A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases.
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.
Organische und Bioorganische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
Christian
C
Schnepel
Organische und Bioorganische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
Hannah
H
Minges
Organische und Bioorganische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.
Marcel
M
Frese
Organische und Bioorganische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany. norbert.sewald@uni-bielefeld.de.
Norbert
0000-0002-0309-2655
N
Sewald
D004926:Escherichia coli / Q000201:enzymology; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D015394:Molecular Structure; D010088:Oxidoreductases / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D014364:Tryptophan; D010088:Oxidoreductases; C407363:tryptophan halogenase
cross-coupling; directed evolution; halogenases; high-throughput screening; one-pot processes