Improving the Thermostability and Catalytic Activity of <i>Rg</i>DAAO by a Combinatorial Strategy Using Sequence Consensus Design and SpyTag/SpyCatcher Self-Cyclization

d-Amino acid oxidase from <i>Rhodotorula gracilis</i> (<i>Rg</i>DAAO) is valuable for pharmaceutical and chemical synthesis due to its high enantioselectivity, but its poor thermostability limits extensive application. This study proposed a synergistic strategy of "sequence consensus design coupled with structure modification" to enhance <i>Rg</i>DAAO thermostability. Through homologous sequence analysis and greedy algorithm-based optimization, a triple mutant M3 (S18T/V7I/Y132F) was obtained, showing a 3.7-fold extension in half-life at 50 and a 5.13 °C increase in melting temperature (<i>T</i>_m) versus wild-type (WT). Furthermore, N/C-terminal autocyclized variants (TDC-WT, CDT-WT, etc.) were constructed using the SpyTag/SpyCatcher system, demonstrating 2-3-fold higher half-life at 50 °C than WT. The combinatorial strategy integrated M3 with cyclization technology, yielding a synergistically significant combinatorial enzyme, LCDT-M3, which exhibited a 12.8-fold longer half-life, a 9.42 °C increase in <i>T</i>_m, and a 2.2-fold greater specific activity compared to WT at 50 °C. Molecular dynamics simulations and structural analyses revealed that M3 was stabilized by optimizing the enhanced local hydrogen-bond networks and reduced global conformational fluctuations. The cyclized <i>Rg</i>DAAO exhibited reduced conformational freedom, promoting correct folding governed by fusion order and flexible linkers. In summary, this work provides an effective strategy for the modification of the thermostability of enzymes required in industry.