The lipases from Candida antarctica : cloning, expression and their application in the synthesis of structured lipids

• Main Author: Pfeffer, Jan Christoph
• Other Authors: Schmid, Rolf D. (Prof. Dr.)
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: 2008
• Subjects: Lipide; Klonierung; Heterologe Genexpression; 570; structured lipids; lipase; expression; cloning; Candida antarctica; Antarc*; Antarctica
• Online Access: 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

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 ...