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Enzyme activity detection system and method of Fe and alpha-oxoglutarate dependence dioxygenases

A ketoglutarate and dioxygenase technology, which is applied in the field of biotechnology detection, can solve the problems of cumbersome detection methods, lack of versatility and the like

Active Publication Date: 2016-04-20
SHENZHEN BEINUOBO BIOTECH CO LTD
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  • Application Information

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Problems solved by technology

These detection methods are cumbersome and not suitable for high-throughput drug screening
Furthermore, existing assays are only applicable to one specific iron and α-ketoglutarate-dependent dioxygenase and are not universal

Method used

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  • Enzyme activity detection system and method of Fe and alpha-oxoglutarate dependence dioxygenases
  • Enzyme activity detection system and method of Fe and alpha-oxoglutarate dependence dioxygenases
  • Enzyme activity detection system and method of Fe and alpha-oxoglutarate dependence dioxygenases

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Embodiment 1

[0049] In this embodiment, prolyl hydroxylase (PHD) is taken as an example to illustrate the enzyme activity detection method provided in the present application.

[0050] 1. Obtaining succinyl-CoA synthase

[0051] Succinyl-CoA synthase is commercially available, for example, from Megazyme Company (Cat. No. E-SCOAS), or prepared by the following method:

[0052] (1) Gene cloning

[0053] a. PCR amplification: for sucC and sucD genes encoding succinyl-CoA synthase β and α chains (such as figure 2 As shown) for PCR amplification, the PCR reaction system and conditions are shown in Table 1 and Table 2, respectively. The electrophoresis results of PCR product detection are shown in image 3 .

[0054] Table 1

[0055]

[0056] The primer sequences used are as follows:

[0057] Primer-F: 5’-GGGAATTCCATATG(NdeI)AACTTACATGAATATCAGGC-3’

[0058] Primer-R: 5’-CGCGAAGCTT(HindIII)TTATTTCAGAACAGTTTTCAGTGC-3’

[0059] Table 2

[0060]

[0061] b. Restriction enzyme digestion and enzyme linkage: The DNA...

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Abstract

The invention provides an enzyme activity detection method of Fe and alpha-oxoglutarate dependence dioxygenases, comprising a step of detecting the enzyme activity of the enzyme by a color reaction of inorganic phosphate generated by a coupled reaction of Fe and oxoglutarate dependence dioxygenases and succinyl-coenzyme A synthetase and ammonium molybdate malachite green dye. In addition, the invention also provides an enzyme activity detection system of dioxygenase, a kit and application of thereof.

Description

Technical field [0001] This application belongs to the field of biotechnology detection, and specifically relates to a general detection method for dioxygenase activity, and more specifically, to all dioxygenase enzymes that use α-ketoglutarate and iron ions as cofactors (ie, iron and α- A general detection method for ketoglutarate-dependent dioxygenase) activity. Background technique [0002] Dioxygenase is a general term for oxidase that binds all two oxygen atoms in an oxygen molecule to a substrate. Iron and α-ketoglutarate-dependent dioxygenases [α-oxoglutarate / Fe(II)-dependentdioxygenases(α-ODDs)] belong to the non-heme ferritin which is widely distributed in nature and is a kind of The important enzymes in redox reactions have various reaction substrates, which can be proteins, such as prolyl hydroxylase (PHD), hypoxia-induced factor (hypoxia induced factor) and collagen prolyl hydroxylase (collagenprolyl hydroxylase), etc. It can also be small molecule compounds, such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26C12Q1/25
Inventor 张雁杨春雨劳滔滔张宥偲
Owner SHENZHEN BEINUOBO BIOTECH CO LTD
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