Figure 4

Protection of laccase3 activity by various protease inhibitors. Brown coloration of DMOP substrate by preincubated ura^- selective media and colormetric observation was as described in Figure 1. Lanes 1-3 contain the 3-day-old ura^- culture supernatant of a mock transformant, TYEGLAC3-1, and TYEGLAC3-5, respectively. YEPD and SCB represent the additions of fresh YEPD media and supernatant of TYEGLAC3-1-cultured YEPD broth, respectively. + indicates various protease-inhibiting treatments to SCB. Six different protease inhibitors [aprotinin (0.8 μM), E-64 (84.0 μM), EDTA (0.8 mM), leupeptin (10.0 μM), pepstatin (7.3 μM), and PMSF (2.0 mM)] were tested and incubation at 65°C for 30 min was conducted as heat treatment [41]. Note that the addition of pepstatin maintains the browning, whereas no other protease inhibitors do.