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A robust and stereocomplementary panel of ene-reductase variants for gram-scale asymmetric hydrogenation

DOI: 10.1016/j.mcat.2021.111404 DOI Help

Authors: Nathalie Nett (Philipps-Universität Marburg) , Sabine Duewel (Philipps-Universität Marburg) , Luca Schmermund (Philipps-Universität Marburg) , Gerrit E. Benary (Philipps-Universität Marburg) , Kara Ranaghan (University of Bristol) , Adrian Mulholland (University of Bristol) , Diederik J. Opperman (University of the Free State) , Sabrina Hoebenreich (Philipps-Universität Marburg)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Molecular Catalysis , VOL 502

State: Published (Approved)
Published: February 2021
Diamond Proposal Number(s): 12633

Abstract: We report an engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 (TsER) that combines control over facial selectivity in the reduction of electron deficient Cdouble bondC double bonds with thermostability (up to 70 °C), organic solvent tolerance (up to 40 % v/v) and a broad substrate scope (23 compounds, three new to literature). Substrate acceptance and facial selectivity of 3-methylcyclohexenone was rationalized by crystallisation of TsER C25D/I67T and in silico docking. The TsER variant panel shows excellent enantiomeric excess (ee) and yields during bi-phasic preparative scale synthesis, with isolated yield of up to 93 % for 2R,5S-dihydrocarvone (3.6 g). Turnover frequencies (TOF) of approximately 40 000 h−1 were achieved, which are comparable to rates in hetero- and homogeneous metal catalysed hydrogenations. Preliminary batch reactions also demonstrated the reusability of the reaction system by consecutively removing the organic phase (n-pentane) for product removal and replacing with fresh substrate. Four consecutive batches yielded ca. 27 g L−1 R-levodione from a 45 mL aqueous reaction, containing less than 17 mg (10 μM) enzyme and the reaction only stopping because of acidification. The TsER variant panel provides a robust, highly active and stereocomplementary base for further exploitation as a tool in preparative organic synthesis.

Journal Keywords: Biocatalysis; Biphasic; Stereoselective; Old yellow enzyme; Protein-ligand docking

Diamond Keywords: Eznymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography

Added On: 27/01/2021 10:24

Discipline Tags:

Biotech & Biological Systems Biotechnology Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)