Crosslinked carbonic anhydrase as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to cross-linked carbonic anhydrase as well as a preparation method and application thereof. The preparation method of the cross-linked carbonic anhydrase comprises the following steps: S1, mixing a carbonic anhydrase solution with a precipitant, and carrying out precipitation reaction to obtain a first mixed solution; the precipitant is ethanol or acetonitrile; s2, adding a cross-linking agent into the first mixed solution, mixing, and carrying out a cross-linking reaction to obtain a second mixed solution; and S3, centrifuging the second mixed solution to obtain a precipitate, namely cross-linked carbonic anhydrase. The invention solves the problems of high cost and tedious steps in the carrier immobilized enzyme technology, and the problems of easy reduction of enzyme activity and instability of the enzyme aggregate in the preparation process of the enzyme aggregate; the cross-linked carbonic anhydrase can be applied to screening and development of carbonic anhydrase inhibitors, and active ingredients in a mixed system can be rapidly and accurately captured.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a cross-linked carbonic anhydrase and a preparation method and application thereof. Background Art

[0002] Carbonic anhydrase (CA) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, a reaction that plays a key role in many physiological and pathological processes. Carbonic anhydrase inhibitors (CAIs) are used to treat various diseases such as glaucoma, retinopathy, cardiogenic edema, and acute mountain sickness. However, the current clinical use of CAIs (acetazolamide, brininamide, dorzolamide, etc.) is not selective and is prone to adverse side effects. Therefore, the screening and development of CAIs is of great significance.

[0003] Traditional Chinese medicine contains abundant active ingredients, which are important resources for CAI screening and development. The discovery of traditional Chinese medicine active ingredients is based on activity screening models, mainly cells and animals. Although this method is effective, it has problems such as long modeling time and high cost.

[0004] In recent years, the "ligand fishing" method that integrates separation and analysis has been widely used in the screening of effective substances in traditional Chinese medicine. Ligand fishing is a method that identifies interacting ligands and receptors based on intermolecular affinity, and then combines it with modern analytical methods to screen and determine the bioactive molecules bound to proteins. Common ligand fishing methods include affinity ultrafiltration, affinity magnetic method, cell membrane chromatography, bioreactor method, etc. The above-mentioned ligand fishing method requires the use of a carrier to immobilize the enzyme. The carrier used is, for example, magnetic nanoparticles, carbon nanotubes, hollow fibers, etc. For example, a Chinese patent with publication number CN104404024B discloses an immobilized carbonic anhydrase and a preparation method thereof, wherein the immobilized carbonic anhydrase includes magnetic nanoparticles, an aldehyde-rich composite material, and carbonic anhydrase; the aldehyde-rich composite material is wrapped on the surface of the magnetic nanoparticles to form an aldehyde-rich shell, and the surface aldehyde group of the aldehyde-rich shell is covalently bonded to the carbonic anhydrase. Although these carriers are helpful for the separation and reuse of enzymes, the introduction of such inactive carriers often leads to higher costs, decreased enzyme activity, and additional physical and chemical modification steps.

[0005] The carrier-free immobilization technology, which forms enzyme aggregates and uses cross-linking agents to achieve immobilization between enzyme aggregates, is expected to become an alternative carrier immobilization technology to achieve ligand fishing due to its advantages of low cost and high enzyme activity. However, how to maintain the activity of the enzyme and the stability of the enzyme aggregates during the preparation of the enzyme aggregates is a technical difficulty. At the same time, cross-linked carbonic anhydrase and cross-linked carbonic anhydrase-based use for screening and development of CAI have not been reported in the prior art. Summary of the invention

[0006] In view of the problems of high cost and complicated steps in the prior art of carrier-immobilized enzyme technology, easy decrease in enzyme activity during the preparation of enzyme aggregates, and instability of enzyme aggregates, the present invention provides a cross-linked carbonic anhydrase and a preparation method and application thereof. Experimental verification shows that the cross-linked carbonic anhydrase of the present invention can be used in the screening and development of carbonic anhydrase inhibitors, and can quickly and accurately capture the active ingredients of traditional Chinese medicine, greatly shortening the cycle of discovery of traditional Chinese medicine effective substances. At the same time, the cost is low, which lowers the threshold for use of the ligand fishing method.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a method for preparing a cross-linked carbonic anhydrase, comprising the following steps:

[0009] S1, mixing the carbonic anhydrase solution and the precipitant to perform a precipitation reaction to obtain a first mixed solution; the precipitant is ethanol or acetonitrile;

[0010] S2, adding a cross-linking agent to the first mixed solution and mixing, performing a cross-linking reaction, to obtain a second mixed solution;

[0011] S3. The second mixed solution is used to obtain a precipitate, which is the cross-linked carbonic anhydrase.

