Immobilized Enzymes: Methods and Applications

  • Wilhelm Tischer
  • Frank Wedekind
Part of the Topics in Current Chemistry book series (volume 200)


Immobilized enzymes are used in organic syntheses to fully exploit the technical and economical advantages of biocatalysts based on isolated enzymes. Immobilization enables the separation of the enzyme catalyst easily from the reaction mixture, and can lower the costs of enzymes dramatically. This is true for immobilized enzyme preparations that provide a well-balanced overall performance, based on reasonable immobilization yields, low mass transfer limitations, and high operational stability. There are many methods available for immobilization which span from binding on prefabricated carrier materials to incorporation into in situ prepared carriers. Operative binding forces vary between weak multiple adsorptive interactions and single attachments through strong covalent binding.Which of the methods is the most appropriate is usually a matter of the desired applications. It is therefore the intention of this paper to outline the common immobilization methods and reaction technologies to facilitate proper applications of immobilized enzymes.


Enzyme immobilization Mass transfer effects Operational stability Immobilization methods 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Hartmeier W (1986) Immobilisierte Biokatalysatoren, Springer, Berlin Heidelberg New York, pp 18–20Google Scholar
  2. 2.
    Buchholz K, Kasche V (1997) Biokatalysatoren und Enzymtechnologie. VCH, Weinheim, pp 7–11Google Scholar
  3. 3.
    Silman ICH, Katchalski E (1966) Ann Rev Biochem 35:873CrossRefGoogle Scholar
  4. 4.
    Zelinski T, Waldmann H (1997) Angew Chem 109:746CrossRefGoogle Scholar
  5. 5.
    Katchalski-Katzir E (1993) TIBTECH 11:471Google Scholar
  6. 6.
    Schmidtke JL, Wescott CR, Klibanov AM (1996) J Am Chem Soc 118:3360CrossRefGoogle Scholar
  7. 7.
    Ottolina G, Carrea G, Riva S, Sartore L, Veronese FM (1992) Biotechnol Lett 14:947CrossRefGoogle Scholar
  8. 8.
    Buchholz K (ed) (1979) Characterization of immobilized biocatalysts. VCH, WeinheimGoogle Scholar
  9. 9.
    Multiple authors (1983) The Working Party on Immobilized Biocatalysts Enzyme Microb Technol 5:304Google Scholar
  10. 10.
    Buchholz K, Kasche V (1997) Biokatalysatoren und Enzymtechnologie, VCH, Weinheim, pp 248–256Google Scholar
  11. 11.
    Mosbach K (ed) (1976) Methods Enzymol 44Google Scholar
  12. 12.
    Mosbach K (ed) (1987) Methods Enzymol 135Google Scholar
  13. 13.
    Mosbach K (ed) (1987) Methods Enzymol 136Google Scholar
  14. 14.
    Kennedy JF, Cabral JMS (1983) Chemical analysis. In: Scouten WH (ed) Solid phase biochemistry, analytical and synthetic aspects, Vol 66. Wiley, New York, pp 253–392Google Scholar
  15. 15.
    Clark DS (1994) Trends Biotechnol 12:439CrossRefGoogle Scholar
  16. 16.
    Balcao VM, Paiva AL, Malcata FX (1996) Enzyme Microb Technol 18:392CrossRefGoogle Scholar
  17. 17.
    Abdul Mazid M (1993) Bio/Technology 11:690CrossRefGoogle Scholar
  18. 18.
    Bahulekar R, Ayyangar NR, Ponranthnam S (1993) Enzyme Microb Technol. 13:858CrossRefGoogle Scholar
  19. 19.
    Rao ZP, Raju DR, Baradarajan A, Satyanarayana M (1984) Indian Chem Eng 26:11Google Scholar
  20. 20.
    List D, Knechtel W (1980) Industrielle Obst-und Gemueseverwertung 65:415Google Scholar
