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Advances in enzyme immobilisation

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Abstract

Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery and stabilisation, other advantages to enzyme immobilisation have emerged, such as enhanced enzyme activity, modification of substrate selectivity and enantioselectivity, and multi-enzyme reactions. These advances promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.

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References

  • Abraham TE, Bindhu LVA (2009) Method for the preparation of cross linked protein crystals. US Pat 2009035828

  • Aloulou A, Rodriguez JA, Fernandez S, van Oosterhout D, Puccinelli D, Carrière F (2006) Exploring the specific features of interfacial enzymology based on lipase studies. Biochem Biophys Acta 1761:995–1013

    PubMed  CAS  Google Scholar 

  • Ansorge-Schumacher MB, Slusarczyk H, Schümers J, Hirtz D (2006) Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide. FEBS J 273:3938–3945

    Article  PubMed  CAS  Google Scholar 

  • Betancor L, Fuentes M, Dellamora-Ortiz G, López-Gallego F, Hidalgo A, Alonso-Morales N, Mateo C, Guisán JM, Fernández-Lafuente R (2005) Dextran aldehyde coating of glucose oxidase immobilized on magnetic nanoparticles prevents its inactivation by gas bubbles. J Mol Catal B 32:97–101

    Article  CAS  Google Scholar 

  • Betancor L, Berne C, Luckarift HR, Spain JC (2006) Coimmobilization of a redox enzyme and a cofactor regeneration system. Chem Commun 3640–3642

  • Bode ML, van Rantwijk F, Sheldon RA (2003) Crude aminoacylase from Aspergillus sp. is a mixture of hydrolases. Biotechnol Bioeng 84:710–713

    Article  PubMed  CAS  Google Scholar 

  • Bolivar JM, Mateo C, Rocha-Martin J, Cava F, Berenguer J, Fernandez-Lafuente R, Guisan JM (2009) The adsorption of multimeric enzymes on very lowly activated supports involves more enzyme subunits: stabilization of a glutamate dehydrogenase from Thermus thermophilus by immobilization on heterofunctional supports. Enzyme Microb Technol 44:139–144

    Article  CAS  Google Scholar 

  • Boller T, Meier C, Menzler S (2002) Eupergit oxirane acrylic beads: how to make enzymes fit for biocatalysis. Org Process Res Dev 6:509–519

    Article  CAS  Google Scholar 

  • Bommarius AS, Riebel BR (2004) Biocatalysis: fundamentals and applications. Wiley-VCH, Weinheim, 611 pp

  • Brady D, Steenkamp L, Reddy S, Skein E, Chaplin J (2004) Optimisation of the enantioselective biocatalytic hydrolysis of naproxen ethyl ester using ChiroCLEC-CR. Enzyme Microb Technol 34:283–291

    Article  CAS  Google Scholar 

  • Brady D, Jordaan J, Simpson C, Chetty A, Arumugam C, Moolman FS (2008) Spherezymes: a novel enzyme immobilisation technology. BMC Biotechnol 8:8

    Article  PubMed  CAS  Google Scholar 

  • Brazeau BJ, De Souza ML, Gort SJ, Hicks PM, Kollmann SR, Laplaza JM, McFarlan SC, Sanchez-Riera FA, Solheid C (2008) Polypeptides and biosynthetic pathways for the production of stereoisomers of monatin and their precursors. US Pat 20080020434

  • Bruns N, Tiller JC (2005) Amphiphilic network as nanoreactor for enzymes in organic solvents. Nano Lett 5:45–48

    Article  PubMed  CAS  Google Scholar 

  • Bulawayo BT, Dorrington RA, Burton SG (2007) Enhanced operational parameters for amino acid production using hydantoin-hydrolysing enzymes of Psuedomonas putida strain RUKM3s immobilised in Eupergit® C. Enzyme Microb Technol 40:533–539

    Article  CAS  Google Scholar 

  • Cabrera Z, Fernandez-Lorente G, Fernandez-Lafuente R, Palomo JM, Guisan JM (2009) Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports. J Mol Catal B (in press)

  • Cao L, Langen LM, Janssen MHA, Sheldon RA (2001) Crosslinked enzyme aggregates. European Pat EP1088887

  • Chaplin JA, Gardiner NS, Mitra RK, Parkinson CJ, Portwig M, Dickson MD, Brady D, Marais SF, Reddy S (2002) Process for preparing (-)-menthol and similar compounds. US Pat 2004058422

