双酚A高效降解菌的筛选及其降解机理的初步研究
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摘要
当今，环境内分泌干扰物对人类健康造成了危害。双酚A(bisphenol A，Bpa)是其中重要的一种。随着Bpa的大量使用，在世界各地各种水体中都有发现，对Bpa污染的治理势在必行。为此，我们开展了下面一些研究。
1．Bpa降解菌的筛选及鉴定。采用经典的细菌筛选技术，得到了Bpa高效降解菌(W2)，该菌可在饱和的Bpa溶液里以其为唯一碳源和能源生长。具有较强的Bpa降解能力。经过种属鉴定，该菌株属于假单胞菌属。
2．Pseudomonas sp.W2培养条件的优化。在实验室里探讨碳源、氮源、通气量、起始pH值、温度等因素对菌株生长及对Bpa降解能力的影响。菌体最佳生长条件为：pH7.0，250ml摇瓶装液量为50ml，温度30℃；Bpa最佳降解条件为：pH7.0，250ml摇瓶装液量为100ml，35℃。
3．Pseudomonas sp.W2降解Bpa机理初探。首先，利用分子生物学技术对该菌株降解Bpa起始关键基因与关键酶进行了初步研究，发现降解Bpa的起始关键基因定位在一个19kb左右的质粒上，起始关键酶是一种胞外酶。其次，利用现代分析化学技术研究Pseudonomas sp.W2对Bpa的代谢途径，发现对羟基苯甲酸、对羟基苯甲醛、对羟基苯乙酮为三种中间代谢产物。最后对该菌株的原儿茶酸双加氧酶基因pcaG进行了扩增测序。
本研究成果将为进一步开发Bpa治理微生物资源及发现Bpa降解关键基因研究奠定基础。
Nowadays, endocrine disrupter chemicals are endangering human health. Of all EDCs, Bisphenol A (Bpa) is very important one. With Bpa used in great quantity,it is detected in all kinds of waters all over the world. Control of Bpa pollution is very necessary. So we did some correlative work.
First of all, the isolation of strain able to degrade Bpa effectively and classify. A strain (W2) that was isolated ,which can degrade BPA very effectively and grow on saturation solution of Bpa as a sole carbon and energy sources. It was identified as pseudonomas sp.
Secondly, study of the cultivation condition of pseudonomas sp. W2. In lab, we researched the effect of cabon source, nitrogen source, pH at beginning, temperature and aerate to the growth of strain W2 and degradation of Bpa. The optimal growth condition: pH7, 30℃, 50ml culture medium in 250ml flask. The optimal Bpa degradation condition: pH7,35℃, 100ml culture medium in 250ml flask.
Thirdly, study of Bpa-degrading mechanism of pseudonomas sp.W2. Through the moleculor biology technique, we acknowledged that the Bpa-degrading enzyme is out-cell and the key gene is located at a plasmit about 19kb. Then, we studied the pathway of bacterial metabolism of Bpa and identified three kinds of metabolon of p-Hydroxy benzoic acid , p-Hydroxy benzaldehyde and p-Hydroxy acetophenone.Finally,we amplified and sequenced the pcaG gene.
Our study result laid a foundation for exploitation of environmental Bpa-remeding microorganism and for building a gene-engineering microorganism to promote bioremediation of Bpa contamination.
1．Bpa降解菌的筛选及鉴定。采用经典的细菌筛选技术，得到了Bpa高效降解菌(W2)，该菌可在饱和的Bpa溶液里以其为唯一碳源和能源生长。具有较强的Bpa降解能力。经过种属鉴定，该菌株属于假单胞菌属。
2．Pseudomonas sp.W2培养条件的优化。在实验室里探讨碳源、氮源、通气量、起始pH值、温度等因素对菌株生长及对Bpa降解能力的影响。菌体最佳生长条件为：pH7.0，250ml摇瓶装液量为50ml，温度30℃；Bpa最佳降解条件为：pH7.0，250ml摇瓶装液量为100ml，35℃。
3．Pseudomonas sp.W2降解Bpa机理初探。首先，利用分子生物学技术对该菌株降解Bpa起始关键基因与关键酶进行了初步研究，发现降解Bpa的起始关键基因定位在一个19kb左右的质粒上，起始关键酶是一种胞外酶。其次，利用现代分析化学技术研究Pseudonomas sp.W2对Bpa的代谢途径，发现对羟基苯甲酸、对羟基苯甲醛、对羟基苯乙酮为三种中间代谢产物。最后对该菌株的原儿茶酸双加氧酶基因pcaG进行了扩增测序。
本研究成果将为进一步开发Bpa治理微生物资源及发现Bpa降解关键基因研究奠定基础。
Nowadays, endocrine disrupter chemicals are endangering human health. Of all EDCs, Bisphenol A (Bpa) is very important one. With Bpa used in great quantity,it is detected in all kinds of waters all over the world. Control of Bpa pollution is very necessary. So we did some correlative work.
