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Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Inorganic Chemistry.
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2013 (English)In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 35, no 4, 585-590 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2013. Vol. 35, no 4, 585-590 p.
Keyword [en]
Alcohol oxidase, Enzyme reactor, Horseradish peroxidase, Immobilization, Kinetics, Nanoporous aluminum oxide
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-197956DOI: 10.1007/s10529-012-1110-5ISI: 000316081800015OAI: oai:DiVA.org:uu-197956DiVA: diva2:615103
Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2014-01-23Bibliographically approved
In thesis
1. Nanoporous Aluminum Oxide – A Promising Support for Modular Enzyme Reactors
Open this publication in new window or tab >>Nanoporous Aluminum Oxide – A Promising Support for Modular Enzyme Reactors
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nanoporous alumina is a rather newly characterized material that so far has found limited use in the construction of bioreactors. The material has many advantages compared to conventional immobilization matrices. I have investigated its use in flow-through bioreactors. The rigidity and porous structure of the material makes it an excellent choice for multienzyme reactor construction. The total activity in a reactor is easily controlled by the number of membranes since the porosity makes the material less prone to increase flow system pressure. This bioreactor is suitable for characterization of new enzymes since the amount of immobilized enzyme is standardized and the enzyme may be reused many times.

We designed a simple stepwise technique for covalent immobilization on this matrix in a monolayer to minimize mass transfer effects in the reactor function. The kinetic parameters for ten different substrates were investigated for immobilized alcohol oxidase and, as a second step, a two-step reactor was also designed by addition of horseradish peroxidase. This bienzymatic reactor was, in turn, employed for measuring injected alcohol concentrations. The use of the matrix for substrate specificity screening was proven for two new epsilon-class glutathione transferases from Drosophila melanogaster. Immobilized trypsin showed a substantially prolonged lifetime and its potential use as an on-line digestion unit for peptide mass fingerprinting was also demonstrated. Finally, I investigated the immobilization of the model enzyme lactate dehydrogenase by adsorption mediated by metal ion chelation similar to IMAC. Regeneration was here possible multiple times without loss of capacity. In conclusion, immobilization of enzymes on nanoporous alumina is a convenient way to characterize, stabilize and reuse enzymes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 51 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1098
nanoporous aluminum oxide, immobilized enzymes, bioreactor
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:uu:diva-210120 (URN)978-91-554-8807-9 (ISBN)
Public defence
2013-12-13, Sal: B42, BMC, Husargatan 3, Uppsala, 14:00 (English)
Available from: 2013-11-21 Created: 2013-10-31 Last updated: 2014-01-23

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Kjellander, MarcusLiljeruhm, JosefineBoman, MatsJohansson, Gunnar
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BiochemistryDepartment of Chemistry - BMCInorganic Chemistry
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