The most commonly used example of the former is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, while NHS iswidely used to generate the NHS ester on the protein. Thisactive ester can then react with amine-bearing supports. Theadvantage of this combination of reagents is that both arewater-soluble and may be used in aqueous media, althoughthe instability of carbodiimides and the subsequently generat-ed active esters under these conditions means that the reactionyields are often rather low. There is also the risk that the NHSesters formed on the protein molecule may then couple toother protein molecules to give poorly defined polymers.

Oxidative cleavage of the 1,2-diols on the oligosaccharides(usually with periodate) generates aldehyde moieties that canthen be used for attachment in a semispecific manner tohydrazine or hydroxylamine functionalized supports via theirrespective hydrazone or oxime. In addition to antibodies, thisstrategy has also been applied to a range of other proteins thatfeature post-translational glycosylation, including several pro-tease and oxidase enzymes [26]. However, it should be notedthat, apart from the multiple potential attachment points on apolysaccharide chain, random orientation may also occur if

Fig. 3 Hydrogen bonds between carbonyl groups and the –NH groups ofthe polypeptidic chain

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the desired protein has more than one site of glycosylation onits surface.

In recent years, several selective immobilization methodsable to proceed under mild physiological conditions havereceived increasing attention. Typically these methods relyon the labeling of the protein of interest with an azide moiety.In the Staudinger ligation, the reaction of an azide with aphosphine forms an intermediate iminophosphorane (aza-ylide) that can then react with electrophiles to give a varietyof products [27, 28]. The generation of an iminophosphoraneis typically followed by reaction with an ester to form a stableamide bond. In the first version of this approach, the electro-philic ester is incorporated into the phosphine to give a finalproduct with the phosphine oxide attached to the linkage. TheStaudinger ligation has been exploited in a large number ofapplications from the labeling of proteins and cell-surfaceglycans to the chemical synthesis of proteins. More recently,the Staudinger ligation has also been applied for the immobi-lization of peptides and proteins [29]. In all cases, a two-stepprocess was required: labeling of the protein of interest withthe azide followed by the immobilization reaction. Thus, inorder to achieve site-selective immobilization, a method forselectively introducing the required azide moiety must first beemployed, such as an enzymatic reaction that recognizesspecific protein sequences. The general strategy is, therefore,first to introduce the DNA sequence coding for the tag adja-cent to the gene encoding for the protein of interest. Subse-quently, expression of the engineered synthetic gene thenyields a fusion protein of the original protein of interestattached to the tagging protein or peptide containing theattachment site. This fusion protein is then used for the im-mobilization procedure.

Another method of selective immobilization is derivedfrom the Huisgen 1,3-dipolar cycloaddition of azides with an

alkyne, where the covalent link is formed through the forma-tion of a 1,4-disubstituted 1,2,3-triazole [30]. This reaction hasbeen popularized as “click” chemistry [31], and in the mostwell-known version, a terminal alkyne and azide are reactedwith Cu(I) catalysts to give near-quantitative conversions tothe triazole. The alkyne moiety is also rarely present in bio-logical pathways and adds further versatility to this reactionsince the alkyne may be introduced to the biomolecule insteadof the azide. This type of “click” chemistry has been used in avariety of applications to enable the attachment of biomole-cules bearing either the azide or alkyne with various polymers,fluorophores, or biochemical labels functionalized with theircounterpart moieties [32]. Similar to the Staudinger chemistry,the site-selective attachment of proteins also requires its use intandem with an enzymatic site-selective labeling method.

Another related family of reactions is “photoclick chemis-try,” which relies on photoirradiation to trigger pyrazolineformation between tetrazoles and alkenes [33]. An addedadvantage of this method is that the newly formed heterocycleis fluorescent, enabling the monitoring of the reaction prog-ress. Although these are interesting developments, at the mo-ment these have not yet been applied for the purpose of proteinimmobilization (Figs. 5 and 6).

Physical entrapment

The best means of avoiding any negative influence on thestructure of an enzyme is to encapsulate it. Proteins or en-zymes can be extremely fragile and easily aggressed by ex-ternal agent such as proteases. Encapsulation of these fragilemacromolecules is a possible strategy for preventing theiraggression and denaturation. A well-established technique toencapsulate biological species such as enzymes, antibodies,

Fig. 4 Reaction of maleimideforming chemical conjugation toa sulfhydryl

Fig. 5 The “click” chemistryreaction of alkyne (1) with azide(2) to form a 1,4-disubstituted1,2,3-triazol

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and other proteins in a functional state is based on the sol–gelchemistry method.

Sol–gels are silica materials that are highly porous andreadily prepared. The sol–gel is a chemically inert glass thatcan be shaped in any desired way. It can be designed to bethermally and mechanically very stable, but the standard sol–gel is brittle. Sol–gels have been used extensively in theimmobilization of proteins and in particular for the develop-ment of biosensors. Although sol–gels are porous, diffusion ofsubstrate to the enzyme can be restricted and care has to betaken to minimize this effect. The synthesis of sol–gels isrelatively mild for enzymes. In the first step, a tetra-alkoxysilane is hydrolyzed via acid catalysis. Hydrolysis isfollowed by condensation where the sol is formed. This is amixture of partially hydrolyzed and partially condensed. Allthe pores of this gel are filled with water and alcohol; it istherefore known as aquagel. When the aquagel is dried byevaporation, a xerogel is obtained. Due to the action of capil-lary forces during the evaporation process, the aquagel shrinksand part of the structure collapses. The xerogel consequentlydoes not have the same structure as the aquagel. To avoid suchcapillary action, the water in the aquagel can be exchangedwith acetone and then with supercritical carbon dioxide. Onevaporation of the carbon dioxide, the structure of the aquagelis maintained and a brittle aerogel is obtained. In this manner,hydrophilic aqua-, xero-, and aerogels are made. By addingalkyltrialkoxysilanes to the synthesis mixture, sol–gels with ahydrophobic surface can be obtained. Overall the sol–gelmethod can generate gels with very different properties [6].

