来源于Yersinia kristensenii的新型植酸酶基因YkAPPAS在毕赤酵母中的表达研究及其酶学特性分析

发布时间：2018-04-17 19:40

本文选题：植酸 + 植酸酶　；参考：《上海海洋大学》2017年硕士论文

【摘要】：通常情况下,粮食中的肌醇和磷大多以植酸的状态存在。以植酸盐状态存在的有机磷酸化合物被称为植酸磷。动物要吸收饲料中以植酸磷状态存在的磷,只有将植酸磷催化成无机磷酸盐的形式。植酸酶属磷酸单酯水解酶,是最近几年出现的一种新型绿色添加剂,它通过催化过程,将植酸以及植酸盐催化成磷酸(或盐)和肌醇。有很多实验研究证明,使用植酸酶作为饲料添加剂时,磷的吸收率可被提高,无机磷的补充量被减少,同时动物粪便中无机磷的比重也下降;在饲料中植酸酶的加入能有效的抑制机体内的植酸与金属离子和蛋白质生成络合物,提高多种微量元素和蛋白质的利用率。天然来源的植酸酶普遍存在表达水平低、提取困难、成本高等问题,这些问题都导致其难以满足现实生产的需要。所以构建基因工程菌作为微型生物反应器来实现异源高水平分泌表达,成为解决这一问题的关键。通过基因工程手段能够解决天然植酸酶分泌量低及抗逆性和热稳定性差等问题。利用巴斯德毕赤酵母作为生物反应器诱导重组植酸酶表达的方法是最具有优势的,该方法操作简便、表达量大且适宜工业化生产。凭借微生物工程技术的不断发展,对植酸酶结构和功能的探索正逐渐的成熟与深化。本文对来源于Yersinia kristensenii的植酸酶基因YkAPPA进行改造,并成功实现了在大肠杆菌(Escherichia coli)EG60和毕赤酵母(Pichia pastoris)GS-115中异源表达,同时对其重组植酸酶相关酶学特性和动力学参数等进行了研究分析,主要研究结果如下:1.利用NCBI数据库公布的来源于Yersinia kristensenii(基因登录号为JQ394763.1)的植酸酶基因序列,按照毕赤酵母的密码子偏爱性对目的基因进行优化,优化前与优化后的氨基酸序列完全一致。设计合成长度为60bp的小片段引物序列,利用PTDS基因合成技术合成了新的重组植酸酶基因YkAPPAS,重组植酸酶基因全长为1266bp,依据密码子偏好性将GC含量调整至51.9%。本实验获得的植酸酶YkAPPAS经过对比后发现,其氨基酸序列中含有植酸酶的最具保守的两个区域:分别是与植酸酶底物结合位点RHG×R×P和酶的催化中心HD。将合成的目的基因与pMD18-T载体连接克隆及测序,随后将测序成功的目的基因与表达载体相连,合成pYPX88-YkAPPAS载体。利用电击法将载体导入酵母GS-115中,筛选具有高活性的转化株,用终浓度为1%的甲醇诱导培养,发酵上清液中酶的分泌量可达到106.73μg/mL,完成了重组植酸酶YkAPPAS在毕赤酵母中的高活性、高产量的表达。在重组植酸酶YkAPPAS基因的5’端添加了6个His标记,所以利用硫酸铵分级沉淀法进行蛋白的初步纯化后再利用Ni离子亲和层析分离出单一的植酸酶重组蛋白。聚丙烯酰氨凝胶电泳结果显示,重组植酸酶YkAPPAS的蛋白分子量约为46kDa,实际结果与生物信息学预测理论值一致,说明YkAPPAS几乎没有糖基化现象的发生。2.将纯化后的重组植酸酶进行酶学特性分析,研究结果显示,其最适pH分别是pH4.5和pH6,pH3-pH12时的活性都在30%以上,最适温度在45℃,在100℃处理5min后其活性为47.55%,90℃处理5min后其活性为56.75%。EDTA及Mn2+对酶的活性有一定的激活作用;Cu2+以及Al3+、Pb2+、Zn2+等对酶活有一定的抑制作用,尤其是SDS抑制力最强。综上所述,本实验获得的相关酶活数据为应用基因工程和蛋白质工程等方法解决植酸酶分泌量少、活性低等问题提供了重要的借鉴和参考价值。
[Abstract]:Usually, in the grain of inositol and phytic acid phosphorus mostly exist in the state. Organic phosphate compounds exist in phytate state called phytate phosphorus. Animal feed to absorb Phytic Acid P state of phosphorus, only the catalytic phytate into inorganic phosphate form. Phytic acid enzyme is phosphoric monoester hydrolases. Is a kind of new green additives in recent years, it will be through catalytic process, phytic acid and phytate catalyzed into phosphoric acid (or salt) and inositol. There are a lot of experiments, the use of phytase as feed additives, phosphorus absorption rate can be improved, adding amount of inorganic phosphorus was reduced, while inorganic phosphorus the proportion of animal manure also decreased; phytic acid with metal ions and protein complexes within the body to effectively inhibit phytase added in the feed, improve the utilization ratio of trace elements and protein. Day However, the source of phytase widespread low expression extraction difficulty, high cost, all these problems make it difficult to meet the real needs of production. So the construction of genetic engineering bacteria as micro bioreactor to achieve a high level of heterologous expression, is the key to solve this problem. By means of genetic engineering can solve the problems of natural phytase the secretion and low resistance and poor thermal stability. As a bioreactor induced by recombinant phytase expression in Pichia pastoris by Pasteur is the most has the advantage of this method is simple, the expression