Quantitative detection of heavy metal mercury by using luciferase and FMN:NADH oxido-reductase
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A fluorescent light and reductase technology, applied in the field of mercury detection, achieves the effects of high sensitivity, high luminescence intensity, and simple and feasible substrate ratio
Inactive Publication Date: 2010-01-13
OCEAN UNIV OF CHINA
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[0007] At present, there is no report on the quantitative detection of heavy metal mercury using luciferase and FMN:NADH oxidoreductase
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Embodiment 1
[0020] Embodiment 1 enzyme liquid preparation
[0021] Collect 20mL of the cell suspension, centrifuge at 4°C (5000rpm, 5min), discard the supernatant, add a certain amount of PBS (5mL), mix with a magnetic oscillator, and sonicate on ice. The sonication time for each sample is 15s, and each interval is 30s. , Smash 15 times, power 300W. Extract to obtain crude enzyme liquid.
Embodiment 2
[0022] Embodiment 2 Enzyme system enzyme activity assay
[0023] To 1mL crude enzyme solution, quickly add substrate 12 alkanal 100μL (27mM), FMN-Na 53μL (10mM), Na 2 S 2 o 4 100μL (34mM), NADH 100μL (0.14mM), for enzyme activity determination.
[0024] The luminescence detection is carried out in a weak luminescence instrument, and the detection conditions are: after adding the solution or reagent, the timing tracking measurement is carried out immediately, and the continuous measurement interval is 1s.
Embodiment 3
[0025] Example 3 Enzyme system is sensitive to heavy metal screening
[0026] Add 500 μL of heavy metal solution to 1 mL of enzyme solution, place it at 4°C for 15 minutes, and quickly add 100 μL (27 mM) of substrate 12 alkanal, 53 μL (10 mM) of FMN-Na, Na 2 S 2 o 4 100μL (34mM), NADH 100μL (0.14mM), for enzyme activity determination. Screen enzyme sensitive heavy metals.
[0027] Heavy metal ions can combine with bacterial proteins to denature them or combine with sulfhydryl groups of certain proteins to inactivate enzymes [13] , the effects of different concentrations of heavy metal ions on the enzyme activity are shown in Figure 5, and the difference is very large. Enzyme to Hg 2+ Most sensitive, followed by Cr 6+ 、Cd 2+ , when the concentration is 0.5μg / L, Hg 2+ The inhibition rate of enzyme activity is over 94%, Cr 6+ The inhibition rate of enzyme activity is 13.69%, Cd 2+ was 3.51%. Also select the Hg that has the greatest influence on the enzyme activity 2+...
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Abstract
The invention aims at using luminous bacteria strain Photobacterium leiognathi YL endoenzyme luciferase derived from marine environment and FMN:NADH oxido-reductase to detect mercury quantitatively. A strain of luminous bacterial YL derived from marine environment separated and purified in the invention is gram-negative bacilli and is verified as Photobacterium leiognathi. China Center for Type Culture Collection (CCTCC for short) is authorized to preserve the bacteria strain on Dec 27, 2006, with the preservation number being M 206139. The 16SrDNA gene sequence of the bacteria strain has already been submitted to the GenBank nucleotide sequence database with the accession number being EF017227. By extracting luminous bacteria endoenzyme luciferase and FMN-NADH oxido-reductase coarse enzyme preparation, the invention establishes a luciferase: FMN-NADH oxido-reductase invitro luminous system, realizes the expression of the luciferase: FMN-NADH oxido-reductase dual-enzyme system outside a living cell, and measures the concentration of mercury in virtue of the inhibition of the heavy metal mercury against the luminescence of the dual-enzyme system. The invention realizes the expression of the luciferase: FMN-NADH oxido-reductase outside the living cell and establishes the luciferase: FMN-NADH oxido-reductase invitro luminous system. The system has stable luminous performance, high luminous and strength and simple and feasible substrate mixture ratio, and can realize the quantitative detection of mercury. The use of the invention to detect mercury is characterized by low cost, simple and convenient operation, high sensitivity and the like, and can meet the requirement of rapid quantitative detection of mercury.
Description
technical field [0001] The invention relates to a method for detecting mercury, in particular to a method for quantitatively detecting heavy metal mercury by using intracellular luciferase and FMN:NADH oxidoreductase of marine luminescent bacteria Photobacterium leiognathi YL. Background technique [0002] According to the inventor's information and literature search, there are only three known genera of marine luminescent bacteria, which are Vibrio, Photobacterium, and Shewanella. On land, there are different short bacillus (Xenorhabdus). In an aerobic natural environment, luminescent bacteria can produce luciferase (LE), catalyze flavin mononucleotide (FMNH 2 ) and long-chain aliphatic aldehydes undergo oxidation reactions to emit blue-green fluorescence. [0003] At present, Photobacterium phosphoreum is widely used in our country. There are some reports on the use of Photobacterium phosphoreum to detect environmental pollutants. Except for Luminescent bacteria, there ...
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