  21. 21.
    Means G, Feeney R (1990) Bioconjugate Chem 1:2CrossRefGoogle Scholar
  22. 22.
    Edwards JO, Pearson RG (1962) J Chem Soc 84:26Google Scholar
  23. 23.
    Sharon N (1993) TIBS 18:221Google Scholar
  24. 24.
    Elgavish S, Shaanan B (1997) TIBS 22:462Google Scholar
  25. 25.
    Bobbitt JM (1956) Adv Carbohydr Chem 11:1Google Scholar
  26. 26.
    Roxer GP (1987) Methods Enzymol 135:141CrossRefGoogle Scholar
  27. 27.
    Cabacungan JC, Ahmed A, Feeney RE (1982) Anal Biochem 124:272CrossRefGoogle Scholar
  28. 28.
    Kurzer F, Douraghi-Zadeh K (1967) Chem Rev 67:107CrossRefGoogle Scholar
  29. 29.
    Fernandezlafuente R, Rosell CM, Rodriguez V, Santana C, Soler G, Bastida A, Guisan JM (1993) Enzyme Microb Technol 15:546CrossRefGoogle Scholar
  30. 30.
    Gurd FRN (1967) Methods Enzymol 11:532CrossRefGoogle Scholar
  31. 31.
    Gounaris AD, Perlman GEJ (1967) Biol Chem 242:2739Google Scholar
  32. 32.
    Solomon B, Levin Y (1974) Biotechnol Bioeng 16:1161CrossRefGoogle Scholar
  33. 33.
    Kondo A, Teshima T (1995) Biotechnol Bioeng 46:421CrossRefGoogle Scholar
  34. 34.
    Ljungquist C, Breitholz A, Brink-Nilsson H, Moks T, Uhlen M, Nilsson B (1989) Eur J Biochem 186:563CrossRefGoogle Scholar
  35. 35.
    Stempfer G, Hoell-Neugebauer B, Kopetzki E., Rudolph R (1996) Nature Biotechnol 14:481–484CrossRefGoogle Scholar
  36. 36.
    Ong E, Gilkes NR, Antony R, Warren J, Miller RC Jr, Kilburn DG (1989) Bio/Technology 7:604CrossRefGoogle Scholar
  37. 37.
    Huang X, Wals MK, Swaisgood HE (1996) Enzyme Microb Technol 19:378CrossRefGoogle Scholar
  38. 38.
    Wong SS (1991) Chemistry of protein conjugation and crosslinking. CRC Press IncGoogle Scholar
  39. 39.
    Stanley WL, Watters GG, Kelly SH, Chan BG, Garibaldi JA, Schade JE (1976) Biotech Bioeng 18:439CrossRefGoogle Scholar
  40. 40.
    Jaworek DJ, Botsch H, Maier J (1976) Methods Enzymol 44:195CrossRefGoogle Scholar
  41. 42.
    Reetz MT, Zonta A, Simpelkamp J (1996) Biotechnol Bioeng 49:527CrossRefGoogle Scholar
  42. 43.
    Urabe I, Yamamoto M, Yamada Y, Okada H (1978) Biochem Biophys Acta 524:435Google Scholar
  43. 44.
    Inada Y, Furukawa M, Sasaki H, Kodera Y, Hiroto M, Nishimura H, Matsushima A (1995) Trends Biotechnol 13:86CrossRefGoogle Scholar
  44. 45.
    Ito Y, Fujii H, Imanashi Y (1992) Biotechnol Lett 14:1149CrossRefGoogle Scholar
  45. 46.
    Takahashi K, Ajima A, Yoshimoto T, Inada Y (1984) Biochem Biophys Res Commun 125:761CrossRefGoogle Scholar
  46. 47.
    Buchholz K (1979) DECHEMA Monographs Vol. 84, Verlag ChemieGoogle Scholar
  47. 48.
    Haller W (1983) Solid phase biochemistry. In: Scouten, W.H. (ed.) Analytical and synthetic aspects. Wiley, New York, pp 535–599Google Scholar
  48. 49.
    Burteau N, Burton S, Crichton RR (1989) FEBS Lett 258:185–189CrossRefGoogle Scholar
  49. 50.
    Richards M, Knowles JR (1968) J Mol Biol 37:232Google Scholar
  50. 51.
    Giesecke U, Wedekind F, Tischer W (1992) DECHEMA Biotechnology Conferences 5:609Google Scholar
  51. 52.
    Guiseley KB (1989) Enzyme Microb Technol 11:706CrossRefGoogle Scholar
  52. 53.
    Tosa T, Sato T, Mori T, Yamamoto K, Takata I, Nishida Y, Chibata I (1979) Biotechnol Bioeng 21:1697CrossRefGoogle Scholar
  53. 54.
    Gerbsch N, Buchholzm R (1995) FEMS Microbiology Rev 16:259–269CrossRefGoogle Scholar