  • Gao S, Wang Y, Wang T, Luo G, Dai Y (2009) Immobilization of lipase on methyl-modified silica aerogels by physical adsorption. Bioresour Technol 100:996–999

    Article  PubMed  CAS  Google Scholar 

  • Grazú V, Abian O, Mateo C, Batista-Viera F, Fernández-Lafuente R, Guisán JM (2003) Novel bifunctional epoxy/thiol-reactive support to immobilize thiol containing proteins by the epoxy chemistry. Biomacromolecules 4:1495–1501

    Article  PubMed  CAS  Google Scholar 

  • Grazú V, Abian O, Mateo C, Batista-Viera F, Fernández-Lafuente R, Guisán JM (2005) Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports. Biotechnol Bioeng 90:597–605

    Article  PubMed  CAS  Google Scholar 

  • Heyman A, Levy I, Altman A, Shoseyov O (2007a) SP1 as a novel scaffold building block for self-assembly nanofabrication of submicron enzymatic structures. Nano Lett 7:1575–1579

    Article  PubMed  CAS  Google Scholar 

  • Heyman A, Barak Y, Caspi J, Wilson DB, Altman A, Bayer EA, Shoseyov O (2007b) Multiple display of catalytic modules on a protein scaffold: nano-fabrication of enzyme particles. J Biotechnol 131:433–439

    Article  PubMed  CAS  Google Scholar 

  • Ho M, Mao X, Gu L, Li P (2008) Facile route to enzyme immobilization: core-shell nanoenzyme particles consisting of well-defined poly(methyl methacrylate) cores and cellulase shells. Langmuir 24:11036–11042

    Article  PubMed  CAS  Google Scholar 

  • Hwang S, Ahn J, Lee S, Lee TG, Haam S, Lee K, Ahn I-S, Jung J-K (2004) Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization. Biotechnol Lett 26:603–605

    Article  PubMed  CAS  Google Scholar 

  • Jordaan J, Simpson C, Brady D, Gardiner NS (2009a) Emulsion-derived particles. Patent WO2009/057049

  • Jordaan J, Mathye SF, Simpson C, Brady D (2009b) Improvement of chemical and physical stability of laccase using spherezymes self-immobilisation technology (unpublished)

  • Katchalski-Katzir E, Kraemer DM (2000) Eupergit C, a carrier for immobilization of enzymes of industrial potential. J Mol Catal B 10:157–176

    Article  CAS  Google Scholar 

  • Kaul P, Stolz A, Banerjee UC (2007) Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis. Adv Synth Catal 349:2167–2176

    Article  CAS  Google Scholar 

  • Kaulpiboon J, Pongsawasdi P, Zimmermann W (2007) Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with γ-cyclodextrin. FEBS J 274:1001–1010

    Article  PubMed  CAS  Google Scholar 

  • Khalaf N, Govardhan CP, Lalonde JJ, Persichetti RA, Wang Y-F, Margolin AL (1996) Cross-linked enzyme crystals as highly active catalysts in organic solvents. J Am Chem Soc 118:5494–5495

    Article  CAS  Google Scholar 

  • Kim J, Grate JW (2003) Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Lett 3:1219–1222

    Article  CAS  Google Scholar 

  • Kim J, Grate JW, Wang P (2008) Nanobiocatalysis and its potential applications. Trends Biotechnol 26:639–646

    Article  PubMed  CAS  Google Scholar 

  • Kouisni L, Rochefort D (2008) Confocal microscopy study of polymer microcapsules for enzyme immobilisation in paper studies. J Appl Polym Sci 111:1–10

    Article  CAS  Google Scholar 

  • Křenkova J, Foret F (2004) Immobilized microfluidic enzymatic reactors. Electrophoresis 25:3550–3563

    Article  PubMed  CAS  Google Scholar 

  • Kubáč D, Kaplan O, Elisakova V, Patek M, Vejvoda V, Slamova K, Tothova A, Lemaire M, Gallienne E, Lutz-Wahl S, Fischer L, Kuzma M, Pelantova H, van Pelt S, Bolte J, Kren V, Martinkova L (2008) Biotransformation of nitrile to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4. J Mol Catal B 50:107–113

    Article  CAS  Google Scholar 

  • Kunamneni A, Ghazi I, Camarero S, Ballesteros A, Plou FJ, Alcalde M (2008) Decolorization of synthetic dyes by laccase immobilized on epoxy-activated carriers. Process Biochem 43:169–178

