Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures

• Published in: Microbial Cell Factories
• Main Authors: Garcia Percival, Bollok Monika, Wallberg Fredrik, Jahic Mehmedalija, Enfors Sven-Olof
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2003
• Subjects: Microbiology; QR1-502; Antarc*; Antarctica
• Online Access: https://doi.org/10.1186/1475-2859-2-6; https://doaj.org/article/f2734f7d093841e398ed1a48db0096a6

Abstract Background A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut + strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain. Results A protein concentration of about 1 g L -1 was produced in the MLFB process. However, this product was considerably degraded by protease(s). By applying the TLFB process, the yield was increased to 2 g L -1 full-length product and no proteolytic degradation was observed. Flow cytometry analysis showed that the percentage of dead cells increased rapidly during the initial methanol feed phase in the MLFB process and reached a maximum of about 12% after about 40–70 hours of methanol feeding. In the TLFB process, cell death rate was low and constant and reached 4% dead cells at the end of cultivation (about 150 hours methanol feeding time). The lower cell death rate in the TLFB correlated with a lower protease activity in the culture supernatant. The specific alcohol oxidase (AOX) activity in the TLFB process was 3.5 times higher than in the MLFB process. Conclusion Three mechanisms that may contribute to the much higher accumulation of product in the TLFB process are: 1) reduced proteolysis due to lower temperature, 2) reduced proteolysis due to lower cell death and protease release to the medium, 3) increased synthesis rate due to higher AOX activity.