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Aufreinigung und Charakterisierung des rekombinant hergestellten Enzyms Glycerokinase aus Pichia farinosa

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons86478

Seidemann,  J.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;
University of Applied Sciences Jena, Germany;

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Zitation

Seidemann, J. (2010). Aufreinigung und Charakterisierung des rekombinant hergestellten Enzyms Glycerokinase aus Pichia farinosa. Diploma Thesis, Jena.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0013-900E-6
Zusammenfassung
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.