Specific Release of Bacteriochlorophylls B800 of LH2 from Rhodobacter azotoformans Induced by Sodium Dodecyl Sulfate

采用吸收光谱法研究了十二烷基硫酸钠(SdS)诱导rHOdObACTEr AzOTOfOrMAnS外周捕光复合体lH2细菌叶绿素(bACTErIOCHlOrOPHyllS,bCHlS)的解离行为和规律.结果表明:室温下,在10MMOl？l-1TrIS-HCl(PH8.0)缓冲液中,低浓度SdS只诱导lH2中b800细菌叶绿素解离生成游离bCHlS,而b850不受影响;当浓度达到0.08%(W/V)时,能特异性地诱导b800缺失,其缺失过程和游离bCHlS的生成过程均符合单指数模型,且二者的速率常数近似相等.高浓度SdS能同时诱导b800和b850解离生成游离bCHlS,其中b800可发生缺失,而b850则不完全解离,解离过程均符合单指数模型;b800对SdS更敏感,其解离速率常数约是b850的4倍,游离bCHlS生成速率常数明显低于b800解离速率常数,而与b850解离速率常数相接近.The release behaviors of bacteriochlorophylls of peripheral light-harvesting complex LH2 from Rhodobacter azotoformans induced by sodium dodecyl sulfate (SDS) were investigated using absorption spectroscopy.The results indicated that bacteriochlorophylls of B800 band are released from their binding sites and transformed into free bacteriochlorophylls by incubating LH2 sample in 10 mmol?L-1 Tris-HCl (pH 8.0) buffer containing SDS of low concentration at room temperature.However, the bacteriochlorophylls of B850 band are not released.The dynamics of B800 release and free BChl formation induced by 0.08% (w/V) SDS can be well fitted by the monoexponential model.The rate constant of B800 release is nearly equal to that of free BChls formation.The release of both B800 and B850 of LH2 can be induced by high concentration SDS, simultaneously.The bacteriochlorophylls of B800 band can be completely transformed into free BChls, but not for B850.Although both of their release processes show monoexponential models in 1% SDS solution, the release rate constant of B850 is remarkably lower than that of B800 and close to that of free BChls formation.国家自然科学基金(No.30970068);国家科技基础条件平台建设(No.2005DKA21209);厦门大学近海海洋环境科学国家重点实验室高级访问学者基金(No.MELRS0907);山西省回国留学人员科研(No.200713)资助项

Preparation and surface modification of high dispersive lamellar magnesium hydroxide particles from magnesite

氢氧化镁阻燃剂具有无毒、热稳定性高、抑烟效果好等特点，是近年来广泛应用的一类新型无机阻燃剂。综合考虑氢氧化镁阻燃剂的发展前景以及我国菱镁矿资源的优势，本文采用菱镁矿为原料，通过煅烧、湿法球磨、水热处理、表面改性制备高分散的片状MH阻燃剂，并将其应用至室温硫化硅橡胶（RTVSR）中考察其阻燃性能。本文较系统地研究了湿法球磨与水热处理对MH颗粒晶体结构、形貌、分散性等性能的影响，初步探讨了本工艺MH颗粒的晶体生长机理。研究了湿法球磨工艺条件对MH颗粒表面改性效果的影响以及MH颗粒性质对改性效果及阻燃效果的影响。 通过考察煅烧温度和时间对轻烧氧化镁活性以及活性氧化镁含量的影响得到菱镁矿的最佳煅烧工艺。当煅烧温度为650℃，煅烧时间为3.5h，轻烧氧化镁的活性较好，活性氧化镁的含量最高，约为89.33%。 通过对轻烧氧化镁进行湿法球磨处理，发现湿法球磨可以加速MgO的水化速率，使大部分轻烧MgO快速水化成MH，为水热晶化处理提供分散性较好的前躯体。以聚乙二醇400(PEG400)或聚乙烯吡咯烷酮K30(PVP)为助磨剂，球料比质量为20:1?30:1时，对轻烧MgO湿法球磨2?4h，可明显改善水热产物MH颗粒的分散性，减小其粒径，缩小粒度分布。 通过对湿法球磨后的浆料进行水热处理，可快速地使浆料中残留的MgO水化成MH，且进一步完善原有片状MH颗粒的晶体结构和形貌。水热温度、水热时间、碱浓度均是影响MH颗粒晶体结构和粒径的重要因素。 初步探讨了通过湿法球磨-水热处理工艺制备的MH颗粒的晶体生长机理：在湿法球磨过程中，大部分的MgO快速溶解、水化成MH初级粒子，这些初级粒子通过取向连接形成片状MH颗粒。在水热处理过程中片状MH颗粒在原有基础上不断发育完全，而残余的MgO可能通过溶解作为MH晶粒进一步生长的原料。 为改善MH颗粒的有机相容性，采用湿法球磨的方式对市售的氢氧化镁和自制的氢氧化镁进行表面改性。研究发现球磨工艺条件，如球磨时间、球料比、水料比和表面改性剂用量等均是影响MH颗粒改性效果的重要因素。此外，MH颗粒的自身性质，如表面电性、极性和比表面积也严重影响着MH颗粒的表面改性效果。MH颗粒经过表面改性后表面吸附有改性剂，且MH颗粒与改性剂之间形成了新的化学键，MH颗粒的表面极性减弱，在RTVSR基底材料中具有较好的相容性和阻燃性。MH颗粒的自身性质，如粒径和形貌，影响其阻燃效果，在分散性好的前提下，片状的、粒径小的MH颗粒阻燃效果好

