Date

2013

Author

Gökbudak, Burcu

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In this study, the aim is to develop an effective feeding strategy for therapeutically important protein, recombinant human growth hormone (rhGH) production by Bacillus subtilis scoC knockout (scoC-) strain carrying pMK4::pre(subC)::hGH plasmid. In the first part of the research program, laboratory scale air filtered shake bioreactor experiments were conducted to optimize the production conditions. Thereafter, semi-batch production processes at pilot scale bioreactor system were performed by designing exponential feeding strategies in order to enhance the rhGH production capacity. In this context, the effects of glutamine and medium composition on the cell concentration and rhGH production were separately investigated in batch bioreactor experiments at laboratory scale. The maximum rhGH concentration was obtained in the defined medium containing 0.05 g L-1 glutamine (G-0.05) as 76 mg L-1 at t=28 h. In the second part of the air filtered shake bioreactor experiments, the effects of medium composition were investigated in seven different media in order to achieve high cell cultivations leading to rhGH productions at high levels by semi-batch operation. The maximum cell and rhGH concentrations were obtained as 2.47 g L-1 and 75 mg L-1 in the medium P-6 which basically contained 8 g L-1 peptone and 5 mL L-1 trace salt solutions different than that of the defined medium, respectively. Based on these results, the medium containing peptone and trace salt solutions in the presence of glucose and (NH4)2HPO4 was used as the new feeding substrate medium for the semi-batch experiments. In the pilot scale bioreactor experiments, eight different feeding strategies were designed for rhGH production in semi-batch processes. The effects of exponential feeding strategies with the pre-determined specific growth rates chosen as 0.04, 0.10, 0.17 and 0.26 h-1 on rhGH production were investigated. The highest cell and rhGH concentrations were achieved as 4.43 g L-1 and 366 mg L-1, respectively, by the glucose based semi-defined medium feeding strategy conducted at a pre-determined specific growth rate µ0=0.17 h-1 at t=28 h. By this feeding strategy, the overall product and cell yield on total substrate were acquired as 62.92 g kg-1 and 0.73 g g-1, respectively.

Subject Keywords

Recombinant human somatotropin., Bacillus subtilis., Metabolism.

URI

http://etd.lib.metu.edu.tr/upload/12616267/index.pdf
https://hdl.handle.net/11511/23069

Collections

Graduate School of Natural and Applied Sciences, Thesis