Figure 4

Influence of temperature, pH and various additives on EngU activity. All assays were performed in triplicate, the error bars represent standard deviations. (a) Temperature-vs-activity profile. The relative activity of EngU (0.06 mg/ml) with barley β-glucan was measured at the indicated temperatures in McIlvaine buffer, pH 6 and 25 min incubation time. (b) Dependence of activity on the pH. Incubations were performed with 0.06 mg/mL purified protein at 90 °C for 45 min in McIlvaine (pH 4–6), phosphate buffer (pH 6–8) and Tris-HCl buffer (pH 8–9). (c) Influence of metal ions, SDS, EDTA and β-mercaptoethanol (β-ME) on the activity of EngU with barley β-glucan. Incubations were performed with 0.06 mg/ml EngU at 90 °C for 12 min in McIlvaine buffer, pH 6. The activity without additions is set to 100%. (d) Kinetics of thermoinactivation of EngU. After incubation of recombinant EngU (0.2 mg/mL) in 50 mM McIlvaine buffer pH 6 for the indicated time, the remaining activity was measured at 80 °C in 50 mM McIlvaine buffer pH 6 for 20 min, using barley β-glucan as the substrate. The activity at t = 0 was set to 100%.