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Appl Microbiol Biotechnol. 2017 Feb;101(3):1163-1174. doi: 10.1007/s00253-016-7880-2. Epub 2016 Oct 11.

Regioselective hydroxylation of cholecalciferol, cholesterol and other sterol derivatives by steroid C25 dehydrogenase.

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Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.
Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, 31343, Krakow, Poland.
Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30060, Krakow, Poland.
Department of Chemical Engineering, Silesian University of Technology, Ks. M. Strzody 7, 44100, Gliwice, Poland.
Institute of Chemical Engineering, Polish Academy of Sciences, Bałtycka 5, 44100, Gliwice, Poland.
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60627, Poznan, Poland.
Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.


Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D3 used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6-9 % (w/v) 2-hydroxypropyl-β-cyclodextrin as a solubilizer and 1.25-5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8-15 mM K3[Fe(CN)6] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min-1 mg-1), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH2 groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.


25-hydroxycholesterol; Calcifediol; Molybdenum enzyme; Regioselective hydroxylation; Steroid C25 dehydrogenase; Sterol

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