Effects of in Situ Cobalt Ion Addition on the Activity of a GFP-OPH Fusion Protein: The Fermentation Kinetics

Abstract

The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E. coli BL21 expressing a GFP (green fluorescent protein)-OPH (organophosphorus hydrolase) fusion protein. It was found that cobalt ion addition improved the OPH activity significantly. When 2 mM of CoCl(2) was supplied during the IPTG-induction phase, OPH activity was enhanced approximately 10-fold compared to the case without cobalt or by the addition of cobalt to the cell extracts. Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity. Also, the maximum OPH activity was not linearly related to inducer concentration. A mathematical model was then constructed to simulate these phenomena. Model parameters were determined by constrained least-squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity. Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor.

Polyhistidine-containing organophosphorus hydrolase with outstanding properties

“…Researchers have tried to increase the enzyme yield per cell biomass unit, and enhance the specific catalytic activity of the enzyme (Raushel 2002;Efremenko et al 2004;McDaniel 2004;Cho et al 2006). Some investigators focused their efforts on the improvement of downstream processing, and used OPH modified with various amino acid affinity tag sequences, simplifying purification (Wang et al 2001;Wu et al 2001;Shimazu et al 2003;Efremenko et al 2006). A hexahistidine (His 6 ) tag has been the most frequently used amino acid sequence for this purpose.…”