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题名	极端嗜热热解纤维素菌糖苷水解酶热稳定性及底物特异性机制研究
作者	孟冬冬
学位类别	博士
答辩日期	2014-11
授予单位	中国科学院研究生院
授予地点	北京
导师	李福利
关键词	木质纤维素 嗜热菌 糖苷水解酶 热稳定性 晶体结构
学位专业	生物化学与分子生物化学
中文摘要	极端嗜热厌氧细菌Caldicellulosiruptor sp. F32可以在75 °C条件下，直接降解未经预处理的木质纤维素，并利用水解产生的五碳糖、六碳糖生长、发酵，主要产物是乙酸、乳酸、H2等，产生少量乙醇。在水解木质纤维素过程中，菌株产生大量的糖苷水解酶（Glycoside hydrolyase，GH），高效的把纤维素、半纤维素转化为单糖。不同于真菌分泌的游离酶及一些细菌分泌的纤维小体，这些酶往往由多个模块组成。研究多模块酶组装机制，对于酶热适应性进化及底物特异性机制，具有重要的生物学意义。本文首次对Caldicellulosiruptor sp. F32基因组中的73个CAZymes（Carbohydrate-Active Enzymes，碳水化合物活性酶）进行归类研究。结果显示，该菌中包含44个糖苷水解酶，分布于22个不同的家族，至少包含4个内切葡聚糖酶以及3个木聚糖酶；22个β-或α-糖苷酶；6个碳水化合物酯酶及3个多糖裂解酶；另有多达23个CBM模块，分属于11个不同的家族。上述分析表明，F32含有极其丰富的CAZymes资源，它们协同作用赋予菌株极强的木质纤维素降解能力。对Caldicellulosiruptor sp. F32的GH10和GH11家族的木聚糖酶进行表征，缺失CBM36的GH11家族的XynATM1与全长蛋白质XynA相比，比酶活提高2.3倍，75 °C半衰期提高8.7倍。与之相反，含有两个CBM22模块的GH10家族的XynB，在缺失CBM模块后，比酶活及热稳定性都急剧降低。N端氨基酸突变体XynATM1-M在75 °C下的半衰期为48 h，较XynATM1提高1.5倍，表明木聚糖酶末端非规则区域氨基酸对蛋白质的热稳定性具有重要作用。对蛋白质热动力学稳定性进行研究，差示扫描量热法 (Differential Scanning Calorimetry, DSC)结果表明，木聚糖酶溶解温度Tm与其热稳定性正相关。同源模建及化学交联法分析显示，XynB存在分子内相互作用，而XynA没有。我们推断模块间刚性linker氨基酸组成造成蛋白质XynA采取伸展构象，不具有分子内相互作用，而XynB与之相反。因此，木聚糖酶热稳定性取决于模块本身的酶学性质和模块间相互作用。F32EG5是GH5家族糖苷水解酶，与来源于解纤维梭菌的内切葡聚糖酶CelCCA具有最高的序列相似性53%。酶学性质表明，该蛋白质具有1284 U/mg的大麦葡聚糖酶活，几乎不具有CMCase活力，最适温度80 °C，最适pH 5.5，在80 °C的半衰期为8 h。上述分析显示，F32EG5是典型的地衣聚糖酶。通过核磁共振分析、寡糖酶解产物分析，F32EG5具有强的切断β-1,3糖苷键的能力，同时具有弱的β-1,4糖苷键水解能力，这与以往β-1,3-1,4-葡聚糖酶不同，是一种新型地衣聚糖酶。晶体结构显示，F32EG5与其他GH5家族蛋白质采取相同的(α/β)8桶状折叠，具有相同的催化机制。同1EDG、3NDZ等CMCase/地衣聚糖酶等双功能酶相比，F32EG5底物结合位点+1、+2、+3位氨基酸残基的特异性，决定其独特的底物选择性。另外，+3位底物结合位点的E247和R275形成的盐键极大增加了野生型蛋白质的热稳定性。分子对接结果显示，F32EG5对葡四糖的催化效率依次为Li4C>Li4B>G4>Li4A，进一步阐明了F32EG5的催化机制和底物选择性机制。本论文首次对GH5家族的嗜热地衣聚糖酶结构—功能关系进行研究，揭示了β-1,3-1,4-葡聚糖的降解机制。
英文摘要	Caldicellulosiruptor sp. F32, can degrade un-pretreated lignocellulosic biomass at 75 °C directly, and ferment C5 and C6 sugars to acetic acid, lactic acid, H2, and a small amount of ethanol. Cellulose and hemicellulose were converted into monosaccharides efficiently, facilitated by a diverse set of glycoside hydrolases (GHs). Differ with free enzymes secreted by fungi and cellulosome produced by some bacteria, cellulases from Caldicellulosiruptor sp. F32 usually are multi-modular enzymes. Research on assembly mechanism of multi-modular cellulases, have important biological significance for thermal adaptation evolution and substrate specificity of these enzymes. In the present study, 73 carbohydrate-active enzymes (CAZymes) from Caldicellulosiruptor sp. F32 were classified. The results showed that there are 44 glycoside hydrolases distributed in 22 different families, including at least 4 endoglucanases, 3 xylanases, 22 β- or α- glycosidases, 6 polysaccharide lyases, 2 esterases, and 23 CBMs belong to 11 different families. The above analysis showed that F32 contains abundant CAZymes and their synergistic effect confers it strong lignocellulosic biomass degradation ability. The xylanases GH family 11 XynA and GH10 XynB from Caldicellulosiruptor sp. F32 were overexpressed in Escherichia coli, purified and characterized. GH11 XynATM1 lacking CBM36 exhibited a considerable improvement in specific activity and thermal stability compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM22. Site-directed mutagenesis at N-terminal amino acid resulted in a XynATM1-M with 50% residual activity improvement at 75 °C for 48 h, revealing that the non-regular region disturbed protein thermostability. