(442c) The Use of Fed Batch Cultivation for Achieving High Cell Densities for the Pilot Scale Production of a Recombinant Protein (Phenylalanine Dehydrogenase) in Escherichia Coli | AIChE

(442c) The Use of Fed Batch Cultivation for Achieving High Cell Densities for the Pilot Scale Production of a Recombinant Protein (Phenylalanine Dehydrogenase) in Escherichia Coli

Authors 

Faulkner, E. M. - Presenter, University College Dublin
Barrett, M. - Presenter, University College Dublin
Okor, S. - Presenter, University College Dublin
Paradisi, F. - Presenter, University College Dublin
Engel, P. - Presenter, University College Dublin
Glennon, B. - Presenter, University College Dublin


The most frequently employed technique used to obtain high cell densities in E. coli cultures is fed-batch fermentation. During fed-batch operation, it is critical to control the specific growth rate as the formation of inhibitory by-products such as acetic acid, cell productivity and plasmid stability are related. To obtain high protein expression levels in bacterial cultures, it is important to obtain a state of well-balanced, exponential growth. Balanced growth is achieved when all nutrients are supplied in optimum amounts above the limiting concentration and below the toxic concentration or the concentration which promotes wasteful utilization. If balanced growth is not achieved oxygen limitation occurs and harmful levels of toxic products, such as acetate, are accumulated which can prevent high-density growth of the bacteria and limit recombinant protein expression. In fed-batch culture, acetate secretion is reduced by controlled nutrient feeding (i.e. one of the ingredients is growth rate limiting- usually the carbon source) while growing cells at lower specific growth rate.

The objective of this work was to design a feeding profile which would achieve a high cell density cultivation of E. coli TG1 and produce the recombinant protein, phenylalanine dehydrogenase (PheDH). PheDH is a commercially valuable biocatalyst which introduces a chiral centre in converting keto acids to L-alpha amino acids. A model, based on Monod kinetics with overflow metabolism and including acetate production and re-utilisation, was used to predict biomass, glucose and acetate levels at various feed rates over time. A feed profile was then designed which would promote glucose limited growth with minimal acetic acid in the cell free medium. Cell concentrations of 53 g l-1 dry cell weight (DCW) were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/ mg DCW and the product formation rate of 0.41 U/ mg DCW, h.

The specific activity of the unpurified enzyme was 1.18 U/mg DCW. This PheDH, produced in the bioreactor was subsequently, used to catalyse the reaction from phenylpyruvic acid to L-phenylalanine. The enzyme was used in whole cell form minimising cost and time consumption. 85% conversion to product was achieved.

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