title: Regioselective hydroxylation of cholecalciferol, cholesterol and other sterol derivatives by steroid C25 dehydrogenase.
abstract: 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 D3used 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-1mg-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 NH2groups 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.
authors.affiliation: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Rugor
authors.affiliation: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Tataruch
authors.affiliation: 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.
authors.forename: J
authors.initials: J
authors.lastname: Staroń
authors.affiliation: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Dudzik
authors.affiliation: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.
authors.forename: E
authors.initials: E
authors.lastname: Niedzialkowska
authors.affiliation: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.
authors.forename: P
authors.initials: P
authors.lastname: Nowak
authors.affiliation: 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.
authors.forename: A
authors.initials: A
authors.lastname: Hogendorf
authors.affiliation: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Michalik-Zym
authors.affiliation: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland.
authors.forename: D B
authors.initials: DB
authors.lastname: Napruszewska
authors.affiliation: 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.
authors.forename: A
authors.initials: A
authors.lastname: Jarzębski
authors.affiliation: Department of Chemical Engineering, Silesian University of Technology, Ks. M. Strzody 7, 44100, Gliwice, Poland.
authors.forename: K
authors.initials: K
authors.lastname: Szymańska
authors.affiliation: Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60627, Poznan, Poland.
authors.forename: W
authors.initials: W
authors.lastname: Białas
authors.affiliation: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, PL-30239, Krakow, Poland. ncszalen@cyfronet.pl.
authors.forename: M
authors.identifier: http://orcid.org/0000-0002-7650-9263
authors.initials: M
authors.lastname: Szaleniec
mesh_terms: D020562:Betaproteobacteria / Q000201:enzymology; D055162:Biocatalysis; D019149:Bioreactors; D002112:Calcifediol / Q000378:metabolism; D002762:Cholecalciferol / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D006888:Hydroxycholesterols / Q000378:metabolism; D006900:Hydroxylation; D060847:Metabolic Engineering; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism*
chemical_list: D006888:Hydroxycholesterols; D013261:Sterols; D002762:Cholecalciferol; C007997:25-hydroxycholesterol; D002784:Cholesterol; D010088:Oxidoreductases; D002112:Calcifediol
keywords: 25-hydroxycholesterol; Calcifediol; Molybdenum enzyme; Regioselective hydroxylation; Steroid C25 dehydrogenase; Sterol