本文選題：無花果蛋白酶　切入點：過氧化物模擬酶　出處：《西南大學》2017年碩士論文　論文類型：學位論文

【摘要】：酶是生物有機體進行自我復制和新陳代謝必不可少的生物催化劑。無論是從對應的底物類型還是執(zhí)行的反應類型來看,他們都是最具特異性的催化劑。在大多數情況下,一種特定的酶只有幾種對應的底物,并且對于不同底物執(zhí)行的是同一反應類型。天然酶由于易變性和降解,來源有限,需要嚴苛的儲存條件,熱穩(wěn)定性低,費用昂貴,易受環(huán)境影響以及保存時間短,導致其應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、過氧化氫酶的模擬、超氧化物歧化酶的模擬、過氧化物酶的模擬,等等。其中研究最為深入和廣泛的是過氧化物模擬酶,至今,越來越多的納米過氧化物模擬酶被發(fā)現或合成,有力地促進了過氧化物模擬酶的發(fā)展。但美中不足的是,這些非生物催化劑時常需要復雜的制備過程,并且需要表面修飾以防止團聚,這難免會導致低的重復性和低的催化活性。此外,相比于非生物催化劑,生物催化劑反應條件更加溫和,且在良好的生理條件下?lián)碛邢喈敻叩拇呋屎头磻俾�。因�?生物型過氧化物模擬酶的開發(fā)和研究仍然不可或缺。本文發(fā)現無花果蛋白酶不僅具有蛋白酶性質還具有過氧化物模擬酶性質。無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,其應用前景非常廣泛�；谄溥^氧化物模擬酶性質,我們實現了一些分析應用:檢測細胞中釋放的H_2O_2、檢測半胱氨酸以及檢測汞離子。(1)我們用足夠的證據證明了無花果蛋白酶固有的過氧化物模擬酶性質,無花果蛋白酶可以催化H_2O_2氧化有機底物(如TMB、ABTS、OPD)發(fā)生對應的顯色反應。同時,優(yōu)化了無花果蛋白酶作為過氧化物模擬酶的催化條件(pH、溫度、反應時間、底物濃度),設計實驗證實了該催化活性在各種情景中的有效性,研究了其催化機理,并與HRP的催化活性作了對比。此外,實驗結果顯示無花果蛋白酶作為過氧化物模擬酶時的活性位點與其作為蛋白酶是的活性位點不同,說明一種酶可能催化多種類的底物執(zhí)行不同類型的反應。為進一步深入認識酶的功能提供了新依據。(2)基于無花果蛋白酶的過氧化物模擬酶性質,結合過氧化物酶底物TMB,我們建立了簡便、靈敏、實用的比色法檢測H_2O_2,并成功應用于人乳腺癌細胞中釋放的H_2O_2的檢測。用無花果蛋白酶的過氧化物模擬酶性質檢測H_2O_2不需要復雜的合成和修飾過程,且綠色環(huán)保。無花果蛋白酶實現了H_2O_2的檢測,說明也能用于與H_2O_2相關的葡萄糖、黃嘌呤、尿酸、肌氨酸、膽堿、乙酰膽堿等的檢測。為今后無花果蛋白酶作為模擬酶在免疫分析、生物技術和環(huán)境監(jiān)測中的應用打下基礎。(3)我們利用無花果蛋白酶的過氧化物模擬酶性質建立了一種簡單、靈敏、綠色的比色法高選擇性定量檢測半胱氨酸。無花果蛋白酶作為過氧化物模擬酶,能夠催化TMB-H_2O_2的反應。半胱氨酸的引入,競爭了與TMB反應的H_2O_2,使得顯色反應受到抑制。其在652 nm處吸光度值的降低與半胱氨酸的濃度成線性關系,以此來檢測半胱氨酸。該方法不需要昂貴的儀器,不需要材料制備過程、不用引入有機溶劑或重金屬,結果可視化,并成功應用于生物樣品中半胱氨酸的檢測。表明無花果蛋白酶在生物樣品分析中潛在的應用。我們的工作促進了無花果蛋白酶的模擬酶性質在醫(yī)藥診斷和生物化學方面的應用。(4)以汞為代表的重金屬污染嚴重威脅著人類健康和社會環(huán)境,建立經濟、高效的汞離子檢測方法任重道遠�；跓o花果蛋白酶的過氧化物模擬酶性質以及半胱氨酸中巰基與汞離子的高親和力實現了“抑制-恢復”比色法定量檢測汞離子。無花果蛋白酶能夠催化H_2O_2氧化TMB發(fā)生明顯的藍色反應,而半胱氨酸能夠消耗H_2O_2,使顯色反應受到抑制,溶液藍色變淺。由于汞離子能夠與半胱氨酸中的巰基結合,使溶液中半胱氨酸的濃度降低,溶液藍色恢復。該比色法檢測汞離子無需任何標記、經濟、綠色,說明無花果蛋白酶可以作為過氧化物模擬酶應用于環(huán)境分析,展示了無花果蛋白酶的過氧化物模擬酶性質在環(huán)境監(jiān)測中的巨大潛力。
[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.

