Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines


The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Schematic representation of the strategy pursued for the identification of novel TAs active towards bulky substrates.
Figure 2: Selected compounds of interest for the present study.
Figure 3: Quinonoid of compounds 1a and 2a accommodated in the active site of 3FCR.


  1. Truppo, M. D., Rozzell, J. D. & Turner, N. J. Efficient production of enantiomerically pure chiral amines at concentrations of 50 g/L using transaminases. Org. Process Res. Dev. 14, 234–237 (2010).

    CAS  Article  Google Scholar 

  2. Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

    CAS  Article  Google Scholar 

  3. Fuchs, M., Farnberger, J. E. & Kroutil, W. The industrial age of biocatalytic transamination. Eur. J. Org. Chem. 2015, 6965–6982 (2015).

    CAS  Article  Google Scholar 

  4. Steffen-Munsberg, F. et al. Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications. Biotechnol. Adv. 33, 566–604 (2015).

    CAS  Article  Google Scholar 

  5. Koszelewski, D., Tauber, K., Faber, K. & Kroutil, W. ω-Transaminases for the synthesis of non-racemic α-chiral primary amines. Trends Biotechnol. 28, 324–332 (2010).

    CAS  Article  Google Scholar 

  6. Cassimjee, K. E., Branneby, C., Vahak, A., Wells, A. & Berglund, P. Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration. Chem. Commun. 46, 5569–5571 (2010).

    CAS  Article  Google Scholar 

  7. Koszelewski, D., Lavandera, I., Clay, D., Rozzell, D. & Kroutil, W. Asymmetric synthesis of optically pure pharmacologically relevant amines employing ω-transaminases. Adv. Synth. Catal. 350, 2761–2766 (2008).

    CAS  Article  Google Scholar 

  8. Green, A. P., Turner, N. J. & O'Reilly, E. Chiral amine synthesis using ω-transaminases: an amine donor that displaces equilibria and enables high-throughput screening. Angew. Chem. Int. Ed. 53, 10714–10717 (2014).

    CAS  Article  Google Scholar 

  9. Höhne, M., Schätzle, S., Jochens, H., Robins, K. & Bornscheuer, U. T. Rational assignment of key motifs for function guides in silico enzyme identification. Nature Chem. Biol. 6, 807–813 (2010).

    Article  Google Scholar 

  10. Desai, A. A. Sitagliptin manufacture: a compelling tale of green chemistry, process intensification, and industrial asymmetric catalysis. Angew. Chem. Int. Ed. 50, 1974–1976 (2011).

    CAS  Article  Google Scholar 

  11. Han, S.-W., Park, E.-S., Dong, J.-Y. & Shin, J.-S. Mechanism-guided engineering of ω-transaminase to accelerate reductive amination of ketones. Adv. Synth. Catal. 357, 1732–1740 (2015).

    CAS  Article  Google Scholar 

  12. Sayer, C. et al. The substrate specificity, enantioselectivity and structure of the (R)-selective amine: pyruvate transaminase from Nectria haematococca. FEBS J. 281, 2240–2253 (2014).

    CAS  Article  Google Scholar 

  13. Jiang, J., Chen, X., Feng, J., Wu, Q. & Zhu, D. Substrate profile of an ω-transaminase from Burkholderia vietnamiensis and its potential for the production of optically pure amines and unnatural amino acids. J. Mol. Catal. B 100, 32–39 (2014).

    CAS  Article  Google Scholar 

  14. Nobili, A. et al. Engineering the active site of the amine transaminase from Vibrio fluvialis for the asymmetric synthesis of aryl–alkyl amines and amino alcohols. ChemCatChem 7, 757–760 (2015).

    CAS  Article  Google Scholar 

  15. Genz, M. et al. Alteration of the donor/acceptor spectrum of the (S)-amine transaminase from Vibrio fluvialis. Int. J. Mol. Sci. 16, 26953–26963 (2015).

    CAS  Article  Google Scholar 

  16. Steffen-Munsberg, F. et al. Connecting unexplored protein crystal structures to enzymatic function. ChemCatChem 5, 150–153 (2013).

    CAS  Article  Google Scholar 

  17. Middelfort, K. S. et al. Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin. Protein Eng. Des. Sel. 26, 25–33 (2013).