[0012] Preferably, the preparation method of the carbonic anhydrase solution in step S1 is: dissolving carbonic anhydrase in a buffer solution, and the concentration of the carbonic anhydrase solution is 0.1-10 mg / mL, preferably 1 mg / mL.

[0013] Preferably, in step S1, the volume ratio of the carbonic anhydrase solution to the precipitant is 1-1000 μL:90-9000 μL.

[0014] Preferably, in step S1, the volume ratio of the carbonic anhydrase solution to the precipitant is 100 μL:900 μL.

[0015] Preferably, the precipitant in step S1 is ethanol.

[0016] Preferably, the conditions of the precipitation reaction in step S1 are: temperature of 2-10° C. and time of 0.5-10 h.

[0017] Preferably, the conditions of the precipitation reaction in step S1 are: temperature of 6° C. and time of 2 h.

[0018] Preferably, the cross-linking agent in step S2 is glutaraldehyde, and the final concentration of the glutaraldehyde is 1-6 nM.

[0019] Preferably, the final concentration of glutaraldehyde in step S2 is 2-4 nM.

[0020] Preferably, the conditions of the cross-linking reaction in step S2 are: temperature of 5-45° C. and time of 1-3 h.

[0021] Preferably, the conditions of the cross-linking reaction in step S2 are: temperature of 20-30° C. and time of 2-2.5 h.

[0022] Preferably, the conditions of the cross-linking reaction in step S2 are: temperature of 25° C. and time of 2.5 h.

[0023] Preferably, the centrifugal conditions in step S3 are: centrifugal force of 1000-14000×g, time of 5-

[0024] 30min.

[0025] Preferably, the centrifugal conditions in step S3 are: centrifugal force of 12000×g, time of 10 min.

[0026] Preferably, step S3 further comprises washing the precipitate with a buffer solution, the number of washing times being 1-3 times.

[0027] Preferably, the buffer is 3-morpholinepropanesulfonic acid (MOPS).

[0028] The second aspect of the present invention provides a cross-linked carbonic anhydrase obtained by the above preparation method.

[0029] The third aspect of the present invention provides a use of the cross-linked carbonic anhydrase obtained by the above preparation method in the screening of carbonic anhydrase inhibitors.

[0030] Preferably, the application is to screen carbonic anhydrase inhibitors from a traditional Chinese medicine extract, and the traditional Chinese medicine extract is salvianolic acid.

[0031] The fourth aspect of the present invention provides a method for screening carbonic anhydrase inhibitors from salvianolic acid, comprising the following steps:

[0032] Step 1: Prepare sample eluent:

[0033] Step 1.1, mixing 0.1 mg of the cross-linked carbonic anhydrase obtained by the above preparation method with the salvianolic acid solution, and incubating the mixture to obtain an incubation solution;

[0034] Step 1.2, centrifuging the incubation solution to obtain a precipitate, and washing the precipitate to obtain a washed precipitate;

[0035] Step 1.3, adding a desorption solution to the washed precipitate for desorption, oscillation, and centrifugation, and collecting the supernatant as the sample eluent;

[0036] Step 2: Prepare the negative control group eluate:

[0037] Step 2.1, 0.1 mg of cross-linked carbonic anhydrase is placed in an oil bath and heated to inactivate it, and the inactivated cross-linked carbonic anhydrase is mixed with the salvianolic acid solution and incubated to obtain a negative control group incubation solution;

[0038] Step 2.2: Repeat steps 1.2 and 1.3 for the negative control group incubation solution, and collect the supernatant as the negative control group eluate;

[0039] Step 3: Structural identification:

[0040] Step 3.1, performing HPLC-MS analysis on the sample eluate and the negative control group eluate, respectively, and comparing the peak areas of each peak in the total ion current graphs of the two groups of samples;

[0041] Step 3.2, if the difference in peak area in step 3.1 is greater than 10%, further comparing the peak area of ​​the extracted ion map corresponding to the mass-to-charge ratio shown in one of the primary mass spectra;

[0042] Step 3.3: If the peak area difference in step 3.2 is greater than 10%, further UPLC-QTOF analysis is performed to confirm the structure.

