汽油添加剂甲基叔丁基醚(MTBE)污染的植物修复

甲基叔丁基醚(MTBE)是北美燃料市场最常用的汽油添加剂。由于其在土壤中的不吸附性和极高的水溶性,MTBE已成为一种蔓延性的地下水污染物。本实验用一自行设计的植物反应器来观察和测定在不同温度条件下柳树(Salix alba)对MTBE污染水溶液的修复潜力。长出新根须和嫩叶的柳树枝条在一容积500mL的锥型瓶中生长12d(其中MTBE溶液450mL)来观察MTBE对柳树生长的影响,同时测定柳树对MTBE的吸收和降解。MTBE及其主要降解产物叔丁基醇(TBA)用气相色谱来检测。本实验结果表明在为期12d的时间内,水溶液中24．84～53．27％MTBE可以通过柳树的蒸腾作用去除。同时在15℃～25℃的温度范围内,：MTBE的去除率(％)和柳树的蒸腾量(g)之间存在着明显的线性关系。由于没有发现TBA和其它可能的降解产物,植物挥发是MTBE植物修复技术中主要的作用机理。尽管植物在MTBE污染的修复过程中只是起着中间传输媒介的作用,大量的MTBE通过植物的蒸腾作用以气态的形式释放到大气中,但由于MTBE在气态下的光氧化非常快,MTBE不会造成空气污染。我们认为植物修复技术对MTBE污染的土壤和地下水仍不失为一个有效的修复手段。     

云芝NADPH-细胞色素P450还原酶在大肠杆菌中表达及酶学特性分析

云芝Trametes versicolor具有很强的环境有机污染物降解能力,其烟酰胺腺嘌呤二核苷酸-细胞色素P450还原酶(NADPH-cytochrome P450 reductase,CPR)为细胞色素P450酶(Cytochrome P450s,CYPs)提供电子,参与有机污染物的降解过程。序列分析显示,云芝基因组拥有1个潜在CPR序列和多个潜在CYP序列。为深入研究云芝CPR参与细胞降解有机污染物的分子机制,实验进行了云芝CPR在大肠杆菌中异源表达和酶学特性分析。结果表明,经IPTG诱导后,去除预测的N端膜锚定区域(氨基酸残基1–24)的CPR蛋白(CPRΔ24)可在重组菌中实现可溶性表达,且表达蛋白的分子量与理论值(78 kDa)一致。镍离子亲和层析和分子筛层析纯化后测得其比活性为5.82 U/mg。酶学性质分析显示,重组CPRΔ24的最适温度和pH分别为35℃和8.0,并对一些金属离子及有机溶剂具有不同程度的耐受性。酶在35℃、pH 8.0反应条件下对NADPH的动力学参数K_m和k_(cat)分别为19.7μmol/L、3.31/s;对底物细胞色素c的动力学参数K_m和k_(cat)分别为25.9μmol/L、10.2/s。以上研究为探究云芝CPR在环境有机污染物降解途径中的功能机制奠定基础。     

甲基叔丁基醚微生物降解研究进展*

汽油添加剂甲基叔丁基醚(Methyl tert-Butyl Ether,MTBE)的水体污染问题近年引起广泛关注,因而微生物降解MTBE的研究也渐成热点。对MTBE的微生物降解研究现状进行简要综述,总结好氧条件下降解菌对MTBE的降解情况,以关键中间代谢物-叔丁基醇(tert-butyl alcohol,TBA)为分界点探讨微生物降解MTBE的两步可能途径,浅论MTBE微生物降解的影响因素。     

产绿原酸内生菌细胞色素P450基因的克隆和功能研究

以一株产绿原酸的内生枯草芽孢杆菌为基础,对其绿原酸途径的关键酶基因进行克隆和功能研究。克隆该内生菌的细胞色素P450基因并进行原核表达,对重组蛋白进行肉桂酸羟基化酶(C4H)酶活测定。结果显示,从内生菌中克隆了4个细胞色素P450酶系基因并进行了原核表达,其中P-4重组蛋白具有C4H活性,产生的香豆酸与LC-MS检测的结果一致,该酶的最适温度范围较宽,产物香豆酸对该酶有抑制作用。该内生菌可能利用其细胞色素P450酶系作为C4H的同工酶从而将产物导入绿原酸合成途径。     

植物细胞色素P450

对植物细胞色素P450(CYP450)基因的分离,植物CYP450在苯丙烷类物质、芥子油苷及IAA和萜类等物质的生物合成中的功能,以及对天然生物合成与人工合成物质的解毒功能等研究进展作了简要的综述。指出分离植物细胞色素P450基因,并对其生物学功能进行分析以及植物细胞色素P450降解除草剂的机制及其在环境生物修复等方面的应用是今后一段时间内植物CYP450领域的研究热点。     

