The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans

Cholest-4-en-3-one Delta(1)-dehydrogenase (AcmB) from Sterolibacterium denitrificans, a key enzyme of the central degradation pathway of cholesterol, is a protein catalyzing Delta 1-dehydrogenation of a wide range of 3-ketosteroids. In this study, we demonstrate the application of AcmB in the synthesis of 1-dehydro-3-ketosteroids and investigate the influence of reaction conditions on the catalytic performance of the enzyme. The recombinant AcmB expressed in E. coli BL21(DE3)Magic exhibits a broad pH optimum and pH stability in the range of 6.5 to 9.0. The activity-based pH optimum of AcmB reaction depends on the type of electron acceptor (2,6-dichloroindophenol DCPIP, phenazine methosulfate PMS or potassium hexacyanoferrate K-3[Fe(CN)(6)]) used in the biocatalytic process yielding the best kinetic properties for the reaction with a DCPIP/PMS mixture (k(cat)/K-m = 1.4 center dot 10(5) s(-1).M-1 at pH 9.0) followed by DCPIP (k(cat)/K-m = 1.0 center dot 10(5) s(-1).M-1 at pH = 6.5) and K3[Fe(CN)6] (kcat/Km = 0.5 center dot 10(2) s(-1).M-1 at pH = 8.0). The unique feature of AcmB is its capability to convert both testosterone derivatives (C20-C22) as well as steroids substituted at C17 (C27-C30) such as cholest-4-en-3-one or (25R)-spirost4-en-3-one (diosgenone). Apparent steady-state kinetic parameters were determined for both groups of AcmB substrates. In a batch reactor synthesis, the solubility of water-insoluble steroids was facilitated by the addition of a solubilizer, 2-hydroxypropyl-beta-cyclodextrin, and organic co-solvent, 2-methoxyethanol. Catalytic properties characterization of AcmB was tested in fed-batch reactor set-ups, using 0.81 mu M of isolated enzyme, PMS and aerobic atmosphere resulting in > 99% conversion of the C17-C20 3-ketosteroids within 2 h. Finally, the whole cell E. coli system with recombinant enzyme was demonstrated as an efficient biocatalyst in the synthesis of 1-dehydro-3-ketosteroids.