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Nanoporous Aluminum Oxide – A Promising Support for Modular Enzyme Reactors
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1098
Keyword [en]
nanoporous aluminum oxide, immobilized enzymes, bioreactor
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-210120ISBN: 978-91-554-8807-9 (print)OAI: oai:DiVA.org:uu-210120DiVA: diva2:661111
Public defence
2013-12-13, Sal: B42, BMC, Husargatan 3, Uppsala, 14:00 (English)
Opponent
Supervisors
Available from: 2013-11-21 Created: 2013-10-31 Last updated: 2014-01-23
List of papers
1. Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina
Open this publication in new window or tab >>Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina
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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.

Keyword
Alcohol oxidase, Enzyme reactor, Horseradish peroxidase, Immobilization, Kinetics, Nanoporous aluminum oxide
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-197956 (URN)10.1007/s10529-012-1110-5 (DOI)000316081800015 ()
Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2017-12-06Bibliographically approved
2. Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening and stability
Open this publication in new window or tab >>Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening and stability
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2014 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, 59-63 p.Article in journal (Refereed) Published
Abstract [en]

The previously uncharacterized Drosophila melanogaster Epsilon class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ∊-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that for enzymes in solution and no leakage of enzyme from the matrix could be observed.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-210099 (URN)10.1016/j.ab.2013.10.004 (DOI)000329949500010 ()
Available from: 2013-10-31 Created: 2013-10-31 Last updated: 2017-12-06Bibliographically approved
3. A flow-through trypsin bioreactor for peptide fingerprinting based on nanoporous alumina.
Open this publication in new window or tab >>A flow-through trypsin bioreactor for peptide fingerprinting based on nanoporous alumina.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Trypsin was immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptide mass fingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groups were activated with carbonyl diimidazole to allow coupling of trypsin via e-amino groups. The function was assessed for the artificial substrate BAPA, bovine ribonuclease A and bovine serum albumin. The ribonuclease A was correctly identified from the peptide pattern by a Mascot database search. The activity in a 10-membrane stack was in the range required for on-line ESI-MS peptide mass fingerprinting. The reactor was found to retain 76% of the initial activity after 14 days of storage and repeated use at room temperature.

Keyword
nanoporous aluminum oxide, immobilization, trypsin, peptide mass fingerprinting, enzyme stability
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-210118 (URN)
Available from: 2013-10-31 Created: 2013-10-31 Last updated: 2014-01-23
4. In situ generation of IDA groups on nanoporous alumina for reversible immobilization of enzymes and other biomolecules.
Open this publication in new window or tab >>In situ generation of IDA groups on nanoporous alumina for reversible immobilization of enzymes and other biomolecules.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) ions allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis-Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-210119 (URN)
Available from: 2013-10-31 Created: 2013-10-31 Last updated: 2014-01-23

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