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.     

甲烷氧化菌素的产生和铜捕获作用

甲烷氧化菌素(mb)是由甲烷氧化菌产生的与铜捕获和吸收有关的生物螯合剂。采用NMS/CAS-Cu劈半平板法检测发现甲基弯菌 Methylosinus trichosporium IMV3011在以甲烷和甲醇为碳源时均具有向外界分泌mb的能力。甲基弯菌 Methylosinus trichosporium IMV3011在限铜胁迫下能够在培养介质中积累mb,甲醇为碳源时mb的积累量明显高于甲烷为碳源时mb的积累量,推测mb的合成与细胞中NADH含量有关。mb的专一性研究发现从甲基弯菌 Methylosinus trichosporium IMV3011发酵上清液中分离到的mb能够提高其它3种甲烷氧化菌由无铜培养基向含铜培养基转移时的pMMO活性的表达,并明显缩短它们由无铜培养基转移到含铜培养基时生长的延滞期,提高生长速度。表明其它3种甲烷氧化菌同样能够吸收来自于甲基弯菌Methylosinus trichosporium IMV3011的mb捕获的铜。     

含有甲烷氧化菌的混合菌群特性研究

为获得高效甲烷氧化微生物体系,从农业土壤中采样,以甲烷作为唯一碳源进行好氧选择性传代培养,得到生长性能稳定、生长优于Methylosinus trichosporium OB3b纯培养的具有甲烷单加氧酶(Methane Monooxygenase,MMO)活性甲烷氧化混合菌。利用MMO的共代谢特性,分别以苯酚和环氧丙烷作为目标对象,考察该混合菌对有机污染物的降解及用于生产有用化学物质的催化特性。结果表明,所得混合菌具有高效降解苯酚能力,对初始浓度为600mg/L的苯酚,经过11h培养,苯酚降解率可达99%。另外,以该混合菌为催化剂可以实现丙烯氧化生产环氧丙烷。通过降低磷酸盐浓度可以有效提高环氧丙烷的积累浓度,最大可至5mmol/L。此外,采用纯种分离方法结合PCR扩增、16SrRNA和MMO功能基因分析技术对混合菌群结构进行解析。结果表明,该混合菌群由Ⅱ型甲烷氧化菌及其它至少4种非甲烷氧化菌组成,它们分别属于Methylosinus trichosporium和Acinetobacter junii、Cupriavidusme tallidurans、Comamonas testosteroni和Stenotrophomonas maltophilia。采用PCR方法从混合菌及纯化菌株M.trichosporiums Y9总DNA中都能扩增得到mmoB、mmoX和pmoA基因片段,表明该甲烷氧化菌同时具有sMMO和pMMO两种形式的MMO。通过对从甲烷氧化混合菌中分离纯化得到的甲烷氧化菌进行PCR产物测序,结果发现其与Methylosinus trichosporium的同源性为99.9%。     

土壤大气甲烷氧化菌研究进展

土壤微生物催化是大气中痕量甲烷(约1.8ppmv)氧化的唯一生物途径。目前的研究表明好氧土壤中存在专性和选择性大气甲烷氧化菌2种类型:前者(USCα和USCγ)广泛分布于各种好氧旱地土壤,其甲烷氧化酶对低浓度甲烷亲和力极高,属真正的寡营养型,但至今尚未获得该种类的纯培养菌株。后者属于传统甲烷氧化菌Methylocystis/Methylosinus属,广泛分布于各种周期性排放高浓度甲烷的土壤环境中。该属大部分菌株含有亲和力不同的2套甲烷单加氧酶系统,其中的高亲和力甲烷单加氧酶使这些菌株可以在相当长的时间内(3个月)保持大气浓度甲烷氧化活性,但其生长和繁殖还需依赖于土壤内部阶段性产生的高浓度甲烷。本文详细阐述了2类大气甲烷氧化菌的发现历程及其可能的生存策略,最后系统梳理了几种关键的环境因子(土壤温度及湿度、土壤pH、植被、土地利用及氮输入)对大气甲烷氧化菌群落结构和甲烷氧化活性的影响,提出并展望了土壤大气甲烷氧化菌研究的重要方向。     

