Figure 9.

Determination of active components from culture supernatant by chromatography and SDS-PAGE. (A) Ion exchange chromatography on DEAE A-50 column. The column was pre-equilibrated with 50 mM sodium phosphate buffer pH 7.5. Elution was performed with the same buffer for the first 40 mL and then by a gradient of 0.5 M NaCl added to the buffer. Fractions of 2 mL were collected in tubes at a flow rate of 0.4 mL/min. (B) Molecular sieve chromatography on Sephadex G-100 column. The column was pre-equilibrated with 50 mM sodium phosphate buffer pH 7.5 containing 0.2 M NaCl. Proteins were eluted with the same buffer. Fractions of 2 mL were collected in tubes at a flow rate of 0.2 mL/min. (C) SDS-PAGE analysis of AFB₁ biotransformation enzyme after purification by chromatography. Lane 1, Calibration marker proteins (Pharmacia LKB), 5 μL/lane; lane 2, peak fractions with AFB₁ biotransformation activity from Sephadex G-100 chromatography, 20 μL/lane.