Biotechnology Letters

, Volume 35, Issue 4, pp 585–590 | Cite as

Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina

  • Marcus Kjellander
  • Kathrin Götz
  • Josefine Liljeruhm
  • Mats Boman
  • Gunnar JohanssonEmail author
Original Research Paper


Alcohol oxidase from Pichia pastoris was immobilized on nanoporous aluminium oxide membranes by silanization and activation by carbonyldiimidazole to create a flow-through enzyme reactor. Kinetic analysis of the hydrogen peroxide generation was carried out for a number of alcohols using a subsequent reaction with horseradish peroxidase and ABTS. The activity data for the immobilized enzyme showed a general similarity with literature data in solution, and the reactor could generate 80 mmol H2O2/h per litre reactor volume. Horseradish peroxidase was immobilized by the same technique to construct bienzymatic modular reactors. These were used in both single pass mode and circulating mode. Pulsed injections of methanol resulted in a linear relation between response and concentration, allowing quantitative concentration measurement. The immobilized alcohol oxidase retained 58 % of initial activity after 3 weeks of storage and repeated use.


Alcohol oxidase Enzyme reactor Horseradish peroxidase Immobilization Kinetics Nanoporous aluminum oxide 

Supplementary material

10529_2012_1110_MOESM1_ESM.docx (19 kb)
Supplementary material 1 (DOCX 19 kb)


  1. Anderson GW, Paul RN (1958) N,N′-carbonyldiimidazole, a new reagent for peptide synthesis. J Am Chem Soc 80:4423CrossRefGoogle Scholar
  2. Ayala M, Pickard MA, Vazquez-Duhalt R (2008) Fungal enzymes for environmental purposes, a molecular biology challenge. J Mol Microbiol Biotechnol 15:172–180PubMedCrossRefGoogle Scholar
  3. Badir N (2009) Nanoporous alumina based bioreactor. Magister thesis, Uppsala University, UppsalaGoogle Scholar
  4. Barsan MM, Brett CMA (2008) An alcohol oxidase biosensor using PNR redox mediator at carbon film electrodes. Talanta 74:505–1510CrossRefGoogle Scholar
  5. Beeckmans S (1999) Chromatographic methods to study protein–protein interactions. Methods 19:278–305PubMedCrossRefGoogle Scholar
  6. Chen XB, Sui Y, Cheng YP, Lee HP, Yu P, Winoto SH, Low HT (2010) Mass transport in a microchannel enzyme reactor with a porous wall hydrodynamic modeling and applications. Biotechnol Bioeng 52:227–235Google Scholar
  7. Couderc R, Baratti J (1980) Oxidation of methanol by the yeast, Pichia pastoris. Purification and properties of the alcohol oxidase. Agric Biol Chem 44(10):2279–2289CrossRefGoogle Scholar
  8. Hearn MTW (1987) 1,1′-Carbonyldiimidazole-mediated immobilization of enzymes and affinity ligands. Methods Enzymol 135:102–117PubMedCrossRefGoogle Scholar
  9. Jian G, Pei-Sheng M, Gao-Xiang L (1990) Immobilization of glucose oxidase and peroxidase and their application in flow-injection analysis for glucose in serum. Appl Biochem Biotechnol 23:15–24CrossRefGoogle Scholar
  10. Katchalski-Katzir E (1993) Immobilized enzymes-learning from past successes and failures. Trends Biotechnol 11:471–478PubMedCrossRefGoogle Scholar
  11. Kato N, Omori Y, Tani Y, Ogata K (1976) Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha catalytic properties and subunit structures. Eur J Biochem 64:341–350PubMedCrossRefGoogle Scholar
  12. Mangos TJ, Haas MJ (1996) Enzymatic determination of methanol with alcohol oxidase, peroxidase, and the chromogen 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and its application to the determination of the methyl ester content of pectins. J Agric Food Chem 44:2977–2981CrossRefGoogle Scholar
  13. Mille C (2007) Biochemical applications of nanoporous alumina. Magister thesis, Uppsala University, UppsalaGoogle Scholar
  14. Oliveira GB, Lima JL, Chaves MEC, Azevedo WM, Carvalho LB (2008) Enzyme immobilization on anodic aluminum oxide/polyethyleneimine or polyaniline composites. React Funct Polym 68:27–32CrossRefGoogle Scholar
  15. Palopää D (2007) Biocatalysis in nanoporous AAO membranes. Master of Technology thesis, Uppsala University, UppsalaGoogle Scholar
  16. Patel RN, Hou CT, Laskin AI, Derelanko P (1981) Microbial oxidation of methanol: properties of crystallized alcohol oxidase from a yeast, Pichia sp. Arch Biochem Biophys 210:481–488PubMedCrossRefGoogle Scholar
  17. Schneider JJ, Engstler J (2006) Carbon and polymer filaments in nanoporous alumina. Eur J Inorg Chem 9:1723–1736CrossRefGoogle Scholar
  18. Staab HA (1957) Reaktionsfahige n-carbonsaureester und n-carbonsaureamide des imidazols und triazols. Liebigs Ann 609:83–88CrossRefGoogle Scholar
  19. Stroeve P, Ileri N (2011) Biotechnical and other applications of nanoporous membranes. Trends Biotechnol 29(6):259–266PubMedCrossRefGoogle Scholar
  20. Tanvir S, Pantigny J, Boulnois P, Pulvin S (2009) Covalent immobilization of recombinant human cytochrome CYP2E1 and glucose-6-phosphate dehydrogenase in alumina membrane for drug screening applications. J Membr Sci 329:85–90CrossRefGoogle Scholar
  21. Yang ZP, Si SH, Dai HJ, Zhang CJ (2007) Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes. Biosens Bioelectron 22:3283–3287PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Marcus Kjellander
    • 1
  • Kathrin Götz
    • 1
  • Josefine Liljeruhm
    • 1
  • Mats Boman
    • 2
  • Gunnar Johansson
    • 1
    Email author
  1. 1.Department of Chemistry-BMCUppsala UniversityUppsalaSweden
  2. 2.Department of Chemistry-ÅngströmUppsala UniversityUppsalaSweden

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