【作者】 周峻沛；

【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， 2010， 博士

【摘要】 纤维素酶及半纤维素酶在多种工业领域,包括在饲料工业中都具有巨大的应用价值。天牛幼虫以木材为食,是研究纤维素酶及半纤维素酶的高价值材料。胃肠道内共生细菌蕴藏着丰富多样的纤维素酶及半纤维素酶,但天牛胃肠道细菌来源纤维素酶及半纤维素酶的相关报道却极为有限。本研究使用简并引物同源克隆的方法,从云斑天牛(Batocera horsfieldi)幼虫胃肠道分离培养的34株细菌中,总共获得26个纤维素酶或半纤维素酶编码基因片段,包括糖苷水解酶(GH)第3家族β-葡糖苷酶、48家族纤维二糖水解酶、10和11家族内切木聚糖酶、52家族β-木糖苷酶、67家族α-葡糖醛酸糖苷酶、16家族β-葡聚糖酶、2家族β-半乳糖苷酶和36家族α-半乳糖苷酶。同时,从云斑天牛幼虫胃肠道环境基因组中直接获得了纤维素酶和半纤维素酶编码基因片段,包括第5和45家族内切纤维素酶,48家族纤维二糖水解酶,10家族内切木聚糖酶,39和52家族β-木糖苷酶和67家族α-葡糖醛酸糖苷酶。选取第5和第10家族的氨基酸片段进行多样性分析,Shannon指数分别为2.68和2.09。系统发育分析指出这些氨基酸序列可能并不来源于多样的细菌门类,而主要是源于单个细菌门下的多样的细菌属或种类。通过与松墨天牛(Monochamus alternatus)虫洞环境、瘤胃和白蚁胃肠道来源序列的比较分析(第10家族序列仅与虫洞环境序列比较),揭示了云斑天牛胃肠道环境第5和第10家族的氨基酸序列的特异性(p<0.001)。改进了常规热不对称交错聚合酶链式反应(TAIL-PCR)方法以使操作更简便、更高效,同时建立了针对鸟嘌呤(G)和胞嘧啶(C)含量高的基因的GC TAIL-PCR方法,结果获得了由25个基因构成的木聚糖和木糖代谢染色体片段、6个纤维素酶或半纤维素酶编码基因和2个高GC含量(xynA119中GC含量为67.8%,xynB119中GC含量为67.3%)的木聚糖酶基因。选取其中11个酶作进一步研究,包括来源于Sphingobacterium sp. TN111的第16家族β-葡聚糖酶GluA111,Serratia sp. TN49的第3和家族β-葡糖苷酶BglA49,Sphingobacterium sp. TN19的2个第10家族内切木聚糖酶XynA19和XynB19、43家族α-阿拉伯呋喃糖苷酶Gh43A19和36家族α-半乳糖苷酶GalA19,Streptomyces sp. TN119的第10和11家族内切木聚糖酶XynA119和XynB119,Sphingobacterium sp. TN105的第2家族β-半乳糖苷酶BgaA105和36家族α-半乳糖苷酶GalA105及Flavobacterium sp. TN17的第36家族α-半乳糖苷酶GalA17。这11个酶与数据库中已发表序列的氨基酸序列最高一致性为38.8–80.1%。系统发育分析发现内切木聚糖酶XynA19和α-半乳糖苷酶GalA17与其同分类地位细菌来源的序列亲缘关系较远,而与胃肠道共生细菌来源的序列相近;但第3家族β-葡糖苷酶BglA49却未具有这样的特征。进一步的分析发现胃肠道环境共生细菌来源的第36家族α-半乳糖苷酶具有潜在催化域KWD和SDXXDXXXR,不同于已知具有晶体结构的α-半乳糖苷酶催化域KXD和RXXXD。突变分析表明KWD中的D480,SDXXDXXXR中的S548、D549和R556是GalA17具有酶活的必要氨基酸,其中,D480和D549可能是催化残基。将上述11个酶编码基因都重组到pET载体,并转化到Escherichia coli BL21(DE3)中表达,除了GluA111未能检测到活性外,其余10个都检测到了相应的酶活。利用组氨酸标签纯化了重组的XynA119、XynB119、XynA19、BglA49及GalA17。对这些纯化酶进行酶学性质测定,结果显示:最适pH都在中性范围内(pH5.5–7.5),pH稳定性在中性pH内最佳,最适温度较低(35–60°C),热稳定性较差(低于50°C但都能在37°C稳定)。这些共同特征可能与天牛生存环境及肠道生理环境密切相关。同时,GalA17具多种蛋白酶抗性(胰蛋白酶、胶原蛋白酶、α-糜蛋白酶和蛋白酶K);XynB119在很宽的pH范围内稳定(pH1.0–11.0)并对多种蛋白酶(胃蛋白酶、胰蛋白酶、胶原蛋白酶、α-糜蛋白酶和蛋白酶K)有抗性;XynA19在低温具有活性(10°C时~40%的活性),同时具有低温内切木聚糖酶中相对较好的热稳定性(40°C稳定);BglA49也在低温具有活性(10°C时酶活>20%);与嗜热或中温第10家族内切木聚糖酶和第3家族β-葡糖苷酶结构相比,XynA19和BglA49在结构上的离子键和氢键数量大幅降低。GalA17、XynB119、XynA19和BglA49的性质特点使其在食品和饲料添加剂方面具一定的应用潜力。本文首次研究了云斑天牛胃肠道这一特殊生态环境中共生细菌来源的纤维素酶和半纤维素酶,从酶的多样性、系统发育、高级结构和酶学性质方面进行了相关探讨,增进了对食木昆虫胃肠道内的纤维素酶和半纤维素酶的基础认识,发掘了具有应用潜力的纤维素酶及半纤维素酶。更多还原

