Increased thermal stability and catalytic efficiency of 3-ketosteroid Δ-dehydrogenase5 from Arthrobacter simplex significantly reduces enzyme dosage in prednisone acetate biosynthesis.

The 3-ketosteroid-Δ-dehydrogenase5 (KsdD) from Arthrobacter simplex converts cortisone acetate to prednisone acetate, an important step in steroid catabolism. To achieve sustainable and efficient enzyme production, we employed computer-aided screening, structural analysis, and combinatorial experiments to identify engineered KsdD variants (M1 and M3) with dual advantages of stability and active sites. M1 had a 8.2-fold longer half-life (19.6 h at 30 °C) than KsdD-WT, an 11.8 °C higher half-inactivation temperature (T), and a 10.6 °C higher melting temperature (T). M3 had 3.82-fold higher catalytic activity than WT, a 3.9-fold longer half-life at 30 °C, and higher T and T by 14 °C and 6.9 °C, respectively. Furthermore, kinetic and microscale thermophoresis analyses revealed M3 exhibited higher catalytic efficiency due to its larger enzymatic channel. Molecular dynamics simulations showed M1 promoted tighter secondary structure packing, reduced residue flexibility, and increased hydrogen bond formation, ensuring enzyme stability and activity at elevated temperatures. Under industrial conditions, M1 converted >96 % cortisone acetate within 12 h at 30 °C with a 60 g·L substrate dosage and 6 g·L cell mass, whereas the M3 conversion rate was 95 %. This study demonstrates a robust strategy for developing efficient enzyme mutants, facilitating sustainable industrial production of prednisone acetate with a minimal enzyme dosage.