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Zusammenfassung:
In principle, enzyme activities can be determined either by direct assays in which the enzyme-catalyzed reaction is monitored by measuring product accumulation or substrate depletion over time or by indirect assays, such as enzymatic cycling systems that increase significantly the sensitivity of enzymatic assays [1,2]. In case changes in product or substrate concentrations cannot be observed directly, it is common practice to use one or more coupling enzymes to generate detectable amounts of product. For example, yeast glycerokinase (GK; EC 2.7.1.30) was used as a coupling enzyme in a range of assays measuring ATP- or UTP-generating enzymes with the G3P cycling system [1,2]. One of the difficulties is the replacement of the enzyme by its commercially available homologues from bacteria, as these do not react with UTP.
In this study, the GUT1 gene of the halotolerant yeast Pichia farinosa, encoding GK, was expressed in Pichia pastoris [3]. The yeast was grown in a 5 L bioreactor operated in fed-batch mode using an exponential feeding strategy. Purification of the recombinant enzyme was done by nickel affinity chromatography and anion exchange chromatography. Afterwards, the purified enzyme was characterized biochemically (pH, temperature optima, substrate kinetics). Finally, GK was applied as a coupling enzyme to determine the specific pyruvate kinase (EC 2.7.1.40) activity in stationary-phase Madin-Darby canine kidney (MDCK) cells grown in pyruvate-containing GMEM medium and serum-free Episerf medium.
[1] Gibon, Y. et al. (2004). The Plant Cell, 16, 3304-3325.
[2] Janke, R., Genzel, Y., Wahl, A., Reichl, U. (2010). Biotech Bioeng 107, 566-581.
[3] Janke, R. et al. (2010). Journal of Biotechnology 150, 396–403.
