论文总字数：25848字

摘 要

来自一种嗜热莱斯菌依赖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。对大肠杆菌进行自诱导培养，将纯化的目的蛋白进行酶

学性质的研究。本文叙述了亮氨酸脱氢酶的诱导表达、蛋白纯化以及对酶学性质的研究。该酶由Ni-NTA柱纯化，C末端有组氨酸标签的重组LS菌亮氨酸脱氢酶的分子质量大约为44.3 KD，产率为74.51%。该酶需要以NAD 或NADH作为辅酶因子分别用于氧化脱氨基作用和还原胺化作用，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。该酶还具有良好的热稳定性，在70 °C下，该酶的半衰期为1.75h。酶在pH在7 - 11下显示相当大的活性，接近100%。最适反应温度是60 °C，最适反应pH是10.5。在25 °C，pH为10.5下LS菌的亮氨酸脱氢酶的比活为113.06U/mg。

关键词:亮氨酸脱氢酶；诱导表达；酶纯化；酶学性质

Expression and enzymology properties of leucine dehydrogenase in thermophilic Bacillus

Abstract

An NAD dependent L-leucine dehydrogenase (LeuDH, EC 1.4.1 .9) from Laceyella sacchari was over-expressed in E. coli. The purification of the target protein was studied by self-induced culture of E.coli.The expression and purification of leucine dehydrogenase and the properties of the enzyme were described in this paper.The molecular weight of the recombinant Ls-LeuDH with a C-terminal His-tag was approximately 44.3KDa, which was purified by a Ni-NTA column, and the yield is 74.51%. 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. The enzyme also has good thermal stability .At 70 °C, the half-life of enzyme is approaching 1.75 h. The enzyme showed a great relative activity approaching 100% at pH 7 - 11. The optimal reaction condition is 60 °C pH 10.5. In 25 °C pH 10.5, the Specific activity of Ls-LeuDH is 113.06 U/mg.

Keyword:Leucine dehydrogenase enzymes; Induced expression; Enzyme purification; Enzyme properties

目 录

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

ABSTRACT.............................................................................................................Ⅱ

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

1.1亮氨酸脱氢酶简介.................................................................................................1

1.2亮氨酸脱氢酶氨化还原三羟甲基丙烷生成L型叔亮氨酸的催化机...................1

1.3亮氨酸脱氢酶催化产物.........................................................................................2

1.3.1亮氨酸........................................................................................................2

1.3.2叔亮氨酸.....................................................................................................2

1.3.3氨基丁酸.....................................................................................................2

1.4酶促反应的医疗应用.............................................................................................3

1.4.1利用脲酶和亮氨酸脱氢酶偶联的反应测定血清和尿液中尿素...................................................................................................................................3

1.5工业生产应用..........................................................................................................3

1.6本课题研究意义......................................................................................................5

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

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

2.2序列与菌株..............................................................................................................7

2.3诱导与表达..............................................................................................................7

2.4蛋白纯化..............................................................................................................8

2.41粗酶液制备..................................................................................................8

2.42亮氨酸脱氢酶的纯化..................................................................................9

2.43 SDS-PAGE电泳分析..................................................................................9

2.5 酶的活力测定.......................................................................................................9

2.51 蛋白含量测定以及蛋白含量标准曲线绘制...........................................10

2.52 NADH标准曲线绘制................................................................................10

2.6 酶学性质 ............................................................................................................10

2.61 最适温度和最适pH..................................................................................10

2.62 温度稳定性和PH稳定性.........................................................................11

2.63 底物特异性...............................................................................................11

2.7 同源建模...............................................................................................................11

第三章 结果与讨论..............................................................................................13

3.1诱导与表达............................................................................................................13

3.2蛋白纯化................................................................................................................14

3.2.1蛋白含量标准曲线...................................................................................14

3.2.2 NADH标准曲线.......................................................................................15

3.2.3亮氨酸脱氢酶的纯化...............................................................................17

3.2.4纯化结果...................................................................................................17

3.3酶学性质................................................................................................................18

3.31最适温度和最适pH..................................................................................18

3.32 温度稳定性和PH稳定性........................................................................20

3.33 酶的底物特异性测定..............................................................................22

3.4 同源建模...............................................................................................................23

小结............................................................................................................................25

展望.............................................................................................................................25

参考文献...................................................................................................................26

致谢.............................................................................................................................29

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