Fig. 3.

Time course hydrolysis of lactose by wild-type enzyme and Y364F mutant. The reactions were performed at 50 °C for 0, 1 h, 3 h, 6 h, 10 h, 24 h, 36 h, and 48 h in 0.1 M citrate/phosphate buffer (pH 5.2) containing 240 mg /ml lactose, with or without the addition of 24 mg/ml galactose, and equal aliquots of enzyme (40 µg/ml). The reactions were halted by incubation at 100 °C for 10 min, and the lactose concentration was determined by high-performance liquid chromatography (HPLC, Waters, Boston, MA, USA)