Biotechnology Letters

, 31:1639 | Cite as

Advances in enzyme immobilisation

  • Dean Brady
  • Justin Jordaan


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.


Biocatalyst Biocatalysis Enzyme Immobilisation 



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.


  1. Abraham TE, Bindhu LVA (2009) Method for the preparation of cross linked protein crystals. US Pat 2009035828Google Scholar
  2. 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 1761:995–1013PubMedGoogle Scholar
  3. 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 273:3938–3945PubMedCrossRefGoogle Scholar
  4. 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 32:97–101CrossRefGoogle Scholar
  5. Betancor L, Berne C, Luckarift HR, Spain JC (2006) Coimmobilization of a redox enzyme and a cofactor regeneration system. Chem Commun 3640–3642Google Scholar
  6. Bode ML, van Rantwijk F, Sheldon RA (2003) Crude aminoacylase from Aspergillus sp. is a mixture of hydrolases. Biotechnol Bioeng 84:710–713PubMedCrossRefGoogle Scholar
  7. 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 44:139–144CrossRefGoogle Scholar
  8. Boller T, Meier C, Menzler S (2002) Eupergit oxirane acrylic beads: how to make enzymes fit for biocatalysis. Org Process Res Dev 6:509–519CrossRefGoogle Scholar
  9. Bommarius AS, Riebel BR (2004) Biocatalysis: fundamentals and applications. Wiley-VCH, Weinheim, 611 ppGoogle Scholar
  10. 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 34:283–291CrossRefGoogle Scholar
  11. Brady D, Jordaan J, Simpson C, Chetty A, Arumugam C, Moolman FS (2008) Spherezymes: a novel enzyme immobilisation technology. BMC Biotechnol 8:8PubMedCrossRefGoogle Scholar
  12. 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 20080020434Google Scholar
  13. Bruns N, Tiller JC (2005) Amphiphilic network as nanoreactor for enzymes in organic solvents. Nano Lett 5:45–48PubMedCrossRefGoogle Scholar
  14. 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 40:533–539CrossRefGoogle Scholar
  15. 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)Google Scholar
  16. Cao L, Langen LM, Janssen MHA, Sheldon RA (2001) Crosslinked enzyme aggregates. European Pat EP1088887Google Scholar
  17. 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 2004058422Google Scholar
  18. Gao S, Wang Y, Wang T, Luo G, Dai Y (2009) Immobilization of lipase on methyl-modified silica aerogels by physical adsorption. Bioresour Technol 100:996–999PubMedCrossRefGoogle Scholar
  19. 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 4:1495–1501PubMedCrossRefGoogle Scholar
  20. 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 90:597–605PubMedCrossRefGoogle Scholar
  21. 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 7:1575–1579PubMedCrossRefGoogle Scholar
  22. 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 131:433–439PubMedCrossRefGoogle Scholar
  23. 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 24:11036–11042PubMedCrossRefGoogle Scholar
  24. 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 26:603–605PubMedCrossRefGoogle Scholar
  25. Jordaan J, Simpson C, Brady D, Gardiner NS (2009a) Emulsion-derived particles. Patent WO2009/057049Google Scholar
  26. Jordaan J, Mathye SF, Simpson C, Brady D (2009b) Improvement of chemical and physical stability of laccase using spherezymes self-immobilisation technology (unpublished)Google Scholar
  27. Katchalski-Katzir E, Kraemer DM (2000) Eupergit C, a carrier for immobilization of enzymes of industrial potential. J Mol Catal B 10:157–176CrossRefGoogle Scholar
  28. Kaul P, Stolz A, Banerjee UC (2007) Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis. Adv Synth Catal 349:2167–2176CrossRefGoogle Scholar
  29. Kaulpiboon J, Pongsawasdi P, Zimmermann W (2007) Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with γ-cyclodextrin. FEBS J 274:1001–1010PubMedCrossRefGoogle Scholar
  30. 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 118:5494–5495CrossRefGoogle Scholar
  31. Kim J, Grate JW (2003) Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Lett 3:1219–1222CrossRefGoogle Scholar
  32. Kim J, Grate JW, Wang P (2008) Nanobiocatalysis and its potential applications. Trends Biotechnol 26:639–646PubMedCrossRefGoogle Scholar
  33. Kouisni L, Rochefort D (2008) Confocal microscopy study of polymer microcapsules for enzyme immobilisation in paper studies. J Appl Polym Sci 111:1–10CrossRefGoogle Scholar
  34. Křenkova J, Foret F (2004) Immobilized microfluidic enzymatic reactors. Electrophoresis 25:3550–3563PubMedCrossRefGoogle Scholar
  35. 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 50:107–113CrossRefGoogle Scholar
  36. 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 43:169–178CrossRefGoogle Scholar
  37. 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 163–184Google Scholar
  38. Lee W-F, Huang C-T (2008) Immobilization of trypsin by thermal-responsive hydrogel for the affinity separation of trypsin inhibitor. Desalination 234:195–203CrossRefGoogle Scholar
  39. 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 1:744–753PubMedCrossRefGoogle Scholar
  40. López-Serrano P, Cao L, van Rantwijk F, Sheldon RA (2002) Cross-linked enzyme aggregates with enhanced activity: application to lipases. Biotechnol Lett 24:1379–1383CrossRefGoogle Scholar
  41. Luarent N, Haddoub R, Flitsch SL (2008) Enzyme catalysis on solid surfaces. Trends Biotechnol 26:328–337CrossRefGoogle Scholar
  42. Majumder AB, Mondal K, Singh TP, Gupta MN (2008) Designing cross-linked lipase aggregates for optimum performance as biocatalysts. Biocatal Biotransformation 26:235–242CrossRefGoogle Scholar
