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

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. H 2 O 2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using substoichiometric amounts of 1 mol% of the transaminase cosubstrate 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 %).

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

“…Furthermore, conditions were defined for the co‐immobilization of all three enzymes, at which they all had high specific activities, with main focus on ATA‐Vfl and hcLAAO4 (Table S2). Based on the required concentration of each enzyme determined previously for the soluble cascade, [17] the required activities were defined for the standard reaction volume of 500 μL: 0.318 U ATA‐Vfl, 0.476 U hcLAAO4, 50‐100 U hCAT (Table S3).…”

“…However, hcLAAO4 and hCAT were stable for at least 12 cycles while soluble catalase had a tendency to precipitate and to coprecipitate the other two enzymes in the soluble cascade at higher concentrations. [17] Finally, the engineered ATA-Vfl-8M and the recycling system were co-immobilized and used to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate. Importantly, compared to the original study that used ATA-Vfl-8M alone and in soluble form, a 1,000-fold lower input of the co-substrate was required for complete conversion.…”

“…The use of the immobilized cascade in more than 3 cycles was not efficient as the high amine to ketone ratio in these co‐substrate limited conditions destabilized ATA‐Vfl. However, hcLAAO4 and hCAT were stable for at least 12 cycles while soluble catalase had a tendency to precipitate and to coprecipitate the other two enzymes in the soluble cascade at higher concentrations [17] . Finally, the engineered ATA‐Vfl‐8M and the recycling system were co‐immobilized and used to produce ( R )‐1‐(3‐ethoxy‐4‐methoxyphenyl)‐2‐(methylsulfonyl)ethanamine, an apremilast‐intermediate.…”

“…It was previously shown in the enzyme cascade using soluble enzymes that hCAT inefficiently removed the hydrogen peroxide (H 2 O 2 ), leading to the loss of the α-keto acids, thus interrupting the reaction when the co-substrate concentration was too low (< 1 mol %). [17] To analyze whether close proximity by co-immobilization of hCAT and hcLAAO4 also leads to a better channeling of H 2 O 2 and thus enhances the removal of H 2 O 2 , kinetic resolutions of rac-1-PEA were performed with only 0.1 mol % of the co-substrate and with the enzyme cascade being immobilized separately or together on HA GA -beads (Figure 1B). In case of the single immobilized cascade, a similar pattern to the soluble cascade was observed, with an incomplete but slightly higher conversion at 35 %.…”

“…For example, high enzyme concentrations are required to accelerate reaction rates due to low substrate flux, [26] and proteins can become unstable under operating conditions. hCAT tended to precipitate in the presence of the substrate 1‐PEA and the product acetophenone and caused precipitation of the entire enzyme cascade at high concentrations [17] . A popular strategy to overcome these obstacles is to immobilize the enzymes.…”