【作者】 钊倩倩；

【导师】 张玉臻；

【作者基本信息】 山东大学 ， 微生物学， 2010， 博士

【摘要】 植酸(phytic acid or myo-inositol hexakisphosphate,缩写为IP6)即肌醇六磷酸酯,属于维生素B族,广泛存在于自然界的各种植物组织和粮食产品中,其盐类(phytate)是磷和肌醇的主要存储形式。单胃动物和人类体内由于缺乏能够水解植酸盐的酶类而无法利用植酸中的磷,植酸盐经过肠道,最后通过粪便排出体外；而为了补充动物食料中的无机磷,需要向饲料中加入额外的无机磷。这一方面提高了饲料的生产成本,另一方面,难于利用的植酸酶及过量添加的无机磷,还对牲畜饲喂地区的环境造成了严重的污染。同时,植酸是一种抗营养因子,对金属离子具有很强的络合作用,能阻止动物对磷、二价矿质元素以及蛋白质、脂肪等的利用。植酸酶的出现则解决了这些问题,它可以将植酸及其盐类降解为肌醇和磷酸,这不仅可以避免补加无机磷到饲料中,间接减少了粪便中磷的排放,而且解决了植酸的抗营养问题,有利于动物对矿物元素及金属离子的吸收和利用。植酸酶(myo-inositol hexakisphosphate phosphohyrolases, EC 3.1.3.8 and 3.1.3.26)即肌醇六磷酸水解酶,是将植酸及其盐类水解为肌醇与磷酸(或磷酸盐)的一类酶的总称。在自然界中,植酸酶广泛的存在于动物、植物及微生物体内,其中应用最为广泛的是来自于微生物特别是一些真菌(如霉菌、酵母等)和细菌(如大肠杆菌、芽孢杆菌等)的植酸酶。由于植酸酶具有极高的应用价值,性质优良的植酸酶基因被越来越多的克隆、研究和利用。植酸酶的分类也很广泛,依据植酸中磷酸基团的降解位点可分为3-型植酸酶和6-型植酸酶；依据作用条件的不同又可分为酸性、中性和碱性植酸酶。其中酸性植酸酶类尤其是组氨酸酸性磷酸酶类由于作用条件接近动物体内环境而应用最为广泛。本课题以从土壤中筛选到的一株能分泌植酸酶活性的青霉菌株为基础。采用了CODEHOP PCR和TAIL PCR相结合的方法克隆到了未知的青霉植酸酶基因。本方法首先选取10个已发表的来源于不同种属的真菌植酸酶家族的蛋白(HAPs)序列,ClustalW比对寻找适合引物设计的保守区(blocks),应用CODEHOP程序设计一对引物CDE1和CDE2,以青霉的总RNA反转录得到的cDNA为模板,扩增出植酸酶cDNA的部分序列。然后设计三组嵌套引物,及高(G+C)%含量的AD引物(arbitrary degenerate primer),以青霉基因组为模板,采用TAIL-PCR分别扩增得到植酸酶基因的5’和3’端未知序列。对克隆得到的序列进行拼接得到了植酸酶基因的全长及部分5’UTR和3’UTR的序列。此种克隆方法相对于传统的通过测定植酸酶蛋白的部分氨基酸序列来设计杂交探针筛选DNA文库或cDNA文库的基因克隆方法,更加简单方便。通过对克隆的植酸酶基因序列进行分析得知,植酸酶基因长1504 bp,内含子位置为43-160 bp之间,cDNA为1386 bp,得到蛋白为461 aa,理论分子量为50.42 KDa,无信号肽序列存在,理论等电点为6.10,蛋白含有6个潜在的N-糖基化位点。氨基酸序列中存在典型的RHGXRXP及HD序列,说明来自青霉的植酸酶序列属于组氨酸酸性磷酸酶家族。将克隆得到的植酸酶序列与已发表的真菌植酸酶序列进行比对发现,在含有共同的保守序列RHGXRXP及HD序列的基础上,与来自一株黑曲霉(GenBank的序列号是EF197825)的植酸酶序列DNA相似性为65%,与一株草酸青霉(GenBank的序列号是AY071824)的植酸酶序列DNA相似性为99%,这些结果在一定程度上代表了植酸酶基因序列的进化。天然的植酸酶蛋白通常表达量比较低,无法达到生产的要求,为此,很多真核表达系统被用来进行蛋白的高通量表达,比如酿酒酵母、毕赤酵母、黑曲霉及米曲霉等。其中毕赤酵母由于体内含有受甲醇严格诱导的强启动子,对外源蛋白的翻译后折叠、修饰及糖基化更接近高等真核生物甚至人类,而且毕赤酵母具有生长快速和操作简便的特点,从而越来越成为一种很有潜力的表达系统。在本课题中我们将植酸酶成熟肽的编码序列连入表达载体pPIC9,采用PEG1000的方法整合插入真核表达系统毕赤酵母GS115的基因组上。利用表达系统中的甲醇诱导型强启动子AOXI promoter及信号肽a-factor,得到了高表达量的分泌型重组植酸酶蛋白。性质测定得知,经发酵后获得的发酵液蛋白含量为302mg/L,蛋白的最适作用温度为50℃,最适pH值为5.5-6.0,由于存在糖基化,经SDS-PAGE检测到的表观分子量为67-75 KDa,采用Endoglycosidase Hf去糖基化后的蛋白大小为50 KDa左右,与氨基酸序列推导出的理论值相符。重组蛋白对胃蛋白酶表现出很好的抗性,当蛋白酶／植酸酶的质量比为0.01时,37℃处理2hr后,酶活仍然可保持原来的96.7%,SDS-PAGE检测显示没有植酸酶蛋白的降解。重组植酸酶对胰蛋白酶比较敏感,当蛋白酶／植酸酶的质量比为0.001时,37℃处理2 hr后,酶活损失约80%,SDS-PAGE检测发现明显的蛋白降解。青霉植酸酶对胃蛋白酶的高抵抗特性为其在动物胃内发挥作用奠定了很好的基础。在工业生产中,植酸酶的加工存在一个几秒到几分钟的高温制粒过程,温度通常达到65-90℃,这就要求植酸酶必须具有良好的热稳定性。另外,做为一种饲料添加剂,植酸酶对于植酸的降解作用主要发生在单胃动物的胃内,降解时的环境温度37℃,pH 3.5-5.0,并且存在大量的胃蛋白酶,因此植酸酶的最适作用温度和pH,以及对胃蛋白酶的抗性在一定程度上也可以影响到的它的应用。针对这些问题,我们采用Mn2+-dITP介导的随机突变技术,构建了青霉植酸酶基因突变体库,突变基因转入毕赤酵母,通过对突变蛋白的性质测定,最终筛选到两株热稳定性提高,而最适作用温度和pH有所下降的突变株,对突变基因进行测序,结果分别为Mut2-28(T11A, G56E, L65F, Q144H和L151S),Mut2-249(T11A, H37Y, G56E, L65F, Q144H, L151S和N354D)。两个突变株所产生的重组植酸酶蛋白在保持了突变前的良好的胃蛋白酶抗性的基础上,蛋白的活性,最适温度,pH,及热稳定性都有了很大的改善。突变蛋白的活性分别为133.3 U及121.5U/mg蛋白,最适温度分别为37-55℃和37-50℃。经过80℃,5 min的热处理,突变蛋白的残余酶活分别为70.36%和88.96%。Mut2-28保持了突变前的最适pH5.5-6.0,而Mut2-249的最适pH则降至pH4.8。所有改进的性质使得本课题得到的两个突变体蛋白能很好的耐受加工制粒过程中的高温,并且能够适应单胃动物胃内的低温酸性环境,因此具有很高的商业价值。目前一些常用的植酸酶蛋白如黑曲霉植酸酶,烟曲霉植酸酶的晶体结构被公布以后,对其后进行的植酸酶性质与功能的研究提供了帮助。我们以烟曲霉植酸酶蛋白的晶体结构为参照,应用SWISS MODEL (http://swissmodel.expasy.org)在线软件预测了青霉植酸酶的三级结构,初步确定了它的活性中心的组成,其中催化中心包含如下氨基酸：Arg71, His72, Arg75, Argl55, His352和Asp353,底物结合中心包含如下氨基酸：Thr78,Lys81,Lys84,Asp215,Asp252和Lys291。通过结构分析可以发现,突变G56E,L65F,Q144H和L151S均增加了突变位点与其它氨基酸残基之间的氢键数量,这增强了相邻的二级结构之间的相互作用,从而增加了整个蛋白分子的热稳定性；而突变N354D的侧链pKa由7.0下降到3.86,这个变化影响了催化中心的pKa,从而造成了植酸酶最适pH的改变；另外,突变L151S侧链与His352侧链的空间距离的增大(2.96A→4.35A)有利于底物与催化中心的自由结合作用,从而提高了植酸酶的催化活性。