Fig. 3. The effect of pH, temperature on the activity of recombinant NDM-1. (A) pH. The optimal pH was determined at 30 °C using various 50 mM buffers, including citrate buffer (pH 2.2, 3.0, 4.0, 5.0), Bis–Tris buffer (pH 6.0, 7.2), Tris–HCl buffer (pH 8.0, 9.0) and glycine–NaOH buffer (pH 9.0, 10.0, 11.0). Relative activity (RA) was calculated and the maximum activity was regarded as 100%. To determine the pH stability, the enzyme was pre-incubated in above buffers with different pH (pH 3.0–12.0) at 30 °C for 18 h, and the RAs were determined in BTB buffer (50 mM Bis–Tris buffer (pH 7.2) containing 50 μM Zn²⁺, 1 mM 1,4-dithio-dl-threitol and 10% (v/v) glycerol) at 30 °C. (B) Temperature. The optimal temperature was determined in 50 mM Bis–Tris buffer (pH 7.2) at a temperature range from 5 to 70 °C. To determine temperature stability, the NDM-1 was pre-incubated at different temperature (5–70 °C) for 60 min in 50 mM Bis–Tris buffer (pH 7.2), and the residual activity was then determined in the BTB buffer at 30 °C.