Co‐Immobilization of a Multi‐Enzyme Cascade: (<i>S</i>)‐Selective Amine Transaminases, <scp>l</scp>‐Amino Acid Oxidase and Catalase
Tobias Heinks, Simon Koopmeiners, Nicolai Montua, Norbert Sewald, Matthias Höhne, Uwe T. Bornscheuer, Gabriele Fischer von Mollard
Abstract
Abstract 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 2 O 2 ‐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.