Study On The Role Of Aromatic Pollutants Removal With Vitreoscilla Hemoglobin And Its Mutants

With the development of social economy and productive forces, uncontrolled discharge of three industrial wastes, erosion caused by dust and acid rain and extensive application of fertilizers, pesticides and insecticides become more and more serious. A large number of pollutants are discharged into the air, soil and water. These pollutants are difficult to be completely degrated by microorganism in the nature. Aromatic compound in water and accumulated organic nutrients lead to variation, death and extinction of aquatic animal.The pollution of benzene series is very extensive for wildlife habitats in china, which seriously threatens the survial of wildlife in hydrophytic habitat. At present, the treatment for water pollution is divided into three categories:physical, chemical and biological method. Biological method consist of microbial method and enzyme method. The removal of benzene series by enzyme method is still at its preliminary stage in china.Enzyme is often used as a catalyst to accelerate the degradation of organic compounds. Peroxidases is a sort of enzyme which can remove the pollutions in water. In 2002, Bhunia found that the removal rate of methylene blue and phenol mediated by horseradish peroxidase became slower, because native horseradish peroxidase suffered form the inactivation of dyes molecules. How to solve this problem? The challange we face is to obtain enzyme source which resists the activation caused by dye. Vitreoscilla hemoglobin is a bacterial globin originated from the obligatory aerobic bacterium Vitreoscilla. A variety of aromatic substrates can be catalyzed by Vitreoscilla hemoglobin depending on its peroxidase activity. Therefore, Vitreoscilla hemoglobin, acting as peroxidase, has tremendous potential in the treatment of effluents. So, the following studies were conducted by us.1. We transfered Vitreoscilla hemoglobin gene into Escherichia coli (E.coli) BL21 (DE3) competent cells which were cultured on Luria-Bertani (LB) medium plates in an aerobic environment. A bright band at 14.3KD was found by SDS-gel electrophoresis. The cell lysate was purified by ammonium sulfate precipitation, ion-exchange chromatography and molecular sieve chromatography. Resulting Vitreoscilla hemoglobin exhibted UV absorption peak at 402 nm and 280 nm, Rz>3.0. The obtained Vitreoscilla hemoglobin was treated by potassium ferricyanide to prepare fully oxidized state of Vitreoscilla hemoglobin. UV spectrum demonstrated the presence of fully oxidized state of Vitreoscilla hemoglobin.2. We further designed Vitreoscilla hemoglobin variants Q53H, P54C, Q53H/P54C. These Vitreoscilla hemoglobin variants were purified to investigate its preference and removal efficiency for different substrates (a series of benzene series). Differential scanning calorimetry analysis demonstrated that the melting temperature of Vitreoscilla hemoglobin variant Q53H/P54C was higher than horseradish peroxidase, Vitreoscilla hemoglobin and the other Vitreoscilla hemoglobin variants (Q53H, P54C).3. Considering that methylene blue is commmon pollution, we optimized the reaction conditions for the removal of methylene blue such as pH, temperature and H2O2 concerntration. The results showed that the activity of Vitreoscilla hemoglobin was 5.7-fold higher than that of horseradish peroxidase using methylene blue as substrate at pH 7.5 when 7.5 mM of H2O2 was used. While Vitreoscilla hemoglobin variant Q53H/P54C exhibited a 1.4-fold increase in activity as compared with Vitreoscilla hemoglobin using methylene blue as substrate. Moreover, Vitreoscilla hemoglobin variant Q53H/P54C possessed better thermal stability, pH stability and H2O2-resistance compared with horseradish peroxidase. UV-Vis, circular dichroism (CD) and fluorescence spectra also verified that VHb variant Q53H/P54C had a compact structure. We believed the enhancement of removal efficiency for Vitreoscilla hemoglobin variant Q53H/P54C indicated that replacement of Q53 with a histidine residue causes an increased in the helical content (CD data), moving histidine into an orientation where it faced the heme surface. Accordingly, this structural change likely also made porphyrin-surrounding structures more proximal to the reaction site.4. In benzene series, aniline,2,4,5-trichloroaniline, phenol, and 2,4,6-trichlorophenol are representative discharges from industrial and agricultural production and human life pollution. We screened the optimal pH and optimal temperature of Vitreoscilla hemoglobin and its variants Q53H, P54C, Q53H/P54C when using aniline,2,4,5-trichloroaniline, phenol, and 2,4,6-trichlorophenol as substrate, respectively. Then, we compared the removal efficiency of Vitreoscilla hemoglobin and its variants Q53H, P54C, Q53H/P54C for four different substrates under optimum reaction conditons. The experimental results indicated that Vitreoscilla hemoglobin variants Q53H gave the highest removal efficiency when removing phenol, aniline, 2,4,5-trichloroaniline compared with other proteins, and that Vitreoscilla hemoglobin variants P54C presented the highest removal efficiency when removing 2,4,6-trichlorophenol compared with other proteins. The results illustrated that the stereo-selectivity of Vitreoscilla hemoglobin for different substrates could be improved by site-directed mutagenesis in its distal heme pocket. In the future, we hope that the specific Vitreoscilla hemoglobin variants will be employed for removing specific aromatic pollutants.In conclusion, the removal of methylene blue, aniline, 2,4,5-trichloroaniline, phenol and 2,4,6-trichlorophenol mediated by horseradish peroxidase, Vitreoscilla hemoglobin and Vitreoscilla hemoglobin variants was investigated. The protein secondary structure in near distal heme pocket of Vitreoscilla hemoglobin was modified by site-directed mutagenesis. The change of protein secondary structure might cause the change of ligand-binding properties, which might lead to the improvment of its catalytic activity. In this work, the different Vitreoscilla hemoglobin variants with enhanced removal efficiency for different aromatic pollutants were obtained. Vitreoscilla hemoglobin is a kind of peroxidase originated from microorganism. Compared with typical chemical, physical and biological method, the removal of pollutants mediated by Vitreoscilla hemoglobin is ideal strategy owing to Vitreoscilla hemoglobin’s low cost, easy availability and high resistance to inactivation aroused by pollutants. This study will pave the way for the removal of benzene series pollution in wildlife habitats by Vitreoscilla hemoglobin.