【作者】 王国增；

【导师】 姚斌；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 木聚糖是植物细胞壁的主要组成成分,也是自然界中除纤维素之外第二丰富的再生资源。木聚糖的结构复杂,其完全降解需要多种酶的共同作用。木聚糖酶能够有效地降解木聚糖主链的β-1,4-糖苷键,是木聚糖降解中的关键酶。木聚糖酶广泛分布在微生物中,后者通过对木聚糖的降解利用而在自然界中的碳循环中起到很重要的作用。木聚糖酶在饲料、食品、造纸和生物能源等多个工业领域中也有着重要的应用价值而被广泛研究。现在获得的绝大部分的木聚糖酶都是来自分离纯化的微生物,而越来越多的实验表明环境微生物中绝大多数是未培养的。怎样发掘未培养微生物的基因资源,找到具有应用价值的木聚糖酶成为新的研究热点。木聚糖酶主要划分在糖苷水解酶的第10和11家族。本研究通过对这两个家族的木聚糖酶蛋白序列的比对,确定了四个靠近活性中心或底物结合位点的保守区。基于一致-简并杂交寡核苷酸引物(CODEHOP)的3’简并5’保守的原理,设计了两套简并引物,X10-F/X10-R和X11-F/X11-R。该引物可从具有木聚糖降解能力的细菌和真菌代表菌株中高效扩增到目的基因片段,因此用于环境中木聚糖酶基因多样性的研究。以雪莲根际土壤、冰川土壤、温泉高温土壤、菜园土、池塘底泥、和红树林土壤的宏基因组DNA为模板,以X10-F/X10-R或X11-F/X11-R为引物,分别从6个不同性质的土壤环境中扩增木聚糖酶基因片段。除温泉高温土壤中只能扩增到第10家族的木聚糖酶基因片段外,从其他土壤环境中都能扩增得到相应大小的两个家族木聚糖酶的基因片段(第10家族大约260 bp,第11家族210 bp左右)。将PCR产物纯化后连接载体转化构建了11个片段文库。从各个文库中随机挑选了共2115个阳性克隆,经测序及序列分析表明1075个序列是第10家族的木聚糖酶基因片段,684个序列是第11家族的木聚糖酶基因片段。经去冗余分析后,得到了490个相似性低于95%的特异基因片段,其中368个为第10家族的序列,122个第11家族的序列。序列比对分析表明75%的基因片段与已知序列相似性在80%以下,54%的序列与已知基因的相似性低于65%。该结果说明土壤环境中存在大量的木聚糖酶基因,且大部分具有很高的序列新颖性。通过进化,多样性和丰度的分析发现,6个环境中的木聚糖酶基因的分布和多样性都有很大的差别,而且各个环境中丰度较高的基因也不一样,说明不同环境中降解木聚糖的优势菌群存在显著差异。表明土壤环境中的木聚糖酶基因的分布和环境有很大的关系,而土壤环境因子(pH、含氧量、温度、有机质的含量等)可能是造成木聚糖酶基因的分布和多样性差异的主要原因。土壤环境来源的第10家族木聚糖酶绝大多数来源于细菌,而第11家族木聚糖酶序列中真菌来源的比细菌来源的要多。此外,土壤环境中第11家族的木聚糖酶的基因的多样性要远低于第10家族的。这种分布和多样性的不同暗示这两个糖苷水解酶家族在木聚糖的降解过程中可能发挥着不同的作用。瘤胃内存在大量的微生物,是一个植物细胞壁多糖高效降解的环境。通过使用上述方法,从山羊瘤胃和绵羊瘤胃中一共获得了173个特异的木聚糖酶基因片段,其中107个片段为第10家族的木聚糖酶基因片段,其他属于第11家族。序列比对分析表明这些片段与已知木聚糖酶的相似性整体上要比土壤环境来源的相似性要低,说明瘤胃环境中存在大量的更为新颖的木聚糖酶。进一步的基因多样性,丰度和进化分析指出两大家族的木聚糖酶基因来源于不同的微生物,第10家族的木聚糖酶基因主要集中在非纤维素降解微生物,而第11家族的基因主要来源于纤维素降解微生物。该结果表明两个家族的木聚糖酶在瘤胃环境内木质纤维素降解过程中发挥的作用是不一样的。此外,不同瘤胃来源的木聚糖酶基因比较分析发现瘤胃中木聚糖降解微生物的分布是类似的,但也存在一定的差异。每个瘤胃环境中发挥主要木聚糖降解作用的菌物不同,且都存在特有的木聚糖降解微生物。而这种差异可能与宿主对微生物的选择有一定的关系。基于10个山羊瘤胃环境来源的木聚糖酶基因片段序列,采用改进的TAIL-PCR方法,以山羊瘤胃宏基因组DNA为模板,最终获得了7个全长基因。其中,xynGR40,xynGR67,xynGR77,xynGR112和xynGR117编码的木聚糖酶属于第10家族,xynR8和xynR127编码第11家族的木聚糖酶。序列比对分析发现这些基因与已知基因的相似性为45–75%,表明这些基因都具有很高的新颖性。除XynR8无信号肽外,其它的蛋白都含有信号肽,可以分泌表达。通过结构域分析,发现XynGR40,XynGR77,XynGR117和XynR127是多结构域蛋白,而且其中的XynGR77和XynR127具有特殊的结构,这为以后的木聚糖酶的结构与功能研究提供了很好的实验材料。其中的四个木聚糖酶基因xynGR40,xynGR67,xynR8和xynR127经原核表达后,重组酶进行了纯化和酶学性质分析。这四个酶的比活相对于大多数的木聚糖酶都高,这与瘤胃来源的酶具有相对较高的催化活性是一致的。它们的最适pH在5.5到6.5之间,与瘤胃液的pH(5.8)接近。最适温度差异很大,低至30°C (XynGR40),高至55°C (XynR8)。XynGR40有低温活性,在0°C还剩余10%的酶活。氨基酸的组成、盐桥和氢键的分析以及与嗜热酶在三维结构上的比较说明XynGR40是一个低温木聚糖酶。XynGR40是第一个从瘤胃环境中报道的低温木聚糖酶,它的特殊性质和结构为研究木聚糖酶的结构与功能的关系提供了很好的材料。同时,XynGR40在中温下的热稳定性、高比活、较好的抗离子能力以及产物简单等特性表明XynGR40在工业上有良好的应用前景。总而言之,本研究建立了一个快速、有效的不需要微生物培养的分子生物学方法用于分析复杂环境中的木聚糖酶基因多样性。从特殊环境中获得了大量的新颖的木聚糖酶基因片段。结果表明这些木聚糖酶基因的分布和环境有很大的关系。基于部分片段序列,通过TAIL-PCR的改进方法,可以直接从环境宏基因组DNA中获得了木聚糖酶全长基因。本研究为环境中木聚糖酶的基因多样性和分布提供了一个新的视点,对理解它们在木聚糖自然降解很有意义,也为未培养微生物木聚糖酶基因资源的发掘提供了一个新的途径。更多还原

【Abstract】 Xylan is one of the major components of plant cell wall, and is the second most abundant renewable material after celluose in nature. A large variety of cooperatively acting enzymes are required for complete hydrolysis of xylan because of its complex structure. Among them, endo-1,4-?-D-xylanase (EC 3.2.1.8) is a crucial enzyme that cleaves the ?