One-step immobilization of mutant TvDAAO yields an active and stable biocatalyst in extreme conditions.

D-Amino acid oxidase (DAAO) is an important enzyme in modern biotechnology, used for synthetic, analytical, and medical purposes. Numerous mutant forms of DAAO with different properties have been described previously. Recently, we combined six beneficial amino acid substitutions in DAAO from Trigonopsis variabilis (TvDAAO) to create the multipoint mutant TvDAAO E32R/F33D/F54S/C108F/M156L/C298N (mut-TvDAAO). Compared to the wild-type TvDAAO, the new mutant enzyme showed a fourfold higher catalytic constant in the oxidation of cephalosporin C, an eightfold higher stability against hydrogen peroxide oxidation, and a twentyfold greater thermal stability. In the present work, we demonstrate a one-step immobilization procedure of mut-TvDAAO on strong anion exchange beads Sepabeads EC-QA, yielding an immobilized biocatalyst with enhanced resistance to thermal, oxidative, alkaline, and aeration-induced stress. After immobilization, mut-TvDAAO retains approximately 40% of its initial activity even after incubation for 5 h at 90 °C. Immobilized mut-TvDAAO (imm-mut-TvDAAO) retains approximately 20% of its initial activity in the presence of 0.1 M hydrogen peroxide after 5 h of incubation. The immobilized enzyme remains fully active after 5 h of incubation at pH 13.0. Importantly, immobilization improves catalyst performance under intensive oxygen aeration required for cephalosporin C oxidation, and the activity of imm-mut-TvDAAO does not decrease over 10 consecutive cycles of use. These results show that simple carrier-based immobilization of an engineered TvDAAO variant provides an operationally robust biocatalyst for oxidative transformations under process-relevant stress conditions.