纤维化纤维菌中木糖苷酶基因的克

发布时间：2018-03-08 05:27

  本文选题：纤维化纤维菌　切入点：克隆表达　出处：《东北师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：木聚糖是植物半纤维素的主要成分,在自然界中含量丰富,是自然界中最主要的可再生资源之一,在造纸、食品、医药等行业应用广泛。木聚糖生物质的彻底降解需要一个完善的水解酶系,包括木聚糖酶和β-木糖苷酶等。其中,β-木糖苷酶是木聚糖降解的关键酶之一,与木聚糖酶协同作用将木聚糖彻底降解为木糖。因此,获得高活性的β-木糖苷酶并进行稳定制备,用其与木聚糖酶协同降解木聚糖,是木聚糖降解领域研究重点之一,具有重要的理论研究意义和实际应用价值,也是本论文的研究重点。本论文的主要研究成果如下:1.本研究从纤维化纤维菌中克隆得到一个编码糖苷水解酶家族3(GH3)蛋白的基因ccxyl3a,将该基因与pET-28a(+)载体连接,得到的重组质粒pET-28a/ccxyl3a转化到大肠杆菌BL21(DE3)感受态细胞中,得到重组菌株BL21/pET-28a/ccxyl3a。2.重组菌株BL21/pET-28a/ccxyl3a在16℃条件下、0.5 mM IPTG诱导20 h,实现了重组蛋白CcXyl3A的过表达。将重组酶CcXyl3A从大肠杆菌细胞裂解液上清中通过Ni sepharose fastflow亲和柱层析进行纯化,得到纯度大于90%的重组酶CcXyl3A。3.对纯化的重组酶CcXyl3A进行酶学性质研究。SDS-PAGE结果表明,CcXyl3A的分子量为95 KDa。以对硝基苯基-β-D-木糖苷(pNPβXyl)为底物,测定CcXyl3A的酶活力。结果表明,CcXyl3A的最适pH值为8.5,最适反应温度为45 ℃。离子耐受实验结果表明K+和Na+能够促进CcXyl3A的酶活性,在50 mM K~+或Na~+缓冲体系中,CcXyl3A酶活力分别提高到132.2%和132.6%。底物专一性结果表明CcXyl3A能水解对硝基苯基-β-D-木糖苷(pNPβXyl)、对硝基苯基-β-D-葡萄糖苷(pNPβGlc)和对硝基苯基-α-L-呋喃阿拉伯糖苷(pNPαAraf)。其中,CcXyl3A对水解pNPβXyl的酶活力最高,对pNPαAraf和pNPβGlc的水解活性较低。以寡聚木糖为水解底物时,CcXyl3A能够完全水解木二糖、木三糖、木四糖和木六糖,生成唯一产物木糖。4.对重组酶CcXyl3A和真菌木聚糖酶联合降解木聚糖的能力进行研究,结果表明CcXyl3A和噬热真菌木聚糖酶能够协同作用,降解榉木木聚糖。其中,噬热真菌木聚糖酶将榉木木聚糖降解为木寡糖和木二糖,CcXyl3A进而将其完全降解成木糖。本研究为纤维化纤维菌的糖苷酶在半纤维素降解中的应用奠定了理论基础。
[Abstract]:Xylan is the main component of hemicellulose in plants, rich in nature, is one of the most important renewable resources in nature, in paper, food, The complete degradation of xylan biomass requires a complete hydrolase system, including xylanase and 尾 -xylosidase. Among them, 尾 -xylosidase is one of the key enzymes in the degradation of xylan. Therefore, it is one of the important research points in the field of xylan degradation to obtain high activity 尾 -xylosidase and prepare it stably, and use it to co-degrade xylan with xylanase. Has the important theory research significance and the practical application value, The main research results of this thesis are as follows: 1. A gene ccxyl3aencoding glycoside hydrolase family 3 (GH3) was cloned from Fibrofibria and linked with pET-28a () vector. The recombinant plasmid pET-28a/ccxyl3a was transformed into Escherichia coli BL21DE3) competent cells. The recombinant strain BL21 / pET-28a / ccxyl3a.2. the recombinant strain BL21/pET-28a/ccxyl3a was induced by 0.5 mm IPTG at 16 鈩，

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