quantity and is suitable for industrial production. With the continuous development of microbial engineering technology, to explore the structure and function of phytase is gradually mature with the deepening of YkAPPA. The phytase gene from Yersinia kristensenii was improved, and the successful implementation in Escherichia coli ( Escherichia coli) and EG60 (Pichia pastoris) in Pichia pastoris heterologous expression in GS-115, and the recombinant phytase enzymatic characteristics and kinetic parameters were analyzed, the main results are as follows: 1. using the NCBI database published from Yersinia kristensenii (GenBank accession No. JQ394763.1) of the phytase gene sequence, according to the purpose optimized gene of Pichia pastoris codon preference, optimized amino acid sequence and the optimized length is exactly the same. The design and synthesis of small fragment primers of 60BP, recombinant phytase gene YkAPPAS was prepared by the PTDS gene synthesis technology, the full-length recombinant phytase gene is 1266bp, based on the codon preference of the phytase YkAPPAS GC the content of adjustment to obtain 51.9%. this experiment by comparison found that two Contained Phytase in the amino acid sequence of the most conservative Area: are with phytase substrate binding site RHG * R * P and enzyme catalytic center HD. synthesis gene and cloned into pMD18-T vector cloning and sequencing, then gene sequencing and expression vector successfully connected to pYPX88-YkAPPAS vector synthesis. Using electroporation. The carrier is introduced into yeast GS-115, screening with high activity the transformants were cultured for 1%, with the final concentration of methanol induced secretion in the supernatant enzyme can reach 106.73 u g/mL, the recombinant phytase YkAPPAS in Pichia pastoris expressed high activity and high yield. The recombinant phytase YkAPPAS gene 5 'end with 6 His markers, so the use of ammonium sulfate fractionation of protein purified by Ni ion affinity chromatography to isolate a single recombinant phytase protein. Polyacrylamide gel electrophoresis showed that the recombinant phytase YkAPPAS egg White molecular weight is about 46kDa, the actual results and the theoretical value of bioinformatics prediction, indicates that.2. YkAPPAS almost no glycosylation phenomenon will be carried out after the purification of recombinant phytase enzyme characteristics analysis, the results of the study showed that the optimum pH is respectively pH4.5 and pH6, pH3-pH12 activity in more than 30%, the most optimum temperature at 45 degrees, 100 degrees in the treatment of 5min after its activity was 47.55%, 90 5min after the treatment to activate the activity of 56.75%.EDTA and Mn2+ on the activity of enzyme; Cu2+ and Al3+, Pb2+, Zn2+ has an inhibitory effect on the enzyme activity, especially SDS. To sum up the strongest inhibition in this experiment, enzymes related to live data for the application of gene engineering and protein engineering methods to solve the phytase secretion of small, low activity provides an important reference value.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q55

【参考文献】