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Ultrahigh-flux screening method for phosphotriesterase

A phosphotriesterase and screening method technology, applied in the field of enzyme engineering, can solve problems such as inability to accurately reflect enzyme activity, affect screening accuracy and efficiency, etc. Effect

Active Publication Date: 2016-01-06
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The existing IVC-FACS system based on the cyanocoumarin phosphotriesterase substrate DEPCyC is due to the reaction product cyanocoumarin ( figure 1 , 3-cyano-7-hydroxycoumarin, CYHC) has strong hydrophobicity, and it is easy to quickly enter the outer water phase through the oil phase of the "water-in-oil-in-water" secondary micro-droplet, causing the fluorescence signal to quickly diffuse out of the micro-droplet, so Unable to accurately reflect the enzyme activity, thereby affecting the accuracy and efficiency of screening

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1 substrate HCCA-diethyl phosphate (DEPHCCA) is synthesized

[0044] 7-Hydroxycoumarin-3-carboxylic acid (7-Hydroxycoumarin-3-carboxylic acid, HCCA, compound2,) was purchased from Sigma-Aldrich Company. The synthesis method is as follows:

[0045]

[0046] Dissolve 700mg HCCA (3.4mmol) and 568mg DBU (1,8-diazabicycloundec-7-ene, 3.74mmol) in 20mL DMF (dimethylformamide), slowly add 635mg benzyl bromide (3.74mmol ). The mixture was stirred at room temperature for 14 hours. TLC (EtOAc:PE=1:1) detection confirmed that the reaction was complete. 100 mL of water was added to the reaction system, filtered, and the precipitate was dried to obtain 700 mg of compound 3 (yield 70%). Dissolve 700mg of compound 3 (2.36mmol) in 30mL of a 1:1 mixture of carbon tetrachloride and tetrahydrofuran, slowly add 358mg of triethylamine (3.55mmol) at 0°C, and then add 391mg of compound 4 (diethyl phosphate , 2.84mmol). The mixture was stirred at room temperature for 3 hour...

Embodiment 2

[0048] The expression of embodiment 2GkaP and mutant thereof

[0049] Using GkaP and its mutants 26A8, 26A8Y, 26A8C ( Zhang, Y.; An, J.; Ye, W.; Yang, G.; Qian, Z.G.; Chen, H.F.; Cui, L.; Feng, Y. Appl. Environ. Microbiol. 2012, 78, 6647–6655. ) for experiments, similarly, other sources of GkaP and its mutants can also be used.

[0050] The enzyme gene was constructed in pET28a and expressed in the host E.coliBL21(DE3)CodonPlus. Expression conditions: Gradiently streak the GkaP strain on the LB agar plate containing kanamycin, culture and grow a single colony; pick a single colony and inoculate it in 5 mL of LB liquid medium containing kanamycin, 37°C, Cultivate at 220 rpm until OD600 is 0.6-0.8, add IPTG to a final concentration of 1 mM, and culture at 25° C. at 220 rpm overnight. Harvest bacteria: take 1mL of culture solution and centrifuge at 5000rpm for 2min, remove the supernatant, add 1mL of PBS (pH7.4) to resuspend, then centrifuge at 5000rpm for 2min, remove the ...

Embodiment 4

[0058] Example 4 Retention of Fluorescent Molecules in Droplets

[0059] Water-oil-water secondary micro-droplets were prepared by using PBS solution (pH7.4) containing fluorescent molecules HCCA or CYHC as the inner aqueous phase. The two kinds of micro-droplets were diluted 100 times with the external aqueous phase respectively, and then placed at room temperature (simulating the state of micro-droplets during IVC-FACS sorting), and samples were taken at regular intervals for flow cytometry fluorescence intensity measurement.

[0060]The droplet retention of carboxycoumarin HCCA was compared with cyanocoumarin CYHC (a fluorescent product of DEPCyC, purchased from Sigma-Aldrich). We used the solutions of the two compounds (100 μM, dissolved in PBS) as the inner aqueous phase to prepare w / o / w secondary micro-droplets, diluted 100 times with the outer aqueous phase and incubated at room temperature for different times to measure the fluorescence intensity of flow cytometry ana...

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Abstract

The invention belongs to the field of enzyme engineering and discloses a carboxyl coumarin based phosphotriesterase fluorogenic substrate and a substrate based high-flux screening method for phosphotriesterase. The method is an in vitro compartmentalization based fluorescence-activated cell sorting ultrahigh-flux screening technology for phosphotriesterase. According to the method, a reacted fluorescent product of the carboxyl coumarin based phosphotriesterase substrate has a very good droplet retention property and has the advantages of high sensitivity and accuracy in quantification in the quantifying and screening of enzymatic activity. Due to the hydrophilic difference between the substrate and a reaction product, the substrate can be added into an external water phase and seep through an oil phase for enzymatic reaction; and the produced product is retained in liquid droplets due to improved hydrophilicity, so that the effect of liquid droplet fluorescence capture is achieved, the reaction time is accurately controlled, the fluorescent background interference is lowered, and the accuracy of screening is improved.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and relates to an ultra-high-throughput screening method for phosphotriesterases, in particular to the design and synthesis of a fluorescent substrate for phosphotriesterases, and a method for targeting phosphotriesterases based on the substrate. Enzyme in vitro compartmentalization-fluorescence activated cell sorting (invitrocompartmentalizationbasedfluorescence-activatedcellsorting, IVC-FACS) ultra-high-throughput screening technology. Background technique [0002] Phosphotriesterase (EC3.1.8.1) is an effective biocatalyst for the degradation of organophosphorus compounds, and has important applications in the detection, degradation and detoxification of organophosphorus pesticides and organophosphorus chemical warfare agents (Afriat-Jurnou, L.; Roodveldt, C.; Manco, G.; Tawfik, D.S. Biochemistry 2006, 45, 13677–13686. Afriat-Jurnou, L.; Jackson, C.J.; Tawfik, D.S. Biochemistry 2012, 51, 6047–...

Claims

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Application Information

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IPC IPC(8): C07F9/655C09K11/06C12Q1/44G01N21/64
Inventor 杨广宇马富强冯雁韩云宾
Owner SHANGHAI JIAO TONG UNIV
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