右旋糖酐蔗糖酶分子改造及其催化性质研究
发布时间：2018-07-25 17:16
【摘要】：本实验室前期慈宁宫肠膜状明串珠菌Leuconostoc mesenteroides 0326的右旋糖酐蔗糖酶dextransucrase(EC 2.4.1.5)中克隆获得基因dex-YG,并以该基因为基础构建了右旋糖酐蔗糖酶大肠杆菌表达体系的工程菌。右旋糖酐蔗糖酶以蔗糖为底物通过水解转移葡萄糖基合成高分子葡聚糖。本文通过对右旋糖酐蔗糖酶基因dex-YG进行系列分子截短,分析不同片段的右旋糖酐截短突变酶的特性,以探究右旋糖酐蔗糖酶结构区域与催化功能的关系,揭示其催化机制;在此基础上选定特定区域能进行氨基酸定点突变、嵌入突变,获得不同酶学性质的正突变酶,探究其控制产物特异性的催化机制,获得催化合成不同枝化度的新型右旋糖酐产物,扩大该酶的应用领域。1、以右旋糖酐蔗糖酶基因序列dex-YG为基础,通过生物信息学的比对分析,对其二级结构以及三级结构进行预测分析,对其C端序列进行一系列的截短,分析其结构功能的关系。通过片段截短的方法对其糖链延伸的控制区,寡聚糖合成区域,以及完全保守区域进行了研究分析,探讨右旋糖酐蔗糖酶结构区域与催化功能的关系。结果表明:对其末端重复序列进行删除,会极大的破坏右旋糖酐蔗糖酶合成葡聚糖的能力。随截短片段长度增加,其合成高分子葡聚糖的能力急剧下降,在蛋白367aa个氨基酸的截短后,其右旋糖酐的合成能力完全丧失,相对应的其受体反应的催化功能会明显增强,从而导致寡聚糖的合成能力显著提升。随着更进一步的片段截短直至其保守序列motifⅠ,其受体反应的催化性能也极具下降,其酶活力几乎完全丧失。2、不同类型的糖酐水解酶其合成的葡聚糖其糖苷键的组成却又很大的区别,包括α(1-2)、α(1-3)、α(1-4)、α(1-6)糖苷键。通过对分子对接以及动力学的模拟分析,对受体以及底物结合区域的关键氨基酸进行替换,通过分析其对合成产物的影响结合分子模拟的分析结构,探究其控制产物的催化机制,合成不同键型的右旋糖酐产物,扩大该酶的应用领域。通过对关键位点的氨基酸替换,相对于原始的右旋糖酐蔗糖酶的催化产物右旋糖酐5%α(1-3)以及95%α(1-6)键型组成,突变后的键型组成变为1-9%α(1-3)和90-98%α(1-6)键型组成,部分突变产生了额外的α(1-2)键和α(1-4)键。模拟分析可以发现,替换氨基酸其侧链的大小、电荷状况以及疏水性等都会较大的影响受体结合最稳定构象,从而影响酶学性质以及产物特异性等。3、对催化口袋中的特定氨基酸进行替换会在一定程度的影响产物的键型,但其变化有一定的局限性。通过对不与底物或受体直接作用的保守序列的关键位点进行氨基酸插入突变,会更大程度改变产物右旋糖酐的键型。以同源重组的方式对663以及553位点进行氨基酸的饱和嵌入,通过对活性菌株的筛选以及协同突变,获得了超高分支葡聚糖产物突变株。实验结果得到氨基酸嵌入的突变方式虽然会在一定程度影响酶活性,但其得到的突变体催化性质变化显著。更进一步的协同突变表明,其产物特异性变化更加显著。综上,本文通过对右旋糖酐蔗糖酶基因的分子截短、定点突变和嵌入突变,探讨了右旋糖酐蔗糖酶结构区域与催化功能的关系,揭示其催化机制;为获得特异性的正突变酶以及新型右旋糖酐的催化合成打下了基础,扩大该酶的应用领域。
[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.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.8
本文编号：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.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.8
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