Lc 20ap pump

Cholesterol-producing yeast RH6829 strain^{25 (link)} co-expressing PpCYP90G4 and an Arabidopsis CYP reductase (Supplementary Table 3, strain JKW-35.29) was initially grown in YP medium containing 2% raffinose, and subsequently induced in 12 L of YP medium containing 2% galactose and 100 µm CuSO₄. Cells were collected by centrifugation once the OD reached 5. Cell pellets were freeze-dried and resuspended in 1 L of 80% ethanol containing 10% KOH and 0.5 mg/mL BHT, sonicated for 15 min, and incubated at 50 °C with constant mixing. The extracts were mixed with 2 L of H₂O and partitioned with hexane (3 × 1 L). The hexane fractions were pooled, washed twice with 2 L of H₂O, and dried using a rotary evaporator. The dried extracts were resuspended in a minimal amount of chloroform/hexane (1:1) and separated using a silica column packed with Silica Gel 60 (EMD Chemicals Inc.). A gradient of hexane/chloroform/methanol (from 1:1:0 to 0:1:0 to 0:0:1) was used as the mobile phase. The eluate was collected with a fraction collector and subjected to LC–MS analysis as described above. The fractions enriched with 1 were pooled, dried using a rotary evaporator, and further purified using a preparative HPLC system consisting of a Shimadzu system with a LC-20AP pump, a SPD-20A UV-VIS detector and a FRC-10A fraction collector. 1.5 mg of 1 was obtained.

The mycotoxin-containing extracts dissolved in methanol were purified by preparative HPLC. The LC system was equipped with a LC-20APpump, SIL-10AP injector, FRC-10A fraction collector and a diode array detector (DAD) (Shimadzu, Kyoto, Japan), and a reverse-phase SHIM-pack PRC-ODS C18 column (20 mm × 250 mm, 5 μm, Shimadzu, Kyoto, Japan ) was used for mycotoxin seperation. The mobile phases were acetonitrile/methanol (50:50, v/v, solvent A) and H₂O (solvent B) at a flow rate of 8 mL/min. The detector was set at λ₁ = 220 nm for DON and its acetylated derivatives, and λ₂ = 236 nm for ZEN. The linear gradient program started from 20% A for 8 min and increased to 80% A within 1 min, then held for 11 min. Finally, the initial composition of 20% A was re-established followed by equilibration for 5 min. LC retention times and UV absorbance profiles of the purified mycotoxins were compared to those of standard solutions. The targeted fractions were collected by a fraction collector. The LC-purified mycotoxin fractions were concentrated using a rotary evaporator at 45 °C, frozen overnight at −20 °C and then dried in a freeze–dryer for 48 h.

Optical rotations were determined using a Perkin-Elmer model 343 polarimeter. UV and CD spectra were recorded on an Applied Photophysics Chirascan spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR microscope spectrometer (FT-IR microscope transmission). 1D- and 2D-NMR spectra were obtained at 600 MHz for ¹H and 150 MHz for ¹³C, respectively, on a Bruker AVANCE III HD 600 MHz spectrometers in DMSO-d₆ (δ_H 2.500 and δ_C 39.520), and MeOH-d₄ (δ_H 3.310 and δ_C 49.000) with solvent peaks used as references. HRESIMS data were obtained using a Thermo LTQ Orbitrap XL mass spectrometer. LC-MS analysis was performed using an Agilent 1290 Infinity II LC coupled with a 1100 LC/MSD Model G1946D mass spectrometer. Preparative HPLC was conducted on a Shimadzu LC-20AP pump with a SPD-M20A photodiode array detector. TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 7% H₂SO₄ in 95% aqueous EtOH followed by heating.