  54. 55.
    Itoyama K, Tokura S, Hayashi T (1994) Biotechnol Progress 10:225CrossRefGoogle Scholar
  55. 56.
    Vorlop KD, Klein J (1987) Methods Enzymol 135:259CrossRefGoogle Scholar
  56. 57.
    Scouten WH (1987) Methods Enzymol 135B:30CrossRefGoogle Scholar
  57. 58.
    Takata I, Tosa T, Chibata I (1978) J Solid-Phase Biochem 2:225CrossRefGoogle Scholar
  58. 59.
    Porath J, Axen R (1976) Methods Enzymol 44:19CrossRefGoogle Scholar
  59. 60.
    Kohn J, Wilchek M (1984) Appl Biochem Biotechnol 9:285CrossRefGoogle Scholar
  60. 61.
    Kohn J, Wilchek M (1983) FEBS Lett 154:209CrossRefGoogle Scholar
  61. 62.
    Guisan JM (1988) Enzyme Microbiol Technol 10:375CrossRefGoogle Scholar
  62. 63.
    Gemeiner P, Stefuca V, Bales V (1993) Enzyme Microb Technol 15:551CrossRefGoogle Scholar
  63. 64.
    Carleysmith SW, Dunnill P, Lilly MD (1980) Biotechnol Bioeng 22:735CrossRefGoogle Scholar
  64. 65.
    Koilpillai L, Gadre RA, Bhatnagar S, Raman RC, Ponrathnam S, Kumar KK, Ambekar GR, Shewale JG (1990) J Chem Tech Biotechnol 49:173Google Scholar
  65. 66.
    Boccu E, Gianferrara T, Gardossi L, Veronese FM (1990) IL Farmaco 45:203Google Scholar
  66. 67.
    Malcata FX, Reyes HR, Garcia HS, Hill CG, Amundson CH (1992) Enzyme Microb Technol 14:426CrossRefGoogle Scholar
  67. 68.
    Malcata FX, Reyes HR, Garcia HS, Hill CG, Amundson CH (1990) J Amer Oil Chem Soc 67:890CrossRefGoogle Scholar
  68. 69.
    Bosley J (1997) Biochem Soc Trans 25:174Google Scholar
  69. 71.
    Mattiasson B, Adlercreutz P (1991) Tibtech 9:394Google Scholar
  70. 72.
    Adlercreutz P (1992) Eur J Biochem 199:609CrossRefGoogle Scholar
  71. 73.
    Mustranta A, Forssell P, Poutanen K (1993) Enzyme Microb Technol 15:133CrossRefGoogle Scholar
  72. 74.
    Valivety RH, Halling PJ, Peilow AD, Macrae AR (1992) Biochim Biophys Acta 1122:143Google Scholar
  73. 75.
    Shaw J-F, Chang R-C, Wang FF, Wang YJ (1990) Biotechnol Bioeng 35:132CrossRefGoogle Scholar
  74. 76.
    Svec F, Hradil J, Coupek J, Kalal J (1975) Angew Makromol Chem 48:135CrossRefGoogle Scholar
  75. 78.
    Kolarz BN, Trochimczuk A, Bryjak J, Wojaczynska M, Dziegielewski K, Noworyta A (1990) Angew Makromol Chem 179:173CrossRefGoogle Scholar
  76. 79.
    Erarslan A, Güray AJ (1991) Chem Tech Biotechnol 51:181Google Scholar
  77. 80.
    Dbrobnik J, Saudek V, Svec F, Kalal J, Vojtisek V, Barta M (1979) Biotechnol Bioeng 21:1317CrossRefGoogle Scholar
  78. 81.
    Goldstein L (1976) Methods Enzymol 44:397CrossRefGoogle Scholar
  79. 82.
    Gloger M, Tischer W (1983) In: Bergmeyer HU, Bergmeyer J, Graßl M (eds) Methods of enzymatic analysis, 3rd edn. VCH, Weinheim, vol 1, pp 142–163Google Scholar
  80. 83.
    Tischer W (1995) In: Drauz K, Waldmann H (eds) Enzyme catalysis in organic synthesis. VCH, Weinheim, pp 73–87Google Scholar
  81. 84.
    Lee YY, Tsao GT (1974) J Food Sci 39:667CrossRefGoogle Scholar
  82. 85.
    Yamane T (1981) J Ferment Technol 59:375Google Scholar
  83. 86.
    Kasche V (1983) Enzyme Microb Technol 5:2CrossRefGoogle Scholar
  84. 87.
    Schlothauer RC (1996) Thesis TU Hamburg-HarburgGoogle Scholar
  85. 88.
    Engasser J-M, Horvath C (1974) Biochemistry 13:3845CrossRefGoogle Scholar
  86. 89.
    Horvath C, Engasser J-M (1974) Biotechnol Bioeng 16:909CrossRefGoogle Scholar