Many encapsulation methods have been developed, andthis task can be achieved with lipid vesicles (also calledliposomes ), which are polymolecular aggregates formed inaqueous solution on the dispersion of certain bilayer-formingamphiphilic molecules, such as phospholipids. Under osmot-ically balanced conditions, the vesicles are spherical in shapeand contain one or more (concentric) lamellae that are com-posed of amphiphilic molecules. These shells are curved andself-closed molecular bilayers in which the hydrophobic partof the amphiphiles forms the hydrophobic interior of thebilayer and the hydrophilic part (the polar head group) is incontact with the aqueous phase. The interior of the lipidvesicles is an aqueous core, the chemical composition ofwhich corresponds in a first approximation to the chemicalcomposition of the aqueous solution in which the vesicles areprepared. Depending on the method of preparation, lipidvesicles can bemulti-, oligo-, or unilamellar, containingmany,

a few, or one bilayer shell(s), respectively. The diameter of thelipid vesicles may vary between about 20 nm and a fewhundred micrometers. Lipid vesicles are generally not a ther-modynamically stable state of amphiphiles and do not form“spontaneously” (without input of external energy); they areonly kinetically stable, kinetically trapped systems. The phys-ical properties of lipid vesicles depend on how and underwhich conditions lipid vesicles of a certain amphiphile (or ofa mixture of amphiphiles) are prepared. The mean size, thelamellarity, and the physical stability of the vesicles not onlydepend on the chemical structure of the amphiphiles used, butin general particularly on the method of vesicle preparation.Physical instabilities of lipid vesicle systems involve vesicleaggregation and fusion, possibly leading to vesicle precipita-tion and flat bilayer formation.

The basic principles for the preparation of enzyme-containing lipid vesicles in general do not so depend on thechemical structure of the amphiphiles used. Care should betaken to that all mechanical treatments used during vesiclepreparation, such as lipid dispersion, sonication, or filtrationthrough polycarbonate membranes, have to be carried outabove the transition temperature (Tm). Below Tm, the saturat-ed hydrophobic chains exist predominately in a rigid, extend-ed all trans conformation, similar to their crystalline state.Above Tm, the chains are rather disordered, making the bilay-er fluid (mechanically treatable), characterized by increasedlateral and rotational lipid diffusion rather similar to a liquid. Itis this fluid state of the lipids in biological membranes whichrepresents a fluid matrix for membrane proteins.

The involvement of the highly reproducible extrusion tech-nique, particularly with polycarbonate membranes containingpores with a mean diameter of tenth of nanometers, results inhomogenous and mainly unilamellar vesicles. In addition tothe high degree of reproducibility of the preparation, oneadvantage of the extrusion technique is certainly the fact thatno organic solvent is involved. One possible disadvantage isthe shear force present when the vesicles are squeezed throughthe approximately cylindrical pores, which can inactivate theenzyme. The encapsulation yield at a fixed lipid concentrationmay also be dependent on the enzyme concentration and onthe nature and ionic strength of the buffer used. In comparisonwith liposomes prepared by the extrusion technique, vesiclesprepared by dehydration–rehydration are generally notunilamellar and not monodisperse, which may be an advan-tage or a disadvantage, depending on the type of applicationon the enzyme-containing vesicles. One should also consider

Fig. 6 The “photoclickchemistry” between tetrazolesand alkenes to form pyrazoline

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that the dehydration–rehydration cycle may inactivate theenzyme. It certainly depends on the type of applications andon the cost of the enzyme, whether achieving high encapsu-lation efficiencies is important or not. In the case of drugdelivery, e.g., high EE values are usually desired.

From all these considerations on interactions of water-soluble enzymes with the vesicle membrane, it is clear thatthis aspect is important to be taken into account if one likes tounderstand the behavior of the entrapped enzymes. There aretwo main areas of applications of enzyme-containing lipidvesicles: (a) in the medical or biomedical field, particularlyfor the enzyme-replacement therapy, or (b) in the cheeseripening process (acceleration of the process and control overflavor development). For both of these types of applications,the lipid vesicles are just carriers for the enzymes, containerswhich protect the enzymes from getting in immediate contactwith the medium to which they are added, the blood circula-tion. In the case of the enzyme-containing lipid vesicle-assisted cheese ripening process, the entrapped enzyme isgradually released, allowing catalyzing degradation and mod-ification reactions in the cheese matrix during the ripeningperiod. In the medical applications, the vesicles are used as thedrug delivery system, the drug being the entrapped enzyme. Inmost cases, the vesicles carry enzyme molecules with the aimof replacing- or supporting-endogenous enzymes in the treat-ment of particular diseases (enzyme-replacement therapy).The entrapped enzyme molecules have to be released at aparticular site in the body where they are needed [8].

Biodegradable polymers nanosystems are an attractive al-ternative to liposomes since they have the advantages oflonger circulation in the blood stream and generally higherdrug carrying capacity. Polymers such as poly(lactic acid) andpoly(lactic-co-glycolic acid) have been extensively investigat-ed for their biocompatibility and potential capability of releas-ing therapeutically proteins in a controlled way even over aprolonged period of time. These polymers are degradable bybulk erosion through hydrolysis of the ester bonds. The hy-drolysis rate depends on several nanoparticles physicochemi-cal parameters and can be tailored according to the desiredrelease pattern of the protein to be incorporated. These twopolymers were approved by FDA as excipients to achievecontrolled release of the active ingredient. However, theirapplication in protein delivery systems is often characterizedby low entrapment efficiencies, burst release, instability ofencapsulated hydrophilic protein, and partial protein release.To improve the performance of these polymer nanoparticles,polysaccharides such as alginate (ALG) and chitosan (CS)could be applied. CS and its derivatives have been intensivelystudied as carriers for proteins and drugs. More specificallythese nanoparticles can be totally made by CS or used inseveral copolymer combinations. Copolymers made by thecombination of CS/ALG are able to generate a more “friend-ly” environment which protects peptides and proteins from

stressing conditions and allows their stabilization during en-capsulation, storage, and release.

