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摘要：使用腈水解酶产生有机羧酸来合成制药中间体的方法已得到越来越多的关注。腈水解酶作为生物催化剂，不仅具有反应条件温和性、高效性(比一般催化剂高106 ～1013倍)、污染少、易处理和成本低等优点，而且更重要的是能实现一般化学法不能达到的高度化学、区域和立体选择性，因此具有重要的工业应用价值。腈水解酶催化的水解反应中生成的NH3可在酸性溶液中生成铵盐，所以可用比色法来分析腈水解酶的活性。本论文中介绍了用苯酚-次氯酸盐比色法(Berthelot比色法)的测定方法，并通过紫外可见分光光度计来测定产物中铵盐的浓度，从而得到腈水解酶的活性。经过系统优化得到的最佳反应条件为：扫出的最佳波长为：630nm，水浴的最佳时间为：25min，水浴的最佳温度为：45℃，最佳显色剂的体积：A液是500l，B液的体积为300l。而通过与HPLC的比较、回收率以及精密度的验证表明这个比色方法是灵敏可靠的。6079
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关键字：腈水解酶；生物催化剂；选择性；分光光度法
Spectrophotometric determination of the activity of nitrilase
Abstract: The use of nitrilases to produce carboxylic acids has become an increasingly attractive approach for the synthesis of pharmaceutical intermediates. As biocatalyst, the nitrilases not only has the mild reaction conditions, efficient (106 to 1013 times higher than the average catalyst), less pollution, easy handling and low cost, but also has higher regioselective and/or enantioselective that the general chemical method cannot achieve. Therefore, nitrilases has the important value in industrial application. The NH3 which can be generated by nitrilase-catalyzed hydrolysis reaction can form ammonium salt in an acidic solution. So the activity of the nitrilase can be analysed by the sensitive and reliable colorimetry. In this paper we describe the phenol-hypochlorite colorimetry (Berthelot colorimetry). The concentration of the ammonium salt in the product was determined by UV spectrophotometric. Thereby obtaining the nitrilase activity. After systematic optimization, the best reaction condition is the following: The best absorption wavelength was 630 nm, 25 min was the most suitable time of heating in water bath and the optimum temperature was 45 °C, the optimum volume of A was 500 l, B was 300 l. By the comparison with HPLC, the verification of recoveries and accuracy, it can that indicates that this colorimetric method is sensitive and reliable.So Berthelot colorimetric method can be used to measure nitrilase’s activity.
Key Words：Nitrilases; biocatalyst; selective; spectrophotometric
目 录
1 研究背景 1
1.1 生物催化 1
1.2 腈化合物及腈水解酶 1
1.3 腈水解酶的选择性 2
1.3.1 水解酶的立体选择性 2
1.3.2 水解酶的区域选择性 3
1.3.3 水解酶的化学选择性 3
1.4 腈水解酶在有机合成中的应用 4
1.4.1 脂肪腈和芳基脂肪腈的生物转化 4
1.4.2 脂环腈和杂环腈的生物转化 4
1.4.3 二腈的生物转化 4
1.4.4 芳腈的生物转化 5
1.4.5 水－有机溶剂体系中腈的酶促水解 5
1.5 腈水解酶在生物降解与生物修复中的应用 5
1.6 本课题的研究意义和内容 6
1.6.1 课题的研究意义 6
1.6.2 课题的研究内容 6
2 实验原理和流程 7
2.1实验原理 7
2.1.1 腈水解酶 7
2.1.2 腈水解酶的作用原理 7
2.1.3 分光光度法 8