无花果蛋白酶的过氧化物模拟酶性质及其分析应用

发布时间：2018-03-18 12:16

本文选题：无花果蛋白酶　切入点：过氧化物模拟酶　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：酶是生物有机体进行自我复制和新陈代谢必不可少的生物催化剂。无论是从对应的底物类型还是执行的反应类型来看,他们都是最具特异性的催化剂。在大多数情况下,一种特定的酶只有几种对应的底物,并且对于不同底物执行的是同一反应类型。天然酶由于易变性和降解,来源有限,需要严苛的储存条件,热稳定性低,费用昂贵,易受环境影响以及保存时间短,导致其应用仍然受到诸多限制。因此,目前的研究兴趣大多集中于模拟酶的研究。如:氧化酶的模拟、蛋白酶的模拟、过氧化氢酶的模拟、超氧化物歧化酶的模拟、过氧化物酶的模拟,等等。其中研究最为深入和广泛的是过氧化物模拟酶,至今,越来越多的纳米过氧化物模拟酶被发现或合成,有力地促进了过氧化物模拟酶的发展。但美中不足的是,这些非生物催化剂时常需要复杂的制备过程,并且需要表面修饰以防止团聚,这难免会导致低的重复性和低的催化活性。此外,相比于非生物催化剂,生物催化剂反应条件更加温和,且在良好的生理条件下拥有相当高的催化效率和反应速率。因而,生物型过氧化物模拟酶的开发和研究仍然不可或缺。本文发现无花果蛋白酶不仅具有蛋白酶性质还具有过氧化物模拟酶性质。无花果蛋白酶是天然植物蛋白酶,兼顾了生物催化剂和模拟酶的优点,其应用前景非常广泛。基于其过氧化物模拟酶性质,我们实现了一些分析应用:检测细胞中释放的H_2O_2、检测半胱氨酸以及检测汞离子。(1)我们用足够的证据证明了无花果蛋白酶固有的过氧化物模拟酶性质,无花果蛋白酶可以催化H_2O_2氧化有机底物(如TMB、ABTS、OPD)发生对应的显色反应。同时,优化了无花果蛋白酶作为过氧化物模拟酶的催化条件(pH、温度、反应时间、底物浓度),设计实验证实了该催化活性在各种情景中的有效性,研究了其催化机理,并与HRP的催化活性作了对比。此外,实验结果显示无花果蛋白酶作为过氧化物模拟酶时的活性位点与其作为蛋白酶是的活性位点不同,说明一种酶可能催化多种类的底物执行不同类型的反应。为进一步深入认识酶的功能提供了新依据。(2)基于无花果蛋白酶的过氧化物模拟酶性质,结合过氧化物酶底物TMB,我们建立了简便、灵敏、实用的比色法检测H_2O_2,并成功应用于人乳腺癌细胞中释放的H_2O_2的检测。用无花果蛋白酶的过氧化物模拟酶性质检测H_2O_2不需要复杂的合成和修饰过程,且绿色环保。无花果蛋白酶实现了H_2O_2的检测,说明也能用于与H_2O_2相关的葡萄糖、黄嘌呤、尿酸、肌氨酸、胆碱、乙酰胆碱等的检测。为今后无花果蛋白酶作为模拟酶在免疫分析、生物技术和环境监测中的应用打下基础。(3)我们利用无花果蛋白酶的过氧化物模拟酶性质建立了一种简单、灵敏、绿色的比色法高选择性定量检测半胱氨酸。无花果蛋白酶作为过氧化物模拟酶,能够催化TMB-H_2O_2的反应。半胱氨酸的引入,竞争了与TMB反应的H_2O_2,使得显色反应受到抑制。其在652 nm处吸光度值的降低与半胱氨酸的浓度成线性关系,以此来检测半胱氨酸。该方法不需要昂贵的仪器,不需要材料制备过程、不用引入有机溶剂或重金属,结果可视化,并成功应用于生物样品中半胱氨酸的检测。表明无花果蛋白酶在生物样品分析中潜在的应用。我们的工作促进了无花果蛋白酶的模拟酶性质在医药诊断和生物化学方面的应用。(4)以汞为代表的重金属污染严重威胁着人类健康和社会环境,建立经济、高效的汞离子检测方法任重道远。基于无花果蛋白酶的过氧化物模拟酶性质以及半胱氨酸中巯基与汞离子的高亲和力实现了“抑制-恢复”比色法定量检测汞离子。无花果蛋白酶能够催化H_2O_2氧化TMB发生明显的蓝色反应,而半胱氨酸能够消耗H_2O_2,使显色反应受到抑制,溶液蓝色变浅。由于汞离子能够与半胱氨酸中的巯基结合,使溶液中半胱氨酸的浓度降低,溶液蓝色恢复。该比色法检测汞离子无需任何标记、经济、绿色,说明无花果蛋白酶可以作为过氧化物模拟酶应用于环境分析,展示了无花果蛋白酶的过氧化物模拟酶性质在环境监测中的巨大潜力。
[Abstract]:The enzyme is the test organisms for catalyst essential self replication and The new supersedes the old.. Whether it is the type of reaction from the view of the substrate type or execution, they are a catalyst for the most specific. In most cases, a specific enzyme only several corresponding substrates, and for different substrates is the implementation of the same reaction type. Because of the variability and degradation of natural enzymes, the limited source of need harsh storage conditions, low thermal stability, high cost, easy to be influenced by environment and the preservation time is short, the application is still subject to many restrictions. Therefore, the research of current research interest focused on the simulation of enzyme oxidase simulation, such as:. The simulation of protease simulation, catalase, superoxide dismutase mimetic peroxidase, simulation, and so on. One of the most in-depth study and extensive is mimetic So far, peroxide enzyme, nano enzyme was found more and more simulation or synthesis, effectively promoted the development of mimetic peroxidase. But these non in want of perfection is that biological catalyst often requires complicated preparation process, and surface modifications are required to prevent agglomeration, which will inevitably lead to low repeatability and low catalytic activity. In addition, compared to the non biological catalyst, the reaction conditions of biological catalyst more moderate, and has very high catalytic efficiency and reaction rate in good physical condition. Therefore, the research and development of bio mimetic enzyme is still indispensable. The paper found that not only has the properties of proteinase ficin has mimetic peroxidase properties of ficin. Is a natural plant proteinase, both the advantages and the simulation of enzyme biological catalyst, its application prospect is very