A 1000-L Scale-Up Fermentation of Escherichia Coli Containing PVSEOP7 for Production of Organophosphorus Hydrolase

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 U/mL). 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 mg/L 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. Prepared in collaboration with Geo-Centers, Inc., Aberdeen Proving Ground, MD. See also ADM001851, Proceedings of the 2003 Joint Service Scientific Conference on Chemical & Biological Defense Research, 17-20 November 2003. , The original document contains color images.