极端嗜热厌氧热解纤维素菌F32木质纤维素降解机制研究

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	极端嗜热厌氧热解纤维素菌F32木质纤维素降解机制研究
	英瑜
导师	李福利
	2013-01
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	生物化学与分子生物学
关键词	木质纤维素纤维素酶木聚糖酶热解纤维素菌蛋白表达及鉴定蛋白质组学
摘要	木质纤维素是地球上含量最丰富的可再生生物质资源，由于石油，天然气等石化资源的大量消耗和逐步枯竭及其燃烧所带来的环境污染等问题日益严重，开发可持续利用的新能源已成为人类社会面临的紧迫任务，以秸秆等木质纤维素材料生产生物燃料已受到越来越多的关注。在木质纤维素材料转化为生物燃料的过程中，首先需要转化为发酵微生物可利用的单糖。使用具有优良性状的纤维素酶和半纤维素酶对于进行木质纤维素的高效转化具有十分重要的意义。自然界中存在大量具有纤维素降解能力的微生物，这些微生物既是我们获得纤维素酶的重要来源，也为构建合适的木质纤维素同步糖化发酵系统提供了有力保障。同时，耐高温纤维素降解菌来源的纤维素酶因具有活性高，热稳定性好，使用过程中不易被污染等优势而受到广泛关注。本工作首先筛选得到一株极端嗜热厌氧纤维素降解菌Caldicellulosiruptor sp. F32，16S rRNA分析表明该菌株与模式菌C. saccharolyticus DSM 8903具有99.0%的相似性。通过对这两株菌利用葡萄糖、木糖、木聚糖、微晶纤维素及未经预处理小麦秸秆的生长及代谢产物分析，发现与C. saccharolyticus DSM 8903相比，菌株F32表现出较强的降解纤维素能力，能有效利用未经预处理的小麦秸秆，在利用葡萄糖为碳源时，具有产乳酸较多，而产氢量较少的特点。通过比较分析微晶纤维素、未经预处理小麦秸秆和木聚糖诱导的Caldicellulosiruptor sp. F32与C. saccharolyticus DSM 8903胞外蛋白的酶活性差异，进一步证明了Caldicellulosiruptor sp. F32具有降解效率较高的纤维素酶系和半纤维素酶系。木聚糖酶谱分析及差异蛋白的鉴定也表明Caldicellulosiruptor sp. F32与C. saccharolyticus DSM 8903具有不同的木聚糖降解酶系，通过对其差异蛋白的质谱鉴定，为这两株菌木聚糖酶基因簇的划分提供了新的依据。基于前期的研究结果及Caldicellulosiruptor sp. F32基因组序列草图的相关信息，克隆表达了Caldicellulosiruptor sp. F32来源的一个具有较高外切酶活性的持续性内切纤维素酶JX030399，以羧甲基纤维素钠为底物测得其内切纤维素酶活为17.11 IU/mg，以Avicel PH-101为底物测得其外切纤维素酶活为10.11 IU/mg。通过对JX030399蛋白部分活性位点的预测，为进一步通过对这些活性位点的突变分析，以解析蛋白结构与功能的关系，对纤维素酶分子进行改造以提高其对纤维素的降解活性或持续降解能力提供了研究基础。同时，表达并鉴定了Caldicellulosiruptor sp. F32来源的两个具有较高热稳定性的木聚糖酶。酶学性质分析表明，与C. bescii DSM 6725基因组中注释为木聚糖酶的蛋白Athe_0089具有94%的氨基酸相似性的JX030400蛋白比活力较高，其木聚糖酶活比活力为942.1 IU/mg，而与C. saccharolyticus DSM 8903中注释为木聚糖酶的蛋白Csac_0696具有97%的氨基酸相似性的JX030401蛋白的木聚糖酶活比活力为103.6 IU/mg。该结果为解释Caldicellulosiruptor sp. F32所具有的优于C. saccharolyticus DSM 8903的木聚糖降解能力提供了支持。本工作通过比较蛋白质组学方法，对微晶纤维素诱导的Caldicellulosiruptor sp. F32胞外蛋白中与纤维素降解相关的部分蛋白进行了鉴定，为进一步解析该菌株与纤维素降解相关的蛋白组分，并深入研究其高效纤维素降解机制奠定了基础。
其他摘要	Lignocellulosic biomass is the most abundant renewable resource on earth. Utilization of lignocellulosic biomass to produce second-generation biofuels such as bioethanol has attracted much attention because of the shortage of fossil fuels, emission of greenhouse gasses and air pollution caused by imcomplete combustion of fossil fuels.The conversion of lignocellulosic biomass to fermentable sugars remains a big challenge due to the recalcitrance of insoluble starting materials. Therefore, the discovery of novel microorganisms with the ability to covert lignocellulosic biomass into sugars is very important. Moreover, employing active biomass hydrolyzing enzymes at extreme temperature and pH may be advantageous for industrial scale production because these conditions facilitate overcoming biomass recalcitrance and prevent the growth of contaminating microorganisms. In the present study, we isolated a