Method for real-time fluorescence monitoring of taxodienase reaction

Abstract

The invention discloses a method for real-time fluorescence monitoring of a dienase reaction of taxus chinensis. Artificial macrocyclic receptor calixarene is utilized to screen artificial receptor-fluorescent dye subject-object pairs, and fluorescence signal difference is generated through the characteristics of the artificial receptor-fluorescent dye subject-object pairs to monitor the progress of the dienase reaction of taxus chinensis. The method comprises the following steps: preparing a buffer solution and a standard solution to be titrated; adding a taxodienase stock solution into the standard solution to be titrated, carrying out a titration experiment, and recording and finishing a fluorescence intensity curve; and diluting the to-be-detected sample, and determining the concentration of the taxadiene enzyme in the to-be-detected solution based on the fluorescence intensity curve. The method has the advantages that the detection accuracy is improved, high-throughput screening is facilitated, the detection error is small, and the sample amount can be reduced.

Description

Technical Field

[0001] This invention relates to a method for real-time fluorescence monitoring of taxadiene enzyme reactions, belonging to the fields of biochemistry and biotechnology, and particularly to a monitoring technology for key enzyme reactions in the biosynthesis of paclitaxel. Background Technology

[0002] Paclitaxel, a widely used drug for treating various cancers, is in increasing demand. Traditional methods of extracting paclitaxel cannot meet the large-scale market demand. The yield of paclitaxel extracted from yew plants is very low, the all-chemical synthesis of paclitaxel involves many steps and has a generally low yield and is costly, while plant cell culture technology has unstable yields and impurities interfere with the product purification process.

[0003] For the industrial production of paclitaxel, its synthesis has become one of the mainstream methods. It involves multiple biosynthetic steps, with geranylgeranyl pyrophosphate synthase (GGPP) being a key enzyme. GGPP then undergoes a series of enzymatic reactions to ultimately convert into paclitaxel. Currently, there is no efficient method to detect the reaction progress of paclitaxel and geranylgeranyl pyrophosphate synthase, while traditional monitoring methods suffer from low accuracy, complex operation, and long processing times. Summary of the Invention

[0004] The present invention aims to propose an innovative method for real-time fluorescence monitoring of taxadienase reactions, in order to improve the accuracy and efficiency of monitoring.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for real-time fluorescence monitoring of taxadienase reactions includes the following steps:

[0007] Prepare buffer solutions and standard solutions to be titrated;

[0008] Add taxadienase stock solution to the standard solution to be titrated and perform titration experiment, then record the fluorescence intensity curve.

[0009] The sample was diluted, and the concentration of taxadienase in the solution was determined based on the fluorescence intensity curve.

[0010] Furthermore, this includes using artificial macrocyclic receptors screened with calixarenes to form host-guest pairs with fluorescent dyes, enabling specific recognition of substrates and products; the artificial macrocyclic receptor is CAC4A or QAAC4A-12C, and the fluorescent dye is RhB or Fl.

[0011] The structure of CAC4A is as follows: The structural formula of QAAC4A-12C is:

[0012] Further, the steps of preparing the buffer solution and the standard solution to be titrated include: preparing the HEPES buffer solution; and preparing the standard solution to be titrated.

[0013] Furthermore, at 25°C, using HEPES buffer solution as a solvent, a concentrated solution of fluorescent dye prepared with this buffer solution was added, followed by a concentrated solution of artificial macrocyclic acceptor prepared with HEPES buffer solution, to obtain the standard solution to be titrated.

[0014] Further, before, during, or after the preparation of the buffer solution and the standard solution to be titrated, the following steps are included: preparing a taxadienease stock solution by weighing a fixed weight of taxadienease and preparing a taxadienease stock solution with a fixed molar concentration using DMSO.

[0015] Further, the steps of adding taxadienase stock solution to the standard solution to be titrated and performing a titration experiment, and recording the completed fluorescence intensity curve include: adding taxadienase stock solution; recording fluorescence intensity; and plotting the completed fluorescence intensity curve.