Research progresses of marine Roseobacter lineage

海洋玫瑰杆菌类群(rOSEObACTEr lInEAgE)是属于α-变形菌纲中的一类系统发育相近,但生理代谢功能多样的细菌类群,包含40多个不同的细菌种属。它们在海洋中丰度较高,且分布极为广泛,尤其在近海与极地海洋中,其丰度约占整个浮游细菌群落的15%—25%。玫瑰杆菌类群通过其多样化的生理代谢功能(如好氧不产氧光合作用、一氧化碳氧化、硫化物降解等)在海洋碳、硫循环和全球气候调节中发挥着重要作用。此外,玫瑰杆菌类群还能产生多种具生物活性的次生代谢物质。简要综述了海洋玫瑰杆菌类群的生态分布特征、生存方式、生理代谢功能、基因组特征等的一些研究进展,并结合作者的工作对未来的研究进行了展望。Marine Roseobacter clade is a phylogenetically coherent,but physiologically diverse group of α-Proteobacteria,with members sharing > 89% identity of the 16 S rRNA gene.It contains more than 40 different genera.Roseobacter was found to be abundant in marine environment,especially in coastal and polar oceans,where Roseobacter clade comprises 15% —25% of the total bacterioplankton communities.Roseobacters also exist ubiquitously in the ocean.Based on culture collections,16 S rRNA clone libraries, and single-cell analyses, roseobacters have been identified in most marine environment sampled,such as sea ice,sea floor,hypersaline microbial mats,sponges,sea grasses,and coastal biofilms,etc.whereas,this bacterial group is noticeable absent in analogous freshwater and terrestrial soil environment.Quantitative 16 S rRNA gene analyses show that Roseobacter populations fall off with depth in the ocean,and are often most abundant in bacterial communities associated with marine algae.Roseobacter lineage is the only abundant marine group,whose cultivated representatives are closely related to the not-yet-cultivated environmental clone sequences.Diverse life styles have been found in Roseobacter clade, such as free living, particle associated, or in commensal relationships with marine phytoplankton,invertebrates,and vertebrates.Also,diverse physiological metabolic traits were found in this bacterial group.For example, some roseobacters can generate metabolic energy from light using the ancient purple bacterial mechanism of anaerobic photosynthesis without production of oxygen( anaerobic anoxygenic photosynthesis),while someroseobacters have the physiological metabolic traits of degradation of sulfur compounds or aromatics,oxidation of carbon monoxide,or reduction of trace metals,etc.Thus,it is considered that roseobacters may play important roles in marine carbon and sulfur cycles,as well as the global climate regulation.There are more than forty roseobacters whose whole genome sequences are available in the current public databases.Analysis of these genomes also reflected the metabolic versatility of the Roseobacter lineage.The cellular requirements of roseobacters for nitrogen are largely satisfied by regenerated ammonium and organic compounds( polyamines,allophanate,and urea),while the requirement for carbon sources are satisfied by amino acids,glyoxylate,and aromatic metabolites.Also,a large number of genes are predicted to encode proteins involved in the production,degradation,and efflux of toxins and metabolites,suggesting the potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop.Laboratory experiments found that the various bioactive secondary metabolites of roseobacters include antagonist against fish larval bacterial pathogens,antibiotic against marine bacteria and algae,shellfish poison,and the bioactive LuxR- activating acylated homoserine lactones( AHLs),which are a class of signaling molecules involved in bacterial quorum sensing,etc.Recently,the interactions between roseobacters and their phages caused widespread attention.Several host-phage interaction systems of the roseobacters were set up in the laboratory.Laboratory experiments revealed that roseophages play important roles in mediating the physiology and promoting the evolution of roseobacters in marine environment.This review described the recent research progresses of Roseobacter lineage in terms of their ecological distribution,lifestyle,physiological functions,and genome features.Finally we suggested future research directions based on our understanding of the literature and our own work.国家重大科学研究计划(2013CB955700); 国家自然科学基金项目(41006087;31070054); 厦门市科技计划项目(3502Z20132014); 厦门大学近海海洋环境科学国家重点实验室访问学者基金(MELRS1206

