REVIEWEnzyme immobilization: an updateAhmad Abolpour Homaei & Reyhaneh Sariri &Fabio Vianello & Roberto StevanatoReceived: 5 June 2013 / Accepted: 31 July 2013 / Published online: 29 August 2013#Springer-Verlag Berlin Heidelberg 2013Abstract Compared to free enzymes in solution, immobilizedenzymes are more robust and more resistant to environmentalchanges. Moreimportantly, theheterogeneityof theimmo-bilizedenzymesystemsallowsaneasyrecoveryofbothen-zymesandproducts,multiplere-useofenzymes,continuousoperationofenzymaticprocesses, rapidterminationofreac-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 differentapplicationsinindustry, coveringthelast twodecades. Themost recent papers, patents, andreviewsonimmobilizationstrategies and application are reviewed.KeywordsEnzyme immobilization .Biocatalyst .Enzyme reuseAbbreviationsALG 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 chromatographyKdDissociation constantKMMichaelis constantLCMS Liquid chromatographymass 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 alcoholTmTransition temperatureUSPIO Ultrasmall superparamagnetic iron oxideA. A. Homaei (*)Department of Biology, Faculty of Science, University ofHormozgan, Bandarabbas, Irane-mail: A.Homaei@Hormozgan.ac.irR. Sariri (*)Reyhaneh Sariri, Department of Microbiology, Lahijan Branch,Islamic Azad University, Lahijan, Irane-mail: sariri@guilan.ac.irF. VianelloDepartment of Comparative Biomedicine and Food Science,University of Padua, Padua, ItalyR. Stevanato (*)Department of Molecular Sciences and Nanosystems, University ofVenice, Venice, Italye-mail: rstev@unive.itJ Chem Biol (2013) 6:185205DOI 10.1007/s12154-013-0102-9IntroductionThe relationship between humans and enzymes hasevolvedover time. Evenduringhistorical times, wheretherewasnoconcept of enzymes, ancient Egypt peopleproduced beer and wine by enzymatic fermentation.After several thousand years, enzymatic studies havesignificantly progressed. Enzymes are proteins that ac-celeratemanybiochemical andchemical reactions. Theyare natural catalysts and are ubiquitous, in plants, ani-mals, andmicroorganisms, wheretheycatalyzeprocess-es that are vital to living organisms. The growing knowledgeand technique improvement about protein extraction and pu-rificationleadtotheproductionof manyenzymesat ananalytical grade purity for research and biotechnological ap-plications. Enzymes are intimately involved in a wide varietyof traditional foodprocesses, suchascheesemaking, 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. Thishasledtoestablishment of novel, tailor-madeenzymesfor completelynewapplications, whereenzymeswere not previously used.Application of enzymes in different industries is continu-ouslyincreasing, especiallyduringthelast twodecades.Applicationsofenzymesinfoodindustriesincludebaking[1], dairy products [2, 3], starch conversion [4] and beverageprocessing (beer, wine, fruit and vegetable juices). In textilesindustries,enzymeshavefoundaspecialplaceduetotheireffect on end products [5]. In industries such as pulp and papermaking [6] and detergents [7], the use of enzymes has becomean inevitable processing strategy when a perfect end product isdesired.Applicationofenzymesinmoremodernindustriesincluding biosensors is improving rapidly due to the specific-ity of enzymes which is of prime importance in biosensor [8].Manyother important industriesincludinghealthcareandpharmaceuticals[9] andchemical [10] manufacturearein-creasinglytaking advantages of thesenaturesamazing cata-lysts. During thelast fewyears, enzymeshave widely beenused in biofuels, such as biodiesel and ethanol [11].One of thebest use of enzymes in the modern life is their application intreatmentofwastesingeneral[12]andespeciallyforsolidwastes treatment [13] and waste water purification [14].In some cases, industrial applications of enzymes in organ-ic solventsarealso developed[15]. Moreover,enzymesareproducedfromrenewablerawmaterialsandarecompletelybiodegradable. In addition,themildoperatingconditionsofenzymatic processes mean that they can be operated in rela-tively simple and totally controlled equipment. In short, theyreduce environmental drawbacks of manufacturingbyStructural abstract. A summary of enzyme immobilization techniques investigated in this review work showing their advantage,disadvantages, and various applications.ImmobilizationprincipleAdvantage Disadvantage Application ReferenceDeposition on solid Retaining almost allactivityLow enzyme loading Inversion of carbohydrates [1]Adsorption on mesoporoussilicatesSupport is chemically andmechanically stableand resistant to microbialattackVariable pore size preparationin harsh conditions causingdenaturation of enzymeScaffold for mesoporouscarbon materials[2, 3]Immobilization on polyketoneby hydrogen bondsEasy immobilization, highbinding capacity,extraordinary stableimmobilizationOnly small increase inKM valueApplicable for largeenzymes, peroxidase,and amine oxidases[46]Classical covalentimmobilizationRelatively stable to hydrolysisat neutral pHEsters are unstable in aqueousconditionsImmobilization of antibodies,proteases, and oxidases[4, 5]Physical entrapment Avoid negative influence onenzyme surface, thermallyand mechanically verystableDiffusion of substrate to theenzyme is restrictedApplicable for most enzymesand antibodies,development of biosensors[6]Immobilization usingaffinity tagPossibility 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 forceMedical, biomedical fields,enzyme-replacementtherapy, ripening process[8]Immobilization onbiodegradable polymersLonger circulation in theblood streamLow entrapment efficiencies,burst release, instability ofencapsulated enzymeControl release for enzymereplacement therapy[9]186 J Chem Biol (2013) 6:185205reducing 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-storagelifeandbytheir cumbersomerecoveryandre-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 backgroundThefirst scientificobservationthat ledtothediscoveryofimmobilized enzymes was made in 1916 [1]. Itwas demon-strated that invertase exhibited the same activity when absorbedonasolid, suchascharcoal oraluminumhydroxide, at thebottomofthereactionvessel aswhenuniformlydistributedthroughout the solution. This discovery was later developed tothe currently available enzyme immobilization techniques.Early immobilization techniques provided very lowenzymeloadings, relative to available surface areas. During 1950s and1960s, different covalent methods of enzyme immobilizationweredeveloped. Continuingfrom1960s, todatemorethan5,000 publications and patents have been published on enzymeimmobilizationtechniques. Several hundredenzymeshavebeenimmobilizedindifferentformsandmorethan adozenimmobilized enzymes; for example, penicillin G acylase, in-vertase, lipases, proteases, etc. have been used as catalysts invariouslarge-scaleprocesses. Whileenzymeimmobilizationhasbeenstudiedforanumberofyears, theappearanceofrecent published research and review papers indicates a con-tinuedinterest inthisarea[1618]. AccordingtoPubMeddatabase 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-tagesoverenzymesinsolution,includingeconomicconve-nience, higher stability, andthepossibilitytobeeasilyre-movedfromthereactionmixtureleadingtopureproductisolation.Animmobilizedenzymeis, therefore,attachedtoan 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 strategiesDespite of many advantages in the use of enzymes comparedto traditional catalysts, there are few practical problems asso-ciated with their utilization in industrial applications. Enzymesaregenerallyexpensive,whichmeansthatthecostoftheirisolationandpurificationismanytimeshigherthanthatofordinarycatalysts. Beingproteininstructure, theyarealsohighly sensitive to various denaturing conditions when isolat-ed from their natural environments. Their sensitivity to pro-cessconditions,suchastemperature,pH,andsubstancesattrace 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 formfrommost of the reaction mixtures.One of the most successful methods proposed to overcomethese limitations is the use of an immobilization strategy (vandeVelde2002). Immobilizationis atechnical processinwhich enzymes are fixed to or within solid supports, creatingaheterogeneousimmobilizedenzymesystem. Immobilizedformofenzymesmimictheirnatural modeinlivingcells,wheremost of themareattachedtocellular cytoskeleton,membrane, andorganellestructures. Thesolidsupport sys-tems generally stabilize the structure of the enzymes and, as aconsequence, maintain their activities. Thus, as compared tofreeenzymesinsolution, immobilizedenzymesaremorerobust 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, rapidterminationof reactions, andgreater varietyof bioreactordesigns. Ontheother hand, comparedwithfreeenzymes,most commonlyimmobilizedenzymesshowloweractivityand, generally, higher apparent Michaelis 