[0043] Preferably, the preparation method of the salvianolic acid is as follows: after crushing the salvia miltiorrhiza, adding 80% ethanol to swell, percolating, filtering and collecting the salvia miltiorrhiza extract; after the ethanol is removed by rotary evaporation of the salvia miltiorrhiza extract, hydrochloric acid is added to adjust the pH to 3.0, standing overnight, filtering to remove the filter residue, loading the filtrate on D101 macroporous adsorption resin, using 7 column volumes of pure water and 30% ethanol to remove impurities, using 6 column volumes of 50% ethanol to elute, collecting the eluate, rotary evaporation concentration, and freeze-drying to obtain the salvianolic acid.

[0044] Preferably, the salvianolic acid solution in step 1.1 and step 2.1 is a MOPS solution of salvianolic acid.

[0045] Preferably, the volume of the salvianolic acid solution in step 1.1 is 0.1-10 mL, and the volume of the salvianolic acid solution in step 2.1 is 0.1-10 mL.

[0046] Preferably, the volume of the salvianolic acid solution in step 1.1 is 1 mL, and the volume of the salvianolic acid solution in step 2.1 is 1 mL.

[0047] Preferably, the concentration of the salvianolic acid solution in step 1.1 is 0.5-20 mg / mL, and the concentration of the salvianolic acid solution in step 2.1 is 0.5-10 mg / mL.

[0048] Preferably, the concentration of the salvianolic acid solution in step 1.1 is 10 mg / mL, and the concentration of the salvianolic acid solution in step 2.1 is 10 mg / mL.

[0049] Preferably, the incubation conditions in step 1.1 and step 2.1 are: incubation temperature is 15-45° C., and incubation time is 0.5-5 h.

[0050] Preferably, the incubation conditions in step 1.1 and step 2.1 are: the incubation temperature is 40° C., and the incubation time is 1 h.

[0051] Preferably, in step 1.2, the precipitate is washed with a buffer solution for 1-3 times.

[0052] Preferably, the desorption liquid in step 1.3 is methanol, and the volume of the desorption liquid is 50-5000 μL, preferably 400 μL.

[0053] Preferably, the desorption time in step 1.3 is 0.5-5 h, preferably 1 h.

[0054] Preferably, the centrifugation time in step 1.3 is 5-30 min.

[0055] Preferably, the heating conditions in step 2.1 are: heating temperature is 90-150° C., and heating time is 0.5-5 h.

[0056] Preferably, the heating conditions in step 2.1 are: heating temperature is 120° C., and heating time is 2 h.

[0057] Preferably, the conditions of HPLC-MS in step 3.1 are:

[0058] Chromatographic conditions:

[0059] Chromatographic column: Acquity UPLC HSS T3 column (2.1×100 mm, 1.8 μm);

[0060] Mobile phase: Mobile phase A is 0.05-0.15% formic acid in water, and mobile phase B is acetonitrile;

[0061] Elution method: gradient elution, the gradient elution process is set as shown in Table 1 below:

[0062] Table 1

[0063] Time (min) Mobile phase A (%) Mobile phase B (%) 0-10 95-85 5-15 10-20 85-77 15-23 20-25 77-60 23-40 25-35 60-45 40-55 35-45 45-40 55-60 45-55 40-30 60-70 55-60 30-0 70-100

[0064] Flow rate: 0.2-0.4 mL / min;

[0065] Column temperature: 30-40°C;

[0066] Injection volume: 4-6 μL.

[0067] Mass spectrometry conditions:

[0068] Ion source: ESI ion source, negative ion mode scanning, ion scanning range is m / z 100-1000;

[0069] Fragmentation voltage: step-up, 75m / z-90V, 250m / z-100V, 450m / z-110V, 750m / z-120V, 1000m / z-130V;

[0070] Scan / dwell time: 563ms;

[0071] Dryer flow rate: 11.0L / min, dryer temperature: 325℃;

[0072] Atomizing gas pressure: 50.0psi;

[0073] Capillary voltage: 3.5 kV (positive ion mode), -3.5 kV (negative ion mode);

[0074] Capillary current: 5 nA, chamber current: 0.04 μA.

[0075] Preferably, the conditions of UPLC-QTOF in step 3.3 are:

[0076] Chromatographic conditions: Same as those for HPLC-MS in step 3.1.

[0077] Mass spectrometry conditions:

[0078] Ion source: ESI ion source, scanning in negative ion mode;

[0079] Atomizing gas: Nitrogen, Curtain gas is 30psi; Gas 1 is 55psi; Gas 2 is 55psi;

[0080] ISVF is -4.5kV; CE is -10V; DP is -80V; TEM is 550℃;

[0081] Ion scan range: m / z 50-1500.

