Skip to main content

Table 3 Systematic optimization of process variables for the permeabilization of P. pastoris cells expressing Tv DAO.

From: Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

   Accessible activity after permeabilization
Isopropanol concentration Incubation time Tv1a_mc Tv1_mc
[%, by vol.] [h] [U/g wcw] η [U/g wcw] η
- - 6 ~0.01 5 <0.01
0 0.3 100 0.20 321 0.23
4 0.3 118 0.23   
8 0.3 141 0.28   
10 0.0 100 0.20   
10 0.3 184 0.36 430 0.33
10 0.7 193 0.38   
10 1.5 206 0.41   
10 2.5    579 0.45
10 3.0 226 0.45   
10 5.3    627 0.49
10 10.7    621 0.48
12 0.3 176 0.35 424 0.33
15 0.3 170 0.34   
20 0.3 64 0.13   
40 0.3 14 0.03   
  1. The accessible activity is the activity of the whole-cell system after permeabilization, as measured in U/g wcw and expressed as η, the effectiveness factor (= apparent activity/total intracellular activity), whereas the total intracellular activity was determined after cell disruption with a French Press. Strains Tv1a_mc and Tv1_mc contained 550 and 1283 U/g wcw of total intracellular activity, respectively. Lyophilized Pichia cells were used for permeabilization with a concentration being constant at 4 mg/mL. Results in the first row referred to experiments carried out with intact cells obtained from one standard bioreactor cultivation. Permeabilization was performed three times per defined condition, with a standard deviation of the effectiveness factors of < 5%.