First of all, the isolation of strain able to degrade Bpa effectively and classify. A strain (W2) that was isolated ,which can degrade BPA very effectively and grow on saturation solution of Bpa as a sole carbon and energy sources. It was identified as pseudonomas sp.
Secondly, study of the cultivation condition of pseudonomas sp. W2. In lab, we researched the effect of cabon source, nitrogen source, pH at beginning, temperature and aerate to the growth of strain W2 and degradation of Bpa. The optimal growth condition: pH7, 30℃, 50ml culture medium in 250ml flask. The optimal Bpa degradation condition: pH7,35℃, 100ml culture medium in 250ml flask.
Thirdly, study of Bpa-degrading mechanism of pseudonomas sp.W2. Through the moleculor biology technique, we acknowledged that the Bpa-degrading enzyme is out-cell and the key gene is located at a plasmit about 19kb. Then, we studied the pathway of bacterial metabolism of Bpa and identified three kinds of metabolon of p-Hydroxy benzoic acid , p-Hydroxy benzaldehyde and p-Hydroxy acetophenone.Finally,we amplified and sequenced the pcaG gene.
Our study result laid a foundation for exploitation of environmental Bpa-remeding microorganism and for building a gene-engineering microorganism to promote bioremediation of Bpa contamination.
引文
[1] Aloisi AM, Della Seta D, Ceccarelli I, Farabollini F. Related Articles. Bisphenol-A differently affects estrogen receptors-alpha in estrous-cycling and lactating female rats[J]. Neurosei Lett, 2001, 310, 49-52.
[2] Koponen PS, Kukkonen JV. Effects of bisphenol A and artificial UVB radiation on the early development of Rana temporaria[J]. J Toxicol Environ Health A, 2002,65:947-959.
[3] Nieminen P, Lindstrom-Seppa P, Juntunen M, Asikainen, Mustonen AM, Karonen SL, Mussalo-Rauhamaa H, Kukkonen JV. In vivo effects of bisphenol A on the polecat (mustela putorius) [J].J Toxicol Environ Health A, 2002,65:933-945.
[4] Aloisi AM, Della Seta D, Rendo C, Ceccarelli I, Scaramuzzino A, Farabollini F. Exposure to the estrogenic pollutant bisphenol A affects pain behavior induced by subcutaneous formalin injection in male and female rats[J]. Brain Res,2002,937:1-7.
[5] Al-Hiyasat AS, Darrnani H, Elbetieha AM. Effects of bisphenol A on adult male mouse fertility[J]. Eur J Oral Sci, 2002,110(2): 163-7.
[6] Sehonfelder G, Flick B, Mayr E, Talsness C, Paul M, Chahoud I. In utero exposure to low doses of bisphenol A lead to long-term deleterious effects in
the vagina[J].Neoplasia,2002,4:98-102.
[7] Moon DG, Sung DJ, Kim YS, Cheon J, Kim JJ. Bisphenol A inhibits penile erection via alteration of histology in the rabbit[J]. Int J Impot Res, 2001,13:309-316.
[8] Sogawa N, Onodera K, Sogawa CA, Mukubo Y, Fukuoka H, Oda N, Furuta H. Bisphenol A enhances cadmium toxicity through estrogen receptor[J]. MethOds Find Exp Clin Pharmacol, 2001,23:395-399.
[9] Kim JC, Shin HC, Cha SW, Koh WS, Chung MK, Han SS. Evaluation of developmental toxicity in rats exposed to the environmental estrogen bisphenol A during pregnancy[J].Life Sei,2001,69:2611-2625.
[10] Takahashi O, Oishi S. Testicular toxicity of dietary 2,2-bis(4-hydroxyphenyl)propane (bisphenol A) in F344 rats[J]. Arch Toxicol, 2001,75:42-51.
