MECHANISTIC STUDIES ON HORSERADISH PEROXIDASE AND ITS ROLE IN THE CATALYSIS OF OXIDATION REACTIONS
The catalyzed oxidation reactions by horseradish peroxidase (HRP) comprise a conversion of the ferric state of the heme to the ferryl heme by hydrogen peroxide (H2O2). However, studies barely investigate the effect of other components in the reaction mixtures on HRP function. Thus, we desire to investigate the HRP-catalyzed oxidation reactions in exposure to histidine (His). Herein, we report the first use of His and HRP to initiate the photocatalytic oxidation reactions without the exogenous addition of hydrogen peroxide. We demonstrate that the oxidation reactions of organic molecules proceed 2-times faster using HRP/His system in comparison with HRP/H2O2 system. During this work, we noticed that non-aromatic amino acids are fluorescent in solid state. We also demonstrate that the polymorph A crystal of L-His contains hydrophobic domains within the structure’s interior, which can serve as vehicles for the highly efficient entrapment and transport of hydrophobic small molecules. Despite the importance of H2O2 in the HRP-catalyzed reactions, the detection and quantification of low concentrations of H2O2 is still limited using current methods. In this regard, we demonstrate that high-valent iron-oxo intermediates of HRP are well suited to detect ultratrace amount of H2O2 impurities in alcohols in the range of 0.001–1000 µM using just a UV/Vis spectrophotometer. We monitor the optical spectra of low concentrations of HRP (0.1-1 µM) for the red shift in the Soret and Q-band regions upon the addition of alcohols, as well as the reversibility of this shift to the original wavelength over time due to the spontaneous decay of ferryl intermediates to the ferric state. The motivation for this thesis lie in an industrially-funded project on Cheddar cheese. The aim of this project was to obtain white whey from the yellow whey proteins recovered from the Cheddar cheese making process without using oxidative agents. To address this issue, we conducted several experiments which resulted in all fascinating chapters of this thesis based on our observations, while none of them solved the problem. Thus, we designed microcapsules, which release the cargo only when exposed to lipase during the ripening step, selectively coloring the cheese matrix and obtaining the white whey.
amino acid crystals; catalysis; Fluorescence; horseradish peroxidase; oxidation reaction; photoluminescence
Baird, Barbara A.; Cerione, Richard A.
Food Science and Technology
Ph. D., Food Science and Technology
Doctor of Philosophy
dissertation or thesis