在青霉植酸酶蛋白分子表面Loop上存在的胃蛋白酶切割位点中,Met1、Phe2、Ala5、Leu6和Phe32处于氨基酸序列的N末端,它们的降解对蛋白质的结构及功能没有明显的影响；其余的切割位点具有一个共同的特点,即处于糖基化位点的周围,例如Leu53、Pro373、Gln431处于糖基化位点Asn367周围,Thr112、Leu113处于糖基化位点Asn110周围,Thr241、Leu242处于糖基化位点Asn95周围,糖链在阻止蛋白酶的降解过程中发挥了重要的作用。胰蛋白酶切割位点中,Argl36、Arg142、Lys212、Arg376和Arg407的羧基所组成的肽键,由于暴露在分子表面,远离糖基化位点,因此容易被胰蛋白酶识别而被切割,这可能是造成Penicilliumsp.植酸酶蛋白对胰蛋白酶敏感的关键原因。另外,在活性位点RHGXRXP序列存在的胰蛋白酶切割位点(Arg71和Arg75),可能对植酸酶结构的进一步被破坏和活性的丧失具有一定作用。本文中植酸酶突变后性质的改变及结构的变化进一步证明了蛋白质的结构与功能之间存在着必然的联系。更多还原

【Abstract】 Phytic acid (myo-inositol hexakisphosphate, IP6) belonged to the subfamily of vitamine B. It was the major storage form of phosphorus and inositol in different plants and foods in the nature. The monogastric animals (e.g., poultry and pig) including human beings could not use the phosphorus in phytate because they had little or no phytate-degrading enzymes in their gastrointestinal tracts, so the phytate was metabolized with the dejecta. In another hand, inorganic phosphorus had to be added to the commercial animal feed in order to balance the inorganic phosphorus lackage, which increased the feed’s cost. Moreover, excessive addition of inorganic phosphorus to feed caused environmental pollution. In addition, phytic acid was antinutritive because it could chelate important minerals such as copper, iron, magnesium, calcium and zinc, which prevented the absorption of inorganic phosphorus, minerals, proteins and fats. The addition of phytase might solve this problem because they could hydrolyze phytate into myoinositol and inorganic phosphate in the gastrointestinal tracts of monogastric animals. This could decrease the dejecta of unabsorbed inorganic phosphorus, avoid adding excess inorganic phosphorus, and facilitate the absorption of minerals.Phytase (myo-inositol hexakisphosphate phosphohyrolases, EC 3.1.3.8 and 3.1.3.26) was defined as the enzyme which hydrolyzed phytic acid or phytate into myoinositol and inorganic phosphate. It existed broadly in animals, plants and microbes, and was widely used in several aspects, especially those from fungi (e.g., molds and yeasts) and bacteria (e.g., Escherichia coli and bacillius). More and more phytase genes were studied and applied due to their high value. The categories of phytase were differed as different classification standard. Phytase contained 3-style and 6-style according to the hydrolysis position, and it contained acidic, neutral and alkaline styles according to the catalysis pH. The family of histidine acid phosphatases (HAPs) was most widely applied.Our research based on the selection of Penicillium sp. with phytase activity from soil. The phytase gene was cloned utilizing the combination of CODEHOP PCR and TAIL PCR. Based on the conserved regions recognized by ClustalW online, CODEHOP primers were designed and used to amplify a conserved partial sequence of the phytase gene using total cDNA as template. TAIL PCR was then employed to clone the 5’ and 3’ flanking sequences, and the whole sequence including 5’UTR and 3’UTR was obtained. Compared with the traditional cloning method which a hybridization probe is usually designed to screening DNA libraries or cDNA libraries, the method using in our research was more simple and convenient. Sequence analysis suggested that the gene had a length of 1,504 bp, with an intron sequence at the position