专一营养与兼性甲烷氧化菌降解氯代烃的研究现状、动力学分析及展望

生物修复技术被认为是氯代烃类污染物处理处置的最有效途径之一,而甲烷氧化菌在该领域表现出较大的应用潜力。近期研究发现,突破了仅能利用单碳化合物的局限,兼性甲烷氧化菌能够利用多种底物降解氯代烃,这一独特的新陈代谢特性,使其在污染物生物处置领域逐渐受到关注。结合本课题组研究成果,对甲烷氧化菌降解氯代烃进行了全面总结,主要包括:分析了不同菌株(纯菌株和混合菌株)对不同氯代烃的降解效果;比较了不同类型甲烷单加氧酶在不同底物体系中的活性表达和催化特性;总结了模型菌株甲基弯菌Methylosinus trichosporium OB3b降解氯代烃的动力学特性;概述了兼性甲烷氧化菌株降解氯代烃的特性及其应用潜力;最后讨论了甲烷氧化菌降解氯代烃存在的问题及未来发展方向。     

生物分子机器——甲烷单加氧酶的研究进展

甲烷氧化菌中的甲烷单加氧酶能够在生理条件下选择性地以甲烷和氧气为底物生成甲醇,麻省理工学院的Lippard教授称它为"神奇的生物分子机器"。本文重点对生物分子机器甲烷单加氧酶的结构、编码基因及调控机制、催化反应机理等进行了综述,此外也简要介绍了甲烷单加氧酶的产生菌甲烷氧化菌的研究历史及分类。生物分子机器甲烷单加氧酶可催化甲烷氧化成甲醇,不仅为甲醇的生产提供了一种新颖的生产方法,而且对生物分子机器的设计也有借鉴意义。     

外源电子给体对甲烷单加氧酶催化丙烯环氧化反应活性的影响

以丙烯氧化反应为指标研究了不同外源电子给体对甲烷细菌(Methylomonas sp．GYJ30)休止细胞催化活性的影响。结果表明甲烷、甲醇、甲醛和甲酸盐作为电子给体加入反应中,将甲烷单加氧酶催化丙烯环氧化反应活性分别提高5．3,12．7,10和12．4倍。以甲烷和甲醛作为外源电子给体时提高初始浓度对甲烷单加氧酶具有抑制作用;而以甲醇和甲酸盐作为电子给体时提高初始浓度对甲烷单加氧酶催化活性无明显抑制作用。研究了甲醇作为电子给体时它的代谢、环氧丙烷的积累以及催化反应活性与反应时间的关系     

可降解TCE的甲烷氧化菌16S rDNA与pmoCAB基因簇序列分析

甲烷氧化菌可高效催化甲烷和多种氯代烃降解,开展甲烷单加氧酶的基因簇序列分析研究有助于深入了解催化机理,并提升其在污染物生物降解领域中的应用价值。以甲烷为唯一碳源富集分离甲烷氧化菌,取5种氯代烃作为共代谢基质考察其降解情况,利用MEGE5.05软件构建基于16S r DNA的系统发育树对所选菌株进行初步鉴定,用半巢式PCR法分段扩增菌株的颗粒性甲烷单加氧酶(p MMO)基因簇并进行T-A克隆测序,通过Ex PASy计算p MMO三个亚基的理论分子量。筛选到了一株甲基孢囊菌Methylocystis sp.JTC3,在三氯乙烯(TCE)初始浓度为15.64μmol/L条件下,反应5 d后降解率可达93.79%。经扩增、测序、拼接得到了3 227 bp的pmo CAB基因簇序列,包括771 bp的pmo C基因、759 bp的pmo A基因、1 260 bp的pmo B基因和2个非编码中间序列,所对应γ、β、α亚基理论分子量分别为29.1 k Da、28.6 k Da和45.6 k Da。通过Blast比对发现Methylocystis sp.JTC3的pmo CAB基因簇序列与Methylocystis sp.strain M的pmo CAB一致性较高,其中pmo A的序列相对保守。JTC3菌株可高效降解TCE,对pmo CAB基因簇序列的详细分析可为p MMO活性中心特征、氯代烃类底物选择性等方面的深入研究提供信息数据基础。     

