The melting point was analyzed as described before . In a total volume of 25 µL, 0.2 g L -1 enzyme and 5x SYPRO orange were incubated in 70 mM Tris-HCl pH 7, 8 or 9 with 0 or 5 mM DTT. The sealed PCR plate was incubated at 50 °C for 30 s and then heated in steps of 0.5 °C per 5 s to a final temperature of 95 °C in a Biorad CFX96 Real-Time system. Fluorescence was measured at λex = 470 nm and λem = 570 nm. To estimate protein stability at elevated temperatures, proteins were further incubated at 95°C for 20 min. Fluorescence was measured again afterwards. The melting point was determined as the inflection point of the fluorescence intensity over the temperature.