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。     

颗粒性甲烷单加氧酶的研究

从甲基弯菌(Methylosinus trichosporium)IMV3011(简称M3011)的膜中分离纯化出颗粒性甲烷单加氧酶(Particulate Methane monooxygenase,简称pMMO)和NADH脱氢酶。Cu2+、不同外源电子给体、EDTA等对pMMO活性的都有影响;测定了pMMO分子量,以及其活性中心金属离子含量。对于M3011,培养基中Cu2+浓度对pMMO的生成和活性没有影响;但在分离纯化过程中对pMMO稳定性有明显影响。电镜结果显示,Cu2+浓度对pMMO的数量有较明显的影响。对于纯化的pMMO,对苯二酚仍是有效的电子供体,而NADH却是无效的电子供体。纯化过程中采用对苯二酚作为pMMO活性分析时的电子供体,排除了共纯化NADH脱氢酶的必要,有利于对pMMO活性中心进行深入研究。     

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     

甲烷利用细菌降解三氯乙烯的研究

GYJ3菌株细胞微细结构的电镜观察结果表明：它具有Ⅱ型甲烷利用细菌的特征,应归属于Ⅱ型菌。考察了Cu2+浓度、培养气相中甲烷浓度对菌株细胞中甲烷单加氧酶（EC1．14．13．25,简称MMO）活性的影响。结果表明,培养液中Cu2+浓度为1．5μmol／L,培养气相中甲烷：空气比为2∶1时,可溶性甲烷单加氧酶占细胞中MMO总量的95％。研究了GYJ3菌株细胞悬浮液降解三氯乙烯过程。实验结果表明,GYJ3菌株能够降解不同浓度的三氯乙烯,较高浓度的三氯乙烯对降解反应没有明最的抑制作用。加入甲酸盐作为电子给体能够提高三氯乙烯降解反应速率。实验中观察到GYJ3菌株降解三氯乙烯过程中反应速率随着反应的进行而下降,在三氯乙烯降解过程中三氯乙烯氧化产物是导致细胞失活的主要原因。实验室中测定了GYJ3菌株单位重量细胞降解三氯乙烯极限量,它可作为评价细菌降解三氯乙烯能力的重要指标。     

甲烷氧化细菌Methylosinus trichosporium 3011甲醇累积条件的研究

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。     

Interstitial stem cell proliferation in hydra: evidence for strain-specific regulatory signals.

We have examined the growth behavior of small numbers of interstitial stem cells transplanted into tissue of genetically unrelated strains of Hydra magnipapillata. We show that such stem cells, which are at low density following transplantation, proliferate more rapidly than the stem cells of the host, which are at normal density. The rapid proliferation is similar to the proliferation rate of stem cells transplanted into interstitial cell free tissue. The results suggest that stem cells transplanted into heterotypic tissue are unable to "sense" the presence of host stem cells and to adopt their growth rate to that of the surrounding cells. Thus, the feedback signal which negatively regulates stem cell growth as a function of stem cell density must be strain specific.     

Improving performance of mammalian cells in fed-batch processes through "bioreactor evolution"

The amount of recombinant product obtained from mammalian cells grown in a bioreactor is in part limited by achievable cell densities and the ability of cells to remain viable over extended periods of time. In an attempt to generate cell lines capable of better bioreactor performance, we subjected the DG44 Chinese Hamster Ovary (CHO) host cell line and a recombinant production cell line to an iterative process whereby cells capable of surviving the harsh conditions in the bioreactor were selected. This selective process was termed "bioreactor evolution". Following the selective process, the "evolved" host cells attained a 2-fold increase in peak cell density and a 72% increase in integral cell area. Transient transfection experiments demonstrate that the evolved cells have the same transfection efficiency and the same secretory potential as the initial cells. The "evolved" host was also found to contain a large subpopulation of cells that did not require insulin for growth. From this, a new population of growth-factor-independent cells was obtained. These improvements in host properties should prove beneficial in the expression of recombinant proteins in fed-batch processes. The selective process was also applied to a recombinant production cell line. The evolved cells from this selection exhibited a 38% increase in peak cell density, a 30% increase in integral cell area, and a 36% increase in product titer. These increases were obtained without any appreciable impact on product quality, demonstrating the usefulness of this simple approach to improve the performance of recombinant cell lines.     

