右旋糖酐蔗糖酶分子改造及其催化性質(zhì)研究
發(fā)布時(shí)間：2018-07-25 17:16
【摘要】：本實(shí)驗(yàn)室前期慈寧宮腸膜狀明串珠菌Leuconostoc mesenteroides 0326的右旋糖酐蔗糖酶dextransucrase(EC 2.4.1.5)中克隆獲得基因dex-YG,并以該基因?yàn)榛A(chǔ)構(gòu)建了右旋糖酐蔗糖酶大腸桿菌表達(dá)體系的工程菌。右旋糖酐蔗糖酶以蔗糖為底物通過(guò)水解轉(zhuǎn)移葡萄糖基合成高分子葡聚糖。本文通過(guò)對(duì)右旋糖酐蔗糖酶基因dex-YG進(jìn)行系列分子截短,分析不同片段的右旋糖酐截短突變酶的特性,以探究右旋糖酐蔗糖酶結(jié)構(gòu)區(qū)域與催化功能的關(guān)系,揭示其催化機(jī)制;在此基礎(chǔ)上選定特定區(qū)域能進(jìn)行氨基酸定點(diǎn)突變、嵌入突變,獲得不同酶學(xué)性質(zhì)的正突變酶,探究其控制產(chǎn)物特異性的催化機(jī)制,獲得催化合成不同枝化度的新型右旋糖酐產(chǎn)物,擴(kuò)大該酶的應(yīng)用領(lǐng)域。1、以右旋糖酐蔗糖酶基因序列dex-YG為基礎(chǔ),通過(guò)生物信息學(xué)的比對(duì)分析,對(duì)其二級(jí)結(jié)構(gòu)以及三級(jí)結(jié)構(gòu)進(jìn)行預(yù)測(cè)分析,對(duì)其C端序列進(jìn)行一系列的截短,分析其結(jié)構(gòu)功能的關(guān)系。通過(guò)片段截短的方法對(duì)其糖鏈延伸的控制區(qū),寡聚糖合成區(qū)域,以及完全保守區(qū)域進(jìn)行了研究分析,探討右旋糖酐蔗糖酶結(jié)構(gòu)區(qū)域與催化功能的關(guān)系。結(jié)果表明:對(duì)其末端重復(fù)序列進(jìn)行刪除,會(huì)極大的破壞右旋糖酐蔗糖酶合成葡聚糖的能力。隨截短片段長(zhǎng)度增加,其合成高分子葡聚糖的能力急劇下降,在蛋白367aa個(gè)氨基酸的截短后,其右旋糖酐的合成能力完全喪失,相對(duì)應(yīng)的其受體反應(yīng)的催化功能會(huì)明顯增強(qiáng),從而導(dǎo)致寡聚糖的合成能力顯著提升。隨著更進(jìn)一步的片段截短直至其保守序列motifⅠ,其受體反應(yīng)的催化性能也極具下降,其酶活力幾乎完全喪失。2、不同類(lèi)型的糖酐水解酶其合成的葡聚糖其糖苷鍵的組成卻又很大的區(qū)別,包括α(1-2)、α(1-3)、α(1-4)、α(1-6)糖苷鍵。通過(guò)對(duì)分子對(duì)接以及動(dòng)力學(xué)的模擬分析,對(duì)受體以及底物結(jié)合區(qū)域的關(guān)鍵氨基酸進(jìn)行替換,通過(guò)分析其對(duì)合成產(chǎn)物的影響結(jié)合分子模擬的分析結(jié)構(gòu),探究其控制產(chǎn)物的催化機(jī)制,合成不同鍵型的右旋糖酐產(chǎn)物,擴(kuò)大該酶的應(yīng)用領(lǐng)域。通過(guò)對(duì)關(guān)鍵位點(diǎn)的氨基酸替換,相對(duì)于原始的右旋糖酐蔗糖酶的催化產(chǎn)物右旋糖酐5%α(1-3)以及95%α(1-6)鍵型組成,突變后的鍵型組成變?yōu)?-9%α(1-3)和90-98%α(1-6)鍵型組成,部分突變產(chǎn)生了額外的α(1-2)鍵和α(1-4)鍵。模擬分析可以發(fā)現(xiàn),替換氨基酸其側(cè)鏈的大小、電荷狀況以及疏水性等都會(huì)較大的影響受體結(jié)合最穩(wěn)定構(gòu)象,從而影響酶學(xué)性質(zhì)以及產(chǎn)物特異性等。3、對(duì)催化口袋中的特定氨基酸進(jìn)行替換會(huì)在一定程度的影響產(chǎn)物的鍵型,但其變化有一定的局限性。通過(guò)對(duì)不與底物或受體直接作用的保守序列的關(guān)鍵位點(diǎn)進(jìn)行氨基酸插入突變,會(huì)更大程度改變產(chǎn)物右旋糖酐的鍵型。以同源重組的方式對(duì)663以及553位點(diǎn)進(jìn)行氨基酸的飽和嵌入,通過(guò)對(duì)活性菌株的篩選以及協(xié)同突變,獲得了超高分支葡聚糖產(chǎn)物突變株。實(shí)驗(yàn)結(jié)果得到氨基酸嵌入的突變方式雖然會(huì)在一定程度影響酶活性,但其得到的突變體催化性質(zhì)變化顯著。更進(jìn)一步的協(xié)同突變表明,其產(chǎn)物特異性變化更加顯著。綜上,本文通過(guò)對(duì)右旋糖酐蔗糖酶基因的分子截短、定點(diǎn)突變和嵌入突變,探討了右旋糖酐蔗糖酶結(jié)構(gòu)區(qū)域與催化功能的關(guān)系,揭示其催化機(jī)制;為獲得特異性的正突變酶以及新型右旋糖酐的催化合成打下了基礎(chǔ),擴(kuò)大該酶的應(yīng)用領(lǐng)域。
[Abstract]:The gene dex-YG was cloned from the dextran sucrase dextransucrase (EC 2.4.1.5) of Leuconostoc mesenteroides 0326 in the early stage of tning Gong, and based on this gene, the engineering bacteria of the expression system of E. coli sucrase in dextran was constructed. Sucrase was hydrolyzed with sucrose as the substrate. In this paper, the glucose based polymer glucan was synthesized. In this paper, a series of molecules of dextran sucrase gene dex-YG were truncated to analyze the characteristics of different segments of dextran truncated mutase, in order to explore the relationship between the structure area of dextran sucrase and the catalytic function, and to reveal its catalytic mechanism. On this basis, the specific region can be selected. Amino acid site directed mutagenesis, embedding mutation, obtaining different enzyme properties of positive mutagenesis enzymes, exploring the catalytic mechanism of controlling product specificity, obtaining new dextran products with different dendrite degrees, and expanding the application field.1 of the enzyme, based on the sequence dex-YG of dextran sucrase gene, through bioinformatics ratio On the analysis, the secondary structure and the three stage structure are predicted and analyzed. A series of truncation of the C end sequence is made and the relationship between the structure and function is analyzed. Through the