Isolation and characterization of pigment-protein complexes from Rhodobacter azotoformans

【目的】为揭示不产氧光合细菌产氢菌株色素蛋白复合体(PPC)色素组成和含量与光合放氢的关系奠定基础。【方法】以PPC特征光谱为检测指标,采用硫酸铵分级分离、dEAE-纤维素层析、吸收光谱和SdS-PAgE等方法进行了固氮红细菌(rHOdObACTEr AzOTOfOrMAnS,r.AzOTOfOrMAnS)r7产氢菌株PPC的分离纯化、纯度分析和鉴定;采用表面增强激光解吸电离离子飞行时间质谱、HPlC-MS和荧光光谱法对其中一种PPC进行了组成分析和能量传递活性测定。【结果】从r7菌株获得了3种纯化的PPC,1种为反应中心与中心捕光色素蛋白复合体(rC-lH1),2种为外周捕光色素蛋白复合体(lH2),其中一种lH2的吸收光谱具有异常的423nM强吸收峰,其蛋白的两种亚基的分子量分别为5356.8dA和5697.8dA,类胡萝卜素属球形烯系,分子量为562dA,激发光能够从类胡萝卜素向细菌叶绿素以及细菌叶绿素向细菌叶绿素传递。【结论】固氮红细菌产氢菌株含有2种不同光谱特性的lH2,其中一种具有新光谱特性。ObjectiveIn order to reveal the relationships of compositions and content of pigment in pigment-protein complexes(PPC ) and hydrogen photoevolution from anoxygenic phototrophic bacteria.Methods We isolated and identified pigment-protein complexes using a separation strategy of subsequent fractionated ammonium-sulfate precipitation,ion exchange column chromatography,absorption spectra and sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE ) from hydrogen-producing Rhodobacter azotoformans R7.We investigated the characterizations of the peripheral light-harvesting complex(LH2 ) with an unusual absorption spectrum by surface enhanced laser desorption / ionization time of flight mass spectrometry(SELDI-Tof-MS ),high performance liquid chromatography-mass spectrometry(HPLC-MS) and fluorescence spectra.ResultsWe acquired three types of PPC, the reaction center and core light-harvesting complex(RC-LH1 ) and two kinds of LH2,from strain R7 incubated anaerobically in the light.The two LH2 showed the different absorption spectra,one of them displayed unusual absorption spectrum with the maximum absorption band at 423 nm.The unusual LH2 consisted of two kinds of protein subunits with the molecular weight of 5556.8 Da and 5697.8 Da and carotenoid of spheroidene series with the molecular weight of 562 Da.It was also capable of transferring energy from carotenoid to bacterialcholorophyll and from B800 bacterialcholorophyll to B850 bacterialcholorophyll.Conclusions Rhodobacter azotoformans R7 with hydrogen-producing capacity could photosynthesize two types of LH2 under anaerobically in the light,one of them presented novel spectral property.国家自然科学基金(30970068);国家科技基础条件平台建设项目(2005DKA21209);近海海洋环境科学国家重点实验室(厦门大学)高级访问学者基金(MELRS0907)---