A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate.

Seven mutants of Escherichia coli were isolated that are sensitive to methyl methane sulfonate but not to UV light. They exhibited decreased host cell reactivation capacity for methyl methane sulfonate-treated phage lambda. Five of the mutations were mapped in the same region as alkA (previously called alk) and may indeed be identical to known mutations. Another mutation was found near nalA, and the gene responsible was named alkB. Its phenotype was different from that of ada, since the alkB mutant exhibited a normal adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine. A third type of mutation was mapped near polA, but this mutant contained an almost normal level of DNA polymerase I activity.     

一株甲烷利用菌的分离及其在甲烷气体测定中的应用

从山西太原晋阳湖水样中分离得到一株能以甲烷为唯一碳源生长的菌株ME16.气相色谱分析表明ME16菌株能利用甲烷.ME16菌株的16S rDNA 序列与铜绿假单胞菌(Pseudomonasaeruginosa,ATCC 10145,AF094713)相似性为99%.该菌株最适培养条件为30℃、2%接种量、25%甲烷含量和培养基pH为6.0.用电化学法研究了ME16固定化细胞体系中不同含量甲烷对溶氧的响应时间以及溶氧变化与甲烷含量的关系.结果表明,加入固定化细胞后.溶氧变化在100s内达到平衡,溶氧消耗量与通入甲烷气体含量在0～16%呈线性关系,相关系数为0.9954.对样品气体8次测量,RSD为3.34%,表明该反应体系重现性良好,为该菌株进一步研究甲烷传感器奠定基础.     

Host range studies of FLOPC-1 murine myeloma C particles.

The host range of the C particle produced by FLOPC-1 myeloma cells, FLOPC-1 murine myeloma-associated virus (FL-MuMAV), was assessed in terms of its ability to productively infect and/or induce new viral antigens in a variety of different cell lines. Production of C particle-like structures by cells exposed to FL-MuMAV) was determined by incorporation of [3H]uridine into particles with a density of 1.16 g/ml and/or measurement of RNA-dependent DNA polymerase activity in concentrated culture medium. to FL-MuMAV was capable of infecting NIH/3T3, normal rat kidney (NRK) cell, BALB/c 3T3, and the A31 clone of BALB/3T3 cells but not rabbit cell line, SIRC. Thus, it is an N, B-tropic murine virus as replication in NRK cells has been shown not to delineate a group of murine viruses with a separate host range (M. M. Lieber, C. J. Sherr, and G. J. Todero, 1974). Further neoantigens, reactive with anti-FL-MuMAV serum, were detected on infected cells. Production of the MuMAV-like particle and MuMAV-associated cell antigens in infected NIH/3T3 and NRK cells persisted for three subcultures. The limited production could not be explained by the lack of an RNA-dependent DNA polymerase or high-molecular-weight RNA as the particles possessed both of these properties. The particles produced by infected NIH/3T3 or NRK cells were antigenically and physicochemically similar to FL-MuMAV and not K-MuLV. The MuMAV-like particles produced by infected NIH/3T3 were capable of limited replication in NIH/3T3 and and BALB/3T3 cells, whereas NRK-MuMAV replicated for a limited period in NIH/3T3, NRK, and SIRC cells; i.e., they had a different host range than FL-MuMAV. The particles produced by infected BALB/3T3 and A31 cells had the same host range as FL-MuMAV. In certain situations, isotopically labeled particles with a density of 1.16 g/ml were produced which appeared to lack RNA-dependent DNA polymerase.     

