Co-Immobilization of a Multi-Enzyme Cascade: (S)-Selective Amine Transaminases, l-Amino Acid Oxidase and Catalase

• Published in: Chembiochem : a European journal of chemical biology Vol. 24; no. 19; p. e202300425
• Main Authors: Heinks, Tobias, Koopmeiners, Simon, Montua, Nicolai, Sewald, Norbert, Höhne, Matthias, Bornscheuer, Uwe T, Fischer von Mollard, Gabriele
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 04-10-2023
• Subjects: Amines; Amino acid oxidase; Amino acids; Catalase; Enzymes; Hydrogen peroxide; Immobilization; Recycling; Recycling systems; Substrates; Transaminases; substrate channeling; recycling; chiral amines; apremilast; biocatalysis
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202300425

An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co-substrate was required and l-amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1 mol % most likely due to a more efficient H
O
-removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate.