Enhancing the thermal stability of L-threonine dehydrogenase through computational-driven combinatorial mutagenesis

L-Threonine dehydrogenases (L-TDHs) are crucial industrial enzymes that catalyze the dehydrogenation of L-threonine to produce L-2-aminoethyl acetate, demonstrating significant application value in the food additive and pharmaceutical industries. Although L-threonine dehydrogenase derived from Escherichia coli (EcTDH) exhibits high catalytic efficiency, its poor thermal stability limits its widespread industrial application. In this study, we employed a semi-rational design strategy to successfully construct a combinatorial mutant N221W/T248L of EcTDH. Experimental results revealed a remarkable improvement in the thermal stability of the N221W/T248L mutant compared to the wild-type enzyme, the half-life of N221W/T248L mutant at 50 °C (t_1/2 (50 °C)) reached 115 min, which is 3.2 times that of the wild-type enzyme (35 min). This enhancement significantly increases the suitability of EcTDH for high-temperature industrial environments. Meanwhile, the N221W/T248L mutant also displayed similar specific activity (12.01 U mg^-1) and catalytic efficiency (4.25 s^-1 mM^-1) to the wild-type enzyme. Moreover, the protein engineering strategy adopted in this study provides an effective reference for the directed evolution and optimization of other industrial enzymes.