Properties and Kinetics of Kex2 Protease Mutants K291

Two kinds of Kex2 protease mutants, Kex2-K291L and Kex2-K291H were successfully expressed in Pichia pastoris, which were induced by methanol and purified with anion exchange chromatography (Q-FF). Finally, the enzymatic characteristics of these Kex2 proteases were characterized. Compared with the wild-type Kex2, the degradation of the two mutants Kex2-K291H and Kex2-K291L was significantly improved. The wild-type Kex2 protease was degraded during the purification, and a non-single band appeared, while the mutants were not degraded during the purification, and it was still a single band. The optimum pH and temperature of these two mutants were the same as those of the wild-type Kex2. Their optimal pH and temperature were pH 9.0 and 37 ℃, respectively. Compared with the wild-type Kex2 protease, the pH stability range of the mutant Kex2-K291H was expanded, from the range of pH 5.0 to 6.0 to the range of pH 5.0 to 7.0. Compared with the wild-type Kex2, Kex2-K291H was more stable at the temperature range of 4 ℃ to 37 ℃. Enzymatic reaction kinetics studies showed that the K_cat/K_m values of mutant Kex2-K291H and Kex2-K291L were 1.85 and 2.05 fold higher than that of the wild-type Kex2 protease, respectively.