Stability and stabilization of D-amino acid oxidase from the yeast Trigonopsis variabilis.

The use of DAO (D-amino acid oxidase) for the conversion of cephalosporin C has provided a significant case for the successful implementation of an O(2)-dependent biocatalyst on an industrial scale. Improvement of the operational stability of the immobilized oxidase is, however, an important goal of ongoing process optimization. We have examined DAO from the yeast Trigonopsis variabilis with the aim of developing a rational basis for the stabilization of the enzyme activity at elevated temperature and under conditions of substrate turnover. Loss of activity in the resting enzyme can occur via different paths of denaturation. Partial thermal unfolding and release of the FAD cofactor, kinetically coupled with aggregation, contribute to the overall inactivation rate of the oxidase at 50 degrees C. Oxidation of Cys(108) into a stable cysteine sulfinic acid causes both decreased activity and stability of the enzyme. Strategies to counteract each of the denaturation steps in DAO are discussed. Fusion to a pull-down domain is a novel approach to produce DAO as protein-based insoluble particles that display high enzymatic activity per unit mass of catalyst.