【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O629.8

【相似文獻】

相關期刊論文 前10條

1 ;無花果蛋白酶的制備[J];技術與市場;2008年11期

2 郭冬青;紀付江;程紹杰;宋淑亮;;無花果蛋白酶的研究進展[J];北方園藝;2010年07期

3 王文友;楚惠民;宮本芝;王富國;;無花果蛋白酶對育肥豬的影響試驗[J];中國畜牧獸醫(yī)文摘;2011年03期

4 郭冬青;紀付江;高健;周燕霞;宋淑亮;吉愛國;;無花果蛋白酶的不同提取方法比較及酶學性質研究[J];湖北農業(yè)科學;2011年06期

5 楊萌,呂源玲,王洪新;無花果蛋白酶和果膠的綜合提取[J];食品科學;1998年07期

6 劉德富，董文彥，，白虹，張東平，于穎，樊麗彬;幾丁質固定化無花果蛋白酶的研究[J];食品科學;1994年11期

7 ;技術薈萃[J];適用技術市場;1999年05期

8 李雨林;周海英;申琳;生吉萍;吳顯榮;;無花果蛋白酶與木瓜蛋白酶對牛肉嫩化的研究[J];肉類工業(yè);2006年11期

9 馮自立;馬娜;;無花果蛋白酶在PEG/(NH_4)_2SO_4雙水相體系中的分配行為[J];食品科學;2010年19期

10 姚淑琴,郭滿棟;模擬酶研究進展[J];理化檢驗(化學分冊);2004年08期

相關會議論文 前2條

1 趙慧暉;王忠輝;張立春;呂弋;;基于金納米團簇模擬酶檢測尿酸的研究[A];中國化學會第28屆學術年會第9分會場摘要集[C];2012年

2 劉星;王琦;張立春;呂弋;;基于BSA為模板合成二氧化錳納米粒子的模擬酶的研究[A];中國化學會第28屆學術年會第9分會場摘要集[C];2012年

相關博士學位論文 前1條

1 石文兵;基于納米微粒模擬酶的光分析方法構建及其應用研究[D];西南大學;2011年

相關碩士學位論文 前9條

1 黃露;無花果蛋白酶的分離純化與性質研究[D];吉林大學;2007年

2 劉晴晴;模擬酶Cu(Ⅱ)配合物的制備及其對肽鍵的水解作用[D];中國海洋大學;2015年

3 熊裕豪;新型納米模擬酶的設計合成及應用研究[D];廣西師范大學;2016年

4 郝翠婷;蛋白質控制合成的銅納米簇用于模擬酶研究[D];吉林大學;2017年

5 劉琳;X射線聯(lián)合含碲模擬酶抗乳腺癌作用的研究[D];吉林大學;2017年

6 余翔;胰凝乳蛋白酶模擬酶的制備及其磁性固定化研究[D];四川農業(yè)大學;2010年

7 王瑋;自組裝納米簇模擬過氧化物酶：光譜學及電化學研究[D];河南大學;2012年

8 張成新;基于過氧化物模擬酶及核酸工具酶技術的高靈敏光學生物傳感器構筑及性能研究[D];青島科技大學;2013年

9 劉亞麗;含鐵金屬有機骨架化合物的可控合成及其模擬酶性質的應用研究[D];西南大學;2014年

本文編號：1629535