Caldicellulosiruptor菌属嗜热木聚糖水解酶系的筛选与研究

发布时间：2018-01-22 01:50

本文关键词： 半纤维素水解作用木聚糖酶乙酰木聚糖酯酶 β-木糖苷酶　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：农林业废弃物主要由纤维素、半纤维素、木质素等大分子化合物构成,木聚糖作为其组成成分中含量最多的半纤维素,对其完全酶解可产生木糖,这不仅能够减少其焚烧带来的环境污染,也实现了废物额资源化利用。天然木聚糖结构中通常含有不同的侧链取代基(如阿拉伯糖、O-乙酰基、阿魏酸和4-O-甲基葡萄糖醛酸等),所以,对其完全降解需要一系列酶系协同作用。通常可降解半纤维素的酶制剂主要有木聚糖酶、β-木糖苷酶、乙酰木聚糖酯酶、阿拉伯呋喃糖苷酶和阿魏酸酯酶等。本文通过基因组序列注释、比对以及蛋白结构预测的方法,分别在嗜热厌氧微生物热解纤维素菌属Caldicellulosiruptor sp.F32基因组中发现一个热稳定性良好的乙酰木聚糖脂酶Axe7以及在同菌属Caldicellulosiruptor saccharolyticus基因组中发现两个β-木糖苷酶CsXyl39A和CsXyl39B,利用基因克隆、质粒构建以及在大肠杆菌中表达目标蛋白并纯化等实验方法,获得了这几种酶的重组蛋白并分别用不同方法对其进行酶学性质测定并探究了在实际中的应用。Axe7是Caldicellulosiruptor sp.F32所表达的具有最适温度高,热稳定性好、酶催化效率高等诸多优点的、具有一定商业化潜力的乙酰木聚糖脂酶。以4-甲基乙酸伞形酯(4-Methylumbelliferyl-acetate)作为底物时,Axe7的最适反应pH在6.5-7.0之间,最适反应温度为85°C,在最适的温度和pH条件下,Axe7活性半衰期(Half-life)超过4 h。不同金属离子对其酶活有一定的抑制影响。通过测定酶动力学发现Axe7对底物的催化效率较好。众多优良特性使得Axe7为木质纤维素的高温糖化和生物炼制提供了一个可工业化的潜在选择。CsXyl39A(GenBank登录号为Csac_2404)和CsXyl39B(GenBank登录号为Csac_2409)为从热解纤维素菌Caldicellulosiruptor saccharolyticus中获得的β-木糖苷酶,该两种酶位于C.saccharolyticus基因组中与半纤维素水解有关的同一基因簇中,但序列相似性有很大差异。经酶学性质研究表明,两者均为热稳定的β-木糖苷酶,最适温度和pH较为相似,CsXyl39A具有很强的有机溶剂耐受性,而CsXyl39B则有极强的木糖以及葡萄糖耐受性。在实际应用过程中,CsXyl39A经大孔树脂进行固定化后发现,固定化并未明显改变其热稳定性,但进一步提升了其对有机溶剂的耐受性,另外,该酶可应用于水解三七皂苷R1中,反应产物经HPLC分析确定生成了药理作用更好的人参皂苷Rg1。而CsXyl39B与木聚糖酶有较好的协同作用,两者共同反应能够明显提高木聚糖的水解效率。这些优良特性使这两种β-木糖苷酶在工业化应用中潜力巨大。
[Abstract]:Agricultural and forestry wastes are mainly composed of cellulose, hemicellulose, lignin and other macromolecular compounds. Xylan is the most abundant hemicellulose in its composition, and its complete enzymatic hydrolysis can produce xylose. This can not only reduce the environmental pollution caused by incineration, but also realize the recycling of waste. Natural xylan structures usually contain different side chain substituents (such as arabinose O- acetyl). Ferulic acid and 4-O- methylglucuronic acid, etc., therefore, the complete degradation of hemicellulose requires a series of enzymatic synergism. Usually, xylanase and 尾 -xylanase are the main enzymes that can degrade hemicellulose. Acetylxylanesterase, arabinofuran glucosidase, ferulic esterase, etc. In this paper, the methods of genome sequence annotation, alignment and protein structure prediction were used. In addition to thermophilic anaerobic microorganism pyrolytic cellulose bacteria Caldicellulosiruptor. The discovery of a thermostable acetylxylanlipase Axe7 in sp.F32 genome and its presence in synbacteria Caldicellulosiruptor. Two 尾 -xylosidase CsXyl39A and CsXyl39B were found in saccharolyticus genome. The methods of gene cloning, plasmid construction, expression and purification of the target protein in Escherichia coli were used. The recombinant proteins of these enzymes were obtained, and their enzymatic properties were determined by different methods, and the application of .Axe7 to Caldicellulosiruptor was explored. The expression of sp.F32 has the optimum temperature. Good thermal stability, high enzyme catalytic efficiency and many other advantages. Acetylxylanlipase with certain commercial potential. Using 4-Methylumbelliferyl-acetate4-Methylumbelliferyl-acetate4-Methylumbelliferyl-acetate4-Methylumbelliferyl-acetate4 as substrate. The optimum reaction pH of Axe7 is between 6.5-7.0 and the optimum reaction temperature is 85 掳C. Axe7 active half-life. More than 4. The enzyme activity was inhibited by different metal ions. Through the determination of enzyme kinetics, it was found that the catalytic efficiency of Axe7 to substrate was better. Many excellent properties made Axe7 saccharify lignocellulose at high temperature. Bio-refining offers a potential option for industrialization. CsXyl39A (. GenBank login number Csacs 2404) and CsXyl39B(GenBank login number Csacs 2409). 尾 -xylosidase obtained from pyrolytic cellulose strain Caldicellulosiruptor saccharolyticus. The two enzymes are located in the same gene cluster related to hemicellulose hydrolysis in the genome of C. saccharolyticus, but the sequence similarity is very different. Both of them were thermostable 尾 -xylosidase, and the optimum temperature and pH were similar to those of CsXyl39A. However, CsXyl39B had strong tolerance to xylose and glucose. In practical application, CsXyl39A was immobilized with macroporous resin. The immobilization did not change its thermal stability, but further enhanced its tolerance to organic solvents. In addition, the enzyme could be used in the hydrolysis of Panax notoginsenoside R1. The reaction product was determined by HPLC analysis to produce a better pharmacological effect of ginsenoside Rg1.And CsXyl39B and xylanase had a better synergistic effect. These excellent properties make these two 尾 -xylosidases have great potential in industrial application.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936

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