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黑曲霉植酸酶基因的定点突变及其在毕赤酵母的表面展示

发布时间:2019-01-12 16:14  文章来源:笔耕文化传播
【摘要】:黑曲霉植酸酶PHYA目前被公认为最具应用前景的饲用植酸酶之一,但是野生型菌株所产生的植酸酶不能满足工业生产的需求,本研究以Aspergillus niger ZJUY中的植酸酶基因phyA为亲本,借助定点突变的方法,对植酸酶的关键位点的密码子进行优化,并引入氢键(T295S,Q296R和V43N)和对二硫键Cys196-Cys446进行缺失突变,以解决植酸酶在制粒过程中易变性失活的问题。最终将絮凝素Flo1p与改造过的植酸酶以N端融合的方式,展示在Pichia pastoris GS115细胞表面,用1%的甲醇进行诱导表达,通过细胞免疫荧光染色和流式细胞仪检测,证实植酸酶在毕赤酵母细胞表面成功展示。本研究的主要研究结果如下:(1)以黑曲霉基因组为模板,通过三轮PCR获得去除内含子的植酸酶基因phyA,该法的优势在于在所提取的RNA质量较差的情况下仍可获得完整的去除内含子的植酸酶基因。(2)以野生型的PHYA为亲本,通过PCR最终成功引入R62、H63、G64、R66、P68、R146、H342、D343、S295、R296、N43和S446等12个突变位点,突变效率较高,操作简便易行。(3)借助无缝克隆的方法,成功将锚定片段FS、携带标签Flag的目的片段phyA插入到酵母表达载体pPICZαC中,相比于采用传统的载体构建方法,该法阳性克隆效率较高。(4)采用氯化锂转化的方法,成功将构建的8种表达载体pPICZαC-FS/phyA通过同源重组的方式整合到毕赤酵母GS115基因组上,并通过甲醇的诱导成功展示到GS115细胞表面。(5)通过展示酶特性的研究发现,二硫键Cys196-Cys446的缺失能够使展示酶的内源荧光的发射波长发生红移,改变了酶分子的构象。此外,二硫键Cys196-Cys446的缺失突变能够提高展示酶对底物的亲和力和底物专一性,但会使催化效率下降。(6)展示植酸酶引入氢键和缺失二硫键会导致酶促反应的最适温度和最适pH发生一定程度的改变。(7)通过热稳定性的研究发现,重组菌株A61在90℃水浴处理的过程中,展示酶活丧失比较缓慢,这一特性可以克服制粒过程中(60~90℃)植酸酶变性失活的不足。(8)通过酸碱耐受性研究发现,重组菌株A31、A61、A84在pH 1.6-4.0的范围内,残余酶活均保持在80%以上,可以较好的满足动物饲料用酶的要求。(9)金属离子Na~+、K~+、Fe~(2+)、Zn~(2+)在低浓度时可以激活展示植酸酶的活性,而Cu2+、Mn2+、Co2+对展示植酸酶主要表现为抑制作用。
[Abstract]:The phytase PHYA of Aspergillus Niger is recognized as one of the most promising forage phytase, but the phytase produced by wild-type strains can not meet the needs of industrial production. In this study, phytase gene phyA in Aspergillus niger ZJUY was used as parent. With the help of site-directed mutation, the codon at the key site of phytase was optimized, and the deletion mutation of hydrogen bond (T295SZQ296R and V43N) and disulfide bond Cys196-Cys446 were introduced to solve the problem of deactivation of phytase in granulation process. Finally, the flocculant Flo1p and the modified phytase were displayed on the surface of Pichia pastoris GS115 cells by N terminal fusion. The expression was induced by 1% methanol, and detected by cell immunofluorescence staining and flow cytometry. It was confirmed that phytase was successfully displayed on the surface of Pichia pastoris cells. The main results of this study are as follows: (1) using Aspergillus Niger genome as template, the phytase gene phyA, was obtained by three rounds of PCR. The advantage of this method is that the phytase gene can be obtained when the quality of the extracted RNA is poor. (2) the wild-type PHYA was used as the parent, and the R62H63G63G64G64H6C6H68P68R146H342was successfully introduced through PCR. There are 12 mutation sites, such as D343H S295N, R296N 43 and S446, which have high mutation efficiency and are easy to operate. (3) by means of seamless cloning, the target fragment phyA of anchoring fragment FS, carrying label Flag was successfully inserted into the yeast expression vector pPICZ 伪 C. Compared with the traditional method of vector construction, the positive clone efficiency of this method was higher. (4) the method of lithium chloride conversion was used. The eight expression vectors pPICZ 伪 C-FS/phyA were successfully integrated into Pichia pastoris GS115 genome by homologous recombination, and successfully displayed on the surface of GS115 cells by methanol induction. The absence of disulfide bond Cys196-Cys446 can make the emission wavelength of endogenous fluorescence of the enzyme red shift and change the conformation of enzyme molecule. In addition, the deletion mutation of disulfide bond Cys196-Cys446 can improve the enzyme affinity to substrate and substrate specificity. However, the catalytic efficiency is decreased. (6) it is shown that the introduction of hydrogen bond and the absence of disulfide bond in phytase lead to some changes in the optimum temperature and pH of the enzymatic reaction. (7) through the study of thermal stability, it is found that, The loss of enzyme activity of recombinant strain A61 in 90 鈩,

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