辣根过氧化物酶的生物印迹及其催化低聚苯胺的合成
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摘要
辣根过氧化物酶(horseradish peroxidase)是一种从辣根中提取的过氧化物酶(E,C,1.11.1.7),分子量为44000D的糖蛋白,含糖量为18%。它的分子包含一个亚铁血红素辅基,其卟啉环是原卟啉,中心离子是Fe(Ⅱ)。由于原料易得,商品价格低廉,作用底物广泛,成为过氧化物酶中使用最广泛的酶之一。在有机合成中作为催化剂使用,其催化机理为过氧化循环过程,对苯胺及其衍生物,酚类等有很高的催化效率。在酶免疫测定中,由于HRP具有发光底物,因此而建立的化学发光免疫测定在临床中得到广泛应用。近年来,HRP在有机合成、生物传感器、食品分析、生物医学检测、荧光分析、污水处理等方面的应用更为突出。尤其在免疫诊断试剂术、生物传感器、木素降解和工业“三废”处理等方面的应用研究进展飞速。
本课题以辣根过氧化物酶为研究对象,以对苯二胺和过氧化氢为底物,在25%的二氧六环溶液中,从酸碱性、温度、底物浓度等几个方面,初步研究了辣根过氧化物酶催化氧化反应的一般特性,测定了部分酶促反应动力学参数,同时对反应产物进行表征。结果发现HRP热稳定性较差,酸碱适应范围较窄,有机相催化效率低,且酶易失活等不良特性。因此,本文以提高有机相酶催化效率、水相酶热稳定性为出发点,结合分子印迹和生物印迹两种技术达到对辣根过氧化物酶的印迹。利用底物对苯二胺诱导酶构象,获得天然印迹酶(NIP),并加入酰化试剂马来酸酐制备乙酰化印迹酶(DIP),冷冻干燥后加入交联剂和引发剂进行紫外光聚合,聚合完毕后用二氧六环洗脱配体,最后干燥就得到了所需的交联印迹酶聚合物(CLIPs),即为水相生物印迹酶。
为了考察三种酶(天然印迹酶、乙酰化印迹酶、交联印迹酶聚合物)的性质,研究内容设计如下:
(1)考察印迹酶在有机相中的催化活性,并分别考察pH值、温度、水含量、表观Km四因素对印迹酶催化活性的影响。同时与有机相游离酶进行比较;
(2)考察乙酰化印迹酶在水相中的催化活性,并分别考察pH值、温度、表观Km三因素对乙酰化印迹酶在水相中的催化活性的影响,同时与水相游离酶进行比较;
(3)考察交联印迹酶聚合物在水相中的催化活性,并分别考察pH值、温度、表观Km三因素对交联印迹酶聚合物在水相中的催化活性的影响,同时与水相游离酶进行比较。结果表明,三种酶酸碱稳定范围及最适pH保持不变;印迹后酶活性提高约10倍,表观Km值减小约30倍;交联后酶热稳定性显著提高,80℃时酶活性仍然很高。但催化效率仍然很低。
由此可见,生物印迹技术在改良酶特性、提高有机相酶促反应催化效率等方面是行之有效的。但交联印迹酶聚合物催化效率仍然很低,可望从以下几个方面提高未来生物印迹酶催化效率:印迹模板的选择要恰当;最大可能地减小印迹微粒的粒径,以得到可溶的催化剂;酶催化机理研究有待进一步深入。
The horseradish peroxidase (horseradish peroxidase) is (E , C 1.11.1.7) one kind ofperoxidase drawed in horseradish, whose molecular weight is 44000 D glycoprotein andsugar content is 18%.Its molecule contains Heme prosthetic group, whose porphyrin link isoriginal porphyrin and the centre ion is Fe (II). Since material is available and its price ischeap and the substrate is broad, then it is becoming one of the broadest enzyme. Useedin organic synthesis as catalysator, whose catalyzes mechanism is peroxidationcirculation, it has very high catalysis efficiency on aniline and their derivative.the kindwaits for phenol to. Since HRP has the glowing substrate, among enzyme immunoassayestablishes the chemiluminescence immunoassay obtains the widespread application in clinical.Inrecent years,the application of HRP is more outstanding in organic synthesis, biosensor,food analysis, biomedicine examination, fluorescence analysis, sewage treatment etc. Especiallythe applied research progress is rapid in the immunity diagnosis reagent technique, the biosensor, thelignin degeneration, the industry“the three wastes”and so on.
