Development of an Enzyme Cascade System for the Synthesis of Enantiomerically Pure D‐Amino Acids Utilizing Ancestral L‐Amino Acid Oxidase

Abstract Enantiomerically pure D‐amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide‐based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L‐amino acids into their D‐isomers. The system integrates four enzymes: ancestral L‐amino acid oxidase (AncLAAO‐N4), D‐amino acid dehydrogenase (DAADH), D‐glucose dehydrogenase (GDH), and catalase. AncLAAO‐N4 initiates the process by converting L‐amino acids to corresponding keto acids, which are then stereo‐selectively aminated to D‐amino acids by DAADH using NADPH and NH 4 Cl. Concurrently, any generated H 2 O 2 is decomposed into O 2 and H 2 O by catalase, while GDH regenerates NADPH from D‐glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D‐amino acids, including a D‐Phe derivative, three D‐Trp derivatives, and D‐phenylglycine, all with high enantiopurity (>99 % ee ) at a preparative scale (>100 mg). This system demonstrates a versatile approach for producing a diverse array of D‐amino acids.