    Article  Google Scholar 

  18. Kaulmann, U., Smithies, K., Smith, M. E. B., Hailes, H. C. & Ward, J. M. Substrate spectrum of ω-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis. Enzyme Microb. Technol. 41, 628–637 (2007).

    CAS  Article  Google Scholar 

  19. Cassimjee, K. E., Humble, M. S., Land, H., Abedi, V. & Berglund, P. Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4′-substituted acetophenones, and follows Swain–Lupton parameterisation. Org. Biomol. Chem. 10, 5466–5470 (2012).

    CAS  Article  Google Scholar 

  20. Steffen-Munsberg, F. et al. Revealing the structural basis of promiscuous amine transaminase activity. ChemCatChem 5, 154–157 (2013).

    CAS  Article  Google Scholar 

  21. Cassimjee, K. E., Manta, B. & Himo, F. A quantum chemical study of the ω-transaminase reaction mechanism. Org. Biomol. Chem. 13, 8453–8464 (2015).

    CAS  Article  Google Scholar 

  22. Yu, H., Zhao, Y., Guo, C., Gan, Y. & Huang, H. The role of proline substitutions within flexible regions on thermostability of luciferase. Biochim. Biophys. Acta Prot. Proteomics 1854, 65–72 (2015).

    CAS  Article  Google Scholar 

  23. Deszcz, D. et al. Single active-site mutants are sufficient to enhance serine:pyruvate α-transaminase activity in an ω-transaminase. FEBS J. 282, 2512–2526 (2015).

    CAS  Article  Google Scholar 

  24. Wilke, A. et al. The M5nr: a novel non-redundant database containing protein sequences and annotations from multiple sources and associated tools. BMC Bioinformatics 13, 141 (2012).

    CAS  Article  Google Scholar 

  25. Levin, K. B. et al. Following evolutionary paths to protein–protein interactions with high affinity and selectivity. Nature Struct. Mol. Biol. 16, 1049–1055 (2009).

    CAS  Article  Google Scholar 

  26. Shafee, T., Gatti-Lafranconi, P., Minter, R. & Hollfelder, F. Handicap-recover evolution leads to a chemically versatile, nucleophile-permissive protease. ChemBioChem 16, 1866–1869 (2015).

    CAS  Article  Google Scholar 

  27. Grishin, N. V., Phillips, M. A. & Goldsmith, E. J. Modeling of the spatial structure of eukaryotic ornithine decarboxylases. Protein Sci. 4, 1291–1304 (1995).

    CAS  Article  Google Scholar 

  28. Schätzle, S., Höhne, M., Redestad, E., Robins, K. & Bornscheuer, U. T. Rapid and sensitive kinetic assay for characterization of ω-transaminases. Anal. Chem. 81, 8244–8248 (2009).

    Article  Google Scholar 

Download references


The authors thank J.F. Kabisch for preparing the M5nr database, M. Althaus and I. Duffour for developing the chiral and achiral analysis methods, J. Joerger and M. Rothe for the preparative separation of the chiral amines, C. Wyss-Gramberg for the NMR analysis of the Mosher amides, I. Menyes for support with HPLC and gas chromatography analyses and P. Meier for performing the preparative asymmetric synthesis experiments.

Author information

Authors and Affiliations



U.T.B., H.I. and B.W. initiated the study and directed the project. P.S. undertook the substrate and product syntheses. I.V.P. performed the bioinformatics analysis. I.V.P., M.S.W. and M.G. prepared and characterized all the variants. I.V.P, S.P.H. and H.I. performed the preparative asymmetric synthesis experiments. I.V.P., H.I. and U.T.B. prepared the manuscript, which was revised and approved by all authors.

Corresponding authors

Correspondence to Hans Iding or Uwe T. Bornscheuer.

Ethics declarations

Competing interests

The biocatalysis group of Roche has a committed interest over the long term in establishing a set of technically applicable TAs with broad substrate acceptance to assist devising more attractive, shorter, economical and greener synthetic routes to investigational drugs and beyond.

Supplementary information

Supplementary information

Supplementary information (PDF 1963 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pavlidis, I., Weiß, M., Genz, M. et al. Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines. Nature Chem 8, 1076–1082 (2016).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing