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Luciferase: construction of oxidoreductases in vitro luminous system

A technology of luciferin and reductase, applied in the field of luciferase

Inactive Publication Date: 2009-08-05
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no luciferase in China: the establishment of an in vitro luminescence system for FMN-NADH oxidoreductase

Method used

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  • Luciferase: construction of oxidoreductases in vitro luminous system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] The luminescent bacteria seed solution with good properties was inoculated into 300mL enriched liquid medium according to the inoculation amount of 5%, and cultivated at a constant temperature of 25°C and 150r / min. Collect 20 mL of the cell suspension, centrifuge at 4°C (5000 rpm, 5 min), discard the supernatant, add a certain amount of PBS (5 mL), mix with a magnetic oscillator, and sonicate on ice for 15 seconds for each sample, with an interval of 30 seconds , Smash 15 times, power 300W. Extract luciferase crude enzyme solution.

Embodiment 2

[0016] To 1 mL of crude enzyme solution, quickly add 100 μL (27 mM) of substrate 12 alkanal, 3 μL (10 mM) of FMN-Na5, and 100 μL (0.14 mM) of NADH at one time for luminescence detection.

[0017] Luminescence detection is carried out in a weak luminescence instrument. The detection conditions are: set the wavelength to 474nm, after adding the solution or reagent, immediately perform timing tracking measurement, and the continuous measurement interval is 1s. When the crude luciferase and FMN-NADH oxidoreductase crude enzyme preparation extracted from the cell are added to the enzyme reaction substrate, the luciferase catalyzes the substrate to emit light.

Embodiment 3

[0019] Take 1mL crude enzyme solution each, and compare the luminescence properties of the luciferase single-enzyme in vitro luminescence system and the luciferase: FMN-NADH oxidoreductase two-enzyme in vitro luminescence system.

[0020] (1) Luciferase single enzyme in vitro luminescence system

[0021] Add different concentrations of substrates to 1mL of crude enzyme solution: dodecanal 100μL (27mM), FMN-Na53μL (10mM) and Na 2 S 2 o 4 100μL (34mM), the pH of the system is 7.0. The detection of luminescence is carried out in a weak luminescence instrument, and the detection conditions are as follows: set the wavelength to 474nm, quickly add each reaction substrate, immediately track and measure at regular intervals, and the continuous measurement interval is 1s.

[0022] (2) Luciferase: FMN-NADH oxidoreductase dual-enzyme in vitro light-emitting system

[0023] See Example 2

[0024] Comparing the luminescence intensity and stability of single and double enzymes, the res...

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Abstract

The invention relates to a luciferase, namely the determination of FMN-NADH oxido reductase luminescent conditions in vitro, in particular to a luciferase existing in ocean luminescent bacteriocyte, namely the luminescent expression of FMN-NADH oxido reductase outside cells. The invention aims to establish the luciferase, namely a FMN-NADH oxido reductase luminescent system in vitro for achieving the luciferase, and the expression of the FMN-NADH oxido reductase outside the living cells for achieving the enzyme activity. The invention establishes the optimal luciferase luminescent system in vitro through extracting the luciferase in the luminescent bacteriocyte and FMN-NADH oxido reductase crude enzyme preparations and optimizing the effect of various substrates on enzymatic reaction. The system has the advantages of stable luminescent performance, high luminescent intensity, simple and feasible substrate mixture ratio, and can achieve the quantitative determination.

Description

technical field [0001] The present invention relates to the determination of luciferase: FMN-NADH oxidoreductase in vitro luminescence conditions, in particular to the extracellular luminescence of luciferase and FMN-NADH oxidoreductase present in marine luminescent bacteria Express. Background technique [0002] According to the knowledge of the inventors through consulting data and literature search, luminescent bacteria refer to a type of bacteria accompanied by visible fluorescence emission during the growth process of bacteria under normal physiological conditions. The visible fluorescence wavelength is between 450-490nm , visible to the naked eye in the dark. [0003] The research on the luminescent mechanism of luminescent bacteria shows that the luminescent mechanism of different types of luminescent bacteria is the same, which belongs to enzymatic oxidation reaction. It is catalyzed by intracellular luciferase (LE) through the action of molecular oxygen, and the r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/66C12Q1/26G01N21/76
Inventor 王静雪林洪朱兰兰
Owner OCEAN UNIV OF CHINA