[0082] In a fifth aspect, the present invention provides a carbonic anhydrase inhibitor obtained by the above method, wherein the carbonic anhydrase inhibitor comprises one or more of lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B and salvianolic acid C.

[0083] A sixth aspect of the present invention provides a use of the carbonic anhydrase inhibitor obtained by the above method in the preparation of a drug for treating and / or preventing vascular diseases, altitude sickness, eye diseases or osteoporosis.

[0084] Preferably, the eye disease comprises ocular hypertension.

[0085] The present invention has no particular limitation on the altitude sickness, and those well known to those skilled in the art may be used, such as chronic altitude sickness, acute altitude sickness, and altitude sickness caused by increased altitude, such as headache, dizziness, nausea, drowsiness, shortness of breath, palpitations, vomiting, fatigue, insomnia, numbness of hands and feet, or arrhythmia.

[0086] The present invention has no particular limitation on the ocular hypertension, which can be any disease well known to those skilled in the art, such as glaucoma, cataract or macular edema.

[0087] The medicine for treating and / or preventing vascular diseases, altitude sickness, eye diseases or osteoporosis comprises salvianolic acid C and at least one pharmaceutical excipient.

[0088] The pharmaceutical excipients mentioned above refer to conventional pharmaceutical excipients in the pharmaceutical field, including fillers, adhesives, wetting agents, absorption enhancers, surfactants, flavoring agents or sweeteners, etc.

[0089] The filler can be starch, sucrose or microcrystalline cellulose; the binder can be starch slurry, hydroxypropyl cellulose, gelatin or polyethylene glycol; the wetting agent can be magnesium stearate, micropowder silica gel or polyethylene glycol; the absorption enhancer can be polysorbate or lecithin; the surfactant can be poloxamer, fatty acid sorbitan or polysorbate.

[0090] The dosage forms of the drug for treating and / or preventing vascular diseases, the drug for treating and / or preventing altitude sickness, the drug for treating and / or preventing ocular hypertension and eye diseases caused by it, and the drug for treating and / or preventing osteoporosis and its complications can all be tablets, pills, powders, dispersible tablets, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft capsules, hard capsules, sterile injections, liniments or suppositories; and can all be made into conventional, immediate-release, sustained-release or delayed-release preparations.

[0091] The drugs for treating and / or preventing vascular diseases, drugs for treating and / or preventing altitude sickness, drugs for treating and / or preventing ocular hypertension and eye diseases caused by it, and drugs for treating and / or preventing osteoporosis and its complications can also be administered through various routes, including oral, nasal, intramuscular, subcutaneous or intravenous injections.

[0092] Compared with the prior art, the present invention has the following beneficial effects:

[0093] 1. Compared with the carrier-based immobilized enzyme technology, the cross-linked carbonic anhydrase provided by the present invention has a simple preparation method, low cost, good stability and high enzyme activity.

[0094] 2. The method for screening carbonic anhydrase inhibitors from salvianolic acid based on cross-linked carbonic anhydrase provided by the present invention can quickly and accurately capture the active ingredients in the salvianolic acid component that can inhibit carbonic anhydrase, and identify them with the assistance of LC-MS technology, which greatly shortens the discovery cycle of carbonic anhydrase inhibitors in active ingredients of traditional Chinese medicine and lowers the threshold for using ligand fishing methods.

[0095] 3. The present invention provides a method for screening carbonic anhydrase inhibitors from salvianolic acid based on cross-linked carbonic anhydrase, and for the first time discovered that salvianolic acid C can inhibit carbonic anhydrase, providing a basis for the development of new carbonic anhydrase inhibitory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 It is the relative activity of the cross-linked carbonic anhydrase of Example 1, Example 2, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, Comparative Example 1-4 and Comparative Example 1-5.

[0097] Figure 2 The relative activities of the cross-linked carbonic anhydrases of Example 1, Example 3, Comparative Example 2-1 and Comparative Example 2-2, wherein a represents Example 3, b represents Example 1, c represents Example 4, and d represents Comparative Example 2.

[0098] Figure 3 The relative activities of the cross-linked carbonic anhydrases of Example 1, Example 5, Comparative Example 3-1 and Comparative Example 3-2. In the figure, a represents Comparative Example 3-1, b represents Example 5, c represents Example 1, and d represents Comparative Example 3-2.

[0099] Figure 4 The relative activity of the cross-linked carbonic anhydrase was obtained by adopting the preparation method of Example 1 and only changing the relative centrifugal force in step S3.

