A Comprehensive Quantitative Assay for Amine Transaminases

Carins, Ryan; Gomm, Andrew; Peel, Christopher; Sharkey, Michael; O'Reilly, Elaine

Abstract

The development of effective high-throughput screening assays has contributed greatly to the wealth of designer enzymes available, by enabling rapid identification of desired variants from large mutant libraries. Here, we report a general and operationally simple end-point assay for transaminases that enables the screening of both amine donors and acceptors in liquid phase. The spectrophotometric-based screen exploits the amine donor 2aminoethylaniline (2-AEA) and relies on reaction of in situ generated indole with Ehrlich’s reagent. The assay has also been adapted to allow screening in the reverse direction by addition of indole and subsequent spectrophotometric analysis. Importantly, the screen provides qualitative information on the enantio-preference of the individual biocatalysts. To increase the assay throughput, an engineered expression strain (E. coli BL21(DE3) ΔtnaA) lacking tryptophanase activity, was generated to enable reliable and direct evaluation of individual colonies arrayed on agar plates.

Citation

Carins, R., Gomm, A., Peel, C., Sharkey, M., & O'Reilly, E. (2019). A Comprehensive Quantitative Assay for Amine Transaminases. ChemCatChem, 11(19), 4738-4743. https://doi.org/10.1002/cctc.201901430

• Journal Article Type: Article
• Acceptance Date: Aug 9, 2019
• Online Publication Date: Aug 9, 2019
• Publication Date: Oct 7, 2019
• Deposit Date: Sep 13, 2019
• Publicly Available Date: Aug 10, 2020
• Journal: ChemCatChem
• Print ISSN: 1867-3880
• Electronic ISSN: 1867-3899
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 11
• Issue: 19
• Pages: 4738-4743
• DOI: https://doi.org/10.1002/cctc.201901430
• Keywords: Physical and Theoretical Chemistry; Inorganic Chemistry; Organic Chemistry; Catalysis
• Public URL: https://nottingham-repository.worktribe.com/output/2613209
• Publisher URL: https://onlinelibrary.wiley.com/doi/full/10.1002/cctc.201901430
• Additional Information: This is the peer reviewed version of the following article: Cairns, R. , Gomm, A. , Peel, C. , Sharkey, M. and O'Reilly, E. (2019), A Comprehensive Quantitative Assay for Amine Transaminases. ChemCatChem, which has been published in final form at https://doi.org/10.1002/cctc.201901430. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.