Enhanced Biomass Production of Recombinant Pfu DNA Polymerase Producer Escherichia coli BL21(DE3) by Optimization of Induction Variables Using Response Surface Methodology.

Abstract

Pfu DNA polymerase is one of the most preferred molecular enzymes that is isolated from the hyperthermophilic Pyrococcus furiosus and used for high-throughput DNA synthesis by the polymerase chain reaction. Therefore, an efficient Pfu DNA polymerase production method is necessary for molecular techniques. In the present study, Pfu DNA polymerase was expressed in recombinant Escherichia coli BL21(DE3) and significant parameters for the biomass production were optimized using the central composite design which is the most popular method of response surface methodology. Induction conditions including cell density prior induction (OD600nm), post-induction temperature, IPTG concentration, and post-induction time and their interactions on biomass production were investigated. The maximum biomass production (14.1g/L) in shake flasks was achieved using the following predicted optimal conditions: OD600nm before induction of 0.4 and the induction at 32°C for 7.7h, with 0.6mM IPTG. Optimized culture conditions were implemented to scale up experiments. 22% and 70% increase in biomass production was achieved in 3 L and 10 L bioreactors, respectively as compared to initial biomass production observed in unoptimized conditions. Similary, a 30% increase of Pfu DNA polymerase production was obtained after the optimization. The polymerase activity of the purifed Pfu DNA polymerase was assessed by PCR amplification and determined as 2.9 U/μl by comparison with commercial Pfu DNA polymerase. The findings of this study indicated that the proposed fermentation conditions will contribute to further scale‑up studies to enhance the biomass for the production of other recombinant proteins.