Figure 5

Gel-filtration chromatography of ultrafiltrate. One ml of the ultrafiltrate prepared from the cell-free extract of induced C. guilliermondii cells was applied to a Bio-Gel P-2 column (1.0 cm × 50 cm) previously washed with distilled water and eluted with distilled water. The column was calibrated with cyanocobalamin (a, Mr = 1,355), cellobiose (b, Mr = 342), and glucose (c, Mr = 180), as indicated by vertical arrows. The elution pattern was monitored by measuring at 240 nm (dotted line). The fractions containing the activator of FAD activity (filled circle) were examined for color reactions with DTNB solution at 412 nm (open circle) and ninhydrin solution at 570 nm (open triangle). The activation was expressed as ΔU mg^-1 [(activity + eluate)-(control activity)] after 20-min incubation at 25°C. For the color reactions, 0.1-ml aliquots were added to 0.9 ml of 5 mM DTNB in 50 mM phosphate buffer (pH 7.5) and ninhydrin solution, respectively.