【作者】 高兆明；

【导师】 陈秀兰； 徐洵； 张玉忠；

【作者基本信息】 山东大学 ， 微生物学， 2011， 博士

【摘要】 红树林(mangrove)是自然分布于热带和亚热带海岸潮间带、受周期性潮水浸淹、并以红树植物为主体的常绿灌木或乔木组成的潮滩湿地木本植物群落,它位于陆地和海洋交界的滩涂地带,是陆地向海洋过度的特殊生态系统。由于处于周期性遭受海水浸淹的潮间带环境,红树林土壤兼有海洋和陆地的性质却与二者不同,具有沼泽化、盐渍化、强酸性、有机质含量高和部分厌氧等特征。其独特的生境特征造就了丰富独特的微生物和相应的基因及酶资源。由于红树落叶等木质纤维素的存在,纤维素降解微生物也是该区域重要的生物组成部分。现今工业社会中,纤维素酶用途广泛,可用于食品、纺织、饲料、石油勘探、中药成分提取等众多领域,特别是在纺织、洗涤、造纸和生物能源等工业应用上具有重要价值。因此,探索红树林这一特殊生境中的纤维素酶产生菌和相应的酶基因资源具有重要的基础研究和工业应用价值。在本论文中,我们从厦门红树林泥样中筛选到一株纤维素酶产生菌G21,通过16S rRNA基因序列的同源性分析,确定该菌株为弧菌属一个潜在的新种,随后对其分类学地位进行了系统的分析鉴定。同时,克隆了菌株G21的纤维素酶基因,并在大肠杆菌中进行了重组表达和性质分析。此外,用采集于海南清澜港的红树林泥样经富集和传代培养,获得了一个高效稳定的厌氧纤维素降解菌群SQD-1.1,并通过PCR-DGGE、16S rRNA基因文库构建、宏基因组测序等方法对其进行了分析,对红树林厌氧条件下的纤维素降解菌群有了一定的认识。1.菌株G21的分离与鉴定菌株G21是以厦门白鹭洲红树林区的泥土为样品、以CMC-Na为底物筛选产纤维素酶菌株时获得的一个菌株。革兰氏染色为阴性,菌体呈弯曲的杆状,具极生鞭毛,其宽度为0.7-0.8μm,长度为1.4-1.55μm。37℃下,在2216E平板上培养24h后,菌落为圆形或梭形、白色、半透明。生长盐度范围为0.5-10% NaCl (w/v),生长温度范围为15-40℃。菌株在含10mM葡萄糖的厌氧条件下生长,不产生气体。氧化酶和触酶阳性,Voges Proskauer实验阴性,硝酸盐还原阳性。水解淀粉、CMC和吐温80,不水解明胶、酪蛋白、海藻糖和DNA。主要脂肪酸为sum in feature 3 (C16:1ω7c and/or iso-C15:0 2-OH), C16:0和C18：1ω7c。16S rRNA基因的系统发育树分析表明菌株G21属于弧菌属,并且与菌株V.furnissii ATCC 350116T和V.fluvialis LMG 7894T处于同一分枝,其16S rRNA基因与两个近缘菌株的同源性分别97.4%和97.1%。DNA-DNA杂交实验表明该菌株与两个近缘菌株的全基因组DNA杂交率分别为59.5%和58.6%,低于70%的判定标准,可以确认是新的菌种。对rpoA、recA、ftsZ、mreB和gapA等多位点序列进行分析,进一步确认菌株G21为新的菌种。其DNA G+C含量为46.0 mO1%。根据系统进化、表型特征、生理生化特征和DNA-DNA杂交分析,可以确定菌株G21为弧菌属一个新的菌种,命名为Vibrio xiamenensis sp. nov.,模式菌株为G21T (=DSM 22851T=CGMCC 1.10228T)。2.内切纤维素酶Cel5A的基因克隆、表达和性质分析截至目前,还没有来自弧菌属菌株的纤维素酶基因克隆表达的相关研究报道。本部分,我们克隆了菌株G21的纤维素酶基因,获得一个全长1362 bp、编码453个氨基酸的内切-p-1,4-葡聚糖酶基因,命名为cel5A。其蛋白由一个糖水解酶5家族(GH5)的催化结构域和2家族的纤维素结合结构域(CBM2)组成。该酶的催化结构域与菌株Teredinibacter turnerae T7902的双功能酶beta 1,4-endoglucanase/cellobiohydrolase同源性最高,为69%。将纤维素酶Cel5A在大肠杆菌中进行重组表达、纯化和性质分析。其最适反应pH值和温度分别为pH6.5-7.5和50℃。该酶具有很好的耐碱性,在pH 5.5-10.5的范围内处理1h,仍保持90%以上的活性。同时,分析发现碱溶液对该酶具有激活作用,经弱碱性溶液处理1h后,酶的活性提高了15%。Cel5A也具有很好的耐盐性,添加NaCl至0.5 M后,活性提高了60%,在4 M NaCl条件下,活性仍高于未添加NaCl时的酶活,进一步的研究表明Cel5A热稳定性在0.4 M NaCl下也有所升高。Cel5A特异性作用于木质纤维素的无定形区域,最终水解产物主要为纤维二糖。鉴于其耐盐、耐碱性质,Cel5A在工业应用上具有潜在的应用价值。另外,氯化钠提高酶的稳定性及碱液对该酶的激活作用等,也让该酶成为研究酶的性质和结构关系的理想材料。3.红树林厌氧纤维素降解菌群的富集传代培养及鉴定自然界中,木质纤维素在很大程度上是被厌氧环境下的微生物菌群降解掉的,这些菌群在自然条件下保持稳定,并表现出高效的纤维素降解效率,研究这种状态下的纤维素降解特点和机制,将有助于纤维素的开发利用。在本研究中,我们以采集于海南红树林的泥样为材料,富集厌氧纤维素降解菌群。通过27代的连续传代培养,共有8个菌群样品可以保持高效稳定的纤维素降解效率。菌群SQD-1.1可以在三天中有效地降解滤纸底物,并且能以滤纸为唯一碳源生长,另外还可有效降解木聚糖底物,因此我们对该菌群做了进一步的研究分析。PCR-DGGE分析表明,菌群SQD-1.1在10天的连续培养过程中,其结构保持稳定,不同转接代数之间的菌群结构也基本稳定；16S文库测序分析发现,该菌群有27个OUT序列,分布于7个细菌门中的21个属,包括一个可能的纤维素降解菌M117；通过平板分离获得了12个不同的菌株,其中包括5个可能的新种,对其进行酶活性检测,发现菌株P2具有纤维素酶活性,推断在菌群降解纤维素过程中发挥重要作用。菌群SQD-1.1的研究分析为探索自然环境下厌氧纤维素降解机制提供了很好的材料,为进一步在工业上高效利用木质纤维素生产乙醇提供了参考。4.SQD-1.1宏基因组测序为了从整体上了解菌群结构,获得相关的酶基因序列,我们对菌群SQD-1.1进行了宏基因组测序。共获得1.2 Gbp的DNA序列,拼接组装1 kbp以上的contig1240个。根据EGTs序列分析结果,该菌群主要的微生物类群为变形菌门、螺旋菌门、拟杆菌门和厚壁菌门,其中,与纤维素降解相关的厚壁菌门梭菌属的微生物是主要的组成部分。根据宏基因组注释的结果,找到了包括外切纤维素酶、内切纤维素酶、β-葡萄糖苷酶在内的丰富的纤维素酶基因序列。另外通过blastall比对找到了纤维小体相关的dockerin和cohesin结构域序列片段,证明有纤维小体类似结构的复合物存在。从菌群中预测的纤维素酶基因序列许多都与已知蛋白同源性较低,为新的纤维素酶基因,对这些基因的异源表达和深入研究可以增加纤维素酶资源,也可能揭示新的纤维素降解机制。我们的研究表明,红树林中仍有许多新的纤维素降解微生物和酶资源有待于开发。更多还原