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]. Comparedtotheir freeforms,immobilizedenzymesaregenerallymorestableandeas-ier tohandle. Inaddition, thereactionproducts arenotcontaminatedwiththe enzyme (especiallyuseful inthefoodandpharmaceutical industries), andinthe case ofproteases, the rate of the autolysis process canbe dra-maticallyreduceduponimmobilization[22].These alterations result from structural changes introducedintotheenzymemoleculebytheappliedimmobilizationprocedureandfromthecreationofamicroenvironment inwhichtheenzymeworks, different fromthebulksolution.Theresult wouldbeapureproduct not contaminatedwithother environmental ingredients and easy to be isolated fromthe solution. The attached enzyme is then ready for the sub-sequent reactions without the needfor repeated, time-consuming, and costly extraction and purification procedures.Enzymes may be immobilized by a variety of methods, whichmaybebroadlyclassifiedasphysical, whereweakinterac-tions between support and enzyme exist, and chemical, whereJ Chem Biol (2013) 6:185205 187covalent bonds areformedbetweenthesupport andtheenzyme.Inparticular, thedevelopment andapplicationsof site-selectiveproteinimmobilizationhaveundergonesignificantadvances 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, andcontinuousflowreactorsystems.Theadvent of high-densityarrayprintingtechnologyandimproved methods for high-throughput production and puri-ficationoflargenumbersofproteinshave, inrecent years,allowed the preparation and exploitation of protein microar-rays. However, many of the applications reported have reliedonproteinattachment methodsthat result innon-specificimmobilization, either covalentlythroughamine, aldehyde,andepoxy-derivatizedsurfaces or throughadsorptiononnitrocellulose-, hydrogel-, or polylysine-coated slides. Anum-ber of moreselectiveapproacheshavebeendemonstratedemploying affinity reagents that bind specific epitopes or tagson proteins and expose them in a correctly orientated manner,suchasnickel nitrilotriaceticacid(Ni-NTA)-coatedslides,whichare usedtobindhistidine-taggedproteins andstreptavidin(oravidin).Figure1schematicallyexplainsthemechanismby whicha protein is firsttagged by twomole-culesof histidinefollowedbyinteractionwithaNi-NTAnanogold. It canbeseeninthis figurethat this typeofinteraction can only provide non-covalent attachment [3].Bioconjugationisanimportant aspect of thebiologicalsciences and critical to the applications discussed above; as aresult, manymethodshavebeendescribedthroughrecentyears. Of these, a small number have become widely appliedduetotheireaseof use, flexibility,andreagentcommercialavailability. In general, these methods rely on physicochemi-cal adsorptionphenomenaoronfunctional groupsthat arenaturallypresentinproteins.Theyare,therefore,extremelystraightforward to employ and applicable to all proteins, bothnative and modified.Modes of immobilizationTraditionally, 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 bindingcanbephysical orchemical, involving weak or covalent bonds. In general, phys-ical bonding is comparatively weak and is hardly able to keeptheenzymefixedtothecarrierunderindustrial conditions.Thesupportcanbeasyntheticresin,aninorganicpolymersuch as zeolite or silica, or a biopolymer. Entrapment involvesinclusion of an enzyme in a polymer network (gel lattice) suchasanorganicpolymer or asilicasolgel, or amembranedevice 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].Manyothermethods,whichareeitheroriginalcombina-tions of the ones listed, sometimes specific for a given supportor enzyme have been developed. However, no single methodandsupport isthebest for all enzymesandtheir variousapplications. This is because of the widely different chemicalcharacteristicsandcompositionof enzymes, thedifferentproperties of substrates and products, and the various uses oftheproduct. However, all of themethods maypresent 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 problemsareinherent inmembranereactor-confinement,entrapment, and micro-encapsulations.Protein adsorptionNon-covalent methods of protein immobilization are widelyemployed and involve either passive adsorption onto hydro-phobic surfaces or electrostatic interactions with charged sur-faces. Theuseof nitrocellulosemembranesor polylysine-Fig. 1 Selective approachesemploying affinity reagents188 J Chem Biol (2013) 6:185205coatedslidesfor electrostaticbindingisperhapsthemostwidely familiar. As noted above, the major advantage of thiskindof immobilizationisthat 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 proteinontosurfaces oftenresults inconformationalchanges 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 silicatesInthepastdecade, muchworkhasbeenundertakeninthesynthesis of mesoporous silicates and in the immobilization ofenzymesontothesesupports. In1992, theMobil researchgroup [24] discovered a family of mesoporous silicates whichhad a narrowpore size distribution, amorphous silica surfaces,and pore sizes in the range of 20300 . These new regularrepeatingmesoporousstructuresofferedthepossibilityofadsorbingorentrappinglargemoleculeswithintheirpores.Sincetheirdevelopment, thesematerialshavepromisedanimprovement about catalyticresearchof immobilizeden-zymes. It wasanticipatedthat mesoporoussilicateswouldprovide a sheltered protected environment in which reactionswith selected substrates could proceed. Following their initialdiscovery, mesoporous structures have been synthesized usingcationic, neutral, andblockcopolymer surfactants. Thesecopolymerscontainorganicfunctional groupsandmetalslocatedwithintheir frameworkor graftedonto theirsurfaceandhavebeenusedasascaffoldtodevelopmesoporouscarbon 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 solgels, display similar stability tomesoporous silicates and have been used to encapsulate pro-teinsforthedevelopmentofbiosensors. However, solgelssuffer from the disadvantage of possessing a highly variableporesizedistribution(10400). Moreimportantly, theirpreparationcaninvolvetheuseof harshconditionsor re-agents, whichcancausedenaturationof proteinsandaredetrimentaltoenzymeactivity. Usingmesoporoussilicates,protein encapsulation occurs after the synthesis of the support,avoiding this difficulty [25].Protein immobilization on polyketone polymer by hydrogenbondsAn innovative immobilization process has been recently pro-posed [46] which utilizes polyketone polymer, a completelynewsupport. Polyketonepolymer [CO-CH2CH2]n,obtained by copolymerization of ethane and carbon monox-ide, hasbeenutilizedfor immobilizationofthreedifferentFig. 2 Some methods used forenzyme immobilizationJ Chem Biol (2013) 6:185205 189enzymes, oneperoxidasefromhorseradishandtwoamineoxidasesfrombovineserumandlentil seedlings. Theeasyimmobilization procedure was carried out in diluted aqueousbuffer,gentlymixingtheproteinswiththepolymer.Nobi-functional agents or spacer arms are required for the immobi-lization, whichoccursexclusivelyviaalargenumber 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, activitymeasurementsdemonstratethat immo-bilizedenzymesretaintheir fullycatalyticcharacteristics,whereonlyasmall increaseofKMvalueisobserved. Theperoxidase activated polymer was used as active packed bedof an enzymatic reactor for continuous flow conversion andflowinjectionanalysis of hydrogenperoxidecontainingsolutions.Classical covalent immobilization methodsFor 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 oflysineresiduesreadilyreact withsupportsbearingactiveesters, with the most common being N-hydroxysuccinimide(NHS) esters, toformstableamidebonds. However, onedisadvantage of using NHS esters is that they are unstable inaqueousconditions, andthus, theattachmentofproteinsinaqueous buffers will compete with ester hydrolysis, possiblyresulting in a modest immobilization yield. As an alternative,aldehyde groups can be coupled with exposed amino groupsfollowedbyreductionusingsodiumcyanoborohydride, orother reagent, to form a stable secondary amine linkage.The nucleophilicity of the amine group also allows reactionwithepoxide-functionalizedmaterials (diglycidyl ethers).These epoxides have the advantage of being relatively stableto hydrolysis at neutral pH, which allows easy handling of thematerials but can result in slowor incomplete coupling. More-over, it is necessary to have supports with the highest degreeof epoxy groups, in conditions where they may be very stabletopermit longenzymesupport reactionsandhavingtheshortest possible spacer arms (mainly in the third generationof epoxy supports) to effectively freeze the enzyme structure.Newheterofunctional supportsor newusesof theabove-described supports may speed up the implementation of manyindustrial bioprocesses, at present hamperedbythelackofsuitable 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) toformstablethioether bonds. It hasbeenshownthat maleimidegroupsstronglyfavorconju-gate