truncation of fragments, the control area of its sugar chain extension, the oligosaccharide synthesis area, and the completely conservative region are studied and analyzed, and the structure area of dextran sucrase is discussed. The relationship between the domain and the catalytic function shows that the ability of dextran sucrase to synthesize dextran can greatly destroy the ability of dextran sucrase to synthesize dextran. As the length of the truncated fragment increases, the ability to synthesize the polymer dextran sharply decreases. After the truncation of the protein 367aa amino acids, the synthesis of dextran is completely bereaved. The catalytic function of the receptor reaction was significantly enhanced and the synthesis capacity of oligosaccharides was significantly enhanced. As further fragments were truncated until its conservative sequence motif I, the catalytic performance of its receptor reaction was also greatly reduced, and its enzyme activity almost completely lost.2, and different types of glycic anhydride hydrolase was synthesized. The composition of glucoside bonds is very different, including alpha (1-2), alpha (1-3), alpha (1-4), and alpha (1-6) glycoside bonds. By simulating the docking and kinetics of molecular docking, the key amino acids in the receptor and the substrate binding region are replaced, and the analysis of their effects on the synthetic products and the analytical structure of the molecular simulation are carried out to explore its control. The catalytic mechanism of the product to synthesize the product of different bond forms of dextran to expand the application field of the enzyme. By replacing the amino acid at the key site, the mutation is changed to 1-9% a (1-3) and 90-98% alpha (1-6) bonds, which are composed of 5% alpha (1-3) and 95% alpha (1-6) bond forms of the catalytic product of the original dextran sucrase. The partial mutation produces an additional alpha (1-2) bond and alpha (1-4) bond. It is found that the size of the side chain, the charge status, and the hydrophobicity of the substituted amino acids will greatly influence the most stable conformation of the receptor binding, thus affecting the enzyme properties and the specificity of the product, such as.3, for the specific amino acids in the catalytic pockets. The change has a certain degree of influence on the bond type of the product, but the change has some limitations. By inserting the amino acid into the key site of the conservative sequence that does not directly interact with the substrate or the receptor, the bond type of the product of the product will be changed to a greater extent. The amino acid saturation of the 663 and the 553 loci of the homologous recombination is carried out. The mutant strains of ultra high branching glucan products were obtained by screening and co mutation of active strains. The results showed that the mutation mode embedded in the amino acid could affect the enzyme activity to a certain extent, but the change of the catalytic properties of the mutant was significant. A further synergistic mutation showed that the specific change of the product was specific. To sum up, the relationship between the structure area of dextran sucrase and the catalytic function of dextran sucrase was explored through the molecular truncation, fixed-point mutation and embedding mutation of dextran sucrase gene, and its catalytic mechanism was revealed, and the basis for the catalytic synthesis of specific positive mutaginase and new dextran was established, and the enzyme was expanded. Application field.