of 43 bp to 160 bp, and had no signal peptide sequence. The codon sequence of mature protein had a length of 1,386 bp, with a protein length of 461 aa acorrdingly. The theoretical isoelectric point (pⅠ) was 6.10, and there were 6 potential positions of N- glycosylation. The cloned gene contained the RHDXRXP and HD sequences which existed typically in the family of histidine acid phosphatases (HAPs). The gene showed a similarity of 65% with phytase gene from Aspergillus niger (accession number EF197825 in GenBank), and 99% with PJ3 phytase gene (accession number AY071824 in GenBank) from Penicillium oxalicum. The resulst might indicate the revolution of phytase gene.The phytase produced in nature always could not satisfy the requirement of industry because of its low expression. In order to solve this problem, lots of eukaryotic expression systems were used to obtain high protein expression, such as Saccharomyces cerevisiae, Pichia pastoris, A. niger, Aspergillus oryzae and so on. The P. pastoris strains have been a potential expression system owing to its strong promoter induced by methanol, rapid growth, simple manipulation and the protein modification style which similar to the high eukaryotic cells even including human beings. The cDNA sequence (1,386 bp) was inserted into the plasmid pPIC9 and integrated into the genome of P. pastoris GS115. Under the control of the alcohol oxidaseⅠpromoter and facilitated by a-factor in the yeast host, the cDNA sequence produced a highly-expressed recombinant protein with phytase activity in the culture supernatant. Property analysis suggested that the protein yield was 302 mg/L culture, with optimal catalyzing temperature at 50℃, optimal pH at 5.5-6.0. SDS-PAGE analysis showed a molecular size of 67-75KDa, and the size decreased to about 50 KDa after deglycosylation by endoglycosidase Hf. The recombinant enzyme showed a high resistance to pepsin. The enzyme activity lost no more than 5% after 2 hr of digestion under various protease/protein (w/w) ratios, and no degradation detected by SDS-PAGE analysis. The phytase enzyme was sensitive to the trypsin digestion. Almost 80% of the original activity was lost after 2 hr of digestion at the trypsin/protein (w/w) ratio of 0.001. SDS-PAGE analysis showed that the protein was extensively digested. The high resistance to pepsion enabled the phytase protein be particularly suitable for working in the stomach of animals.In industry, a good thermal stability was required for phytase to sustain the heat denaturation process of regular feed pelleting process in which the temperature was up to 65-90℃for several seconds or minutes. In addition, the catalysis of phytate hydrolysis occured in the animals’stomach and small intestine at the body temperature (37℃) and low pH (about 3.5-5.0), in the presence of proteases like pepsin and trypsin. Therefore, the low catalytic temperature and pH and the presence of pepsin and trypsin seriously affected the activity of phytase so as to limit its application. To solve these problems, random mutation laboratory of the phytase gene was constructed by Mn2+-dITP mutation method, and the mutant sequences were transformed to P. pastoris. The properties of the recombinant proteins were detected and two mutants were screened with improved thermal stability and reduced optimal temperature and pH. The