RESUMEN
The effects of culturing conditions on D-hydantoinase production by a recombinant Escherichia coli strain were investigated using a controlled fed-batch fermentation system. Glucose concentration and pH of the culture broth were maintained at less than 3.3 g/L and at 7.0, respectively, in a 5 L jar fermentor. The optimal composition of the batch medium was glucose, 0.25%; yeast extract, 0.75%; (NH4)2SO4, 0.25%; KH2PO4, 0.2%. The optimal feeding solution was glucose, 60%; yeast extract, 30%; ammonia water, 9%. Following 25-h cultivation, 0.02 mM isopropyl-beta-D-thiogalactopyranoside was added to induce dht gene expression and the temperature was shifted from 32 to 27 degrees C to avoid inclusion body formation. The plasmid-harboring dht gene was found to be stably maintained in the E. coli. Under optimal conditions, a cell density of about 25 g dry cell weight/L and a high volumetric productivity of 8300 U/L/h could be achieved after 48 h culture at agitation and aeration rates of 1000 revolutions per minute and 1 vvm, respectively.
Asunto(s)
Amidohidrolasas/biosíntesis , Escherichia coli/crecimiento & desarrollo , Proteínas Recombinantes/metabolismo , Amidohidrolasas/genética , Medios de Cultivo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Concentración de Iones de Hidrógeno , Cuerpos de Inclusión , Plásmidos , Proteínas Recombinantes/genéticaRESUMEN
An improved expression plasmid pET-insulin-like growth factor-2 (IGF2) was constructed and transferred into Escherichia coli BL21(DE3) for the expression of tilapia insulin-like growth factor-2. The recombinant insulin-like growth factor-2 was produced as inclusion bodies, and the recombinant insulin-like growth factor-2 content was as high as 10.3% of the total protein content. For production of recombinant insulin-like growth factor-2 in E. coli, pH-stat fed-batch cultures were used to achieve a high cell density culture. A cell concentration 183gl(-1) dry cell weight (DCW) was obtained after 30h cultivation and plasmid stability was maintained at high levels. Expression of insulin-like growth factor-2 was induced at three different cell concentrations, 50, 78.5, and 114.5gl(-1) dry cell weight. When cells were induced at a cell concentration of 114.5gl(-1) dry cell weight, the amount of insulin-like growth factor-2 produced was 9.69gl(-1) (11.3% of the total protein). Using a simple purification process including inclusion body isolation, denaturation, refolding and Ni-NTA affinity chromatography, 19.51mg of insulin-like growth factor-2 was obtained from a 22.5ml of culture, and the recovery yield was 20.5%. The biological activity of the purified IGF-2 was demonstrated as promoting the growth of four different cell lines by the colorimetric bioassay and the best growth stimulation ratio was obtained for the Balb/3T3 clone 31A cell line.