Fluorescence-Based Kinetic Assay for High-Throughput Discovery and Engineering of Stereoselective omega-Transaminases
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
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.
biocatalysis, chiral amines, high-throughput screening, protein engineering, reductive amination
IdentifiersURN: urn:nbn:se:kth:diva-172236DOI: 10.1002/adsc.201500215ISI: 000355235700013ScopusID: 2-s2.0-84930226708OAI: oai:DiVA.org:kth-172236DiVA: diva2:848444
QC 201508252015-08-252015-08-142016-10-17Bibliographically approved