The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans
Agnieszka M. Wojtkiewicz , Patrycja Wójcik , Magdalena Procner , Monika Flejszar , Maria Oszajca , Mateusz Hochołowski , Mateusz Tataruch , Beata Mrugała , Tomasz Janeczko , Maciej Szaleniec
AbstractCholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans, a key enzyme of the central degradation pathway of cholesterol, is a protein catalyzing Δ1-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 - K3[Fe(CN)6]) used in the biocatalytic process yielding the best kinetic properties for the reaction with a DCPIP/PMS mixture (kcat/Km = 1.4•105 s-1•M-1 at pH 9.0) followed by DCPIP (kcat/Km = 1.0•105 s-1•M-1 at pH = 6.5) and K3[Fe(CN)6] (kcat/Km = 0.5•102 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)-spirost-4-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-β-cyclodextrin, and organic co-solvent, 2-methoxyethanol. Catalytic properties characterization of AcmB was tested in fed-batch reactor set-ups, using 0.81 μM 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.
|Journal series||Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, (N/A 100 pkt)|
|Publication size in sheets||0.5|
|Keywords in English||3-ketosteroid Δ1-dehydrogenase, Δ1-dehydrogenation, 1,2-dehydrogenation, steroid biotransformation, phenazine methosulfate, diosgenone|
|ASJC Classification||; ; ; ; ; ;|
|Score||= 100.0, 18-12-2020, ArticleFromJournal|
|Publication indicators||= 0; = 0; : 2018 = 1.131; : 2019 = 3.813 (2) - 2019=3.889 (5)|
|Citation count*||5 (2021-05-06)|
* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.