Quillaja saponin

Total saponins contents of A. lebbeck seeds were estimated by colorimetric methods [17 (link)]. Gross saponins extract (10 mg) was dissolved in 5 mL of 80% aqueous methanol and 50 μL of this solution was taken in different test tubes to which 0.25 mL of vanillin reagent (8%, w/v in 99.9% ethanol) was added. Test tubes were placed in ice-cold water bath and 2.5 mL of 72% (v/v) sulphuric acid was added slowly on the inner side of the wall. After mixing the content in each tube, then left as such for 3 min, then warmed the tubes at 60°C for 10 min using water bath and cooled in ice-cold water bath. Absorbance was measured at 544 nm using spectrophotometer against the reagent blank and standard curve was prepared. Quillaja saponin (Sigma-Aldrich) was used as a reference standard [18 ] and the concentration of total saponins was expressed as Quillaja saponin equivalents (QS μg/mg extract).

Folin-Ciocalteu reagent, HPLC grade methanol, tannic acid ACS reagent, Quillaja saponin, 3,5-dinitrosalicilic acid (98 %), aluminum trichloride, sulfuric acid (18 M), glucose (99.5 %), stigmasterol (95 %), hydroquinone (99 %), ursolic acid (98.5 %), digitoxin (92 %) and bromocresol green (95 %) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Quercetin dihydrate (98 %) and dragendorff’s reagent were obtained from Aldrich (Milwaukee, WI, USA). Ferric Chloride (FeCl₃•6H₂O) (97.0-102.0 %) was obtained from Spectrum Chemical and Lab Products (Gardena, California, USA) and potassium ferrocyanide (K₄Fe(CN)₆•3H₂O), HCl (12 M), sodium hydrogen phosphate, ethyl acetate (EtOac), acetic anhydride and dichloromethane were obtained from Thermo Fisher Scientific (New Jersey, USA). Nicotine (99 %) was obtained from VWR (New Jersey, USA). All chemicals were used without further purification.

Either LB- (25 g/L, Becton–Dickinson) or TB- (24 g/L yeast extract, 12 g/L tryptone, 0.4% glycerol, 100 mM potassium phosphate buffer, pH 7.4) media were used for the cultivation of B. megaterium. For the preculture, 50 mL of medium either containing 10 µg/mL tetracycline (cells harboring a variant of pSMF2.1) or 10 µg/mL chloramphenicol (cells harboring a variant of pSMF3) were inoculated with cells from an agar plate or glycerol stock. For the main culture, 50 mL of medium containing 10 µg/mL of the required antibiotic were inoculated with 500 µL of the preculture. The main culture was grown until an optical density of ~0.4 was reached, protein expression was induced by addition of 0.25 g xylose dissolved in 1 mL distilled water. For whole-cell conversion experiments, substrates were dissolved in a 45% 2-hydroxypropyl-β-cyclodextrin/4% Quillaja saponin-solution. 2.5 mL of the solution were added to the culture directly after protein induction. A final substrate concentration of 300 µM was used for all conversion experiments. Cholesterol from sheep wool (purity ≥99%), 7-dehydrocholesterol (≥98%), β-sitosterol from plant extracts (purity ≥70%, containing residual campesterol), 2-hydroxypropyl-β-cyclodextrin (average molecular weight ~1,460) and Quillaja saponin (Sapogenin content ≥10%) were purchased from Sigma-Aldrich.