Advancing the Synthosome system towards industrial application : introduction of trigger systems and enzyme quantification

  • Synthosomes are copolymer vesicles which harbour a transmembrane channel in the polymer membrane and are functionalised in the interior of the vesicle, for example by encapsulating an enzyme. Protein engineering can introduce trigger systems to channel or encapsulated enzyme, making the system highly specific for the desired task. The aim of the work was to advance the synthosome system towards industrial applications by introducing trigger systems and investigating encapsulation efficiencies. For efficient characterisation of synthosomes, we aimed at developing a method for quantifying the encapsulated enzyme. The principle was based on a fusion protein and a reporter oligonucleotide, both of which were optimised and shown to be fully functional. Although the single steps were successfully completed, final proof of principle could not be shown due to encapsulation efficiencies below the detection limit. In the second part of the work, pH triggers for a channel protein (OmpF) and a protease (Subtilisin E) are described. Protease activation only after pH change could be used to overcome low stability of the enzyme in liquid detergents. Subtilisin variants with a narrowed pH activity profile were therefore generated by Directed Evolution. The mutations contributing to the altered characteristics can be found in the prosequence, on the protease surface and close to the catalytic triad. Furthermore, we report for the first time a reversible pH-controlled release system based on OmpF. Six histidine mutations were introduced to the channel constriction site. By varying the pH (5 to 7), it was possible to monitor the pH dependent release of the fluorophore acridine orange through the engineered OmpF His. Molecular modelling studies suggest that the principle of pH-switchability is based on a combination of changes in channel diameter and electrostatics. Since the changes occur near physiological pH, the system is especially interesting for drug delivery.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Saskia Maria Ihle
Referee:Ulrich Schwaneberg, Bernhard Hauer, Danilo Roccatano
Advisor:Ulrich Schwaneberg
Persistent Identifier (URN):urn:nbn:de:101:1-201305237551
Document Type:PhD Thesis
Date of Successful Oral Defense:2009/08/20
Date of First Publication:2009/08/27
PhD Degree:Biochemical Engineering
School:SES School of Engineering and Science
Library of Congress Classification:T Technology / TP Chemical technology / TP248.13-248.65 Biotechnology / TP248.3 Biochemical engineering. Bioprocess engineering
Call No:Thesis 2009/25

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