【學(xué)位授予單位】：合肥工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O629.8
本文編號(hào)：2144497
[Abstract]:The gene dex-YG was cloned from the dextran sucrase dextransucrase (EC 2.4.1.5) of Leuconostoc mesenteroides 0326 in the early stage of tning Gong, and based on this gene, the engineering bacteria of the expression system of E. coli sucrase in dextran was constructed. Sucrase was hydrolyzed with sucrose as the substrate. In this paper, the glucose based polymer glucan was synthesized. In this paper, a series of molecules of dextran sucrase gene dex-YG were truncated to analyze the characteristics of different segments of dextran truncated mutase, in order to explore the relationship between the structure area of dextran sucrase and the catalytic function, and to reveal its catalytic mechanism. On this basis, the specific region can be selected. Amino acid site directed mutagenesis, embedding mutation, obtaining different enzyme properties of positive mutagenesis enzymes, exploring the catalytic mechanism of controlling product specificity, obtaining new dextran products with different dendrite degrees, and expanding the application field.1 of the enzyme, based on the sequence dex-YG of dextran sucrase gene, through bioinformatics ratio On the analysis, the secondary structure and the three stage structure are predicted and analyzed. A series of truncation of the C end sequence is made and the relationship between the structure and function is analyzed. Through the truncation of fragments, the control area of its sugar chain extension, the oligosaccharide synthesis area, and the completely conservative region are studied and analyzed, and the structure area of dextran sucrase is discussed. The relationship between the domain and the catalytic function shows that the ability of dextran sucrase to synthesize dextran can greatly destroy the ability of dextran sucrase to synthesize dextran. As the length of the truncated fragment increases, the ability to synthesize the polymer dextran sharply decreases. After the truncation of the protein 367aa amino acids, the synthesis of dextran is completely bereaved. The catalytic function of the receptor reaction was significantly enhanced and the synthesis capacity of oligosaccharides was significantly enhanced. As further fragments were truncated until its conservative sequence motif I, the catalytic performance of its receptor reaction was also greatly reduced, and its enzyme activity almost completely lost.2, and different types of glycic anhydride hydrolase was synthesized. The composition of glucoside bonds is very different, including alpha (1-2), alpha (1-3), alpha (1-4), and alpha (1-6) glycoside bonds. By simulating the docking and kinetics of molecular docking, the key amino acids in the receptor and the substrate binding region are replaced, and the analysis of their effects on the synthetic products and the analytical structure of the molecular simulation are carried out to explore its control. The catalytic mechanism of the product to synthesize the product of different bond forms of dextran to expand the application field of the enzyme. By replacing the amino acid at the key site, the mutation is changed to 1-9% a (1-3) and 90-98% alpha (1-6) bonds, which are composed of 5% alpha (1-3) and 95% alpha (1-6) bond forms of the catalytic product of the original dextran sucrase. The partial mutation produces an additional alpha (1-2) bond and alpha (1-4) bond. It is found that the size of the side chain, the charge status, and the hydrophobicity of the substituted amino acids will greatly influence the most stable conformation of the receptor binding, thus affecting the enzyme properties and the specificity of the product, such as.3, for the specific amino acids in the catalytic pockets. The change has a certain degree of influence on the bond type of the product, but the change has some limitations. By inserting the amino acid into the key site of the conservative sequence that does not directly interact with the substrate or the receptor, the bond type of the product of the product will be changed to a greater extent. The amino acid saturation of the 663 and the 553 loci of the homologous recombination is carried out. The mutant strains of ultra high branching glucan products were obtained by screening and co mutation of active strains. The results showed that the mutation mode embedded in the amino acid could affect the enzyme activity to a certain extent, but the change of the catalytic properties of the mutant was significant. A further synergistic mutation showed that the specific change of the product was specific. To sum up, the relationship between the structure area of dextran sucrase and the catalytic function of dextran sucrase was explored through the molecular truncation, fixed-point mutation and embedding mutation of dextran sucrase gene, and its catalytic mechanism was revealed, and the basis for the catalytic synthesis of specific positive mutaginase and new dextran was established, and the enzyme was expanded. Application field.
【學(xué)位授予單位】：合肥工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O629.8
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