[0016] Further, the step of adding taxadienase stock solution to the standard solution to be titrated and performing a titration experiment, and recording the completed fluorescence intensity curve, further includes: gradually adding taxadienase stock solution to the standard solution to be titrated; measuring the fluorescence intensity at different concentrations of taxadienase, calculating the ratio of fluorescence intensity to reference fluorescence intensity to determine the relative fluorescence intensity, and plotting fluorescence intensity curves of different relative fluorescence intensities and different concentrations of taxadienase.

[0017] Further, the steps of diluting the test sample and determining the concentration of taxadienase in the test solution based on the fluorescence intensity curve include: diluting the test solution; adding an artificial macrocyclic acceptor and a fluorescent dye to the test solution; and measuring the fluorescence intensity of the test solution.

[0018] Further, the steps of diluting the test sample and determining the concentration of taxadienase in the test solution based on the fluorescence intensity curve include: diluting the test solution with HEPES buffer; adding a concentrated solution of fluorescent dye prepared with HEPES buffer and a concentrated solution of fluorescent dye to the diluted test solution; detecting the fluorescence intensity of the test sample, calculating the relative fluorescence intensity, and comparing it with the fluorescence intensity curve to determine the concentration of taxadienase in the sample.

[0019] Furthermore, the progress of the taxadienase reaction is monitored in real time by monitoring the concentration of the taxadienase in the test solution.

[0020] Compared with existing technologies, the method for real-time fluorescence monitoring of taxadienase reactions proposed in this invention has significant innovations and advantages. First, by screening suitable artificial acceptor-fluorescent dye host-guest pairs using artificial macrocyclic acceptors (calixarenes), specific recognition of substrates and products is achieved, generating detectable differences in fluorescence signals and greatly improving monitoring accuracy. For example, QAAC4A-12C has a strong binding affinity to both enzyme substrates and products. Using the Fl & QAAC4A-12C host-guest pair, a significant difference in signal response was achieved for the same concentrations of GGPP and TAX, with a detection error less than half that of traditional methods. Second, the method of this invention is relatively simple to operate, transferring the enzyme reaction process to a microplate reader equipped with well plates, reducing complex operational steps, lowering technical difficulty and cost, and improving the operability of the method. Third, it is beneficial for saving samples during high-throughput screening. Due to the strong binding affinity of QAAC4A-12C to enzyme substrates and products, fluorescence detection can be performed at lower concentrations. For example, in high-throughput screening experiments, traditional methods require a large amount of sample, while the method of this invention can save more than half of the sample volume at the same screening scale. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 This is a flowchart of the real-time fluorescence monitoring of taxadienease reaction according to the present invention;

[0023] Figure 2 The relationship between relative fluorescence intensity and GGPP concentration. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The geranylgeranyl pyrophosphate synthase (GGPP) reaction plays a central role in the synthesis of paclitaxel. Paclitaxel is an important anticancer drug and is widely used in the clinical treatment of various cancers. The geranylgeranyl pyrophosphate synthase (GGPP) can catalyze the cyclization reaction of geranylgeranyldiphosphate (GGDP) to generate taxadiene (TAX). Taxadiene (TAX) undergoes a series of functional group reactions to generate baccatin, which in turn synthesizes paclitaxel (PTX). It can be said that the geranylgeranyl pyrophosphate (GGPP) reaction is a key step in the biosynthesis of paclitaxel. Synthesis of CAC4A (5,11,17,23-tetra[(p-carboxyphenyl)azo]-25,26,27,28-tetrahydroxycalix[4]arene)

