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Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0003-3073-5641
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
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2015 (English)In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 357, no 8, 1721-1731 p.Article in journal (Refereed) Published
Abstract [en]

omega-Transaminases are a valuable class of enzymes for the production of chiral amines with either (R)- or (S)-configuration in high optical purity and 100% yield by the biocatalytic reductive amination of prochiral ketones. A versatile new assay was developed to quantify omega-transaminase activity for the kinetic characterization and enantioselectivity typing of novel or engineered enzymes based on the conversion of 1-(6-methoxynaphth-2-yl)alkylamines. The associated release of the acetonaphthone product can be monitored by the development of its bright fluorescence at 450 nm with very high sensitivity and selectivity. The assay principle can be used to quantify omega-transaminase catalysis over a very broad range of enzyme activity. Because of its simplicity and low substrate consumption in microtiter plate format the assay seems suitable for liquid screening campaigns with large library sizes in the directed evolution of optimized transaminases. For assay substrates that incorporate structural variations, an efficient modular synthetic route was developed. This includes racemate resolution by lipase-catalyzed transacylation to furnish enantiomerically pure (R)and (S)-configured amines. The latter are instrumental for the rapid enantioselectivity typing of omega-transaminases. This method was used to characterize two novel (S)-selective taurine-pyruvate transaminases of the subtype 6a from thermophilic Geobacillus thermodenitrificans and G. thermoleovorans.

Place, publisher, year, edition, pages
John Wiley & Sons, 2015. Vol. 357, no 8, 1721-1731 p.
Keyword [en]
biocatalysis, chiral amines, high-throughput screening, protein engineering, reductive amination
National Category
Chemical Sciences
URN: urn:nbn:se:kth:diva-172236DOI: 10.1002/adsc.201500215ISI: 000355235700013ScopusID: 2-s2.0-84930226708OAI: diva2:848444

QC 20150825

Available from: 2015-08-25 Created: 2015-08-14 Last updated: 2016-10-17Bibliographically approved
In thesis
1. Amine Transaminases in Biocatalytic Amine Synthesis
Open this publication in new window or tab >>Amine Transaminases in Biocatalytic Amine Synthesis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of enzymes, nature´s own catalysts, both isolated or as whole cells to perform chemical transformations is called biocatalysis. As a complement to classical chemical catalysis, biocatalysis can be an environmentally friendly and more economical option in the production and synthesis of chemicals. Research on the application of amine transaminases in synthesis of chiral amines have exploded over the last two decades and interest from the industry is increasing. Amine transaminases are promising catalysts due to their ability to perform reductive amination of ketones with excellent enantioselectivity.

For a process to be efficient, high substrate specificity of the applied enzyme is an important factor. A variant of Chromobacterium violaceum amine transaminase that was obtained through rational design has an increased specific activity toward (S)-1-phenylethylamine and a set of 4´-substituted acetophenones. This result makes this variant a promising catalyst for the asymmetric synthesis of similar amines.

Amine transaminase catalyzed asymmetric synthesis of amines generally suffers from unfavorable equilibrium. Two methods that include spontaneous tautomerization and biocatalytic amidation for equilibrium displacement have therefore been developed.

Efficient assays and screening methods are demanded for the discovery and development of novel amine transaminases. For this purpose, a sensitive fluorescence-based assay that holds promise as a high-throughput screening method was developed.

One of the major obstacles for application of enzymes in industrial processes is the instability of the enzyme toward harsh conditions. The stability of Chromobacterium violaceum amine transaminase was investigated and improved using co-solvents and other additives. Co-lyophilization with surfactants was also applied to improve the performance of the same enzyme in organic solvents.

Place, publisher, year, edition, pages
Stockholm: Henrik Land, 2016. 101 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:18
Amine Transaminase, Biocatalysis, Transamination, Reductive Amination, Enzyme, Enzyme Engineering, Equilibrium Displacement, Screening, Enzyme Stability
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-194112 (URN)978-91-7729-164-0 (ISBN)
Public defence
2016-11-25, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (English)

QC 20161017

Available from: 2016-10-17 Created: 2016-10-17 Last updated: 2016-10-17Bibliographically approved

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Land, HenrikAnderson, MattiasBerglund, Per
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