A 1000-L Scale-Up Fermentation of Escherichia Coli Containing PVSEOP7 for Production of Organophosphorus Hydrolase
EDGEWOOD CHEMICAL BIOLOGICAL CENTER ABERDEEN PROVING GROUND MD
Pagination or Media Count:
Four 20-L scale batch fermentations in LB medium were performed to investigate time-dependent yields of organophosphorus hydrolase OPH in E. coli XL1 containing pVSEOP7. The specific OPH activity was monitored during each 20-L batch fermentation culture by assaying several times between the IPTG induction point OD of 0.4 to 0.5 at 600 nm and the cell harvest point near-stationary phase of each 20-L batch culture. From the four 20-L batch fermentations, only the 20-L batch cultures with 1 mM of cobalt chloride added 3-hr prior to cell harvest resulted in a drastic increase in the specific OPH activity 369 to 1809 UmL. A similar strategy of 1 mM cobalt chloride addition was successfully used to obtain a 20.7 mg OPH per liter of culture at the 1000-L scale batch fermentation in LB, as compared to 5 to 10 mgL in flask cultures. The specific OPH activity was shown to be stable through additional process time associated with downstream processing steps such as cell concentration by continuous disk centrifugation, cell paste preparation by bottle centrifugation, cell disruption by Microfluidizer homogenization, and lysed cell clarification by 0.22 m tangential flow microfiltration.
- Organic Chemistry