摘要本文通过基因融合表达策略对Arabidopsis thaliana羟基腈裂解酶的蛋白结构进行定向改造，在酶的基因序列前加上一段螺旋基因，使Arabidopsis thaliana羟基腈裂解酶的pH稳定性和温度稳定性能够得到较大幅度的提升。实验结果证明：经过定向改造后，得到的1USE-AtHNL和1Y66-AtHNL的酶产物主要以活性包涵体的形式存在，在热稳定性和pH稳定性上，都有了很明显的提升；1YBK-AtHNL的酶产物主要以可溶性蛋白的形式存在，在热稳定性上有明显的提升，但是在pH稳定性上有所降低。总体而言，重组1USE-AtHNL和1Y66-AtHNL的稳定性得到提高，证明该策略在酶稳定改造中的应用潜力。88931

In this work, the protein structure of hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) was directional modified by gene fusion expression strategy。 A coiled-coil domain was added to the enzyme's gene sequence to improve the pH stability and temperature stability of recombinant HNL from Arabidopsis thaliana。 The results showed that after the directional modification, recombinant HNLs with coiled-coil domain 1USE and 1Y66 (1USE-AtHNL and 1Y66-AtHNL, respectively) were expressed in the form of catalytically-active inclusion bodies (CatIBs), and their thermal stability and pH stability have been significantly improved compared with wild AtHNL。 However, the recombinant HNL with coiled-coil domain 1YBK (1YBK-AtHNL) was produced in the form of soluble protein, and its thermal stability and pH stability have been reduced。 Summary: 1USE-AtHNL and 1Y66-AtHNL were successfully constructed and expressed as the form of CatIBs for improving the stability of AtHNL。

毕业论文关键词：定向改造； 羟基腈裂解酶; 活性包涵体； pH稳定性； 温度稳定性

Keywords: Directed modification; Hydroxynitrile lyase; Active inclusion body; pH stability; Thermal stability

目  录

1 绪论 4

1。1羟基腈裂解酶 4

1。2 酶的定向改造技术 5

1。3 活性包涵体(CatIBs) 5

1。4反应条件对酶的影响 6

1。4。1 温度的影响 6

1。4。2 pH值的影响 6

1。5 本论文研究内容 6

2实验材料与方法 7

2。1实验材料 7

2。1。1菌种源Y于U优I尔O论P文W网wwW.yOueRw.com 原文+QQ75201-8766 和质粒 7

2。1。2主要实验仪器 8

2。1。3 主要药品与试剂 9

2。2实验方法 10

2。2。1培养基与试剂配置 10

2。2。2 重组质粒的构建 12

2。2。3 PCR操作 12

2。2。4 菌种的诱导表达及保存 13

2。2。5 稳定性的测定 13

3 实验结果与讨论 14

3。1 PCR产物验证结果 14

3。2 重组质粒的诱导与表达 15

3。3 经过定向改造后得到的酶是具有催化活性的包涵体（CatIBs） 17

3。4 重组质粒的稳定性 17

3。4。1 热稳定性 17

3。4。2 最适pH值