BPUL - Laccase from Bacillus pumilus DSM 27 (ATCC7061)
After the measurement of activity of BPUL we made a scale-up and fermented E. coli KRX with <partinfo>BBa_K863000</partinfo> in Braun Biostat B with a total volume of 3 L. Agitation speed, pO2 and OD600 were determined and illustrated in Figure 1. We got a long lag phase of 2 hours due to a relativly old preculture. The cell growth caused a decrease in pO2 and after 3 hours the value fell below 50 %, so that the agitation speed was increased automatically. After 8,5 hours the deceleration phase started and therefore the agitation speed was descreased. The maximal OD600 of 3,53 was reached after 10 hours. The cells were harvested after 11 hours.
Figure 1: Fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in Braun Biostat B, scale: 3 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold a pO2 of 50 %, OD600 taken every 30 minutes.
Figure 2: Chromatogramm of Wash and Elution from FLPC Ni-NTA-Histag Purification of BPUL produced by 3 L fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>.
Another scale-up of the fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> was made up to a final working volume of 6 L in Bioengineering NFL22. Agitation speed, pO2 and OD600 were determined and illustrated in Figure 3. There was no noticeable lag phase.
Figure 3: Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (BPUL) in Bioengineering NFL22, scale: 6 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation increased when pO2 was below 30 %, OD600 taken every hour.
Figure 4: Chromatogramm of Wash and Elution from FLPC Ni-NTA-Histag Purification of BPUL produced by 6 L fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>.
ECOL - Laccase from Escherichia coli BL21 (DE3)
Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in Infors Labfors Bioreactor, scale: 3 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold a pO2 of 50 %, OD600 taken every 30 minutes.
Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in Bioengineering NFL22, scale: 6 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation increased when pO2 was below 30 %, OD600 taken every hour.
XCCL - Laccase from Xanthomonas campestris pv. campestris B100
BHAL - Laccase from Bacillus halodurans C-125
TTHL - Laccase from Thermus thermophilus HB27
Fermentation of E. coli KRX with <partinfo>BBa_K863012</partinfo> (TTHL) in Bioengineering NFL22, scale: 6 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation increased when pO2 was below 30 %, OD600 taken every hour.