Fluorescent detecting method of epoxide hydrolase producing bacterium
A technology for epoxy and fluorescence detection, applied in chemical instruments and methods, chemiluminescence/bioluminescence, luminescent materials, etc., can solve time-consuming and complex problems, and achieve simple operation process, simple equipment, and improved detection rate Effect
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Embodiment 1
[0019] Taking commercial epoxide hydrolase (71832, purchased from Sigma-Aldrich) as an example, the coumarin compound shown in formula (I) prepared from 4-methyl-7-hydroxycoumarin raw material was used as a fluorescent probe. Needle detection of epoxide hydrolase activity:
[0020] (1) Preparation of coumarin compounds:
[0021] Dissolve 10mmol of 4-methyl-7-hydroxycoumarin in 30ml of tetrahydrofuran and dimethyl sulfoxide (1:1), stir in an ice bath and slowly add NaH, continue stirring for 15min until fully dissolved, then add 20mmol of 4- Bromo-1-butene was reacted at 20°C for 4h. The reaction solution was added with saturated sodium chloride, extracted with ethyl acetate, washed with saturated sodium bicarbonate and saturated sodium chloride in turn, dried over magnesium sulfate, and then evaporated to dryness with a rotary evaporator. Separation and purification by column chromatography (cyclohexane: ethyl acetate = 2:1) gave the target intermediate. This intermediate w...
Embodiment 2
[0026] Dissolve the fluorescent probe in DMSO to make a solution with a concentration of 1 mM, take 1 μl dissolved in 9 μl buffer solution (Tris-HCl buffer solution with pH = 7.3) and mix it with styrene or phenyl-shrunk solution from various places in Zhejiang Province. Glyceryl ether is the only carbon source to screen and obtain 21 kinds of bacterial strains to be tested (thalline cell concentration is 20-50mg / ml), after reacting for 2.5 hours, add the existing NaIO 4 (Concentration: 140mM) 1μl, react for 10 minutes, add boric acid buffer (pH=8) 80μl, then add 20mg / ml 10μl BSA (concentration: 20mg / ml), to reach the final system 100μl. Aspirate the reaction solution from the 1.5ml EP tube, add it to a 96-well plate, and use a fluorescence spectrophotometer at λ ex / λ em Measure its fluorescence intensity at 360 / 460, get figure 2 . The fluorescence intensity data showed that the bacterial cells of No. 16 and No. 18 strains had strong epoxide hydrolase activity, and the co...
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