-1,4 backbone of xylan. Xylanases are widely distributed in microorganisms, thus the latter can degrade xylan, utilize it as energy source and consequently play an important role in carbon cycle on earth. Xylanases have been extensively studied because of their great applications in animal feed, baking, pulp and paper, biofuel, and other industries. So far most xylanases are from cultured microorganisms, which is limited since vast majority of microorganisms are uncultivable. How to retrieve novel xylanases from those uncultured microbes is a hot point in the xylanase study.Xylanases are mainly confined into glycoside hydrolase (GH) family 10 and 11. Based on the sequence alignments of GH 10 and 11 xylanases, two conserved regions in close proximity to active sites or substrate binding sites were identified, respectively. According to the principles of COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP), two sets of primers, X10-F/X10-R and X11-F/X11-R, were designed. The primer sets were verified to be specific and efficient by successful amplification of objective gene fragments from xylan-degrading bacteria and fungi of various taxa, and used to study the diversity of xylanases in environments.By using the metagenomic DNA of rhizosphere soil of snow lotus, glacier soil, hot spring soil, farmland soil, pond sediment, and Mangrove soil as templates, X10-F/X10-R or X11-F/X11-R were used to amplify objective gene fragments. Except for the hot spring soil that had only GH 10 xylanase gene fragments, gene fragments of both GH 10 and 11 were amplified from other soils (about 260 bp for GH 10 and about 210 bp for GH 11). PCR products were purified and used to construct 11 clone libraries. A total of 2115 positive clones were randomly picked up from 11 libraries and sequenced. Of them, 1075 sequences were confirmed to be GH 10 xylanase gene fragments, and 684 belong to GH 11. After removing the redundant sequences, 490 distinct xylanase gene fragments (368 GH 10 and 122 GH 11) shared similarities of below 95%. Based on BLASTx analysis, 75% of the distinct fragments had similarities of below 80% with known xylanases and 54% shared similarities of below 65% with known ones. This result suggested that there are abundant xylanases in environments and most of them are novel. Based on phylogenesis, diversity and abundance analysis, these xylanase gene fragments are different in distribution and diversity, and vary in predominance. Thus the predominant xylan-degrading microorganisms in each soil environment are assumed to be different. This variance might be related to the soil factors, such as pH, oxygen content, temperature, organic matter content and so on. The vast majority of GH 10 xylanase gene fragments are from bacteria, and more GH 11 sequences are related to fungi. Moreover, the diversity of GH 11 xylanase in these soil environments is much lower than that of GH 10. The variance of diversity and distribution of GH 10 and 11 xylanases may imply their different roles in the xylan degradation in nature.Rumen harbors immensely diverse microorganisms and is a special environment in which the microbial-mediated hydrolysis of plant cell wall polysaccharides is highly efficient. By using the same method described above, a total of 173 distinct xylanase gene fragments, 107 GH 10 and 66 GH 11, were obtained from the metagenomic DNA of goat and sheep rumen. Sequence analysis showed that the similarities of these fragments with known sequences are much lower than those of soil environments with known sequences, which suggested that numerous novel xylanases exist in the rumen microenvironment. Further gene diversity, abundance and phylogenetic analysis indicated that xylanase genes of