[11] 李林，周玲，张杨，胡森科，程占胜．西安市饮用水中六六六、滴滴涕污染的调查研究[J]．环境与健康杂志，1998,15：208．
[12] 吴平谷，韩关根，王惠华，赵莹．饮用水中邻苯二甲酸酯类的调查[J]．环境与健康杂志，1999,16：338-339．
[13] 邵兵，胡建英，杨敏．重庆流域嘉陵江和长江水环境中壬基酚污染状况调查[J]．环境科学学报，2002，22：12-16．
[14] 梁增辉，何世华，孙成均,王福玉，战威，贾凌志，张宏，王福俭，吴德生．引起青蛙畸形的环境内分泌干扰物的初步研究[J]．环境与健康杂志,2002，19：419-421．
[15] 齐文启，孙宗光．痕量有机污染物的监测．化学工业出版社，2001，8-13
[16] Khim JS, Lee KT, Kannan K, Villeneuve DL, Giesy JP, Koh CH. Trace organic contaminants in sediment and water from Ulsan Bay and its vicinity, Korea[J].Arch Environ Contam Toxicol,2001,40:141-150.
[17] Stachel B, Ehrhorn U, Heernken OP, Lepom P, Reincke H, Sawal G, Theobald N. Xenoestrogens in the River Elbe and its tributaries. Environ
Pollut, 2003, 124:497-507.
[18] Mountfort KA, Kelly J, Jickells SM, Castle L.Investigations into the potential degradation of polycarbonate baby bottles during sterilization with consequent release of bisphenol A[J]. Food-Addit-Contam, 1997,14: 737-740.
[19] Hirano T, Honda Y, Watanabe T, Kuwahara M. Degradation of bisphenol A by the lignin-degrading enzyme, manganese peroxidase, produced by the white-rot basidiomycete, Pleurotus ostreatus[J]. Biosci Biotechnol Biochem, 2000,64:1958-1962.
[20] Lobos JH, Leib TK, Su TM. Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium[J]. Appl Environ Microbiol, 1992,58:1823-1831.
[21] Kang JH, Kondo F. Bisphenol A degradation by bacteria isolated from river water[J]. Arch Environ Contam Toxicol,2002,43:265-269.
[22] Ohko Y, Ando I, Niwa C, Tatsuma T, Yamamura T, Nakashima T, Kubota Y, Fujishima A. Degradation of bisphenol A in water by TiO2 photocatalyst[J]. Environ-Sci-Technol, 2001, 35: 2365-2368.
[23] Kuramitz H, Nakata Y, Kawasaki M, Tanaka S.Electrochemical oxidation of bisphenol A. Application to the removal of bisphenol A using a carbon fiber electrode[J]. Chemosphere ,2001,45:37-43.
[24] Hesselsoe M, Jensen D, Skals K, Olesen T, Moldrup P, Roslev P, Mortensen GK, Henriksen K.Degradation of 4-nonylphenol in homogeneous and nonhomogeneous mixtures of soil and sewage sludge[J]. Environ Sci Technol, 2001,35:3695-3700.
[25] anghe T, Dhooge W, Verstraete W. Isolation of a bacterial strain able to degrade branched nonylphenol[J]. Appl Environ Microbiol, 1999,65:746-755.
[26] Fujii K, Urano N, Ushio H, Satomi M, Kimura S. Sphingomonas cloacae sp.
nov., a nonylphenol-degrading bacterium isolated from wastewater of a sewage-treatment plant in Tokyo[J]. Int J Syst Evol Microbiol, 2001,51:603-610.
[27] Fujii K, Urano N, Ushio H, Satomi M, Iida H, Ushio-Sata N, Kimura S. Profile of a nonylphenol-degrading microflora and its potential for bioremedial applications[J]. J Biochem (Tokyo), 2000,128:909-916.
[28] Yuan SY, Yu CH, Chang BV. Biodegradation of nonylphenol in river sediment. Environ Pollut, 2004,127:425-430.
[29] Tsutsumi Y, Haneda T, Nishida T. Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes[J]. Chemosphere,2001,42: 271-276.
[30] Ridgway TJ, Wiseman H. Removal of oestrogens and oestrogen mimics from the environment. Biochem Soc Trans, 1998,26:675-678.
