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A flow-through trypsin bioreactor for peptide fingerprinting based on nanoporous alumina.
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 - BMC.
(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 [en]
nanoporous aluminum oxide, immobilization, trypsin, peptide mass fingerprinting, enzyme stability
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-210118OAI: oai:DiVA.org:uu-210118DiVA: diva2:661109
Available from: 2013-10-31 Created: 2013-10-31 Last updated: 2014-01-23
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1098
Keyword
nanoporous aluminum oxide, immobilized enzymes, bioreactor
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
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)
Opponent
Supervisors
Available from: 2013-11-21 Created: 2013-10-31 Last updated: 2014-01-23

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