【作者】 王敏；

【导师】 王晓云；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 在饲料中添加植酸酶可提高植物性饲料中磷的利用率和单胃动物对矿质元素的吸收率,并减轻动物排泄物中磷对环境的污染。植酸酶的饲喂效果已经在全世界范围内得到了确证,但目前还未能得到很好的推广应用。其主要原因是植酸酶在天然材料中含量太低;另一方面,酶的稳定性不能完全满足工业化生产的要求。因此,提高植酸酶基因的表达水平,改善植酸酶的稳定性是一个亟待解决的问题。以往研究者往往通过体外添加酶的保护剂、酶的固定化等手段提高植酸酶的稳定性,这些措施都取得了一定的效果,但不是十分的理想。随着基因工程的发展,在分子水平上对酶基因进行改造为改良酶蛋白的性质开辟了一条新的途径。二硫键对蛋白质的结构稳定性及功能具有重要的作用,植酸酶分子中含有5对二硫键,目前关于二硫键对植酸酶的催化活性和构象稳定性的研究较少,仅限于二硫键整体上在植酸酶的去折叠过程中对维持酶的空间结构和催化活性的影响。本研究以自行筛选的产胞外植酸酶的黑曲霉Aspergillus niger N-J为出发菌株,克隆了植酸酶phyA基因,将其在毕赤酵母(Pichia pastoris)中实现了高效表达,酶学性质研究表明表达的植酸酶具有良好的应用特性。在此基础上,首次对植酸酶(PHYA)5对二硫键分别进行了缺失研究,系统地比较了缺失突变体与野生型植酸酶在结构和功能上的区别。本实验的主要结果如下:1.以自行筛选的产胞外植酸酶的黑曲霉Aspergillus niger N-J基因组DNA为模板,PCR扩增得到植酸酶phyA基因片段。序列分析表明phyA开放阅读框长度为1347 bp,包含编码448个氨基酸的植酸酶成熟肽的完整序列;在其编码的氨基酸序列中,存在组氨酸酸性磷酸酶的活性位点保守序列、11个潜在的N-糖基化位点及10个半胱氨酸残基。将phyA基因构建了酵母表达载体pPICZαA/phyA,转化毕赤酵母Pichia pastoris GS115,得到了高表达工程菌株。诱导后发酵液植酸酶比活力较出发菌株相比提高了30,000倍,酶学性质研究表明表达的植酸酶具有优良的工业应用特性,从而实现了植酸酶的高效表达。2.利用定点突变技术将植酸酶分子中的5对二硫键分别进行了突变,构建了5个二硫键分别缺失的植酸酶突变体(C12S, C263S, C395S, C417S和C446S)的表达质粒。5个突变体均在毕赤酵母中得到了高效表达。3.对纯化后酶蛋白的酶学性质研究表明,5个缺失突变体在酸性pH范围内均具有较高的酶活性,比较适合在动物酸性胃肠环境中发挥作用,但二硫键Cys12-Cys21和Cys245-Cys263的缺失造成了酶蛋白pH适用范围的缩小;同时Cys12-Cys21的缺失降低了酶蛋白的最适作用温度。酶促反应动力学研究发现,二硫键Cys52-Cys395、Cys196-Cys446、Cys245-Cys263和Cys417-Cys425的缺失增大了酶对底物的亲和力及催化效率;而Cys12-Cys21的缺失降低了酶与底物的结合能力,同时也降低了酶的催化效率。4.对野生型及突变体植酸酶的空间构象分析表明,5对二硫键的缺失均对酶蛋白的空间结构产生了或多或少的影响,其中Cys245-Cys263和Cys417-Cys425缺失突变体的分子结构变得尤为松散。5.对酶抵抗变性剂稳定性的分析发现,二硫键缺失突变体抗变性剂的能力较野生型均降低,但不同缺失突变体的表现各异。其中二硫键Cys12-Cys21、Cys245-Cys263和Cys417-Cys425的缺失极大地降低了酶在盐酸胍溶液中的稳定性,而Cys52-Cys395和Cys196-Cys446缺失的突变体降低较少。6.对野生型及突变体植酸酶的热稳定性分析表明,5对二硫键的缺失均造成突变体活性中心的构象比野生型更容易发生变化,二硫键Cys245-Cys263及Cys417-Cys425的缺失极大地降低了酶分子的热稳定性;而在高温下Cys196-Cys446的缺失提高了酶分子的耐热能力。此外我们还推测,在受热过程中酶分子的结构存在自我调整以对抗热变性的过程。更多还原

【Abstract】 Phytase catalyzes the hydrolysis of phytate to myo-inositol and inorganic phosphate. The supplementation of animal feed with phytase increases the bioavailability of phosphorus in monogastric animals besides reducing the phosphorus pollution. Despite its corroborated effect, the low yield and stability remain a critical issue in the extensive application of phytase. Therefore, increasing the yield and improving the stability of phytase are of great urgency.Studies of enhancing the stability of phytase are mainly focused on adding protecting agent or the immobility. As the development of gene engineering, it is to be a promising approach to improve enzyme property via protein engineering. Disulfide bonds are indispensable for the stability and function of enzyme. Phytase contains five disulfide bonds. However, comparatively few studies regard the contribution of disulfide bonds to the catalytic activity and conformational stability.In this paper, we selected an A. niger strain which produces extracellular