水稻几丁质酶定向进化概要1.docVIP

分类号： 密级： 硕 士 研 究 生 学 位 论 文 论文题目：专 业： 研究方向： 研 究 生： 指导老师： 论文起止日期 年月至 年 月E.coli TOP10F′构建突变体文库；利用几丁质平板法筛选到了突变体CHA-P。 通过正交试验优化得到CHA-P重组蛋白的最佳表达条件：pH6.5，IPTG浓度0.8mM，33℃，200rpm，培养5h；CHA-P胞外酶活力为80umol/min, 较野生酶活力提高了2倍。重组蛋白主要以包涵体形式存在。运用尿素梯度透析复性法对包涵体进行复性，复性后的酶比活力为0.24U/mg。 对突变体进行序列分析发现：CHA-P的核苷酸序列较野生几丁质酶核苷酸序列发生了318位C→T，368位A→G，374位A→G三个碱基的改变，相应的氨基酸序列发生了天冬氨酸→甘氨酸（123位D→G），酪氨酸→半胱氨酸（125位Y→C）的改变。在突变序列中也发生了一段 (4位-129位) 碱基缺失和插入 (736位-768位)，插入使得终止密码子提前出现，造成蛋白质翻译的提前终止。 关键词：几丁质酶 易错PCR DNA改组 定向进化 酶活 DIRECTED EVOLUTION OF RICE CHITINASE ABSTRACT Random mutagenesis on rice chitinase gene chAB was conducted by using error-prone PCR and DAN-shuffing strategy．The mutation product was recombinated in expression vector pMBP-P, and into E.coli TOP10F′ to construct the mutant library. The mutant strain CHA-P was selected using the method of chitin plate. The optimal expression conditions of the mutant CHA-P recombinant protein were obtained by orthogonal test which involve: 0.8mM IPTG induction, the pH of the medium is 6.5, oscillate fermenting 5h with 200rpm and 33℃. CHA-P extracellular enzyme activity is 80umol/min, which increased twice times compared with that of the wild-type enzyme. The recombinant proteins mainly existed in inclusion bodies. The inclusion bodies were refolded by urea gradient dialysis refolding method. The enzymatic activity of the renatured recombinant proteins is 0.24U/mg. Sequence analysis of the mutant CHA-P showed that three nucleotides substitution 318C/T，368A/G and 374A/G occurred，and caused two amino acids of 123D/G and 125Y/C. Furthermore, the mutation includes the absence of 126 bases (4 to 129) and 2 bases insertion (736 to 738), which makes the termination codon appeared in advance, and causing premature termination of the protein translation. KEYWORDS: chitinase; error-prone PCR; DNA shuffling; directed evolution; enzymat