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摘要：腈水解酶（腈转化酶或腈水合酶/酰胺酶）作为一种生物催化剂，已经被广泛应用在制药行业中有机羧酸及其衍生物的生产，而传统有机腈化学水解的方法，通常是需要高温、强酸、碱等相对严苛的条件，所以建立一个简单、快速且适用范围广的测定酶活性的方法是非常必要的。本次试验用盐酸羟胺和N,N'-二环己基碳二亚胺（DCC）与被腈水解酶催化有机腈得到的有机羧酸溶液反应生成羟肟酸，基于羟肟酸在氯化铁溶液中生成紫红色羟肟酸铁络合物显色的性质，通过紫外-可见分光光度法测量该溶液的吸光度来判断羧酸浓度。本研究将盐酸羟胺、DCC与显色剂的浓度、显色反应的温度、水浴时间以及反应时间对吸光度的影响进行了优化，对其抗干扰因素进行了测定，并且利用羟肟酸铁和高效液相色谱法（HPLC）的相对标准偏差（RSD）和加样回收率的比对，验证了此方法的准确性。结果表明,显色反应的最佳条件为：盐酸羟胺浓度为4mmol/L，DCC浓度为0.2mol/L，氯化铁浓度为0.5mol/L，反应时间为10min，水浴时间为15min。22463
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毕业论文关键词：生物催化剂； 腈水解酶；分光光度法；显色反应；有机羧酸
Studies on the determination of the content of carboxylic acids in aqueous solution by spectrophotometry
Abstract: Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase / amidase) as one kind of the biocatalyst, which have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, however, the traditional chemical hydrolysis method of nitriles, which usually requires high temperature, strong base or acid and other harsh condit- ions. So, it is important to build a simple, rapid and widely used method of determination of enzyme activity. The ferric hydroxamate spectrophotometry was developed fordetermination of carboxylic acid in aqueous solutions was developed by the formation hydroxamic acid from ca- rboxylic acid in the presence of hydroxylamine hydrochloride and DCC as well as the formati- on of purple ferric hydroxamate from hydroxamic acid in the presence of acidic ferric chloride solution. In this assay we optimize the concentration of hydroxylamine hydrochloride, DCC and chromogenic agent, the temperature of the color reation, bath time and reaction time for the ab- sorbance, and determine the interference factors. Compared with the relative standard deviation (RSD) and the ratio of recovery between ferric hydroxamate spectrophotometry and HPLC, we can verify the accuracy of this method. The results illustrate that the best condition of color rea- ction is: concentration of hydroxylamine hydrochloride is 4mmol/L, concentration of DCC is 0.2mol / L, concentration of ferric chloride is 0.5mol/L, reaction time is 10 min, bath time is 15 min.
KeyWords: Biocatalyst; Nitrilase; Spectrophotometry; Color reaction; Carboxylic acids
目 录
1 研究背景 1
1.1生物催化剂 1
1.1.1生物催化剂概述 1
1.1.2生物催化剂的起源 1
1.1.3生物催化剂的筛选 1
1.1.4酶的概述——常用生物催化剂 2
1.2 生物催化剂在制药方面的应用 3
1.3腈水解酶 3
1.3.1腈水解酶概述 3
1.3.2催化腈水解的酶系的类型 3
1.3.3腈水解酶的应用 4
1.3.4腈水解酶的筛选方法现状研究 4
1.3.5羧酸含量的测定方法 5
1.4本课题研究的意义及思路 6
1.4.1本课题研究的意义 6
1.4.2本课题研究的思路 6
2 实验原理及流程 8
2.1实验原理 8