Kinetics properties of enzymes can be also altered duringthe entrapment process allowing potential change of the pro-tein specificity to its substrate. Gaining a clear picture of thesebasics knowledge will definitely lead to a change of objectdesign to increase the protein load, to control the proteinrelease, and to retain the protein integrity and efficacy [9].

Bioaffinity interactions

Over the years, a number of protein–protein and protein–smallmolecule binding interactions have been harnessed for immo-bilization. These strategies exploit the selectivity of suchinteractions and are, therefore, highly specific with respect tothe identity of binding partners as well as the location on themolecules at which binding occurs. Historically, many of thetags that have been described were developed for proteinpurification by affinity chromatography, but few have beenwidely co-opted for immobilization in other applications.Perhaps the most well-known genetically encoded affinitytag is the polyhistidine tag. This small tag, usually consistingof six sequential histidine residues, chelates transition metalsincluding Cu(II), Co(II), Zn(II), or Ni(II), although the latter ismost commonly employed. Here, a support bearing a chelat-ingmoiety such as nitrilotriacetic acid or imino-diacetic acid istreated with a solution of the relevant metal salt to produce asupport presenting the metal ions. This metal-activated sup-port is then used for protein immobilization through chelationwith the histidine residues of the tag. This method of immo-bilization is widely used for the temporary capture of proteinsduring purification and is often termed immobilized metalaffinity chromatography (IMAC) [7].

However, the general level of selectivity of this method isrelatively low since several endogenous proteins have beenidentified that are also able to bind to metal ions, thus com-peting with the desired histidine-fused protein. As a result, formost applications the desired histidine-fusion protein must bepurified prior to use. The strength of the binding interaction isalso relatively weak (Kd≈1–10 μM), although proteins bear-ing tags with 10–12 His residues or two separate histidine tagshave been shown to give improvements of up to 1 order ofmagnitude, enabling in situ immobilization of the target pro-tein on Ni-NTA slides directly from cell lysates [34].

Antibodies, apart from being the target of immobilizationfor use in microarrays and biosensors, may also be used as ameans of immobilizing other proteins due to the selectivity oftheir binding interactions. This concept is regularly applied forpurification through the use of columns with immobilizedantibodies acting to trap their epitope target and is known asimmunoaffinity chromatography. The method is, however,rarely used for other applications for several reasons. Inorder to achieve uniform immobilization, a well-defined

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monoclonal antibody is needed; polyclonal antibodies areunsuitable since they are not a single species but a heteroge-neous population of antibodies that bind their epitope in avariety of conformations. Indeed, without in-depth structuralknowledge of the binding interaction, it is impossible todetermine strength of the binding or if the binding may blockthe active site of the protein of interest. Furthermore, therelatively weak nature of the binding means that it is oftenunsuitable for many applications that require longer-term ormore robust immobilization of proteins.

Undoubtedly the most well-known and extensivelyresearched protein-mediated immobilization technique relieson the non-covalent interaction of either avidin or streptavidinto proteins functionalized with biotin [35]. The interactionbetween biotin and (strept) avidin is extremely strong (Kd≈10−15 M), and this combined with the fact that these proteinsare unusually stable to heat, denaturants, extremes of pH, andproteolysis means that the binding is essentially irreversible.The widespread availability of supports such as microtiterplates, microarray substrates, and magnetic particles that arecoated with these proteins has also greatly contributed to thepopularity of this method as a means of protein immobiliza-tion. However, in order to exploit this attachment for proteinimmobilization, the protein of interest must first be labeledwith biotin. Classically, this can be achieved with a number ofnonselective chemical biotinylation reagents such as biotinNHS ester [36].

One conceptually straightforward method of protein im-mobilization is through the use of enzymatically active fusionproteins. In this case, a protein of interest is fused to anenzyme (capture protein) that reacts selectively with animmobilized substrate analogue or inhibitor. This reactionforms a covalent bond between the enzyme and the substrateon the surface. This strategy was reported in 2002 byMrksich,using the serine esterase cutinase from the filamentous fungusFusarium solani pisi [37]. Cutinase is selectively inhibited byalkylphosphonate para-nitrophenol esters through esterifica-tion of the active site serine residue, resulting in the formationof a covalent bond. This protein and ligand were chosenbecause they possessed a number of desirable characteristicsfor an immobilization technique. The enzyme was relativelysmall (210 amino acid residues), globular, and monomeric,which would minimize any steric interactions with the fusedprotein. Both the termini are opposite to the active site andhence would be amenable to the generation of both N - and C-terminal fusions in which the fused protein would be orientedaway from the support surface. Furthermore, the phosphonatediester inhibitor was relatively stable toward hydrolysis, andwhen bound in the cutinase active site, the alkyl tail of thephosphonate protruded out of the enzyme and offered anaccessible location for attachment to the support.

There are also a number of other site-selective immobili-zation methods that do not require enzymatically mediated

attachment. These rely on the recognition of specific function-al groups present on amino acids in the protein or an associ-ated peptide tag. One example of this employs phenolic oxi-dative cross-linking, a phenomenon that is widely observed innature, which includes protein cross-linking through tyrosineresidues [38]. Such dityrosine crosslinks occur in structuralproteins such as elastin and silk and are catalyzed bymetalloenzymes, although a simple complex of Ni(II) withthe tripeptide glycine–glycine–histidine can also catalyze the-se reactions. Since IMAC and immobilization with Ni(II) viasix-histidine tags and NTA-functionalized supports are al-ready widely used, it was rationalized that this complex couldbe used as the cross-linking catalyst instead [39]. By bringingtwo tyrosine residues, one from the protein to be immobilizedand another from the support, into close proximity to theNi(II) complex, it would be possible to cross-link the twoand form a covalent bond between the support and a specificlocation on the protein. Once the cross-linking had beenachieved, the nickel was removed. The extent of dityrosinecross-linkage could also be easily determined since this moi-ety displays a characteristic fluorescence emission at 420 nm.

