论文总字数：22844字

摘 要

一种来自嗜热放线菌依赖NAD 的L型亮氨酸脱氢酶（LeuDH EC,1.4.1.9）在大肠杆菌中得以表达。C末端有组氨酸标记的重组嗜热放线菌亮氨酸脱氢酶的分子质量大约为44.3 KD，该酶由Ni-NTA柱纯化，产率为81.27 %。该酶在市场上应用十分的广泛。该酶需要以NAD 和NADH作为辅酶因子，分别用于氧化脱氨作用和还原氨化作用，不能用NADP 或者NADPH代替。该酶对于另一种氨基酸的降解和合成路径有潜在的应用能力。我们使用纯化后的酶液研究其酶学性质。酶学性质研究表明：在70 ℃下，该酶的半衰期为1.75 h。该酶在pH 9-11下显示相当大的活性，接近100%。最适反应温度为60 ℃，最适反应pH为10。在25℃，pH为10下嗜热放线菌的亮氨酸脱氢酶的比活为78.05 U/mg。

关键词: 亮氨酸脱氢酶 酶纯化 酶学性质 大肠杆菌

Abstract

An NAD dependent L-leucine dehydrogenase (LeuDH,EC 1.4.1.9) from Thermophilic actinomycetes was over-expressed in E. coli. The molecular weight of the recombinant Ls-LeuDH with a C-terminal His-tag was approximately 44.3 KDa, which was purified by a Ni-NTA column, and the yield is 81.27 %.The enzyme is widely applied in the market.The enzyme required NAD and NADH as cofactors for oxidative deamination and reductive amination respectively, which cannot be replaced by NADP or NADPH. This enzyme has a potential application for an alternative amino acid reduction and synthesis pathway. We use the purified enzyme fluid study its enzymology properties.Enzymology properties studies have shown that the half-life of enzyme is approaching 1.75 h at 70 °C,. The enzyme showed a great relative activity approaching 100 % at pH 9 - 11. The optimal reaction condition is 60 °C pH 10. In 25 ℃ pH 10, the Specific activity of Ti-LeuDH is 78.05 U/mg。

Key Words: Leucine dehydrogenase; Enzyme purification; Enzymology properties; E.coli

目录

摘要...............................................................I

Abstract...........................................................II

第一章 文献综述....................................................1

1.1 亮氨酸脱氢酶的研究进展.........................................1

1.1.1亮氨酸脱氢酶简介..............................................1

1.1.2亮氨酸脱氢酶参与的反应........................................1

1.1.2.1脲酶-亮氨酸脱氢酶偶联法测定尿素的方法学评价..................1

1.1.2.2亮氨酸脱氢酶的反应机理......................................1

1.2 亮氨酸脱氢酶催化产物作用.......................................2

1.2.1亮氨酸作用....................................................2

1.2.2叔亮氨酸......................................................3

1.2.3氨基丁酸......................................................3

1.2.4三甲基丙酮酸..................................................3

1.3 亮氨酸脱氢酶的工业化生产及其应用...............................3

1.3.1辅酶再生策略..................................................4

1.3.2酶膜反应器....................................................5

1.3.3发酵法........................................................5

1.3.4持续补料和分批补料............................................5

1.3.5分批发酵......................................................6

第二章材料与方法.........................................6

2.1 试剂和器材.................................................... 6

2.2序列与菌株......................................................8

2.3诱导与表达..............................................8

2.3.1诱导时间的确定................................................8

2.4 蛋白纯化 ......................................................9

2.4.1 粗酶的制备....................................................9

2.4.2 LeuDH的纯化..................................................9

2.4.3 SDS一PAGE电泳分析..........................................9

2.5酶活力测定 ....................................................10

2.5.1 蛋白含量标准曲线绘制. ........................................10

2.5.2 NADH标准曲线绘制............................................11

2.6亮氨酸脱氢酶的酶学性质研究.....................................11

2.6.1最适温度和热稳定性...........................................11

2.6.2 最适PH和稳定性...............................................11

2.6.3 底物特异性...................................................12

1. 结果与分析................................................12

3.1序列分析.......................................................12

3.2诱导与表达.....................................................14

3.3蛋白纯化 ......................................................15

3.3.1分级纯化表....................................................15

3.3.2 SDS-PAGE电泳.................................................15

3.3.3蛋白含量标准曲线..............................................16

3.3.4 NADH标准曲线................................................16

3.4亮氨酸脱氢酶的酶学性质研究.....................................18

3.4.1 亮氨酸脱氢酶的最适反应温度...................................18

3.4.2温度对酶稳定性的影响..........................................18

3.4.3亮氨酸脱氢酶的最适pH.........................................19

3.4.4亮氨酸脱氢酶的pH稳定性.......................................20

3.4.5 底物特异性...................................................21

小结.............................................................22

展望..........................................................................................................22

致谢....................................................23

参考文献................................................23

第一章 文献综述

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