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Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.ORCID-id: 0000-0003-3073-5641
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
Vise andre og tillknytning
2015 (engelsk)Inngår i: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 357, nr 8, s. 1721-1731Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
John Wiley & Sons, 2015. Vol. 357, nr 8, s. 1721-1731
Emneord [en]
biocatalysis, chiral amines, high-throughput screening, protein engineering, reductive amination
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-172236DOI: 10.1002/adsc.201500215ISI: 000355235700013Scopus ID: 2-s2.0-84930226708OAI: oai:DiVA.org:kth-172236DiVA, id: diva2:848444
Merknad

QC 20150825

Tilgjengelig fra: 2015-08-25 Laget: 2015-08-14 Sist oppdatert: 2022-06-23bibliografisk kontrollert
Inngår i avhandling
1. Amine Transaminases in Biocatalytic Amine Synthesis
Åpne denne publikasjonen i ny fane eller vindu >>Amine Transaminases in Biocatalytic Amine Synthesis
2016 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Henrik Land, 2016. s. 101
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:18
Emneord
Amine Transaminase, Biocatalysis, Transamination, Reductive Amination, Enzyme, Enzyme Engineering, Equilibrium Displacement, Screening, Enzyme Stability
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-194112 (URN)978-91-7729-164-0 (ISBN)
Disputas
2016-11-25, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20161017

Tilgjengelig fra: 2016-10-17 Laget: 2016-10-17 Sist oppdatert: 2022-06-27bibliografisk kontrollert

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