addition with thiols at physiological pH(6.57.5)since under these conditions amines are predominantlyprotonatedand un-reactive(Fig. 4). As proteinsgenerallyhave very fewsurface-exposed cysteine residues, it ispossible to achieve site-selective immobilization, espe-cially if the protein of interest can be engineered to removeall but onesurfacecysteineresidueor toinsert asinglecysteineonthesurfacewherenonepreviouslyexisted.Thenucleophilicity of the thiol group also means that it can reactwith epoxides and NHS esters, although in practical terms thislatter reactionisrelativelyslowandtheresultant 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 acarbodiimidecouplingagent andanauxiliarynucleophile.The most commonly used example of the former is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, whileNHSiswidelyusedtogeneratetheNHSesterontheprotein.Thisactive ester can then react with amine-bearing supports. Theadvantageof thiscombinationof reagentsisthat botharewater-solubleandmaybeusedinaqueousmedia,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 NHSestersformedontheproteinmoleculemaythencoupletoother protein molecules to give poorly defined polymers.Oxidative cleavage of the 1,2-diols on the oligosaccharides(usually with periodate) generates aldehyde moieties that canthenbeusedfor attachment inasemispecificmanner 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 apolysaccharidechain,randomorientationmayalsooccurifFig. 3 Hydrogen bonds between carbonyl groups and the NHgroups ofthe polypeptidic chain190 J Chem Biol (2013) 6:185205the desired protein has more than one site of glycosylation onits surface.In recent years, several selective immobilization methodsabletoproceedunder mildphysiological conditionshavereceivedincreasingattention. Typicallythesemethodsrelyon the labeling of the protein of interest with an azide moiety.IntheStaudinger ligation, thereactionof anazidewithaphosphineformsanintermediateiminophosphorane(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 exploitedin a large number ofapplicationsfromthelabelingof proteinsandcell-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 withtheazidefollowedby theimmobilization reaction.Thus, inordertoachievesite-selectiveimmobilization, amethodforselectively introducing the required azide moiety must first beemployed, suchasanenzymaticreactionthat 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, expressionof theengineeredsyntheticgenethenyieldsafusionproteinof theoriginal proteinof interestattachedtothetaggingproteinor peptidecontainingtheattachment site. This fusion protein is then used for the im-mobilization procedure.Another methodof selectiveimmobilizationisderivedfrom the Huisgen 1,3-dipolar cycloaddition of azides with analkyne, 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,whichreliesonphotoirradiationtotrigger pyrazolineformationbetweentetrazolesandalkenes[33]. Anaddedadvantage 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 entrapmentThebest meansofavoidinganynegativeinfluenceonthestructureofanenzymeistoencapsulateit. Proteinsoren-zymes can be extremely fragile and easily aggressed by ex-ternal agent such as proteases. Encapsulation of these fragilemacromoleculesisapossiblestrategyfor preventingtheiraggression and denaturation. A well-established technique toencapsulatebiologicalspeciessuchasenzymes,antibodies,Fig. 4 Reaction of maleimideforming chemical conjugation toa sulfhydrylFig. 5 The click chemistryreaction of alkyne (1) with azide(2) to form a 1,4-disubstituted1,2,3-triazolJ Chem Biol (2013) 6:185205 191and other proteins in a functional state is based on the solgelchemistry method.Solgelsaresilicamaterialsthat arehighlyporousandreadily prepared. The solgel is a chemically inert glass thatcan be shaped in any desiredway.It can be designed to bethermally and mechanically very stable, but the standard solgel isbrittle. Solgelshavebeenusedextensivelyintheimmobilization of proteins and in particular for the develop-ment of biosensors. Although solgels are porous, diffusion ofsubstrate to the enzyme can be restricted and care has to betakentominimizethiseffect. Thesynthesisof solgelsisrelativelymildfor enzymes. Inthe first step, a tetra-alkoxysilaneishydrolyzedviaacidcatalysis. Hydrolysisisfollowed by condensation where the sol is formed. This is amixture of partially hydrolyzed and partially condensed. Alltheporesofthisgelarefilledwithwaterandalcohol;itisthereforeknownasaquagel. Whentheaquagel isdriedbyevaporation, 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 suchcapillaryaction,thewaterintheaquagelcanbeexchangedwith 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,hydrophilicaqua-, xero-,andaerogels aremade.Byaddingalkyltrialkoxysilanes to the synthesis mixture, solgels with ahydrophobicsurfacecanbeobtained. Overall thesolgelmethod can generate gels with very different properties [6].Manyencapsulationmethodshavebeendeveloped, andthistaskcanbeachievedwithlipidvesicles (alsocalledliposomes), whicharepolymolecular aggregatesformedinaqueous 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 theamphiphilesformsthehydrophobicinterior of thebilayer and the hydrophilic part (the polar head group) is incontact withtheaqueousphase. Theinterior of thelipidvesiclesisanaqueouscore, thechemical compositionofwhichcorrespondsinafirstapproximationtothechemicalcomposition of the aqueous solution in which the vesicles areprepared. Dependingonthemethodof preparation, lipidvesicles can be multi-, oligo-, or unilamellar, containing many,a few, or one bilayer shell(s), respectively. The diameter of thelipidvesiclesmayvarybetweenabout 20nmandafewhundred micrometers. Lipid vesicles are generally not a ther-modynamically stablestate of amphiphiles and do notformspontaneously (without input of external energy); they areonly kinetically stable, kinetically trapped systems. The phys-ical propertiesof lipidvesiclesdependonhowandunderwhich conditions lipid vesicles of a certain amphiphile (or ofamixtureofamphiphiles)areprepared.Themeansize,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 basicprinciples for the preparationof enzyme-containing lipid vesicles in general do not so depend on thechemicalstructureoftheamphiphilesused.Careshouldbetakentothat all mechanical treatmentsusedduringvesiclepreparation, such as lipid dispersion, sonication, or filtrationthroughpolycarbonatemembranes, havetobecarriedoutabove the transition temperature (Tm). Below Tm, the saturat-ed hydrophobic chains exist predominately in a rigid, extend-edall trans conformation, similartotheircrystallinestate.Above Tm, the chains are rather disordered, making the bilay-erfluid(mechanicallytreatable), characterizedbyincreasedlateral 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 tothehighdegreeof reproducibilityof thepreparation, 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, vesiclespreparedbydehydrationrehydrationare generallynotunilamellar 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 considerFig. 6 The photoclickchemistry between tetrazolesand alkenes to form pyrazoline192 J Chem Biol (2013) 6:185205that thedehydrationrehydrationcyclemayinactivatetheenzyme. It certainly depends on the type of applications andon the cost of the enzyme, whether achieving high encapsu-lationefficienciesisimportant or not. Inthecaseof drugdelivery, e.g., high EE values are usually desired.Fromall theseconsiderationsoninteractionsof water-solubleenzymeswiththevesiclemembrane,itisclearthatthis aspect is important to be taken into account if one likes tounderstand the behavior of the entrapped enzymes. There aretwomainareasof applicationsof enzyme-containinglipidvesicles:(a)inthemedicalorbiomedicalfield,particularlyfor theenzyme-replacement therapy, or (b) inthecheeseripening 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. Inthecaseof theenzyme-containinglipidvesicle-assistedcheeseripeningprocess, theentrappedenzymeisgradually released, allowing catalyzing degradation and mod-ificationreactionsinthecheesematrixduringtheripeningperiod. 