Fermentation and recovery of the EcoRI restriction enzyme with a genetically modified Escherichia coli strain

Abstract

The fermentation and recovery of the EcoRl restriction endonuclease with a genetically modified Escherichia coli strain is investigated. Vast amounts of product could be obtained after cultivation in a 20-L computer-coupled pilot fermentor and purification of the recovered wet cells. It was found that in the end the product is at least inhibitory and probably lethal to the cells (the lethality has been proven with genetic experiments) so that optimum yield requires an optimized choice for the time instant of induction. Growth after induction and product formation require substantial amounts of oxygen, which must be supplied if a high population level is to be achieved. pH control may alleviate the burden of high oxygen supply. Quantitative assessment after the different purification stages indicate that approximately 15% active enzyme can be obtained from the total amount produced.