生物质合成气催化制取甲烷研究进展

天然气的供需矛盾促使人们去寻找新的天然气资源,其中利用生物质热化学催化制取生物质基天然气的技术受到了全世界的广泛关注。而生物质合成气催化制取甲烷是该工艺流程的核心步骤之一。分别从甲烷化反应器和甲烷化催化剂两个方面阐述了国际上生物质合成气催化制取甲烷的研究现状,并综述了关于甲烷化催化剂积碳现象的研究进展。同时分析了目前生物质合成气催化制取甲烷面临的主要问题,并指明了未来的发展方向。     

甲烷氧化菌Methylomonas sp.GYJ3中甲烷单加氧酶基因与16S rRNA序列分析

甲烷氧化细菌中的关键酶系甲烷单加氧酶是一个含双核铁的多组份氧化酶,常温、常压下能够催化甲烷转化为甲醇。对甲烷氧化细菌Methylomonas sp.GYJ3中溶解性甲烷单加氧酶基因和16SrDNA进行了测序与分析。利用已知相关基因数据库信息,设计了PCR引物和测序引物,获得了满意的测序结果。全长的溶解性甲烷单加氧酶基因为5690bp,部分16S rDNA的序列长度为1280bp。与已发表的甲烷氧化细菌中甲烷单加氧酶进行了比较,结果表明MMOX组份中氨基酸序列的同一性为78%到99%,基因序列的同一性为71%到97%,6个组份中orfY片段的同一性相对较低。MMOX氨基酸序列的多序列联配表明,MMOX序列具有高度保守性,特别是在双核铁中心区域。16S rDNA进化分析显示Methylomonas sp.GYJ3与γ蛋白细菌是相关联的,基于MMOX氨基酸序列的进化分析证明,与Methylomonas sp.GYJ3最近似的菌株是Ⅰ型甲烷氧化细菌Methylomonas sp.KSWⅢ。综合分析表明,菌株GYJ3属于Ⅰ型甲烷氧化细菌Methylomonas sp.属。这个结果为Ⅰ型甲烷氧化细菌也能表达溶解性甲烷单加氧酶提供了新的证据。羟基化酶的理论等电点是6.28,理论分子量为248874.41Da。     

甲烷氧化菌及甲烷单加氧酶的研究进展

甲烷氧化菌是以甲烷作为唯一碳源和能源进行同化和异化代谢的微生物,其关键酶之一是甲烷单加氧酶(MMOs),可以在氧气的作用下催化甲烷等低碳烷烃或烯烃羟基化或环氧化,甲烷氧化菌在自然界碳循环和工业生物技术中具有重要的应用价值.因此,近20年来对于甲烷氧化菌和MMOs的研究一直倍受生物学家的关注.以下从现代生物技术的角度,对近年来国内外在甲烷氧化菌的分类与分布,MMOs的结构与功能、甲烷氧化菌与MMOs的基因工程等方面取得的研究成果进行了总结,全面综述了甲烷氧化菌及MMOs的应用基础研究现状,并对其今后的研究和应用方向提出了展望.     

甲烷单加氧酶的研究进展

甲烷氧化细菌在转化甲烷制造新型燃料、单细胞蛋白和新功能酶生产、污水处理等方面有着潜在的应用前景,因此,甲烷单加氧酶作为其代谢过程中重要的酶系也受到人们的广泛关注。我们简要综述了近年来对甲烷单加氧酶的性质、结构、催化机理等方面的研究,特别是对颗粒性甲皖单加氧酶的相关性质进行了详细的阐述。     

甲烷氧化过程中铜的作用研究进展

甲烷生物氧化在全球甲烷平衡和温室效应控制中扮演着重要的角色,而铜是甲烷生物氧化过程中的重要影响因子.一方面,铜是调控不同类型甲烷单加氧酶表达的主要影响因子,是组成颗粒性甲烷单加氧酶的必需金属元素;另一方面,在自然环境体系中,铜含量及其形态的变化对甲烷氧化菌的分布、代谢甲烷和非甲烷类有机化合物的能力以及甲烷氧化菌的特异性铜捕获系统也会产生较大影响.准确把握铜在甲烷生物氧化过程中发挥的作用将有助于全面了解甲烷生物氧化过程,进而更好地指导甲烷氧化微生物在温室气体减排及非甲烷有机物污染修复中的应用.本文主要从铜的角度,概述了铜对甲烷氧化菌的分布和活性的影响,介绍了铜在调控甲烷单加氧酶的表达和活性以及调节甲烷氧化菌铜捕获系统方面的作用,并展望了其研究方向.     

