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Abstract:
Glycerokinase can be used as a coupling enzyme for sensitive enzyme activity
measurements.
The main goal of this work was the development of a purification strategy for recombinant
Glycerokinase produced in Pichia pastoris. Subsequently, the purified enzyme was
characterized with regard to pH- and temperature optima, as well as maximum reaction
rate (Vmax) and Michaelis-Menten constant (Km) for ATP.
The purification cascade included the following steps: cell disruption, solid liquid phase
separation, immobilized metal chelate affinity chromatography, anion exchange
chromatography, and a subsequent buffer exchange. The specific enzyme activity could be
increased by a factor of 60 to 201.6 U/mg.
A pH optimum of 7.0 was determined for purified Glycerokinase, whereas the temperature
optimum was found to be 45°C. The enzyme kinetic constants Vmax 277.8 U/mg and Km
2.47 mM were obtained using Lineweaver-Burk plotting.