In 25% dioxane ring solutions, takeing the HRP catalyzed oxidation ursol's responseas the basis, the article has studied the HRP general enzymology nature, and mensuratedthe partial enzymatic reaction kinetics parameter. The result suggested that the HRP hassome bad characteristic,such as thermal stability is poor, the adaptation scope is narrowto the acid and alkali, the organic catalytic efficiency is low, and the enzyme isdeactivation and so on.Therefore takeing improving organic Organic phase enzymaticefficiency and the hot stability of aqueous phase enzyme as starting point, the articleobtains native imprinted protein(NIP), derivatized imprinted protein( DIP) andcrosslinkeinked imprinted proteins (CLIPs) by taking the substrate ursol as the templateby bio-imprinting and crosslinking method. And the modification enzyme has beenreviewed from the organic water content, the acid and alkali stability, thermostable aswell as the apparent Km three aspects. At the same time the enzyme carries outcomparison with free enzyme. The result suggested that the acid and alkali stable regionand most suitable pH of three kind of modification enzyme maintain invariable;aftersignature the enzyme activity enhances approximately 10 times, and the apparent Kmvalue reduces approximately 20 times; After the crossing linking, the enzymethermostability obviously enhances the enzymatic activity is still very high time 80℃. But the catalyze efficiency is still very low.
From preliminary study result, the bio-imprinting technology is effective inameliorating enzyme characteristic, improving organic Organic phase enzymaticefficiency and so on. But the efficiency of the crosslinkeinked imprinted proteins is stillvery low, which can be improved in the future from the following several aspects:theimprinted templates's choice must be appropriate; imprint particle grain diameter is mostgreatly possibly diminuted, which is to get soluble catalyst; the enzyme catalysismechanism research waits for further thoroughly.
本课题以辣根过氧化物酶为研究对象,以对苯二胺和过氧化氢为底物,在25%的二氧六环溶液中,从酸碱性、温度、底物浓度等几个方面,初步研究了辣根过氧化物酶催化氧化反应的一般特性,测定了部分酶促反应动力学参数,同时对反应产物进行表征。结果发现HRP热稳定性较差,酸碱适应范围较窄,有机相催化效率低,且酶易失活等不良特性。因此,本文以提高有机相酶催化效率、水相酶热稳定性为出发点,结合分子印迹和生物印迹两种技术达到对辣根过氧化物酶的印迹。利用底物对苯二胺诱导酶构象,获得天然印迹酶(NIP),并加入酰化试剂马来酸酐制备乙酰化印迹酶(DIP),冷冻干燥后加入交联剂和引发剂进行紫外光聚合,聚合完毕后用二氧六环洗脱配体,最后干燥就得到了所需的交联印迹酶聚合物(CLIPs),即为水相生物印迹酶。
为了考察三种酶(天然印迹酶、乙酰化印迹酶、交联印迹酶聚合物)的性质,研究内容设计如下:
(1)考察印迹酶在有机相中的催化活性,并分别考察pH值、温度、水含量、表观Km四因素对印迹酶催化活性的影响。同时与有机相游离酶进行比较;
(2)考察乙酰化印迹酶在水相中的催化活性,并分别考察pH值、温度、表观Km三因素对乙酰化印迹酶在水相中的催化活性的影响,同时与水相游离酶进行比较;
(3)考察交联印迹酶聚合物在水相中的催化活性,并分别考察pH值、温度、表观Km三因素对交联印迹酶聚合物在水相中的催化活性的影响,同时与水相游离酶进行比较。结果表明,三种酶酸碱稳定范围及最适pH保持不变;印迹后酶活性提高约10倍,表观Km值减小约30倍;交联后酶热稳定性显著提高,80℃时酶活性仍然很高。但催化效率仍然很低。
由此可见,生物印迹技术在改良酶特性、提高有机相酶促反应催化效率等方面是行之有效的。但交联印迹酶聚合物催化效率仍然很低,可望从以下几个方面提高未来生物印迹酶催化效率:印迹模板的选择要恰当;最大可能地减小印迹微粒的粒径,以得到可溶的催化剂;酶催化机理研究有待进一步深入。
