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Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening and stability
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Department of Neurochemistry, Stockholm University, SE-10691, Sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Inorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
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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.

Place, publisher, year, edition, pages
2014. Vol. 446, 59-63 p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-210099DOI: 10.1016/j.ab.2013.10.004ISI: 000329949500010OAI: oai:DiVA.org:uu-210099DiVA: diva2:660998
Available from: 2013-10-31 Created: 2013-10-31 Last updated: 2014-02-20Bibliographically 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, MarcusBoman, MatsMannervik, BengtJohansson, Gunnar
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