机械球磨对煅烧菱镁矿制备纳米片状氢氧化镁颗粒的影响

以菱镁矿为原料,通过煅烧、湿法球磨、水热处理制备高分散纳米片状氢氧化镁(MH)颗粒,研究了助磨剂、球磨时间、球料质量比对所制MH颗粒的分散性、粒度和形貌的影响.结果表明,机械球磨可加速煅烧菱镁矿的水化过程,为水热晶化提供分散性较好的前躯体;以聚乙二醇400(PEG400)或聚乙烯吡咯烷酮K30(PVP)为助磨剂,球料比质量为20:1～30:1时,对轻烧MgO湿法球磨2～4h,可明显改善MH颗粒的分散性,减小其粒径,缩小粒度分布

Deletion of the DGAAT gene with a bleomycin resistance cassette in the oleaginous yeast Rhodosporidium toruloides

The red yeast Rhodosporidium toruloides is oleaginous with a great propensity to accumulate intracellular lipids over 70% of its dry cell weight when carbohydrates were fed as carbon sources. Therefore, this yeast has been intensively studied recently in order to produce microbial lipid for a sustainable biodiesel industry. On the other hand, the biochemical machinery of lipid metabolism may be explored for production of value-added metabolites. For example, diacylglycerols, the precursor to triacylglycerols, are attractive because of their special biological activities. In this study, we isolated the genomic DNA sequence of the acyl-coenzyme A:diacylglycerol acyltransferase gene (DGAAT). We generated a knockout construction with bleomycin resistance cassette replacing the 1858 bp open reading frame of the DGAAT gene. The haploid strain R. toruloides NP11 was electroporated with the cassette, and transformant was observed on YEPD medium containing 50 μg/ml Zeocine. It should be noted that the wild-type strain were killed by 5 μg/ml Zeocine. Further PCR analysis and genome walking showed that the bleomycin cassette was integrated into the chromosome of the yeast. These results demonstrated that cassette was functional in R. toruloides under the control of the DGAAT promoter and the terminator. We will discuss more details and progress of this project during the conference.The red yeast Rhodosporidium toruloides is oleaginous with a great propensity to accumulate intracellular lipids over 70% of its dry cell weight when carbohydrates were fed as carbon sources. Therefore, this yeast has been intensively studied recently in order to produce microbial lipid for a sustainable biodiesel industry. On the other hand, the biochemical machinery of lipid metabolism may be explored for production of value-added metabolites. For example, diacylglycerols, the precursor to triacylglycerols, are attractive because of their special biological activities. In this study, we isolated the genomic DNA sequence of the acyl-coenzyme A:diacylglycerol acyltransferase gene (DGAAT). We generated a knockout construction with bleomycin resistance cassette replacing the 1858 bp open reading frame of the DGAAT gene. The haploid strain R. toruloides NP11 was electroporated with the cassette, and transformant was observed on YEPD medium containing 50 μg/ml Zeocine. It should be noted that the wild-type strain were killed by 5 μg/ml Zeocine. Further PCR analysis and genome walking showed that the bleomycin cassette was integrated into the chromosome of the yeast. These results demonstrated that cassette was functional in R. toruloides under the control of the DGAAT promoter and the terminator. We will discuss more details and progress of this project during the conference

保护层对汽车尾气氧传感器响应特性影响

汽车尾气净化用电势型氧传感器的电极表面通常涂覆有一层保护层,以＂过滤＂废气中的杂质。考察了保护层制作工艺和保护层物理参数对电势型氧传感器稳态输出电压、使用寿命和响应时间的影响,结果表明：1）当制备保护层所使用的材料（主要是高温粘结剂）和传感器电极或电解质发生化学反应时,测量得到的稳态输出电压也会和真实值相差甚远。选定质量分数为20%硅胶作为高温粘结剂可制备得到性能优良的传感器保护层;2）在保护层制备过程中加入适当比例的YSZ粉末材料,可使电极保护层的使用寿命提高;3）传感器的总响应时间与传感器保护层的物理性能（厚度、孔径等）和传感器的工作温度密切相关

