Semi-rational design in simultaneous engineering of the substrate specificity and thermostability of yeast alcohol dehydrogenase

Abstract Background Yeast alcohol dehydrogenase I (YADH) catalyzes the reversible reduction of aldehydes to primary alcohols. Thermostable YADH variants with broad substrate specificities at elevated temperatures are desirable for industrial biocatalysis. To simultaneously alter substrate specificity and enhance thermostability, we performed site-directed mutagenesis of the YADH substrate-binding pocket using a semi-rational design approach. Results We aimed to alter the substrate specificity of YADH to efficiently catalyze the reduction of the non-native substrate, trans-2-nonenal, to trans-2-nonenol, while simultaneously enhancing thermostability. A small mutant library was generated using site-directed mutagenesis guided by docking simulations of YADH with trans-2-nonenal. Screening identified the YADH-Y119F variant, which exhibited both enhanced catalytic efficiency for trans-2-nonenal and thermostability. Specifically, Y119F exhibited a 1.1-fold increase in catalytic efficiency (Kcat/Km) and more than a three-fold improvement in thermostability (Tm and half-life at 65–70°C) compared to wild-type YADH (WT-YADH). Conclusions Our findings demonstrate that semi-rational design can effectively improve the substrate specificity and thermostability of YADH. The YADH-Y119F variant combines high catalytic efficiency with excellent thermostability, making it a promising candidate for industrial applications, such as high-temperature bioconversion processes and the synthesis of fragrances or specialty chemicals. This study highlights the broader potential of structure-guided semi-rational design for tailoring robust enzymes for biotechnological applications.