Abstract: The recombinant Escherichia coli producing pullulanase was studied. After analysis of the data in the shake flask culture, the strategy of producing of pullulanase by high cell density cultivation of recombinant E.coli was investigated. The data of the cell optical density, cell dry mass, separation of soluble and insoluble cell fractions, SDS-PAGE were analyzed cell growth and production of pullulanase. Shake flask experiments were done in the defined medium and LB medium. After 5h of induction, the specific pullululanase concentration obtained on defined medium was considerably higher than that on LB medium, whereas the cell growth was slower. The pullulanase produced on defined medium accumulated in the insoluble cell fraction less than that on LB medium. A pre-determined feeding (μset=0.12) strategy was chosen to maintain carbon-limited growth using a defined medium. Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor in order to prevent the accumulation of acetic acid. Expression of pullulanase was induced when cell concentration OD600 was 70.0. Applying the feeding strategy, final cell concentration of 53.3g per liter dry cell weight (g/L DCW) and production of pullulanase in the soluble cell fraction in a volumetric concentration of 1.35 g per liter were obtained.

Key words: high cell density cultivation, recombinant Escherichia coli, pullulanase