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摘要：腈类化合物是一种化工原料，也是药品和精细化工生产中的重要中间体。腈类化学水解通常需要强酸或强碱条件和较高的反应温度，通常产生有害副产品。而腈水解酶是一类重要的水解酶,它能够催化腈类化合物生成相对应的羧酸或者酰胺类物质。作为生物催化，具有反应条件温和、高度选择性、产物纯度高等优点。因此，腈水解酶不仅应用于工农业生产，在药物合成过程中也有着不可忽视的作用。本文我们对重组腈水解酶产生菌的发酵优化进行了研究，根据发酵终止时的OD值可计算出其细胞量（g/L），并用高效液相色谱法，通过产物峰面积从而最终算出酶活。系统研究了碳源、氮源、金属离子、初始pH值、诱导剂条件以及温度对发酵产酶的影响。我们发现最适培养条件为：可溶性淀粉20g/L、酵母粉15g/L、硫酸镁1.0g/L、初始pH 7.0、温度为30℃。最终经过发酵优化后，腈水解酶酶活由最初的30.19 U/L提升到260.22 U/L，较优化前提高了约8.6倍。30595
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毕业论文关键词： 腈水解酶；酶活性；发酵优化
Fermentation optimization and catalytic performance of Nitrilase in Escherichia coli
Abstract: Nitrile compound is a kind of chemical raw material, which is also important intermediate in the production of pharmaceuticals and fine chemicals. Chemical hydrolysis of nitriles typically requires strongly acidic or basic conditions and higher reaction temperature, and usually produces unwanted byproducts. Nitrilase is an important kind of hydrolase, which can catalyze nitrile compounds and generate the corresponding carboxylic acid or amide substance. As a biological catalyst, it has the advantages of mild reaction condition, high selectivity, high product purity. Therefore, nitrilase is not only used in industrial and agricultural production, which also plays an unignorable role in the process of drug synthesis. This paper studied the fermentation optimization of producing strain of recombinant nitrilase. According to the OD value at the end of the fermentation, we can calculate the cell volume (g/L), and finally calculate enzyme activity through product peak area by high performance liquid chromatography. Carbon source, nitrogen source, metal ion, pH value and inducing conditions on the fermentation were systematically studied. We found the optimum culture conditions were: 20g/L of soluble starch, yeast powder 15g/L, magnesium sulfate 1.0g/L, initial pH value 7.0, temperature 30℃. After optimization, the enzyme activity was increased about 8.6times (from 30.19 U/L to 260.22 U/L).
Keywords: Nitrilase; fermentation optimization; enzyme activity
目录
1 研究背景 1
1.1 腈水解酶简介 1
1.2 产腈水解酶的微生物 1
1.3 腈水解酶的作用机理 1
1.4腈水解酶的应用 2
1.4.1光学活性及其衍生物方面 2
1.4.2工农业生产方面 2
1.4.3环境保护方面方面 3
1.4.4食品医药生产方面 3
1.5腈水解酶的底物专一性 4
1.6重组大肠杆菌的简介 4
1.7重组腈酶的发酵条件优化 5
2 材料与方法 8
2.1 实验材料 8
2.1.1 菌种 8
2.1.2 培养基与试剂 8
2.1.3 仪器和设备 8
2.2 实验方法 8
2.2.1 基本操作 8
图2.2.1 腈水解酶催化底物二腈水解反应液相色谱图 9
2.2.2 分析方法 9
3 实验过程及结果分析 11
3.1碳源优化 11
3.1.1碳源种类对生长及酶活的影响 11