Interfacing Pichia pastoris cultivation with expanded bed adsorption
Abstract For improved interfacing of the Pichia pastoris fed‐batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating th...
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crwiley:10.1002/bit.20811 2024-06-02T07:57:15+00:00 Interfacing Pichia pastoris cultivation with expanded bed adsorption Jahic, Mehmedalija Knoblechner, Josef Charoenrat, Theppanya Enfors, Sven‐Olof Veide, Andres 2006 http://dx.doi.org/10.1002/bit.20811 https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fbit.20811 https://onlinelibrary.wiley.com/doi/pdf/10.1002/bit.20811 en eng Wiley http://onlinelibrary.wiley.com/termsAndConditions#vor Biotechnology and Bioengineering volume 93, issue 6, page 1040-1049 ISSN 0006-3592 1097-0290 journal-article 2006 crwiley https://doi.org/10.1002/bit.20811 2024-05-03T10:57:12Z Abstract For improved interfacing of the Pichia pastoris fed‐batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one‐step recovery and purification procedure for a fusion protein composed of a cellulose‐binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium‐stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM–CALB. In the EBA process, no cell‐adsorbent interaction was detected but the cell density had to be reduced by a two‐times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L · h. © 2006 Wiley Periodicals, Inc. Article in Journal/Newspaper Antarc* Antarctica Wiley Online Library Biotechnology and Bioengineering 93 6 1040 1049 |
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Abstract For improved interfacing of the Pichia pastoris fed‐batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one‐step recovery and purification procedure for a fusion protein composed of a cellulose‐binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium‐stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM–CALB. In the EBA process, no cell‐adsorbent interaction was detected but the cell density had to be reduced by a two‐times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L · h. © 2006 Wiley Periodicals, Inc. |
format |
Article in Journal/Newspaper |
author |
Jahic, Mehmedalija Knoblechner, Josef Charoenrat, Theppanya Enfors, Sven‐Olof Veide, Andres |
spellingShingle |
Jahic, Mehmedalija Knoblechner, Josef Charoenrat, Theppanya Enfors, Sven‐Olof Veide, Andres Interfacing Pichia pastoris cultivation with expanded bed adsorption |
author_facet |
Jahic, Mehmedalija Knoblechner, Josef Charoenrat, Theppanya Enfors, Sven‐Olof Veide, Andres |
author_sort |
Jahic, Mehmedalija |
title |
Interfacing Pichia pastoris cultivation with expanded bed adsorption |
title_short |
Interfacing Pichia pastoris cultivation with expanded bed adsorption |
title_full |
Interfacing Pichia pastoris cultivation with expanded bed adsorption |
title_fullStr |
Interfacing Pichia pastoris cultivation with expanded bed adsorption |
title_full_unstemmed |
Interfacing Pichia pastoris cultivation with expanded bed adsorption |
title_sort |
interfacing pichia pastoris cultivation with expanded bed adsorption |
publisher |
Wiley |
publishDate |
2006 |
url |
http://dx.doi.org/10.1002/bit.20811 https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fbit.20811 https://onlinelibrary.wiley.com/doi/pdf/10.1002/bit.20811 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Biotechnology and Bioengineering volume 93, issue 6, page 1040-1049 ISSN 0006-3592 1097-0290 |
op_rights |
http://onlinelibrary.wiley.com/termsAndConditions#vor |
op_doi |
https://doi.org/10.1002/bit.20811 |
container_title |
Biotechnology and Bioengineering |
container_volume |
93 |
container_issue |
6 |
container_start_page |
1040 |
op_container_end_page |
1049 |
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1800740192605175808 |