The horseradish peroxidase (horseradish peroxidase) is (E , C 1.11.1.7) one kind ofperoxidase drawed in horseradish, whose molecular weight is 44000 D glycoprotein andsugar content is 18%.Its molecule contains Heme prosthetic group, whose porphyrin link isoriginal porphyrin and the centre ion is Fe (II). Since material is available and its price ischeap and the substrate is broad, then it is becoming one of the broadest enzyme. Useedin organic synthesis as catalysator, whose catalyzes mechanism is peroxidationcirculation, it has very high catalysis efficiency on aniline and their derivative.the kindwaits for phenol to. Since HRP has the glowing substrate, among enzyme immunoassayestablishes the chemiluminescence immunoassay obtains the widespread application in clinical.Inrecent years,the application of HRP is more outstanding in organic synthesis, biosensor,food analysis, biomedicine examination, fluorescence analysis, sewage treatment etc. Especiallythe applied research progress is rapid in the immunity diagnosis reagent technique, the biosensor, thelignin degeneration, the industry“the three wastes”and so on.
In 25% dioxane ring solutions, takeing the HRP catalyzed oxidation ursol's responseas the basis, the article has studied the HRP general enzymology nature, and mensuratedthe partial enzymatic reaction kinetics parameter. The result suggested that the HRP hassome bad characteristic,such as thermal stability is poor, the adaptation scope is narrowto the acid and alkali, the organic catalytic efficiency is low, and the enzyme isdeactivation and so on.Therefore takeing improving organic Organic phase enzymaticefficiency and the hot stability of aqueous phase enzyme as starting point, the articleobtains native imprinted protein(NIP), derivatized imprinted protein( DIP) andcrosslinkeinked imprinted proteins (CLIPs) by taking the substrate ursol as the templateby bio-imprinting and crosslinking method. And the modification enzyme has beenreviewed from the organic water content, the acid and alkali stability, thermostable aswell as the apparent Km three aspects. At the same time the enzyme carries outcomparison with free enzyme. The result suggested that the acid and alkali stable regionand most suitable pH of three kind of modification enzyme maintain invariable;aftersignature the enzyme activity enhances approximately 10 times, and the apparent Kmvalue reduces approximately 20 times; After the crossing linking, the enzymethermostability obviously enhances the enzymatic activity is still very high time 80℃. But the catalyze efficiency is still very low.
From preliminary study result, the bio-imprinting technology is effective inameliorating enzyme characteristic, improving organic Organic phase enzymaticefficiency and so on. But the efficiency of the crosslinkeinked imprinted proteins is stillvery low, which can be improved in the future from the following several aspects:theimprinted templates's choice must be appropriate; imprint particle grain diameter is mostgreatly possibly diminuted, which is to get soluble catalyst; the enzyme catalysismechanism research waits for further thoroughly.
引文
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[1]Xie,Y.C.Liu,H.Z. and Chen,J.Y,kinetcs of lipase catalyzed enantioselective esterification ofracemic ibuoprofen in isooctane,Chinese J,of Chem.Eng.2000,8,6-14
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[3]龚伟,宋锡瑾,王杰.脂肪酶构象刻录及稳定性研究.浙江大学学.2006,5(40):842~844
[4] Mustafa T,Mustafa K S,Erhan D,et al.A bio-imprinted urease biosensor: Improved thermaland operational stabilities.Talanta,2007,6:1~5.
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