    Article  CAS  Google Scholar 

  • Lalonde J, Margolin A (2002) Immobilization of enzymes. In: Drauz K, Waldmann H (eds) Enzyme catalysis in organic chemistry, 2nd edn. Wiley-VCH, Weinheim, pp 163–184

    Google Scholar 

  • Lee W-F, Huang C-T (2008) Immobilization of trypsin by thermal-responsive hydrogel for the affinity separation of trypsin inhibitor. Desalination 234:195–203

    Article  CAS  Google Scholar 

  • Lee J, Kim J, Kim J, Jia H, Kim MI, Kwak JH, Jin S, Dohnalkova A, Park HG, Chang HN, Wang P, Grate JW, Hyeon T (2005) Simple synthesis of hierarchically ordered mesocellular mesoporous silica materials hosting crosslinked enzyme aggregates. Small 1:744–753

    Article  PubMed  CAS  Google Scholar 

  • López-Serrano P, Cao L, van Rantwijk F, Sheldon RA (2002) Cross-linked enzyme aggregates with enhanced activity: application to lipases. Biotechnol Lett 24:1379–1383

    Article  Google Scholar 

  • Luarent N, Haddoub R, Flitsch SL (2008) Enzyme catalysis on solid surfaces. Trends Biotechnol 26:328–337

    Article  CAS  Google Scholar 

  • Majumder AB, Mondal K, Singh TP, Gupta MN (2008) Designing cross-linked lipase aggregates for optimum performance as biocatalysts. Biocatal Biotransformation 26:235–242

    Article  CAS  Google Scholar 

  • Margolin AL (1996) Novel crystalline catalysts. Trends Biotechnol 14:223–230

    Article  CAS  Google Scholar 

  • Mateo C, Fernández-Lorente G, Abian O, Fernández-Lafuente R, Guisán JM (2000) Multifunctional epoxy supports: a new tool to improve the covalent immobilization of proteins. The promotion of physical adsorptions of proteins on the supports before their covalent linkage. Biomacromolecules 1:739–745

    Article  PubMed  CAS  Google Scholar 

  • Mateo C, Torres R, Fernández-Lorente G, Ortiz C, Fuentes M, Hidalgo A, López-Gallego F, Abian O, Palomo JM, Betancor L, Pessela BCC, Guisan JM, Fernández-Lafuente R (2003) Epoxy-amino groups: a new tool for improved immobilization of proteins by the epoxy method. Biomacromolecules 4:772–777

    Article  PubMed  CAS  Google Scholar 

  • Mateo C, Fernandes B, Van Rantwijk F, Stolz A, Sheldon RA (2006) Stabilisation of oxygen-labile nitrilases via co-aggregation with poly(ethyleneimine). J Mol Catal B 38:154–157

    Article  CAS  Google Scholar 

  • Mateo C, Fernandez-Lafuente R, Archelas A, Guisan JM, Furstoss R (2007a) Preparation of a very stable immobilized Solanum tuberosum epoxide hydrolase. Tetrahedron Asymmetry 18:1233–1238

    Article  CAS  Google Scholar 

  • Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007b) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 40:1451–1463

    Article  CAS  Google Scholar 

  • Mateo C, Grazú V, Pessela BCC, Montes T, Palomo JM, Torres R, López-Gallego F, Fernández-Lafuente R, Guisán JM (2007c) Advances in the design of new epoxy supports for enzyme immobilization-stabilization. Biochem Soc Trans 35:1593–1601

    Article  PubMed  CAS  Google Scholar 

  • May O, Nguyen PT, Arnold FH (2000) Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nat Biotechnol 18:317–320

    Article  PubMed  CAS  Google Scholar 

  • Miletić N, Vuković Z, Nastasović A, Loos K (2009) Macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethylacrylate) resins—versatile immobilisation supports for biocatalysts. J Mol Catal B 56:196–201

    Article  CAS  Google Scholar 

  • Moolman S, Brady D, Sewlall AS, Rolfes H, Jordaan J (2005) Stabilization of enzymes. Patent WO 2005/080561

  • Nahálka J, Gemeiner P (2006) Thermoswitched immobilization—a novel approach in reversible immobilization. J Biotechnol 123:478–482

    Article  PubMed  CAS  Google Scholar 

  • Ozyilmaz G (2009) The effect of spacer arm on hydrolytic and synthetic activity of Candida rugosa lipase immobilized on silica gel. J Mol Catal B 56:231–236