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (Tm) determined by using differential scanning calorimetry (DSC). Through homology modeling and cross-linking experiments analysis, we demonstrated that for GH10 XynB, the resistance against thermoinactivation was generally contributed by both domain properties and inter-domain interactions, whereas for GH11 XynA, no inter-domain interactions observed. We speculated optimized intra-molecular interaction is determined by catalytic domain and CBM through fine tuning rigidity and flexibility of linker, which provides microbes a powerful evolution strategy to assemble catalysts adapted to various ecological conditions. F32EG5 has the highest identity of 53% with endoglucanase CelCCA, which from Clostridium cellulolyticum. Enzymatic properties showed that, F32EG5 have 1284 U/mg specific activity against barley β-glucanase, and the half-life was 8 h at its optimal conditions of 80 °C and pH 5.5. All the results indicated that F32EG5 was a novel thermostable specific lichenase. Through NMR and gluco-oligosaccharides hydrolysis, we demonstrated that F32EG5 had strong ability to break the β-1,3 linkage after a β-1,4 linkage, as well as the β-1,4 linkage before a β-1,3 linkage, which is completely different from the extensively studied GH family 16 lichenase with a cleavage site on the β-1,4 linkage after a β-1,3 linkage. The structure of F32EG5 is a typical (α/β)8 barrel fold, and has same catalytic mechanism as other GH5 enzymes. Compared with CMCase/lichenase (1EDG and 3NDZ), F32EG5 exhibited distinct exit sites (+1, +2, +3) in the substrate binding cleft, which play key roles in the substrate specificity. A salt bridge formed by Glu247 and Arg275 occupy the +3 position have vigorous effect on enzyme catalytic and thermostability ability. The molecular docking of different glucotetraoses on F32EG5 further confirmed the catalytic mechanism and substrate selectivity of F32EG5. Therefore, F32EG5 exhibits a novel type lichenase with high thermostability and activity with a distinct the cleavage pattern for β-1,3-1,4-mixed linkage glucan. This dissertation for the first time expounded the structure−function relationship of a GH5 lichenase, which revealed the degradation mechanism of β-1,3-1,4-glucan.
语种	中文
学科主题	微生物学
公开日期	2016-06-30
内容类型	学位论文
源URL	[http://ir.qibebt.ac.cn/handle/337004/8077]
专题	青岛生物能源与过程研究所_微生物资源团队
作者单位	中国科学院青岛生物能源与过程研究所
推荐引用方式 GB/T 7714	孟冬冬. 极端嗜热热解纤维素菌糖苷水解酶热稳定性及底物特异性机制研究[D]. 北京. 中国科学院研究生院. 2014.

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