A wide range of biologically mediated methods are avail-able for site-specific protein immobilization. This area ofresearch is notable because it is the culmination of a majormultidisciplinary research effort spanning molecular biology,protein engineering, organic chemistry, and materials science.The more recent introduction of biologically mediatedmethods for site-specific, particularly enzyme-mediated, im-mobilization of proteins from cell lysates should enable thefuture fabrication of more highly sensitive protein microarraysand biosensors as wells as finding new applications in nano-technology and single molecule enzymology.

Support materials

The properties of supported enzyme preparations aregoverned by the properties of both the enzyme and the carriermaterial. The interaction between the two provides animmobilized enzyme with specific chemical, biochemical,mechanical, and kinetic properties. The support (carrier) canbe a synthetic organic polymer, a biopolymer, or an inorganicsolid [40, 41]. Avariety of matrixes have been used as supportmaterials for enzyme immobilization [42].

Materials proposed in many examples are not suitable forindustrial processing procedures due to their low mechanicalstrength [43]. This type of supporting matrixes is sometimescalled soft gel.

Synthetic polymers

The most common and widely used synthetic polymers usedas support for enzyme immobilization are represented by

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acrylic resins, such as Eupergit®-C (Evonik Industries). Theyare macroporous copolymers of N ,N ′-methylene-bi-(methacrylamide), glycidyl methacrylate, allyl glycidyl ether,and methacrylamide with average particle size around170 mm and a pore diameter of about 20–30 nm (Fig. 7).They are highly hydrophilic and stable, both chemically andmechanically, over a pH range from 0 to 14, and does notswell or shrink even upon drastic pH changes. A majordrawback of hydrophilic resins is diffusion limitations, theeffects of which, as would be expected, are more pro-nounced in kinetically controlled processes. Immobilizationby covalent attachment to acrylic resins has been success-fully applied to a variety of enzymes for industrial applica-tion [44, 45].

As above reported, polyketone polymers –[–CO-CH2CH2–]n– appear promising supports for protein immobi-lization for their mechanical properties, high immobilizationcapacity, simplicity of the immobilization procedure by sim-ple reactant mixing, and absence of bi-functional reagents orspacer arms. Polyketone polymers are obtained by palladium-catalyzed copolymerization of ethane and carbon monoxide[46]. Due to the close presence of polar keto groups, thepolymers present a high liquid volume value (8.2 ml/g for apolymer not exceeding 25,000 Da), larger than twice than thatshown by commercial sepharose, because the high possibilityof hydrogen bonds between the keto groups and the watermolecules and no restriction due to cross linking are present.Water accessible carbonyl groups were quantified in about10 meq/g polymer, and the apparent concentration of theimmobilized enzyme on the polyketone was more than2.36 mg/ml [46].

Biopolymers

Avariety of biopolymers, mainly water-insoluble polysaccha-rides such as cellulose, starch, agarose, carragenans, and chi-tosan, have been widely used as supports for immobilizingenzymes (Fig. 8). These matrixes form very inert aqueousgels, characterized by high gel strength, even at low concen-tration [47]. Moreover, due to their chemical structure, whichcan be easily activated, they may be prepared to bind proteinsand enzymes in a reversible or irreversible way. Likewise,with the use of bi-functional reagents, it is possible to formcross-links which strengthen their structure increasing theirmechanical and thermal resistance. Due to these particularcharacteristics, biopolymers are resins which are almost idealto be used in purification (reversible binding) and in immobi-lization processes (irreversible binding) of biomolecules. Cur-rent techniques to conjugate polysaccharides to proteins in-clude aldehyde, carbodiimide, epoxide, hydrazide, active es-ter, radical, and addition reactions, some of which are suitablefor in situ conjugation of materials, while others are bettersuited for ex situ conjugation and purification.

Hydrogels

Enzymes can also be immobilized in natural or synthetichydrogels or cryogels in non-aqueous media. Polyvinyl alco-hol (PVA) cryogels formed by the freeze-thawing method, forexample, have been widely used for immobilization of wholecells [48]. However, free enzymes, owing to their smaller size,can diffuse out of the gel matrix and are, consequently,leached in an aqueous medium. In order to entrap free

Fig. 7 Structure ofpolyacrylamide gel matrix

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enzymes, the size of the enzyme must be increased, e.g., bycrosslinking. An alternative method to increase the size ofthe enzyme is to form a complex with a polyelectrolyte.Owing to their ampholytic character, proteins exist aspolycations or polyanions, depending on the pH of themedium. Hence, they can often form complexes with op-positely charged polyelectrolytes.

Inorganic supports

On the other hand, rigid supports may be preferable. Avarietyof inorganic solids can be used for the immobilization ofenzymes, e.g., alumina, silica, zeolites, and mesoporous sil-icas [49]. Silica-based supports are the most suitable matricesfor enzyme immobilization in industrial manufacturing ofenzyme-processed products [18, 50, 51], as well as for re-search purposes [52]. The most important advantages of silicagels compared to soft gels are their higher mechanical strengththat allow their utilization for a much wider range of operatingpressures and experimental conditions, as evidenced by theirpreferential use in the preparation of high performance liquidchromatography media [53–55]. Additionally, the surface ofsilica gel can be readily modified by chemical methods toprovide various types of functional groups for facilitatedligand attachment. It has been shown that an extremely highloading of invertase was achieved by chemical modificationof gel surface using amino group [56]. The overall surfacearea offered by silica gel matrixes is unparalleled. Further-more, the silica gel can be easily fabricated to provide desir-able morphology, pore structures, andmicro-channels to allowsubstrate–ligand interaction. Furthermore, silica gel is me-chanically stable and chemically inert, and it is thereforeenvironmental- and solvent-friendly for industrialmanufacturing and processing.