[0026] In a round-bottom flask, 1.37 g (10 mmol) of 4-aminobenzoic acid and 2 mL (20 mmol) of concentrated hydrochloric acid were added to 15 mL of water. The solution was cooled to 2 °C by an ice-water bath, and 0.80 g (10 mmol) of sodium nitrite in 10 mL of water was slowly added while keeping the temperature below 5 °C. The resulting solution was slowly added to a solution of 25,26,27,28-tetrahydroxycalix[4]arene (1.0 g, 2.36 mmol) and sodium acetate (2.46 g, 30 mmol) in 26 mL of MeOH-DMF (5:8, v:v) to obtain a red suspension. After stirring at room temperature for 2 h, 150 mL of 0.25% hydrochloric acid solution was added. After heating to 60 °C and holding for 30 min, the mixture was filtered, washed with water and MeOH, and the reddish solid of CAC4A was obtained, with a quantitative yield. NMR (400MHz, DMSO-d6) δ 8.04 (d, 8H, Ar-H), 7.83 (d, 8H, ArH), 7.82 (s, 8H, calix-H), 4.41 and 3.71 (s, 8H, Ar-CH2-Ar); MS (MALDI-TOF): calcd.for[MH]-1015.269, found 1015.195.

[0027] Synthesis of QAAC4A-12C:

[0028] To a THF (10 mL) solution of aminocalix[4]arene pentadecyl ether (NH2C4A-12C, 1) (0.9 g, 0.78 mmol), HCl (1 M, 9 mL) and NaNO2 (0.38 g, 5.5 mmol, 7 mL in H2O) were added sequentially at 0 °C. After stirring at room temperature for 1 h, 0.2 g phenol (2.12 mmol) in pyridine (18 mL) was added dropwise to the solution mixture, and the mixture was stirred at room temperature for another 16 h. The reaction solution was added dropwise to 200 mL H2O to precipitate, collected by vacuum filtration, and then purified by column chromatography (DCM:MeOH = 40:1) to obtain the azo-linked intermediate. Glycidyltrimethylammonium chloride (1.38 g, 9 mmol) was added to the isopropanol (20 mL) solution of the intermediate. The mixture was refluxed for 18 h and stirred at room temperature for another 2 h. A yellow precipitate was obtained (if it is still a clear liquid, stir in an ice-water bath for 10 min). The precipitate was then filtered under vacuum and washed with isopropanol to give QAAC4A-12C (265 mg, 80%). ¹H NMR (400 MHz, DMSO-d6) 7.52 (s, 8H, ArH), 4.38 (d, J = 13.62 Hz, 4H, Ar-CH₂-Ar), 3.65 (t, J = 7.28 Hz, 8H, CH₂-O-Ar), 3.34 (d, J = 13.85 Hz, 4H, Ar-CH₂-Ar), 1.68 (dt, J = 14.23 Hz, J = 7.45 Hz, 8H, -CH₂-CH₂-CH⁻), 1.42 (m, 4H, -CH⁻), 1.12 (m, 8H, -CH₂-CH₂-CH⁻), 0.82 (d, J = 6.32 Hz, 24H, -(CH₃)₂).

[0029] Example 1:

[0030] In this embodiment, a fluorescence competitive titration method was used to apply the prepared artificial macrocyclic receptor QAAC4A-12C to the ultrasensitive and selective quantitative detection of taxadienase (GGPP) reaction. Using the artificial macrocyclic receptor (QAAC4A-12C) and the fluorescent dye (Fl) as the sensing pair for detecting the substrate taxadienase (GGPP), a system for quantitatively detecting GGPP concentration was constructed. The specific determination method includes three steps: S101-S103.

[0031] Step S101: Preparation of buffer solution and standard solution to be titrated. This step uses hydroethylpiperazine ethanesulfonic acid (HEPES) buffer. HEPES has good buffering capacity in the pH range of 6.8 to 8.2, effectively maintaining pH stability in the biological reaction system. An artificial macrocyclic acceptor (QAAC4A-12C) and a fluorescent dye (FI) are added to the HEPES buffer to obtain the standard solution to be titrated. The fluorescent dye (FI) is fluorescein, a common fluorescent label widely used in bioimaging and analytical chemistry. Fluorescent dyes (FI) emit strong fluorescence under appropriate conditions and are used to label biomolecules or as probes to detect specific chemicals.

[0032] The specific amounts of the prepared components and the process are given below to further clarify the steps expressed in step S101.

[0033] S1011 Preparation of HEPES buffer solution: Accurately weigh 2.383g of HEPES and use ultrapure water to prepare a 1L solution with a concentration of 10mmol / L and a pH of 7.4. Use this solution as a buffer solution (referred to as HEPES buffer solution).

