论文总字数：23199字

摘 要

目前主要研究的多聚磷酸激酶家族中，PPK2-class Ⅲ 酶能够以多聚磷酸盐作为底物催化AMP到ATP的磷酸化。由于以多聚磷酸盐作为底物具有廉价、稳定、无毒等特点，因此，利用多聚磷酸激酶以多聚磷酸盐为底物合成ATP极具吸引力，对于ATP再生具有重要的研究意义。本课题以节杆菌的基因组作为模板，通过PCR扩增出多聚磷酸激酶基因ppk，将其连接到表达载体pET-28a上，再转化至大肠杆菌Rosetta(DE3)中，从而构建出了含ppk基因的重组菌株Rosetta(DE3）/pET-28a-ppk。用IPTG诱导表达后，SDS-PAGE分析结果显示，重组菌株表达的目的蛋白大小约为34kD，且基本以可溶形式存在于上清液中。然后利用镍柱亲和层析得到了电泳纯的多聚磷酸激酶，经检测纯酶液蛋白含量为297.5µg/mL，酶活为3.92U/mg。进一步对PPK酶反应条件优化，结果表明，最适反应温度为37℃，最适反应pH为8.0，反应底物中AMP的最适浓度为1mM，polyP6的最适浓度为2mM。

关键词：多聚磷酸激酶；过表达；酶活测定

Construction of polyphosphate kinase overexpressing strains and determination of enzyme activity

ABSTRACT

Among the major polyphosphate kinase families currently studied, the PPK2-class III enzyme is able to catalyze the phosphorylation of AMP to ATP using polyphosphate as a substrate. Because polyphosphates are inexpensive, stable, and non-toxic, the use of polyphosphate kinase to synthesize ATP using polyphosphate as a substrate is extremely attractive and has important research significance for ATP regeneration. In this study, the polyphosphate kinase gene ppk was amplified by PCR using the genome of Arthrobacter as a template, which was then ligated into expression vector pET-28a and then transformed into E. coli Rosetta (DE3). Thus, the recombinant strain Rosetta (DE3)/pET-28a-ppk containing the ppk gene was constructed. After induction by IPTG, the results of SDS-PAGE analysis showed that the size of the expressed protein of the recombinant strain was approximately 34 kD, and was basically present in the supernatant in a soluble form. Then electrophoretically pure polyphosphate kinase was obtained by affinity chromatography on nickel column. The protein content of the purified enzyme was 297.5μg/mL, and the enzyme activity was 3.92U/mg. Further optimization of the PPK enzyme reaction conditions showed that the optimal reaction temperature was 37°C, the optimal reaction pH was 8.0, the optimal concentration of AMP in the reaction substrate was 1 mM, and the optimal concentration of polyP6 was 2 mM.

Key Words: Polyphosphate kinase; Overexpression; Enzyme activity assay

目 录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1研究背景 1

1.2 ATP研究进展及意义 2

1.2.1研究进展 2

1.2.2研究意义 3

1.3多聚磷酸激酶（PPK）与ATP 4

1.3.1多聚磷酸激酶概述 4

1.3.2多聚磷酸激酶与ATP 5

1.4论文研究意义、内容及路线 6

1.4.1研究意义及目的 6

1.4.2研究内容 7

1.4.3实验流程 7

第二章 实验材料与方法 8

2.1实验材料 8

2.1.1菌株与质粒 8

2.1.2培养基及培养方法 8

2.1.3试剂盒与工具酶 9

2.1.4实验试剂 9

2.1.5仪器与设备 11

2.2实验方法 12

2.2.1克隆载体的制备与线性化 12

2.2.2目的基因ppk的扩增 12

2.2.3感受态细胞的制备 13

2.2.4重组质粒的构建 13

2.2.5 ppk基因在大肠杆菌中的表达与鉴定 15

2.2.6粗酶液制备及纯化 15

2.2.7蛋白质含量检测 15

2.2.8酶活测定 16

2.2.9产物ATP的测定 16

2.2.10 pH对酶活力的影响 16

2.2.11温度对酶活力的影响 16

2.2.12反应底物AMP及polyP6最适浓度 16

第三章 结果与讨论 18

3.1表达载体pET -28a-ppk的构建与转化 18

3.2 重组多聚磷酸激酶的诱导表达与纯化 19

3.3 纯酶酶活检测 20

3.4 pH对酶活力的影响 20

3.5温度对酶活力的影响 20

3.6反应底物AMP及polyP6最适浓度 21

第四章 结论与展望 23

4.1结论 23

4.2展望 23

参考文献 24

致 谢 28

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