Mushroom polyphenol oxidase (EC 1.14.18.1) was investigated to determine its potential for application as a biocatalyst in the synthesis of o-quinones, in organic medium. In order to determine the kinetic properties of the biocatalyst, a system was devised which comprised an immobilised polyphenol oxidase extract, functioning in chloroform. The system was hydrated by the addition of buffer. A simple method for the consistent measurement of reaction rates in this heterogenous system was designed and used to obtain detailed enzyme kinetic data relating to optimisation of reaction conditions and substrate specificity. The aqueous content of the system was optimised using p-cresol as a substrate. A crude, immobilised extract of Agaricus bisporus was used to hydroxylate and oxidise a range of selected p-substituted phenolic substrates, yielding, as the sale products, o-quinones. These products were efficiently reduced to catechols by extracting the reaction mixtures with aqueous ascorbic acid solution. The biocatalytic system was also successfully utilised to produce L-DOPA, the drug used to treat Parkinson's disease, from L-acetyl tyrosine ethyl ester (ATEE). Michaelis-Menten kinetics were used to obtain apparent Km and V values with respect to the selected phenolic substrates, and the kinetic parameters obtained were found to correlate well with the steric requirements of the substrates and with their hydrophobicity. In the course of the investigation, a novel ¹H NMR method was used to facilitate measurement of the UV molar absorption coefficients of the o-quinones in reaction mixtures, thus avoiding the necessity to isolate these unstable, water-sensitive products. The biocatalytic system was extended to a continuous process, in which the immobilised enzyme was shown to function successfully in the chloroform medium for several hours, with high conversion rates. Modifications, involving partial purification and the addition of a surfactant, were investigated to determine their effect on the kinetic parameters. The results obtained using partially purified enzyme indicated that the removal of extraneous protein and/or melanoid material lead to a reduced capacity for conversion of sterically demanding substrates. The addition of the anionic detergent, sodium dodecyl sulphate (SOS), enhanced the ability of the biocatalyst to bind and oxidise sterically demanding substrates. These effects are attributed to changes in the polar state of groups within the protein binding pocket, which result in altered flexibility and hydrophobicity. Computer modelling of several biomimetic dinuclear copper complexes also indicated the importance of flexibility for effective biocatalysis. Novel binuclear copper (II complexes, containing a flexible biphenyl spacer and imidazole or benzimidazole donors, were prepared and analysed using NMR, UV, AA and cyclic voltammetric techniques. The complexes were also shown, in a detailed kinetic study, to mimic the catecholase activity of polyphenol oxidase by oxidising 3,5-di-tertbutylcatechol, and to catalyse the coupling of the phenolic substrate 2,4-di-tert-butylphenol. However, the complexes were apparently too flexible to react with smaller substrates. These biomimetic complexes provided valuable insights into the nature of the dinuclear copper binding site.

No description available.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Thesis (doctoral)--University of Helsinki, 2007. / Includes bibliographical references. Also available on the World Wide Web.

No description available.

No description available.

Thesis (Ph. D.)--Illinois State University, 1975. / Title from title page screen, viewed Nov. 15, 2004. Dissertation Committee: Anthony E. Liberta (chair), Robert Chasson, Derek McCracken, Fritz Schwalm, David Weber. Includes bibliographical references (leaves 84-90) and abstract. Also available in print.

Thesis (doctoral)--Helsinki University of Technology, 2008. / Includes bibliographical references. Also available on the World Wide Web.

No description available.

The biocatalytic activity of mushroom tyrosinase was optimized in chloroform medium, using five selected phenolic substrates, including catechin (CT), vanillin (VA), chlorogenic acid (CA), p-aminophenol (pAP) and hydroquinone (HQ). The specific activity (SA) of tyrosinase determined as the change in absorbance at the selected wavelength per $ mu$g protein per sec ($ delta$A/$ mu$g protein/sec) in chloroform was much higher than that obtained in aqueous media. The optimal amount of enzymatic protein for tyrosinase biocatalysis in chloroform was found to be 44.0 mg protein/L for CT and VA, 31.6, 180.5 and 90.3 mg protein/L, respectively, for CA, pAP and HQ. The optimal pH for the oxidative activity of tyrosinase in chloroform was 6.0 for all the substrates; however, the optimal temperature for enzymatic activity was 30$ sp circ$C for CT and 25$ sp circ$C for the other four substrates. The use of 1.25 and 6.65 mM catechol in chloroform medium activated the tyrosinase activity maximally by 56.2% and 267.2%, respectively for CT and CA as substrates; however, no effect from catechol (0 to 7 mM) was found with VA, pAP or HQ. In addition, the use of 4.25, 2.25 and 5.39 mM ethylenediamine tetraacetic acid (EDTA) in chloroform, with CT, VA and pAP as substrates, inhibited the tyrosinase activity maximally by 44.3, 84.7 and 67.0%, respectively; however, the use of 4.75 and 1.60 mM EDTA activated the enzyme by 101.9% and 115.9%, respectively, for CA and HQ. The use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) demonstrated the phenolic substrate bioconversion, whereas the spectrophotometric scanning showed the product formation during the enzymatic reaction. (Abstract shortened by UMI.)