Spectrophotometric Determination Of Environmental Pollutants Using Lignin Peroxidase

As the rapid development of world economy and the increase of population, more and more contaminations are released in the environment. The pollution of aromatic amine, phenols compounds and organic peroxides get more concern among the numerous contaminations. Sources of aromatic compounds in the environment include the abiotic and biotic degradation of nitroaromatic compounds, azo dyes, several classes of pesticides like carbamates, phenylureas. Phenolic compounds can be found in industrial waste from for example coal mining, oil refinery, paint, plastic, and pharmaceutical industry. The peroxides in atmosphere mostly roots in the second contamination of photochemical smog which is an important photochemical oxidant. The fats also automatically produce hydrogen peroxide oxidation. So it is necessary to set up simple analytic methods to determine those compounds in the environment.Lignin peroxidase is a novel fungal peroxidase found in the early 1980s, it shares many structural and mechanistic features with other peroxidases, yet its oxidizing power (1.36 V vs. NHE) is much stronger. A number of phenolic and nonphenolic compounds have been shown to be substrates for this enzyme, and among them veratryl alcohol (VA) is the optimum one. However, there are few reports about the determination of environmental pollutants. Traditionally, aqueous solvents are used as the media for enzymatic determination, and H₂O₂ was supplied externally in one batch at the beginning to initiate the reaction. In this way, we got poor reproducibility of data and selectivity, and we had difficulty in determining the hydrophobic compounds. To overcome these drawbacks, we used glucose oxidase (GOD) and glucose generating H₂O₂ in this paper, so a sustainable constant activity of LiP was observed due to the controlled release of H₂O₂. According to the two-substrate mechanism of LiP, the selectivity of determination of phenolic compounds can be improved by choosing appropriate indicator substrate. Here we determined organic peroxides in H₂O/AOT/Brij30/octane system. Our aim was to overcome the shortcomings of the existing enzyme determination by using these three measures, and also this was the originality of the paper.This paper was divided in four sections:In the first section, we introduce the pollution and analysis methods of aromatic amine, phenols and organic peroxides, and summarized recent developments of micellar enzymology and LiP study, and 124 references are cited.In the second section, we studied the analytical characteristics of the determination of aromatic amine in which LiP coupled with GOD using spectrophotometric method. We used glucose oxidase (GOD) and glucose generating H₂O₂due to the controlled release of H₂O₂, so a sustainable constant activity of LiP was observed due to the controlled release of H₂O₂. To optimize the conditions, we studied the effect of pH, concentrations of GOD, glucose and hydrogen peroxide to get the analytical characteristic of the method. The determination of aromatic amine in carrots was carried out by using the standard additions method. To demonstrate the advantages of the controlled release of H₂O₂ supply strategy, an external H₂O₂ supply strategy was also used to initiate LiP-catalyzed oxidation of OPD. Results showed that the use of the proposed H₂O₂ supply strategy had some advantages. Not only the initial rate increased greatly, but also the linear range of the A-t curve widened.In the third section, based on the effect of phenols on the kinetics of oxidation of aromatic amine catalyzed by LiP, an attempt was made to determine environmental pollutants-phenols selectively. The influence of phenols on the kinetics of enzymatic oxidation of aromatic amine with H₂O₂ was found to depend on a correlation between redox properties of phenols and the indicator-substrate of peroxidase. Using o-tolidine oxidation catalyzed by LiP as an indicator, the rate of the indicator reaction decreased in the presence of phenol whose redox potentials higher than o-tolidine. Howerer, phenols with lower potentials led to an induction period on kinetic curves of the indicator reaction, the duration of the induction period is directly proportional to the concentration of phenolic compounds. Different analytical signals-the initial rate and the lag time of the indicator reaction-were used for the determination of phenols with various groups. It was expedient to study the possibility of the individual determination of isomers with different groups. When the reaction of p-anisidine (PAD) oxidation catalyzed by LiP was used as indicator, the rate of the indicator reaction increased and was proportionally to the concentration of phenol whose redox potentials higher than that of PAD. Whereas, the rate of the indicator reaction decreased in the presence of phenol with lower redox potentials and was proportionally to their concentration. But it was impossible to determine phenols selectively.In the fourth section, a mothod for spectrophotometric determination of organic peroxides in the reversed micelles using o-phenylenediamine (OPD) as the substrate of LiP was reported. The initial rate is proportional to the concentration of peroxides in solution. In the paper, a new mixed reversed micelle formed by anionic surfactant AOT and non-ionic surfactant polyoxyethylene lauryl ether (Brij30) was used to determination organic peroxides. The media allows the enzymatic determination of substances scarcely soluble in water. Furthermore, the content of the water needed for the hydration of the enzyme, and therefore, for enzyme operation, is easy to control and optimize. To optimize the conditions of OPD oxidation with 2-butanone peroxide in the presence of LiP, we studied the effect of pH andω₀ to get the analytical characteristic of the method. The determination of organic peroxides in olive oil was carried out by using the standard additions method. In order to avoid the disturbance of water-soluble substrate, we dissolved the olive oil in 0.5mmol·L^-1 HC1 solution. Later the organic phase was extracted using ethyl acetate, and the extraction was added to reversed micellar to determine. Compared with the existing method, this way not only decreased the steps of operation, but also avoided the pollution and loss of samples. The method could satisfy the the limits of detection and sensitivity in the determination of organic peroxides in the olive oil.