【Abstract】 Mangrove ecosystems are diverse intertidal wetlands commonly situated in tropical, sub-tropical and temperate coastal systems. The degree of bacterial diversity in these systems is expected to be high due to the combination of the mixing of seawater and freshwater and the resuspension of sediments and particles from many sources. The unique swampy, saline and partially anaerobic environment makes it a special source for obtaining microorganisms as well as enzyme and gene resource. Some reports have shown that a large number of bacteria and fungi participate in the cellulolytic processing of the abundant mangrove lignocellulose. In modern industries, cellulases are widely used in textile industry, laundry detergents, pulp and paper industry, and pharmaceutical applications. Cellulase is also used in the fermentation of biomass into biofuels, although this process is relatively experimental now. Thus, exploration of the cellulase-producing bacteria and relative enzymes in the specific mangrove ecosystem is important for further research and industrial applications.In this study, we isolated a cellulase-producing bacterium G21 from the mangrove soil samples.16S rRNA analysis indicated that it belonged to genus Vibrio, and polyphasic taxonomic approach was used to characterize it. Meanwhile, a cellulase gene was cloned from strain G21 and expressed in E. coli BL21, and the recombinant enzyme was characterized. Besides, a high-stable cellulolytic consortium SQD-1.1 was obtained by enrichment from mangrove soil samples and successive subcultivation. The consortium was further analyzed by PCR-DGGE,16S rRNA gene library construction and plate cultivation and metagenomic sequencing.1. Isolation and characterization of strain G21Strain G21 was isolated from mangrove soil samples collected from Xiamen Fujian province of China, when cellulas-producing bacteria were screened using CMC-Na as substrate. The cells of G21 are Gram-negative, slightly curved rods (0.7-0.8μm×1.4-1.55μm) and motile with a single polar flagellum. Its colonies on marine 2216E agar plate are circular to a little fusiform, white and half-translucent after incubation at 37℃for 24 h. The strain grows in the range of 15-40℃and 0.5-10% NaCl(w/v).Growth of G21 in anaerobic condition is detected in the presence of 10 mM glucose, but no gas was produced. Positive for oxidase and catalase. Negative for Voges Proskauer. Nitrate is reduced to nitrite. Starch, CMC and Tween 80 are hydrolyzed. Gelatine, casein, alginate and DNA are not hydrolyzed. The major cellular fatty acids of G21 were sum in feature 3 (C16:1ω7c and/or iso-C15:0 2-OH), C16:0 and C18:1ω7c.Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain G21 belonged to the genus Vibrio and formed a cluster with V. furnissii ATCC 350116T (sequence similarity,97.4%) and V.fluvialis LMG 7894T (97.1%). However, multilocus sequence analysis (rpoA, recA, ftsZ, mreB and gapA) and DNA-DNA hybridization experiments indicated that it was distinct from related Vibrio species. The DNA G+C content was 46.0 mol%.Based on the phylogenetic, phenotypic, chemotaxonomic and DNA-DNA hybridization analysis, it is concluded that strain G21 represents a novel species of the genus Vibrio, for which the name Vibrio xiamenensis sp. nov. is proposed. The type train is G21 (=DSM 22851T=CGMCC 1.10228T).2. Gene cloning and characterization of cellulase Cel5AAlthough cellulases have been isolated from various microorganisms, no functional cellulase gene has been reported from the Vibrio genus until now. In this study, a novel endo-β-1,4-glucanase gene, cel5A,1362 bp in length, was cloned from the newly-identified bacterium, Vibrio sp. G21. The deduced protein of cel5A contains a catalytic domain of glycosyl hydrolase family 5 (GH5), followed by a cellulose binding domain (CBM2). The catalytic domain of Cel5A shows the highest sequence similarity (69%) to the bifunctional beta 1,4-endoglucanase/cellobiohydrolase from Teredinibacter turnerae T7902.The gene cel5A was overexpressed in Escherichia coli and the mature Cel5A enzyme was purified to homogeneity. The optimal pH and temperature of the recombinant enzyme were determined to be 6.5-7.5 and 50℃, respectively. Cel5A was stable over a wide range of pH and retained more than 90% of its total activity even after treatment in pH 5.5-10.5 for 1 h, indicating its high alkali resistance. Moreover, the enzyme was activated after pretreatment with mild alkali, a novel characteristic that has not been previously reported in other cellulases. Cel5A also showed a high level of salt tolerance. Its activity rose to 1.6-fold in 0.5 M NaCl and remained elevated even in 4 M NaCl. Further experimentation demonstrated that the thermostability of Cel5A was improved in 0.4 M NaCl. In addition, Cel5A showed specific activity towardsβ-1,4-linkage of amorphous region of lignocellulose, and the main final hydrolysis product of carboxymethylcellulose sodium and cellooligosaccharides was cellobiose.Considering the salt-tolerant and alkali-resistant properties of the enzyme, Cel5A of Vibrio sp. G21 has the potential to be used in industrial applications. Additionally, the characteristics of the enzyme, including thermostability improvement in salt solution and activation by alkaline, make it a good candidate for further research on the structure-function relationship and enzyme directed evolution.3. Enrichment and characterization of anaeraobic cellulolytic microbial consortiumIn nature, lignocellulose is partly decomposed by microbial communities inhabiting in anaerobic environments. These communities can keep stable and show high cellulolytic efficiency in native