Recombinant l-Amino Acid Oxidase with Broad Substrate Spectrum for Co-substrate Recycling in (S)-Selective Transaminase-Catalyzed Kinetic Resolutions

Tobias Heinks; Jannik Paulus; Simon Koopmeiners; Tobias Beuel; Norbert Sewald; Matthias Höhne; Uwe T Bornscheuer; Gabriele Fischer von Mollard

doi:10.1002/cbic.202200329

Recombinant l-Amino Acid Oxidase with Broad Substrate Spectrum for Co-substrate Recycling in (S)-Selective Transaminase-Catalyzed Kinetic Resolutions

Chembiochem. 2022 Aug 17;23(16):e202200329. doi: 10.1002/cbic.202200329. Epub 2022 Jul 5.

Authors

Tobias Heinks¹, Jannik Paulus², Simon Koopmeiners¹, Tobias Beuel¹, Norbert Sewald², Matthias Höhne³, Uwe T Bornscheuer⁴, Gabriele Fischer von Mollard¹

Affiliations

¹ Faculty of Chemistry, Biochemistry, Bielefeld University, Universitätsstr. 25, 33615, Bielefeld, Germany.
² Faculty of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
³ Protein Biochemistry, Institute of Biochemistry, University of Greifswald, Felix Hausdorff-Str. 4, 17487, Greifswald, Germany.
⁴ Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix Hausdorff-Str. 4, 17487, Greifswald, Germany.

Abstract

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co-substrate. A dual-enzyme recycling system overcomes this limitation: l-amino acid oxidases (LAAO) recycle the accumulating co-product of (S)-selective transaminases in the kinetic resolution of racemic amines to produce pure (R)-amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H₂ O₂ as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub-stoichiometric amounts of 1 mol% of the transaminase co-substrate as well as the initial application of l-amino acids instead of α-keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)-methylbenzylamine (>99 %ee) at complete conversion (50 %).

Keywords: biocatalysis; co-substrate recycling; enantiopure (R)-amines; kinetic resolution; l-amino acid oxidase (LAAO); transaminases.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amines / chemistry
Catalysis
L-Amino Acid Oxidase*
Oxidoreductases
Stereoisomerism
Substrate Specificity
Transaminases* / metabolism

Substances

Amines
Oxidoreductases
L-Amino Acid Oxidase
Transaminases