270342 Effect of pH and Aeration On Plasmid Stability and Phytase Expression in Escherichia Coli BL21(DE3) During Batch Cultivations in Semi-Scale Bioreactor
Othman, N.Z1, Ramli.S1., Masri, J.H1., Sarmidi, M.R1, Aziz, R1, Tran, T.T2 , Hatikaul.R2., El Enshasy, H.A1,3.
1.Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 Skudai Johor, Malaysia
2. Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund, Sweden.
3.Bioprocess Development Department, City for Scientific Research and Technology Applications (CSAT), New Burg Al Arab, Alexandria, Egypt;
Phytase production by recombinant Escherichia coli BL21 (DE3) is seen as cost-effective alternative for industrial production. In this cultivation process, aeration is an important factor in achieving high cell density cultivation and the expression of foreign protein by the host cells. Phytase production was growth associated process and the enzyme expression was dependent on plasmid stability. In this study, the optimized medium was used for batch cultivation of E. coli BL21(DE3) that growth under controlled pH 7 and uncontrolled pH in 16-L stirred tank bioreactor. Furthermore studies were conducted to investigate the kinetics of cell growth, phytase expression and plasmid stability when E. coli BL21(DE3) cultivated under different aeration rates of 1.0 v v-1 min-1, 2.0 v v-1 min-1 , and 3 v v-1 min-1 under controlled pH in 16-L stirred tank bioreactor. The results showed that the total phytase production obtained in batch cultures was 2.507 U mL-1 and 1.198 U mL-1 for controlled and uncontrolled pH when induced with 30 mM of lactose, respectively with approximately 2.5 g L-1 cell mass. When cell cultivated at lower aeration rate of 1.0 v v-1 min-1, significant decrease in plasmid stability was observed after 4 h of induction as a result of oxygen limitation in culture concomitant with acetate accumulation up to 8 g L-1. On the other hand, cultivation in the 16-L stirred-tank bioreactor under controlled pH and aeration of 2.0 v v-1 min-1and 3 v v-1 min-1 supported cell growth of 3.43 g L-1 and 3.82 g L-1, respectively. Highest total phytase production of 14.24 U mL-1 was obtained in 3.0 v v-1 min-1 bioreactor aerated culture. The maximal specific growth rate (µmax) and maximum specific phytase activity obtained in 3.0 v v-1 min-1 aerated culture were 0.333 h-1 and 5088.21 U g-1, respectively. Cultivation at aeration of 3.0 v v-1 min-1 can maintained the plasmid stability more than 60% after 7 h in the post-induction phase and accumulation of acetic acid in this culture was only 2 g L-1.
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