The identification and application of a robust ω-transaminase with high tolerance towards substrates and isopropylamine from a directed soil metagenome?

Catalysis Science & Technology Pub Date: 2022-02-14 DOI: 10.1039/D1CY02032C

Abstract

ω-Transaminase-mediated asymmetric amination of a ketone substrate has gained significant attention for its immense potential to synthesize chiral amine pharmaceuticals and precursors. However, few of these have been authentically applied in industry due to inherent limitations such as low catalytic efficiency, unfavorable equilibrium, and poor tolerance towards high concentrations of substrate and isopropylamine (IPA). In this study, by specially screening a metagenomic library from amidogen-enriched environments established to retrieve class III transaminases, a robust ω-transaminase, ATA1012, was identified that exhibited high industrial potential. First, it showed relative stability at 30–50 °C and even at 30 °C for 800 h with residual activity >50%, which greatly benefits a continuous industrial process operation. Second, it was capable of tolerating IPA concentrations as high as 2 M. IPA is one of the most industrially favored amine donors because it is inexpensive and achiral; however, it is not widely accepted by most ω-transaminases. Third, it also showed high substrate tolerance towards the target ketones 1-Boc-3-piperidone (2t) and 1-Boc-3-pyrrolidone (2s) at concentrations up to 750 mM, and 2 IPA equivalents were sufficient to efficiently shift the equilibrium to the desired production side with up to 100% conversion. After systematic optimization of the reaction parameters, including the substrate loading, reaction temperature, IPA dosage and pyridoxal-5′-phosphate (PLP) concentration in the amination process, up to 0.75 M 1-Boc-3-piperidone (2t) (150 g L?1) and 1-Boc-3-pyrrolidone (2s) (139 g L?1) were efficiently converted to the corresponding chiral amines with ee values of >99.9% in 12 h. The hectogram reaction process was readily scaled up, producing a green productive amination process for the efficient production of chiral amines. The molecular basis of the outstanding catalytic efficiency of ATA1012 was also elucidated by molecular docking and molecular dynamics analysis.

Graphical abstract: The identification and application of a robust ω-transaminase with high tolerance towards substrates and isopropylamine from a directed soil metagenome
Recommended Literature