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).Theentrappedenzymemoleculeshavetobereleasedat aparticular site in the body where they are needed [8].Biodegradable polymers nanosystems are an attractive al-ternativetoliposomessincetheyhavetheadvantagesoflongercirculationinthebloodstreamandgenerallyhigherdrug 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-calparametersandcanbetailoredaccordingtothedesiredreleasepatternoftheproteintobe incorporated.ThesetwopolymerswereapprovedbyFDAasexcipientstoachievecontrolledreleaseof theactiveingredient. However, theirapplication in protein delivery systems is often characterizedbylowentrapment efficiencies, burst release, instabilityofencapsulated hydrophilic protein, and partial protein release.To improve the performance of these polymer nanoparticles,polysaccharidessuchasalginate(ALG) andchitosan(CS)could be applied. CS and its derivatives have been intensivelystudiedascarriersforproteinsanddrugs. MorespecificallythesenanoparticlescanbetotallymadebyCSor usedinseveral copolymer combinations. Copolymersmadebythecombination of CS/ALG are able to generate a more friend-lyenvironment whichprotectspeptidesandproteinsfromstressing 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 thesebasicsknowledgewilldefinitelyleadtoachangeofobjectdesigntoincreasetheproteinload, tocontrol theproteinrelease, and to retain the protein integrity and efficacy [9].Bioaffinity interactionsOver the years, a number of proteinprotein and proteinsmallmolecule binding interactions have been harnessed for immo-bilization. Thesestrategies exploit theselectivityof 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 thetagsthat havebeendescribedweredevelopedfor proteinpurificationbyaffinitychromatography, butfewhavebeenwidelyco-optedfor immobilizationinother applications.Perhapsthemost well-knowngeneticallyencodedaffinitytag 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-ing moiety such as nitrilotriacetic acid or imino-diacetic acid istreated with a solution of the relevant metal salt to produce asupportpresenting the metalions.Thismetal-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 proteinsduringpurificationandisoftentermedimmobilizedmetalaffinity chromatography (IMAC) [7].However, the general level of selectivity of this method isrelativelylowsinceseveralendogenousproteinshavebeenidentified 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 (Kd110 M), although proteins bear-ing tags with 1012 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 forpurificationthroughtheuseof columnswithimmobilizedantibodies acting to trap their epitope target and is known asimmunoaffinitychromatography. Themethodis, however,rarelyusedfor other applicationsfor several reasons. Inorder toachieveuniformimmobilization, awell-definedJ Chem Biol (2013) 6:185205 193monoclonal antibodyisneeded; polyclonal antibodiesareunsuitable since they are not a single species but a heteroge-neouspopulationofantibodiesthat bindtheir epitopeinavariety of conformations. Indeed, without in-depth structuralknowledgeof thebindinginteraction, it is impossibletodetermine strength of the binding or if the binding may blocktheactivesiteof theproteinof interest. Furthermore, therelativelyweaknatureofthebindingmeansthatitisoftenunsuitable for many applicationsthatrequire longer-termormore robust immobilization of proteins.Undoubtedlythe most well-knownandextensivelyresearched protein-mediated immobilization techniquerelieson the non-covalent interaction of either avidin or streptavidintoproteinsfunctionalizedwithbiotin[35]. Theinteractionbetween biotin and (strept) avidin is extremely strong (Kd1015M), 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.Thewidespreadavailabilityof supportssuchasmicrotiterplates, 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, theproteinofinterestmustfirstbelabeledwith biotin. Classically, this can be achieved with a number ofnonselectivechemical biotinylationreagentssuchasbiotinNHS ester [36].Oneconceptuallystraightforwardmethodofproteinim-mobilization is through the use of enzymatically active fusionproteins. Inthiscase, aproteinof interest isfusedtoanenzyme(captureprotein) that reacts selectivelywithanimmobilizedsubstrateanalogueor inhibitor. Thisreactionforms a covalent bond between the enzyme and the substrateon the surface. This strategy was reported in 2002 by Mrksich,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 acovalent bond. Thisproteinandligandwerechosenbecause they possessed a number of desirable characteristicsfor an immobilization technique. The enzyme was relativelysmall (210aminoacidresidues), globular, andmonomeric,which would minimize any steric interactions with the fusedprotein.Boththeterminiareoppositetotheactivesiteandhence 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, andwhenboundinthecutinaseactivesite,thealkyltailofthephosphonateprotrudedout of theenzymeandofferedanaccessible location for attachment to the support.There are also a number of other site-selective immobili-zationmethodsthat donot requireenzymaticallymediatedattachment. 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].Suchdityrosinecrosslinksoccurinstructuralproteins suchas elastinandsilkandare catalyzedbymetalloenzymes, althoughasimplecomplexofNi(II)withthe tripeptide glycineglycinehistidine can also catalyze the-se reactions. Since IMAC and immobilization with Ni(II) viasix-histidinetagsandNTA-functionalizedsupportsareal-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 immobilizedandanother fromthesupport, intocloseproximitytotheNi(II) complex, it wouldbepossibletocross-linkthetwoand form a covalent bond between the support and a specificlocationontheprotein. Oncethecross-linkinghadbeenachieved,thenickelwasremoved.Theextentofdityrosinecross-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-ablefor site-specificproteinimmobilization. Thisareaofresearchisnotablebecauseitistheculminationofamajormultidisciplinary research effort spanning molecular biology,protein engineering, organic chemistry, and materials science.The more recent introductionof biologicallymediatedmethods for site-specific, particularly enzyme-mediated, im-mobilizationofproteinsfromcelllysatesshouldenablethefuture fabrication of more highly sensitive protein microarraysand biosensors as wells as finding new applications in nano-technology and single molecule enzymology.Support materialsThe properties of supportedenzyme preparations aregoverned by the properties of both the enzyme and the carriermaterial. The interactionbetweenthe twoprovides animmobilizedenzymewithspecificchemical, 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 polymersThe most common and widely used synthetic polymers usedassupport for enzymeimmobilizationarerepresentedby194 J Chem Biol (2013) 6:185205acrylic 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 around170mmandaporediameterofabout 2030nm(Fig. 7).They are highly hydrophilic and stable, both chemically andmechanically, overapHrangefrom0to14, anddoesnotswell or shrinkevenupondrastic pHchanges. Amajordrawbackofhydrophilicresinsisdiffusionlimitations, theeffects of which, as would be expected, are more pro-nouncedinkineticallycontrolledprocesses. Immobilizationbycovalent attachment toacrylicresinshasbeensuccess-fullyappliedtoavarietyofenzymesforindustrialapplica-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-catalyzedcopolymerizationofethaneandcarbonmonoxide[46]. Duetotheclosepresenceof polar ketogroups, 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 possibilityofhydrogenbondsbetweentheketogroupsandthewatermolecules and no restriction due to cross linking are present.Wateraccessiblecarbonyl groupswerequantifiedinabout10meq/gpolymer, andtheapparent concentrationof theimmobilizedenzymeonthepolyketonewas morethan2.36 mg/ml [46].BiopolymersAvariety of biopolymers, mainly water-insoluble polysaccha-rides such as cellulose, starch, agarose, carragenans, and chi-tosan,havebeenwidelyusedassupportsforimmobilizingenzymes(Fig. 8). Thesematrixesformveryinert 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 proteinsandenzymesinareversibleorirreversibleway. Likewise,with the use of bi-functionalreagents, it is possible toformcross-linkswhichstrengthentheirstructureincreasingtheirmechanical andthermal resistance. Duetotheseparticularcharacteristics, biopolymers are resins which are almost idealto be used in purification (reversible binding) and in immobi-lization processes (irreversible binding) of biomolecules. Cur-renttechniquestoconjugatepolysaccharidestoproteinsin-clude aldehyde, carbodiimide, epoxide, hydrazide, active es-ter, radical, and addition reactions, some of which are suitableforinsituconjugationofmaterials, whileothersarebettersuited for ex situ conjugation and purification.HydrogelsEnzymescanalsobeimmobilizedinnatural 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,candiffuseout of thegel matrixandare, consequently,leachedinanaqueous medium. Inorder toentrapfreeFig. 