טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentCohen Yaniv Vered
SubjectPolymerization of Brominated Phenol Compounds by Peroxidases
DepartmentDepartment of Civil and Environmental Engineering
Supervisor Professor Emeritus Carlos Dosoretz
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Peroxidases are heme enzymes which catalyze the oxidation of wide variety of phenolic compounds to phenoxy radicals that couple to form oligomeric and polymeric products. The increase in molecular weight increases the hydrophobicity and decreases the solubility of products formed, which eventually precipitate, and therefore can be easily separated from the treated waters. Therefore, a reduction in toxicity of water contaminated with toxic phenols, following the peroxidase-catalyzed polymerization process, is expected. Nevertheless, even at very low concentrations, some soluble oxidation products may exhibit a relative toxicity higher than the original substrate.

This study examined the oxidation of the three monobromophenols (BP) congeners by horseradish peroxidase (HRP), which were characterized by high catalytic rate and low affinity: 4BP has the highest kcat, 3BP has the lowest KM and kcat. The highest KM values achieved for the 2BP suggests the occurrence of steric hindrance. In accordance, to the kinetic constants, rate of transformation was 4BP>2BP> 3BP.

A substantial decrease in the catalytic efficacy of the reaction is attributed to HRP inactivation during the reaction, soon after its initiation, most probably due to product inactivation. Different protein content analysis procedures clarified that the main reason for HRP inactivation was due to precipitation from the reaction medium, mainly by hydrophobic interaction. In the absence of polyethylene glycol (PEG), approx. 5% protein only remained in the soluble fraction whereas 20-30% of soluble enzyme was detected with PEG. It appears that the molecular weight bands of protein in the precipitate had shifted toward the higher molecular weights, compared to the enzyme control. These findings suggest an occurrence of a persistent bond between reaction products and HRP, a bond that resisted protein denaturation during SDS-PAGE analysis. To the best of our knowledge, this is the first report regarding a persistent interaction (covalent bond?) between HRP and products in the precipitate.

The oxidation and polymerization of the reactions were accompanied by extensive debromination, especially 4BP which achieved the maximum events of dehalogenation possible per substrate. Both co-polymerization and an additional HRP dose should be implemented in order to achieve a maximum extent of organic matter removal and highest degree of precipitation and detoxification.