耐热木聚糖酶基因改造及对酶学性质的影响
耐热木聚糖酶基因改造及对酶学性质的影响(任务书,开题报告,外文翻译,论文13500字)
摘 要
木聚糖酶是木聚糖降解中的关键酶，分布很广，目前已在造纸、食品、饲料、能源、材料、医药等领域得到广泛应用。尤其是耐热耐碱特性的木聚糖酶在造纸等行业具有很大的应用潜力，是目前研究的热点。
本论文以实验室已有的基因工程菌JM109/pET28a-xynB64为出发菌株，检测了目的基因xynB64的序列，分析了该酶N端的信号肽序列和C端疏水氨基酸序列。通过分子生物学手段将该酶进行了改造，获得了不含信号肽和组氨酸标签的耐热木聚糖酶XynBⅡ以及不含信号肽序列的耐热木聚糖酶XynBⅢ，其对应的产酶菌株分别为Rosetta(DE3)/pET28a(+)-xynB64(Δ4-60)和Rosetta(DE3)/pET28a(+)-xynB64（Δ4-60）- His.Tag。将改造前后的耐热木聚糖酶产生菌在相同条件诱导产酶后对比发现，改造后可溶耐热木聚糖酶表达量增加了近20%，其包涵体表达量降低约10%。
将XynB、XynBⅡ和XynBⅢ的酶学性质进行对比发现，耐热木聚糖酶的最适温度依然在100℃以上，但XynBⅡ和XynBⅢ的温度稳定性有的少量提高；XynB、XynBⅡ、XynBⅢ的最适pH分别在6.5、5.7、6.0；XynBⅡ、XynBⅢ与XynB相比，其pH稳定性几乎没有变化，但前两者能够在更宽的pH范围内保持很高的酶活力。金属离子对XynB、XynBⅡ、XynBⅢ三者影响相近。总的来说，高浓度的Mg2+ 、Mn2+对本耐热木聚糖酶有显著的激活作用，能够将酶活提高50%~60% ；Cu2+、Zn2+、Fe3+及低浓度的Ca2+、Mn2+、Mg2+有很强的抑制作用，可以将酶活降低20%~80%不等；K+、Na+对酶活力基本无影响。 [资料来源：http://doc163.com]
关键词：耐热木聚糖酶 信号肽 酶学性质 包涵体
ABSTRACT
Xylanase, the key component of xylanolytic enzyme system, was widely applied in paper manufacture, food, feed, energy, materials and medicine industries. Especially the thermostable xylanase with alkali tolerance is of particular interest.
Using the recombinant engineering bacteria JM109/pET28a-xynB64, conserved in the laboratory, as the original strain, sequence of xynB64 was analyzed. And the signal peptide sequence in N-terminal domain was predicted. By molecular biological means, we acquired two enzymes: one is XynBⅡ not containing signal peptide and histidine label, and the other XynBⅢ not containing signal peptide, whose corresponding production strains are Rosetta(DE3)/pET28a(+)-xynB64(Δ4-60) and Rosetta(DE3)/pET28a(+)-xynB64(Δ4-60)- His.Tag, respectively. Compared with heat-resistant xylanase before modification, the soluble expression of XynBⅡ and XynBⅢ were found to increase almost 20%, while their inclusion body expressions decreased about 10% after induced in the same conditions.
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Comparing the enzymatic properties of XynB, XynBⅡ and XynBⅢ, it is found that the optimum reaction temperature remains above 100℃,but the temperature stability of XynB Ⅱ and XynB Ⅲ increase a little; the optimum pH of XynB, XynB Ⅱand XynB Ⅲ are 6.5, 5.7, 6.0, respectively. Compared with XynB, XynB Ⅱand XynB Ⅲ keep high enzyme activity within a wider pH range. Metal ions have similar effects on the enzyme activity of XynB, XynB Ⅱ and XynB Ⅲ. In general, high concentrations of Mg2+ , Mn2+ significantly activate the enzyme activity of these heat-resistant xylanase by 50%~ 60%; Cu2+, Zn2+, Fe3+and low concentration of Ca2+, Mn2+and Mg2+ can reduced these heat-resistant xylanase by 20%~ 80%; K+ and Na+ barely have any effects on the enzyme activity.
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目录
摘 要 I
ABSTRACT II
目录 III
第一章 文献综述 2
1.1 引言 2
1.2 耐热木聚糖酶 2
1.2.1 耐热木聚糖酶的性质 2
1.2.2 耐热木聚糖酶的耐热机理 3
1.2.3 耐热木聚糖酶的应用 4
1.3 包涵体 6
1.3.1 包涵体形成原因 6
1.3.2 包涵体复性的条件 7
1.4 本课题主要研究内容 7
第二章 耐热木聚糖酶的改造及其酶学性质 9
2.1 引言 9
2.2 材料与方法 9
2.2.1 菌种与质粒 9
2.2.2 培养基 10
2.2.3 实验仪器 10
2.2.4 实验试剂 11
2.2.5 实验方法 12
2.3 实验结果与讨论 20
2.3.1 测序结果及其信号肽分析与实验设计 20
2.3.2 重组表达质粒的构建 22
2.3.3 改造前后耐热木聚糖酶表达量对比 23
2.3.4 木糖标准曲线 25
2.3.5 耐热木聚糖酶改造前后酶学性质 25
第三章 结论与展望 31
3.1 结论 31
3.2 展望 31
参考文献 32 [资料来源：http://Doc163.com]
致谢 35