Amine transaminases (ATAs) are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the (R)-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the (S)-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee) of both (R)- and (S)-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of <10% (without affecting the enantioselectivity). In contrast, the commercially engineered V. fluvialis variant, ATA-256, still enabled an 87% conversion, yielding a corresponding amine with >99% ee. Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results.
Recebido em 05/11/2019; aceito em 19/03/2020; publicado na web em 24/04/2020 DIRECTED EVOLUTION OF ENZYMES: SMALL CHANGES, BETTER BIOCATALYSTS. Biocatalysis is now a mature field, both in the laboratory and industrial scale. This approach counts on enzyme high selectivity, biodegradability, elegant control over the outcome of reaction conditions and as a possible solution to address some challenges in Green Chemistry faced by synthetic organic chemists. However, many wild-type ready-to-use enzymes were not designed to accommodate the organic substrates needed by today's demands. Or they do not fit a predefined optimum process condition or even, useful reactions are not accessible because there is no enzyme counterpart for metallo-and organocatalysts. This review will give a brief introduction on the protein engineering tools (directed evolution, rational design, and semi-rational design) and will focus on directed evolution of enzymes, their impact in chemistry with examples from production of commodity chemicals to pharmaceutical intermediates and its potential as a tool to achieve greener chemistry criteria will be discussed.
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