A High‐Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases. Issue 45 (13th September 2016)

• Record Type: Journal Article
• Authors: Schnepel, Christian; Minges, Hannah; Frese, Marcel; Sewald, Norbert
• Is Part Of: Angewandte Chemie international edition. Volume 55:Issue 45(2016)
• Page Start: 14159
• Page End: 14163
• Publication Date: 2016-09-13
• Subjects: cross-coupling -- directed evolution -- halogenases -- high-throughput screening -- one-pot processes; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201605635
• Languages: English
• ISSNs: 1433-7851

Abstract

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. Abstract : A needle in a haystack : The synthesis of a fluorogenic aryltryptophan by Suzuki–Miyaura coupling enabled the specific detection of 5‐, 6‐ and 7‐brominated tryptophan in E. coli lysate. This reaction constituted the basis for a high‐throughput assay to determine the activity of tryptophan halogenases in protein engineering, which yielded a thermostable tryptophan 6‐halogenase variant with higher activity and thermostability.