One of the simplest and most inexpensive methods toimmobilize an enzyme on silica is by simple absorption. It is

used, for example, to formulate enzymes for detergent pow-ders which release the enzyme into the washing liquid duringwashing.

Smart polymers

A novel approach to immobilize enzymes is via covalentattachment to stimulus-responsive or mart polymer, whichundergo dramatic conformational changes in response tosmall changes in their environment, e.g., temperature, pH,and ionic strength [57]. The most studied example is thethermo-responsive and biocompatible polymer, poly-N -isopropylacrylamide (polyNIPAM) [58]. Aqueous solutionsof polyNIPAM exhibit a critical solution temperature (LCST)around 32 °C, below which the polymer readily dissolves inwater while, above the LCST it becomes insoluble owing toexpulsion of water molecules from the polymer network.Hence, the biotransformation can be performed under condi-tions where the enzyme is soluble, thereby minimizing diffu-sional limitations and loss of activity owing to protein confor-mational changes on the surface of a support. Subsequently,raising the temperature above the LCST leads to precipitationof the immobilized enzyme, thus facilitating its recovery andreuse. An additional advantage of using such thermo-responsive immobilized enzymes is that runaway conditionsare avoided because when the reaction temperature exceeds theLCST, the catalyst precipitates and the reaction shuts down.Two methods are generally used to prepare the enzyme–polyNIPAM conjugates: (a) introduction of polymerizable vi-nyl groups into the enzyme followed by copolymerization withNIPAM or (b) reaction of NH2 groups on the surface of theenzyme with a copolymer of NIPAM containing reactive estergroups or the homopolymer containing anN-succinimide esterfunction as the end group. More recently, an alternativethermo-responsive polymer has been described [59–61]. Itconsists of random copolymers derived from 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol)methacrylate and combines the positive features of poly(ethyl-ene glycol), non-toxicity, and anti-immunogenicity, withthermo-responsive properties similar to polyNIPAM. TheLCST could be varied between 26 and 90 °C depending onthe relative amounts of OEGMA used. These properties makethis a potentially interesting support for biocatalysts.

Conducting polymers

Polymers that exhibit electrical conductivity have now beensuccessfully synthesized, and the past two decades havewitnessed unending interest in the synthesis and characteriza-tion of such conducting polymers due to the potential techno-logical applications of these materials. Conducting polymersalso receive a great interest in biotechnology. A large numberof organic compounds, which effectively transport charge, are

Fig. 8 Chemical structures of cellulose and its amino derivatives

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roughly divided into three groups, i.e., charge transfercomplexes/ion radical salts, organometallic species, and con-jugated organic polymers. A new class of polymers known asintrinsically conducting polymers or electroactive conjugatedpolymers has recently emerged. Such materials exhibit inter-esting electrical and optical properties previously found onlyin inorganic systems. Electronically conducting polymers dif-fer from all the familiar inorganic crystalline semiconductors,e.g., silicon in two important features that polymers are mo-lecular in nature and lack long range order. A key requirementfor a polymer to become intrinsically electrically conductingis that there should be an overlap of molecular orbitals toallow the formation of delocalized molecular wave function.Besides this, molecular orbitals must be partially filled so thatthere is a free movement of electrons throughout the lattice.Enzyme immobilization and biosensor construction are someof these applications [62].

Various conducting polymers have been considered forimmobilization of enzymes. Of these, polypyrrole hasgained most interest for the entrapment of protein mole-cules due to its low oxidation potential. This unique char-acteristic enables the growth of film from aqueous solutionsthat are compatible with most biological systems. Thisapproach is usually based on entrapment of an enzyme intothe structure of polypyrrole film by potentiostatic orgalvanostatic polymerization of pyrrole in enzyme solution,which contains a supporting electrolyte.

Conducting polymers can be prepared by chemical orelectrochemical polymerization. Although the chemical meth-od offers mass production at a reasonable cost, the electro-chemical method involves the direct formation of conductingpolymer thin films with better control of thickness andmorphology, which are more suitable for direct application.It is established that the conducting properties of the poly-mer films greatly depend on the mode of synthesis and alsoon the number of parameters, such as type of counter-ion,the type of electrolyte and its concentration, synthesistemperature, electrochemical voltage, pH of the electrolyte,etc. Thus, in order to improve the properties of polymerfilms suitable for a desired application, it is necessary tocritically control and optimize the various synthesis param-eters [63].

The other electrochemically prepared conducting polymersused for the biomolecule immobilization are polyacetylene,polythiophene, polyindole, and polyaniline. Also few biosen-sors based on insulating electropolymerized films polyphenol,poly(o -phenylenediamine), polydichlorophenolindophenol,and overoxidized polypyrrole have also been elaborated[64]. Many theoretical models have also been coupled withthe electrochemical entrapment of enzyme for the evaluationof the role of the thickness of polymeric layers, enzymelocation, enzyme loading, etc. on the biosensor functioning[65].

Gold nanoparticles

The progress of nanotechnology in the 1990s was followed bythe rapid growth of nanobiotechnology. “Nanobiocatalysis” isone typical example. In the early approaches to nanobio-catalysis, enzymes were immobilized on various nanostruc-tured materials using conventional approaches, such as simpleadsorption and covalent attachment. This approach gatheredattention by immobilizing enzymes onto a high surface area ofnanostructured materials, such as nanoporous materials,electrospun nanofibers, and magnetic nanoparticles. Thislarge surface area resulted in improved enzyme loading,which in turn increased the apparent enzyme activity per unitmass or volume compared to that of enzyme systemsimmobilized onto conventional materials. One of the particu-larly advantageous features of nanostructured materials is thecontrol over size at the nanometer scale, such as the pore sizein nanopores, thickness of nanofibers or nanotubes, and theparticle size of nanoparticles. The uniform size distribution ofnanomaterials and their similarity in size with enzyme mole-cules, together with other advantageous nanomaterial proper-ties such as conductivity and magnetism, have revolutionizednanobiocatalytic approaches in various areas of enzyme tech-nology and led to improved enzyme properties in nanobio-catalytic systems, particularly with regard to enzyme stabilityand activity. Recently, nanobiocatalytic approaches haveevolved beyond simple enzyme immobilization strategies toinclude enzyme stabilization, wired enzymes, the use of en-zymes in sensitive biomolecular detection, artificial enzymes,nanofabrication, and nanopatterning [66].