[0034] S1012 Preparation of the standard solution to be titrated: Then, at 25°C, using 10 mmol / L HEPES buffer solution with pH 7.4 as the solvent, add a 100 μmol / L concentrated Fl solution prepared with this buffer solution. At this time, a clear fluorescence signal can be observed. Then, add a 100 μmol / L concentrated QAAC4A-12C solution prepared with HEPES buffer solution to obtain the standard solution to be titrated.

[0035] While GGPP stock solution is not required in step S101, it can be optionally prepared in S101 for direct use in subsequent steps. The preparation of the GGPP stock solution can be done before or after preparing the HEPES buffer solution, or before or after preparing the standard solution to be titrated. The method for preparing the GGPP stock solution is to weigh 0.35 mg of GGPP and dissolve it in DMSO to prepare 3 mL of a 250 μmol / L GGPP stock solution.

[0036] The volume of the standard solution to be titrated was set to 10 times that of the diluted test solution to ensure sufficient reactants and accurate measurement results during titration. In this standard solution, the concentration of Fl was 0.5 μmol / L, and the concentration of QAAC4A-12C was 3 μmol / L. This concentration setting was to ensure that Fl emitted a sufficient fluorescence signal at this concentration, and that the concentration of QAAC4A-12C was sufficient for effective interaction with Fl.

[0037] In the experiment, a concentrated Fl solution was first added to the standard solution to be titrated. A clear fluorescence signal was observed, indicating that the fluorescence properties of Fl were well utilized under these conditions. Subsequently, a concentrated QAAC4A-12C solution was added, and a significant decrease in the fluorescence signal was observed. This decrease typically indicates an interaction between QAAC4A-12C and Fl, caused by fluorescence quenching or binding.

[0038] The fluorescence intensity at this point is recorded as a baseline fluorescence intensity for subsequent detection and analysis. This baseline fluorescence intensity provides a reference point for adding different concentrations of QAAC4A-12C, allowing researchers to quantitatively analyze the concentration changes of QAAC4A-12C and their impact on the fluorescence properties of Fl by comparing changes in fluorescence signals.

[0039] Step S102: Add taxadienease stock solution to the standard solution to be titrated and perform a titration experiment, recording the fluorescence intensity curve. In this step, the standard solution to be titrated prepared in the previous step is mixed with GGPP stock solution. During the mixing process, the GGPP concentration and fluorescence intensity are recorded, and a fluorescence intensity curve is plotted.

[0040] S1021 Addition of GGPP Stock Solution: GGPP stock solution was gradually added to the standard solution to be titrated, and the change in fluorescence signal was observed. In the experiment, the stock solution was gradually added to the standard solution to be titrated, and the fluorescence signal was observed to gradually recover. This phenomenon indicates that the addition of GGPP can effectively interact with the previous QAAC4A-12C, weakening the quenching effect of QAAC4A-12C on the fluorescence signal, thereby restoring the fluorescence intensity.

[0041] S1022 Recording Fluorescence Intensity: Relative fluorescence intensity was measured, and a standard curve of fluorescence intensity versus GGPP concentration was plotted to confirm its linear relationship. This fluorescence competitive titration method allows us to plot a standard curve of relative fluorescence intensity as a function of GGPP concentration. Relative fluorescence intensity is calculated as the ratio between the fluorescence intensity value and the reference fluorescence intensity (i.e., the fluorescence intensity measured after adding QAAC4A-12C), providing a reference for the changes in fluorescence signal corresponding to different GGPP concentrations.

[0042] In the experiment, the detection limit for GGPP was set at 3.5 mol / L, demonstrating the high sensitivity of this method at such a low concentration. The GGPP concentration in the standard curve was calculated based on the molar amount of GGPP added relative to the volume of the test solution, ensuring the accuracy of the concentration under different experimental conditions and contributing to the reliability of the experimental results.

