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摘要成功从实验室保藏菌株H2中分离纯化出NADH氧化酶。经16S rDNA测序、MUG培养基等方法鉴定菌株H2是大肠埃希氏杆菌，最适培养温度为37℃，最佳发酵时间为12h。通过超声破碎法从菌体提取粗酶，再通过硫沉、Sephadex G50层析、DEAE 650离子交换层析、AF-Blue TOYOPEARL 650ML 串联AF-Red TOYOPEARL 650ML亲和层析对酶逐步纯化。纯化后的NADH氧化酶的比活为5U/mg，纯化倍数为13.2。由Gel Analysis 软件分析SDS-PAGE电泳图，得出NADH氧化酶的分子量为47～48kDa。其最适pH为8.0，最适温度为40℃，在50℃温育10min失活。Zn2+、Cu2+、Ni2+对酶活有显著的抑制作用。酶可能属于NADH过氧化酶，在催化NADH氧化为NAD+时产H2O2，但需进一步验证。用纤文素包裹的磁铁对酶进行固定化，由于该酶易失活，固定化效果差。19534
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关键词 NADH氧化酶 大肠埃希氏杆菌 纯化 表征 产H2O2 固定化
毕业设计说明书（论文）外文摘要
Title The research of NADH oxidase’s immobilization
Abstract
A NADH oxidase was purified from laboratory preserved strains H2. H2 strains was identified as Escherichia coli by microbial morphology、 physiology and biochemistry、 16S rDNA. For H2 strains, the optimal cultivation temperature is 37℃ and the best fermentation time is 12h.We extracted the crude enzyme from the bacteria by ultrasonic crushing. The enzyme was further purified by salt fractionation、Sephadex G50 chromatography、DEAE 650 ion exchange chromatography、AF-Blue TOYOPEARL 650 ML series AF-Red TOYOPEARL 650 ML affinity chromatography.The specific activity of enzyme after purification was 5U/mg and the purification fold was 13.2. The molecular weight of the enzyme was 31 000，according to the analysis of SDS-PAGE electrophoresis by the Gel Analysis software. The temperature and pH optimum of the NADH oxidase was 40℃ and 8.0. The enzyme lost activity after incubated 10 min at 50℃. Zn2+、Cu2+、Ni2+were strong inhibitors of the enzyme. The enzymes may be NADH peroxidase , which could produce H2O2 in the process of redox reaction，but further verifications were needed. The NADH oxidase was immobilized by the cellulose covered magnets. Because the enzyme deactivated easily, the interest of the immobilization was very poor.
Keywords NADH oxidase Escherichia coli Purification Characterization H2O2 producing Immobilization
目 次
1 引言 1
1.1 NADH氧化酶 1
1.1.1 NADH氧化酶简介 1
1.1.2 NADH氧化酶研究进展 2
1.2 固定化酶技术 3
1.2.1 固定化酶的研究进展 3
1.2.2 酶固定化方法 3
1.3 本毕业论文课题的研究意义 4
1.4 本论文的主要研究思路: 4
2 菌株的活化与鉴定 6
2.1 引言 6
2.2 实验材料 6
2.2.1 菌种来源 6
2.2.2 实验仪器与设备 6
2.2.3 主要试剂 6
2.2.4 培养基制备 7
2.3 实验方法 7
2.3.1 菌株活化 7
2.3.2 菌种保藏 7
2.3.3 平板上菌落形态 7
2.3.4 细菌革兰氏染色 7
2.3.5 菌株16S rDNA鉴定 8
2.3.6 MUG法鉴定 8
2.3.7 产气型鉴定 9
2.4 实验结果 9