甲基叔丁基醚微生物降解研究进展

汽油添加剂甲基叔丁基醚（Methyl tert—Butyl Ether,MTBE）的水体污染问题近年引起广泛关注,因而微生物降解MTBE的研究也渐成热点。对MTBE的微生物降解研究现状进行简要综述,总结好氧条件下降解菌对MTBE的降解情况,以关键中间代谢物-叔丁基醇（tert-butyl alcohol,TBA）为分界点探讨微生物降解MTBE的两步可能途径,浅论MTBE微生物降解的影响因素。     

北柴胡细胞色素P450酶基因的克隆及其植物过量表达载体的构建

目的:克隆北柴胡中可能参与柴胡皂苷生物合成的细胞色素P450酶基因,构建其过量表达载体,为通过转基因验证其功能奠定基础。方法:在454高通量测序获得5'和3'端部分cDNA序列的基础上,利用LD-PCR方法获得全长cDNA,根据全长cDNA序列设计含有酶切位点的PCR引物,利用高保真酶,以RNA反转录产物为模板PCR扩增细胞色素P450酶基因的开放读框,扩增产物与pEASY-T1 Simple载体连接,转化大肠杆菌DH5α;重组质粒pT1-P450经菌液PCR和酶切方法验证后测序,采用NCBI在线Blastx、DNAman和MEGA4软件对序列进行生物信息学分析,随后将pT1-P450的酶切产物插入双元载体pCAMBIA-SUPER 1300,菌液PCR和酶切验证重组质粒p1300-P450。结果:扩增到了北柴胡细胞色素P450酶基因BcCYP87E,构建了这一基因的过量表达载体。结论:细胞色素P450酶基因的克隆和转基因载体的构建,为后续开展转基因研究,验证其生物功能奠定了基础。     

细胞色素P450酶系与除草剂代谢

细胞色素P450是广泛存在于动物、植物和微生物体内的一类具有混合功能的血红素氧化酶系。它不但能够催化苯丙烷类、萜类化合物和脂肪酸等内源性物质的生物合成 ,而且参与许多外源性物质包括除草剂等的生物氧化。综述了代谢除草剂的细菌、哺乳动物和植物细胞色素P450酶系 ,概述了细胞色素P450酶系参与除草剂代谢的作用方式 :脱烷基化作用、环甲基化羟基化作用和芳环的羟基化作用等。这些细胞色素P450酶系在培育除草剂抗性作物、生物安全和生物修复方面表现出了巨大的潜能     

飞蝗解毒酶系活力测定方法

飞蝗Locusta migratoria是重要的农业害虫,代谢抗性是飞蝗主要的农药抗性机制之一。与代谢抗性相关的解毒酶系主要有:非专一性酯酶系(Non-specficesterases,ESTs)、谷胱甘肽S-转移酶系(Glutathione S-transferases,GSTs)和细胞色素P450单加氧酶系(Cytochrome P450 monooxygenases,P450s),解毒酶系活力的测定是研究飞蝗农药代谢机制的重要途径。本文详细介绍了飞蝗解毒酶系的测定方法,为蝗虫及其他昆虫解毒酶系的测定提供参考。     

二元酸发酵过程中流加H2O2对细胞色素P450酶及产物生成的影响

在二元酸发酵过程中流加H2O2对热带假丝酵母发酵生产二元酸有明显的促进作用,2mmol/L的H2O2对产酸的促进作用最为明显,比对照提高了26%。对细胞色素P450酶的分析表明,流加H2O2对细胞色素P450酶的活性有明显的促进作用,并且细胞色素P450酶的活性跟产酸成正相关。此外,还进一步分析了流加H2O2对产酸的促进机理。     

Field note phytovolatilization of oxygenated gasoline-impacted groundwater at an underground storage tank site via conifers

A stand of five conifers (Pinus sp.) bordering a gasoline service station was studied to estimate the methyl tert-butyl ether (MTBE) emission rate from gasoline-impacted groundwater. Groundwater was impacted with gasoline oxygenates MTBE and tert-butyl alcohol (TBA) at combined concentrations exceeding 200,000 microg/L. Condensate from trees was collected in sealed environmental chambers and analyzed. Concentrations of MTBE in condensate ranged from 0.51 to 460 microg/L; TBA ranged from 12 to 4100 microg/L (n=19). Transpirate concentrations were derived from MTBE air-liquid partitioning data exhibited in controls spiked with known concentrations of analyte. Tree emissions were estimated by multiplying average transpirate concentrations by transpiration rates derived from evapotranspiration data. Stand evapotranspiration was calculated using meteorological data from the California Irrigation Management Information System (CIMIS) applied in the Standardized Reference Evapotranspiration Equation.     

