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Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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2003 (English)In: Microbial cell factories, ISSN 1475-2859, Vol. 2, no 1, 6- p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2003. Vol. 2, no 1, 6- p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-13465DOI: 10.1186/1475-2859-2-6PubMedID: 12871597OAI: oai:DiVA.org:kth-13465DiVA: diva2:325425
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2010-06-18Bibliographically approved
In thesis
1. Process techniques for production of recombinant proteins with Picha pastoris
Open this publication in new window or tab >>Process techniques for production of recombinant proteins with Picha pastoris
2003 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 2003. 51 p.
Keyword
p. Pastoris, fed-batch culture, proteolysis, modeling
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-3572 (URN)91-7283-522-2 (ISBN)
Public defence
2003-06-13, 00:00
Note
QC 20100618Available from: 2003-06-26 Created: 2003-06-26 Last updated: 2010-06-18Bibliographically approved

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