Tyrosinase inactivation in non -aqueous media

• Main Author: Warrington, James Claude
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2000
• Subjects: Chemical engineering; Organic chemistry
• ISBN: 9780612498358; 0612498352

Tyrosinase has practical utility in the preparation of o-diphenolics, o-quinones, or products requiring such precursors for synthesis. Such compounds are more easily prepared in organic solvents, and thus, a further understanding of tyrosinase performance in non-aqueous media is advantageous. The inactivation of the catecholase activity of mushroom tyrosinase was studied under partially aqueous and non-aqueous conditions. Partially aqueous conditions were investigated using a perfluorochemical emulsion. Fully non-aqueous studies were conducted in chloroform, toluene, amyl acetate, isopropyl ether and butanol. The effect of product partition coefficients on tyrosinase stability was investigated. Also, the relative effects of reaction inactivation and solvent denaturation on tyrosinase performance were quantified. At least two forms of tyrosinase contributed significantly to product formation. Kinetic models were developed to account for reaction inactivation, solvent denaturation and the two observed forms of tyrosinase. The stability of soluble tyrosinase in a 30v/v% perfluorochemical emulsion consisting of perfluorooctane, buffer and pluronic-F68 was investigated. Catechol was used as a substrate. Tyrosinase performance in the perfluorochemical emulsion was statistically the same as in aqueous buffer. The stability of tyrosinase in fully non-aqueous systems was investigated. The enzyme was immobilized on glass beads, and assays were conducted over a range of substrate (4-methylcatechol) concentrations. The solvent/water and solvent/salt hydrate partition coefficients of 4-methylcatechol and 4-methyl-o-benzoquinone were found independently. The reaction components were pre-equilibrated for two weeks with a saturated salt solution. No distinct relationship was observed between the product partition coefficients and enzyme stability. The initial reaction velocity varied between 1.3 × 103 mol product/((mol enzyme)(min)) in toluene and 8.7 × 103 mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 × 103 mol product/mol enzyme in toluene and 7.2 × 10 4 mol product/mol enzyme in amyl acetate. There is evidence that more than one enzymatic form exists. The first form demonstrates a greater inactivation rate than the second and contributes to the majority of turnover in amyl acetate (∼70%) and toluene (∼90%), whereas the second form is responsible for the majority of turnover in chloroform (∼60%). Both forms are subject to reaction inactivation and denaturation by the solvent.