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

An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different & alpha;-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 & alpha;-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 H2O2-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.

Automated summary

An enzyme cascade consisting of l-amino acid oxidase (hcLAAO4) and catalase (hCAT) was immobilized together with the (S)-selective amine transaminase (ATA-Vfl) to achieve higher reaction rates and more efficient H2O2-removal. The co-immobilized enzyme cascade showed good stability and high enantiomeric purity in preparative kinetic resolutions. However, further recycling was limited due to the instability of ATA-Vfl. The use of an engineered ATA-Vfl-8M in the immobilized enzyme cascade allowed a significant reduction in the amount of co-substrate required.