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Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes

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Abstract

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood β-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q O/q O,max), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90–95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.

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Abbreviations

AOX:

Enzyme alcohol oxidase

AOX1 :

Alcohol oxidase gene 1

CPR:

Carbon dioxide production rate (mol h−1)

DOT:

Dissolved oxygen tension (%)

MLFB:

Methanol limited fed-batch

OLFB:

Oxygen limited fed-batch

OUR:

Oxygen uptake rate (mol h−1)

PI:

Propidium iodide

q o :

Specific oxygen uptake rate (mol g −1cell h−1)

q o,max :

Maximum specific oxygen uptake rate (mol g −1cell h−1)

q p :

Specific β-glucosidase productivity (U g −1cell h−1)

Q i :

Inlet air flow rate (L h−1)

Q o :

Outlet air flow rate (L h−1)

RRR:

Relative rate of respiration

V :

Medium volume (L)

V m :

Molar volume of gas (L mol−1)

X :

Biomass concentration from dry weight (g L−1)

Y C CO2/S :

Carbon yield coefficient of carbon dioxide from methanol (mol mol−1)

Y C X/S :

Carbon yield coefficient of biomass from methanol (mol mol−1)

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Acknowledgements

TC is supported by the university lecturer development program from the Ministry of Education and Suranaree University, Thailand. This work is part of the BiMaC Enzyme Factory programme financed by the Södra Skogsägarnas Stiftelse för Forskning, Utveckling och Utbildning.

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Authors and Affiliations

  1. School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, Roslagstullsbacken 21, 106 91, Stockholm, Sweden

    Theppanya Charoenrat, Helle Stendahl-Andersen, Mehmedalija Jahic & Sven-Olof Enfors

  2. School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand

    Theppanya Charoenrat & Mariena Ketudat-Cairns

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  1. Theppanya Charoenrat
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  2. Mariena Ketudat-Cairns
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  3. Helle Stendahl-Andersen
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  4. Mehmedalija Jahic
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  5. Sven-Olof Enfors
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Correspondence to Sven-Olof Enfors.

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Charoenrat, T., Ketudat-Cairns, M., Stendahl-Andersen, H. et al. Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes. Bioprocess Biosyst Eng 27, 399–406 (2005). https://doi.org/10.1007/s00449-005-0005-4

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  • DOI: https://doi.org/10.1007/s00449-005-0005-4

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