The enzymatic degradation of polysaccharides is a major goal of the biotechnology industry, most notably for both first (starch) and second (cellulose/chitin) generation biofuels. The classical model for enzymatic polysaccharide breakdown involves the action of endo- and exo- acting glycoside hydrolases which act in synergy; endo-acting enzymes generating free chain-ends for the processive exo-glycosidases. In 2010 a new enzyme class was discovered which overturned the classical hydrolase model. These enzymes, now known as Lytic Polysaccharide Monooxygenases (LPMOs), are understood to be copper-dependent oxygenase enzymes that cause chain cleavage within polysaccharides, facilitating their degradation by classical hydrolases. This research showcases the findings for an LPMO of the “Auxiliary Activity” class AA10 from the bacterium Bacillus amyloliquefaciens (BaAA10). Methods of gene expression and protein production are described, including the incorporation of a SUMO-tag which resulted in increased yields of BaAA10. The nature of the copper binding was assessed using Isothermal Titration Calorimetry and Differential Scanning Fluorimetry, which demonstrated the extremely tight binding of copper to the enzyme and its increased stability when copper is bound. Additionally, the detection of lactone and aldonic acid products of oxidative degradation, in MALDI-TOF Mass Spectrometry results, determined for the first time that BaAA10 was active on both alpha and beta forms of chitin. Finally, a Cu(II) structure of BaAA10 was able to be obtained following the use of a spiral data collection technique as a method of preventing photoreduction of Cu(II) to Cu(I) in the X-ray beam. This structure can now provide us with details regarding the copper active site and, together with potential spectroscopic studies in the future, can help us to determine the mechanism of action of these LPMO enzymes, so that we can better exploit their use within the biofuel industry.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:698340 |
| Date | January 2016 |
| Creators | Gregory, Rebecca Charlotte |
| Contributors | Davies, Gideon ; Walton, Paul |
| Publisher | University of York |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.whiterose.ac.uk/15659/ |
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