[0043] Experimental results show a good linear relationship between GGPP concentration and relative fluorescence intensity, further confirming the feasibility of this system for ultrasensitive quantitative detection of GGPP concentration. Fluorescence intensity curves showing the change in relative fluorescence intensity as a function of GGPP concentration were obtained using a blank sample without added GGPP.

[0044] Step 103: Dilute the test sample and determine the concentration of taxadienase in the test solution based on the fluorescence intensity curve. In this step, FI and QAAC4A-12C are added to the diluted test sample, the fluorescence intensity of the mixed solution is measured, and the GGPP concentration in the solution is determined based on the aforementioned fluorescence intensity curve.

[0045] S1031 Dilution of the test solution: Dilute the test solution with HEPES buffer. Under specific temperature conditions of 25°C, dilute the test solution using a 10 mmol / L HEPES buffer solution with a pH of 7.4. HEPES, as a biological buffer, maintains the pH stability of the solution; dilution of the test solution ensures that the concentration of GGPP remains within a measurable range in subsequent fluorescence assays.

[0046] S1032 adds an artificial macrocyclic acceptor and a fluorescent dye to the test solution: then, a concentrated Fl solution prepared with the HEPES buffer and a concentrated QAAC4A-12C solution are added to obtain the test solution to be titrated. The volume of this test solution to be titrated is 10 times the volume of the diluted test sample, wherein the concentration of Fl is 0.5 μmol / L and the concentration of QAAC4A-12C is 3 μmol / L.

[0047] S1033 Determination of fluorescence intensity of the test solution: The fluorescence intensity of the test sample is detected, the relative fluorescence intensity is calculated, and compared with the fluorescence intensity curve to determine the concentration of GGPP in the sample. After thorough mixing, the fluorescence intensity of the test solution to be titrated is detected using equipment such as a fluorescence spectrometer. The relative fluorescence intensity is calculated from the detected fluorescence intensity. This process requires comparing the detected absolute fluorescence intensity with the reference fluorescence intensity to eliminate background noise or other interference. There is a certain relationship between the relative fluorescence intensity and the concentration of GGPP, which can be represented by a fluorescence intensity curve. Figure 2 ).

[0048] The calculated relative fluorescence intensity was compared with the fluorescence intensity curve established in step two. The fluorescence intensity curve was obtained from a GGPP sample of known concentration, providing a quantitative relationship between fluorescence intensity and GGPP concentration. Based on the relative fluorescence intensity, the concentration of GGPP in the test solution can be determined.

[0049] Finally, the progress of the taxadienase reaction was monitored in real time by monitoring the concentration of taxadienase in the test solution. During the taxadienase reaction, the substrate GGPP and the product TAX exhibit different competitive binding abilities to the fluorescent dye. When the fluorescent dye (F1) binds to the artificial macrocyclic acceptor (QA), the artificial acceptor can effectively quench the fluorescence of the encapsulated dye through a photoinduced electron transfer mechanism. During the enzyme reaction, the presence of the substrate and product affects the binding of the dye to the artificial acceptor, resulting in differences in fluorescence signals. For example, when the concentration of substrate GGPP is high, it competes with the fluorescent dye (F1) for binding to the artificial acceptor (QA), leading to an enhanced fluorescence signal; when the concentration of product TAX is high and the concentration of GGPP is low, the binding of GGPP to the artificial acceptor (QA) weakens, enhancing the binding of the artificial acceptor (QA) to FI, resulting in a weakened fluorescence signal. By detecting these differences in fluorescence signals, the progress of the taxadienase reaction can be monitored in real time.

[0050] By using artificial macrocyclic acceptors (calixarenes) to screen suitable artificial acceptor-fluorescent dye F1 host-guest pairs, specific recognition of substrates and products can be achieved, resulting in detectable differences in fluorescence signals. This method transfers the enzyme monitoring process from cuvettes to microplate readers equipped with well plates, establishing a high-throughput screening method for taxadiene enzymes and providing strong support for the biosynthesis and production of paclitaxel.

