【作者】 林白雪；

【导师】 谢联辉；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 温泉与地球早期环境比较接近,其中微生物生态系统相对简单、稳定和封闭,对其进行微生物多样性的研究不仅有助于认识高温环境中的微生物遗传多样性与功能多样性,揭示温泉中嗜热菌的多样性及其与环境的关系,而且对认识生命的起源和进化具有十分深远的意义。嗜热菌是地球独特的生物资源和珍贵的科研素材。温泉中存在丰富的嗜热菌资源,它们有着奇特的耐高温特性。由嗜热菌产生的热稳定酶在工业和分子生物学等领域有重要的应用。研究和开发纤维素酶在生物质能源领域具有重要的作用,特别是热稳定的纤维素酶具有良好的应用前景。本研究利用分子生物学技术,以非培养的方式对厦门近海温泉沉积物的细菌和古菌的群落组成进行了研究。采用细菌16S rDNA基因通用引物Euba16F/1492R扩增细菌16S rDNA,分别构建了3个温度梯度的细菌16S rDNA基因文库,并进行ARDRA分析和系统发育分析。结果发现：三种温度温泉的细菌主要分布在变形杆菌门(Proteobacteria)、产水菌门(Aquificae)、拟杆菌门(Bacteroidetes)、绿屈挠杆菌门(Chloroflexi)、异常球菌-栖热菌门(Deinococcus-Thermus)、热袍菌门(Thermotogales)、厚壁菌门(Firmicutes)、酸杆菌门(Acidobacteria)等类群,以及一些未知的类群。Proteobacteria在三个温度沉积物中的种类及数量最为丰富,所占比例在70%-87%之间。三个温度沉积物中都是以变形杆菌为优势群落,其中Alphaproteobacteria在90℃温泉中属于优势类群,比例占37.5%。Betaproteobacteria是70℃和80℃的温泉的优势菌群,比例分别为43.9%和50.6%。采用古菌16S rDNA基因通用引物Arch21F和Arch958R扩增古菌16S rDNA,构建了两个温度的温泉样品的古菌16S rDNA文库,结果表明,温泉样品的古菌属于两大类群：泉古菌(Crenarchaeota)和广古菌(Euryarchaeota)。泉古菌是近海温泉的优势菌群。80℃样品中泉古菌的比例占89.2%,70℃样品中泉古菌占91.3%。大多数克隆与来自陆地温泉和深海热泉的不可培养克隆具有同源性。70℃样品中还有一个克隆SA一61,属于古菌界的未知类群。古菌主要分布在泉古菌的热变形菌目(Thermoproteales)和硫还原球菌目(Desulfurococcales)和未知的亚群、以及广古菌的热球菌目(Thermococcales)和古丸菌目(Archaeoglobales)。在已知的类群中,70℃和80℃温泉中硫还原球菌目(Desulfurococcales)属于优势类群。采用纯培养的方法,从美国内达华州温泉和中国福建永泰温泉分离得到100多株嗜热细菌。从中筛选得到产纤维素酶和半纤维酶的嗜热细菌并进行了16SrDNA鉴定。LY7和LY8是产纤维素酶的嗜热细菌,菌株LY7属于脂环酸芽孢杆菌属(Alicyclobacillus sp.);菌株LY8属于土芽孢杆菌属(Geobacillus sp.)生长范围在40～70℃之间,最适温度65℃=LY-1、LY-2、LY-3和LY-4是产半纤维素酶的嗜热菌,属于土芽孢杆菌属(Geobacillus sp.),生长范围在40～70℃之间,最适温度55℃。在许多微生物降解纤维素过程中,p-葡萄糖苷酶是限速酶,若p-葡萄糖苷酶酶活低,会导致纤维二糖的累积从而抑制内切葡聚糖和外切葡聚糖酶活,影响整个纤维素降解进程。为了大量获得p-葡萄糖苷酶并研究热稳定p-葡萄糖苷酶的性质,从温泉嗜热细菌(Geobacillus sp. LY8中克隆了热稳定的p-葡萄糖苷酶基因。从氨基酸序列分析,该酶属于糖苷水解酶类第一家族。将Geobacillus sp. LY8的p-葡萄糖苷酶基因与pGEX-4T-2连接,转化大肠杆菌构建了重组菌,进行了p-葡萄糖苷酶的高效表达。重组β-葡萄糖苷酶在大肠杆菌中以可溶的形式在细胞周质大量表达。纯化了重组p-葡萄糖苷酶,并对其酶学性质进行了研究。研究表明：(1)重组p-葡萄糖苷酶的最适反应温度为65℃,反应温度的范围非常广泛：30—90℃均能发挥良好的酶活性,且在90℃仍有良好的酶活力。在55—75℃下处理120min,仍有很好的酶活力,有良好的温度耐受性。说明该重组p-葡萄糖苷酶是一种优良的高温酶。(2)pH范围广：pH4-pH8范围内均表现出良好的酶活性。(3)具有很强的抗抑制剂和去污剂的能力。因此,该重组β-葡萄糖苷酶是一种优良的高温酶,在中温、高温(特别是高温),以及酸性和中性pH条件下,均能发挥良好的酶活性,具有很大的应用潜力。真菌是纤维素降解的主要微生物。本研究采用纯培养的方法从温泉中分离得到21株产纤维素酶的嗜热真菌。对其中8株进行18S rDNA鉴定,C1与WJM-4-WG属于芽枝霉属(Cladosporium sp.)。W3W、CC-1、C2F1和C2F2属于曲霉属(Aspergillus sp.)。CC-8属于毛孢酵母属(Trichosporon sp.)。WJM-l-G的ITS序列与已知序列的相似性很低,可能是新种。对纤维素酶活较高的菌株CC-1、CC-8以及WJM-4-WG,研究了温度、pH、碳源、氮源、摇床转速、接种量、装液量等对嗜热真菌产内切葡聚糖酶、外切葡聚糖酶以及β—葡萄糖苷酶的影响。成功克隆了Aspergillus sp.CC-1的外切葡聚糖酶基因(1713bp),该酶属于糖苷水解酶类第六家族。该酶的克隆为将来构建工程菌进行外切葡聚糖酶的表达和嗜热酶的结构和功能的研究奠定了基础。更多还原

