来源于Kosakonia radicincitans新型植酸酶基因通过密码子优化在毕赤酵母里高效表达
发布时间：2018-08-08 12:50
【摘要】：植酸(肌醇六磷酸)是豆类及谷类等作物种子中磷的主要储存形式,但单胃动物(如家禽、猪等)由于体内缺乏分解植酸的酶而难以有效利的用植酸磷,大部分以植酸形式存在的磷被动物直接排出体外,造成严重的环境污染;同时,植酸磷还是一种抗营养因子,它可以和多种金属离子如Ca2+、Mn2+、Mg2+、Zn2+、Cu2+、Fe2+等以及许多蛋白质螯合成相应的不溶性络合物,降低了这些营养物质的生物有效利用率以及动物对营养物质的有效利用。植酸酶(phytase),即肌醇六磷酸水解酶(myo-inositol hexakisphosphate phosphohydrolase,EC 3.1.3.8),是催化植酸(肌醇六磷酸)及其植酸盐水解生成肌醇与磷酸(或磷酸盐)的一类酶的总称,属于组氨酸磷酸酶家族。作为单胃动物饲料添加剂,植酸酶能有效的提高植物性饲料中磷的利用率,进而提高单胃动物对矿质元素的吸收,同时也减轻了动物排泄物中磷对环境的污染。多种微生物如细菌、酵母、丝状真菌等都能够产生植酸酶,但天然生物体中植酸酶含量极其低,难以获得大批量产品。通过基因工程技术构建的植酸酶基因工程菌即可实现植酸酶的高效异源表达,在分子水平上对植酸酶基因进行遗传操作,改善植酸酶的酶学性质如p H最适性、热温度性、催化活性等,提高其在动物体内的有效生物活性,特别是植酸酶的稳定性等,这将对植酸酶的大规模生产和应用开辟更为广阔的前景。本文将来源于Kosakonia radicincitans的植酸酶基因(Kr APPA)在Pichia pastoris表达系统高效表达了有生物活性的胞外植酸酶Kr APPAS。具体实验过程如下:首先根据Kosakonia radicincitans的植酸酶蛋白序列和毕赤酵母密码子偏爱性设计新的植酸酶基因的核苷酸序列,然后再根据此序列设计一系列的引物,用来合成这个目的基因。将合成好的基因经过大肠杆菌克隆并测序鉴定,结果显示设计的基因序列与野生型的完全一致。新的植酸酶基因与野生型基因之间的核苷酸序列比对结果显示77.7%的同源性。将测序正确的植酸酶基因Kr APPAS连接到p PIC9K表达载体上,利用电击法将基因导入到毕赤酵母GS115细胞中,然后筛选具有高活性的重组酵母转化株,甲醇诱导(浓度为1%)24h后蛋白表达量达到最高,约为45μg/ml,发酵上清液中植酸酶活性达82.27U/m L,比活性为1828.18 U/mg,从而实现了植酸酶Kr APPAS基因在Pichia pastoris中的高效分泌表达。通过硫酸铵分级沉淀和Ni离子亲和层析分离纯化出单一的植酸酶重组蛋白,SDS-PAGE结果显示,其分子量约为45k Da,与生物信息学预测理论值大小相当,几乎没有糖基化现象的发生。酶学特性研究结果显示,重组植酸酶的Vmax和Km值分别为1735μmol?min-1mg-1和0.236 m M,最适p H值为3.5,最适温度为55℃,在30-65℃之间,酶相对活性都很高,温度高于60℃后,相对酶活随温度的升高逐渐降低。重组植酸酶的热稳定性比较好,在温度低于65℃时非常稳定,在65℃温育30分钟后酶活剩余90%,当温度超过70℃时,重组植酸酶的稳定性急剧下降,温育仅仅15min时酶活就只剩50%左右。Cu2+和Pb2+对重组植酸酶抑制作用较为强烈,在其终浓度仅为2 m M时,重组植酸酶的活性分别为40%和70%左右。金属离子Mg2+、Ca2+(终浓度为2 m M)对重组植酸酶有一定的激活作用,尤其是Mg2+的激活作用最为明显,可使酶活提高约15%。重组漆酶对SDS极其敏感,在2 m M终浓度处理后重组植酸酶的残余活性几乎为零。综上所述,Kosakonia radicincitans来源的植酸酶非常成功的在Pichia pastoris里高效表达,为植酸酶的结构与功能研究提供了优良的素材。本研究获得了一个具有优良性状并具有潜在商业价值的植酸酶Kr APPAS,确定为HAP植酸酶家族并且有同样的活性位点保守序列RHGXRXP。因此,本研究具有重要的理论意义和实践应用价值。
[Abstract]:Phytic acid (inositol six phosphoric acid) is the main form of phosphorus in seeds of crops such as legumes and cereals, but single stomach animals (such as poultry, pigs, etc.) are difficult to efficiently use phytate phosphorus because of the lack of enzymes that decompose phytate acid in the body. Most of the phosphorus in the form of phytic acid is discharged directly from animals, causing serious environmental pollution. At the same time, phytate phosphorus is also used. It is an anti nutrient factor, which can synthesize insoluble complexes with a variety of metal ions such as Ca2+, Mn2+, Mg2+, Zn2+, Cu2+, Fe2+, and many protein chelates, which reduce the bioavailability of these nutrients and the effective use of animals for nutrients. The phytase (phytase), that is, inositol six phosphoric acid hydrolase (myo-inosit) Ol hexakisphosphate Phosphohydrolase, EC 3.1.3.8), a class of enzymes that catalyze phytic acid (inositol six phosphoric acid) and its phytate hydrolysis to produce inositol and phosphoric acid (or phosphate), belonging to the histidine phosphatase family. As a feed additive for single stomach animals, phytase can effectively improve the utilization of phosphorus in plant feed, and then increase the utilization of phosphorus in plant feed. One stomach animal absorbs mineral elements and reduces the pollution of phosphorus to the environment in animal excrement. A variety of microbes, such as bacteria, yeast, filamentous fungi, can produce phytase, but the content of phytase in natural organisms is extremely low, and it is difficult to obtain large quantities of products. Through the genetic engineering technology of phytase gene engineering bacteria The effective heterologous expression of phytase can be realized, the genetic operation of phytase gene is carried out at the molecular level, the enzymatic properties of phytase, such as P H, thermal temperature, catalytic activity and so on, improve its effective bioactivity in the animal, especially the stability of phytase, which will be produced and applied to the phytase in a large scale. In the future, this paper deriving from the Kosakonia radicincitans phytase gene (Kr APPA) in the Pichia pastoris expression system highly expressed the biological activity of the extracellular phytase Kr APPAS. specific experiment process as follows: first according to the sequence of the phytase protein of Kosakonia radicincitans and the preference for Pichia pastoris codon Design a new phytase gene nucleotide sequence, and then design a series of primers based on this sequence to synthesize this target gene. The synthesized gene is