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The angle of a phenol side-chain decides regio- and enantioselectivity in Rhodococcus ruber alcohol dehydrogenase A
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.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
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(English)Article in journal (Other academic) Submitted
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-318982OAI: oai:DiVA.org:uu-318982DiVA: diva2:1085657
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2017-04-01
In thesis
1. Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity
Open this publication in new window or tab >>Characterization and Directed Evolution of an Alcohol Dehydrogenase: A Study Towards Understanding of Three Central Aspects of Substrate Selectivity
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many different chemicals are used in the everyday life, like detergents and pharmaceuticals. However, their production has a big impact on health and environment as much of the raw materials are not renewable and the standard ways of production in many cases includes toxic and environmentally hazardous components. As the population and as the life standard increases all over the planet, the demand for different important chemicals, like pharmaceuticals, will increase. A way to handle this is to apply the concept of Green chemistry, where biocatalysis, in the form of enzymes, is a very good alternative. Enzymes do not normally function in industrial processes and needs modifications through protein engineering to cope in such conditions. To be able to efficiently improve an enzyme, there is a need to understand the mechanism and characteristics of that enzyme.

Acyloins (α-hydroxy ketones) are important building blocks in the synthesis of pharmaceuticals. In this thesis, the enzyme alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber has been in focus, as it has been shown to display a wide substrate scope, also accepting aryl-substituted alcohols. The aim has been to study the usefulness of ADH-A as a biocatalyst towards production of acyloins and its activity with aryl-substituted vicinal diols and to study substrate-, regio-, and enantioselectivity of this enzyme.

This thesis is based on four different papers where the focus of the first has been to biochemically characterize ADH-A and determine its mechanism, kinetics and its substrate-, regio-, and enantioselectivity. The second and third paper aims towards deeper understanding of some aspects of selectivity of ADH-A. Non-productive binding and its importance for enantioselectivity is studied in the second paper by evolving ADH-A towards increased activity with the least favored enantiomer through protein engineering. In the third paper, regioselectivity is in focus, where an evolved variant displaying reversed regioselectivity is studied. In the fourth and last paper ADH-A is studied towards the possibility to increase its activity towards aryl-substituted vicinal diols, with R-1-phenyl ethane-1,2-diol as the model substrate, and the possibility to link ADH-A with an epoxide hydrolase to produce acyloins from racemic epoxides.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 95 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1497
Keyword
Alcohol dehydrogenase, ADH-A, Biocatalysis, Directed evolution, Enantioselectivity, Enzyme kinetics, Enzyme mechanisms, Protein Engineering, Regioselectivity, Substrate selectivity
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-318984 (URN)978-91-554-9875-7 (ISBN)
Public defence
2017-05-19, BMC A1:111A, Husargatan 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2017-04-28 Created: 2017-03-30 Last updated: 2017-05-05

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