Catalytic kinetics of recombinant nitrile hydratase coupling terminal salt bridge and point-mutation
Chen Ji · 2014
Nitrile hydratase(NHase) is a double-subunit enzyme widely used for industrial production of acrylamide from acrylonitrile. By Discovery Studio 2.5, salt-bridge network simulation toward different recombinant NHases was carried out. A new mutant, NHaseM-TH-SBM(SBM), was obtained, which couples the salt-bridge mutation in C-terminal(SB, S344K-S346K-L347E-435DT436(+)) with the point-mutation of N362S. Simulation results show that the total number of salt-bridges in SBM decreases but the number in ?-? interface increases to 7 pairs. Using both the original NHase TH and SB mutant as controls, SBM is successfully expressed in recombinant E. coli. The activity is 543.9 U·mg?1, increased by 31.0% with respect to TH. The reaction and inactivation kinetics was also investigated. The catalytic rate constant Kcat of Michaelis-Menten equation is increased by 20% and 60% compared to TH and SB, respectively. The apparent inactivation constant KD is 68.0% of TH at 42℃, indicating that SBM also improves thermal stability.