The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids
This work was part of the cooperation "Sturctured Lipids by Bio-Engineering" between the Institute of Technical Biochemistry at the University of Stuttgart and the Nestlé Research Center in Lausanne, Switzerland. In the framework of this project new enzymatic methods for the synthesis of s...
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ftunivstutt:oai:elib.uni-stuttgart.de:11682/918 2024-06-09T07:40:20+00:00 The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids Die Lipasen aus Candida antarctica : Klonierung, Expression und ihre Anwendung in der Synthese von strukturierten Lipiden Pfeffer, Jan Christoph Schmid, Rolf D. (Prof. Dr.) 2008 https://doi.org/10.18419/opus-901 http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-34375 http://elib.uni-stuttgart.de/handle/11682/918 en eng 277783151 http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-34375 http://elib.uni-stuttgart.de/handle/11682/918 http://dx.doi.org/10.18419/opus-901 info:eu-repo/semantics/openAccess Lipide Klonierung Heterologe Genexpression 570 structured lipids lipase expression cloning Candida antarctica doctoralThesis 2008 ftunivstutt https://doi.org/10.18419/opus-901 2024-05-14T03:07:08Z This work was part of the cooperation "Sturctured Lipids by Bio-Engineering" between the Institute of Technical Biochemistry at the University of Stuttgart and the Nestlé Research Center in Lausanne, Switzerland. In the framework of this project new enzymatic methods for the synthesis of structured triacylglycerides and 2-monoacylglycerides were established and scaled up to a technical scale (> 100 gram product). Additionally a lipase showing high sn2-preference which is an ideal prerequisite for the direct synthesis of 2-monoacylglycerides and structured lipids was expressed in technical scale (5 litre fermenter), purified and generally characterised. For this lipase the prerequisites for an error-prone PCR (epPCR)-based mutagenesis aiming at improvement of the biocatalyst were generated. The chemical synthesis of 2-monoacylglycerides is challenging due to several reaction steps, a costly purification and often results in very low yields. In contrast 2-monoacylglycerides and structured triacylgylcerides are relatively easy to synthesise using lipases with regard to yield and purity. Lipase A from Candida antarctica seemed to be a promising catalyst for the direct esterification of fatty acids and glycerol yielding in 2-monoacylglycerides. This enzyme displays the highest preference for the sn2-position published in scientific literature. The immobilised enzyme showed only a very weak esterification potential and the esterification was - in contrast to the hydrolysis, where CalA showed a clear sn2-preference - unspecific. Different parameters (temperature, solvent, water activity, substrates of different chain lengths, and use of ionic liquids) were changed, but no improvement in activity or specificity could be observed. Therefore optimisation of CalA via directed evolution was planned. As the crystal structure of CalA is not solved yet and also the homology with other lipases is quite low, the only possibility to optimize the enzyme was directed evolution: design of a mutant library by epPCR followed by a ... Doctoral or Postdoctoral Thesis Antarc* Antarctica OPUS - Publication Server of the University of Stuttgart |
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OPUS - Publication Server of the University of Stuttgart |
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ftunivstutt |
language |
English |
topic |
Lipide Klonierung Heterologe Genexpression 570 structured lipids lipase expression cloning Candida antarctica |
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Lipide Klonierung Heterologe Genexpression 570 structured lipids lipase expression cloning Candida antarctica Pfeffer, Jan Christoph The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids |
topic_facet |
Lipide Klonierung Heterologe Genexpression 570 structured lipids lipase expression cloning Candida antarctica |
description |
This work was part of the cooperation "Sturctured Lipids by Bio-Engineering" between the Institute of Technical Biochemistry at the University of Stuttgart and the Nestlé Research Center in Lausanne, Switzerland. In the framework of this project new enzymatic methods for the synthesis of structured triacylglycerides and 2-monoacylglycerides were established and scaled up to a technical scale (> 100 gram product). Additionally a lipase showing high sn2-preference which is an ideal prerequisite for the direct synthesis of 2-monoacylglycerides and structured lipids was expressed in technical scale (5 litre fermenter), purified and generally characterised. For this lipase the prerequisites for an error-prone PCR (epPCR)-based mutagenesis aiming at improvement of the biocatalyst were generated. The chemical synthesis of 2-monoacylglycerides is challenging due to several reaction steps, a costly purification and often results in very low yields. In contrast 2-monoacylglycerides and structured triacylgylcerides are relatively easy to synthesise using lipases with regard to yield and purity. Lipase A from Candida antarctica seemed to be a promising catalyst for the direct esterification of fatty acids and glycerol yielding in 2-monoacylglycerides. This enzyme displays the highest preference for the sn2-position published in scientific literature. The immobilised enzyme showed only a very weak esterification potential and the esterification was - in contrast to the hydrolysis, where CalA showed a clear sn2-preference - unspecific. Different parameters (temperature, solvent, water activity, substrates of different chain lengths, and use of ionic liquids) were changed, but no improvement in activity or specificity could be observed. Therefore optimisation of CalA via directed evolution was planned. As the crystal structure of CalA is not solved yet and also the homology with other lipases is quite low, the only possibility to optimize the enzyme was directed evolution: design of a mutant library by epPCR followed by a ... |
author2 |
Schmid, Rolf D. (Prof. Dr.) |
format |
Doctoral or Postdoctoral Thesis |
author |
Pfeffer, Jan Christoph |
author_facet |
Pfeffer, Jan Christoph |
author_sort |
Pfeffer, Jan Christoph |
title |
The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids |
title_short |
The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids |
title_full |
The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids |
title_fullStr |
The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids |
title_full_unstemmed |
The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids |
title_sort |
lipases from candida antarctica : cloning, expression and their application in the synthesis of structured lipids |
publishDate |
2008 |
url |
https://doi.org/10.18419/opus-901 http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-34375 http://elib.uni-stuttgart.de/handle/11682/918 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
277783151 http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-34375 http://elib.uni-stuttgart.de/handle/11682/918 http://dx.doi.org/10.18419/opus-901 |
op_rights |
info:eu-repo/semantics/openAccess |
op_doi |
https://doi.org/10.18419/opus-901 |
_version_ |
1801383763592085504 |