[0051] This invention selects CAC4A and QAAC4A-12C as artificial macrocyclic acceptors because they possess specific structures and properties, enabling them to form stable host-guest pairs with specific fluorescent dyes. The fluorescent dye selected by CAC4A is Rhodamine B (RhB), and the dye selected by QAAC4A-12C is fluorescein (Fl). The fluorescence of these dyes themselves is not affected by the presence of the substrate GGPP and the product TAX, while their corresponding artificial acceptors can effectively quench the fluorescence of the encapsulated dye through a photoinduced electron transfer mechanism. This characteristic allows them to produce significant differences in fluorescence signals during the enzymatic reaction, thereby enabling the monitoring of taxadienase reactions.

[0052] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and incorporate common knowledge or customary techniques in the art disclosed herein. The specification and examples are to be considered exemplary only, and the true scope of this application is indicated by the claims.

Claims

1. A method for real-time fluorescence monitoring of taxadienase reaction, characterized in that... Includes the following steps: Prepare buffer solutions and standard solutions to be titrated; Add taxadienase stock solution to the standard solution to be titrated and perform titration experiment, then record the fluorescence intensity curve. The sample was diluted, and the concentration of taxadienase in the solution was determined based on the fluorescence intensity curve.

2. The method according to claim 1, characterized in that, This includes using artificial macrocyclic receptors (CAC4A or QAAC4A-12C) and fluorescent dyes as host-guest pairs to achieve specific recognition of substrates and products; the artificial macrocyclic receptors are CAC4A or QAAC4A-12C, and the fluorescent dyes are RhB or Fl.

3. The method according to claim 1, characterized in that, The steps for preparing the buffer solution and the standard solution to be titrated include: Prepare HEPES buffer solution; Prepare the standard solution to be titrated.

4. The method according to claim 3, characterized in that, At 25°C, using HEPES buffer solution as a solvent, a concentrated solution of fluorescent dye prepared with this buffer solution was added, followed by a concentrated solution of artificial macrocyclic acceptor prepared with HEPES buffer solution, to obtain the standard solution to be titrated.

5. The method according to claim 3, characterized in that, Before, during, or after the preparation of the buffer solution and the standard solution to be titrated, the following steps are included: preparing a taxadienease stock solution by weighing a fixed weight of taxadienease and preparing a taxadienease stock solution with a fixed molar concentration using DMSO.

6. The method according to claim 1, characterized in that, The steps include adding taxadienase stock solution to the standard solution to be titrated, performing a titration experiment, and recording the fluorescence intensity curve. Add taxadienease stock solution; Record fluorescence intensity; The fluorescence intensity curve has been plotted.

7. The method according to claim 6, characterized in that, The steps include adding taxadienease stock solution to the standard solution to be titrated, performing a titration experiment, and recording the fluorescence intensity curve. Further steps include: Taxadienease stock solution was gradually added dropwise to the standard solution to be titrated. The fluorescence intensity was measured at different concentrations of taxadienase, and the ratio of the fluorescence intensity to the reference fluorescence intensity was calculated to determine the relative fluorescence intensity. Fluorescence intensity curves of different relative fluorescence intensities and different concentrations of taxadienase were plotted.

8. The method according to claim 1, characterized in that, The steps of diluting the test sample and determining the concentration of taxadienase in the test solution based on the fluorescence intensity curve include: Dilute the test solution; Artificial macrocyclic acceptors and fluorescent dyes were added to the test solution; Measure the fluorescence intensity of the test solution.

9. The method according to claim 8, characterized in that, The steps for diluting the test sample and determining the concentration of taxadienase in the test solution based on the fluorescence intensity curve include: diluting the test solution with HEPES buffer; adding a concentrated solution of fluorescent dye prepared with HEPES buffer and a concentrated solution of fluorescent dye to the diluted test solution; detecting the fluorescence intensity of the test sample, calculating the relative fluorescence intensity, and comparing it with the fluorescence intensity curve to determine the concentration of taxadienase in the sample.

10. The method according to any one of claims 1-9, characterized in that, The progress of the taxadienase reaction is monitored in real time by monitoring the concentration of the taxadienase in the test solution.

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