7 Structure ofpolyacrylamide gel matrixJ Chem Biol (2013) 6:185205 195enzymes, the size of the enzyme must be increased, e.g., bycrosslinking. Analternativemethodtoincreasethesizeofthe enzyme is toforma complexwitha polyelectrolyte.Owing to their ampholytic character, proteins exist aspolycations or polyanions, depending on the pHof themedium. Hence, theycanoftenformcomplexeswithop-positelychargedpolyelectrolytes.Inorganic supportsOn the other hand, rigid supports may be preferable. Avarietyof inorganicsolidscanbeusedfor theimmobilizationofenzymes, e.g.,alumina,silica,zeolites,andmesoporous sil-icas [49]. Silica-based supports are the most suitable matricesfor enzymeimmobilizationinindustrial manufacturingofenzyme-processedproducts[18, 50, 51], aswell asforre-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 [5355]. Additionally, the surface ofsilicagel canbereadilymodifiedbychemical methodstoprovidevarioustypesof functional groupsfor facilitatedligand attachment. It has been shown that an extremely highloading of invertase was achieved by chemical modificationofgel surfaceusingaminogroup[56]. Theoverall surfaceareaofferedbysilicagel matrixesisunparalleled. Further-more, the silica gel can be easily fabricated to provide desir-able morphology, pore structures, and micro-channels to allowsubstrateligandinteraction. Furthermore, silicagel isme-chanicallystableandchemicallyinert, andit isthereforeenvironmental- and solvent-friendly for industrialmanufacturing and processing.Oneof thesimplest andmost inexpensivemethodstoimmobilize an enzyme on silica is by simple absorption. It isused, for example, to formulate enzymes for detergent pow-ders which release the enzyme into the washing liquid duringwashing.Smart polymersAnovel approachtoimmobilizeenzymesisviacovalentattachment tostimulus-responsiveor mart polymer, whichundergodramaticconformational changesinresponsetosmall changesintheir environment, e.g., temperature, pH,andionicstrength[57]. Themost studiedexampleisthethermo-responsiveandbiocompatiblepolymer, poly-N-isopropylacrylamide(polyNIPAM) [58]. Aqueoussolutionsof 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 toexpulsionof water moleculesfromthepolymer 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. Anadditional advantage of usingsuchthermo-responsiveimmobilizedenzymesisthatrunawayconditionsare avoided because when the reaction temperature exceeds theLCST,thecatalystprecipitatesandthereaction shutsdown.TwomethodsaregenerallyusedtopreparetheenzymepolyNIPAM conjugates: (a) introduction of polymerizable vi-nyl groups into the enzyme followed by copolymerization withNIPAMor (b) reaction of NH2 groups on the surface of theenzyme with a copolymer of NIPAM containing reactive estergroups or the homopolymer containing an N-succinimide esterfunctionas theendgroup. Morerecently, analternativethermo-responsivepolymer hasbeendescribed[5961]. Itconsists of randomcopolymers derived from2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol)methacrylate and combines the positive features of poly(ethyl-eneglycol), non-toxicity, andanti-immunogenicity, withthermo-responsivepropertiessimilar topolyNIPAM. TheLCST couldbe variedbetween26 and 90Cdependingonthe relative amounts of OEGMA used. These properties makethis a potentially interesting support for biocatalysts.Conducting polymersPolymers that exhibit electrical conductivity have now beensuccessfullysynthesized, andthepast twodecades 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, areFig. 8 Chemical structures of cellulose and its amino derivatives196 J Chem Biol (2013) 6:185205roughlydividedintothreegroups, i.e., chargetransfercomplexes/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 conductingisthat thereshouldbeanoverlapof molecular orbitalstoallow 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 conductingpolymers havebeenconsideredforimmobilization of enzymes. Of these, polypyrrole hasgainedmost interest for the entrapment of proteinmole-culesduetoitslowoxidationpotential. Thisuniquechar-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 filmby potentiostatic orgalvanostaticpolymerizationofpyrroleinenzymesolution,whichcontainsasupportingelectrolyte.Conductingpolymerscanbepreparedbychemical orelectrochemical polymerization. Although the chemical meth-odoffersmassproductionatareasonablecost, theelectro-chemical method involves the direct formation of conductingpolymer thinfilms withbetter control of thickness andmorphology, which are more suitable for direct application.Itisestablished thattheconductingpropertiesofthepoly-mer films greatly depend on the mode of synthesis and alsoonthenumberofparameters, suchastypeofcounter-ion,the type of electrolyte and its concentration, synthesistemperature, electrochemical voltage, pH of the electrolyte,etc. Thus, inorder toimprovetheproperties of polymerfilmssuitablefor adesiredapplication, it isnecessarytocritically control and optimize the various synthesis param-eters[63].The other electrochemically prepared conducting polymersusedforthebiomoleculeimmobilizationarepolyacetylene,polythiophene, polyindole, and polyaniline. Also few biosen-sors based on insulating electropolymerized films polyphenol,poly(o-phenylenediamine), polydichlorophenolindophenol,andoveroxidizedpolypyrrolehavealsobeenelaborated[64].Many theoreticalmodelshave alsobeen coupledwiththe electrochemical entrapment of enzyme for the evaluationof theroleof thethicknessof polymericlayers, enzymelocation, enzymeloading,etc.onthebiosensorfunctioning[65].Gold nanoparticlesThe progress of nanotechnology in the 1990s was followed bythe rapid growth of nanobiotechnology. Nanobiocatalysis isonetypical example. Intheearlyapproachestonanobio-catalysis, enzymeswereimmobilizedonvariousnanostruc-tured materials using conventional approaches, such as simpleadsorption and covalent attachment. This approach gatheredattention by immobilizing enzymes onto a high surface area ofnanostructuredmaterials, suchas nanoporous materials,electrospunnanofibers, andmagneticnanoparticles. Thislargesurfacearearesultedinimprovedenzymeloading,which in turn increased the apparent enzyme activity per unitmass or volume comparedtothat 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, thicknessof nanofibers or nanotubes, andtheparticle 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-nologyandledtoimprovedenzymepropertiesinnanobio-catalytic systems, particularly with regard to enzyme stabilityandactivity. Recently, nanobiocatalyticapproaches 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].Nanosizedparticles of noblemetals, especiallygoldnanoparticles(AuNPs), havereceivedgreatinterestsduetotheirattractiveelectronic, optical, andthermalpropertiesaswellascatalyticpropertiesandpotentialapplicationsinthefields of physics, chemistry, biology, medicine, and materialscience. Therefore, thesynthesis andcharacterizationofAuNPshaveattractedconsiderableattentionfrombothafundamental andapractical point of view. Avarietyofmethods have been developed to prepare AuNPs [67]. How-ever, the high surface energy of AuNPs makes themextremelyreactive, 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 metalsalts in the presence of astabilizer which binds to their surface to impart high stabilityandadesirablelinkingchemistryandprovidethedesiredcharge and solubility properties. Some of the commonly usedmethodsfor surfacepassivationincludeprotectionbyself-assembledmonolayers, themost popular beingcitrateandthiol-functionalized organics; encapsulation in the H2O poolsof reverse microemulsions; and dispersion in polymeric ma-trixes (Fig. 9). Although the synthesis of AuNPs makes greatJ Chem Biol (2013) 6:185205 197progress, thecontrol ofsize, monodispersion, morphology,andsurfacechemistryof AuNPsisstill agreat challenge.Recently, designingnovel protectorsforAuNPshavebeenthefocusofintenseresearchbecausesurfacechemistryofAuNPs will play a key role in future application fieldssuchas nanosensor, biosensor, catalysis, nanodevice, andnanoelectrochemistry.Magnetic nanoparticlesIn 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 fortheefficient separationofmicro-andnanometer 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-spondingbulkmaterial. Forexample, magneticanisotropy,which keeps a particle magnetized in a specific direction, isgenerally proportional to the volume of a particle [68]. Mag-neticnanoparticleswilldisplaysuperparamagnetism, 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 removedfromsolutionusinganexternal magnet, but theydonotagglomerate(i.e.,theystay suspended)afterremovaloftheexternal magnetic field.Amongmagneticcarriers, nano-sizedmagneticparticlespossess manyadvantageous properties. Particles suchascrosslinked iron oxide [69, 70], ultrasmall superparamagneticironoxide[71, 72], andmonocrystallineironoxidenano-particles [73,74]hadallbeen developed as imagingagentsin magnetic resonance imaging. Magnetic particles have beenused to immobilize enzymes in order to enhance bioelementstability, easeseparationfromthereactionmixture, andin-creasecatalyst stability, andtheyhavebeenproposedforbiotechnologicalapplications[75]orforanalytical 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 accumulationincancer tissues. Specifictargetingofmagneticnanoparticlestotumorshasbeenaccomplishedinvarious experimental systems [78].There have been various methods developed for the prep-arationofparamagneticnanoparticles[79]. Themost com-monly used protocol involves co-precipitation of ferrous andferricionsinbasicsolutions. Thedevelopment ofuniformnanometersizedparticleshasbeenintensivelypursuedbe-cause of their technological and fundamental scientific impor-tance. These nanoparticular materials often exhibit very inter-estingelectrical,optical,magnetic,andchemicalproperties,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 aftertheformationof ironoxideparticles[80, 81]. Otherwise,magneticnanoparticlescanbecoatedwithsilicacontainingahighcoverageof silanol groups, whichcaneasilybeFig. 