Figure 5

Western blot of the TRI201 enzyme from Fusarium fujikuroi (FfTRI201) extracted from VA04B-125 mashes. Mash samples weighing 1 g were collected at the end of fermentation (71 hours), and protein extractions were performed. Mashes containing untransformed RW2802 yeast were used as a negative control. For analysis, 4 μl of each extract were loaded onto a 12% acrylamide-SDS-PAGE gel and run at 150 V for 1 hour. A protein standard (Precision Plus Protein Dual Color Standard) was used to determine protein size. The western blot was probed with rabbit anti-FsTRI101 primary antibody, and the probe detected with alkaline phosphatase-conjugated anti-rabbit antibody. Lane 1: purified FfTRI201 from Escherichia coli; lane 2: FfTRI201 from mash 1 containing transformed yeast; lane 3 FfTRI201 from mash 2 containing transformed yeast; lane 4: FfTRI201 from mash 3 containing transformed yeast; lane 5: mash 1 containing untransformed RW2802; lane 6: mash 2 containing untransformed RW2802; and lane 7: mash 3 containing untransformed RW2802.