Enhanced Thermal Stabilization of Yeast d ‐Aspartate Oxidase Through Intersubunit Disulfide Bond Engineering

The flavoenzyme d ‐aspartate oxidase (DDO) is a promising candidate for several biotechnological applications, including the detection of acidic d ‐amino acids and the optical resolution of their racemic mixtures. The DDO from the yeast Cryptococcus humicola strain UJ1 (ChDDO) exhibits high catalytic activity and strict substrate specificity toward d ‐Asp; however, its practical use is hindered by limited stability, particularly upon dilution. In this study, we engineered ChDDO by introducing an intersubunit disulfide bond through structure‐guided substitutions (G87C/Y292C). The resulting variant exhibited improved resistance to heat‐induced loss of activity in residual‐activity assays after defined heat treatments, while retaining comparable apparent K m and k cat values for d ‐Asp. In a residual‐activity assay using the purified enzymes, the apparent (operational) T 50 increased by 21.8°C compared with the wild‐type enzyme. Biochemical analyses confirmed the formation of an intersubunit disulfide bond and suggest that it contributes to improved resistance to heat‐induced loss of activity under the tested conditions, without compromising the enzyme's useful catalytic properties. Collectively, these results indicate that intersubunit disulfide engineering can improve the functional robustness of ChDDO under the assay conditions used, thereby supporting its practical use in DDO‐based applications.