Optimierung der Bisphenol A-Biosorption von Saccharomyces cerevisiae auf Basis eines oberflächen-exprimierten Rezeptorproteins

The elimination of endocrine disruptors, like bisphenol A, from wastewater is an environmental challenge of increasing importance, but no satisfying technical solution has been achieved until now. Thus, it was the aim of the present dissertation to increase the bisphenol A-affinity of yeast by cell surface-expression. Expressing a high affinity protein on its cell surface, the immobilised yeast could serve as a cheap feedstock in a bio filtration system for the elimination of bisphenol A traces from wastewater. Although, a cell own protein should be modified to increase its affinity and should be fused with an effectively anchor able cell wall protein, considering the cell proteome. In the present dissertation the “old yellow enzyme”, natively found in Saccharomyces cerevisiae, was modified by site-directed mutagenesis. A manual docking from the “old yellow enzyme” with bisphenol A was simulated and simulations of punctiform mutagenesis in the protein binding site were done based on the well known crystal structure of the protein. Five simulated mutagenesis products, which should deliver possible new or improved interactions between ligand and protein, were analysed with a secondary structure analysis program and with the Ramachandran plot. The chosen mutations were accomplished at the “old yellow enzyme”- gene by PCR and the belonging proteins were expressed in Escherichia coli. The mutated proteins were purified and investigated by equilibrium dialysis to compare the bisphenol A- binding properties and to determine the dissociation constant. The yield of pure protein was increased to a factor of 2.5 by optimising the induction conditions of Escherichia coli concerning incubation temperature and the amount of inducing substance. The reason for the increased yield was the decreased generation of inclusion bodies. An effective protein purification protocol has been developed, that delivered the required amount of 10 mg pure protein for the equilibrium dialysis with an acceptable effort of time and costs. Four of five investigated, purified proteins showing a mutation that should build an additional hydrogen bond to the beta-OH-group of bisphenol A, bound less bisphenol A than the wild type protein. Only the exchange of the amino acid on position 194, asparagine against aspartate, provoked an increase of the bisphenol A- affinity. This mutant protein had a five times higher affinity than the wild type “old yellow enzyme” and a determined Kd- value of 93 µM. The mutated gene N194D was coupled with three different genes of cell wall anchors from Saccharomyces cerevisiae, Aga, Cwp2 and Sed1, and was transformed into Saccharomyces cerevisiae by two different cell surface expression vectors, pYD and pFB. All fusion proteins were expressed on the cell surface and were localized by fluorescent antibodies. It was determined a significant increase of the bisphenol A binding speed for all modified strains following to cell surface expression of the fusion proteins. The maximum adsorbed bisphenol A amount was scarcely influenced by the expression of the fusion proteins. The strains based on the pFB- plamid, respectively the cell wall anchors Cwp2 and Sed1, were the most effective ones. Almost one minute after the beginning of incubation, 77% of their adsorption capacity, which was 475 respectively 510 µg bisphenol A per gram dry cell matter, was bound. In comparison to the wild type strain bound only 13%. The additional insertion of a 25 amino acid long glycine- serine- linker sequence between the cell wall anchor and the binding protein was ineffective. Less by the choice of the cell wall anchor but by the choice of the expression vector the strains based on the pFB- plasmids were determined as the most useful. The present dissertation delivers several new, optimized strains of Saccharomyces cerevisiae, which show a significant increased bisphenol A binding speed by expression of a cell own, punctiform mutated proteins on their cell surface. The strains show this behaviour under conditions of pH and temperature which are common in the effluent of wastewater treatment plants. The proteome of the cell was scarcely influenced by the genetic modification which could be an advantage for the permission for a technical use. The strains can now be used as a inexpensive feedstock for immobilization experiments which aim the construction of a bio filtration system to eliminate bisphenol A from wastewater.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2008

Author dc:creator

• Schiffers, Jens

Contributors dc:contributor

• Wolf, Klaus

• info:eu-repo/classification/ddc/570
• Saccharomyces
• Saccharomyces cerevisiae
• NADPH
• Endokrin wirksamer Stoff
• Ortspezifische Mutagenese
• Biofilter
• In silico-Methode
• Proteine
• Biowissenschaften, Biologie
• Oberflächenexpression
• OYE
• Biosorption
• Proteinreinigung
• Gleichgewichtsdialyse
• old yellow enzyme
• cell surface expression
• site directed mutagenesis
• inclusion bodies
• endocrine disruptors

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112720%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50165