Expression of a lipase in prokaryote and eukaryote host systems allowing engineering
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 (...
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ftkthstockholm:oai:DiVA.org:kth-11124 2023-05-15T14:01:36+02:00 Expression of a lipase in prokaryote and eukaryote host systems allowing engineering Wittrup Larsen, Marianne 2009 application/pdf http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11124 eng eng KTH, Biokemi Stockholm : KTH TRITA-BIO-Report, 1654-2312 2009:14 http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11124 urn:isbn:978-91-7415-420-7 info:eu-repo/semantics/openAccess pseudozzyma (candida) antarctica lipase B escherichia coli pichia pastoris rational design Doctoral thesis, comprehensive summary info:eu-repo/semantics/doctoralThesis text 2009 ftkthstockholm 2022-08-11T12:33:22Z 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 ... Doctoral or Postdoctoral Thesis Antarc* Antarctica Royal Institute of Technology, Stockholm: KTHs Publication Database DiVA |
institution |
Open Polar |
collection |
Royal Institute of Technology, Stockholm: KTHs Publication Database DiVA |
op_collection_id |
ftkthstockholm |
language |
English |
topic |
pseudozzyma (candida) antarctica lipase B escherichia coli pichia pastoris rational design |
spellingShingle |
pseudozzyma (candida) antarctica lipase B escherichia coli pichia pastoris rational design Wittrup Larsen, Marianne Expression of a lipase in prokaryote and eukaryote host systems allowing engineering |
topic_facet |
pseudozzyma (candida) antarctica lipase B escherichia coli pichia pastoris rational design |
description |
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 ... |
format |
Doctoral or Postdoctoral Thesis |
author |
Wittrup Larsen, Marianne |
author_facet |
Wittrup Larsen, Marianne |
author_sort |
Wittrup Larsen, Marianne |
title |
Expression of a lipase in prokaryote and eukaryote host systems allowing engineering |
title_short |
Expression of a lipase in prokaryote and eukaryote host systems allowing engineering |
title_full |
Expression of a lipase in prokaryote and eukaryote host systems allowing engineering |
title_fullStr |
Expression of a lipase in prokaryote and eukaryote host systems allowing engineering |
title_full_unstemmed |
Expression of a lipase in prokaryote and eukaryote host systems allowing engineering |
title_sort |
expression of a lipase in prokaryote and eukaryote host systems allowing engineering |
publisher |
KTH, Biokemi |
publishDate |
2009 |
url |
http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11124 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
TRITA-BIO-Report, 1654-2312 2009:14 http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11124 urn:isbn:978-91-7415-420-7 |
op_rights |
info:eu-repo/semantics/openAccess |
_version_ |
1766271524540514304 |