Reconstruction of Hyper‐Thermostable Ancestral L‐Amino Acid Oxidase to Perform Deracemization to D‐Amino Acids

L‐amino acid oxidases (LAAOs) with broad substrate specificity can be used in the deracemization of D,L‐amino acids (D,L‐AAs) to their D‐enantiomers. Hyper‐thermostable LAAO (HTAncLAAO) was designed through a combination of manual sequence data mining and ancestral sequence reconstruction. Soluble expression of HTAncLAAO (>50 mg/L) can be achieved using an E. coli system. HTAncLAAO, which recognizes seven L‐AAs as substrates, exhibits extremely high thermal stability and long‐term stability; the t1/2 value was 95 °C and 99 % ee, D‐enantiomer). These results suggest that HTAncLAAO is an excellent biocatalyst to perform this deracemization. BioCATalysis: The hyper‐thermostable ancestral L‐amino acid oxidase (HTAncLAAO), which was screened from a sequence database, converted L‐amino acids to D‐enantiomers at high temperatures using a one‐pot reaction. HTAncLAAO had the highest thermo‐ and long‐term stability compared to previously reported LAAOs. HTAncLAAO is a useful biocatalyst in the deracemization to D‐amino acids.