different GH families are from different taxa. GH 10 rumen xylanases are mostly distributed in noncellulolytic microorganisms, and GH 11 xylanase genes in cellulolytic microorganisms, implying their different roles in xylan degradation. Both rumens harbored similar xylan-degrading microbial communities based on the sequence comparison analysis. Each rumen has different predominant xylan-degrading microorganisms and harbors unique xylanase producers however, implying that some xylan-degrading microorganisms might be host specific.Based on the sequences of 10 xylanase gene fragments obtained from goat rumen, 7 full-length xylanase genes were cloned directly from the metagenomic DNA using a modified TAIL-PCR method. Of them, xynGR40,xynGR67,xynGR77,xynGR112 and xynGR117 encode GH 10 xylanases, and xynR8 and xynR127 are GH 11 xylanase-encoding genes. Sequence analysis showed that these genes have low identities (45–75%) with known xylanases, suggesting their novelty. Except for XynR8 that has no putative signal peptide, all other xylanases were predicted to have signal peptides and can secrete into the environment surroundings. Domain analysis indicated that XynGR40,XynGR77,XynGR117 and XynR127 are multi-domain proteins, and XynGR77 and XynR127have special structures. Thus this study provides valuable materials to study the relationships of xylanase structure and function.Four xylanase genes,xynGR40,xynGR67, xynR8 and xynR127 were expressed in Escherichia coli BL21 (DE3), the recombinant proteins were purified and characterized. Like those xylanases from rumen microenvironments that have high catalytic activities, these four recombinant xylanases had higher specific activity towards xylan than most xylanases. The pH optima ranged from 5.5 to 6.5, which are close to the pH of rumen fluid (5.8). The temperature optima varied from 30°C (XynGR40) to 55°C (XynR8). XynGR40 had low temperature activity, retaining about 10% of the activity even at 0°C. Analysis of the amino acid composition, hydrogen bonds, and salt bridges and structure comparison with thermophilic xylanases indicated that XynGR40 is a cold active xylanase. XynGR40 is the first cold active xylanase from rumen microenvironments. Its special characteristics and structures make XynGR40 a good material to study the relationship of xylanase structure and function of. Moreover, XynGR40 is thermostable at mesophilic temperatures, has high catalytic efficiency at low temperatures, is resistant to most ions, and produces simple hydrolysis products, thus has great potential for industrial applications.In summary, this study developed a rapid and efficient culture-independent molecular method to explore the diversity and distribution of xylanase genes in complex environments. By using this method, a large number of novel xylanase gene fragments were obtained from special microenvironments. The distribution of these genes is environment specific. Full-length xylanase genes can be obtained directly from metagenomic DNA using a modified TAIL-PCR method based on the sequences of some fragments. Our study draws an insight into the diversity and distribution of xylanases in various environments, which is very meaningful to understand their roles in xylan degradation in nature. Moreover, this study also provides a new way to obtain novel xylanase genes from uncultivated microorganisms.更多还原