extensive. Based on the peroxide The properties of enzyme mimics, we realized some application analysis: H_2O_2 release were detected, and the detection of cysteine detection of mercury ions. (1) we use sufficient evidence to prove that the ficin inherent peroxidase like properties, ficin can catalyze the oxidation of H_2O_2 organic substrates (such as TMB, ABTS, OPD) color the corresponding reaction conditions were optimized. At the same time, the catalytic ficin as mimetic peroxidase (pH, temperature, reaction time, substrate concentration), design experiments to demonstrate the effectiveness of the catalytic activity in each scenario, study its catalytic mechanism, and compared with the catalytic activity of HRP. In addition, the experimental results show that the active site active site ficin as mimetic peroxidase when and as protease is different, a variety of catalytic enzymes may not perform substrate The same type of reaction. It provides a new basis for the further understanding of enzyme function. (2) ficin mimetic peroxidase peroxidase substrate binding properties based on TMB, we establish a simple, sensitive and practical colorimetric detection of H_2O_2, detection and successful application in release in human breast cancer cells H_2O_2 by ficin mimetic peroxidase detection properties of H_2O_2 does not require the synthesis and modification of the complex process, and green environmental protection. Ficin implements H_2O_2 detection, that can also associated with H_2O_2 for glucose, xanthine, uric acid, creatine, choline, acetylcholine. Detection for ficin as simulation in enzyme immunoassay, lay the foundation for the application of biotechnology and environmental monitoring. (3) we use ficin mimetic peroxidase properties built a simple, Sensitive, high selective colorimetric method for quantitative detection of cysteine Gree ficin as mimetic enzyme, can catalyze the TMB-H_2O_2 reaction. The introduction of competition with cysteine, TMB reaction of H_2O_2, the color reaction was inhibited. The absorption at 652 nm reduced light value linear relationship with the concentration of cysteine. In order to detect cysteine. This method does not require expensive equipment, does not need the material preparation process, without introducing organic solvents or heavy metals, results visualization, and successfully applied to the detection of cysteine in biological samples. It shows that the application of ficin potential in the analysis of biological samples. We work to promote the simulation of enzyme properties of ficin the application in medical diagnosis and biological chemistry. (4) the heavy metal pollution in mercury represents a serious threat to human health and social environment, The establishment of economic, a mercury ion detecting method with high efficiency. Based on enzyme properties and high affinity for sulfhydryl and mercury ions in simulated cysteine ficin peroxide inhibition recovery than color method. The quantitative detection of mercury ions ficin catalyzed H_2O_2 oxidation to TMB blue reaction obviously, while cysteine can consume H_2O_2. The color reaction was inhibited, the blue light. The solution of mercury ion binds to the cysteine thiol, the concentration of cysteine reduced, blue solution recovery. Without any mark, without the colorimetric detection of mercury ions economy, green, that can be used as mimetic peroxidase ficin used in environmental analysis, display the great potential of ficin mimetic peroxidase properties in environmental monitoring.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.8

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