有机介质与纳米自组装体对次血红素六肽模拟酶活性影响的研究

发布时间：2018-05-18 04:46

本文选题：次血红素 + 电子顺磁共振　；参考：《吉林大学》2017年博士论文

【摘要】：模拟酶是通过人工合成出的一类具有天然酶生物特性的催化剂。模拟酶是一种仿生高分子,通过模拟天然酶的活性中心和作用机理使其具备天然酶的一些催化特性。天然酶是从生物体内提取出来的一类具备催化特性的蛋白质。天然酶有很多优点,比如专一催化一类化学或者生物反应、具有很高的催化效率等。但是天然酶作为蛋白质,在体外进行催化反应时很容易受物理因素或者化学因素的影响,因此在工业生产中天然酶往往并不能取代化学催化剂。模拟酶具有简单的催化结构,可以解决天然酶不稳定的缺点。20世纪60年代至今,多种多样的模拟酶被人工合成出来,并且应用于工业生产和环境化学中。通过研究模拟酶的结构、活性中心、与底物作用的方式等,可以更好的改进模拟酶的催化性能。次血红素(Deuterohemin,Dh)是通过人工合成的一种铁卟啉化合物。次血红素区别于天然的血红素,缺少了两个乙烯基,由于乙烯基易被氧化,因此这样的结构特征可以使次血红素在氧化底物过程中更加稳定。在1971年,就有研究表明次血红素可以在过氧化氢的存在下氧化有机底物,虽然活性不高,但是这个结果说明了次血红素可以模拟过氧化物酶活性,也为研究次血红素化合物的功能奠定了基础。金属卟啉模拟过氧化物酶活力取决于中心金属的电子结构以及额外配体与金属卟啉环之间的相互作用。次血红素虽然存在金属中心,可以通过金属的变价模拟过氧化物酶活性,但是其缺少了一个额外的配体,导致了很低的过氧化物酶活性。根据微过氧化物酶(Microperoxidases,MPs)的侧链设计出了一个含有六个氨基酸的肽段(Ala-His-Thr-Val-Glu-Lys),并将这个肽段共价连接在了次血红素的羧基上。我们将这个人工合成的次血红素肽,命名为次血红素六肽(Dh HP-6)。因为近端的组氨酸可以与活性中心铁进行配位,因此其活性高于没有肽段的次血红素。为了进一步研究组氨酸对次血红素活性的影响,我们用丙氨酸代替了组氨酸,合成了一个新的肽段(Ala-Ala-Thr-Val-Glu-Lys),并将这个肽段连接在了次血红素的羧基上(Dh AP-6)。利用紫外可见吸收光谱,我们对两种次血红素肽进行了结构的分析,并且进行了酶活性的测定。我们发现,Dh AP-6的过氧化物酶活性低于Dh HP-6。因此,组氨酸的配位,对提高次血红素的过氧化物酶活性起到了关键的作用。随后,我们对Dh HP-6的过氧化物酶活性进行了反应条件的优化,并在最适条件下,进行了动力学的研究,发现Dh HP-6相比于辣根过氧化物酶(HRP)具有很高的底物亲和性。酶的催化反应是需要水作为介质才能进行的,而有机溶剂可以破坏天然酶中的氢键和剥夺酶分子表面的必需水,因此在有机介质中天然酶的结构很容易被破坏,导致活性中心的改变,从而使酶活力下降或者使酶失活。由于Dh HP-6结构简单,不具备蛋白质复杂的三维结构,所以在有机溶剂中也表现出了很好的酶活性。我们发现Dh HP-6的过氧化物酶活性与有机溶剂的极性有关,溶剂的极性越大,Dh HP-6的过氧化物酶活性越高。在15%的甲醇溶液中,Dh HP-6的活性最高,是Horseradish Peroxidase(HRP)活性的15倍。为了可以更好的利用Dh HP-6的过氧化物模拟酶活性,通过Dh HP-6与Cu_3(PO_4)_2自组装,将Dh HP-6固定化。发现Dh HP-6与Cu_3(PO_4)_2自组装之后形成了一种类似于花的纳米结构,我们称之为Dh HP-6-Cu_3(PO_4)_2纳米花。利用扫描电镜、固体紫外、固体红外等手段,对Dh HP-6-Cu_3(PO_4)_2纳米花进行了结构的表征。通过电子顺磁共振光谱,我们首次从实验角度解释了纳米花形成的机理。形成纳米花之后,Dh HP-6对底物的亲和性进一步提高。在形成纳米花的过程中,原本聚集的Dh HP-6,分散的固定到了Cu_3(PO_4)_2上,使得Dh HP-6的活性中心暴露,因此Dh HP-6-Cu_3(PO_4)_2纳米花的酶活力和对底物的亲和性都有所提高。我们尝试将Dh HP-6-Cu_3(PO_4)_2纳米花应用于苯酚的检测,并发现其具有很好的LOD值和灵敏度。而且,多次使用和长时间在缓冲溶液中贮存也没有明显的活力损失,因此Dh HP-6-Cu_3(PO_4)_2纳米花是一种具有很好的应用前景的生物催化剂。最后,我们利用理论计算,研究了Dh HP-6氧化苯酚的反应机理。研究发现,在氧化苯酚的过程中,以两态反应模式(TSR)进行,且二重态和四重态的能量和结构都很接近。电子结构分析表明,O-H键的活化是一个PCET机制,电子由苯酚转移到了卟啉环上,氢质子转移到了金属氧上,而金属中心的电子未发生变化。二重态和四重态的不同在于前者是α电子转移,后者是β电子转移。
[Abstract]:A mimic enzyme is a kind of synthetic catalyst with natural enzyme biological properties. The analogue enzyme is a bionic polymer by simulating the active center and mechanism of natural enzyme to make it have some catalytic properties of natural enzyme. Natural enzyme is a kind of protein with catalytic properties extracted from the organism. There are many advantages, such as catalyzing a class of chemical or biological reactions, and having high catalytic efficiency. But natural enzymes are easily affected by physical or chemical factors when they are used as proteins in vitro. Therefore, natural enzymes can not replace chemical catalysts in industrial production. The single catalytic structure can solve the disadvantage of natural enzyme instability..20 century since 60s, a variety of analog enzymes have been synthesized and applied to industrial production and environmental chemistry. By studying the structure of the enzyme, the activity center, the way of the action of the substrate and so on, the catalytic performance of the analogue enzyme can be improved better. Deuterohemin (Dh) is an artificially synthesized iron porphyrin compound. The heme is different from the natural heme and lacks two vinyl groups. Because the vinyl group is easily oxidized, this structural feature can make the heme more stable during the oxidation of the substrate. In 1971, a study showed that heme could be used. The oxidation of organic substrates in the presence of hydrogen peroxide is not highly active, but this result shows that heme can simulate peroxidase activity and also provide a basis for the study of the function of heme compounds. The interaction between porphyrin rings. Although heme exists in the metal center, it can simulate peroxidase activity through the metal variation, but it lacks