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Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris.

Anasontzis GE, Salazar Penã M, Spadiut O, Brumer H, Olsson L - Biotechnol. Prog. (2014)

Bottom Line: We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates.The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction.An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed.

View Article: PubMed Central - PubMed

Affiliation: Industrial Biotechnology, Dept. of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, SE-412 96, Sweden; Wallenberg Wood Science Center, Chalmers University of Technology, Kemigården 4, Gothenburg, SE-412 96, Sweden.

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Volumetric GalOx activity as a function of time in culture using (A) high methanol-feeding rates, following the function F(t) = e0.28t (♦) and F(t) = e0.28EXP(0.28t) (▪), and (B) low methanol-feeding rates following the function F(t) = 6e0.1t (•) and F(t) = 4.76e0.1t (▴). Under mild conditions, the volumetric GalOx activity in the fermentation broth was almost 10 times higher than in the former case. Time refers to the total time in culture. The exponential phase started at 40 h.
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fig02: Volumetric GalOx activity as a function of time in culture using (A) high methanol-feeding rates, following the function F(t) = e0.28t (♦) and F(t) = e0.28EXP(0.28t) (▪), and (B) low methanol-feeding rates following the function F(t) = 6e0.1t (•) and F(t) = 4.76e0.1t (▴). Under mild conditions, the volumetric GalOx activity in the fermentation broth was almost 10 times higher than in the former case. Time refers to the total time in culture. The exponential phase started at 40 h.

Mentions: The first two experiments (experiments A and B; Table2) were performed with high exponential feeding rates (Figure 2A): experiment A at feeding rate that was double the maximum specific growth rate of P. pastoris on methanol which was reported to be 0.14 h−1,43 and experiment B with a double exponential feeding rate with the purpose to increase the stress for the cells. The volumetric productivity was basically the same in these two experiments, i.e., 1.3 and 1.4 kU L−1 h−1, respectively. However, production of active GalOx was found to be 148 U mg−1 in fermentation A, where a lower feeding profile was used, and 132 U mg−1 in fermentation B. These two experiments already indicated that there was a positive effect of a lower methanol-feeding rate on the production of active GalOx. To confirm this hypothesis, we performed two similar fed-batch experiments with only small differences in the initial flow and the same exponential feeding profiles, which were lower than the maximum specific growth rate (experiments C and D; Table2). In fact, the volumetric productivity was almost 8-fold higher when low methanol-feeding rates were applied rather than high feeding rates. Not only was the overall amount of extracellular protein significantly higher, but also the amount of active, correctly folded GalOx increased almost 5-fold, as shown from the values of the specific GalOx activity (Table2). The small differences in the initial flow did not see to affect the yield, although experiment D, with the lower initial flow, resulted in slightly better volumetric productivity and volumetric activity.


Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris.

Anasontzis GE, Salazar Penã M, Spadiut O, Brumer H, Olsson L - Biotechnol. Prog. (2014)

Volumetric GalOx activity as a function of time in culture using (A) high methanol-feeding rates, following the function F(t) = e0.28t (♦) and F(t) = e0.28EXP(0.28t) (▪), and (B) low methanol-feeding rates following the function F(t) = 6e0.1t (•) and F(t) = 4.76e0.1t (▴). Under mild conditions, the volumetric GalOx activity in the fermentation broth was almost 10 times higher than in the former case. Time refers to the total time in culture. The exponential phase started at 40 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4282061&req=5

fig02: Volumetric GalOx activity as a function of time in culture using (A) high methanol-feeding rates, following the function F(t) = e0.28t (♦) and F(t) = e0.28EXP(0.28t) (▪), and (B) low methanol-feeding rates following the function F(t) = 6e0.1t (•) and F(t) = 4.76e0.1t (▴). Under mild conditions, the volumetric GalOx activity in the fermentation broth was almost 10 times higher than in the former case. Time refers to the total time in culture. The exponential phase started at 40 h.
Mentions: The first two experiments (experiments A and B; Table2) were performed with high exponential feeding rates (Figure 2A): experiment A at feeding rate that was double the maximum specific growth rate of P. pastoris on methanol which was reported to be 0.14 h−1,43 and experiment B with a double exponential feeding rate with the purpose to increase the stress for the cells. The volumetric productivity was basically the same in these two experiments, i.e., 1.3 and 1.4 kU L−1 h−1, respectively. However, production of active GalOx was found to be 148 U mg−1 in fermentation A, where a lower feeding profile was used, and 132 U mg−1 in fermentation B. These two experiments already indicated that there was a positive effect of a lower methanol-feeding rate on the production of active GalOx. To confirm this hypothesis, we performed two similar fed-batch experiments with only small differences in the initial flow and the same exponential feeding profiles, which were lower than the maximum specific growth rate (experiments C and D; Table2). In fact, the volumetric productivity was almost 8-fold higher when low methanol-feeding rates were applied rather than high feeding rates. Not only was the overall amount of extracellular protein significantly higher, but also the amount of active, correctly folded GalOx increased almost 5-fold, as shown from the values of the specific GalOx activity (Table2). The small differences in the initial flow did not see to affect the yield, although experiment D, with the lower initial flow, resulted in slightly better volumetric productivity and volumetric activity.

Bottom Line: We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates.The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction.An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed.

View Article: PubMed Central - PubMed

Affiliation: Industrial Biotechnology, Dept. of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, SE-412 96, Sweden; Wallenberg Wood Science Center, Chalmers University of Technology, Kemigården 4, Gothenburg, SE-412 96, Sweden.

Show MeSH
Related in: MedlinePlus