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Expression of a lipase in prokaryote and eukaryote host systems allowing engineering
KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101). (Biochemistry)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pseudozyma (Candida) antarctica lipase B (PalB) was expressed in Escherichia coli facilitating protein engineering. The lack of glycosylation was evaluated for a deeper understanding of the difficulties in expressing PalB in E. coli. Different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3), Origami 2(DE3), and coexpression of groES and groEL. Periplasmic expression resulted 5.2 mg/L active PalB at 16 °C in shake flasks. This expression level was improved by using the EnBase technology, enabling fed-batch cultivation in 24-deep well scale. The feed rate was titrated with the addition of α-amylase, which slowly releases glucose as energy source. Different media were evaluated where the EnBase mineral salt medium resulted in 7.0 mg/L of active PalB.

Protein secreted directly into the media was obtained using the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for screening and production of PalB in P. pastoris. A protease sensitive fusion protein CBM-PalB (cellulose-binding module) was used as a model system. When optimised, the expression system resulted in 46 mg/L lipase in 72 hours in shake flask, 37 mg/L lipase in 28 hours in 96-deep-well plate format, and 2.9 g PalB per 10 L bioreactor cultivation.

The E. coli expression system was used to express a small focused library of PalB variants, designed to prevent water from entering the active site through a hypothesised tunnel. Screening of the library was performed with a developed assay, allowing for simultaneous detection of both transacylation and hydrolytic activity. From the library a mutant S47L, in which the inner part of the tunnel was blocked, was found to catalyse transacylation of vinyl butyrate in 20 mM butanol 14 times faster than hydrolysis. Water tunnels, assisting water in reaching the active sites, were furthermore found by molecular modelling in many hydrolases. Molecular modelling showed a specific water tunnel in PalB. This was supported by experimental data, where the double mutant Q46A S47L catalysed transacylation faster than hydrolysis compared to the wild type PalB.

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , 52 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2009:14
Keyword [en]
pseudozzyma (candida) antarctica lipase B, escherichia coli, pichia pastoris, rational design
Identifiers
URN: urn:nbn:se:kth:diva-11124ISBN: 978-91-7415-420-7 (print)OAI: oai:DiVA.org:kth-11124DiVA: diva2:236156
Public defence
2009-10-02, Svedbergssalen, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20100818

Available from: 2009-09-21 Created: 2009-09-21 Last updated: 2014-09-26Bibliographically approved
List of papers
1. Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems
Open this publication in new window or tab >>Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems
2008 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 62, 90-97 p.Article in journal (Refereed) Published
Abstract [en]

Candida antarctica lipase B (CALB) carrying a point mutation, N74S, resulting in a non-glycosylated protein was actively expressed in Pichia pastoris yielding 44 mg/L which was similar to that of the glycosylated CALB wild type expressed in A pastoris. Hence, the major obstacle in the Escherichia coli expression of CALB is not the lack of glycosylation. To understand and improve the expression of CALB in E. coli, a comprehensive investigation of four different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3) and Origami 2(DE3) as well as co-expression with chaperones groES and groEL in Origami B(DE3), all using the pET-22b(+) vector and the T7lac promoter.

Furthermore the E. coli expression was carried out at three different temperatures (16, 25 and 37 C) to optimise the expression. Periplasmic expression resulted in highest amount of active CALB of the four systems, yielding a maximum of 5.2 mg/L culture at 16 degrees C, which is an improvement to previous reports.

The specific activity of CALB towards tributyrin in E. coli was found to be the same for periplasmic and cytosolic expression. Active site titration showed that the CALB mutant N74S had a lower specific activity in comparison to wild type CALB regardless of expression host. The expected protein identity was confirmed by LC-ESI-MS analysis in E. coli, whereas in P. pastoris produced CALB carried four additional amino acids from an incomplete protein processing.

Keyword
Candida antarctica lipase B; Escherichia coli; Expression; Glycosylation; Pichia pastoris
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11114 (URN)10.1016/j.pep.2008.07.012 (DOI)000260061900013 ()2-s2.0-52949132991 (Scopus ID)
Note
QC 20100818Available from: 2009-09-21 Created: 2009-09-21 Last updated: 2010-12-07Bibliographically approved
2. Controlling nutrient release in cell cultivation
Open this publication in new window or tab >>Controlling nutrient release in cell cultivation
Show others...
2009 (English)In: Genetic Engineering and Biotechnology News, ISSN 1935-472X, Vol. 29, no 11, 50-51 p.Article in journal (Refereed) Published
Keyword
BioSilta, Cell culture, Protein production
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-152468 (URN)000266618300020 ()2-s2.0-77954693400 (Scopus ID)
Note

QC 20140926

Available from: 2014-09-26 Created: 2014-09-26 Last updated: 2014-09-26Bibliographically approved
3. Screening and production of Pseudozyma (Candida) antarctica lipase B in Pichia pastoris using the GAP promoter as alternative to the AOX promoter expression system
Open this publication in new window or tab >>Screening and production of Pseudozyma (Candida) antarctica lipase B in Pichia pastoris using the GAP promoter as alternative to the AOX promoter expression system
(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-24143 (URN)
Note
QC 20100818Available from: 2010-08-18 Created: 2010-08-18 Last updated: 2010-09-08Bibliographically approved
4. Suppression of water asa nucleophile in Pseudozyma (Candida) antarctica lipase B catalysis.
Open this publication in new window or tab >>Suppression of water asa nucleophile in Pseudozyma (Candida) antarctica lipase B catalysis.
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-24144 (URN)
Note
QC 20100818Available from: 2010-08-18 Created: 2010-08-18 Last updated: 2010-09-08Bibliographically approved
5. A tunnel provides the active site of a lipase withsubstrate water.
Open this publication in new window or tab >>A tunnel provides the active site of a lipase withsubstrate water.
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-24146 (URN)
Note
QC 20100818Available from: 2010-08-18 Created: 2010-08-18 Last updated: 2010-09-08Bibliographically approved

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