Nanosized particles of noble metals, especially goldnanoparticles (AuNPs), have received great interests due totheir attractive electronic, optical, and thermal properties aswell as catalytic properties and potential applications in thefields of physics, chemistry, biology, medicine, and materialscience. Therefore, the synthesis and characterization ofAuNPs have attracted considerable attention from both afundamental and a practical point of view. A variety ofmethods have been developed to prepare AuNPs [67]. How-ever, the high surface energy of AuNPs makes them extremelyreactive, and most systems undergo aggregation without pro-tection or passivation of their surfaces. Thus, special precau-tions have to be taken to avoid their aggregation or precipita-tion. Typically, AuNPs are prepared by chemical reduction ofthe corresponding transition metal salts in the presence of astabilizer which binds to their surface to impart high stabilityand a desirable linking chemistry and provide the desiredcharge and solubility properties. Some of the commonly usedmethods for surface passivation include protection by self-assembled monolayers, the most popular being citrate andthiol-functionalized organics; encapsulation in the H2O poolsof reverse microemulsions; and dispersion in polymeric ma-trixes (Fig. 9). Although the synthesis of AuNPs makes great

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progress, the control of size, monodispersion, morphology,and surface chemistry of AuNPs is still a great challenge.Recently, designing novel protectors for AuNPs have beenthe focus of intense research because surface chemistry ofAuNPs will play a key role in future application fieldssuch as nanosensor, biosensor, catalysis, nanodevice, andnanoelectrochemistry.

Magnetic nanoparticles

In recent years, substantial progress has been made in devel-oping technologies in the field of magnetic carriers. Magneticseparation is an emerging technology that uses magnetism forthe efficient separation of micro- and nanometer para- andferro-magnetic particles from different chemical and biologi-cal samples.

With the rapid development of nanotechnology, magneticnanoparticles are currently being widely studied. It has longbeen known that the physicochemical properties of magneticnanoparticles can be vastly different from those of the corre-sponding bulk material. For example, magnetic anisotropy,which keeps a particle magnetized in a specific direction, isgenerally proportional to the volume of a particle [68]. Mag-netic nanoparticles will display superparamagnetism, whichmeans that the particles are attracted by a magnetic field, butretain no residual magnetism after the field is removed. There-fore, suspended superparamagnetic particles can be removedfrom solution using an external magnet, but they do notagglomerate (i.e., they stay suspended) after removal of theexternal magnetic field.

Among magnetic carriers, nano-sized magnetic particlespossess many advantageous properties. Particles such ascrosslinked iron oxide [69, 70], ultrasmall superparamagneticiron oxide [71, 72], and monocrystalline iron oxide nano-particles [73, 74] had all been developed as imaging agentsin magnetic resonance imaging. Magnetic particles have beenused to immobilize enzymes in order to enhance bioelementstability, ease separation from the reaction mixture, and in-crease catalyst stability, and they have been proposed forbiotechnological applications [75] or for analytical devices,such as biosensors [76, 77]. Furthermore, nanomedicine is anemerging field that uses nanoparticles to facilitate the diagno-sis and treatment of diseases. Iron nanoparticles were used intumor treatment relying on tumor vessel leakiness for prefer-ential accumulation in cancer tissues. Specific targeting ofmagnetic nanoparticles to tumors has been accomplished invarious experimental systems [78].

There have been various methods developed for the prep-aration of paramagnetic nanoparticles [79]. The most com-monly used protocol involves co-precipitation of ferrous andferric ions in basic solutions. The development of uniformnanometer sized particles has been intensively pursued be-cause of their technological and fundamental scientific impor-tance. These nanoparticular materials often exhibit very inter-esting electrical, optical, magnetic, and chemical properties,which cannot be achieved by their bulk counterparts. In liter-ature, polymers such as dextran, PVA, and DEAE-starch wereadded to coat the particles for better stability, before or afterthe formation of iron oxide particles [80, 81]. Otherwise,magnetic nanoparticles can be coated with silica containinga high coverage of silanol groups, which can easily be

Fig. 9 Porphyrin–goldnanoparticle (AuNP)

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anchored with defined and generic surface chemistries [82,83]. As a result, magnetic nanoparticles coated with anultrathin layer of silica would be more useful as magneticnanocarrier [84].

Cross-linked enzyme aggregates (CLEAs)

Cross-linked enzyme crystals (CLECs) are highly activeimmobilized enzymes of controllable particle size, varyingfrom 1 to 100 μm. Their operational stability and ease ofrecycling, coupled with their high catalyst and volumetricproductivities, renders them ideally suited for industrial bio-transformations. However, CLECs have an inherent disad-vantage: Enzyme crystallization is a laborious procedurerequiring enzyme of high purity, leading to their high costs.Themore recently developed cross-linked enzyme aggregates(CLEAs), on the other hand, are produced by simple precip-itation of the enzyme from aqueous solution, as physicalaggregates of protein molecules, by the addition of salts, orwater miscible organic solvents or non-ionic polymers [40,41, 85]. These physical aggregates are held together by non-covalent bonding without perturbation of their tertiary struc-ture that is without denaturation. Subsequent cross-linking ofthese physical aggregates renders them permanently insolublewhile maintaining their pre-organized superstructure andhence their catalytic activity. This discovery led to the devel-opment of a new family of immobilized enzymes: CLEAs.This type of immobilized enzyme is very effectivebiocatalysts as they can be produced by inexpensive andeffective method. CLEAs can readily be reused and exhibitsatisfactory stability and performance for selected applica-tions. The methodology is applicable to essentially any en-zyme, including cofactor dependent oxidoreductases [40, 41].The use of CLEAs to penicillin acylase used in antibioticsynthesis has shown large improvements over other type ofbiocatalysts [86].

