Potassium bromide

Material and reagents: Testosterone (17β‐hydroxy‐4‐androsten‐3‐one), yeast extract, ABTS and naphthalene were purchased from AppliChem (Arheilgen, Germany); H₂O₂ (30 % w/v), soybean peptone, agar, α‐d‐glucose, benzyl alcohol, phenol, and sodium azide were from Carl Roth; malt extract, 2‐chlorophenol, and 4‐chlorophenol were obtained from Merck; glucose oxidase from Aspergillus niger was purchased from Sigma–Aldrich (specific activity 215 U mg⁻¹). All other chemicals were purchased from Sigma–Aldrich at the highest purity available.
Peroxygenases of A. aegerita (AaeUPO) and M. rotula (MroUPO) were produced and purified as described previously;14, 16 recombinant UPOs from C. cinerea (rCciUPO) and H. insolens (rHinUPO=rnovo) were gifts from Novozymes A/S (Copenhagen, Denmark).25, 26, 27, 28The specific activities of AaeUPO and MroUPO were 63.5 U mg⁻¹ and 48.1 U mg⁻¹, respectively (1 U is the oxidation of 1 μmol veratryl alcohol to veratraldehyde per 1 min at 23 °C).14Cultivation ofC. globosum: C. globosum (strain DSM 62110) was purchased from the German Collection of Microorganisms and Cell cultures (Braunschweig, Germany) and was routinely grown on malt extract agar medium (malt extract (20 g L⁻¹) and agar (15 g L⁻¹)) at 24 °C. For enzyme production, the fungus was cultured in 500 mL Erlenmeyer flasks containing carbon‐ and nitrogen‐rich basic liquid medium (200 mL; glucose (42 g L⁻¹), peptone (18 g L⁻¹), yeast extract (4.5 g L⁻¹) in deionized water) on a rotary shaker (120 rpm) at 24 °C for four weeks. Liquid cultures were inoculated with a mycelial suspension (5 % v/v) obtained by homogenization of the content of two agar plates fully covered with fungal mycelium in sterile sodium chloride (100 mL, 0.9 % w/v).
Enzyme assays: UPO activities were measured photometrically by monitoring the oxidation of veratryl alcohol (5 mm) into veratraldehyde at 310 nm (ϵ₃₁₀=9300 m⁻¹ cm⁻¹) in McIlvaine buffer at pH 7.14 Reaction was started by the addition of hydrogen peroxide (2 mm). Laccase activity during cultivation was determined by the oxidation of ABTS to the corresponding ABTS cation radical at 420 nm (ϵ₄₂₀=36 000 m⁻¹ cm⁻¹) in McIlvaine buffer at pH 4.5 in the absence of H₂O₂.29 The specific ring‐hydroxylating activity of CglUPO was monitored by the oxygenation of naphthalene (1 mm) to naphthalene oxide and 1‐naphthol at 303 nm (ϵ₃₀₃=2030 m⁻¹ cm⁻¹) in McIlvaine buffer at pH 6.0; the reaction was started by adding hydrogen peroxide (2 mm).30Purification and characterization ofCglUPO: All purification steps were carried out at room temperature. Enzyme fractions were assayed for UPO activity, and protein content was determined with a Pierce BCA protein assay kit (Thermo Fisher) with bovine serum albumin as standard. Protein purification was carried out by using ammonium sulfate precipitation and fast protein liquid chromatography (FPLC) on Q‐Sepharose FF (IEC), Superdex75 (SEC), and Mono Q columns (IEC), successively. All chromatographic steps were accomplished with an ÄKTA purifier FPLC system (GE Healthcare).
The molecular mass of purified CglUPO was analyzed by SDS‐PAGE by using a 10 % Bolt Bis‐Tris Gel (Thermo Fisher Scientific). The separated protein bands were visualized with a Colloidal Blue Staining Kit (Generon Ltd, Berkshire, UK, order code GEN‐QC‐Stain‐1L); a protein marker (#26616, Thermo Fisher Scientific) was used as standard.
Proteomic enzyme identification was performed at the Helmholtz‐Centre for Environmental Research—UFZ, Department of Molecular Systems Biology (Leipzig, Germany). For detailed information (peptide mapping), see the Supporting Information.
