Construction Of The System Based On Enzymatic Degradation Of Environmental Toxicants Chlorine Peroxidase Activity Central Domain Research

Chloroperoxidase (CPO) has a variety of catalytic properties and lots of suitable substrates because of its unique structure of CPO’s active center. However, the structure-activity relationship needs to be further elucidated. In this paper, based on the study of key sites in CPO’s structural domain to deeply understand of the structure-activity relationship of CPO, this enzyme was applied to the degradation of environmental toxicants, in which four degradation systems were conceived for bisphenol A, diclofenac sodium, naproxen and estrone respectively. Its main contents were as follows:Analysis of the active center structural domain:The coordinated combination of heme propionate with Mn2+in active center of chloroperoxidase was confirmed by means of removing the metal ions by EDTA ligand competition reaction and determining Mn2+by graphite furnace atomic absorption and inductively coupled plasmas atomic emissive spectrometry measurement. The exact role of Mn2+ion in CPO has yet to be established, and some studies had shown that the activity of the enzyme was not altered by the presence or absence of Mn2+. However, in our study, the presence of Mn2+in this site was found playing a role in holding the active domain and maintaining preferential conformation of CPO based on the UV absorption spectroscopy, circular dichroism (CD)analysis as well as chlorination activity and peroxidation activity measurement before and after Mn2+being removed from CPO. So it was concluded that the presence of Mn2+in CPO active domain was a necessary factor for maintaining enzyme activity. Furthermore, it was found that the change of micro-environment around heme in CPO active center was a reversible process, and the catalytic activity will be recovered when Mn2+was re-introduced though it was a very slow dynamic process that needed nearly100h. Moreover, the chlorination activity and peroxidation activity of CPO was observed to be improved compared with that of native CPO when exogenous Ag+and Cr3+are introduced instead of Mn2+. Therefore this method would provide a new way for the improvement of CPO catalytic performance by means of chemical modification.Optimization of enzymatic degradation process:The degradation of bisphenol A, diclofenac sodium, naproxen and estrone were studied using the H2O2as oxidant catalyzed by CPO. The effects of various factors (the amount of enzyme, H2O2concentration, pH, reaction time) on degradation efficiency were evaluated, and optimized accordingly. The optimal degradation conditions of bisphenol A:the amount of enzyme:2.0×10-9mol/L; H2O2concentration:0.15mmol/L; pH value:3.0; the reaction time:7min; The optimal degradation conditions of diclofenac sodium:the amount of enzyme:5.0×10-9mol/L; H2O2concentration:0.1mmol/L; pH value:3.0; the reaction time:9min; The optimal degradation conditions of naproxen:the amount of enzyme:20.0×10-9mol/L; H2O2concentration:0.1mmol/L; pH value:3.0; the reaction time:7min; The optimal degradation conditions of estrone:the amount of enzyme:0.8×10-9mol/L; H2O2concentration:25μmol/L; pH value:2.0-6.0; the reaction time:18min. Under their optimal reaction conditions, their degradation efficiency all can reach100%.Determination of degradation products and analysis degradation pathways:The LC-MS technology were employed to determined the degradation products, and the degradation pathway was speculated accordingly. The four environmental toxicants selected in this paper are organic pollutants that are refractory by traditional method. The results showed that the four toxicants molecules were broken into smaller pieces effectively. So this enzymatic oxidation by CPO can be used as an effective pre-treatment stage combined with the microbial degradation process to decontaminate pollutants completely so as to realize the sustainable development strategy.Catalysis cycle involved in enzymatic degradation and degradation mechanism. There are two mechanisms were clarified:the first one is that toxicants went directly into the active center of enzyme, and reactd with compound I, a oxoferryl porphyrin π cation radical containing Fe(IV), which was a intermediates with strong oxidant generated in enzyme cycle. Compound I then transferd to CPO through a two single-electron process. The other is concerned with the chlorination activity of CPO. Some small strong oxidants, such as Cl2, HClO, generated on line in enzymatic cycle using Cl-as a substrate, can oxidize toxicants in the bulk solution, which may get rid of restrictions for substrate’s species and size required by the channel access active center in CPO.Performance evaluation of enzymatic degradation system:After the four environmental toxicants degradation(bisphenol A, diclofenac sodium, naproxen and estrone), their chemical oxygen demand (COD) and total organic carbon (TOC) decreased by5.0%/23.2%,4.3%/25.2%,7.2%/7.5%,1.2%/1.4%,1.2%/1.4%separately. The decline of COD indicated that the content of organic matter in the poison was decreased. The decline of TOC indicated that the biodegradable ability was increased.