Biochemical characterisation of polyphenol oxidase

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dc.contributor.advisor Leung, KHI en
dc.contributor.advisor Kilmartin, P en Li, Yu en 2017-12-20T22:45:21Z en 2017 en
dc.identifier.uri en
dc.description.abstract The work described in this Thesis has focused on the development and application of biochemical techniques to characterise grape polyphenol oxidase (PPO). PPO is a coppercontaining enzyme that uses molecular oxygen as cosubstrate to catalyse two different reactions: the hydroxylation of monophenols to diphenols, and the subsequent oxidation of diphenols to ο-quinones. The mature form of grape PPO (residues 104–443) was produced recombinantly by using an Escherichia coli expression system. The purification protocol was optimised to give active enzymes. Mass spectrometry was used to confirm the identity of the recombinant protein, and ultraviolet/visible (UV/Vis) spectrophotometry was to study the kinetics and inhibition of the enzyme. Overall, the results showed that the recombinant grape PPO possessed similar activity to enzymes that were purified from grapes. A nuclear magnetic resonance (NMR)-based kinetic and inhibition assay was also developed by using grape and mushroom PPOs as model systems. Using the NMR assay, the roles of antioxidants and inhibitors in modulating the effect of PPO-catalysed polyphenol oxidation were investigated. The method was also validated by work in tandem with virtual highthroughput screening and molecular modelling, in which several novel inhibitors of grape PPO were discovered. The substrate selectivity of PPO was also investigated by using grape PPO as a model system. Four different grape PPO mutants were produced and purified, and their ability to accept monophenol and diphenol as substrates was investigated. The results showed that two residues inside the active site may modulate the monophenolase and diphenolase activities of the enzyme although it was not possible to conclusively pin-point the exact roles these amino acid residues play. The extraction, purification and characterisation of grape PPO from New Zealand Sauvignon Blanc grapes were also investigated. It was found that grape juice contained negligible amount of grape PPO. However, grape PPO was successfully extracted and purified from grape berries. The extracted protein was characterised by mass spectrometry. This was the first time grape PPO was purified from Sauvignon Blanc grapes, which may provide useful information to the wine industry as PPO-catalysed oxidation of polyphenols may affect the flavour molecules that give wines their distinctive tastes. en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA99265046004802091 en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. en
dc.rights.uri en
dc.rights.uri en
dc.title Biochemical characterisation of polyphenol oxidase en
dc.type Thesis en Food Science en The University of Auckland en Doctoral en PhD en
dc.rights.holder Copyright: The author en
dc.rights.accessrights en
pubs.elements-id 719490 en
pubs.record-created-at-source-date 2017-12-21 en

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