论文总字数：19660字

摘 要

为获得能耐受较高温度的乳酸脱氢酶，本实验从嗜热菌Thermus thermophiles HB8中提取基因组，设计合适引物，PCR克隆得到嗜热菌L-乳酸脱氢酶基因片段，酶切后连接到pet-22b( )载体上，获得重组质粒pET-22b-LDH。将重组后的质粒pET-22b-LDH转入大肠杆菌BL21中，筛选后得到含有重组质粒的菌株BL-21-LDH。发酵培养转化成功的菌种，离心收集菌体，磷酸钠缓冲液重悬菌体后进行高压破碎，再离心收集上清，得到粗酶液。65 ℃水浴粗酶液30 min使其他不耐热蛋白失活达到第一步纯化目的，得到的酶液进行冷冻干燥，获得干燥的酶粉，可以较长时间保存。用蛋白纯化仪把重新溶解干燥酶粉进一步纯化，利用两步纯化后的酶液进行各项酶学性质的研究。酶学性质研究表明该LDH 的最适反应温度为90 ℃，最适pH 6.5；纯酶在95 ℃的半衰期为8 h，SDS-PAGE 结果显示分子量为32 kDa，与理论推算值相吻合。以丙酮酸和NADH为底物时，相对于丙酮酸Km 值1.92 mmol/L，Vmax 为0.34 U/mg；相对于NADH Km 值0.12 mmol/L，Vmax 值为1.83 U/mg。

关键词：嗜热菌 乳酸脱氢酶 克隆表达 酶学性质

Cloning and characterization of lactate dehydrogenase gene from thermophile bacteria in Escherichia coli

Abstract

For getting heat-resisting lactate dehydrogenase, in this study, we choose Thermus thermophiles HB8 to extract genome. After designing a suitable primer, we successfully get the LDH gene by PCR reaction from Thermus thermophiles HB8.And the LDH gene were ligated to expression vector pET-22b( ) through cutting by the same enzyme. E.coli-BL-21competent cells were transformed with the recombinant plasmid 22b-LDH. After screening, we get strains BL - 21 – LDH which contain recombinant plasmid 22b-LDH. Next culture the strain and collect the cells by centrifuge. Using sodium phosphate buffer to suspend the cells and breaking the cells by high pressure disruptor. We got the crude enzyme by centrifuging the broken cells solution and collecting the supernatant. We put the crude enzyme solution into 65℃water in order to make the heat labile enzyme lose activity. After this step, the enzyme solution would be freeze-dried into dry enzyme powder which can be saved for long time. Next, the dry enzyme powder would be dissolved and purified by Protein purification apparatus. Last, we used the enzyme solution purified by two steps to study the enzymatic properties. The recombinant enzyme had a molecular mass of 332kDa. The optimal temperature and pH of LDH were observed 90 ℃ and 6.5. The purified enzyme had a half-life of 8 h at 95 ℃. The Km and Vmax values were 1.92 mmol/L, 0.34 U/mg of protein for pyruvate, and 0.12 mmol/L and 1.83 U/mg for NADH.

Key Words: Thermophilic bacteria; Lactate dehydrogenase; Cloning and expression; Enzymatic property

目 录

摘 要 I

Abstract II

第一章 文献综述 1

1.1 乳酸简介 1

1.1.1基本介绍 1

1.1.2生产方法 1

1.1.3乳酸的应用 3

1.1.4乳酸生产现状及发展 4

1.2 乳酸脱氢酶简介 4

第二章 实验材料与方法 6

2.1 实验材料 6

2.1.1菌株和质粒 6

2.1.2培养基 6

2.1.3溶液配制 6

2.1.4主要仪器和设备 6

2.1.5工具酶，分子量标准和试剂盒 7

2.1.6主要试剂 8

2.2 实验方法 9

2.2.1 Thermus thermophilus HB8菌种活化及培养 9

2.2.2获取目的基因 9

2.2.3表达载体的构建 9

2.2.4 验证 11

2.2.5发酵培养及酶的获取 11

2.2.6 酶学性质的研究 13

第三章 实验结果 16

3.1 目的基因PCR扩增结果 16

3.2 酶切验证结果 16

3.3 蛋白浓度测定，纯化回收率，蛋白电泳图片 17

3.4 最适温度 18

3.5 温度稳定性 18

3.6 最适pH 19

3.7 酶动力学参数 19

3.7.1丙酮酸 19

3.7.2 NADH 19

3.8 金属离子的影响 20

第四章 结论与展望 21

参考文献 22

致 谢 24

第一章 文献综述

1.1 乳酸简介

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