  43. Margolin AL (1996) Novel crystalline catalysts. Trends Biotechnol 14:223–230CrossRefGoogle Scholar
  44. 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 1:739–745PubMedCrossRefGoogle Scholar
  45. 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 4:772–777PubMedCrossRefGoogle Scholar
  46. 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 38:154–157CrossRefGoogle Scholar
  47. Mateo C, Fernandez-Lafuente R, Archelas A, Guisan JM, Furstoss R (2007a) Preparation of a very stable immobilized Solanum tuberosum epoxide hydrolase. Tetrahedron Asymmetry 18:1233–1238CrossRefGoogle Scholar
  48. 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 40:1451–1463CrossRefGoogle Scholar
  49. 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 35:1593–1601PubMedCrossRefGoogle Scholar
  50. May O, Nguyen PT, Arnold FH (2000) Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nat Biotechnol 18:317–320PubMedCrossRefGoogle Scholar
  51. 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 56:196–201CrossRefGoogle Scholar
  52. Moolman S, Brady D, Sewlall AS, Rolfes H, Jordaan J (2005) Stabilization of enzymes. Patent WO 2005/080561Google Scholar
  53. Nahálka J, Gemeiner P (2006) Thermoswitched immobilization—a novel approach in reversible immobilization. J Biotechnol 123:478–482PubMedCrossRefGoogle Scholar
  54. 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 56:231–236CrossRefGoogle Scholar
  55. Palomo JM (2008) Lipases enantioselectivity alteration by immobilization techniques. Curr Bioact Compd 4:126–138CrossRefGoogle Scholar
  56. 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 56:202–207CrossRefGoogle Scholar
  57. 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 4:107–113PubMedCrossRefGoogle Scholar
  58. Pierre AC (2004) The sol-gel encapsulation of enzymes. Biocatal Biotransformation 22:145–170CrossRefGoogle Scholar
  59. Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF (2007) Stability of biocatalysts. Curr Opin Chem Biol 11:220–225PubMedCrossRefGoogle Scholar
  60. 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 111:3842–3847CrossRefGoogle Scholar
  61. 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 10:361–372CrossRefGoogle Scholar
  62. Reetz MT, Jaeger K-E (1998) Overexpression, immobilization and biotechnological application of Pseudomonas lipases. Chem Phys Lipids 93:3–14PubMedCrossRefGoogle Scholar
  63. 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 5:2195–2200PubMedCrossRefGoogle Scholar
  64. Rochefort D, Kouisni L, Gendron K (2008) Physical immobilization of laccase on an electrode by means of poly(ethyleneimine) microcapsules. J Electroanal Chem 617:53–63CrossRefGoogle Scholar
  65. Roy JJ, Abraham TE (2004) Strategies in making cross-linked enzyme crystals. Chem Rev 104:3705–3721CrossRefGoogle Scholar
  66. 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 354:4823–4826CrossRefGoogle Scholar
  67. 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 52–53:49–57CrossRefGoogle Scholar
  68. Sheldon RA (2007a) Enzyme immobilisation: the quest for optimum performance. Adv Synth Catal 349:1289–1307CrossRefGoogle Scholar
  69. Sheldon RA (2007b) Cross-linked enzyme aggregates (CLEA®s): stable and recyclable biocatalysts. Biochem Soc Trans 35:1583–1587PubMedCrossRefGoogle Scholar
  70. 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 23:141–147CrossRefGoogle Scholar
  71. Spahn C, Minteer SD (2008) Enzyme immobilization in biotechnology. Recent Pat Eng 2:195–200CrossRefGoogle Scholar
  72. St. Clair NL, Navia MA (1992) Cross-linked enzyme crystals as robust biocatalysts. J Am Chem Soc 114:7314–7316CrossRefGoogle Scholar
  73. St. Clair N, Wang YF, Margolin AL (2000) Cofactor-bound cross-linked enzyme crystals (CLEC) of alcohol dehydrogenase. Angew Chem Int Ed Engl 39:380–383PubMedCrossRefGoogle Scholar
  74. Straathof AJ, Panke S, Schmid A (2002) The production of fine chemicals by biotransformations. Curr Opin Biotechnol 13:548–556PubMedCrossRefGoogle Scholar
  75. 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 42:1021–1027CrossRefGoogle Scholar
  76. 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 36:3–9CrossRefGoogle Scholar
  77. Thuku RN, Brady D, Benedik MJ, Sewell BT (2009) Microbial nitrilases: versatile, spiral forming enzymes. J Appl Microbiol 106:703–727PubMedCrossRefGoogle Scholar
  78. 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 15:1107–1114Google Scholar
  79. 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 101:460–469PubMedCrossRefGoogle Scholar
  80. 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 56:189–195CrossRefGoogle Scholar
  81. 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 241:121–125Google Scholar
  82. 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 5:814–817PubMedCrossRefGoogle Scholar
  83. 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 44:322–326CrossRefGoogle Scholar
  84. Yu A, Liang Z (2009) Enzymatically active colloidal crystal arrays. J Colloid Interface Sci 330:144–148PubMedCrossRefGoogle Scholar
  85. 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 20:1041–1048CrossRefGoogle Scholar
  86. 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 57:130–135CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Enzyme TechnologiesCSIR BiosciencesModderfonteinSouth Africa
  2. 2.Department of Biotechnology and Food TechnologyTshwane University of TechnologyPretoriaSouth Africa
  3. 3.Molecular BiomaterialsCSIR BiosciencesBrummeriaSouth Africa

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