cloned and sequenced in Escherichia coli. The results show that the designed gene sequence is complete with the wild type. The new phytase gene is between the wild type and the wild type. The nucleotide sequence alignment results showed the homology of 77.7%. The sequencing of the correct phytase gene Kr APPAS was connected to the P PIC9K expression vector, and the gene was introduced into Pichia pastoris GS115 cells by electric shock. Then the recombinant yeast transformant with high activity was screened and the protein expression was highest after the methanol induction (concentration of 1%) 24h. For 45 mu g/ml, the phytase activity in the fermented supernatant reached 82.27U/m L and the specific activity was 1828.18 U/mg, thus the efficient secretory expression of the phytase Kr APPAS gene in Pichia pastoris was realized. A single phytase recombinant protein was purified by ammonium sulfate fractionation and Ni ion affinity chromatography. The results of SDS-PAGE showed that the molecular weight of the recombinant protein was about the molecular weight of the enzyme. 45k Da is equivalent to the theoretical value of bioinformatics prediction, and there is almost no glycosylation. The results of enzymatic properties study showed that the Vmax and Km values of the recombinant phytase were 1735 Mu mol? Min-1mg-1 and 0.236 m M respectively, the optimum P H value was 3.5, the optimum temperature was 55, the relative activity of the enzyme was high and the temperature was higher than 60 C at 30-65. The relative enzyme activity gradually decreases with the increase of temperature. The thermal stability of the recombinant phytase is better, it is very stable at the temperature below 65 C. The enzyme activity remains 90% after 30 minutes of temperature breeding at 65 Centigrade. When the temperature exceeds 70, the stability of the recombinant phytase decreases sharply. The enzyme activity is only 50%.Cu2+ and Pb2+ to the recombinant phytase when the temperature is only 15min. The inhibitory effect was strong. When the final concentration was only 2 m M, the activity of recombinant phytase was 40% and 70% respectively. Metal ions Mg2+ and Ca2+ (final concentration 2 m M) had certain activation effect on the recombinant phytase, especially the activation of Mg2+ was most obvious, which could make the enzyme activity increase about 15%. recombined laccase sensitive to SDS, and in 2 m M. The residual activity of the recombinant phytase after degree treatment is almost zero. In summary, the phytase from the Kosakonia radicincitans source is highly successful in Pichia pastoris expression, providing a good material for the structure and function of phytase. This study obtained a phytic acid with excellent properties and potential commercial value. The enzyme Kr APPAS is identified as the HAP phytase family and has the same active site conserved sequence RHGXRXP.. Therefore, this study has important theoretical significance and practical application 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】：2015
【分类号】：Q786 ;S816.7
本文编号：2171829
[Abstract]:Phytic acid (inositol six phosphoric acid) is the main form of phosphorus in seeds of crops such as legumes and cereals, but single stomach animals (such as poultry, pigs, etc.) are difficult to efficiently use phytate phosphorus because of the lack of enzymes that decompose phytate acid in the body. Most of the phosphorus in the form of phytic acid is discharged directly from animals, causing serious environmental pollution. At the same time, phytate phosphorus is also used. It is an anti nutrient factor, which can synthesize insoluble complexes with a variety of metal ions such as Ca2+, Mn2+, Mg2+, Zn2+, Cu2+, Fe2+, and many protein chelates, which reduce the bioavailability of these nutrients and the effective use of animals for nutrients. The phytase (phytase), that is, inositol six phosphoric acid hydrolase (myo-inosit) Ol hexakisphosphate Phosphohydrolase, EC 3.1.3.8), a class of enzymes that catalyze phytic acid (inositol six phosphoric acid) and its phytate hydrolysis to produce inositol and phosphoric acid (or phosphate), belonging to the histidine phosphatase family. As a feed additive for single stomach animals, phytase can effectively improve the utilization of phosphorus in plant feed, and then increase the utilization of phosphorus in plant feed. One stomach animal absorbs mineral elements and reduces