【作者】 王超；

【导师】 张洪斌；

【作者基本信息】 合肥工业大学 ， 制药工程（专业学位）， 2017， 硕士

【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。更多还原

【Abstract】 We constructed the engineering bacteria of the dextransucrase in Escherichia coli expression system by cloning the dex-YG gene of dextransucrase(EC 2.4.1.5)from Leuconostoc mesenteroides 0326.Dextransucrase can synthesize α-glucan by hydrolysis of Glucose using sucrose as substrate.In this paper,structure and function of dextransucrase were investigated by truncated,mutated,amino acid insert mutation of the dextransucrase gene dex-YG to expand its application areas.The catalytic functions of dextransucrase were analyzed by different truncation of the fragments of dextransucrase.Mutant enzymes with different catalytic functions were obtained by deleting different fragments.The amino acid of catalytic region were replaced,embedded to investigate different enzymatic properties of mutant enzymes in order to explore the mechanism of product specificity and obtain the dextran products with different properties for expanding the application of the enzyme.1.The gene sequence dex-YG,the secondary structure and the tertiary structure were predicted by the analysis of biological information.The C-terminal sequence was truncated and analyzed to study structure and function of dextransucrase.The region of α-glucan chain extension,the oligosaccharide synthesis,and the completely conserved region were studied by fragment truncation.Repeated sequence deletion at the end of dextransucrase would greatly affect the ability of its synthesis of dextran.As the length of the truncated fragment increases,and its ability to synthesize the polymer dextran decreases sharply.After a certain length of truncation,the synthesis of dextran ability were completely inactive.The ability of synthesis of oligosaccharides was significantly improved.As the further fragment was truncated until its conserved sequence motif I,the catalytic activity of the oligonucleotides was also significantly decreased,almost completely inactive.2.Dextransucrase belongs to the family of GH70,and the catalyzed mechanism of the family is completely conserved.All of GH70 enzymes can synthesize theα-glucan product with sucrose as the substrate,but the glycosidic bonds of α-glucan products from different types of GH70 enzyme have obvious difference,including α(1-2),α(1-3),α(1-4),α(1-6)glycosidic bonds.Based on the analysis of molecular docking and kinetics of dextransucrase,the key amino acids of the receptor and the substrate binding region were replaced by the analysis of its effect on the synthesized product.The mechanism of product specificity was explored by analysis of themolecular modeling structure.More dextran products can expand the application of dextransucrase.By changing the amino acid of the catalytic region,the glycosidic bonds of the dextran 5% α(1-3)and 95% α(1-6)bonds of the original dextran was changed to 1-9% α(1-3)and 90-98% α(1-6)bond,and some mutations dextran produce additional α(1-2)and α(1-4)bonds.Simulation analysis can be found that the size of the side chain of the replacement amino acid,and the nature of the charge hydrophobicity will have a greater impact on the receptor of the most stable conformation,thus affecting the nature of the enzyme and product specificity.3.The amino acid substitution of the catalytic pocket will affect the bond type of the product,but its variation has some limitations.The structure of the product dextran was changed to a greater extent by embedding the amino acid on the critical site of the conserved sequence but not directly with the substrate or receptor.By homologous recombination,663 and 553 site were inserted of amino acids.Through the screening of active strains,as well as synergistic mutations obtained,the highly branched dextran product mutants were obtained.Amino acid embedding method,although it would affect the enzyme activity to a certain extent,but its active mutant its catalytic properties change was more significant.With further synergistic mutantion,the product specific changes more significant.Different properties of dextran products also provide a basis for subsequent research and application.In summary,the structure and function of dextransucrase were studied by molecular means,and the mutant enzymes with different catalytic properties and different mutants were obtained,which laid the foundation for the subsequent research,application and modified dextransucrase.更多还原