本文選題：無(wú)花果蛋白酶　切入點(diǎn)：過(guò)氧化物模擬酶　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：酶是生物有機(jī)體進(jìn)行自我復(fù)制和新陳代謝必不可少的生物催化劑。無(wú)論是從對(duì)應(yīng)的底物類型還是執(zhí)行的反應(yīng)類型來(lái)看,他們都是最具特異性的催化劑。在大多數(shù)情況下,一種特定的酶只有幾種對(duì)應(yīng)的底物,并且對(duì)于不同底物執(zhí)行的是同一反應(yīng)類型。天然酶由于易變性和降解,來(lái)源有限,需要嚴(yán)苛的儲(chǔ)存條件,熱穩(wěn)定性低,費(fèi)用昂貴,易受環(huán)境影響以及保存時(shí)間短,導(dǎo)致其應(yīng)用仍然受到諸多限制。因此,目前的研究興趣大多集中于模擬酶的研究。如:氧化酶的模擬、蛋白酶的模擬、過(guò)氧化氫酶的模擬、超氧化物歧化酶的模擬、過(guò)氧化物酶的模擬,等等。其中研究最為深入和廣泛的是過(guò)氧化物模擬酶,至今,越來(lái)越多的納米過(guò)氧化物模擬酶被發(fā)現(xiàn)或合成,有力地促進(jìn)了過(guò)氧化物模擬酶的發(fā)展。但美中不足的是,這些非生物催化劑時(shí)常需要復(fù)雜的制備過(guò)程,并且需要表面修飾以防止團(tuán)聚,這難免會(huì)導(dǎo)致低的重復(fù)性和低的催化活性。此外,相比于非生物催化劑,生物催化劑反應(yīng)條件更加溫和,且在良好的生理?xiàng)l件下?lián)碛邢喈?dāng)高的催化效率和反應(yīng)速率。因而,生物型過(guò)氧化物模擬酶的開(kāi)發(fā)和研究仍然不可或缺。本文發(fā)現(xiàn)無(wú)花果蛋白酶不僅具有蛋白酶性質(zhì)還具有過(guò)氧化物模擬酶性質(zhì)。無(wú)花果蛋白酶是天然植物蛋白酶,兼顧了生物催化劑和模擬酶的優(yōu)點(diǎn),其應(yīng)用前景非常廣泛�；谄溥^(guò)氧化物模擬酶性質(zhì),我們實(shí)現(xiàn)了一些分析應(yīng)用:檢測(cè)細(xì)胞中釋放的H_2O_2、檢測(cè)半胱氨酸以及檢測(cè)汞離子。(1)我們用足夠的證據(jù)證明了無(wú)花果蛋白酶固有的過(guò)氧化物模擬酶性質(zhì),無(wú)花果蛋白酶可以催化H_2O_2氧化有機(jī)底物(如TMB、ABTS、OPD)發(fā)生對(duì)應(yīng)的顯色反應(yīng)。同時(shí),優(yōu)化了無(wú)花果蛋白酶作為過(guò)氧化物模擬酶的催化條件(pH、溫度、反應(yīng)時(shí)間、底物濃度),設(shè)計(jì)實(shí)驗(yàn)證實(shí)了該催化活性在各種情景中的有效性,研究了其催化機(jī)理,并與HRP的催化活性作了對(duì)比。此外,實(shí)驗(yàn)結(jié)果顯示無(wú)花果蛋白酶作為過(guò)氧化物模擬酶時(shí)的活性位點(diǎn)與其作為蛋白酶是的活性位點(diǎn)不同,說(shuō)明一種酶可能催化多種類的底物執(zhí)行不同類型的反應(yīng)。為進(jìn)一步深入認(rèn)識(shí)酶的功能提供了新依據(jù)。(2)基于無(wú)花果蛋白酶的過(guò)氧化物模擬酶性質(zhì),結(jié)合過(guò)氧化物酶底物TMB,我們建立了簡(jiǎn)便、靈敏、實(shí)用的比色法檢測(cè)H_2O_2,并成功應(yīng)用于人乳腺癌細(xì)胞中釋放的H_2O_2的檢測(cè)。用無(wú)花果蛋白酶的過(guò)氧化物模擬酶性質(zhì)檢測(cè)H_2O_2不需要復(fù)雜的合成和修飾過(guò)程,且綠色環(huán)保。無(wú)花果蛋白酶實(shí)現(xiàn)了H_2O_2的檢測(cè),說(shuō)明也能用于與H_2O_2相關(guān)的葡萄糖、黃嘌呤、尿酸、肌氨酸、膽堿、乙酰膽堿等的檢測(cè)。為今后無(wú)花果蛋白酶作為模擬酶在免疫分析、生物技術(shù)和環(huán)境監(jiān)測(cè)中的應(yīng)用打下基礎(chǔ)。(3)我們利用無(wú)花果蛋白酶的過(guò)氧化物模擬酶性質(zhì)建立了一種簡(jiǎn)單、靈敏、綠色的比色法高選擇性定量檢測(cè)半胱氨酸。無(wú)花果蛋白酶作為過(guò)氧化物模擬酶,能夠催化TMB-H_2O_2的反應(yīng)。半胱氨酸的引入,競(jìng)爭(zhēng)了與TMB反應(yīng)的H_2O_2,使得顯色反應(yīng)受到抑制。其在652 nm處吸光度值的降低與半胱氨酸的濃度成線性關(guān)系,以此來(lái)檢測(cè)半胱氨酸。該方法不需要昂貴的儀器,不需要材料制備過(guò)程、不用引入有機(jī)溶劑或重金屬,結(jié)果可視化,并成功應(yīng)用于生物樣品中半胱氨酸的檢測(cè)。表明無(wú)花果蛋白酶在生物樣品分析中潛在的應(yīng)用。我們的工作促進(jìn)了無(wú)花果蛋白酶的模擬酶性質(zhì)在醫(yī)藥診斷和生物化學(xué)方面的應(yīng)用。(4)以汞為代表的重金屬污染嚴(yán)重威脅著人類健康和社會(huì)環(huán)境,建立經(jīng)濟(jì)、高效的汞離子檢測(cè)方法任重道遠(yuǎn)�；跓o(wú)花果蛋白酶的過(guò)氧化物模擬酶性質(zhì)以及半胱氨酸中巰基與汞離子的高親和力實(shí)現(xiàn)了“抑制-恢復(fù)”比色法定量檢測(cè)汞離子。無(wú)花果蛋白酶能夠催化H_2O_2氧化TMB發(fā)生明顯的藍(lán)色反應(yīng),而半胱氨酸能夠消耗H_2O_2,使顯色反應(yīng)受到抑制,溶液藍(lán)色變淺。由于汞離子能夠與半胱氨酸中的巰基結(jié)合,使溶液中半胱氨酸的濃度降低,溶液藍(lán)色恢復(fù)。該比色法檢測(cè)汞離子無(wú)需任何標(biāo)記、經(jīng)濟(jì)、綠色,說(shuō)明無(wú)花果蛋白酶可以作為過(guò)氧化物模擬酶應(yīng)用于環(huán)境分析,展示了無(wú)花果蛋白酶的過(guò)氧化物模擬酶性質(zhì)在環(huán)境監(jiān)測(cè)中的巨大潛力。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O629.8

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