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Table 1 Optimization of fed-batch cultivation of recombinant PichiaPink™ strains expressing Af Cel12A and Ta Cel5A endoglucanases

From: Expression of endoglucanases in Pichia pastoris under control of the GAP promoter

Run

Strain

Temperature (°C)

Feed

Feed regime

Max dry cell weight (g/l)

Total supernatant protein (g/l)

1

GAP/TaCel5A

30

Glycerol

1714 g over 70 h

109

2.8

2b

GAP/TaCel5A

30

Glycerol

2470 g over 70 h

155 ± 5

3.8 ± 0.4

3

GAP/TaCel5A

30

Glucose

2470 g over 70 h

145

4.9

4

GAP/TaCel5A

25

Glycerol

2470 g over 70 h

155

5.4

5

GAP/AfCel12A

30

Glycerol

1714 g over 70 h

132

3.4

6b

GAP/AfCel12A

30

Glycerol

2470 g over 70 h

146 ± 9

3.3 ± 0.3

7

GAP/AfCel12A

30

Glucose

2470 g over 70 h

100

n.d.a

8

GAP/AfCel12A

25

Glycerol

2470 g over 70 h

170

7.3

  1. aNot determined; bOptimal parameters, fermentations run in 5 parallels.
  2. Fermentations were carried out to analyze the effect of temperature, increased glycerol feed and carbon source on the GAP/TaCel5A strain and the GAP/AfCel12A strain. The PichiaPink™ cells were grown in 15-l scale bioreactors with a 5-l initial volume for 24 hours in batch mode, followed by feeding as indicated; see Methods for more details. Runs were performed as single experiments; however, optimized runs were performed five times, and standard deviations are provided. SDS-PAGE analyses of the culture supernatants are shown in Figure 3.