[0100] Figure 5 The total ion current diagrams of the eluates after incubation of acetazolamide with active cross-linked carbonic anhydrase and inactivated cross-linked carbonic anhydrase are compared. In the figure, a represents the signal of the desorption solution after incubation with active cross-linked carbonic anhydrase, and b represents the signal of the desorption solution after incubation with inactivated cross-linked carbonic anhydrase.

[0101] Figure 6 The total ion current diagram of the eluate after incubation of salvianolic acid with active cross-linked carbonic anhydrase and inactivated cross-linked carbonic anhydrase is compared. In the figure, a represents the signal of the desorption solution after incubation with active cross-linked carbonic anhydrase, and b represents the signal of the desorption solution after incubation with inactivated cross-linked carbonic anhydrase, 1-lithospermic acid, 2-rosmarinic acid, 3-salvianolic acid A, 4-salvianolic acid C, and 5-salvianolic acid B.

[0102] Figure 7The extracted ion diagram of potential carbonic anhydrase inhibitors. In the figure, A represents lithospermic acid, corresponding to Figure 6 1 in the formula, B represents rosmarinic acid, corresponding to Figure 6 2 in the above formula, C represents salvianolic acid A, corresponding to Figure 6 3 in the above formula, D represents salvianolic acid C, corresponding to Figure 6 4 in the equation, E represents salvianolic acid B, corresponding to Figure 6 In Figure 5, a represents the signal of the desorption solution after incubation with active cross-linked carbonic anhydrase, and b represents the signal of the desorption solution after incubation with inactivated cross-linked carbonic anhydrase.

[0103] Figure 8 The results of carbonic anhydrase inhibitory activity determination of potential carbonic anhydrase inhibitors. In the figure, A represents the inhibition rate of lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B and salvianolic acid C on carbonic anhydrase at 100 μM: a represents salvianolic acid A, b represents salvianolic acid C, c represents rosmarinic acid, d represents salvianolic acid B, and e represents lithospermic acid; B represents the inhibition rate of salvianolic acid A at different concentrations on carbonic anhydrase, C represents the inhibition rate of salvianolic acid C at different concentrations on carbonic anhydrase, D represents the inhibition rate of rosmarinic acid at different concentrations on carbonic anhydrase, E represents the inhibition rate of salvianolic acid B at different concentrations on carbonic anhydrase, and F represents the inhibition rate of lithospermic acid at different concentrations on carbonic anhydrase. DETAILED DESCRIPTION

[0104] The present invention is further explained below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0105] In the following examples, unless otherwise specified, all raw materials used were commercially available or prepared by conventional methods in the art.

[0106] Embodiment 1 A method for preparing a cross-linked carbonic anhydrase comprises the following steps:

[0107] S1, 100 μL of carbonic anhydrase solution (carbonic anhydrase is dissolved in MOPS at a concentration of 1 mg / mL) and 900 μL of ethanol are mixed, and the mixture is shaken at 6° C. for 2 h for precipitation reaction to obtain a first mixed solution;

[0108] S2, adding glutaraldehyde to the first mixed solution, mixing, the final concentration of glutaraldehyde is 2 nM, shaking at 25° C. for 2.5 h for cross-linking reaction, to obtain a second mixed solution;

[0109] S3. Centrifuge the second mixed solution at a relative centrifugal force of 12,000×g for 10 min, wash the precipitate with MOPS three times, and the precipitate at the bottom of the centrifuge tube is the cross-linked carbonic anhydrase.

[0110] Example 2

[0111] The difference from Example 1 is that ethanol is replaced by acetonitrile, and the rest are the same.

[0112] Example 3

[0113] The difference from Example 1 is that the final concentration of glutaraldehyde is 1 nM, and the rest are the same.

[0114] Example 4

[0115] The difference from Example 1 is that the final concentration of glutaraldehyde is 4 nM, and the rest are the same.

[0116] Example 5

[0117] The difference from Example 1 is that the cross-linking reaction is carried out at 25° C. with shaking for 2 h, and the rest is the same.

[0118] Comparative Example 1

[0119] The difference between Comparative Example 1-1 and Example 1 is that ethanol is replaced by sodium sulfate, and the rest are the same.

[0120] The difference between Comparative Example 1-2 and Example 1 is that ethanol is replaced by acetone, and the rest are the same.

[0121] The difference between Comparative Example 1-3 and Example 1 is that ethanol is replaced by tert-butyl alcohol, and the rest are the same.

[0122] Comparative Examples 1-4 differ from Example 1 in that ethanol is replaced by ammonium sulfate, and the rest are the same.

[0123] The difference between Comparative Example 1-5 and Example 1 is that ethanol is replaced by isopropanol, and the rest are the same.