9 Porphyringoldnanoparticle (AuNP)198 J Chem Biol (2013) 6:185205anchoredwithdefinedandgenericsurfacechemistries[82,83]. As aresult, magneticnanoparticles coatedwithanultrathinlayerofsilicawouldbemoreuseful asmagneticnanocarrier [84].Cross-linked enzyme aggregates (CLEAs)Cross-linkedenzymecrystals(CLECs) arehighlyactiveimmobilizedenzymesofcontrollableparticlesize, varyingfrom1to100m. Theiroperational stabilityandeaseofrecycling, coupledwiththeir highcatalyst andvolumetricproductivities, renders them ideally suited for industrial bio-transformations. However, CLECshaveaninherent disad-vantage: Enzymecrystallizationisalaboriousprocedurerequiring enzyme of high purity, leading to their high costs.The more recently developed cross-linked enzyme aggregates(CLEAs), on the other hand, are produced by simple precip-itationof theenzymefromaqueoussolution, asphysicalaggregates of protein molecules, by the addition of salts, orwatermiscibleorganicsolventsornon-ionicpolymers[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 insolublewhilemaintainingtheir pre-organizedsuperstructureandhence their catalytic activity. This discovery led to the devel-opmentofanewfamilyofimmobilizedenzymes:CLEAs.This type of immobilized enzyme is very effectivebiocatalystsastheycanbeproducedbyinexpensiveandeffectivemethod.CLEAscanreadilybereusedandexhibitsatisfactorystabilityandperformancefor selectedapplica-tions.Themethodologyisapplicabletoessentiallyanyen-zyme, including cofactor dependent oxidoreductases [40, 41].Theuseof CLEAstopenicillinacylaseusedinantibioticsynthesishasshownlarge improvements over othertype ofbiocatalysts [86].Selected applications of immobilized enzymesVarious applications of immobilized enzymes can be found inindustry, medicine, andresearch. Table1hasgatheredaselectionof currentlyusedimmobilized-enzymeprocesses.Inanycommercial application, thechoiceof aspecificimmobilizedenzymeor modeof immobilizationmust bebasedonaspecificcompromiseabout all advantagesanddisadvantages between free and immobilized enzyme. Otherindustrial applications of immobilizedenzymes includelaboratory-scale organic synthesis and analytical and medicalapplications [87, 88]. Furthermore, since enzymes are able tocatalyzereactionsnotonlyinaqueoussolutionsbutalsoinorganicmedia, immobilizedenzymescancatalyzeorganicsynthesis [89].BiosensorsInrecent analytical applications, immobilizedenzymesareused mainly in biosensors [90]andin diagnostic teststrips.Biosensors are constructed by integrating biological sensingsystems, e.g., enzyme (s), with a transducer. Enzymes for themost cases are immobilized either directly on a transducersworking tip or using a polymer membrane tightly wrapping itup. Inprinciple, duetoenzymespecificityandsensitivity,biosensors canbetailoredfor nearlyanytarget analyte[9194], and these can be both enzyme substrates and enzymeinhibitors. Advantageously, their determination is performedwithout specialpreparationofthesample. Themostexten-sively studiedenzymesfortheapplicationinenzyme-basedbiosensorsarepresentedinTable2. Practically, fourofthesensorslisted,namelyglucose,lactate,urea,andglutamate,havebeenwidelyusedandcommercialized[90]. Recentadvances were reviewed [9597].Enzymebiosensorsaremadebyimmobilizationof en-zymes on the sensing surface [98100]. Due to the presenceof reactive functional groups, organic polymeric carriers suchas poly(2-glucosyloxyethyl methacrylate)-concanavalin[101], polyethylenimine[102], andpolyvinylferrocenium[103] are used for this purpose. However, inorganic materialsprovide a better stability because of their thermal and mechan-ical stabilityandnon-toxicity[104]. Variousenzymeshavebeen 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 beenusedtopreparenanoarrays and nanowires [107109] and biosensors[ 110112] .Table 1 Some important industrial applications of immobilized enzymesystemsEnzyme EC number Substrate Product-Galactosidase 3.2.1.23 Lactose Lactose-free milkLipase 3.1.1.3 Triglycerides Cocoa butter substitutesNitrile hydratase 4.2.1.84 Acrylonitrile NicotinamideAminoacylase 3.5.1.14D,L-AminoacidsL-Amino acidsRaffinase 3.2.1.22 Raffinose Galactose and sucroseInvertase 3.2.1.26 Sucrose Glucose and fructosemixtureThermolysin 3.4.24.27 Peptides AspartamGlucoamylase 3.2.1.3 StarchD-GlucosePapain 3.4.22.2 Proteins Removal of chill hazein beersTyrosinase 4.1.99.2 PyrocathecolL-DOPAJ Chem Biol (2013) 6:185205 199Medical applications of immobilized enzymes include di-agnosis and treatment of diseases. Offering a great potential inthis area, real application of immobilized enzymes has as yetsufferedfromseriousproblemsfromtheir toxicitytothehuman organism, allergenic and immunological reactions, aswell as fromtheir limitedstabilityinvivo. Examplesofpotential medical usesofimmobilizedenzymesystemsarelisted in Table 3.Immobilization of digestive enzymes onto solid supportsThe use of immobilized enzymes finds particular advantagesusing proteolytic enzymes. Papain, a thiol protease, present inthe latex of Carica papayaexhibits a broad proteolytic activ-ityandisanenzymeofindustrialuseandofhighresearchinterest. Papain has a variety of industrial applications, espe-cially in food industry. It is used to tenderize meat and meatproducts, inthemanufactureof proteinhydrolysate, inbrewing industry for clarifying juice and beer, in dairy indus-tryforcheese,inbackingindustry,andintheextractionofflavor and color compounds from plants. Papain can also beused 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, treatmentofredbloodcellsprior touseinantibody-dependent cell-mediated cytotoxicity assays with lymphocytes, and enzymeinhibition-based biosensors for food safety and environmentalmonitoring [115117].Inorganic immobilization supports such as particulate alu-minum oxides (alumina) have also been examined for immo-bilizationof theproteolyticenzyme, papain[118]. Inthisresearch, organic phosphate linkers have been used for crea-tionoffreecarboxyl groupsinatwo-stepprocess. Papainbinding to these alumina derivatives was performed using thewater solublecarbodiimide1-ethyl-3-(dimethylaminopropyl)carbodiimide. It has been shown that immobilized papain hadsimilarkineticconstantscomparedtopapaininsolution.Byfluorescence measurements, it was concluded that the hydro-phobicenvironment of theactivesiteremainedunchanged,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-edbymassspectrometry(LCMS) isnormallyusedasasuitable analytical method. In practice, proteins are subjectedto proteolytic cleavage, and a complex mixture of peptides isproduced. Thepeptidemixtureisthenseparatedbyhigh-resolutionLCMS[119121]. Proteindigestionisnormallyperformed in a homogeneous aqueous solution containing theproteolyticenzymeandthesample(enzyme-to-proteinratio,1:50). However, this method suffers fromseveral 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-basedbiosensorsEnzyme EC No. Substrate ApplicationAcetylcholinesterase 3.1.1.7 Acetylcholine Determination ofcarbamatepesticidesAlcoholdehydrogenase1.1.1.1 Ethanol Food science,biotechnologyAlliinase 4.4.1.4 CysteinesulfoxideFood industry(garlic derivatives)Cholesterol oxidase 1.1.3.6 Cholesterol Medical applicationsCholine oxidase 1.1.3.17 Choline Used withacetylcholineesteraseGlucose oxidase 1.1.3.4 Glucose Diabetes, food science,biotechnologyGlutamate oxidase 1.4.3.11 Glutamate Food science,biotechnologyHorseradishperoxidase1.11.1.7 H2O2Biological andindustrialapplicationsLactatedehydrogenase1.1.1.27 Lactate Sport sciences,food sciencesLactate oxidase 1.13.12.4 Lactate Sport sciences,food sciencesPenicillinase 3.5.2.6 Penicillins PharmaceuticalapplicationsTyrosinase 1.14.18.1 Phenols Determination ofphenolics in foodUrease 3.5.15 Urea Medical diagnosis,artificial kidneysTable 3 Selected potential medical uses of immobilized enzymesEnzyme (EC number) ConditionArginase (3.5.3.1) CancerAsparginase (3.5.1.1) LeukemiaCarbonic dehydratase (4.2.1.1) Artificial lungsCatalase (1.11.1.6) AcatalasemiaGlucoamylase (3.2.1.3) Glycogen storage diseaseGlucose oxidase (1.1.3.4) Artificial pancreasGlucose-6-phosphatedehydrogenase (1.1.1.49)Glucose-6-phosphatedehydrogenase deficiencyHeparinase (4.2.2.7) Extracorporeal therapy proceduresPhenylalanine ammonia lyase(4.3.1.5)PhenylketonureaUrease (3.5.1.5) Artificial kidneyUrate oxidase (1.7.3.3) HyperuricemiaXanthine oxidase (1.1.3.22) LeschNyhan disease200 J Chem Biol (2013) 