Non-significance of rhizosphere degradation during phytoremediation of MTBE

Methyl tertiary butyl ether (MTBE) is a gasoline additive associated with groundwater pollution at gas station sites. Previous research on poplar trees in hydroponic systems suggests that phytovolatilization is an effective mechanism for phytoremediation of MTBE (Rubin and Ramaswami, 2001), but the potential for microbial degradation of MTBE in the rhizosphere of trees had not been assessed. MTBE had largely been considered recalcitrant to microbial processes, but recent fieldwork suggests rapid biodegradation may occur in certain cases. This paper investigates the potential for rhizosphere degradation of MTBE at time frames relevant for phytoremediation. Three experiments were conducted at different levels of aggregation to examine possible degradation of MTBE by rhizosphere microorganisms that had been acclimated to low levels of MTBE for 6 weeks. MTBE soil die-away studies, conducted with both poplar trees and fescue grass, found no significant differences between MTBE concentration in vegetated and unvegetated soils over a two-week attenuation period. Closed chamber tests comparing hydroponic and rhizospheric poplar tree systems also showed essentially complete recovery of MTBE mass in both systems, suggesting an absence of degradation. Finally, rhizosphere microbes tested in aerated bioreactors were found to be thriving and metabolizing root materials, but did not show measurable degradation of MTBE. In all tests, the MTBE degradation product, Tert Butyl Alcohol (TBA), was not detected. The insignificance of MTBE degradation by rhizosphere microorganisms suggests that plant processes be the primary focus of further research on MTBE phytoremediation.     

Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6

Methyl tert-butyl ether (MTBE) is widely used as gasoline oxygenate and octane number enhancer for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health are of major public concern. However, information on the metabolism of MTBE in human tissues is scarce. The present study demonstrates that human cytochrome P450 2A6 is able to metabolize MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and marker for exposure to MTBE. As CYP2A6 is known to be constitutively expressed in human livers, we infer that it may play a significant role in metabolism of gasoline ethers in liver tissue.     

Rate of Natural Attenuation of Tert-Butyl Alcohol at a Chemical Plant

Tert-butyl alcohol (TBA) may be present in groundwater as an original component of leaked gasoline, or as a degradation product of methyl tert-butyl ether (MTBE). Evidence for natural attenuation of TBA in groundwater is presented from a chemical plant in Pasadena, Texas. Shallow groundwater in several areas of the plant has been affected by historic leaks and spills of TBA. A decade of regular groundwater monitoring of one groundwater plume, consisting primarily of TBA, shows generally declining concentrations and a plume area that is shrinking. Natural attenuation mechanisms are limiting the advective transport of TBA. The principal mechanism of attenuation in this case is probably biodegradation as the other physical components of natural attenuation (dilution, dispersion, diffusion, adsorption, chemical reactions, and volatilization) cannot explain the behavior of the plume over time. Biodegradation was also indicated by the enrichment of stable carbon isotope composition (¹³C/¹²C) of TBA along the flow path. Preliminary dissolved gas and electron acceptor analyses indicate the groundwater is at least under sulfate reducing condition in the core of the plume and the process responsible for biodegradation of TBA may include fermentation under aerobic (plume fringes) and possible anaerobic conditions. This case history demonstrates that natural attenuation of TBA is important, and can be used as a groundwater management tool at this site.     

The Comparative and Combined Inhibitory Effects of MTBE and TBA on the Activities of Soil Exoenzymes

Methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) are major soil contaminants, and they have been actively investigated for their toxic effects on living organisms in soil ecosystems. Although previous studies have been used as tools to evaluate the health of soil, they have been limited in scope and ability to analyze the overall microbial activity. In the present study, the effects of MTBE and TBA on the activity of soil exoenzymes including urease, acid phosphatase, arylsulfatase, β-glucosidase, dehydrogenase, and fluorescein diacetate hydrolase, which are involved in nutrient cycles and overall microbial activities, were investigated. Soil samples were treated with 0–2% of MTBE and TBA solutions, and the comparative effects and combined effects on quantity of active soil exoenzymes were determined. The activity of six exoenzymes exposed solely to MTBE and TBA did not significantly change with dose concentration or exposure time, but did show significant changes when exposed to high concentrations of MTBE and TBA combined, with dehydrogenase being the most affected. Therefore, we proposed dehydrogenase as a potential biomarker to assess the risk of co-contamination of MTBE and TBA.     