[0124] Comparative Example 2

[0125] The difference from Example 1 is that the final concentration of glutaraldehyde is 6 nM, and the rest are the same.

[0126] Comparative Example 3

[0127] The difference between Comparative Example 3-1 and Example 1 is that the cross-linking reaction is carried out at 25° C. with shaking for 1.5 h, and the rest is the same.

[0128] The difference between Comparative Example 3-2 and Example 1 is that the cross-linking reaction is carried out at 25° C. with shaking for 3 h, and the rest is the same.

[0129] Test Case

[0130] 1. Relative activity of cross-linked carbonic anhydrase: The relative activity is obtained by comparing the free enzyme activity and the cross-linked carbonic anhydrase activity. 100 μL of free carbonic anhydrase (1 mg / mL) was mixed with 200 μL of p-nitrophenylacetate (PNPA, 6 mM) solution and incubated at 25°C for 20 minutes. The absorbance of the mixed solution at 405 nm was measured using a microplate reader to obtain the absorbance A1 of the free enzyme; 100 μL of free carbonic anhydrase solution was replaced with MOPS buffer, and the rest of the operations were the same as above to obtain the absorbance A2 of the blank control; 0.1 mg of cross-linked carbonic anhydrase was mixed with 100 μL of MOPS buffer, 200 μL of PNPA solution (6 mM) was added, and the rest of the operations were the same as above to obtain the absorbance A3 of the cross-linked carbonic anhydrase; the relative activity of the cross-linked carbonic anhydrase was calculated according to calculation formula ①.

[0131] Relative activity % = [(A3-A2 / (A1-A2)] × 100%①

[0132] The measurement results of Examples 1, 2 and Comparative Example 1 are as follows Figure 1 As shown, it can be seen that the relative activity of the cross-linked carbonic anhydrase is high only when ethanol and acetonitrile are used as precipitants.

[0133] The measurement results of Examples 1, 3-4 and Comparative Example 2 are as follows Figure 2 As shown, it can be seen that the relative activity of the cross-linked carbonic anhydrase is high only when the final concentration of glutaraldehyde in the mixed solution is 2nM-4nM.

[0134] The measurement results of Examples 1, 5 and Comparative Example 3 are as follows Figure 3 As shown, it can be seen that when the cross-linking time is 2h-2.5h, the relative activity of the cross-linked carbonic anhydrase is high.

[0135] 2. Stability of cross-linked carbonic anhydrase: The preparation method of Example 1 was adopted, and only the relative centrifugal force in step S3 was changed to evaluate the relative activity change of the cross-linked carbonic anhydrase. The results are as follows: Figure 4 As shown, it can be seen that the cross-linked carbonic anhydrase of Example 1 still maintains good activity even under higher relative centrifugal force conditions (12000×g), indicating that the cross-linked carbonic anhydrase prepared by the present invention has strong structural stability. Therefore, a larger centrifugal force can be used for preparation to reduce the loss of the cross-linked enzyme during the washing process.

[0136] Based on the results of test examples 1 and 2, it can be concluded that the cross-linked carbonic anhydrase obtained when ethanol or acetonitrile is used as the precipitant, the final concentration of glutaraldehyde is 2nM-4nM, the cross-linking time is 2h-2.5h, and the relative centrifugal force is 12000×g has the best relative activity and stability.

[0137] 3. The cross-linked carbonic anhydrase was used to affinity bind acetazolamide to verify its feasibility for fishing carbonic anhydrase inhibitors:

[0138] The following steps are involved:

[0139] Step 1: Prepare the positive control group eluate:

[0140] Step 1.1, 0.1 mg of the cross-linked carbonic anhydrase prepared from the cross-linked carbonic anhydrase of Example 1 was mixed with 1 mL of 0.5 mg / mL acetazolamide solution (MOPS solution of acetazolamide), and incubated at 40° C. for 1 h to obtain a positive control group incubation solution;

[0141] Step 1.2, centrifuge the incubation solution for 10 minutes, wash the precipitate with MOPS, repeat 3 times to obtain the precipitate;

[0142] Step 1.3, add 400 μL methanol to the precipitate for desorption, shake for 1 hour and then centrifuge, collect the supernatant as the eluate of the positive control group;

[0143] Step 2: Prepare the negative control group eluate:

[0144] Step 2.1, 0.1 mg of the cross-linked carbonic anhydrase of Example 1 was placed in an oil bath and heated at 120° C. for 2 h to inactivate it, the inactivated cross-linked carbonic anhydrase was mixed with 1 mL of 0.5 mg / mL acetazolamide solution, and incubated at 40° C. for 1 h to obtain a negative control group incubation solution;