    Article  CAS  Google Scholar 

  • Palomo JM (2008) Lipases enantioselectivity alteration by immobilization techniques. Curr Bioact Compd 4:126–138

    Article  CAS  Google Scholar 

  • Pchelintsev NA, Youshko MI, Švedas VK (2009) Quantitative characteristic of the catalytic properties and microstructure of cross-linked enzyme aggregates of penicillin acylase. J Mol Catal B 56:202–207

    Article  CAS  Google Scholar 

  • Pessela BCC, Mateo C, Carrascosa AV, Vian A, García JL, Rivas G, Alfonso C, Guisán JM, Fernández-Lafuente R (2003) One-step purification, covalent immobilization, and additional stabilization of a thermophilic poly-His-tagged β-galactosidase from Thermus sp. Strain T2 by using novel heterofunctional chelate–epoxy Sepabeads. Biomacromolecules 4:107–113

    Article  PubMed  CAS  Google Scholar 

  • Pierre AC (2004) The sol-gel encapsulation of enzymes. Biocatal Biotransformation 22:145–170

    Article  CAS  Google Scholar 

  • Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF (2007) Stability of biocatalysts. Curr Opin Chem Biol 11:220–225

    Article  PubMed  CAS  Google Scholar 

  • Prakasham RS, Devi GS, Laxmi KR, Rao CS (2007) Novel synthesis of ferric impregnated silica nanoparticles and their evaluation as a matrix for enzyme immobilization. J Phys Chem C 111:3842–3847

    Article  CAS  Google Scholar 

  • Ran N, Zhao L, Chen Z, Tao J (2008) Recent applications of biocatalysis in developing green chemistry for chemical synthesis at the industrial scale. Green Chem 10:361–372

    Article  CAS  Google Scholar 

  • Reetz MT, Jaeger K-E (1998) Overexpression, immobilization and biotechnological application of Pseudomonas lipases. Chem Phys Lipids 93:3–14

    Article  PubMed  CAS  Google Scholar 

  • Rocchietti S, Ubiali D, Terreni M, Albertini AM, Fernández-Lafuente R, Guisán JM, Pregnolato M (2004) Immobilization and stabilization of recombinant multimeric uridine and purine nucleoside phosphorylases from Bacillus subtilis. Biomacromolecules 5:2195–2200

    Article  PubMed  CAS  Google Scholar 

  • Rochefort D, Kouisni L, Gendron K (2008) Physical immobilization of laccase on an electrode by means of poly(ethyleneimine) microcapsules. J Electroanal Chem 617:53–63

    Article  CAS  Google Scholar 

  • Roy JJ, Abraham TE (2004) Strategies in making cross-linked enzyme crystals. Chem Rev 104:3705–3721

    Article  CAS  Google Scholar 

  • Santos JC, Paula AV, Rocha CGF, Nunes GFM, de Castro HF (2008a) Morphological and mechanical properties of hybrid matrices of polysiloxane–polyvinyl alcohol prepared by sol–gel technique and their potential for immobilizing enzyme. J Non-Cryst Solids 354:4823–4826

    Article  CAS  Google Scholar 

  • Santos JC, Paula AV, Nunes GFM, de Castro HF (2008b) Pseudomonas fluorescens lipase immobilization on polysiloxane–polyvinyl alcohol composite chemically modified with epichlorohydrin. J Mol Catal B 52–53:49–57

    Article  CAS  Google Scholar 

  • Sheldon RA (2007a) Enzyme immobilisation: the quest for optimum performance. Adv Synth Catal 349:1289–1307

    Article  CAS  Google Scholar 

  • Sheldon RA (2007b) Cross-linked enzyme aggregates (CLEA®s): stable and recyclable biocatalysts. Biochem Soc Trans 35:1583–1587

    Article  PubMed  CAS  Google Scholar 

  • Sheldon RA, Schoevaart R, van Langen IM (2005) Cross-linked enzyme aggregates (CLEAs): a novel and versatile method for enzyme immobilization (a review). Biocatal Biotransformation 23:141–147

    Article  CAS  Google Scholar 

  • Spahn C, Minteer SD (2008) Enzyme immobilization in biotechnology. Recent Pat Eng 2:195–200

    Article  CAS  Google Scholar 

  • St. Clair NL, Navia MA (1992) Cross-linked enzyme crystals as robust biocatalysts. J Am Chem Soc 114:7314–7316

