Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Wallace, P.
Discovery of polyesterases using proteomics
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2017. pp. 266
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Birner-Grünberger Ruth
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- Abstract:
- Two approaches to identify novel polyesterases to be used in the hydrolysis and handling of discarded polyesters were pursued. First a novel esterase, PpEst, that hydrolyses the co-aromatic-aliphatic polyester poly(1,4-butylene adipate-co-terephthalate) (PBAT) was identified by proteomic screening of the Pseudomonas pseudoalcaligenes secretome. PpEst was induced by the presence of PBAT in the growth media and had predicted arylesterase (EC 3.1.1.2) activity. PpEst showed polyesterase activity on both whole and milled PBAT film releasing terephthalic acid and 4-(4-hydroxybutoxycarbonyl)benzoic acid while end product inhibition by 4-(4-hydroxybutoxycarbonyl)benzoic acid was observed. Modelling of a PBAT mimicking oligomer into the PpEst active site indicated that the binding pocket could be big enough to accommodate large polymers. This is the first report of a PBAT degrading enzyme being identified by proteomic screening and shows that this approach can contribute to the discovery of new polymer degrading enzymes. Moreover, these results indicate that arylesterases could be an interesting enzyme class for identifications of polyesterases. The second approach was to design, synthesise and use activity based probes (ABPs) that were directed towards polyesterases. These ABPs are inhibitors that would covalently bind polyesterases and be used to enrich these enzymes from either cultivated organisms or environmental samples. The ABPs were designed to mimic the structure of the aromatic polyester poly(ethylene terephthalate), be soluble in water and target only enzymes capable of hydrolysing aromatic ester bonds in polymeric structures. 7 new ABPs that were active towards serine hydrolases were synthesised and tested on commercially available esterases and lipases as well as on known polyesterases. The new ABPs were determined to target enzymes capable of degrading aromatic polyesters, but also enzymes that have hydrolytic abilities only towards small aliphatic compounds. The specificity of the ABPs would have to be improved for these to be applied to the identification of novel polyesterases, but they are novel serine hydrolase ABPs and their specificity towards the human proteome would be interesting to determine as few ABPs with aromatic components are reported.