Kinetic constants (K_m, k_cat) of CglUPO and pH optima were determined for veratryl alcohol, benzyl alcohol, DMP, ABTS, NBD (pH 7),31 and naphthalene (pH 6; Supporting Information). Halogenating activity was tested by incubating CglUPO (0.2 U mL⁻¹, 0.46 μm) in potassium phosphate buffer (100 mm, pH 3 and pH 7) in the presence of phenol (0.1 mm), potassium bromide or chloride (10 mm) and H₂O₂ (2 mm).32 After 10 min, the reaction mixture was analyzed by HPLC for the formation of bromo‐ and chlorophenols against authentic standards.
Enzymatic conversion of testosterone: The reaction mixture (total volume 0.5 mL) contained purified AaeUPO (2 U mL⁻¹, 0.7 μm), MroUPO (2 U mL⁻¹, 1.3 μm) or CglUPO (0.2 U mL⁻¹, 0.46 μm) in potassium phosphate buffer (20 mm, pH 7) with testosterone (5 mm), α‐d‐glucose (2 %, w/v), and acetone (5 %, v/v). Reactions were started by addition of glucose oxidase (GOx, 0.02 U mL⁻¹) and stirred at room temperature for 24 h (after this time, no residual activity of CglUPO was detectable). Kinetic data were determination for CglUPO (2 U mL⁻¹, 4.8 μm) with testosterone (5 mm) in potassium phosphate buffer (20 mm, pH 7). Reactions were initiated by the addition of hydrogen peroxide (2 mm) and stopped after 2 min by adding sodium azide (1 mm). Higher concentrations of hydrogen peroxide were not applied, in order to prevent enzyme inactivation from heme bleaching and the disproportionate increase in the UPO intrinsic catalase activity (both have been reported for other UPOs and heme peroxidases).33, 34, 35 Products were recovered with reversed‐phase SPE cartridges (Strata‐X 33u, Phenomenex), with elution in methanol, and analyzed by HPLC.
At preparative scale, a 500 mL flask was filled with testosterone (100 mg, 0.35 mmol), acetone (10 mL), water (140 mL), potassium phosphate buffer (40 mL 0.1 m, pH 7), and CglUPO stock solution (20 mL 400 U in potassium phosphate buffer (0.1 m, pH 7)). The reaction mixture was stirred at room temperature while hydrogen peroxide (100 mm, 4 mL h⁻¹) was continuously supplied by a syringe pump. Hydrogen peroxide was used instead of glucose/GOx, in order to ensure constant peroxide dosage and to avoid impurities in the reaction mixture (glucose and gluconolactone); the syringe pump system was as effective as the GOx‐based H₂O₂ generation system. Samples (50 μL) were taken from the reaction mixture every 30 min, and the reaction (in the samples) was stopped by adding acetonitrile (50 μL) and sodium azide (10 μL, 10 mm). The samples were centrifuged, and the supernatants were analyzed by HPLC (below). After 7 h, thin layer chromatography (in ethyl acetate/n‐hexane, 9:1) indicated complete conversion of testosterone. The reaction mixture was extracted three times with ethyl acetate (50 mL), then the combined organic fractions were dried with Na₂SO₄ and evaporated to dryness to give 91 mg of crude products 1 a (R_f 0.67) and 1 b (R_f 0.11). The compounds were purified by chromatography on silica gel with ethyl acetate/n‐hexane (9:1) as the eluent to obtain 65 mg (61.1 %) of 1 a (96.3 % purity) and 7 mg (6.6 %) of 1 b (98.7 % purity).
Analytical methods: The HPLC‐MS system (Waters) comprised a 2690 separation module, a 2996 photo diode array detector, and a Micromass ZMD 2000 single quadrupole mass spectrometer. Separation was on a LiChrospher C18 column (125×4 mm, 5 μm, Phenomenex) with mobile phases A (formic acid (0.1 %)) and B (acetonitrile) and at stepwise gradient (20 % B (3 min), increase to 55 % B (20 min), increase to 90 % B (3 min)). The final level was maintained until all analytes had been eluted from the column (flow‐rate 1 mL min⁻¹, column temperature 30 °C). Reaction products were identified by comparison to authentic standards based on retention time, UV absorption spectrum and mass spectra [M+H]⁺ or [M−H]⁻ ions, and quantified by total peak area by using response factors of the same or similar compounds. Data from replicates were averaged. Standard deviations were below 5 % of the mean in all cases.
¹H (400 MHz) and ¹³C (100 MHz) NMR spectra of testosterone and its enzymatic conversion products were obtained on Bruker spectrometer (Bruker Avance II 400 MHz) in the solvent indicated.