一个Ⅱ型甲烷氧化菌中甲烷单加氧酶羟基化酶的分离纯化和理化性质

甲烷氧化细菌能够催化甲烷和一系列小分子烃类化合物的羟基化反应,对控制全球变暖起着重要作用,在工业催化和生物除污中具有非凡的潜能。应用层析方法纯化了Ⅱ型甲烷氧化细菌MethylosinustrichosporiumIMV3011中甲烷单加氧酶的羟基化酶,并对其进行了表征。凝胶过滤法测定了该酶分子量为201.3kD;SDS-PAGE表明羟基化酶含有三个亚基(αβγ),分子量分别为58kD、36kD和23kD,比较两种方法证明该羟基化酶是一个同型二聚体构型(αβγ)2,总分子量为234kD。薄层等电聚焦测定该酶的等电点为5.2。酶的比活力为603.6nmol/(min.mg),活力回收为34.3%。HPLC法测定该酶的纯度在95%以上。原子吸收光谱显示每分子羟基化酶中含有3.02个Fe原子。羟基化酶的稳定性pH值为6.2～7.5,稳定性温度为低于35℃。菌株IMV3011的细胞表观构型呈现了长型、稍微弯曲的杆状形态。     

Methanotrophs: Multifunctional bacteria with promising applications in environmental bioengineering

Methane is an important greenhouse gas which is produced from many natural and anthropogenic sources. It plays an important role in overall global warming. A significant amount of methane is removed through microbiological oxidation by methanotrophic bacteria, which are widespread in the environment, including many extreme environments. The key enzyme of these microorganisms, methane monooxygenase (MMO), especially the soluble MMO, is remarkable in its broad substrate specificity. This unique capability, i.e. catalyzing reactions of environmental importance, has attracted great attention for applied microbiologists and biochemical engineers. In this review, recent advances in the application of methanotrophs to environmental bioengineering are summarized, including biodiversity, catalytic properties, and cultivation, etc. We have focused on two aspects of the application and potential value of methanotrophs in environmental bioengineering, namely methane removal and biodegradation of toxic compounds. The removal of methane produced from landfills has been widely studied, and much of this work can be used as a source of reference for coal mine gas removal. Many bioreactors using methanotrophs in bioremediation have been developed in recent years. These reactors have two forms of configuration, single-stage and multi-stage. Current limitations which may affect the engineering applications of methanotrophs are discussed, such as the lack of suitable methanotrophic isolate, gas transfer limitation, competitive inhibition of MMO, regeneration of reducing equivalents for MMO and product toxicity.     

Epoxypropane biosynthesis by whole cell suspension of methanol-growth Methylosinus trichosporium IMV 3011

Methanotrophs containing methane monooxygenase (MMO) can catalyze the epoxidation of propene to epoxypropane. Methane cannot support dense biomass growth due to its low aqueous solubility. Low growth rate is important limiting factor for the application of methanotrophs. Methanol can act as growth substrate, but direct addition of methanol is toxic to most methanotrophs. The MMO activity during growth on methanol is also uncertain. In this paper, methanol-adapted Methylosinus trichosporium IMV 3011 was successfully cultivated at high cell densities using methanol as sole carbon source. A biomass density of 1.68 g dry weight cell l^?1 was achieved and cells contained almost 80% of the MMO activity measured for cells grown with methane. It has been found that methanol can also act as the electron-donating substrate to regenerate the NADH and drive epoxypropane synthesis. The effect of methanol supply on the epoxidation capacity of Methylosinus trichosporium IMV3011 was studied in batch reactor. 0.016% methanol concentration was found to give the highest propene epoxidation capacity.     

Kinetics and activation thermodynamics of methane monooxygenase compound Q formation and reaction with substrates

The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of methane by Q exceeds that of many other hydrocarbons with weaker C-H bonds.     

Heat-tolerant methanotrophic bacteria from the hot water effluent of a natural gas field.

Methanotrophic bacteria were isolated from a natural environment potentially favorable to heat-tolerant methanotrophs. An improved colony plate assay was developed and used to identify putative methanotrophic colonies with high confidence. Fourteen new isolates were purified and partially characterized. These new isolates exhibit a DNA sequence homology of up to 97% with the conserved regions in the mmoX and mmoC genes of the soluble methane monooxygenase (MMO)-coding gene cluster of Methylococcus capsulatus Bath. The copper regulation of soluble MMO expression in the same isolates, however, differs from that of M. capsulatus Bath, as the new isolates can tolerate up to 0.8 microM copper without loss of MMO activity while a drastic reduction of MMO activity occurs already at 0.1 microM copper in M. capsulatus Bath. The isolates can be cultivated and utilized at elevated temperatures, and their copper- and heat-tolerant MMO activity makes these bacteria ideal candidates for future biotechnological use.     