[31] 刘营，孔繁翔，杨积晴．菌根真菌对环境污染物的降解、转化能力概述．上海环境科学，1998，17(2)：4-6．
[32] 孙雷心．革兰氏阳性多氯联苯降解菌RHODOCOCCUS ERYTHROPOLIS TA421编码2，3-二羟基联苯-1，2-加双氧酶的多基因．生物技术通报，1996，(1)：113．
[33] 岳贵春．混合发酵法降解多氯联苯的试验研究．环境科学学报，1987,(2)：207-211．
[34] 马瑞霞．微生物转化γ-666及短芽胞杆菌(Bacillus brevis)对γ-666的降解．中国环境科学，1984(3)：63-66．
[35] 刘庆余，李悦，齐英，马元中．邻苯二甲酸酯类在土柱模拟试验中的微生物降解．环境卫生工程，1997，(4)：3-6．
[36] 刘庆余，蔡晓冬，杨意东，张林原．污泥发酵处理中微生物对毒性有机物的降解环境卫生工程，1997，(2)：3-6．
[37] Spivack J, Leib TK, Lobos JH. Novel pathway for bacterial metabolism of bisphenol A. Rearrangements and stilbene cleavage in bisphenol A metabolism[J].J Biol Chem,1994,269:7323-7329.
[2] Koponen PS, Kukkonen JV. Effects of bisphenol A and artificial UVB radiation on the early development of Rana temporaria[J]. J Toxicol Environ Health A, 2002,65:947-959.
[3] Nieminen P, Lindstrom-Seppa P, Juntunen M, Asikainen, Mustonen AM, Karonen SL, Mussalo-Rauhamaa H, Kukkonen JV. In vivo effects of bisphenol A on the polecat (mustela putorius) [J].J Toxicol Environ Health A, 2002,65:933-945.
[4] Aloisi AM, Della Seta D, Rendo C, Ceccarelli I, Scaramuzzino A, Farabollini F. Exposure to the estrogenic pollutant bisphenol A affects pain behavior induced by subcutaneous formalin injection in male and female rats[J]. Brain Res,2002,937:1-7.
[5] Al-Hiyasat AS, Darrnani H, Elbetieha AM. Effects of bisphenol A on adult male mouse fertility[J]. Eur J Oral Sci, 2002,110(2): 163-7.
[6] Sehonfelder G, Flick B, Mayr E, Talsness C, Paul M, Chahoud I. In utero exposure to low doses of bisphenol A lead to long-term deleterious effects in
the vagina[J].Neoplasia,2002,4:98-102.
[7] Moon DG, Sung DJ, Kim YS, Cheon J, Kim JJ. Bisphenol A inhibits penile erection via alteration of histology in the rabbit[J]. Int J Impot Res, 2001,13:309-316.
[8] Sogawa N, Onodera K, Sogawa CA, Mukubo Y, Fukuoka H, Oda N, Furuta H. Bisphenol A enhances cadmium toxicity through estrogen receptor[J]. MethOds Find Exp Clin Pharmacol, 2001,23:395-399.
[9] Kim JC, Shin HC, Cha SW, Koh WS, Chung MK, Han SS. Evaluation of developmental toxicity in rats exposed to the environmental estrogen bisphenol A during pregnancy[J].Life Sei,2001,69:2611-2625.
[10] Takahashi O, Oishi S. Testicular toxicity of dietary 2,2-bis(4-hydroxyphenyl)propane (bisphenol A) in F344 rats[J]. Arch Toxicol, 2001,75:42-51.
[11] 李林，周玲，张杨，胡森科，程占胜．西安市饮用水中六六六、滴滴涕污染的调查研究[J]．环境与健康杂志，1998,15：208．
[12] 吴平谷，韩关根，王惠华，赵莹．饮用水中邻苯二甲酸酯类的调查[J]．环境与健康杂志，1999,16：338-339．
[13] 邵兵，胡建英，杨敏．重庆流域嘉陵江和长江水环境中壬基酚污染状况调查[J]．环境科学学报，2002，22：12-16．
[14] 梁增辉，何世华，孙成均,王福玉，战威，贾凌志，张宏，王福俭，吴德生．引起青蛙畸形的环境内分泌干扰物的初步研究[J]．环境与健康杂志,2002，19：419-421．
[15] 齐文启，孙宗光．痕量有机污染物的监测．化学工业出版社，2001，8-13
[16] Khim JS, Lee KT, Kannan K, Villeneuve DL, Giesy JP, Koh CH. Trace organic contaminants in sediment and water from Ulsan Bay and its vicinity, Korea[J].Arch Environ Contam Toxicol,2001,40:141-150.