【Abstract】 Cellulases and hemicellulases have great potential for application in various industries, including the feed industry. The larvae of longhorned beetles (Cerambycidae) can develop deep within sapwood, and are thought to be a potential source harboring diverse and novel symbiotic microorganisms producing cellulases and hemicellulases. So far enzymes from symbiotic microorganisms in the gut of longhorned beetles still remain extremely limited.Using the homologous cloning strategy with degenerate primers, 26 gene fragments encoding cellulases or hemicellulases were cloned from the 34 bacterial strains isolated from the gut of Batocera horsfieldi larvae. These cellulases or hemicellulases are glycosyl hydrolase family (GH) 3β-glucosidases, GH 48 cellobiohydrolases, GH 10 and GH 11 endo-xylanases, GH 52β-xylosidases, GH 67α-glucuronidases, GH 16β-1,3(4)-endoglucanases, GH 2β-galactosidases, and GH 36α-galactosidases. Environmental DNA of luminal contents (TNG) of B. horsfieldi larvae was extracted and used as the template to clone gene fragments encoding cellulases or hemicellulases. As a result, some gene fragments related with GH 5 and GH 45β-1,4-endoglucanases, GH 48 cellobiohydrolases, GH 10 endo-xylanases, GH 39 and GH 52β-xylosidases, and GH 67α-glucuronidases were isolated. Furthermore, gene fragments of GH 5 and GH 10 were chosen for diversity analysis. Their Shannon indexes were 2.68 (GH 5) and 2.09 (GH 10), respectively. Phylogenetic analysis indicated that the GH 10 and GH 5 protein fragments from TNG were novel and diverse, and present at limited breadth of bacterial phyla but much greater in depth in terms of genera and species. Significant difference (p < 0.001) between GH 10 or GH 5 protein fragments from TNG and that from the wormhole niche of Monochamus alternatus, and among GH 5 protein fragments from gastrointestinal habitats (TNG, termite gut or rumen) revealed environment-specific distributions of GH 10 and GH 5 proteins in TNG.In this study, the conventional thermal asymmetric interlaced-PCR (TAIL-PCR) technique was improved for efficacy and ready-to-use, and GC TAIL-PCR specific for GC-rich genes was developed. As a result, a chromosomal segment containing 25 genes related to xylan and xylose metabolism and showing xylanolytic activity, 6 cellulases or hemicellulases encoding genes, and 2 xylanases encoding genes (xynA119 and xynB119) with 67.8% and 67.3 GC content, respectively, were obtained. Among them, 11 enzymes were selected for further study, including the GH 16β-1,3(4)-endoglucanase GluA111 from Sphingobacterium sp. TN111; GH 3β-glucosidase BglA49 from Serratia sp. TN49; GH 10 endo-xylanases XynA19 and XynB19, GH 43 arabinofuranosidase Gh43A19, and GH 36α-galactosidase GalA19 from Sphingobacterium sp. TN19; GH 10 endo-xylanase XynA119 and GH 11 endo-xylanase XynB119 from Streptomyces sp. TN119; GH 2β-galactosidase BgaA105 and GH 36α-galactosidase GalA105 from Sphingobacterium sp. TN105; and GH 36α-galactosidase GalA17 from Flavobacterium sp. TN17. The deduced amino acid sequences of these 11 enzymes showed the highest identity of 38.8–80.1% with their counterparts in public databases. Phylogenetic analysis revealed that endo-xylanase XynA19 andα-galactosidase GalA17 were more closely related to their counterparts from symbiotic bacteria in the gastrointestinal habitats than to that from bacteria belonged to the same phylum. The GH 3β-glucosidase BglA49 had no such characteristic. GH 36α-galactosidases from symbiotic bacteria shared two putative catalytic motifs KWD and SDXXDXXXR instead of KXD and RXXXD motifs of knownα-galactosidases. Site-directed mutagenesis indicated four essential amino acid residues (D480 in KWD; S548, D549, and R556 in SDXXDXXXR) involved in theα-galactosidase activity, among which D480 and D549 were the putative catalytic residues.The eleven genes mentioned above were inserted into pET vector and expressed successfully in Escherichia coli BL21 (DE3). All recombinant enzymes showed activity except for gluA111. The recombinant enzymes XynA119, XynB119, XynA19, BglA49 and GalA17 were purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography and subjected to biochemical characterizations. All of the five enzymes showed optimal activity at neutral pH (pH 5.5–7.5) and low temperature (35–60°C), and were more stable at neutral pH and temperatures lower than 50°C. These common features are putatively dependent on the living environment and gut physiological environment of B. horsfieldi larvae. GalA17 was resistant to various proteases (trypsin, collagenase, proteinase K andα-chymotrypsin). XynB119 remained stable over a broad pH range (pH 1.0–11.0), and had strong protease resistance (pepsin, trypsin, collagenase, proteinase K andα-chymotrypsin). XynA19 remained active at low temperatures (retained ~40% xylanase activity at 10°C), and was more stable than known low-temperature endo-xylanases at 40°C. BglA49 was also active at low temperature (remained >20% activity at 10°C). Compared to the thermophilic and mesophilic counterparts, XynA19 and BglA49 had fewer hydrogen bonds and salt bridges. These properties suggest that GalA17, XynB119, XynA19 and BglA49 have various potential applications in food and feed industries.It is the first report to explore the diversity, phylogenesis, structure and properties of cellulases and hemicellulases from symbiotic bacteria harbored in the gut of B. horsfieldi larvae. The information improves our knowledge of cellulases and hemicellulases in the gut of wood-feeding insect and promotes the development of novel industrial enzymes.更多还原