an extra ligand, which leads to a very low peroxidase activity. A six amino acid is designed according to the side chain of the Microperoxidases (MPs). The peptide segment (Ala-His-Thr-Val-Glu-Lys), which is covalently linked to the carboxyl group of the heme, is named the heme heme six peptide (Dh HP-6), because the near terminal histidine can be coordinated with the active central iron, so its activity is higher than the heme without the peptide. One step was to study the effect of histidine on the activity of heme. We used alanine instead of histidine and synthesized a new peptide segment (Ala-Ala-Thr-Val-Glu-Lys), which was connected to the carboxyl group of heme (Dh AP-6). The structure of two heme peptides was analyzed by UV visible absorption spectrum. The activity of the enzyme was determined. We found that the peroxidase activity of Dh AP-6 was lower than that of Dh HP-6., so the coordination of histidine played a key role in improving the activity of the peroxidase in the heme. Then, we optimized the activity of the peroxidase in Dh HP-6 and carried out the power under the optimum conditions. Studies have found that Dh HP-6 has a very high substrate affinity compared to horseradish peroxidase (HRP). The catalytic reaction of the enzyme is required by water as a medium, and organic solvents can destroy the hydrogen bonds in natural enzymes and the necessary water on the surface of the enzyme molecules. Therefore, the structure of natural enzymes in organic medium is easily destroyed. The changes in the active center make the activity of the enzyme declines or inactivate the enzyme. Because Dh HP-6 has a simple structure and does not have a complex three-dimensional structure of protein, it also shows good enzyme activity in the organic solvent. We found that the peroxidase activity of Dh HP-6 is related to the polarity of organic solvents, the greater the polarity of the solvent, the Dh HP-6 The higher the activity of peroxidase is, the activity of Dh HP-6 is the highest in 15% methanol solution, which is 15 times of the activity of Horseradish Peroxidase (HRP). In order to make better use of the activity of the peroxidase in Dh HP-6, it will be fixed by Dh HP-6 and Cu_3 (PO_4). The nanostructures, which are similar to flowers, are called Dh HP-6-Cu_3 (PO_4) _2 nanoscale flowers. The structure of Dh HP-6-Cu_3 (PO_4) _2 nanoscale was characterized by scanning electron microscopy, solid ultraviolet and solid infrared, and the mechanism of nanoscale formation was explained by the electron paramagnetic resonance spectrum. Then, the affinity of Dh HP-6 to the substrate was further improved. In the process of forming the nanoscale, the original aggregation of Dh HP-6 was fixed to Cu_3 (PO_4) _2, making the active center of Dh HP-6 exposed, so the enzyme activity of Dh HP-6-Cu_3 (PO_4) and the affinity to the substrate were improved. It was applied to the detection of phenol and found that it had good LOD value and sensitivity. Moreover, the storage of Dh HP-6-Cu_3 (PO_4) _2 nanoscale was a kind of biocatalyst with good application prospect. Finally, we used theoretical calculation to study Dh HP. In the process of oxidation of phenol by -6, it is found that in the process of oxidation of phenol, the two state reaction mode (TSR) is carried out, and the energy and structure of the double and four states are close. The electronic structure analysis shows that the activation of the O-H bond is a PCET mechanism, the electron transfer from phenol to the porphyrin ring and the hydrogen proton transfer to the metal oxygen, and gold. The electron in the center is not changed. The difference between the duality and the four heavy States is that the former is alpha electron transfer and the latter is beta electron transfer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O643.36

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