The d-amino acid oxidase (DAAO) from Rhodotorula taiwanensis has proven to have great potential for applications due to its excellent catalytic kinetic parameters. However, its poor thermal stability has limited its performance in biocatalysis. Herein, starting from the variant SHVG of RtwDAAO, this study employed a comprehensive computational design approach for protein stability engineering, resulting in positive substitutions at specific sites (A43S, T45M, C234L, E195Y). The generated variant combination, SHVG/SMLY, exhibited a significant synergistic effect, leading to an extension of the half-life and Tmapp. The ancestral sequence reconstruction revealed the conservation of the variant sites. The association of the variant sites with the highly stable ancestral enzyme was further explored. After determining the contribution of the variant sites to thermal stability, it was applied to other homologous sequences and validated. Molecular dynamics simulations indicated that the increased hydrophobicity of the variant SHVG/SMLY was a key factor for the increased stability, with strengthened intersubunit interactions playing an important role. In addition, the physical properties of the amino acids themselves were identified as crucial factors for thermal stability generality in homologous enzymes, which is important for the rapid acquisition of a series of stable enzymes.
Keywords: ancestral enzymes; d-amino acid oxidase; generality; molecular dynamics simulations; thermal stability.