摘 要非特异性核酸酶是一类能够降解DNA或RNA的酶，不仅具有非特异性，而且具有热稳定性及广泛的适用性。本实验以Y.e.p非特异性核酸酶为研究对象，先用NCBI在线Blast工具比对分析，再用定点突变方法研究影响Y.e.p非特异性核酸酶酶活和热稳定性的因素。通过热不稳定Y.e.e和热稳定Y.e.p非特异性核酸酶的序列对比，定点突变其中15个差异的氨基酸，观察到有三个位点Glu202，Ile203和Asp264的氨基酸改变引起Y.e.p非特异性核酸酶的酶活和热稳定性急剧下降，故推测Glu202，Ile203和Asp264是影响Y.e.p非特异性核酸酶酶活和热稳定性的关键氨基酸残基。本实验说明这三个氨基酸对Y.e.p非特异性核酸酶的酶活和热稳定性具有重要作用。47929

  Abstract  Non-specific nuclease is a kind of enzyme which can degrade DNA and RNA. It not only has non-specific activity, but also has thermal stability, and also has wide applicability. In this experiment, effect of enzyme activity and thermostability of Y.e.p non-specific nuclease was studyed by sequence alignment and site-directed mutagennesis. The amino acid sequences of Y.e.e and Y.e.p non-specific nuclease were analyzed, respectively. 15 amino acids were designed for site-directed mutagenesis. The results showed the mutations of E202A, I203F and D264E significantly affected the enzyme activity and thermal stability of Y.e.p non-specific nuclease, indicating Glu202, Ile203 and Asp264 might be key residues of Y.e.p non-specific nuclease. This experiment showed Glu202, Ile203 and Asp264 play an important role in enzyme activity and thermal stability of Y.e.p non-specific nuclease.

毕业论文关键词：非特异性核酸酶，定点突变，热稳定性，酶活

Key words: non-specific nuclease, site-directed mutagennesis, thermostability，enzyme activity

目  录

引言 4

1材料与方法 4

1.1 实验材料 4

1.1.1 菌株与质粒 4

1.1.2 酶与试剂盒 4

1.1.3 主要试剂 5

1.1.3 培养基和工作液 5

1.1.4 实验仪器 5

1.2实验方法 5

1.2.1 序列比对分析 5

1.2.2 突变位点选择 5

        1.2.3定点突变 6

    1.2.3.1定点突变原理 6

    1.2.3.2定点突变引物设计 6

    1.2.3.3定点突变基因的获得 7

1.2.4突变质粒的提取 8

1.2.5突变酶的诱导表达 8

1.2.6突变酶蛋白质含量测定 9

1.2.7酶活测定 9

1.2.8热稳定性测定 10

2结果与分析 10

2.1序列对比 10

2.2突变位点选择 10

2.3突变酶蛋白含量测定 11

2.4酶活测定 11

2.5热稳性测定 12

3讨论 12

致谢 13

参考文献