【Abstract】 The environment of hot spring was quite similar with early Earth, and the ecology of microbial in hot spring was simple and stable. The exploration of microbial diversity in such ecosystem was important for the understanding of the microbial community structure, physiology and the relationship to the geochemical conditions. It also was helpful for understanding of the origin and the evolution of life.Thermophiles were the unique microbial source and rare material of scientific research. Hot spring had attracted broad interest because of the unique thermophilic properties of the organisms thriving in these biotopes and the description of an increasing number of new thermophilic species. Thermostable enzymes synthesized by thermophilic microorganisms were the important resources in many industrial processes and biochemistry. Cellulases played an important role in the biomass energy, especially thermostable cellulases.In the study, the bacterial and arechaeal diversity of the coastal hot spring in Xiamen were investigated by culture-independent molecular approaches. Three16S rDNA gene libraries of bacterial community from different temperature hot spring sediments were constructed and the analyses were performed by amplified ribosomal DNA restriction analysis (ARDRA) technique. The sequence similarities were analyzed by using the BLAST programs for searching the GenBank DNA databases. The phylogenetic analyses indicated that most of the bacterial16S rDNA sequences belonged to at least eight groups, including Proteobacteria, Aquificae, Bacteroidetes, Chloroflexi, Thermotogales, Firmicutes and Acidobacteria, and a few belonged to unknown groups. All bacterial libraries were dominated by Proteobacteria. In hot spring of90℃,Alphaproteobacteria was the predominant species (37.5%). While Betaproteobacteria was dominant in the hot spring of70℃(43.9%) and80℃(50.6%)。To describe arechaeal diversity, the arechaeal16S rDNA gene libraries of arechaeal community from different temperature (70℃and80℃) hot spring sediments were constructed. The phylogenetic analyses indicated that the arechaeal16S rDNA sequences belonged to Crenarchaeota and Euryarchaeota. Most of the archaeal phylotypes were related to sequences of yet-uncultivated microorganisms retrieved from terrestrial geothermal springs, deep-sea hydrothermal vents and the subsurface. All archaeal libraries were dominated by Crenarchaeota, and the proportion of Crenarchaeota in hot spring of80℃and70℃were89.2%发and91.3%resperctively. Clone SA-61belonged to unknown phylum of archaea. The archaeal phylotypes belonged to Thermoproteales and Desulfurococcales of Crenarchaeota phylum, and Thermococcales and Archaeoglobales of Euryarchaeota phylum, and a few belonged to unknown groups. Desulfurococcales were dominant in the both hot springs.Culture-based approaches were used in isolating thermophiles from Hot spring. In total, more than100thermophilic bacterial strains were isolated from hot springs in Nevada of USA and Yongtai country of China. Superior cellulose and hemicellulose decomposing strains were screened and identified by16S rDNA sequencing analysis. LY7and LY8which degraded cellulose were indentified as Alicyclobacillus sp. LY7nd Geobacillus sp. LY8. Geobacillus