Evolutionary feedback mediated through population density, illustrated with viruses in chemostats

A cornerstone of evolutionary ecology is that population density affects adaptation: r and K selection is the obvious example. The reverse is also appreciated: adaptation impacts population density. Yet, empirically demonstrating a direct connection between population density and adaptation is challenging. Here, we address both evolution and ecology of population density in models of viral (bacteriophage) chemostats. Chemostats supply nutrients for host cell growth, and the hosts are prey for viral reproduction. Two different chemostat designs have profoundly different consequences for viral evolution. If host and virus are confined to the same chamber, as in a predator-prey system, viral regulation of hosts feeds back to maintain low viral density (measured as infections per cell). Viral adaptation impacts host density but has a small effect on equilibrium viral density. More interesting are chemostats that supply the viral population with hosts from a virus-free refuge. Here, a type of evolutionary succession operates: adaptation at low viral density leads to higher density, but high density then favors competitive ability. Experiments support these models with both phenotypic and molecular data. Parallels to these designs exist in many natural systems, so these experimental systems may yield insights to the evolution and regulation of natural populations.     

Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development

Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parasite growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appears as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.     

四种类型启动子控制下空肠弯曲杆菌血红蛋白对大肠杆菌生长速度的影响

细菌血红蛋白可增加细胞在低氧条件下的氧气利用率,提高细胞的增长速率。为提高E. coli在现有发酵水平下的生物量,获得新型的基因工程底盘细胞,基于空肠弯曲杆菌(Campylobacter jejuni)血红蛋白基因,比较分析了组成型和诱导型启动子对血红蛋白在E. coli基因工程菌中的表达状况及对E. coli生长的影响。首先合成了空肠弯曲杆菌血红蛋白基因chb,并让chb基因在诱导型启动子(P_T7和P_vgh)和组成型启动子(P2和P_spoI-Ⅱ)控制下表达;随后,测定了由四种类型启动子控制的血红蛋白工程菌在摇瓶和蓝盖瓶中的生长状况。结果表明四种重组菌可以表达出有活性的血红蛋白,并可显著提高大肠杆菌的生长;在发酵罐中的试验也表明四种工程菌同样对细胞生长具有出促进作用;进一步比较分析了菌体密度、细胞鲜重和CO差式光谱值间的相互关系,讨论了四种类型启动子控制的基因工程菌在诱导表达和生长调控等方面的特点,并为在微氧和好氧等不同发酵条件下选用基因工程底盘细胞提供了参考。     

Separation of Viable Rickettsia typhi from Yolk Sac and L Cell Host Components by Renografin Density Gradient Centrifugation

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.     

Virus–host cell interactions in vaccine production cell lines infected with different human influenza A virus variants: A proteomic approach

A proteomic approach was used to investigate the dynamic cellular host cell response induced by influenza virus infection in two different vaccine production cell lines, MDCK and Vero. For identification of proteins possibly involved in global host cell response mechanisms and virus–host cell interactions in these producer cell lines, quantitative 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were performed. In particular, host cell proteome alterations caused by infection with influenza virus variants showing differences in replication characteristics in MDCK cells were compared. Moreover, the host cell response to virus infection in Vero cells with respect to their deficiency in interferon (IFN) production and the need for virus adaptation to optimize productivity of this cell line were analyzed. Several proteins with differential abundance profiles were identified and Western blot analysis was performed for further confirmation of selected proteins. IFN-induced signal transduction, cytoskeleton remodeling, vesicle transport and proteolysis were the principal pathways that changed in infected MDCK cells. In Vero cells alterations of cell interactions, heat shock and oxidative stress response were detected. The findings will improve understanding of host cell response mechanisms during influenza vaccine production and viral strategies to evade these responses and to replicate efficiently in different cell lines.