Biotreatment of groundwater contaminated with MTBE: interaction of common environmental co-contaminants

Contamination of groundwater with the gasoline additive methyl tert-butyl ether (MTBE) is often accompanied by many aromatic components such as benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene (BTEX). In this study, a laboratory-scale biotrickling filter for groundwater treatment inoculated with a microbial consortium degrading MTBE was studied. Individual or mixtures of BTEX compounds were transiently loaded in combination with MTBE. The results indicated that single BTEX compound or BTEX mixtures inhibited MTBE degradation to varying degrees, but none of them completely repressed the metabolic degradation in the biotrickling filter. Tert-butyl alcohol (TBA), a frequent co-contaminant of MTBE had no inhibitory effect on MTBE degradation. The bacterial consortium was stable and showed promising capabilities to remove TBA, ethylbenzene and toluene, and partially degraded benzene and xylenes without significant lag time. The study suggests that it is feasible to deploy a mixed bacterial consortia to degrade MTBE, BTEX and TBA at the same time.     

石油添加剂甲基叔丁基醚的污染治理技术研究进展

甲基叔丁基醚是一种石油添加剂,广泛应用于中高档汽油中.其对环境造成的污染和对人体健康造成的危害已日益引起人们的高度重视.本文综述了近年来国外有关甲基叔丁基醚的污染治理技术研究进展,主要是高级氧化技术和微生物降解.已用于处理甲基叔丁基醚的高级氧化技术包括;多相光催化氧化法、紫外光加强的过氧化氢氧化法、臭氧法与臭氧-过氧化氢联合氧化法、超声法与超声-臭氧联合氧化法、芬顿法与光芬顿氧化法、氧气的还原性活化和水的γ射线辐射.微生物降解主要涉及有氧代谢和无氧代谢两大途径.     

Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria.

Several propane-oxidizing bacteria were tested for their ability to degrade gasoline oxygenates, including methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). Both a laboratory strain and natural isolates were able to degrade each compound after growth on propane. When propane-grown strain ENV425 was incubated with 20 mg of uniformly labeled [14C]MTBE per liter, the strain converted > 60% of the added MTBE to 14CO2 in < 30 h. The initial oxidation of MTBE and ETBE resulted in the production of nearly stoichiometric amounts of tert-butyl alcohol (TBA), while the initial oxidation of TAME resulted in the production of tert-amyl alcohol. The methoxy methyl group of MTBE was oxidized to formaldehyde and ultimately to CO2. TBA was further oxidized to 2-methyl-2-hydroxy-1-propanol and then 2-hydroxy isobutyric acid; however, neither of these degradation products was an effective growth substrate for the propane oxidizers. Analysis of cell extracts of ENV425 and experiments with enzyme inhibitors implicated a soluble P-450 enzyme in the oxidation of both MTBE and TBA. MTBE was oxidized to TBA by camphor-grown Pseudomonas putida CAM, which produces the well-characterized P-450cam, but not by Rhodococcus rhodochrous 116, which produces two P-450 enzymes. Rates of MTBE degradation by propane-oxidizing strains ranged from 3.9 to 9.2 nmol/min/mg of cell protein at 28 degrees C, whereas TBA was oxidized at a rate of only 1.8 to 2.4 nmol/min/mg of cell protein at the same temperature.     

Involvement of a Novel Enzyme, MdpA, in Methyl tert-Butyl Ether Degradation in Methylibium petroleiphilum PM1

Methylibium petroleiphilum PM1 is a well-characterized environmental strain capable of complete metabolism of the fuel oxygenate methyl tert-butyl ether (MTBE). Using a molecular genetic system which we established to study MTBE metabolism by PM1, we demonstrated that the enzyme MdpA is involved in MTBE removal, based on insertional inactivation and complementation studies. MdpA is constitutively expressed at low levels but is strongly induced by MTBE. MdpA is also involved in the regulation of tert-butyl alcohol (TBA) removal under certain conditions but is not directly responsible for TBA degradation. Phylogenetic comparison of MdpA to related enzymes indicates close homology to the short-chain hydrolyzing alkane hydroxylases (AH1), a group that appears to be a distinct subfamily of the AHs. The unique, substrate-size-determining residue Thr⁵⁹ distinguishes MdpA from the AH1 subfamily as well as from AlkB enzymes linked to MTBE degradation in Mycobacterium austroafricanum.