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Angew Chem Int Ed Engl. 2016 Nov 2;55(45):14159-14163. doi: 10.1002/anie.201605635. Epub 2016 Sep 13.

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

Author information

1
Organische und Bioorganische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
2
Organische und Bioorganische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany. norbert.sewald@uni-bielefeld.de.

Abstract

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.

KEYWORDS:

cross-coupling; directed evolution; halogenases; high-throughput screening; one-pot processes

PMID:
27618794
DOI:
10.1002/anie.201605635
[Indexed for MEDLINE]

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