A New 3-Ketosteroid-Δ-Dehydrogenase with High Activity and Broad Substrate Scope for Efficient Transformation of Hydrocortisone at High Substrate Concentration.

3-Ketosteroid-Δ-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, nine putative genes from different origins were selected and overexpressed in BL21(DE3). These recombinant enzymes catalyzed the Δ-desaturation of a variety of steroidal compounds. Among them, the KstD from sp. (PrKstD) displayed the highest specific activity and broad substrate spectrum. The detailed catalytic characterization of PrKstD showed that it can convert a wide range of 3-ketosteroid compounds with diverse substituents, ranging from substituents at the C9, C10, C11 and C17 position through substrates without C4-C5 double bond, to previously inactive C6-substituted ones such as 11β,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-dione. Reaction conditions were optimized for the biotransformation of hydrocortisone in terms of pH, temperature, co-solvent and electron acceptor. By using 50 g/L wet resting cells harboring PrKstD enzyme, the conversion of hydrocortisone was about 92.5% within 6 h at the substrate concentration of 80 g/L, much higher than the previously reported results, demonstrating the application potential of this new KstD.