Mechanism of methane monooxygenase – a structural and quantum chemical perspective

 The diiron site of methane monooxygenase (MMO) has the unique ability to activate methane. Structural studies of the MMO diiron site have revealed a limited number of coordination sites for dioxygen and dioxygen derived species. Using quantum mechanical studies of the MMO reaction, several possible reaction paths have been investigated. Energetically feasible geometries have been obtained for the different reaction steps, where the substrate activation is best described by an almost pure hydrogen abstraction step, followed by the formation of a metal-carbon bond. Received: 14 October 1997 / Accepted: 20 January 1998     

O-dealkylation: A newly discovered class of reactions catalysed by the soluble mono-oxygenase of the methanotroph Methylosinus trichosporium 0B3b

Summary Cell-free extracts of Methylosinus trichosporium 0B3b (MT 0B3b) containing the soluble, broad specificity methane mono-oxygenase (MMO) have been shown to catalyse yet another type of reaction : O-dealkylation. Several 4-substituted anisoles were investigated as substrates, all showed O-demethylation to varying extents by cell-free extracts of the bacterium. This catalytic ability is common to organisms grown on either methane or methanol as sole carbon source, although the rates of biotransformation are lower for the latter. O-demethylation of anisole itself was inhibited (> 99%) by ethyne, a known MMO inhibitor, strongly indicating that the MMO is the enzyme responsible for this catalysis.     

The soluble methane monooxygenase gene cluster of the trichloroethylene-degrading methanotroph Methylocystis sp. strain M.

In methanotrophic bacteria, methane is oxidized to methanol by the enzyme methane monooxygenase (MMO). The soluble MMO enzyme complex from Methylocystis sp. strain M also oxidizes a wide range of aliphatic and aromatic compounds, including trichloroethylene. In this study, heterologous DNA probes from the type II methanotroph Methylosinus trichosporium OB3b were used to isolate souble MMO (sMMO) genes from the type II methanotroph Methylocystis sp. strain M. sMMO genes from strain M are clustered on the chromosome and show a high degree of identity with the corresponding genes from Methylosinus trichosporium OB3b. Sequencing and phylogenetic analysis of the 16S rRNA gene from Methylocystis sp. strain M have confirmed that it is most closely related to the type II methanotroph Methylocystis parvus OBBP, which, unlike Methylocystis sp. strain M, does not possess an sMMO. A similar phylogenetic analysis using the pmoA gene, which encodes the 27-kDa polypeptide of the particulate MMO, also places Methylocystis sp. strain M firmly in the genus Methylocystis. This is the first report of isolation and characterization of methane oxidation genes from methanotrophs of the genus Methylocystis.     

Intracytoplasmic membranes in oxygen-limited chemostat cultures of Methylosinus trichosporium OB3b: Biocatalytic implications of physiologically balanced growth

Summary The continuous culture growth conditions for induction of intracytoplasmic membranes in Methylosinus trichosporium OB3b are described. During oxygen-limited, nitrate-excess chemostat culture, organisms have an extensive intracytoplasmic membrane system and particulate, cell-free methane mono-oxygenase (MMO). Under methane limitation fewer intracytoplasmic membranes are seen, while under all other conditions tested, membranes are absent and cell-free MMO is entirely soluble. These findings may be important in relation to the development of oxidative biotransformation processes using this bacterium.     

The methane monooxygenase gene cluster of Methylosinus trichosporium: cloning and sequencing of the mmoC gene

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. The soluble MMO enzyme complex from Methylosinus trichosporium also oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study we have used heterologous DNA probes from the type X methanotroph Methylococcus capsulatus (Bath) to isolate mmo genes from the type II methanotroph M. trichosporium. We report here that the gene encoding the reductase component, Protein C of MMO, lies adjacent to the genes encoding the other components of soluble MMO in M. trichosporium but is separated by an open reading frame of unknown function, orfY. The complete nucleotide sequence of these genes is presented. Sequence analysis of mmoC indicates that the N-terminus of Protein C has significant homology with 2Fe2S ferredoxins from a wide range of organisms.Abbreviations MMO methane monooxygenase