Title

Biocatalysis with cross-linked enzyme-polylysine films in microemulsions

Date of Completion

January 2008

Keywords

Chemistry, Organic

Degree

Ph.D.

Abstract

This thesis demonstrates stable biocatalytic films of cheap and commercially available horseradish and soybean peroxidases and myoglobin, capable of biocatalysis at 90°C and pH 2.0 to 12.0 in microemulsions and buffers. In chapter two, stable covalently linked films of myoglobin and polylysine on pyrolytic graphite electrodes reacted with tert-butylhydroperoxide to form ferryloxy protein species according to Michaelis-Menten enzyme kinetics. Rotating disk voltammetry data obtained in microemulsions, micellar solutions and buffers and analyzed with the Michaelis-Menten model gave kinetic parameters that revealed a strong influence of water phase acidity as judged by the catalytic efficiencies. This study demonstrates how microemulsion composition, and especially water phase acidity can be used to control reaction kinetics. ^ Chapters three and four explores thermostability of these cross-linked films of HRP, SBP and Mb enzymes and polylysine on pyrolytic graphite and 500 nm diameter silica colloids at 90°C in buffers and microemulsions. Circular Dichroism and visible spectroscopy and voltammetry showed that these enzymes retained near native structures in the films. Further, these films remained stable up to 9 and 12 hours in buffers and microemulsions respectively at 90°C, conditions which same enzymes in solution denatures within few minutes. Overly, catalytic efficiencies toward reduction of tert-butylhydroperoxide and oxidation of o-methoxyphenol to 3,3'-dimethoxy-4,4'-biphenoquinone were higher at 90°C than 25°C in both media. Microemulsions supported highest catalytic efficiencies than buffers under similar conditions of temperature. These studies strongly demonstates the application of microemulsions in biocatalysis at 90°C employing the enzymes HRP, SBP and Mb. ^ Chapter five examines the integrity of myoglobin structure in DDAB films by voltammetry, visible absorption and CD spectroscopy. Data in this chapter was discussed within the context of previously reported data, confirming that when DDAB films are made from vesicle dispersions at pH 5 to 7 and used in neutral buffers containing NaBr, Mb in the films is in a near-native conformation. This study discredits reports that pyrolytic graphite are catalysts for denaturation and heme loss in Mb-DDAB films. ^