A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases

Schnepel C, Minges H, Frese M, Sewald N (2016)
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 55(45): 14159-14163.

Download
Es wurde kein Volltext hochgeladen. Nur Publikationsnachweis!
Zeitschriftenaufsatz | Veröffentlicht | Englisch
Abstract / Bemerkung
Biocatalytic halogenation with tryptophan halogenases is hampered by severe limitations such as low activity and stability. These drawbacks can be overcome by directed evolution, but for screening large mutant libraries, a facile high-throughput method is required. Therefore, we developed a quantitative halogenase assay based on a Suzuki-Miyaura cross-coupling towards the formation of a fluorescent aryltryptophan. The technique was optimized for application in crude E.coli lysate without intermediary purification steps, and was used for quantitatively monitoring the formation of halogenated tryptophans with high specificity by facile fluorescence screening in microtiter plates. This novel screening approach was exploited to engineer a thermostable tryptophan 6-halogenase. Libraries were constructed by error-prone PCR and selected for improved thermal resistance simply by fluorogenic cross-coupling. Our method led to an enzyme variant with substantially increased thermal stability and 2.5-fold improved activity.
Erscheinungsjahr
Zeitschriftentitel
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Band
55
Zeitschriftennummer
45
Seite
14159-14163
ISSN
eISSN
PUB-ID

Zitieren

Schnepel C, Minges H, Frese M, Sewald N. A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 2016;55(45):14159-14163.
Schnepel, C., Minges, H., Frese, M., & Sewald, N. (2016). A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 55(45), 14159-14163. doi:10.1002/anie.201605635
Schnepel, C., Minges, H., Frese, M., and Sewald, N. (2016). A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 55, 14159-14163.
Schnepel, C., et al., 2016. A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 55(45), p 14159-14163.
C. Schnepel, et al., “A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases”, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, vol. 55, 2016, pp. 14159-14163.
Schnepel, C., Minges, H., Frese, M., Sewald, N.: A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 55, 14159-14163 (2016).
Schnepel, Christian, Minges, Hannah, Frese, Marcel, and Sewald, Norbert. “A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases”. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 55.45 (2016): 14159-14163.

4 Zitationen in Europe PMC

Daten bereitgestellt von Europe PubMed Central.

Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo.
Sharma SV, Tong X, Pubill-Ulldemolins C, Cartmell C, Bogosyan EJA, Rackham EJ, Marelli E, Hamed RB, Goss RJM., Nat Commun 8(1), 2017
PMID: 28794415
Aromatic Halogenation by Using Bifunctional Flavin Reductase-Halogenase Fusion Enzymes.
Andorfer MC, Belsare KD, Girlich AM, Lewis JC., Chembiochem 18(21), 2017
PMID: 28879681

Export

Markieren/ Markierung löschen
Markierte Publikationen

Open Data PUB

Web of Science

Dieser Datensatz im Web of Science®

Quellen

PMID: 27618794
PubMed | Europe PMC

Suchen in

Google Scholar