目录
摘要 III
关键词III
AbstractIV
引言
引言1
1 材料与方法2
1.1 实验材料 2
1.1.1菌株、载体和引物2
1.1.2试剂材料2
1.1.3常用培养基3
1.1.4实验仪器材料3
1.2 珊瑚球菌EGB来源的外切葡聚糖酶基因的克隆表达3
1.2.1 供试菌株的培养3
1.2.2 菌体总DNA的提取3
1.2.3 外切葡聚糖酶基因5817的扩增4
1.2.4 琼脂糖凝胶电泳检测4
1.2.5 扩增片段的回收5
1.3 珊瑚球菌EGB来源的外切葡聚糖酶基因的重组表达菌株构建5
1.3.1 大肠杆菌普通感受态细胞的制备 5
1.3.2 质粒的提取5
1.3.3 载体pET29a(+)的Nde I/Xho I双酶切及纯化6
1.3.4表达载体pET29a(+)5817的构建6
1.3.5 pMD19T5817克隆载体构建6
1.3.6 表达菌株的构建7
1.4 重组外切葡聚糖酶的表达和纯化8
1.4.1 重组外切葡聚糖酶的诱导表达8
1.4.2 重组外切葡聚糖酶的纯化9
1.4.3 SDSPAGE电泳分析及酶谱分析9
1.4.4 蛋白含量测定10
1.5 重组外切葡聚糖酶的酶学性质初步研究11
1.5.1 重组外切葡聚糖酶酶活力测定11
1.5.2 重组外切葡聚糖酶的底物特异性11
1.5.3 温度对重组外切葡聚糖酶活性的影响11
1.5.4 pH对重组外切葡聚糖酶活性的影响11
1.6 生物信息学分析软件和网址12
2 结果与分析12
2.1 重组酶的性质预测12
2.2 系统发育进化树分析13
2.3 蛋白序列分析14
2.4 外切葡聚糖酶基因的克隆表达和表达载体构建15 *51今日免费论文网|www.51jrft.com +Q: @351916072@ 

2.5 重组酶的诱导表达和SDSPAGE分析16
2.6 重组酶的酶学特性17
2.6.1 重组酶的底物特异性分析 17
2.6.2 温度对重组酶活性的影响17
2.6.3 pH对重组酶活性的影响18
3 小结与讨论 19
3.1 实验总结19
3.2 创新和不足19
致谢20
参考文献20
珊瑚球菌EGB来源的外切葡聚糖酶基因的克隆表达与蛋白纯化
摘要
粘细菌中富含糖苷水解酶基因，珊瑚球菌EGB来源的外切葡聚糖酶基因5817是一类通过EGB基因组克隆鉴定得到的新型糖苷水解酶基因，将其与pET29a(+)连接转入大肠杆菌BL21构建重组表达菌株，进行基因克隆表达和蛋白纯化。经过蛋白序列比对及结构域预测分析发现该蛋白同时具有2个DUF4082、1个DUF11和1个Fibronectin 3（FN 3）结构域，不含糖苷水解酶催化结构域，与同类型的多功能水解酶相比，结构上有较大差异。经初步酶学性质分析，重组酶活性为0.0439 U/nmol，底物特异性分析表明该外切葡聚糖酶是一类同时能够水解β1,3、β1,4和β1,6糖苷键的多功能酶，最适底物为木聚糖，最适温度为50℃，最适pH为PBS缓冲液pH 8.0；温度稳定性在3050℃之间较好，pH 8.0时稳定性较好。该研究丰富了新型糖苷水解酶资源，为该类糖苷水解酶基因高效工程菌株的构建奠定了基础。
CLONING EXPRESSION AND PROTEIN PURIFICATION OF EXOGLUCANASE GENE DERIVED FROM CORALLOCOCCUS SP.EGB
ABSTRACT
Myxobacteria are rich in glucoside hydrolase genes. Exoglucanase gene 5817 from Corallococcus sp. strain EGB is a new type of glucoside hydrolase gene identified by gene cloning. It was connected with pET29a(+) and transferred to Escherichia coli BL21 to construct recombinant expression strain for gene cloning and protein purification. After protein sequence alignment and forecast analysis found that the protein contains 2 DUF4082, 1 DUF11 and 1 FN 3 structure domains, without glucoside hydrolase catalytic domain. Therefore, compared with the structures of same type of multifunctional hydrolase, it was great differences. According to the preliminary enzymatic property analysis, the activity of the recombinant enzyme was 0.0439 U/nmol, and the substrate specificity analysis showed that the extracellular glucanase was a kind of multifunctional enzyme capable of hydrolysis to β1,3、β1,4and β1,6 glycoside bonds at the same time. The optimal substrate was xylan, the optimal temperature was 50℃, and the optimal pH was PBS buffer pH 8.0. The protein was stable at temperatures of 30℃50℃, and pH 8.0(PBS buffer). This study enriched the resources of novel glycoside hydrolase and laid a foundation for the construction of highefficient engineered strain with glycoside hydrolase gene.

原文链接：http://www.jxszl.com/swgc/smkx/606485.html