| 摘要: | 研究期間：10108~10207;Long-chain alkylphenols, such as nonylphenols (n- and t-) and t-octylphenol, are used in the formulation and production of mainly plastics, paints, pesticides, phenolic resins, phenolic oximes, stabilizers and detergents. Some of them are used as plasticizers. In addition, they are estrogenic-like compounds and persistent to be degraded by the bacteria in the environments. Due to the structural similarity to estradiol, nonylphenols/t-octylphenol influence the growth, reproduction and sexual development of human and other animals, therefore, causing health and ecological concern. Nonylphenols (normally used as a mixture of isomers on the nonyl side chain) have been applied in many activities/industries worldwide and the amount is greater than t-octylphenol. But the estrogenic activity of t-octylphenol is 10~20 fold higher than that of nonylpheol on the induction of vitellogenin. In this study, 4-t-octylphenol was used as the sole source of carbon for the isolation of bacteria. One bacterial isolate, Acinetobacter sp. OP5, was from the sediment of drainage which runs through campus and rice filed. It was shown to be able to grow on a wide range of alkylphenols. A gene cluster (10.3 kb) from strain OP5 was cloned and shown to contain the genes responsible for (1) Octylphenol hydroxylase (ophA1A2A3A4A5A6), a multi-component alkylphenol hydroxylase to hydroxylate the carbon source; (2) Octylcatechol dioxygenase (ophB), a meta-cleavage dioxygenase to disrupt the aromatic ring. Their functions are suggested by the deduced protein sequences of the cloned genes. With the two continuous reactions catalyzed by the two enzymes, the estrogenic activity of the long-chain alkylphenols will be disrupted. The bacterium can further utilize the ring-fission products and, therefore, use t-octylphenol as sole carbon source to grow. In this three-year grant application, we propose to investigate the function of the individual and different combination of protein components from the multi-component hydroxylase. Also the substrate specificity, enzyme kinetics, products etc. will be confirmed by purified enzymes. The study is anticipated to be the first to demonstrate the octylphenol/nonylphenol-degrading by purified enzyme and in the future to investigate the catalytic mechanism. |
