Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79 g/L and 6.8 u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025 h , DCW increased to 88.39 g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80 mL/h, a recombinant uricase activity was 4.13, 7.2, and 0 u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04 u/mL/h (0.0017 mg/mL/h) to 0.18 u/mL/h (0.0078 mg/mL/h).