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Substrate Promiscuity in In-Vitro Evolved Alcohol Dehydrogenases
(Widersten)ORCID iD: 0000-0001-5915-1514
(Widersten)
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-395439OAI: oai:DiVA.org:uu-395439DiVA, id: diva2:1362302
Available from: 2019-10-18 Created: 2019-10-18 Last updated: 2019-10-21
In thesis
1. Engineered Alcohol Dehydrogenases for Stereoselective Chemical Transformations
Open this publication in new window or tab >>Engineered Alcohol Dehydrogenases for Stereoselective Chemical Transformations
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enzymes are biomolecules built from amino acids and catalyze the chemical transformations in a cell. Enzymes are by nature stereoselective, biodegradable, environmentally friendly, and can perform catalysis in aqueous solutions and at ambient temperatures. Due to these advantages the use of enzymes as biocatalysts for chemical transformations has emerged as an attractive “greener” alternative to conventional chemical synthesis strategies. And, if naturally occurring enzymes cannot carry out the desired chemical transformations, the functional properties of enzymes can be modified by directed evolution or protein engineering techniques. Since enzymes are genetically encoded they can be optimized for desired traits such as substrate selectivity or improved catalytic efficiency. Considering these advantages and also keeping the synthetic and industrial application in mind, we have employed alcohol dehydrogenase-A (ADH-A) from Rhodococcus ruber DSM 44541 as a study object in engineering for new catalytic properties. ADH-A tolerates water miscible organic solvents, accepts a relatively wide range of aromatic sec-alcohols/ketones as substrates and is therefore a potentially useful biocatalyst for asymmetric synthesis of organic compounds.

 

Presented research work in this thesis has been primarily focused on engineering of ADH-A and characterization of resulting enzyme variants. The engineering efforts have aimed for altered substrate scope, as well as stereo- and regioselectivities. Furthermore, possible substrate promiscuity in engineered enzyme variants has also been addressed. In short, i). Paper I: three sub sites, each consisting of two-three amino acid residues within the active-site cavity were exposed to saturation mutagenesis in step-wise manner, coupled to an in vitro selection for improved catalytic activity with the unfavored (R)-1-phenylethanol. The observed stereoselectivity could be explained partly by a shift in nonproductive substrate binding. ii). Paper II is aimed specifically towards the improving the catalytic activity with aryl-substituted vicinal diols, such as (R)-1-phenylethane-1,2-diol, and the possibility to link the ADH-A reaction with a preceding epoxide hydrolysis to produce the acyloin 2-hydroxyacetophenone from rac-styrene oxide. iii). Paper III is mainly focused towards studies of regioselectivity. Here, ADH-A and engineered variants were challenged with a substrate containing two sec-alcohol functions and the cognate di-ketone. The regioselectivity in wild type as well as in engineered variants could in part be explained by a combination of experimental and computer simulations. iv). Paper IV is focused on elucidating possible effects on substrate promiscuities in engineered variants as compared to the wild type parent enzyme, when challenged with a spectrum of potential previously untested substrates.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 79
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1872
Keywords
alcohol dehydrogenase-A, biocatalysts, protein engineering, enzyme kinetics, sec-alcohols, ketones, stereoselectivity, regioselectivity, substrate selectivity and promiscuity.
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-395527 (URN)978-91-513-0788-6 (ISBN)
Public defence
2019-12-06, A1:107a, BMC (Biomedicinskt centrum), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-11-15 Created: 2019-10-21 Last updated: 2019-11-18

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