Temperature limited fed-batch technique for control of proteolysis in Pichia pastorisbioreactor cultures
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 Neocallim...
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ftbiomed:oai:biomedcentral.com:1475-2859-2-6 2023-05-15T13:51:24+02:00 Temperature limited fed-batch technique for control of proteolysis in Pichia pastorisbioreactor cultures Jahic, Mehmedalija Wallberg, Fredrik Bollok, Monika Garcia, Percival Enfors, Sven-Olof 2003-06-18 http://www.microbialcellfactories.com/content/2/1/6 en eng BioMed Central Ltd. http://www.microbialcellfactories.com/content/2/1/6 Copyright 2003 Jahic et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Research 2003 ftbiomed 2007-11-11T15:40:15Z 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. Other/Unknown Material Antarc* Antarctica BioMed Central |
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description |
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. |
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Other/Unknown Material |
author |
Jahic, Mehmedalija Wallberg, Fredrik Bollok, Monika Garcia, Percival Enfors, Sven-Olof |
spellingShingle |
Jahic, Mehmedalija Wallberg, Fredrik Bollok, Monika Garcia, Percival Enfors, Sven-Olof Temperature limited fed-batch technique for control of proteolysis in Pichia pastorisbioreactor cultures |
author_facet |
Jahic, Mehmedalija Wallberg, Fredrik Bollok, Monika Garcia, Percival Enfors, Sven-Olof |
author_sort |
Jahic, Mehmedalija |
title |
Temperature limited fed-batch technique for control of proteolysis in Pichia pastorisbioreactor cultures |
title_short |
Temperature limited fed-batch technique for control of proteolysis in Pichia pastorisbioreactor cultures |
title_full |
Temperature limited fed-batch technique for control of proteolysis in Pichia pastorisbioreactor cultures |
title_fullStr |
Temperature limited fed-batch technique for control of proteolysis in Pichia pastorisbioreactor cultures |
title_full_unstemmed |
Temperature limited fed-batch technique for control of proteolysis in Pichia pastorisbioreactor cultures |
title_sort |
temperature limited fed-batch technique for control of proteolysis in pichia pastorisbioreactor cultures |
publisher |
BioMed Central Ltd. |
publishDate |
2003 |
url |
http://www.microbialcellfactories.com/content/2/1/6 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
http://www.microbialcellfactories.com/content/2/1/6 |
op_rights |
Copyright 2003 Jahic et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. |
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