Enzyme Immobilization - An Update

  • View

  • Download

Embed Size (px)


enzimas inmovilizadas

Text of Enzyme Immobilization - An Update

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). I