Topics in Catalysis

, Volume 57, Issue 5, pp 339–348 | Cite as

Cryostructured and Crosslinked Viable Cells Forming Monoliths Suitable for Bioreactor Applications

  • Oksana Zaushitsyna
  • Dmitriy Berillo
  • Harald Kirsebom
  • Bo MattiassonEmail author
Original Paper


Applications of immobilized biocatalysts in both research and industry require highly active catalysts, preferably at a low cost. In this study, cryogels with high catalyst density were produced through cryostructuration of whole Escherichia coli (E.coli) cells. Prepared cryogels are macroporous materials composed of metabolically active cells crosslinked to each other via polymeric structures. Different macromolecular reagents: oxidized dextran (OxDex), polyvinyl alcohol (PVA) and polyethyleneimine (PEI), the two latter activated with glutaraldehyde (GTA) have been synthesized. Prepared polymers were tested as effective and mild crosslinkers for cells during the cryostructuration procedure. Combination of the two synthetic polymers: PEI+GTA and PVA+GTA was found most suitable for formation of macroporous and stable structures from cells without any toxic effect on them. About 90 % of β-glucosidase activity in cells was retained after crosslinking with a combination of synthetic polymers, whereas E. coli crosslinked using GTA showed complete loss of activity. Preserved viability of cells in cryogel offers possibility to induce protein expression in cells after crosslinking. For β-glucosidase induction post immobilization yielded 50 % activity of that from cells induced in free form before cryo-structurization. The results of the post immobilization studies indicate an interesting potential for handling very sensitive enzymes.


Crosslinked cells Cryogel Macromolecular crosslinkers Cell immobilization Enzyme activity 



To funding of Marie Curie Networks for Initial Training fellowship in the BIOTRAINS project (FP7-PEOPLE-ITN-2008-238531) and the financial support from Ångpanneföreningens Foundation for Research and Development is gratefully acknowledged.


  1. 1.
    Straathof AJJ, Panke S, Schmid A (2002) The production of fine chemicals by biotransformations. Curr Opin Biotechnol 13(6):548–556CrossRefGoogle Scholar
  2. 2.
    Liese A, Villela Filho M (1999) Production of fine chemicals using biocatalysis. Curr Opin Biotechnol 10(6):595–603CrossRefGoogle Scholar
  3. 3.
    Cao L (2005) Immobilised enzymes: science or art? Curr Opin Chem Biol 9(2):217–226CrossRefGoogle Scholar
  4. 4.
    Cao L, Langen LV, Sheldon RA (2003) Immobilised enzymes: carrier-bound or carrier-free? Curr Opin Biotechnol 14(4):387–394CrossRefGoogle Scholar
  5. 5.
    Tischer W, Kasche V (1999) Immobilized enzymes: crystals or carriers? Trends Biotechnol 17(8):326–335CrossRefGoogle Scholar
  6. 6.
    Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym Microb Technol 40(6):1451–1463CrossRefGoogle Scholar
  7. 7.
    Brady D, Jordaan J (2009) Advances in enzyme immobilisation. Biotechnol Lett 31(11):1639–1650. doi: 10.1007/s10529-009-0076-4 CrossRefGoogle Scholar
  8. 8.
    Sheldon RA (2007) Enzyme immobilization: the quest for optimum performance. Adv Synth Catal 349(8–9):1289–1307CrossRefGoogle Scholar
  9. 9.
    Bickerstaff G Jr (1997) Immobilization of enzymes and cells. In: Bickerstaff G (ed) Immobilization of enzymes and cells. Methods in biotechnology, vol 1. Humana Press, Totowa, pp 1–11CrossRefGoogle Scholar
  10. 10.
    Sheldon RA (2010) Cross-linked enzyme aggregates as industrial biocatalysts. Org Process Res Dev 15(1):213–223CrossRefGoogle Scholar
  11. 11.
    Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom APG, van Rantwijk F, van der Wielen LAM, Sheldon RA (2004) Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 87(6):754–762CrossRefGoogle Scholar
  12. 12.
    Svec F, Fréchet JMJ (1996) New designs of macroporous polymers and supports: from separation to biocatalysis. Science 273(5272):205–211CrossRefGoogle Scholar
  13. 13.
    Kirsebom H, Mattiasson B, Galaev IY (2009) Building macroporous materials from microgels and microbes via one-step cryogelation. Langmuir 25(15):8462–8465CrossRefGoogle Scholar
  14. 14.
    Plieva FM, Karlsson M, Aguilar M-R, Gomez D, Mikhalovsky S, Galaev IY (2005) Pore structure in supermacroporous polyacrylamide based cryogels. Soft Matter 1(4):303–309CrossRefGoogle Scholar
  15. 15.
    Lozinsky V, Plieva F, Galaev I, Mattiasson B (2001) The potential of polymeric cryogels in bioseparation. Bioseparation 10(4–5):163–188CrossRefGoogle Scholar
  16. 16.
    Lozinsky VI, Zubov AL, Titova EF (1997) Poly(vinyl alcohol) cryogels employed as matrices for cell immobilization. 2. Entrapped cells resemble porous fillers in their effects on the properties of PVA-cryogel carrier. Enzyme Microb Technol 20(3):182–190CrossRefGoogle Scholar
  17. 17.
    Bečka S, Škrob F, Plháčková K, Kujan P, Holler P, Kyslík P (2003) Cross-linked cell aggregates of trigonopsis variabilis: D-amino acid oxidase catalyst for oxidation of cephalosporin C. Biotechnol Lett 25(3):227–233CrossRefGoogle Scholar
  18. 18.
    Šulek F, Fernández DP, Knez Ž, Habulin M, Sheldon RA (2011) Immobilization of horseradish peroxidase as crosslinked enzyme aggregates (CLEAs). Process Biochem 46(3):765–769CrossRefGoogle Scholar
  19. 19.
    Mateo C, Palomo JM, van Langen LM, van Rantwijk F, Sheldon RA (2004) A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates. Biotechnol Bioeng 86(3):273–276CrossRefGoogle Scholar
  20. 20.
    Sloan JW, Alexander BH, Lohmar RL, Wolff IA, Rist CE (1954) Determination of dextran structure by periodate oxidation techniques. J Am Chem Soc 76(17):4429–4434CrossRefGoogle Scholar
  21. 21.
    Schoevaart R, Siebum A, van Rantwijk F, Sheldon R, Kieboom T (2005) Glutaraldehyde cross-link analogues from carbohydrates. Starch Stärke 57(3–4):161–165CrossRefGoogle Scholar
  22. 22.
    Aragão Börner R, Zaushitsyna O, Berillo D, Scaccia N, Mattiasson B, Kirsebom H (2013) Immobilization of Clostridium acetobutylicum DSM 792 as macroporous aggregates through cryogelation for butanol production. Process Biochem (in press)Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Oksana Zaushitsyna
    • 1
  • Dmitriy Berillo
    • 1
  • Harald Kirsebom
    • 1
  • Bo Mattiasson
    • 1
    Email author
  1. 1.Department of Biotechnology, Center for Chemistry and Chemical EngineeringLund UniversityLundSweden

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