hyperthermophilic anaerobic bacterium Caldicellulosiruptor sp. F32 which could use glucose, xylose, crystalline cellulose, xylan, unpretreated lignocelllulosic biomass as carbon sources. Sequence analysis of 16S rRNA gene of strain F32 showed it was closely related to Caldicellulosiruptor saccharolyticus DSM 8903 with 99.0% identity. In contrast to C. saccharolyticus DSM 8903, strain F32 grows more robustiously on cellulose, even on unpretreated wheat straw and produces more lactate and acetate but less H2 than C. saccharolyticus DSM 8903.The enzyme activities of the secreted proteins of Caldicellulosiruptor sp. F32 and C. saccharolyticus DSM 8903 induced by cellulose, unpretreated wheat straw and xylan were analysed. The higher CMCase, Avicelase and xylanase activities of the secretome of strain F32 showed that it is an attractive candidate for exploring novel cellulase and hemicellulase. Meanwhile, the analysis of xylanase zymograms and characterization of different activated proteins showed that there is significant difference in their xylanase systems. Based on our genomic sequencing raw data, the genome of Caldicellulosiruptor sp. F32 contains two xylanases encoded by JX030400 and JX030401. The amino acid sequence of the protein encoded by JX030400 exhibits 94% identity with xylanase (Athe_0089) of Caldicellulosiruptor bescii DSM 6725 and the amino acid sequence of the protein encoded by JX030401 exhibits 97% identity with xylanase (Csac_0696) of C. saccharolyticus DSM 8903.The two thermostable xylanases from strain F32 were expressed in E. coli. The xylanase activity of JX030400 is unique with almost 9 fold higher specific activity than JX030401. The results showed that Caldicellulosiruptor sp. F32 provides a dual-xylanase system and could more efficiently utilize hemicellulose than C. saccharolyticus DSM 8903.We expressed a bifunctional endo-/exo-type cellulase encoded by the gene JX030399 of Caldicellulosiruptor sp. F32. The protein showed CMCase (17.11 IU/mg) and Avicelase (10.11 IU/mg) activity. We also modeled the structure of JX030399 and performed multiple structure alignment to find the active sites that affect the specificity or catalytic ability of JX030399. We also used two-dimensional gel electrophoresis and LC-MS/MS to identify the components of the secreted cellulolytic system of Caldicellulosiruptor sp. F32. The results offer a large amount of useful information for analyzing the mechanism of cellulose degradation of strain F32.
作者部门	微生物资源
学科领域	微生物资源
公开日期	2013-07-13
学位类型	博士
语种	中文
文献类型	学位论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/1492
专题	分子微生物工程研究组
推荐引用方式 GB/T 7714	英瑜. 极端嗜热厌氧热解纤维素菌F32木质纤维素降解机制研究[D]. 北京. 中国科学院研究生院,2013.

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