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.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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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.
Stichworte
cross-coupling; directed evolution; halogenases; high-throughput; screening; one-pot processes
Erscheinungsjahr
2016
Zeitschriftentitel
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Band
55
Ausgabe
45
Seite(n)
14159-14163
ISSN
1433-7851
eISSN
1521-3773
Page URI
https://pub.uni-bielefeld.de/record/2907432

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.
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