Chloroperoxidase Catalyzed Oxidation Of Cyclic Olefins Research

Chloroperoxidase (CPO) is now considered as the most versatile enzyme in heme peroxidase family because of its various catalytic activities and suitable substrates due to its unique active site structure. The active center in CPO is similar to the one in cytochrome P-450, having a proximal thiolate ligand of cysteine (Cys29) as the fifth axial ligand of porphyrin iron. In contrast to cytochrome P-450, the reaction process catalyzed by CPO is much simpler with no need for coenzyme. So it's a biocatalyst with potential applications for industry.Small cyclenes is a common and abundant co-product in oil refining. The study on "oxygenated" reaction for cyclones to make them to be value-added derivative for develop downstream products would be very meaningful. Therefore, in this paper, cyclohexene and cyclopentene was chosen as model substrates to develop an environmental-friendly route for biotransformation of small cyclenes catalyzed by CPO with H2O2 as oxidant.CPO is a hydrophilic enzyme, but hydrophobic organic substrates are mostly insoluble in water or low solubility, resulting in the difficult access for substrate to CPO efficiently, which is a major drawback that restricted application of CPO in organic synthesis. In this work, the yield of expected products was tuned and enhanced by solvent engineering based on co-solvent effect of organic solvents and the additive effect of hydrophilic imidazole/pyridine ionic liquids (ILs) and hydrophilic quaternary ammonium salt (QAS). Lots of spectrum analysis and reaction kinetic assay were employed to investigate the effect of ILs and the QAS on the microenvironment around the active center of CPO and protein structure to elucidate the reason for the improvement of CPO catalytic performance. Meanwhile, a biological super-thin films immobilized by CPO was prepared with supramolecular self-assembly technology for reuse of CPO in reaction system. The main results are as follows:1. Cyclohexene system:According to factors affecting the reaction conditions (pH, amount of enzyme, substrate and oxidant quantity and reaction time, etc.) to optimize reaction conditions, cyclohexene conversion and cyclohexene yield of 44.5% and 40.3 %, respectively. The conversion of cyclohexene was enhanced 31.0% and 36.5% respectively when only 1.5%(WAdditive/WPBS) of hydrophilic imidazole/pyridine ionic liquids (ILs) or hydrophilic quaternary ammonium salt (QAS) was introduced into the PBS buffer. The decrease of enzyme kinetic parameters Km and increase of Kcat and Kcat/Km indicated that both the affinity and specificity of CPO to the substrate were improved in the presence of ILs or QAS, resulting in the increase of turnover number. This was the main reason of conversion enhancement. Moreover, Uv-vis, fluorescence and circular dichroism assay and analysis of products composition indicated that ILs and QAS played multiple functions in this enzyme-catalyzed reaction:phase transfer catalysis, inducing catalytically favorable conformation of CPO and tuning the composition of products. The most efficient additive was tetrapropylammonium bromide (TPABr). This enzymic-reaction was over within 150min, and enzyme consumption was small. So, it has some potential utility in industry application.2. Cyclopentene system:By selecting the best conditions such as oxidant quantity, pH, reaction time and amount of enzyme to optimize oxidation of cyclopentene catalyzed by CPO. It was found that when hydrophilic ILs and QAS were introduced into cyclohexene system, the substrate conversion was increased; however, epoxidation products would further hydrolyze to glycol meanwhile. So, organic solvent was introduced into reaction medium as co-solvent. This strategy was expected to enhance both the substrate conversion as well as the yield of epoxycyclopentane (CPEO). But the substrate conversion was enhanced only 11.9% due to in pure buffer due to the unstability of CPO in organic solvents. Amyl acetate was the most effective additives in this reaction.Like all native enzyme, CPO has poor stability under acids, alkalis condition, at high temperature or in the presence of organic solvents, which limited the industrial application of CPO to a large extent. So, a stable biological super-thin film of CPO was prepared with ionized polyethylene terephthalate (PET) as support by supramolecular self-assembly technology. The assembly condition was optimized based on the chlorination activity measurements of monochlorodimedon (MCD) catalyzed by immobilized CPO, while UV-visible spectroscopy was employed to investigate the assembly process. The topographies of the film surface were examined by ESEM. Furthermore, this CPO thin films was applied for the epoxidation of cyclohexene/cyclopentene.