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Methylomonassp.GYJ3菌株中经DEAE-SepharoseCL-6B阴离子交换层析和SephacrylS300凝胶层析分离纯化出甲烷加氧酶羟基化酶组分.经HPLC分析,纯度大于90%,分子量为240kD,纯化倍数为3.9,比活为225nmol环氧丙烷每分钟毫克蛋白.SDS-PAGE表明,羟基化酶由三个亚基组成,亚基分子量为56、43、27kD.ICPAES测定羟基化酶的Fe含量为2.1molFe每摩尔蛋白.HPLC法用于甲烷单加氧酶羟基化酶组分的纯化,纯化的羟基化酶组分比活为541nmol(环氧丙烷)每分钟毫克蛋白,是两步LC法纯化的羟基化酶的两倍,Fe含量为3.78molFe每摩尔蛋白.催化性质研究表明羟基化酶能够被化学还原剂还原为还原态羟基化酶,还原态的羟基化酶单独存在时表现出MMO活性,说明它是MMO活性中心,天然态的羟基化酶单独存在时无MMO活性,加入粗酶液中MMO活性明显增加,说明GYJ3菌中MMO是一个复合酶系. 相似文献
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Metylomonassp.GYJ3菌的甲烷单加氧酶(MMO)粗酶提取液经DEAE-SepharoseCL-6B阴离子交换层析、SephadexG-100凝胶过滤层析和DEAE-TSKgelHPLC分离纯化出MMO还原酶组分.经HPLC分析,纯度大于95%,纯化倍数为4.4,加入至MMO羟基化酶和调节蛋白B的体系中表现比活为228nmol环氧丙烷每分钟毫克蛋白.SDS-PAGE电泳表明还原酶由一种亚基组成,分子量42kD.ICP-AES测定还原酶的Fe含量为1.83molFe每mol蛋白.UV-Vis光谱表明还原酶除280nm蛋白质特征峰外在460nm有最大吸收峰,且A280nm/A460nm为2.50,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个FAD辅基和Fe2S2中心.在厌氧条件下,还原酶能够和NADH作用,UV-Vis光谱分析表明还原酶460nm处特征吸收峰消失,说明在MMO催化过程中还原酶接受NADH的电子.DEAE-SepharoseCL-6B阴离子交换层析分离出调节蛋白B,部分纯化的调节蛋白B的分子量大约在20kD,它能够提高MMO比活性40倍,MMO还原酶和调节蛋白B单独存在时不具有MMO 相似文献
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Meylomonas sp.GYJ3菌的甲烷单加氧酶(MMO)粗酶提取液经DEAE-Sepharose CL-6B阴离子交换层析,Sephadex G-100凝胶过滤层析和DEAE-TSKgel HPLC分离纯化出MMO还原酶组分,经HPLC分析,纯度大于95%,纯化倍数为4.4,加入至MMO羟基化酶和调节蛋白B的体系中表现比活为228nmol环氧丙烷每分钟毫克蛋白,SDS-PAGE电泳表明的酶由 相似文献
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甲烷甲基单胞菌的一个新变种 总被引:1,自引:0,他引:1
对甲烷氧化细菌761M菌株做了进一步鉴定。结果表明,该菌株为甲烷甲基单胞菌的一个新变种,命名为甲烷甲基单胞菌成都变种(Methylomonas methanica vas.chengduensis)。 相似文献
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甲烷氧化细菌Methylosinus trichosporium 3011甲醇累积条件的研究 总被引:5,自引:1,他引:5
本文考察了甲烷氧化细菌Methylosi nus trichosporium 3011的生理特性及反应条件对甲烷单加氧酶和甲醇累积的影响。M.3011菌株在4℃保存30~40天内,菌株的细胞生长量和甲烷单加氧酶活性均不受影响。在生长对数期收获细胞,其甲烷单加氧酶活力最高可达125nmol甲醇/mg(细胞干重)·min。在M.3011菌株的生长对数期后期收集细胞,将反应菌悬液浓度控制在0.15—0.3mg(细胞干重/ml,pH为6.7,反应温度为35℃,甲醇累积量可达3.1μmol甲醇/mg(细胞干重)·h。反应液的磷酸缓冲液的最适浓度为60mmol/L。 相似文献
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甲烷利用细菌HG06的筛选鉴定及其对三种有毒物质降解的研究 总被引:1,自引:0,他引:1
通过传统微生物学方法,从广西玉林市广西玉林师范学院西校区池塘里土壤中筛选到一株甲烷利用细菌HG06,能以甲烷为惟一碳源和能源的无机培养基上生长.通过形态观察及16S rRNA编码序列同源性比较分析,该菌株初步鉴定为甲基孢囊菌.同时对HG06菌株在不同温度、pH值的生长条件进行研究,初步确定了HG06的最适生长的温度为32℃,最适pH值为7.0.TCE最高的耐受浓度为30mg/L;苯胺最高的耐受浓度为1 500mg/L;苯酚最高的耐受浓度为800mg/L.该菌在环保上有一定的应用价值. 相似文献
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甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶羟基化酶组分… 总被引:1,自引:0,他引:1
从Meth ylomonas sp.GYJ3菌株中经DNEAE-SepharoseCl-6B阴离子交换层析和SephacrylS300凝胶层析分离出纯化出甲烷加氧酶羟基酶组分,经HPLC分析,纯度大于90%,分子量为240kD,纯化们数为3.9,比活为225nmol环氧丙烷每分钟毫克蛋白,SDS-PAGE表明,羟基化酶由三个亚基组成,亚基分子量为56、43、27kD.ICPAES测定羟基化酶的Fe 相似文献
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甲烷单加氧酶的催化性能和活性中心结构 总被引:3,自引:0,他引:3
甲烷单加氧酶是甲烷利用细菌代谢甲烷过程中的重要酶系,它能够催化烷烃羟基化和烯烃环氧化反应;还能催化降解氯代烃类,可用于环境中氯代烃类化合物污染的治理,是具有广泛应用前景的生物催化剂.甲烷单加氧酶是含有μ-氧桥双核铁催化活性中心的蛋白,它的研究对分子氧的活化、化学催化剂的设计具有重要意义.文章介绍了甲烷单加氧酶催化性能和机理的最新研究进展. 相似文献
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The responses of the trichloroethylene-degrading bacterium Methylocystis sp. M to six different water-pollutants, carbon starvation, and temperature-shock (heat and cold) were examined using 2-dimensional gel electrophoresis. Twenty-eight polypeptides were induced, and these stress-induced proteins were classified into three groups. Some of the chemically induced proteins were the same as those induced by carbon starvation and temperature-shock. Two of the polypeptides were induced by trichloroethylene. Trichloroethylene-stress protein synthesis required 1-2 h at a concentration of trichloroethylene that had no effect on growth. Furthermore, 25 stress-enhanced polypeptides were observed, and one of these was enhanced by trichloroethylene. Based on these results, we discuss applications of chemical-stress induction of proteins to establish effective bioremediation and bioassay by methanotrophs. 相似文献
