Publikationsserver der RWTH Aachen University
Immobilisierung von BPA-Bindeproteinen an der Oberfläche von Hefezellen
Abstract
dc:descriptionThe aim of this work was to construct yeast cells being able to bind the xeno- estrogen Bisphenol A (BPA) at their surface. In order to measure the amount of BPA bound by yeast cells a new method for the quantification of BPA in aqueous solutions was developed in cooperation with Markus Naaßner (Biology V, RWTH Aachen). This method consisted of BPA extraction from aqueous solutions by SPE, elution with acetone, fluorigenic labeling of BPA with Dansyl-Cl, separation by HPLC, detection and quantification of the dansylated BPA using a fluorescence spectrometer. The BPA binding yeast strain was constructed by displaying a BPA binding protein at the cell surface. KYE1 from K. lactis was chosen as BPA binding protein because it is highly homologous to the phenol binding protein OYE from S. cerevisiae and the steroid binding protein EBP1 from C. albicans. To display KYE1 at the cell surface it had to be fused with the C-terminal half of the surface protein alpha-agglutinin (AGalpha1). Therefore the gene KYE1 and the 3'-half of the gene AGalpha1 were isolated by PCR, cloned and fused. Subsequently, the KYE1-3'-AGalpha1-fusion gene was transformed into the yeast strain P. pastoris GS 115 using the expression vector pPIC9K. This gave the new strain P. pastoris pPIKA. In this strain the expression of the fusion gene was controlled by the AOX1-promotor regulated by methanol. BPA sorption assays with the new strain gave the following results: The highest BPA sorption was measured at 9,3. This optimum was similar to that of the interaction between chlorophenol and the KYE1-homologue OYE from S. cerevisiae. When compared to the host strain P. pastoris GS 115 the new strain P. pastoris pPIKA showed a clear increase of BPA sorption dependent on previous cultivation with methanol. BPA sorption was completed after 30 min. Those kinetics are typical for sorption processes at surfaces which are regarded as passive reactions indepependent from metabolism. The dependency of BPA-sorption on BPA concentration followed the model by Langmuir. Furthermore BPA sorption depended on cell mass. Both observations confirmed that BPA was bound at the cell surface of P. pastoris pPIKA.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2003
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Mergler, Magnus
- Contributors dc:contributor
-
- Wolf, Klaus
Subjects
dc:subject × 7Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger
Identifiers
dc:identifier.*- OAI identifier oai:identifier
- oai:publications.rwth-aachen.de:59016