Selected applications of immobilized enzymes

Various applications of immobilized enzymes can be found inindustry, medicine, and research. Table 1 has gathered aselection of currently used immobilized-enzyme processes.In any commercial application, the choice of a specificimmobilized enzyme or mode of immobilization must bebased on a specific compromise about all advantages anddisadvantages between free and immobilized enzyme. Otherindustrial applications of immobilized enzymes includelaboratory-scale organic synthesis and analytical and medicalapplications [87, 88]. Furthermore, since enzymes are able tocatalyze reactions not only in aqueous solutions but also inorganic media, immobilized enzymes can catalyze organicsynthesis [89].

Biosensors

In recent analytical applications, immobilized enzymes areused mainly in biosensors [90] and in diagnostic test strips.Biosensors are constructed by integrating biological sensingsystems, e.g., enzyme (s), with a transducer. Enzymes for themost cases are immobilized either directly on a transducer’sworking tip or using a polymer membrane tightly wrapping itup. In principle, due to enzyme specificity and sensitivity,biosensors can be tailored for nearly any target analyte[91–94], and these can be both enzyme substrates and enzymeinhibitors. Advantageously, their determination is performedwithout special preparation of the sample. The most exten-sively studied enzymes for the application in enzyme-basedbiosensors are presented in Table 2. Practically, four of thesensors listed, namely glucose, lactate, urea, and glutamate,have been widely used and commercialized [90]. Recentadvances were reviewed [95–97].

Enzyme biosensors are made by immobilization of en-zymes on the sensing surface [98–100]. Due to the presenceof reactive functional groups, organic polymeric carriers suchas poly(2-glucosyloxyethyl methacrylate)-concanavalin[101], polyethylenimine [102], and polyvinylferrocenium[103] are used for this purpose. However, inorganic materialsprovide a better stability because of their thermal and mechan-ical stability and non-toxicity [104]. Various enzymes havebeen immobilized on inorganic materials, such as clay [105],porous silica, and alumina powder [106]. It is well-known thatporous alumina membranes with various dimensions could beeasily fabricated via electrical anodization of high purity alu-minum. These membranes have been used to preparenanoarrays and nanowires [107–109] and biosensors[110–112].

Table 1 Some important industrial applications of immobilized enzymesystems

Enzyme EC number Substrate Product

β-Galactosidase 3.2.1.23 Lactose Lactose-free milk

Lipase 3.1.1.3 Triglycerides Cocoa butter substitutes

Nitrile hydratase 4.2.1.84 Acrylonitrile Nicotinamide

Aminoacylase 3.5.1.14 D, L-Aminoacids

L-Amino acids

Raffinase 3.2.1.22 Raffinose Galactose and sucrose

Invertase 3.2.1.26 Sucrose Glucose and fructosemixture

Thermolysin 3.4.24.27 Peptides Aspartam

Glucoamylase 3.2.1.3 Starch D-Glucose

Papain 3.4.22.2 Proteins Removal of “chill haze”in beers

Tyrosinase 4.1.99.2 Pyrocathecol L-DOPA

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Medical applications of immobilized enzymes include di-agnosis and treatment of diseases. Offering a great potential inthis area, real application of immobilized enzymes has as yetsuffered from serious problems from their toxicity to thehuman organism, allergenic and immunological reactions, aswell as from their limited stability in vivo. Examples ofpotential medical uses of immobilized enzyme systems arelisted in Table 3.

Immobilization of digestive enzymes onto solid supports

The use of immobilized enzymes finds particular advantagesusing proteolytic enzymes. Papain, a thiol protease, present inthe latex of Carica papaya exhibits a broad proteolytic activ-ity and is an enzyme of industrial use and of high researchinterest. Papain has a variety of industrial applications, espe-cially in food industry. It is used to tenderize meat and meatproducts, in the manufacture of protein hydrolysate, inbrewing industry for clarifying juice and beer, in dairy indus-try for cheese, in backing industry, and in the extraction offlavor and color compounds from plants. Papain can also be

used in forage industry to increase the utilization and trans-formation rate of proteins and in resolving plant and animalprotein to make high health products [113, 114]. The potentialuses of papain include amino acid esters and peptide synthe-sis, treatment of acute destructive lactation mastitis, treatmentof red blood cells prior to use in antibody-dependent cell-mediated cytotoxicity assays with lymphocytes, and enzymeinhibition-based biosensors for food safety and environmentalmonitoring [115–117].

Inorganic immobilization supports such as particulate alu-minum oxides (alumina) have also been examined for immo-bilization of the proteolytic enzyme, papain [118]. In thisresearch, organic phosphate linkers have been used for crea-tion of free carboxyl groups in a two-step process. Papainbinding to these alumina derivatives was performed using thewater soluble carbodiimide 1-ethyl-3-(dimethylaminopropyl)carbodiimide. It has been shown that immobilized papain hadsimilar kinetic constants compared to papain in solution. Byfluorescence measurements, it was concluded that the hydro-phobic environment of the active site remained unchanged,while the structure of the rest of the protein was perturbed byits association with the negatively charged surface [118].

Identification of proteins by liquid chromatography follow-ed by mass spectrometry (LC–MS) is normally used as asuitable analytical method. In practice, proteins are subjectedto proteolytic cleavage, and a complex mixture of peptides isproduced. The peptide mixture is then separated by high-resolution LC–MS [119–121]. Protein digestion is normallyperformed in a homogeneous aqueous solution containing theproteolytic enzyme and the sample (enzyme-to-protein ratio,1:50). However, this method suffers from several problems thatmay interfere with identification of sample proteins. Some ofthese problems include long digestion times (up to 24 h), auto-digestion by-products, and limited enzyme-to-substrate ratio.