  87. 90.
    Carleysmith SW, Dunnill P, Lilly MD (1980) Biotechnol Bioeng 22:735CrossRefGoogle Scholar
  88. 91.
    Horvath C, Engasser J-M (1973) Ind Eng Chem Fundam 12:229CrossRefGoogle Scholar
  89. 92.
    McLAren AD, Packer L (1970) Adv Enzymol 33:245Google Scholar
  90. 93.
    Goldstein L, Levin Y, Katchalski E (1964) Biochemistry 3:1913CrossRefGoogle Scholar
  91. 94.
    Trevan MD (1980) Immobilized enzymes — an introduction and applications in biotechnology. Wiley, New York, pp 11–55Google Scholar
  92. 95.
    Halwachs W, Wandrey C, Schügerl K (1978) Biotechnol Bioeng 20:541CrossRefGoogle Scholar
  93. 96.
    Ruckenstein E, Sasidhar V (1984) Chem Eng Sci 39:1185CrossRefGoogle Scholar
  94. 97.
    Kasche V, Bergwall M (1977) In: Salmona M, Saranio M, Garattini S (eds) Insolubilized enzymes. Raven Press, New York, pp 77–86Google Scholar
  95. 98.
    Rolinson GN (1988) J Antimicrob Chemother 22:5CrossRefGoogle Scholar
  96. 99.
    Liou JK, Rousseau I (1986) Biotechnol Bioeng 28:1582CrossRefGoogle Scholar
  97. 100.
    Bailey JE, Chow MTC (1974) Biotechnol Bioeng 16:1345CrossRefGoogle Scholar
  98. 101.
    Ruckenstein E, Rajora P (1985) Biotechnol Bioeng 27:807CrossRefGoogle Scholar
  99. 102.
    ChiroCLEC-BL Information booklet (1997), Altus Biologics Inc.Google Scholar
  100. 103.
    Tischer W, Kasche V, Tibtech, submittedGoogle Scholar
  101. 104.
    Dickinson M, Fletcher PDI (1988) Enzyme Microb Technol 11:55CrossRefGoogle Scholar
  102. 105.
    Klibanov AM (1997) Trends Biotechnol 15:97CrossRefGoogle Scholar
  103. 106.
    Carrea G, Ottolina G, Riva S (1995) Tibtech 13:63Google Scholar
  104. 107.
    Tsai S-W, Dordick JS (1996) Biotechnol Bioeng 52:296CrossRefGoogle Scholar
  105. 108.
    Zacharis E, Moore B, Halling PJ (1997) J Am Chem Soc 119:12396CrossRefGoogle Scholar
  106. 109.
    Khalaf N, Govardhan CP, Lalonde JJ, Persichetti RA, Wang Y-F, Margolin AL (1996) J Am Chem Soc 118:5494CrossRefGoogle Scholar
  107. 110.
    Lilly MD, Woodley JM (1985) In: Tramper J, van der Plas HC, Linko P (eds) Biocatalysts in organic synthesis. Elsevier, Amsterdam, pp 179–191Google Scholar
  108. 111.
    Scheper T (1990) Adv Drug Delivery Rev 4:209CrossRefGoogle Scholar
  109. 112.
    Chang HN, Furusaki S (1997) Adv Biochem Eng 44:27Google Scholar
  110. 113.
    Tischer W, Giesecke U, Lang G, Röder A, Wedekind F (1992) Ann N Y Acad Sci 672:502CrossRefGoogle Scholar
  111. 114.
    Schmid RD, Verger R (1998) Angew Chem 110:1694CrossRefGoogle Scholar
  112. 115.
    Trevan M (1987) Tibtech 5:7Google Scholar
  113. 116.
    Naik SS, Karanth NG (1978) J Appl Chem Biotechnol 28:569Google Scholar
  114. 117.
    Lalonde JJ, Govardhan CP, Khalaf N, Martinez AG, Visuri KL, Margolin A (1995) J Am Chem Soc 117:6845CrossRefGoogle Scholar
  115. 118.
    Persichetti RA, Clair NLS, Griffith JP, Navia AL, Margolin AL (1995) J Am Chem Soc 117:2732CrossRefGoogle Scholar
  116. 119.
    Nagayasu T, Miyanaga M, Tanaka T, Sakiyama T, Nakanishi K (1997) Biotechnol Bioeng 43:1118CrossRefGoogle Scholar

Copyright information

© Springer Verlag Berlin Heidelberg 1999

Authors and Affiliations

  • Wilhelm Tischer
    • 1
  • Frank Wedekind
    • 2
  1. 1.Boehringer Mannheim GmbHPenzbergGermany
  2. 2.Boehringer Mannheim GmbHPenzbergGermany

Personalised recommendations