mutant phytase genes were cloned and sequenced as follows. Mutant 2-28 contained T11A, G56E, L65F, Q144H and L151S mutations, and Mutant 2-249 contained T11A, H37Y, G56E, L65F, Q144H, L151S and N354D mutations. Besides of the excellent resistance to pepsin, other properties were improved largely. The catalytic activity at 37℃was up to 133.3 U and 121.5 U per mg protein with broad optimal temperature ranges of 37-55℃and 37-50℃, respectively. After a heat treatment at 80℃for 5 min, the two mutant proteins could retain about 70.36% and 88.96% of the initial activity, respectively. In addition, the optimal pH of Mut2-249 was reduced to 4.8. These improved properties allowed these two mutations to be more active under the stomach conditions than the wild-type phytase. Moreover, the high thermal stability allowed the phytase to tolerate the high temperature in the regular feed pelleting process. Therefore, the recombinant enzymes we identified would have a good commercial potential in animal feed application.At present, the crystal structures of phytase proteins from Aspergillius ficuum, Aspergillus fumigatus and A. niger have been reported, which facilitate the investigation of the properties and functions of this enzyme. We predicted the tertiary structure of phytase from Penicillium sp. using the SWISS MODEL (http://swissmodel.expasy.org) based on the crystal structure of A. fumigatus. The catalytic centre contained six amino acid residues:Arg71, His72, Arg75, Arg155, His352 and Asp353. The substrate binding site distributed around, which contained Thr78, Lys81, Lys84, Asp 215, Asp252 and Lys291. Structure analysis suggested that the replacements of G56E, L65F, Q144H, and L151S improved the thermal stability of the protein by increasing new hydrogen bonds among the adjacent secondary structures. And, the mutation of L151S enhanced the activity in the temperature range of 37-50℃by facilitating the interaction between substrate and catalytic centre. The substitution of N354D influenced the pH profile by weakening the bondage with the side chain of D353, which caused a pKa shift of the catalytic centre. Among all the pepsin cleavage sites in the surface of the phytase protein, degradation at sites of Metl, Phe2, Ala5, Leu6 and Phe32 did not have obvious affection to the structure and function of the protein because they were located on the N-terminal of the phytase. Other cleavage sites all lay besides the glycosylation sites, for example, sites of Leu53, Pro373 and Gln431 were close to the glycosylation site of Asn367, and sites of Thr112 and Leu113 both near the glycosylation site of Asn110, in addition, sites of Thr241 and Leu242 were around the glycosylation site of Asn95. This suggested that the suger chain played important role in preventing the degradation of protease. Among the trypsin cleavage sites, the peptide bonds which formed by the carboxyl of Arg136, Arg142, Lys212, Arg376 and Arg407 were identifed and cleaved easy because they exposed and far from the glycosylation sites. This might be the key reason for phytase to be sensitive to trypsin. In addition, the cleavage sites in the RHGXRXP sequence (Arg71 and Arg75) might lead to the lost of activity. The improvements of the properties and changes of the structure had proved again a relationship between the function and the structure of the protein.更多还原