【作者】 魏威；

【导师】 李弘剑；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 植酸酶作为一种新型的酶制剂，由于能有效地帮助非反刍动物消化谷物、油料作物中的植酸盐，提高磷的利用率，减轻由于磷排放对环境尤其是水源的污染，另外能够改善家畜对无机盐、氨基酸等营养物质的吸收，促进其生长，所以得到了广泛的研究和应用。 本文以研究植酸酶的热稳定性为主要目的，通过致突变PCR的方法，对来源于Aspergillus niger的植酸酶基因phyA进行突变。以突变后的基因为基础，构建了PTE21a(+)-phyA／BL21(DE3)和PTE35b(+)-phyA／BL21(DE3)表达系统。在2个系统中目的蛋白都得到了高效表达。 对PTE21a(+)-phyA／BL21(DE3)和PTE35b(+)-phyA／BL21(DE3)系统表达蛋白的表达量和生物活性进行了比较研究。PTE21a(+)-phyA／BL21(DE3)系统表达的蛋白占菌体总蛋白的30％以上，主要以包涵体形式存在，经复性后酶的比活力达到1500U／mg；PTE35b(+)-phyA／BL21(DE3)系统表达的蛋白约占菌体总蛋白的20％，主要以溶解形式存在，酶的活力较低。经比较后确定利用PTE21a(+)-phyA／BL21(DE3)系统进行后续实验。 针对PTE21a(+)-phyA／BL21(DE3)系统表达蛋白以包涵体形式存在的问题，改进了包涵体蛋白的复性方法，利用分子筛层析进行复性、纯化，取得了良好的效果。 对PTE21a(+)-phyA／BL21(DE3)系统表达蛋白的热稳定性进行了研究，发现了一个突变菌株PTE21a(+)-phyA／BL21(DE3)-2，其表达的突变蛋白phyA-mut2具有良好的热稳定性，经过75℃-95℃、30min的热处理后，保持了酶活性，并有明显的热激活现象，激活后最高比活力达到5000 U／mg。 对突变蛋白phyA-mut2的基因进行了测序，发现了突变蛋白phyA-mut2在一级结构上的变异。更多还原

【Abstract】 Phytase can effectively help monogastric animals digest thephytate in the plant seeds of corn and oil crop,imrove availability of phosphorus, decrease the environment pollution especially water resourses pollution caused by undigested phosphate, and also improve the absorption of nutrition material such as inorganic salt, amino acid and so on, promote the animal growth. So the phytase obtains extentive research and utilize.The major purpose of this thesis is to research on the thermostablity of phytase. Through the method of mutation-PCR, the gene (phyA) for Aspergillus niger phytase was mutated. Building two expression-system PTE21a(+)-phyA/BL21(DE3) and PTE35b(+)-phyA/BL21(DE3) based on mutated-gene, the purpose protein was effectively expressed in two systems. Research on the expression of recombinant protein and the biology activity through comparing the PTE21a(+)-phyA/BL21 and PTE35b(+)-phyA/BL21. In PTE21a(+) -phyA/BL21, the recombinant protein was in the form of inclusion body and reached 30% in thallus protein.The biology activity of renature protein reached 1500U/mg;In PTE35b(+)-phyA/BL2l, the recombinant protein in the form of soluble protein and reached 25% in thallus protein, The biology activity of refolded protein was very low. Through comparing, the PTE21a(+)-phyA/BL21 was used to undertake continue experiment.The method for renaturation was improved.Research on the thermostablity of recombinant protein .Finding a mutated-strain named PTE21a(+)-phyA/BL21-2. The recombinant protein (phyA-mut2) expressed by it has better thermostablity, after heat treatment 75C-95C, 30min, still kept biological activity and has the phenomenon of activation.The highest bio-activity reached 5000 U/mg.Through sequencing,find the mutation of primary structure in phyA-mut2.更多还原