sp. LY8could be cultured at temperature range from40℃to70℃, while the optimal temperature was65℃. LY-1, LY-2, LY-3and LY-4which degraded hemicellulose, were assigned to Geobacillus sp. based on16S rDNA sequencing analysis. They could grow at temperature range from40℃to70℃, while the optimal temperature was65℃.The β-glucosidase was the rate-limiting enzyme in cellulose hydrolysis of many microorganisms and the resulting accumulation of cellobiose inhibits endoglucanase and cellobilhydrolase activities. To increase production of the enzyme and study the character of thermostable enzyme, the gene encoding a thermostable β-glucosidase from Geobacillus sp. LY8was cloned and sequenced. According to the amino sequence, β-glucosidase from Geobacillus sp. LY8belonged to glycosyl hydrolase family1. After PCR amplification, the β-glucosidase gene was cloned into the pGEX4T-2vector. The recombinant plasmid containing β-glucosidase gene was transformed into E. coli BL21.The recombinant β-glucosidase was heterologously overexpressed in the intracellular space. Then it was purified and the biochemical characteristics were also investigated. The results showed that:(1) The optimal temperature for the enzymatic reaction was65℃, and had good enzyme activity at30～90℃. The recombinant β-glucosidase worked well at90℃and was stable when treated at55～75℃for120min. All of these showed that the recombinant β-glucosidase was an excellent thermostable enzyme.(2) The recombinant β-glucosidase had good activity at broad pH range from pH4to pH8.(3) The recombinant β-glucosidase was unusually stable in organic solvents and detergents. In conclusion, the recombinant β-glucosidase was an excellent thermostable enzyme. It had good enzyme activity at high temperature and acid or natural pH.Fungi was the most important microorganism that degrading cellulose. Culture-based approaches were used in isolating thermophilic fungi from hot spring which degraded cellulose. In total,21thermophilic fungi strains were isolated and eight of them were identified by18S rDNA sequencing analysis. C1and WJM-4-WG belonged to Cladosporium sp.; W3W, CC-1, C2F1and C2F2belonged to Aspergillus sp.; CC-8belonged to Trichosporon sp. While WJM-1-G had low homology with sequences in NCBI, and maybe it belonged to a novel species. Fermentation conditions of three superior cellulose decomposing strains CC-1, CC-8and WJM-4-WG were further studied. The gene encoding a thermostable cellobilhydrolase from Aspergillus sp.CC-1was cloned and sequenced. According to the sequence, cellobilhydrolase from Aspergillus sp.CC-1belonged to glycosyl hydrolase family6. Cloning of cellobilhydrolase gene of themophilic fungi founded the base not only for constructing the engineering strain with high-yield cellulose, but also for the study of unusual structure and function of thermostable enzyme.更多还原