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微囊藻毒素(microcystins, MCs)近年来由于蓝藻水华在世界范围内频发而受到广泛关注。从太湖北部蓝藻水华堆积处理池中分离出一株微囊藻毒素降解细菌SW1, 经16S rDNA序列分析鉴定为鞘氨醇单胞菌(Sphingopyxis sp.)。SW1的生长最适pH为中性(pH 6-8), 但也能生长于pH 10条件下。SW1对MCs的两种异构体MC-LR和MC-RR具有高降解活性, 并表现出一级反应动力学特征, 其降解速率常数分别为0.35/h和0.28/h。温度和pH对SW1降解活性有很强影响: 在温度为22-37℃, pH中性或弱碱性条件下(MC-LR, pH 6-9; MC-RR, pH 7-8), SW1具有高降解活性; 而在低温和强碱性条件下其降解活性受到强烈抑制。聚合酶链式反应(PCR)表明SW1及蓝藻水华堆积处理池均含有mlrA的同源基因, 表明处理池中存在MCs的生物 降解。
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生物模拟采样技术的一个典型代表是三油酸酯/半渗透膜采样器件(SPMD),是将于体内的一种典型中性脂封装在一个半渗透膜内,允许水相污染物跨膜在内侧脂相富集,原理和生物富集过程相似。本研究在实验室控制条件的流动体系中,研究了九种典型低疏水性的硝基芳烃化合物在SPMD和金鱼体内的富集动力学过程,探讨利用SPMD模拟硝基芳烃化合物在金鱼体内的富集,并和同期进行的金鱼富集实验结果比较,讨论该类污染物在金鱼体内降解的构效关系。结果表明,SPMD能够用模拟鱼从水体中亲脂性富集硝基芳烃化合物的过程,富集系数和化合物正辛醇/水分配系数相似;当SPMD和鱼暴露于同一浓度水体时,SPMD富集系数和BCF之间的差别能够表征该化合物在鱼体内的酶促降解过程。 相似文献
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Methanotrophs are a group of bacteria that use methane as sole carbon and energy source. Type I methanotrophs are gamma-proteobacterial methanotrophs using the ribulose monophosphate cycle (RuMP) cycle for methane assimilation. In order to facilitate metabolic engineering in the industrially promising Type I methanotroph Methylomicrobium buryatense 5GB1, flux analysis of cellular metabolism is needed and 13C tracer analysis is a foundational tool for such work. This biological system has a single-carbon input and a special network topology that together pose challenges to the current well-established methodology for 13C tracer analysis using a multi-carbon input such as glucose, and to date, no 13C tracer analysis of flux in a Type I methanotroph has been reported. In this study, we showed that by monitoring labeling patterns of several key intermediate metabolites in core metabolism, it is possible to quantitate the relative flux ratios for important branch points, such as the malate node. In addition, it is possible to assess the operation of the TCA cycle, which has been thought to be incomplete in Type I methanotrophs. Surprisingly, our analysis provides direct evidence of a complete, oxidative TCA cycle operating in M. buryatense 5GB1 using methane as sole carbon and energy substrate, contributing about 45% of the total flux for de novo malate production. Combined with mutant analysis, this method was able to identify fumA (METBUDRAFT_1453/MBURv2__60244) as the primary fumarase involved in the oxidative TCA cycle, among 2 predicted fumarases, supported by 13C tracer analysis on both fumA and fumC single knockouts. Interrupting the oxidative TCA cycle leads to a severe growth defect, suggesting that the oxidative TCA cycle functions to not only provide precursors for de novo biomass synthesis, but also to provide reducing power to the system. This information provides new opportunities for metabolic engineering of M. buryatense for the production of industrially relevant products. 相似文献
