來源于Kosakonia radicincitans新型植酸酶基因通過密碼子優(yōu)化在畢赤酵母里高效表達
發(fā)布時間：2018-08-08 12:50
【摘要】：植酸(肌醇六磷酸)是豆類及谷類等作物種子中磷的主要儲存形式,但單胃動物(如家禽、豬等)由于體內(nèi)缺乏分解植酸的酶而難以有效利的用植酸磷,大部分以植酸形式存在的磷被動物直接排出體外,造成嚴(yán)重的環(huán)境污染;同時,植酸磷還是一種抗?fàn)I養(yǎng)因子,它可以和多種金屬離子如Ca2+、Mn2+、Mg2+、Zn2+、Cu2+、Fe2+等以及許多蛋白質(zhì)螯合成相應(yīng)的不溶性絡(luò)合物,降低了這些營養(yǎng)物質(zhì)的生物有效利用率以及動物對營養(yǎng)物質(zhì)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)及其植酸鹽水解生成肌醇與磷酸(或磷酸鹽)的一類酶的總稱,屬于組氨酸磷酸酶家族。作為單胃動物飼料添加劑,植酸酶能有效的提高植物性飼料中磷的利用率,進而提高單胃動物對礦質(zhì)元素的吸收,同時也減輕了動物排泄物中磷對環(huán)境的污染。多種微生物如細菌、酵母、絲狀真菌等都能夠產(chǎn)生植酸酶,但天然生物體中植酸酶含量極其低,難以獲得大批量產(chǎn)品。通過基因工程技術(shù)構(gòu)建的植酸酶基因工程菌即可實現(xiàn)植酸酶的高效異源表達,在分子水平上對植酸酶基因進行遺傳操作,改善植酸酶的酶學(xué)性質(zhì)如p H最適性、熱溫度性、催化活性等,提高其在動物體內(nèi)的有效生物活性,特別是植酸酶的穩(wěn)定性等,這將對植酸酶的大規(guī)模生產(chǎn)和應(yīng)用開辟更為廣闊的前景。本文將來源于Kosakonia radicincitans的植酸酶基因(Kr APPA)在Pichia pastoris表達系統(tǒng)高效表達了有生物活性的胞外植酸酶Kr APPAS。具體實驗過程如下:首先根據(jù)Kosakonia radicincitans的植酸酶蛋白序列和畢赤酵母密碼子偏愛性設(shè)計新的植酸酶基因的核苷酸序列,然后再根據(jù)此序列設(shè)計一系列的引物,用來合成這個目的基因。將合成好的基因經(jīng)過大腸桿菌克隆并測序鑒定,結(jié)果顯示設(shè)計的基因序列與野生型的完全一致。新的植酸酶基因與野生型基因之間的核苷酸序列比對結(jié)果顯示77.7%的同源性。將測序正確的植酸酶基因Kr APPAS連接到p PIC9K表達載體上,利用電擊法將基因?qū)氲疆叧嘟湍窯S115細胞中,然后篩選具有高活性的重組酵母轉(zhuǎn)化株,甲醇誘導(dǎo)(濃度為1%)24h后蛋白表達量達到最高,約為45μg/ml,發(fā)酵上清液中植酸酶活性達82.27U/m L,比活性為1828.18 U/mg,從而實現(xiàn)了植酸酶Kr APPAS基因在Pichia pastoris中的高效分泌表達。通過硫酸銨分級沉淀和Ni離子親和層析分離純化出單一的植酸酶重組蛋白,SDS-PAGE結(jié)果顯示,其分子量約為45k Da,與生物信息學(xué)預(yù)測理論值大小相當(dāng),幾乎沒有糖基化現(xiàn)象的發(fā)生。酶學(xué)特性研究結(jié)果顯示,重組植酸酶的Vmax和Km值分別為1735μmol?min-1mg-1和0.236 m M,最適p H值為3.5,最適溫度為55℃,在30-65℃之間,酶相對活性都很高,溫度高于60℃后,相對酶活隨溫度的升高逐漸降低。重組植酸酶的熱穩(wěn)定性比較好,在溫度低于65℃時非常穩(wěn)定,在65℃溫育30分鐘后酶活剩余90%,當(dāng)溫度超過70℃時,重組植酸酶的穩(wěn)定性急劇下降,溫育僅僅15min時酶活就只剩50%左右。Cu2+和Pb2+對重組植酸酶抑制作用較為強烈,在其終濃度僅為2 m M時,重組植酸酶的活性分別為40%和70%左右。金屬離子Mg2+、Ca2+(終濃度為2 m M)對重組植酸酶有一定的激活作用,尤其是Mg2+的激活作用最為明顯,可使酶活提高約15%。重組漆酶對SDS極其敏感,在2 m M終濃度處理后重組植酸酶的殘余活性幾乎為零。綜上所述,Kosakonia radicincitans來源的植酸酶非常成功的在Pichia pastoris里高效表達,為植酸酶的結(jié)構(gòu)與功能研究提供了優(yōu)良的素材。本研究獲得了一個具有優(yōu)良性狀并具有潛在商業(yè)價值的植酸酶Kr APPAS,確定為HAP植酸酶家族并且有同樣的活性位點保守序列RHGXRXP。因此,本研究具有重要的理論意義和實踐應(yīng)用價值。
[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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q786 ;S816.7
本文編號：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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q786 ;S816.7
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