conditions. In this study, several mangrove soil samples from Hainan province were used to enrichment anaerobic cellulolytic microbial consortia. Eight consortia were transferred continually for 27 times and still could keep stable for efficient cellulose degradation.Consortium SQD-1.1, which can efficiently degrade filter paper in three days, utilize cellulose as the sole carbon source, and degrade xylan efficiently, was further characterized by PCR-DGGE,16S rRNA gene library and plate cultivation methods. Time-course analysis of the community composition within 10 days by PCR-DGGE showed that the members in SQD-1.1 were stable and no obvious change in consortium structure was found. The compostion of different generations was also stable.16S rRNA gene library analysis showed the presence of 27 OUT sequences, belonging to 21 genera of 7 phyla, and a potential cellulolytic bacterium M117 was found. Isolates using plate cultivation method both under anaerobic and aerobic conditions were belonged to Clostridium, Trichococcus and Proteiniclasticum and so on, of which five were potential new species and/or new species of new genus. Further analysis showed that strain P2 showed endoglucanase activity and should played an important role in the consortium in cellulose degradation.The study of SQD-1.1 can provide a candidate for investigating the mechanism of anaerobic cellulose degradation in nature environments, which is in favor of the industrial lignocellulose utilization for ethanol production.4. Metagenomic sequencing and analysis of consortium SQD-1.1In order to mine the gene and enzyme resources of consortium SQD-1.1, the metagenome DNA of consortium SQD-1.1 was sequenced.1.2 Gbp of DNA sequences was obtained and 1240 contigs longer than 1 kbp were assembled. According to the analysis result of EGTs database, the consortium mainly contains microorganisms belonging to Proteobacteria, Spirochaetes, Bacteroidetes and Firmicutes, and the potential anaerobic cellulolytic genus Clostrium of phylum Firmicutes was one of the main members. Annotation result showed that there were all of three types of cellulase, including endoglucanase, exoglucanase andβ-glucanase in the consortium, many of which are novel in sequence. Further analysis using the Blastall tool indicated that the consortium contained gene fragments belonging to dockerin and cohesion domains, the main components of cellulosome in anaerobic cellulolytic bacteria.As the cellulase genes from metagenomic sequences of consortium SQD-1.1 shared low similarity with proteins in GenBank database, the enzymes should be novel. Expression and further study of these novel cellulose genes can expand the source of cellulases, and may reveal new cellulose-degradation mechanism. Meanwhile, our study indicated that there are still many cellulolytic microorganism and enzyme resources in mangrove ecosystems to explore.更多还原