Table 2 Some of the most frequently studied enzymes for enzyme-basedbiosensors

Enzyme EC No. Substrate Application

Acetylcholinesterase 3.1.1.7 Acetylcholine Determination ofcarbamatepesticides

Alcoholdehydrogenase

1.1.1.1 Ethanol Food science,biotechnology

Alliinase 4.4.1.4 Cysteinesulfoxide

Food industry(garlic derivatives)

Cholesterol oxidase 1.1.3.6 Cholesterol Medical applications

Choline oxidase 1.1.3.17 Choline Used withacetylcholineesterase

Glucose oxidase 1.1.3.4 Glucose Diabetes, food science,biotechnology

Glutamate oxidase 1.4.3.11 Glutamate Food science,biotechnology

Horseradishperoxidase

1.11.1.7 H2O2 Biological andindustrialapplications

Lactatedehydrogenase

1.1.1.27 Lactate Sport sciences,food sciences

Lactate oxidase 1.13.12.4 Lactate Sport sciences,food sciences

Penicillinase 3.5.2.6 Penicillins Pharmaceuticalapplications

Tyrosinase 1.14.18.1 Phenols Determination ofphenolics in food

Urease 3.5.15 Urea Medical diagnosis,artificial kidneys

Table 3 Selected potential medical uses of immobilized enzymes

Enzyme (EC number) Condition

Arginase (3.5.3.1) Cancer

Asparginase (3.5.1.1) Leukemia

Carbonic dehydratase (4.2.1.1) Artificial lungs

Catalase (1.11.1.6) Acatalasemia

Glucoamylase (3.2.1.3) Glycogen storage disease

Glucose oxidase (1.1.3.4) Artificial pancreas

Glucose-6-phosphatedehydrogenase (1.1.1.49)

Glucose-6-phosphatedehydrogenase deficiency

Heparinase (4.2.2.7) Extracorporeal therapy procedures

Phenylalanine ammonia lyase(4.3.1.5)

Phenylketonurea

Urease (3.5.1.5) Artificial kidney

Urate oxidase (1.7.3.3) Hyperuricemia

Xanthine oxidase (1.1.3.22) Lesch–Nyhan disease

200 J Chem Biol (2013) 6:185–205


Enzyme immobilization onto solid supports is a possiblealternative to in-solution digestion. Different reactive groupsof the supporting material (–OH, –NH2, and –COOH) can beutilized for covalent protein binding using relatively simplecoupling strategies [122]. These approaches include co-polymerization with polyacrylamide gels [123], binding ontomicrobeads [124], silica-based substrates [125, 126], syntheticpolymers [127], and the inner walls of open capillaries ormicrochannels in microfluidics [128, 129].

Textile industry

Another application of immobilized enzyme systems is foundin textile industry where the main advantage is their low cost.The industrial plant size needed for continuous process is twoorders of magnitude smaller than that required for batchprocess using free enzymes. The total costs are, therefore,considerably much lower. Immobilized enzymes offer greatlyincreased productivity on the basis of enzyme weight and alsooften provide process advantages [130].

Although oxidation reactions are essential in severalindustries, most of the conventional oxidation technolo-gies have the following drawbacks: non-specific or un-desirable side-reactions and use of environmentally haz-ardous chemicals. This has impelled the search for newoxidation technologies based on biological systems suchas enzymatic oxidation. These systems show the follow-ing advantages over chemical oxidation: Enzymes arespecific and biodegradable catalysts and enzyme reac-tions are carried out under mild conditions.

Enzymatic oxidation techniques have been proposed in agreat variety of industrial fields including pulp and paper,textile, and food industries. Recycling oxidizing enzymes onmolecular oxygen as electron acceptor are the most interestingones. Thus, laccases (benzenediol: oxygen oxidoreductase;EC 1.10.3.2) belong to a particularly promising class of en-zymes for the above-mentioned purposes [131]. The laccasemolecule is a dimeric or tetrameric glycoprotein, which usu-ally contains four copper atoms per monomer distributed inthree redox sites. This enzyme catalyzes the oxidation ofortho- and paradiphenols, aminophenols, polyphenols, poly-amines, lignins, and aryl diamines as well as some inorganicions coupled to the reduction of molecular dioxygen to water[132].

Awide variety of supports and methods are constantly usedfor the immobilization of various industrial enzymes, andseveral applications of laccases have been proposed. Specifi-cally, the direct substrate oxidation of phenol derivatives hasbeen investigated in bioremediation efforts to decontaminateindustrial wastewaters. The polymeric polyphenolic deriva-tives that result from the laccase-catalyzed oxidative cou-plings are usually insoluble and can be separated, easily, byfiltration or sedimentation Additionally, a laccase has been

commercialized, recently, for preparing cork stoppers for winebottles, whereby the enzyme oxidatively diminishes the char-acteristic cork taint and/or astringency that is frequentlyimparted to aged bottled wine [133].

At present, the main technological applications oflaccases are in the textile, in processes related to decol-orization of dyes and in the pulp and paper industriesfor the delignification of woody fibers, particularly dur-ing the bleaching process [134]. In most of these appli-cations, laccases are used together with a chemicalmediator [135]. Currently, all marketed laccases are offungal origin, but the recent identification and structuredetermination of a bacterial laccase may eventuallybroaden the horizon for this enzyme class. It remainsto be seen whether bacterial enzymes can be expressedat levels sufficient for their commercialization.

Conclusions

The technology of immobilized enzymes is still goingthrough a phase of evolution and maturation. Evolutionis reflected in the ever-broadening range of applications ofimmobilized enzymes. Maturation is mirrored in the de-velopment of the theory of how immobilized enzymesfunction and how the technique of immobilization is re-lated to their primary structure through the formation andconfiguration of their three dimensional structure. Therestill remains much room for the development of usefulprocesses and materials based on this hard-won under-standing. Immobilized enzymes will clearly be more wide-ly used in the future. This is just the beginning of theimmobilized enzyme technology era.

Acknowledgments We would like to thank the financial support byHormozgan Science and Technology Park.

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2 3 Enzyme immobilization: an update

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