[0145] Step 2.2: Repeat steps 1.2 and 1.3 for the negative control group incubation solution, and collect the supernatant as the negative control group eluate;

[0146] Step 3, structural identification: The eluate from the positive control group and the eluate from the negative control group were subjected to HPLC-MS analysis respectively:

[0147] The HPLC-MS conditions were:

[0148] Chromatographic conditions:

[0149] Chromatographic column: Acquity UPLC HSS T3 column (2.1×100 mm, 1.8 μm);

[0150] Mobile phase: Mobile phase A is 0.1% formic acid in water, and mobile phase B is acetonitrile;

[0151] Elution method: gradient elution, the gradient elution process is set as shown in Table 2 below:

[0152] Table 2

[0153] Time (min) Mobile phase A (%) Mobile phase B (%) 0-6 100-95 0-5 6-7 95-30 5-70 7-10 30-0 70-100

[0154] Flow rate: 0.3 mL / min;

[0155] Column temperature: 35°C;

[0156] Injection volume: 5 μL.

[0157] Mass spectrometry conditions:

[0158] Ion source: ESI ion source, positive ion mode scanning, ion scanning range is m / z 100-500;

[0159] Fragmentation voltage: 120V;

[0160] Scan / dwell time: 563ms;

[0161] Dryer flow rate: 11.0L / min, dryer temperature: 325℃;

[0162] Atomizing gas pressure: 50.0psi;

[0163] Capillary voltage: 3.5kV;

[0164] Capillary current: 5 nA, chamber current: 0.04 μA.

[0165] Comparing the peak areas of the extracted ion maps of m / z 223.2 of the two groups of samples, the results are as follows Figure 5 This result indicates that active cross-linked carbonic anhydrase can bind to the carbonic anhydrase-positive drug acetazolamide, verifying the feasibility of using cross-linked enzymes for ligand fishing.

[0166] Application Examples

[0167] 1. A method for screening carbonic anhydrase inhibitors from salvianolic acid, comprising the following steps:

[0168] Step 1: Prepare sample eluent:

[0169] Step 1.1, 0.1 mg of the cross-linked carbonic anhydrase of Example 1 was mixed with 1 mL of a 10 mg / mL salvianolic acid solution (MOPS solution of salvianolic acid), and the mixture was incubated at 40° C. for 1 h to obtain an incubation solution;

[0170] Step 1.2, centrifuge the incubation solution for 10 min, and wash the precipitate with MOPS solution three times to obtain a precipitate;

[0171] Step 1.3, add 400 μL methanol to the precipitate for desorption, shake for 1 h, centrifuge, and collect the supernatant as the sample eluent;

[0172] Step 2: Prepare the negative control group eluate:

[0173] Step 2.1, 0.1 mg of the cross-linked carbonic anhydrase of Example 1 was placed in an oil bath and heated at 120° C. for 2 h to inactivate it, the inactivated cross-linked carbonic anhydrase was mixed with 1 mL of a 10 mg / mL salvianolic acid solution, and incubated at 40° C. for 1 h to obtain a negative control group incubation solution;

[0174] Step 2.2: Repeat steps 1.2 and 1.3 for the negative control group incubation solution, and collect the supernatant as the negative control group eluate;

[0175] Step 3: Structural identification:

[0176] Step 3.1, the sample eluate and the negative control group eluate were subjected to HPLC-MS analysis respectively, and the conditions of the HPLC-MS were:

[0177] Chromatographic conditions:

[0178] Chromatographic column: Acquity UPLC HSS T3 column (2.1×100 mm, 1.8 μm);

[0179] Mobile phase: Mobile phase A is 0.1% formic acid in water, and mobile phase B is acetonitrile;

[0180] Elution mode: gradient elution, the gradient elution process settings are the same as Table 1;

[0181] Flow rate: 0.3 mL / min;

[0182] Column temperature: 35°C;

[0183] Injection volume: 5 μL.

[0184] Mass spectrometry conditions:

[0185] Ion source: ESI ion source, negative ion mode scanning, ion scanning range is m / z 100-1000;

[0186] Fragmentation voltage: step-up, 75m / z-90V, 250m / z-100V, 450m / z-110V, 750m / z-120V, 1000m / z-130V;

[0187] Scan / dwell time: 563ms;

[0188] Dryer flow rate: 11.0L / min, dryer temperature 325℃;

[0189] Atomizing gas pressure: 50.0psi;

[0190] Capillary voltage: 3.5 kV (positive ion mode), -3.5 kV (negative ion mode);

[0191] Capillary current: 5 nA, chamber current: 0.04 μA.