the pollution of phosphorus to the environment in animal excrement. A variety of microbes, such as bacteria, yeast, filamentous fungi, can produce phytase, but the content of phytase in natural organisms is extremely low, and it is difficult to obtain large quantities of products. Through the genetic engineering technology of phytase gene engineering bacteria The effective heterologous expression of phytase can be realized, the genetic operation of phytase gene is carried out at the molecular level, the enzymatic properties of phytase, such as P H, thermal temperature, catalytic activity and so on, improve its effective bioactivity in the animal, especially the stability of phytase, which will be produced and applied to the phytase in a large scale. In the future, this paper deriving from the Kosakonia radicincitans phytase gene (Kr APPA) in the Pichia pastoris expression system highly expressed the biological activity of the extracellular phytase Kr APPAS. specific experiment process as follows: first according to the sequence of the phytase protein of Kosakonia radicincitans and the preference for Pichia pastoris codon Design a new phytase gene nucleotide sequence, and then design a series of primers based on this sequence to synthesize this target gene. The synthesized gene is cloned and sequenced in Escherichia coli. The results show that the designed gene sequence is complete with the wild type. The new phytase gene is between the wild type and the wild type. The nucleotide sequence alignment results showed the homology of 77.7%. The sequencing of the correct phytase gene Kr APPAS was connected to the P PIC9K expression vector, and the gene was introduced into Pichia pastoris GS115 cells by electric shock. Then the recombinant yeast transformant with high activity was screened and the protein expression was highest after the methanol induction (concentration of 1%) 24h. For 45 mu g/ml, the phytase activity in the fermented supernatant reached 82.27U/m L and the specific activity was 1828.18 U/mg, thus the efficient secretory expression of the phytase Kr APPAS gene in Pichia pastoris was realized. A single phytase recombinant protein was purified by ammonium sulfate fractionation and Ni ion affinity chromatography. The results of SDS-PAGE showed that the molecular weight of the recombinant protein was about the molecular weight of the enzyme. 45k Da is equivalent to the theoretical value of bioinformatics prediction, and there is almost no glycosylation. The results of enzymatic properties study showed that the Vmax and Km values of the recombinant phytase were 1735 Mu mol? Min-1mg-1 and 0.236 m M respectively, the optimum P H value was 3.5, the optimum temperature was 55, the relative activity of the enzyme was high and the temperature was higher than 60 C at 30-65. The relative enzyme activity gradually decreases with the increase of temperature. The thermal stability of the recombinant phytase is better, it is very stable at the temperature below 65 C. The enzyme activity remains 90% after 30 minutes of temperature breeding at 65 Centigrade. When the temperature exceeds 70, the stability of the recombinant phytase decreases sharply. The enzyme activity is only 50%.Cu2+ and Pb2+ to the recombinant phytase when the temperature is only 15min. The inhibitory effect was strong. When the final concentration was only 2 m M, the activity of recombinant phytase was 40% and 70% respectively. Metal ions Mg2+ and Ca2+ (final concentration 2 m M) had certain activation effect on the recombinant phytase, especially the activation of Mg2+ was most obvious, which could make the enzyme activity increase about 15%. recombined laccase sensitive to SDS, and in 2 m M. The residual activity of the recombinant phytase after degree treatment is almost zero. In summary, the phytase from the Kosakonia radicincitans source is highly successful in Pichia pastoris expression, providing a good material for the structure and function of phytase. This study obtained a phytic acid with excellent properties and potential commercial value. The enzyme Kr APPAS is identified as the HAP phytase family and has the same active site conserved sequence RHGXRXP.. Therefore, this study has important theoretical significance and practical application 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】：2015
【分类号】：Q786 ;S816.7
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