[17] Stachel B, Ehrhorn U, Heernken OP, Lepom P, Reincke H, Sawal G, Theobald N. Xenoestrogens in the River Elbe and its tributaries. Environ
Pollut, 2003, 124:497-507.
[18] Mountfort KA, Kelly J, Jickells SM, Castle L.Investigations into the potential degradation of polycarbonate baby bottles during sterilization with consequent release of bisphenol A[J]. Food-Addit-Contam, 1997,14: 737-740.
[19] Hirano T, Honda Y, Watanabe T, Kuwahara M. Degradation of bisphenol A by the lignin-degrading enzyme, manganese peroxidase, produced by the white-rot basidiomycete, Pleurotus ostreatus[J]. Biosci Biotechnol Biochem, 2000,64:1958-1962.
[20] Lobos JH, Leib TK, Su TM. Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium[J]. Appl Environ Microbiol, 1992,58:1823-1831.
[21] Kang JH, Kondo F. Bisphenol A degradation by bacteria isolated from river water[J]. Arch Environ Contam Toxicol,2002,43:265-269.
[22] Ohko Y, Ando I, Niwa C, Tatsuma T, Yamamura T, Nakashima T, Kubota Y, Fujishima A. Degradation of bisphenol A in water by TiO2 photocatalyst[J]. Environ-Sci-Technol, 2001, 35: 2365-2368.
[23] Kuramitz H, Nakata Y, Kawasaki M, Tanaka S.Electrochemical oxidation of bisphenol A. Application to the removal of bisphenol A using a carbon fiber electrode[J]. Chemosphere ,2001,45:37-43.
[24] Hesselsoe M, Jensen D, Skals K, Olesen T, Moldrup P, Roslev P, Mortensen GK, Henriksen K.Degradation of 4-nonylphenol in homogeneous and nonhomogeneous mixtures of soil and sewage sludge[J]. Environ Sci Technol, 2001,35:3695-3700.
[25] anghe T, Dhooge W, Verstraete W. Isolation of a bacterial strain able to degrade branched nonylphenol[J]. Appl Environ Microbiol, 1999,65:746-755.
[26] Fujii K, Urano N, Ushio H, Satomi M, Kimura S. Sphingomonas cloacae sp.
nov., a nonylphenol-degrading bacterium isolated from wastewater of a sewage-treatment plant in Tokyo[J]. Int J Syst Evol Microbiol, 2001,51:603-610.
[27] Fujii K, Urano N, Ushio H, Satomi M, Iida H, Ushio-Sata N, Kimura S. Profile of a nonylphenol-degrading microflora and its potential for bioremedial applications[J]. J Biochem (Tokyo), 2000,128:909-916.
[28] Yuan SY, Yu CH, Chang BV. Biodegradation of nonylphenol in river sediment. Environ Pollut, 2004,127:425-430.
[29] Tsutsumi Y, Haneda T, Nishida T. Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes[J]. Chemosphere,2001,42: 271-276.
[30] Ridgway TJ, Wiseman H. Removal of oestrogens and oestrogen mimics from the environment. Biochem Soc Trans, 1998,26:675-678.
[31] 刘营，孔繁翔，杨积晴．菌根真菌对环境污染物的降解、转化能力概述．上海环境科学，1998，17(2)：4-6．
[32] 孙雷心．革兰氏阳性多氯联苯降解菌RHODOCOCCUS ERYTHROPOLIS TA421编码2，3-二羟基联苯-1，2-加双氧酶的多基因．生物技术通报，1996，(1)：113．
[33] 岳贵春．混合发酵法降解多氯联苯的试验研究．环境科学学报，1987,(2)：207-211．
[34] 马瑞霞．微生物转化γ-666及短芽胞杆菌(Bacillus brevis)对γ-666的降解．中国环境科学，1984(3)：63-66．
[35] 刘庆余，李悦，齐英，马元中．邻苯二甲酸酯类在土柱模拟试验中的微生物降解．环境卫生工程，1997，(4)：3-6．
[36] 刘庆余，蔡晓冬，杨意东，张林原．污泥发酵处理中微生物对毒性有机物的降解环境卫生工程，1997，(2)：3-6．
[37] Spivack J, Leib TK, Lobos JH. Novel pathway for bacterial metabolism of bisphenol A. Rearrangements and stilbene cleavage in bisphenol A metabolism[J].J Biol Chem,1994,269:7323-7329.