Figure 5.

Confirmation of EstDZ4 esterolytic activity. (A) Detection of EstDZ4 esterolytic activity via native PAGE analysis and Fast Red staining using 1-naphthyl acetate as a substrate. L1, clarified lysate of cells overexpressing estDZ4; L2, clarified lysate of cells carrying an empty vector. (B) Substrate profiling of the esterolytic activity of EstDZ4, using purified enzyme. The relative enzymic activity was measured as released pNP at 410 nm (pH 7, 40°C). The reported values correspond to the mean value of three independent experiments performed in triplicate and the error bars to one standard deviation from the mean value. Assaying the esterolytic activity of EstDZ4 within the pH range of 4–10 at 40°C using pNP-octanoate as the substrate, revealed that the optimal pH for the new enzyme is pH 6.5 (Figure 6A). Measurements of its relative catalytic activity at different temperatures, on the other hand, showed that EstDZ4 has a broad temperature range of action as it retains high levels of esterolytic activity at temperatures between 40–85°C, with its optimal temperature of action being at 75°C (Figure 6B). In order to evaluate the thermostability of EstDZ4, the enzyme was incubated for prolonged time periods in high temperatures and its residual activity was measured. As shown in Figure 6C, EstDZ4 exhibited a half-life of ∼5 h when exposed to 80°C, and even after 24 h of incubation at 70 and 75°C, the enzyme retained more than 40% of its initial activity, demonstrating that EstDZ4 is a highly thermostable esterase.