保护层对汽车尾气氧传感器响应特性影响

汽车尾气净化用电势型氧传感器的电极表面通常涂覆有一层保护层,以＂过滤＂废气中的杂质。考察了保护层制作工艺和保护层物理参数对电势型氧传感器稳态输出电压、使用寿命和响应时间的影响,结果表明：1）当制备保护层所使用的材料（主要是高温粘结剂）和传感器电极或电解质发生化学反应时,测量得到的稳态输出电压也会和真实值相差甚远。选定质量分数为20%硅胶作为高温粘结剂可制备得到性能优良的传感器保护层;2）在保护层制备过程中加入适当比例的YSZ粉末材料,可使电极保护层的使用寿命提高;3）传感器的总响应时间与传感器保护层的物理性能（厚度、孔径等）和传感器的工作温度密切相关

Construction and Characterization of B850-Only LH2 Energy Transfer System in Purple Bacteria

构建b800缺失lH2对于阐明光合作用中光能传递的分子机制与捕光复合体组装机制具有重要意义。采用吸收光谱、荧光光谱、分子筛层析、超滤和SdS-PAgE等方法研究了紫细菌两个典型种外周捕光复合体(lH2)约800nM特征光谱(b800)细菌叶绿素(bCHl)缺失能量传递模型的构建方法及性质。结果表明:在PH 8.0TrIS-HCl(10MMOl·l-1)缓冲液中,0.08%SdS能够使来自rHOdObACTEr AzOTOfOrMAnS的lH2b800bCHl特异性解离,解离体系中加入10%(φ)甲醇,通过超滤脱除游离bCHl,构建了b800缺失lH2,但该缺失模型不够稳定。在PH 1.9缓冲液中,来自rHOdOPSEudOMOnAS PAluSTrIS的lH2b800bCHl能够特异性解离,通过层析得到两个组分。一个组分的b800bCHl不能通过层析脱除,能够重新自组装成lH2。另一个组分为b800缺失lH2,该缺失模型稳定。两种lH2均存在2类以上b800bCHl结合位点,并得到了两类rHOdOPSEudOMOnAS PAluSTrIS b800bCHl解离的lH2,但未发现类似紫色硫细菌中的b800吸收光谱劈裂现象。b800缺失lH2均未呈现约800nM特征荧光光谱。采用两种方法构建了两个物种b800缺失lH2能量传递模型。利用bCHl与缺失b800lH2结合能力不同的特性,将rHOdOPSEudOMOnAS PAluSTrIS中的lH2分成两个类型,实现了异质性亚基lH2的分离。To seek microscopic molecular mechanism of energy transfer and complex reconstitution in the photosynthesis,the conditions for construction of B850-only peripheral light-harvesting complex(LH2)and their properties were investigated using absorption,fluorescence spectroscopy,molecular sieve chromatography,ultrafiltration and sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE)from the purple bacteria.The results indicated that bacteriochlorophylls(BChl)of B800 incubated in 10mmo·L-1 Tris-HCl(pH 8.0)buffer are selectively released from their binding sites of LH2 of Rhodobacter azotoformans(A-LH2)by 0.08%(W/V)SDS.B850-only A-LH2 was constructed after removing free BChl mixing with 10% methyl alcohol by ultrafiltration.B850 BChl was released after A-LH2 was incubated for 240 min in dark at room temperature(RT).While BChl of B800 incubated in pH 1.9buffer were selectively released from their binding sites of LH2 of Rhodopseudomonas palustris(P-LH2).The authors acquired two components using molecular sieve chromatography.Free BChl of one component was not removed and self-assembled to P-LH2.The other removed free BChl and B850-only P-LH2 was constructed.B850 unchanged after P-LH2 was incubated.P-LH2αandβsubunits have different molecular weights,but those of A-LH2 are in the contrary.It is concluded that B850-only P-LH2 is more stable than A-LH2.The enigmatic split of the B800 absorption band was not observed in these LH2,but we acquired two kinds of B800-released LH2 from Rhodopseudomonas palustris.The authors' results may provide a new light to separate homogeneous Apoprotein LH2.国家海洋公益性行业科研专项项目(201505026); 国家自然科学基金项目(31070054;31270106); 福建省自然科学基金项目(2012J01136); 近海海洋环境科学国家重点实验室(厦门大学)高级访问学者基金项目(MELRS0907)资