6:185205Enzymeimmobilization ontosolidsupportsis apossiblealternative to in-solution digestion. Different reactive groupsof the supporting material (OH, NH2, and COOH) can beutilizedforcovalentproteinbindingusingrelativelysimplecouplingstrategies [122]. Theseapproaches includeco-polymerization with polyacrylamide gels [123], binding ontomicrobeads [124], silica-based substrates [125, 126], syntheticpolymers[127], andtheinner wallsof opencapillariesormicrochannels in microfluidics [128, 129].Textile industryAnother 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 twoordersof magnitudesmaller thanthat requiredfor batchprocessusingfreeenzymes. Thetotal costsare, 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 theconventional oxidationtechnolo-gies have the followingdrawbacks: non-specific or un-desirableside-reactionsanduseof environmentallyhaz-ardous chemicals. This has impelledthesearchfor newoxidationtechnologiesbasedonbiological systemssuchasenzymaticoxidation. Thesesystemsshowthefollow-ing advantages over chemical oxidation: Enzymes arespecific and biodegradable catalysts and enzyme reac-tions arecarriedout under mildconditions.Enzymatic oxidation techniques have been proposed in agreat varietyof industrial fieldsincludingpulpandpaper,textile, and food industries. Recycling oxidizing enzymes onmolecular oxygen as electron acceptor are the most interestingones. Thus, laccases(benzenediol: oxygenoxidoreductase;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 atomspermonomer distributedinthreeredoxsites. Thisenzymecatalyzestheoxidationofortho- 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 theimmobilizationof variousindustrial 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. Thepolymericpolyphenolicderiva-tivesthat result fromthelaccase-catalyzedoxidativecou-plings are usually insoluble and can be separated, easily, byfiltrationorsedimentationAdditionally, alaccasehasbeencommercialized, recently, for preparing cork stoppers for winebottles, whereby the enzyme oxidatively diminishes the char-acteristiccorktaint and/or astringencythat is frequentlyimparted to aged bottled wine [133].At present, the main technological applications oflaccasesareinthetextile, inprocessesrelatedtodecol-orization of dyes and in the pulp and paper industriesfor thedelignificationof woodyfibers, particularlydur-ingthebleachingprocess[134]. Inmost of theseappli-cations, laccases are used together with a chemicalmediator [135]. Currently, all marketed laccases are offungal origin, but therecent identificationandstructuredetermination of a bacterial laccase may eventuallybroaden the horizon for this enzyme class. It remainstobeseenwhether bacterial enzymes canbeexpressedat levels sufficient for their commercialization.ConclusionsThe technology of immobilized enzymes is still goingthrough a phase of evolution and maturation. Evolutionisreflectedintheever-broadeningrangeofapplicationsofimmobilized enzymes. Maturation is mirrored in the de-velopment of the theory of howimmobilized enzymesfunctionand howthe technique of immobilization is re-latedtotheir primarystructurethroughtheformationandconfiguration of their three dimensional structure. Therestill remains much roomfor the development of usefulprocesses and materials based on this hard-won under-standing. Immobilizedenzymeswill clearlybemorewide-ly used in the future. This is just the beginning of theimmobilizedenzymetechnologyera.Acknowledgments Wewouldliketothankthefinancial supportbyHormozgan Science and Technology Park.References1. Gomes-Ruffi CR, Henrique da Cunha R, Almeida EL, Chang YK,SteelKJ (2012)Effect of the emulsifiersodium stearoyl lactylateand of the enzymemaltogenic amylase on the quality of pan breadduring storage. LWT- Food Sci Technol 49(1):961012. JarosD, RohmH(2011)EnzymesExogenoustoMilkinDairyTechnologyTransglutaminaseEncyclopediaof DairySciences(Second Edition) 2973003. IsmailB, NielsenSS(2010)Invitedreview:plasminproteaseinmilk: current knowledge and relevance to dairy industry. J Dairy Sci93(11):499950094. Bai Y, Huang H, Meng K, Shi P, Yang P, Luo H, Luo C, Feng Y,Zhang W, Yao B (2012) Identification of an acidic -amylase fromAlicyclobacillussp. A4andassessment ofitsapplicationinthestarch industry. Food Chem 113(4):14731478J Chem Biol (2013) 6:185205 2015. Schckel J, MaturaA, vanPeKH(2011)One-copperlaccase-related enzyme from Marasmius sp.: purification, characterizationand bleaching of textile dyes. Enzyme Microb Technol 48(3):2782846. HakalaTK, LiitiT, SuurnkkiA(2013)Enzyme-aidedalkalineextraction of oligosaccharides and polymeric xylan from hardwoodkraft pulp. Carbohydr Polym 93(1):1021087. Rao CS, Sathish T, Ravichandra P, Prakasham RS (2009) Charac-terizationof thermo- anddetergent stableserineproteasefromisolated Bacillus circulans and evaluation of eco-friendly applica-tions. Process Biochem 44(3):2622688. SoldatkinOO, KucherenkoIS, Pyeshkova VM, Kukla AL,Jaffrezic-RenaultN,El'skayaAV,DzyadevychSV,SoldatkinAP(2012) Novel conductometricbiosensor basedonthree-enzymesystemfor selective determination of heavy metal ions.Bioelectrochemistry 83:25309. Apetrei IM, Rodriguez-Mendez ML, Apetrei C, de Saja JA (2013)Enzymesensorbasedoncarbonnanotubes/cobalt(II)phthalocya-nine and tyrosinase used in pharmaceutical analysis. Sens ActuatorsB 177:13814410. Das R, Ghosh S, Bhattacharje C (2012) Enzyme membrane reactorinisolationof antioxidativepeptidesfromoil industrywaste: acomparison with non-peptidic antioxidants. LWT- Food Sci Technol47(2):23824511. Atadashi IM, Aroua MK, Abdul Aziz A (2010) High quality bio-dieselanditsdieselengineapplication:areview. RenewSustainEnergy Rev 14:1999200812. Luo K, Yang Q, Yu J, Li XM, Yang GJ, Xie BX, Yang F, Zheng W,Zeng GM (2011) Combined effect of sodium dodecyl sulfate andenzymeonwasteactivatedsludgehydrolysisandacidification.Bioresour Technol 102(14):7103711013. Tonini D, Astrup T (2012) Life-cycle assessment of a waste refineryprocessforenzymatictreatment ofmunicipal solidwaste. WasteManag 32(1):16517614. Tong Z, Qingxiang Z, Hui H, Qin L, Yi Z (1997) Removal of toxicphenol and 4-chlorophenol from waste water by horseradish perox-idase 34(4):89390315. GuptaA, KhareSK(2009) Enzymesfromsolvent-tolerant mi-crobes: useful biocatalysts for non-aqueous enzymology. Crit RevBiotechnol 29:445416. Hartmann M(2005) Ordered mesoporous materials forbioadsorption and biocatalysis. Chem Mater 17:4577459317. Kallenberg AI, van Rantwijk F, Sheldon RA (2005) Immobilizationof penicillin Gacylase: the key to optimumperformance. Adv SynthCatal 347:90592618. Pierre AC (2004) The solgel encapsulation of enzymes. BiocatalBiotransfor 22:14517019. CherryJR, FidantsefAL(2003)Directedevolutionofindustrialenzymes: an update. Curr Opin Biotechnol 14:43844320. Kress J, Zanaletti R, Amour A, Ladlow M, Frey JG, Bradley M(2002)Enzymeaccessibilityandsolidsupports: whichmolec-ularweightenzymescanbeusedonsolidsupports?Aninves-tigation using confocal Raman microscopy. ChemEur J8:3769377221. Bommarius AS, Riebel BR (2004) Biocatalysis: fundamentals andapplications. Wiley-VCH, Weinheim22. Massolini G, Calleri E (2005) Immobilized trypsin systems coupledon-line to separation methods: recent developments and analyticalapplications. J Sep Sci 28:72123. Cao L, van Langen L, Sheldon RA (2003) Immobilised enzymes:carrier-bound of carrier-free? Curr Opin Biotechnol 14:38739424. Beck JS, Vartuli JC, Roth WJ, Leonowicz ME, Kresge CT, SchmittKD, Chu CT-W, Olson DH, Sheppard EW, McCullen SB, HigginsJB, Schlenkert JL (1992) A new family of mesoporous molecularsievespreparedwithliquidcrystal templates. JAmChemSoc114:108341084325. Deere J, Magner E, Wall JG, Hodnett BK (2002) Mechanistic andstructural features of protein adsorption onto mesoporous silicates. JPhys Chem B 106:7340734726. Cang-RongJT, PastorinG(2009) The influence of carbonnanotubes on enzyme activity and structure: investigation of differ-ent immobilization procedures through enzyme kinetics and circu-lar dichroism studies. Nanotechnology 20:12027. Khn M,Breinbauer R (2004) The Staudinger ligationa gifttochemical biology. Angew Chem 43:3106311628. SoellnerMB, DicksonKA, NilssonBL, RainesRT(2003)Site-specific protein immobilization by Staudinger ligation. J Am ChemSoc 125:117901179129. KaliaJ, Abbott NL, RainesRT(2007)General methodforsite-specific protein immobilization by Staudinger ligation. BioconjugChem 18:1064106930. Kalia J, Raines RT (2006) Reactivity of intein thioesters: appendinga functional group to a protein. Chembiochem 7:1375138331. Moses JE, Moorhouse AD(2007) The growing applications of clickchemistry. Chem Soc Rev 36:1249126232. TronGC, Pirali T, BillingtonRA, CanonicoPL, Sorba G,Genazzani AA(2008) Click chemistry reactions in medicinal chem-istry: applications of the 1,3-dipolar cycloaddition between azidesand alkynes. Med Res Rev 28:27830833. Song W, Wang Y, Qu J, Lin Q (2008) Selective functionalization ofageneticallyencodedalkene-containingproteinviaphotoclickchemistry in bacterial cells. J Am Chem Soc 130:9654965534. LauerSA,NolanJP(2002)DevelopmentandcharacterizationofNi-NTA-bearing microspheres. Cytometry 48:13614535. Hutsell SQ, KimpleRJ, Siderovski DP, WillardFS, KimpleAJ(2010)High-affinityimmobilizationof proteinsusingbiotin-andGST-based coupling strategies. Methods Mol Biol 627:759036. ChattopadhayaS, AbuBakarFB,YaoSQ(2009)Expandingthechemicalbiologiststoolkit:chemicallabellingstrategiesanditsapplications. Curr Med Chem 16:4527454337. Hodneland CD, Lee YS, Min DH, Mrksich M(2002) Selective immo-bilizationofproteinstoself-assembledmonolayerspresentingactivesite-directed capture ligands. Proc Natl Acad Sci 99:5048505238. Stayner RS, MinDJ, Kiser PF, Stewart RJ(2005) Site-specificcross-linkingof proteins throughtyrosinehexahistidinetags.Bioconjug Chem 16:1617162339. Endrizzi BJ, Huang G, Kiser PF, Stewart RJ (2006) Specific cova-lent immobilizationof proteinsthroughdityrosinecross-links.Langmuir 22:113051131040. Sheldon RA(2007) Enzyme immobilization: the quest for optimumperformance. Adv Synth Catal 349:1289130741. Sheldon RA (2007) Cross-linked enzyme aggregates (CLEAs): sta-ble and recyclable biocatalysts. Biochem Soc Trans 35:1583158742. Girelli AM, MatteiE(2005) Application of immobilized enzymereactorinon-linehighperformanceliquidchromatography:are-view. J Chromatogr B 819:31643. Sakaguchi K, Matsui M, Mizukami F (2005) Applications of zeoliteinorganic composites in biotechnology: current state and perspec-tives. Appl Microbiol Biotechnol 67:30631144. BollerT,MeierC, MenzlerS(2002)EUPERGIT oxiraneacrylicbeads: how to make enzymes fit for biocatalysis. Org Process ResDev 6:50951945. Katchalski-Katzir E, Kraemer DM(2000) Eupergit C, a carrier forimmobilizationof enzymesof industrialpotential.JMolCatalBEnzym 10:15717646. AgostinelliE, Belli F,TemperaG, MuraA,FlorisG, TonioloL,Vavasori A, Fabris, Momo F, Stevanato R (2007) Polyketone poly-mer: a new support for direct enzyme immobilization. J Biotechnol127:67067847. vandeVeldeF, LourenoND, PinheiroHM, BakkerM(2002)Carrageenan: a food-grade and biocompatible support for immobi-lisation techniques. Adv Synth Catal 344:815835202 J Chem Biol (2013) 6:18520548. Tripathi A, Sami H, Jain SR, Viloria-Cols M, Zhuravleva N, NilssonG, Jungvid H, Kumar A (2010) Improved bio-catalytic conversionbynovelimmobilizationprocessusingcryogelbeadstoincreasesolvent production. Enzym Microb Technol 47:445149. HudsonS, CooneyJ, MagnerE(2008)Proteinsinmesoporoussilicates. Angew Chem 47:8582859450. BlancoRM, Terreros P, Fernandez-PerezM, OteroC, Diaz-Gonzalez G (2004) Fictionalization of mesoporous silica for lipaseimmobilizationcharacterization of the support and the catalysts. JMol Catal B Enzym 30:839351. HoLF, Li SY, LinSC, Wen-Hwei H(2004)Integratedenzymepurification and immobilization processes with immobilized metalaffinity adsorbents. Process Biochem 39:1573158152. Vianello F, Zennaro L, Di Paolo ML, Rigo A, Malacarne C, ScarpaM (2000) Preparation, morphological characterization and activityof thin films of horseradish peroxidase. Biotech Bioeng 68:48849553. CabreraK(2004)Applicationsofsilica-basedmonolithicHPLCcolumns. J Sep Sci 27:84385254. Majors RE (2006) Developments in HPLC column packing design.LCGC 24:81555. XuL,FengYQ,DaSL,ShiZG(2004)Applicationsoforderedmesoporousmaterialsinseparationscience. ChinJAnal Chem32:37438056. David AE, Wang NS, Yang VC, Yang AJ (2006) Chemically surfacemodified gel (CSMG): an excellent enzyme-immobilization matrixfor industrial processes. J Biotechnol 125:39540757. Klouda L, Mikos AG (2008) Thermoresponsive hydrogels in bio-medical applications. Eur J Pharm Biopharm 68:344558. Klis M, KarbarzM, StojekZ, Rogalski J, BilewiczR(2009)Thermoresponsive poly(N-isopropylacrylamide) gel for immobili-zation of laccaseon indium tin oxide electrodes. J Phys Chem B113:6062606759. Lozinsky VI, Simenel IA, Kulakova VK, Kurskaya EA,Babushkina TA, Klimova TP, Burova TV, Dubovik AS, GrinbergVY, Galaev IY, Mattiasson B, Khokhlov AR (2003) Synthesis andstudiesofN-vinylcaprolactam/N-vinylimidazolecopolymersthatexhibit the protein-like behaviour in aqueous media. Macromol-ecules 36:7308732360. VirtanenJ, TenhuH(2000) Thermal properties of poly(N-isopropylacrylamide)-g-poly(ethyleneoxide)inaqueoussolutions:influence of the number and distribution of the grafts. Macromole-cules 33:5970597561. VirtanenJ, BaronC, TenhuH(2000) Graftingof poly(N-isopropylacrylamide) with poly(ethyleneoxide) under various reac-tion conditions. Macromolecules 33:33634162. Cirpan A, Alkan S, Toppare L, Hepuzer Y, Yagci Y (2003) Immo-bilization of invertase in conducting copolymers of 3-methylthienylmethacrylate. Bioelectrochemistry 59:293363. Kiralpa S, Balika B, Karatasb S, Toppare L, Gungor A (2008) Analternativesupportingelectrolytefor enzymeimmobilizationinconducting polymers. Int J Biol Macromol 42:19119464. LangeU, RoznyatovskayaNV, MirskyVM(2008) Conductingpolymers in chemical sensors and arrays. Anal ChimActa 614:12665. GerardM, ChaubeyA, MalhotraBD(2002) Applicationofconducting polymers to biosensors. Biosens Bioelectron 17:34535966. ChowDC, Johannes MS, Lee W-K, ClarkRL, Zauscher S,Chilkoti A(2005) Nanofabricationwithbiomolecules. MaterToday8:303967. Daniel M-C, Astruc D (2004) Gold nanoparticles: assembly, supra-molecular chemistry, quantum-size-related properties, and applica-tionstowardbiology, catalysis, andnanotechnology. ChemRev104:29334668. Selvan ST, Hayakawa T, Nogami M, Kobayashi Y, Liz-Marzan LM,Hamanaka Y, Nakamura AJ (2002) Solgel derived goldnanoclusters in silica glass possessing large optical nonlinearities.Phys Chem B 106:101571016269. Schellenberger EA, Bogdanov AJ, Hogemann D, Tait J, WeisslederR, JosephsonL(2002) AnnexinV-CLIO: ananoparticlefordetecting apoptosis by MRI. Mol Imaging 2:10210770. Wunderbaldinger P, Josephson L, Weissleder R (2002) Crosslinkediron oxides (CLIO): a new platform for the development of targetedMR contrast agents. Acad Radiol 9:S304S30671. Keller TM, Michel SC, Frohlich J, Fink D, Caduff R, Marincek B,Kubik-HuchRA(2004)USPIO-enhancedMRI forpreoperativestagingofgynecological pelvictumors: preliminaryresults. EurRadiol 14:93794472. Kooi ME, CappendijkVC, CleutjensKB, KesselsAG, KitslaarPJ, BorgersM, FrederikPM, DaemenMJ, vanEngelshovenJM(2003) Accumulation of ultrasmall superparamagnetic particlesof ironoxideinhumanatheroscleroticplaques canbedetectedby in vivo magnetic resonance imaging. Circulation 107:2453245873. Funovics MA, Kapeller B, Hoeller C, Su HS, Kunstfeld R, Puig S,Macfelda K (2004) MR imaging of the her2/neu and 9.2.27 tumorantigens using immunospecific contrast agents. Magn Reson Imag-ing 22:84385074. Krause MH, Kwong KK, Gragoudas ES, Young LH(2004)MRIofbloodvolumewithsuperparamagneticironinchoroidalmelanoma treated with thermotherapy. Magn Reson Imaging22:77978775. Kluchova K, Zboril R, Tucek J, Pecova M, Zajoncova L,Safarik I, Mashlan M, Markova I, Jancik D, Sebela M,Bartonkova H, Bellesi V, Novak P, Petridis D (2009)Superparamagnetic maghemite nanoparticles fromsolid-statesynthesistheir functionalizationtowards peroral MRI contrastagent andmagneticcarrier for trypsinimmobilization. Biomate-rials30:28556376. Kouassi G, Irudayaraj J, McCartyG(2005) Activityofglucoseoxidase functionalized onto magnetic nanoparticles. Biomagn ResTechnol 3:11077. TsangSC, YuCH, GaoX, TamK(2006) Silica-encapsulatednanomagnetic particles as anewrecoverablebiocatalystcarrier. JPhys Chem B 110:169141692278. Simberg D, Duza T, Park JH, Essler M, Pilch J, Zhang L, DerfusAM, YangM, HoffmanRM, BhatiaS, Sailor MJ, Ruoslahti E(2007) Biomimetic amplification of nanoparticle homing to tumors.Proc Anal Nat Sci 104:93293679. Mornet S, Vasseur S, Grasset F, Duguet E (2004) Magnetic nano-particledesignformedicaldiagnosisandtherapy.JMaterChem14:2161217580. Bergemann C, Muller-Schulte D, Oster J, Brassard LA, Lubbe AS(1999) Magnetic ion-exchange nano- and microparticles for medicalbiochemical and molecular biological applications. J Magn MagnMater 194:455281. LeeJ, IsobeT, SennaM(1996) PreparationofultrafineFe3O4particlesbyprecipitationinthepresenceof PVAat highpH. JColloid Interface Sci 177:49049482. Laurent N, Haddoub R, Flitsch SL (2008) Enzyme catalysis on solidsurfaces. Trends Biotechnol 26:3283783. Laurent S, Forge D, Port M, Roch A, Robic C, Elst LV, Muller RN(2008) Magnetic iron oxide nanoparticles: synthesis, stabilization,vectorization, physicochemical characterizationandbiochemicalapplications. Chem Rev 108:2064211084. Li Y, YanB, DengC, YuW, XuX, YangP, ZhangX(2007)Efficient on-chip proteolysis system based on functionalized mag-netic silica microspheres. Proteomics 7:2330233985. Sheldon RA, Schoevaart R, van Langen L (2005) A novel methodfor enzyme immobilization. Biocat Biotrans 2005:14114786. IllanesA,Wilson