| In this dissertation, the degrading crude enzyme and its immobilized enzyme, and the degrading genes in an atrazine high-effeciency degrading strain HB-5 have been taken as the main object to work over. The application of atrazine degrading crude enzyme and its immobilized enzyme in two atrazine polluted soils have been studied to estimate their future use. Degrading enzyme purification has been generally investigated. Furthermore, the degrading genes have been cloned, sequenced and compared with sequences of other atrazine degrading strains in Genebank. The results could be summarized as follows:1. The physical-chemical characteristics, general situations of application, present pollution situation, ecological toxicological characteristics and bioremediation of atrazine, which had been applied extensively all over the world and in China recently, were introduced. And the degrading enzyme, enzyme immobilization and purification have also been recommended. Also, the cloning of degrading genes has been comprehensive summarized, and accordingly the issues that would be investigated was presented by the author.2. Crude enzyme extracted from HB-5 and the immobilized crude enzyme on sodium alginate were introduced to two atrazine-polluted soil types which are brunisolic soil from Taian in Shandong Province and cinnamon soil from Shenyang in Liaoning Province, to evaluate their degrading ability to atrazine in practical use. Atrazine was applied to 10mg·kg-1 of soil. Brunisolic soil or cinnamon soil samples with crude or immobilized enzyme were incubated at 25oC. Samples were collected every 24 hours from 0 h to 144 h at 7 time-points to extract the residual atrazine for detection by gas chromatography.Results showed both crude enzyme and its immobilized enzyme had good ability in removal of atrazine in soils. At 120 h, only about 20% of the initially applied amount was left in the two soils, except control soils without crude enzyme or immobilized enzyme. In brunisolic soil, treatments with crude enzyme have higher degradation amount than that with immobilized enzyme; whereas, in cinnamon soil, treatments with crude enzyme have lower degradation amount than that with immobilized enzyme. In each soil type, the indigenous microorganism took part in degradation of atrazine in the soil, and their effects showed that indigenous microorganism in the cinnamon soil have contributed more than that in the brunisolic soil in atrazine degradation. Crude and immobilized enzyme both can be utilized in practice, whereas the immobilized enzyme is preferred for its stability.3. The purification of degrading enzyme extracted from HB-5 was investigated. After ammonium sulfate precipitation with 30%～70% saturation, dialysis, an ion-exchange chromatography, and gel filtration, the degrading enzyme were roughly purified. Then, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to test the results of purification.The types of ion-exchange chromatography and the pH conditions of washing buffers have been selected. In this selection, CM-Sepharose FF and DEAE-Sepharose FF were tried according to related literatures; washing buffers at pH 3.010.0 were selected, too. After gel filtration with Sephacryl S-300HR, the rough degrading enzyme was achieved.4. The purified degrading enzyme has been detected for their degrading activity with gas chromatography and SDS-PAGE has been operated to achieve the electrophoretograms.5. The degrading genes in the atrazine high-effeciency degrading strain HB-5 which had been identified as Arthrobacter sp. have been cloned. Primers of atzA, atzB, atzC and trzN were designed referring to former literatures. The total DNA of HB-5 acted as the DNA model to carry on PCR (polymerase chain reaction) and atzB,atzC and trzN genes in HB-5 were cloned, sequenced and compared with former atrazine degrading genes in Genebank, and the similarity were almost all over 98%, respectively.
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