phytase and cloned the phyA gene. This gene was transformed into Pichia pastoris and phytase with potential industrial application was overexpressed. We also investigated the effects of the five disulfide bonds on the conformation, thermostability, resistance to denaturant and catalytic activity of phytase for the first time. Comparison of the functions of different disulfide bonds was also discussed comprehensively. The main results were as follows:1. A phytase-producing strain, Aspergillus niger N-J, was isolated and identified. The phyA gene was cloned by PCR with the genomic DNA of A. niger N-J as template. The sequence analysis showed that the ORF comprises 1347 bp, encoding 448 amino acid residues of phytase mature protein. It has the conservative motif RHGXRXP and 11 potential N-glycosylation sites. It also has 10 Cys that form five pairs of disulfide bonds. We transformed phyA into Pichia pastoris and overexpressed phytase with potential industrial application.2. In this study, five expression plasmids of phytase mutants with deletion of the five disulfide bonds respectively (C12S, C263S, C395S, C417S, C446S) were constructed. All the five mutants were expressed at high level in Pichia pastoris.3. Assay of the enzymological characteristics showed that all the five mutants hold high activity in the range of pH 2.5 to 5.5, which are similar to the stomach and intestines of animal. The deletion of Cys12-Cys21 and Cys245-Cys263 shrank the favorable pH range, and also the deletion of Cys12-Cys21 decreased the optimum temperature. The kinetics analysis showed that the deletions of Cys52-Cys395, Cys196-Cys446, Cys245-Cys263 and Cys417-Cys425 increased the kcat and the kcat/ Km; and the deletion of Cys12-Cys21 decreased the the kcat and the kcat/ Km.4. Conformation studies indicated that deletions of the five disulfide bonds changed the stucture of phytase more or less. The structure of C263S and C417S become loose while the C446S becomes more compact than the wild-type.5. The changes of the resistance to guanidine hydrochloride (GdnHCl) were studied by spectroscopic and activity analysis. It turns out the deletions of disulfide bonds decreased the stability of phytase in GdnHCl solution. However the effect varies greatly with different mutants. The removal of Cys12-Cys21, Cys245-Cys263 and Cys417-Cys425 decreased the stability in GdnHCl solution dramatically, while the removal of Cys52-Cys395 and Cys196-Cys446 changed relatively slighter.6. Thermostability analysis indicated that the deletion of disulfide bond caused a easier-to-change conformation of the active-site. The deletion of Cys245-Cys263 and Cys417-Cys425 decreased the thermolstability largely and the mutant C446S is more stable than the wild-type phytase in high temperature.更多还原