D raffinose pentahydrate

Analytical grade silver nitrate (AgNO₃, 99.8%, Merck, KGaA, Darmstadt, Germany), D-(+) raffinose pentahydrate (Alfa Aesar, Karlsruhe, Germany), and sodium hydroxide (NaOH, 99%, Merck, Germany) were used to prepare aqueous dispersion of silver nanoparticles. The stock standard solutions of 500 mg L⁻¹ Cr(VI) and 500 mg L⁻¹ Cr(III) were prepared using K₂Cr₂O₇ (Sigma-Aldrich, GmbH, Sternheim, Germany) and CrCl₃·6H₂O (Merck, Germany), respectively. Working standard solutions for Cr(VI) within the concentration range of 1 × 10⁻⁶–1 × 10⁻⁴ mol L⁻¹ were prepared daily by appropriate dilution of the stock standard solution using 1.5 × 10⁻³ mol L⁻¹ HCl. All diluted Cr(VI) solutions were kept refrigerated at 4 °C. Analytical grade ascorbic acid (AA) and salts of the different cations studied (NaCl, KCl, MgCl₂, CaCl₂, Pb(NO₃)₂, ZnCl₂, CuCl₂, NiCl₂, CdCl₂, CoCl₂, FeCl₃) were purchased from Merck, Germany. Doubly distilled water was used for preparation of all reagent solutions.

Single colonies of each transformants were incubated in 5 mL appropriate selective drop-out medium containing (SC-LEU) 2% glucose over night at 30 ℃, 200 rpm. Overnight cultures were vortexed and cell density was estimated using a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The cells were diluted into a 250 mL baffled shake flasks containing 50 mL selective media with 2% raffinose [D(+)-raffinose pentahydrate, Acros organics, China] to an OD600  = 0.2. The cells were grown for approximately 6–8 h at 30 ℃, 200 rpm until an OD600 of 1 was achieved. Galactose [D(+)-galactose, VWR chemicals, Belgium] was added to a final concentration of 4% in order to induce transcription, by activating the GAL1/10 promoter system in the pESC-vector system. The cultures were maintained at 30℃ and 200 rpm for 48 h to induce expression of the fungal biosynthetic genes. After 48 h, cultures were pelleted and purification were performed on the pellet and supernatant independently, utilizing the methods described in previous work for the pellet and supernatant respectively [28 (link), 29 ]. The dried extracts were dissolved in 1 mL methanol and analyzed on a Hitachi Elite LaChrom HPLC in accordance to chemical analysis performed in [30 (link)].