Journal of Biotechnology 159 (2012) 188–194 Contents lists available at SciVerse ScienceDirect Journal of Biotechnology j ourna l ho me pag e: www.elsevier.com/locate/jbiotec TTC-based screening assay for -transaminases: A rapid method to detect reduction of 2-hydroxy ketones Torsten Sehl a , Robert C. Simon a,1 , Helen C. Hailes b , John M. Ward c , Ursula Schell c,2 , Martina Pohl a , Dörte Rother a,∗ a Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Leo-Brandt-Str. 1, 52425 Jülich, Germany b Department of Chemistry, University College London, 20 Gordon Street, London WC1H OAJ, UK c Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1H 6BT, UK a r t i c l e i n f o Article history: Received 11 October 2011 Received in revised form 22 December 2011 Accepted 23 December 2011 Available online 2 January 2012 Keywords: -Transaminase Chiral amino alcohols Asymmetric reductive amination Colourimetric screening assay TTC 2-Hydroxy ketones a b s t r a c t A rapid TTC-based screening assay for -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 spectrophotomet- rically 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 -transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)--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 -transaminases have been identified. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Chiral vicinal amino alcohols are versatile building blocks for organic synthesis, as ligands and chiral auxiliaries (Ager et al., 1996; Fehr and Galindo, 1996) or as synthons for pharmaceu- tically active compounds (Bergmeier, 2000), such as drugs like ephedrine and norephedrine, the sphingosine derivative sulfobacin B (Kamiyama et al., 1995), the HIV protease inhibitor saquinavir (Ohta and Shinkai, 1997) and the aminopeptidase inhibitor bestatin (Nakamura et al., 1976; Umezawa et al., 1976). Vicinal amino alco- hols can be synthesised by a reductive amination of 2-hydroxy ketones. Numerous substituted 2-hydroxy ketones are accessible biocatalytically in highly enantiopure forms (Hoyos et al., 2009; Müller et al., 2009). Recently, the biocatalytic reductive amination of prochiral ketones by -transaminases (-TAs) has gained considerable ∗ Corresponding author. Tel.: +49 2461616772; fax: +49 2461613870. E-mail address: do.rother@fz-juelich.de (D. Rother). 1 Present address: Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, 8010 Graz, Austria. 2 Present address: Biopharm, Process Research, GlaxoSmithKline Plc., Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK. interest (Höhne and Bornscheuer, 2009; Hopwood et al., 2011; Savile et al., 2010; Ward and Wohlgemuth, 2010). Transaminases with high turnover numbers and excellent selectivities have been described (Koszelewski et al., 2010; Schätzle et al., 2011; Tufvesson et al., 2011). In addition, a few -TAs reactions have already been successfully implemented into industrial processes at a multi ton scales, e.g. at Cambrex (Scarlato, 2009) and DSM (DSM, 2011). Beside reaction engineering strategies, enzymes with improved affinities towards substrates, reduced substrate and/or product inhibition, as well as activities against novel compounds need to be identified for further applications (Hopwood et al., 2011; Koszelewski et al., 2010; Ward and Wohlgemuth, 2010). Therefore, efficient and rapid screening assays need to be developed in order to screen newly discovered enzymes and mutant libraries. Previously, -TAs screening assays with various advantages and disadvantages depending on their respective application have been reported. Among these the quantification of the reaction from pyruvate to alanine by the formation of coloured Cu-alanine com- plexes has been described (Hwang and Kim, 2004). In other assays the accumulating pyruvate is detected by multienzyme cascades based on either a pH-shift (Truppo et al., 2009) or a colourimet- ric oxidase–peroxidase reaction (Hopwood et al., 2011). Further, a spectrophotometric assay based on the activity towards (S)-- methylbenzylamine (MBA) has been published (Schätzle et al., 0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.12.023