Sephadex lh 20 chromatography

The 48-h cultures of S. rochei strains were harvested, and the supernatant was extracted twice with equal volume of ethyl acetate. The crude extracts were purified by Sephadex LH-20 chromatography (1 × 40 cm, GE Healthcare, Chicago, IL) with methanol. Then the fractions containing antibiotics were purified by silica gel chromatography with chloroform-methanol (80:1–10:1, v/v). NMR spectra were recorded on an ECA-500 spectrometer (JEOL, Tokyo, Japan) equipped with a field gradient accessory. Chloroform-d and methanol-d₄ were used as solvents. Chemical shifts were recorded in δ value based on the solvent signals (δ_C = 77.0 in CDCl₃, δ_C = 49.0 in CD₃OD, and δ_H = 3.30 in residual CH₃OH) or an internal standard tetramethylsilane (δ_H = 0). High resolution ESI-MS spectra were measured by a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). The ¹H- and ¹³C-NMR assignments for lankamycin (1), lankacidin C (2), lankacidin A (3), lankacidinol A (4), iso-lankacidinol (5), and lankacidinol (6) have already been reported (Suzuki et al., 2010 (link); Arakawa et al., 2011 (link); Yamamoto et al., 2018 (link)).

Freshly-prepared spores of AM-7161 were inoculated on totally 40 L of R4 solid medium (40 mL of medium for each Petri dish) and incubated for 5 d at 30°C. Then, solid cultures were cut into small pieces and soaked for 6 h in EtoAc at room temperature, followed by 30 min sonication. Finally, 120 L of EtoAc crude extracts were collected after extracting for three times and evaporated. Subsequently, 6.15 g dried sample was subjected to macroporus resin chromatography (MCI-GEL CHP20/P120, Mitsubishi) through a gradient elution with methanol and water. All the fractions showing antibacterial activity measured as descriptions in the section 2.7 were collected respectively and evaporated, followed by further fractionation through silica gel column chromatography (Qingdao Haiyang Chemicals & Silica gel Co. Ltd, China) with a step elution gradient using CHCl₃ and methanol (1000:1–100:1).
After the fractions showing antibacterial activity were collected and re-dissolved in methanol containing 10% DMSO, they were subjected to Sephadex LH-20 chromatography (GE Healthcare Bio-Sciences AB, Sweden) and eluted with 100% methanol to collect the fraction of compound X.

Substrates (10 mg) dissolved in 200 μl DMSO were fed to 2-day old Streptomyces sp. MBT76 culture broth. This biotransformation period lasted for another 3 days. The control experiment included both negative control of feeding 200 μl DMSO solvent to MBT76, and positive control of 10 mg substrates dissolved in 200 μl DMSO added to sterile culture media. Each experiment was done in triplicate. The method used to harvest the biotransformation products was as described for the metabolomics study.
To isolate the biotransformation products of genistein (Sigma), the combined mixture in 50 ml methanol was first defatted twice with 20 ml of n-hexane, which was subsequently fractionated by Sephadex LH-20 chromatography (GE Healthcare Life Sciences, Eindhoven, The Netherlands) eluting with methanol, to give seven fractions fr1–7. These fractions were subjected to NMR profiling to discard fr1–3, and fr6 that gave no isoflavone signals. Fr7 was purified by silica gel (pore size 60 Å, 70–230 mesh, St. Louis, MO, USA) to yield compound 15 (0.7 mg). Fr4 was separated by preparative TLC on a silica gel plate (Si60, Merck, Darmstadt, Germany), migrated with solvent system of CHCl₃-MeOH (20:3) to give pure compound 16 (0.5 mg) and semi-pure compound 18 (0.2 mg). Fr5 was purified by preparative TLC to give pure compound 17 (0.3 mg) using the same TLC conditions.