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Process design for production of Thai rosewood β-glucosidase in Pichia pastoris
KTH, School of Biotechnology (BIO).
2005 (English)Licentiate thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH , 2005. , 50 p.
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-375ISBN: 91-7178-081-5 (print)OAI: oai:DiVA.org:kth-375DiVA: diva2:9598
Presentation
2005-06-10, FB54, AlbaNova, Roslagstullsbacken 21, Stockholm, 11:00
Supervisors
Note
QC 20101126Available from: 2005-08-08 Created: 2005-08-08 Last updated: 2010-11-26Bibliographically approved
List of papers
1. Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes
Open this publication in new window or tab >>Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes
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2005 (English)In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 27, no 6, 399-406 p.Article in journal (Refereed) Published
Abstract [en]

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-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.

Keyword
oxygen-limited fed batch (OLFB), methanol-limited fed batch (MLFB), Pichia pastoris, b-glucosidase, methanol, expression, yeast, growth, yield, oxidation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-15170 (URN)10.1007/s00449-005-0005-4 (DOI)000233136000006 ()2-s2.0-28244476895 (Scopus ID)
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2016-12-21Bibliographically approved
2. Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth
Open this publication in new window or tab >>Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth
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2006 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 122, no 1, 86-98 p.Article in journal (Refereed) Published
Abstract [en]

Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST 1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

Keyword
beta-glucosidase, Pichia pastoris, methanol limited fed-batch, expanded bed adsorption, multi-modal ligand, methylotrophic yeast, protein-purification, chromatography, expression, binding
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-15530 (URN)10.1016/j.jbiotec.2005.08.016 (DOI)000236102300009 ()2-s2.0-32644473166 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2010-11-26Bibliographically approved

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