The 3-ketosteroid-Δ1-dehydrogenase5 (KsdD5) 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 KsdD5 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 KsdD5-WT, an 11.8 °C higher half-inactivation temperature (T5015min), and a 10.6 °C higher melting temperature (Tm). M3 had 3.82-fold higher catalytic activity than WT, a 3.9-fold longer half-life at 30 °C, and higher T5015min and Tm 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-1 substrate dosage and 6 g·L-1 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.
Keywords: Catalytic efficiency; Molecular dynamics; Secondary structure; Steroid bioconversion; Thermostability; Δ(1)-dehydrogenation.
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