TTC-based screening assay for ω-transaminases: A rapid method to detect reduction of 2-hydroxy ketones
A rapid TTC-based screening assay for ω-transaminases was developed to determine the conversion of substrates with a 2-hydroxy ketone motif. Oxidation of the compounds in the presence of 2,3,5-triphenyltetrazolium chloride (TTC) results in a reduction of the colourless TTC to a red-coloured 1,3,5-triphenylformazan. The enzymatic reductive amination of a wide range of various aliphatic, aliphatic–aromatic and aromatic–aromatic 2-hydroxy ketones can be determined by the decrease of the red colouration due to substrate consumption. The conversion can be quantified spectrophotometrically at 510 nm based on reactions, e.g. with crude cell extracts in 96-well plates. Since the assay is independent of the choice of diverse amine donors a panel of ω-transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)-α-methylbenzylamine and benzylamine. The results could be validated using HPLC and GC analyses, showing a deviation of only 5–10%. Using this approach enzymes were identified demonstrating high conversions of acetoin and phenylacetylcarbinol to the corresponding amines. Among these enzymes three novel wild-type ω-transaminases have been identified.
► We established a novel colourimetric TTC-based screening assay for ω-transaminases. ► The conversion of various substrates with a 2-hydroxy ketone motif can be quantified. ► Works with crude cell extract and is independent on the desired amine donor. ► High reliability of the assay is proven by GC- and HPLC analytics. ► A panel of ω-transaminases with different amine donors and amine acceptors was screened. ► Three novel wild-type ω-transaminases were identified.
Journal: Journal of Biotechnology - Volume 159, Issue 3, 15 June 2012, Pages 188–194