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The rate and products of trichloroethylene (TCE) oxidation by Methylomicrobium album BG8 expressing membrane-associated methane monooxygenase (pMMO) were determined using 14C radiotracer techniques. [(14)C]TCE was degraded at a rate of 1.24 nmol (min mg protein)(-1) with the initial production of glyoxylate and then formate. Radiolabeled CO(2) was also found after incubating M. album BG8 for 5 h with [(14)C]TCE. Experiments with purified pMMO from Methylococcus capsulatus Bath showed that TCE could be mineralized to CO(2) by pMMO. Oxygen uptake studies verified that M. album BG8 could oxidize glyoxylate and that pMMO was responsible for the oxidation based on acetylene inactivation studies. Here we propose a pathway of TCE oxidation by pMMO-expressing cells in which TCE is first converted to TCE-epoxide. The epoxide then spontaneously undergoes HCl elimination to form glyoxylate which can be further oxidized by pMMO to formate and CO(2). 相似文献
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Phthalate esters (PEs) are endocrine‐disrupting pollutants that are ubiquitous in the environment and can be degraded by microorganisms. In this study, we investigated the kinetics and pathway of biodegradation of di‐n‐butyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) by cyanobacteria Anabaena flos‐aquae G. S. West (strain 4054) and two strains of Microcystis aeruginosa (Kütz.) Kütz. (strain 2396 and strain SM). Gas chromatography/mass spectroscopy (GC/MS) and a deuterium‐labeled compound were used to analyze the degrading intermediates. The findings revealed that all three organisms were capable of metabolizing PE, and that among these organisms, A. flos‐aquae achieved the highest degradation. Additionally, the biodegradation of DBP, DEP, and DMP followed first‐order kinetics. Moreover, the results of the enzymatic study suggested that PE was degraded through transesterification on the side chains rather than deesterification. Finally, experiments using deuterium‐labeled DBP showed that there were two degradation pathways: C16→ C14→ C12→ C10→ C8 and C16→ C15→ C13→ C11→ C9. Based on our results, the biodegradation pathway of PE for cyanobacteria was suggested. 相似文献
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Bacterial growth rates and the kinetics of detoxication have been followed during the course of breakdown of 2:4-dichlorophenoxyacetic acid in a liquid mineral salt medium by cultures of Bacterium globiforme isolated from garden soil. It was found that toxicity persisted apparently unchanged long after bacterial growth had ceased, and finally disappeared suddenly after a lag period comparable in duration with those obtained in soil perfusion experiments. These results can best be explained by the production of highly phytotoxic intermediates in the first stages of 2:4-D breakdown. Preliminary experiments involving paper partition chromatography indicate that there may be at least two such intermediates. 相似文献
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牧效黄杆菌对蒽菲芘的降解性能研究 总被引:10,自引:0,他引:10
采用定时、定量、逐步提高驯化所用碳源物浓度的方法,以萘为唯一碳源驯化长期被焦化废水污染的泥土浸出液,7周后,平板划线分离出两株黄杆菌FCN1及FCN2。并对这两株菌降解多环芳烃的特性及无机离子对反应的刺激作用进行了研究。结果表明,FCN1及FCN2能降解转化蒽、菲、芘。加入FCN1,反应10h后,蒽、菲、芘去除率分别为84%、69%、80%,而加入FCN2,各物质的去除率分别为76%、40%、71%。反应进行106h,FCN1对蒽、菲、芘所产生的总有机碳(TOC)的去除率分别为70%、54%、69%,而FCN2对相应物的TOC去除率分别为63%、50%、46%。Fe^3 、Mg^2 的加入对FCN1降解多环芳烃有促进作用。 相似文献