    Article  CAS  Google Scholar 

  • St. Clair N, Wang YF, Margolin AL (2000) Cofactor-bound cross-linked enzyme crystals (CLEC) of alcohol dehydrogenase. Angew Chem Int Ed Engl 39:380–383

    Article  PubMed  CAS  Google Scholar 

  • Straathof AJ, Panke S, Schmid A (2002) The production of fine chemicals by biotransformations. Curr Opin Biotechnol 13:548–556

    Article  PubMed  CAS  Google Scholar 

  • Takaç S, Bakkal M (2007) Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production. Process Biochem 42:1021–1027

    Article  CAS  Google Scholar 

  • Temiño DM-RD, Hartmeier W, Ansorge-Schumacher MB (2005) Entrapment of the alcohol dehydrogenase from Lactobacillus kefir in polyvinyl alcohol for the synthesis of chiral hydrophobic alcohols in organic solvents. Enzyme Microb Technol 36:3–9

    Article  CAS  Google Scholar 

  • Thuku RN, Brady D, Benedik MJ, Sewell BT (2009) Microbial nitrilases: versatile, spiral forming enzymes. J Appl Microbiol 106:703–727

    Article  PubMed  CAS  Google Scholar 

  • van Dongen SFM, Nallani M, Cornelissen JJLM, Nolte RJM, van Hest JCM (2009) A three-enzyme cascade reaction through positional assembly of enzymes in a polymersome nanoreactor. Chem Eur J 15:1107–1114

    Google Scholar 

  • Wang P-Y, Tsai S-W, Chen T-L (2008) Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization. Biotechnol Bioeng 101:460–469

    Article  PubMed  CAS  Google Scholar 

  • Wang Z-G, Wan L-S, Liu Z-M, Huang X-J, Xu Z-K (2009) Enzyme immobilization on electrospun polymer nanofibers: an overview. J Mol Catal B 56:189–195

    Article  CAS  Google Scholar 

  • Wilson L, Illanes A, Abián O, Fernández-Lafuente R, Guisán JM (2002) Encapsulation of very soft cross-linked enzyme aggregates (CLEA) in very rigid LentiKats™ Landbauforshung Volkenröde. FAL Agric Res 241:121–125

    CAS  Google Scholar 

  • Wilson L, Betancor L, Fernández-Lorente G, Fuentes M, Hidalgo A, Guisán JM, Pessela BC, Fernández-Lafuente R (2004) Cross-linked aggregates of multimeric enzymes: a simple and efficient methodology to stabilize their quaternary structure. Biomacromolecules 5:814–817

    Article  PubMed  CAS  Google Scholar 

  • Wilson L, Illanes A, Soler L, Henríquez MJ (2009) Effect of the degree of cross-linking on the properties of different CLEAs of penicillin acylase. Process Biochem 44:322–326

    Article  CAS  Google Scholar 

  • Yu A, Liang Z (2009) Enzymatically active colloidal crystal arrays. J Colloid Interface Sci 330:144–148

    Article  PubMed  CAS  Google Scholar 

  • Zhang YF, Wu H, Li J, Li L, Jiang YJ, Jiang Y, Jiang ZY (2008) Protamine-templated biomimetic hybrid capsules: efficient and stable carrier for enzyme encapsulation. Chem Mater 20:1041–1048

    Article  CAS  Google Scholar 

  • Zhang Y, Wu H, Li L, Li J, Jiang Z, Jiang Y, Chen Y (2009) Enzymatic conversion of Baicalin into Baicalein by β-glucuronidase encapsulated in biomimetic core-shell structured hybrid capsules. J Mol Catal B 57:130–135

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank BioPAD and ZA Biotech for financial support in development of SphereZymes, and Novozymes SA for provision of enzymes in ongoing research projects.

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  1. Enzyme Technologies, CSIR Biosciences, Ardeer Road, Private Bag X2, Modderfontein, 1645, South Africa

    Dean Brady

  2. Department of Biotechnology and Food Technology, Tshwane University of Technology, Nelson Mandela Drive, Arcadia Campus, Private Bag X680, Pretoria, 0001, South Africa

    Dean Brady

  3. Molecular Biomaterials, CSIR Biosciences, Meiring Naude Road, Brummeria, 0091, South Africa

    Justin Jordaan

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Brady, D., Jordaan, J. Advances in enzyme immobilisation. Biotechnol Lett 31, 1639–1650 (2009). https://doi.org/10.1007/s10529-009-0076-4

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