Fermentation and recovery of EcoRI endonuclease using two different genetically engineered E.coli strains

Abstract

A small scale procedure developed for the purification of EcoRI restriction enzyme has been applied to two different overproducing E.coli strains. The final purification yields obtained are 1.3x105 U/g cells for E.coli M5248 and 3.3x106 U6g cells for E.coli 294. For the E.coli 294 (pPG430) strain, 0.1 mM IPTG concentration at an optical density of 1.2 at 595 nm over an 6 hours period were determined to be the optimum conditions for induction. For the E.coli M5248 (pSCC2) strain, the induction of EcoRI enzyme was achieved by a temeparuter-shift at an optical density of 1.0 at 590 nm over an 5 hours period. Investigation of the stabiltiy of both recombinant plasmids showed that the enzyme productivity and the plasmid stabilty were effected fro the medium composition and the temperature increase.The unstructured modeling of cell growth has shown that substrate-inhibited kinetics is successful in predicting the growth behavior of both E.coli strains. A three-compartment model was developed to describe the dynamic changes in the intracellular components of E.coli 294 cells. The model gives a good description of the growth kinetics with an initial glucose concentraiton of 10 g/L.