【摘要】：黑曲霉植酸酶PHYA目前被公認為最具應用前景的飼用植酸酶之一,但是野生型菌株所產(chǎn)生的植酸酶不能滿足工業(yè)生產(chǎn)的需求,本研究以Aspergillus niger ZJUY中的植酸酶基因phyA為親本,借助定點突變的方法,對植酸酶的關鍵位點的密碼子進行優(yōu)化,并引入氫鍵(T295S,Q296R和V43N)和對二硫鍵Cys196-Cys446進行缺失突變,以解決植酸酶在制粒過程中易變性失活的問題。最終將絮凝素Flo1p與改造過的植酸酶以N端融合的方式,展示在Pichia pastoris GS115細胞表面,用1%的甲醇進行誘導表達,通過細胞免疫熒光染色和流式細胞儀檢測,證實植酸酶在畢赤酵母細胞表面成功展示。本研究的主要研究結果如下:(1)以黑曲霉基因組為模板,通過三輪PCR獲得去除內(nèi)含子的植酸酶基因phyA,該法的優(yōu)勢在于在所提取的RNA質量較差的情況下仍可獲得完整的去除內(nèi)含子的植酸酶基因。(2)以野生型的PHYA為親本,通過PCR最終成功引入R62、H63、G64、R66、P68、R146、H342、D343、S295、R296、N43和S446等12個突變位點,突變效率較高,操作簡便易行。(3)借助無縫克隆的方法,成功將錨定片段FS、攜帶標簽Flag的目的片段phyA插入到酵母表達載體pPICZαC中,相比于采用傳統(tǒng)的載體構建方法,該法陽性克隆效率較高。(4)采用氯化鋰轉化的方法,成功將構建的8種表達載體pPICZαC-FS/phyA通過同源重組的方式整合到畢赤酵母GS115基因組上,并通過甲醇的誘導成功展示到GS115細胞表面。(5)通過展示酶特性的研究發(fā)現(xiàn),二硫鍵Cys196-Cys446的缺失能夠使展示酶的內(nèi)源熒光的發(fā)射波長發(fā)生紅移,改變了酶分子的構象。此外,二硫鍵Cys196-Cys446的缺失突變能夠提高展示酶對底物的親和力和底物專一性,但會使催化效率下降。(6)展示植酸酶引入氫鍵和缺失二硫鍵會導致酶促反應的最適溫度和最適pH發(fā)生一定程度的改變。(7)通過熱穩(wěn)定性的研究發(fā)現(xiàn),重組菌株A61在90℃水浴處理的過程中,展示酶活喪失比較緩慢,這一特性可以克服制粒過程中(60~90℃)植酸酶變性失活的不足。(8)通過酸堿耐受性研究發(fā)現(xiàn),重組菌株A31、A61、A84在pH 1.6-4.0的范圍內(nèi),殘余酶活均保持在80%以上,可以較好的滿足動物飼料用酶的要求。(9)金屬離子Na~+、K~+、Fe~(2+)、Zn~(2+)在低濃度時可以激活展示植酸酶的活性,而Cu2+、Mn2+、Co2+對展示植酸酶主要表現(xiàn)為抑制作用。
[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 偽 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 偽 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，

本文編號：2407969