Enhancing the Thermostability of an ( <i>R</i> )-Selective ω-Transaminase with Broad Substrate Scope by Integrated Rational Design

(<i>R</i>)-selective ω-transaminases (<i>R</i>-ω-TAs) are essential biocatalysts for chiral amine synthesis, but their natural scarcity and poor thermostability hinder industrial application. In this study, an <i>R</i>-ω-TA from <i>Nocardioides</i> sp. CER19 (<i>Ns</i>TA) with broad substrate scope was identified. To improve its thermostability, an integrated rational design strategy combining folding free energy prediction, consensus sequence analysis and surface charge optimization was applied. The double mutant Y60F/V254L exhibited a 5-fold extended half-life at 45 °C and a 7.82 °C increase in melting temperature (<i>T</i>_m) compared to the wild type, while maintaining a 1.29-fold higher catalytic efficiency. Structural analysis revealed that enhanced stability resulted from synergistic interfacial hydrophobic reinforcement and surface loop rigidification. This study generated a thermostable <i>R</i>-ω-TA mutant with broad substrate scope and confirmed the efficacy of the integrated computer-aided design strategy, providing a framework for engineering robust industrial biocatalysts.