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Optimierung der intrazellulären Expression rekombinanter Glycerokinase in Pichia pastoris


Schaefer,  P.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Schaefer, P. (2009). Optimierung der intrazellulären Expression rekombinanter Glycerokinase in Pichia pastoris. Bachelor Thesis, Hochschule Esslingen, Esslingen.

The methylotrophic yeast Pichia pastoris was used as expression host for the production of recombinant glycerokinase which is applied as a coupling enzyme in enzymatic cycling assays for determination of key enzymes of the central carbon metabolism in MDCK cells. The main goal of this work was the optimisation of expression of recombinant glycerokinase. Shaker flask and bioreactor cultivations under different growth conditions were conducted. For quantification of glycerol a pre-existing enzymatic assay was optimised and validated. In shaker flask cultivations a maximum biomass concentration of 29 g·L-1 and 6.4 g·L-1 was reached for cultivation on glycerol and methanol, respectively. The maximum specific enzyme activity of glycerokinase was 9.84 U·(mg protein)-1. A two step bioreactor cultivation, consisting of a glycerol-batch followed by a methanolfed batch phase was established. A maximum biomass concentration of 133 g·L-1 was reached after 73 h of cultivation. The experiments were performed with a predetermined constant feed rate and exponential methanol-feed. Presets for μ were 0.04 h-1 and 0.06 h-1. This resulted in real growth rates of 0.03 h-1 and 0.05 h-1, respectively. The corresponding maximum specific enzyme activities of recombinant glycerokinase were measured as 6.50 U·(mg protein)-1 and 9.65 U·(mg protein)-1, respectively.