Date

2012

Author

Bozkurt, Bahar

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In this study, recombinant human growth hormone (rhGH) production by Pichia pastoris-Mut+ strain was improved by designing feeding strategies which were applied in the production phase of the bioreactor operations. During the bio-reactor experiments the cell growth, sorbitol and methanol consumptions, recom-binant hGH production, alcohol oxidase (AOX) activity, the by-products protease and organic acid concentrations were followed and analyzed. In this context, in the first part of the study, three bioreactor operations were designed and per-formed. In general, the designed strategies are fundamentally based on simulta-neous feeding of the two substrates starting at t=0 h of the production phase, i.e., batch-wise 50 gL-1sorbitol feeding, together with fed-batch methanol feeding with a specific growth rate of μ0=0.03 h-1 or μ0=0.04 h-1, and fed-batch sorbitol feeding with a specific growth rate of μ0=0.025h-1 which was calculated based on the specific consumption rate qS=0.152 g g-1h-1 of sorbitol. Consequently, sorbitol concentration was kept constant at 50 gL-1 within t=0-15h of the production phase; where, sorbitol feeding was terminated at t=15h. Amongst, in the first strategy (SSM1), methanol was fed to the system with the specific growth rate of μ0=0.03 h-1, and the H+ concentration (pH) in the bioreactor was kept constant at pH=5.0. In the second strategy (SSM2), pH was kept constant at 5.5 until t=24h of the induction phase (production phase), thereafter, was reduced to pH= 5.0; where methanol was fed to the bioreactor with the specific growth rate of μ0=0.03 h-1. In the third strategy (SSM3), methanol was fed with the specific growth rate of μ0=0.04 h-1, and the pH in the bioreactor was kept constant at pH 5.0. The highest rhGH production and cell concentration were achieved in the first strategy SSM1 as CrhGH=640 mg L-1 and CX=105.3 g L-1, and the overall cell and product yields on total substrate were calculated as YX/S =0.21 g g-1 and YCrhGH/S =1.83 mg g-1. In the second part of this study the two-substrates sorbitol and methanol were fed simultaneously in a solution compose of 1.37 mol sorbitol and 6.21 mol methanol in 13.88 mol water, which is named as SM. In this strategy (SM), the two-substrate solution was fed to the medium with the specific growth rate of μ0=0.03 h-1 on sorbitol until t=30h; thereafter, only methanol was fed to the bio-reactor with the specific growth rate of μ0=0.03 h-1. The highest cell and rhGH concentrations obtained in SM were, respectively, Cx=104.7 g L-1 and CrhGH=124 mg L-1; and the overall cell and product yields on the total substrate were calcu-lated as YX/S=0.21 g g-1 and YCrhGH/S=0.39 mg g-1. Although the highest cell con-centration obtained at SM is close to that of the SSM1, the rhGH concentration obtained at SM is 5.2-fold lower than that of the strategy SSM1.

Subject Keywords

Human growth., Hormones., Pichia pastoris., Sorbitol.

URI

http://etd.lib.metu.edu.tr/upload/12614559/index.pdf
https://hdl.handle.net/11511/21722

Collections

Graduate School of Natural and Applied Sciences, Thesis