[0192] The total ion currents of the two groups of eluents are compared. Figure 6 As shown;

[0193] Step 3.2: For the peaks with a peak area change greater than 10%, the mass-to-charge ratio in the primary mass spectrum was investigated, and 5 potential carbonic anhydrase inhibitors with a corresponding extracted ion peak area change greater than 10% were screened out. The results are as follows: Figure 7 As shown;

[0194] Step 3.3, perform UPLC-QTOF analysis to confirm the structure;

[0195] The UPLC-QTOF conditions:

[0196] Chromatographic conditions: Same as those for HPLC-MS in step 3.1;

[0197] Mass spectrometry conditions:

[0198] Ion source: ESI ion source, scanning in negative ion mode;

[0199] Atomizing gas: Nitrogen, Curtain gas is 30psi; Gas 1 is 55psi; Gas 2 is 55psi;

[0200] ISVF is -4.5kV; CE is -10V; DP is -80V; TEM is 550℃;

[0201] Ion scan range: m / z 50-1500.

[0202] Five potential carbonic anhydrase inhibitors were identified, namely lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid C and salvianolic acid B.

[0203] 2. Activity verification of potential carbonic anhydrase inhibitors

[0204] Lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, and salvianolic acid C were prepared into different concentration gradients in the concentration range of 0.5-1000 μM using MOPS buffer as the test solution. 40 μL of the test solution was added to a 96-well plate and mixed with 50 μL of PNPA solution (6 mM), and then 10 μL of carbonic anhydrase solution (100 nM) was added to start the reaction. The well plate was placed in a constant temperature mixer at 37°C.

[0205] Incubate at 600 r / min for 20 min, then use an ELISA reader to detect the sample at 405 nm and record the absorbance value of each well. Calculate the inhibition rate of the test substance according to the inhibition rate calculation formula ②.

[0206] Inhibition rate % = [1-(a1-a2) / (a3-a4)] × 100%②

[0207] Wherein, a1 represents the absorbance of the well containing the analyte solution and the carbonic anhydrase solution; a2 represents the absorbance of the well containing the carbonic anhydrase solution replaced by MOPS buffer; a3 represents the absorbance of the well containing the analyte solution replaced by MOPS buffer; a4 represents the absorbance of the well containing the analyte solution and the carbonic anhydrase solution replaced by MOPS buffer. The measurement results are shown in Figure 8 .

[0208] Depend on Figure 8 It can be seen that the five potential active ingredients obtained by the method for screening carbonic anhydrase inhibitors from salvianolic acid based on cross-linked carbonic anhydrase all have carbonic anhydrase inhibitory activity, among which salvianolic acid C was found for the first time to have the effect of inhibiting carbonic anhydrase activity. These results indicate that the cross-linked carbonic anhydrase provided by the present invention can be used to screen carbonic anhydrase inhibitors from active ingredients of traditional Chinese medicine with high efficiency and accuracy.

[0209] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a cross-linked carbonic anhydrase, characterized in that: The following steps are involved: S1, mixing the carbonic anhydrase solution and the precipitant to perform a precipitation reaction to obtain a first mixed solution; the precipitant is ethanol or acetonitrile; S2, adding a cross-linking agent to the first mixed solution and mixing, performing a cross-linking reaction, to obtain a second mixed solution; S3. Centrifuge the second mixed solution to obtain a precipitate, which is the cross-linked carbonic anhydrase.

2. The preparation method according to claim 1, characterized in that: The cross-linking agent in step S2 is glutaraldehyde, and the final concentration of the glutaraldehyde is 1-6 nM; the time of the cross-linking reaction in step S2 is 1-3 h.

3. The preparation method according to claim 2, characterized in that: The cross-linking agent in step S2 is glutaraldehyde, and the final concentration of the glutaraldehyde is 2-4 nM; the time of the cross-linking reaction in step S2 is 2-2.5 h.

4. A cross-linked carbonic anhydrase obtained by the preparation method according to any one of claims 1 to 3.

5. Use of the cross-linked carbonic anhydrase obtained by the preparation method according to any one of claims 1 to 3 in screening carbonic anhydrase inhibitors.

6. The use according to claim 5, characterized in that: The application is to screen carbonic anhydrase inhibitors from traditional Chinese medicine extracts, and the traditional Chinese medicine extracts are salvianolic acid.

7. A method for screening carbonic anhydrase inhibitors from salvianolic acid, characterized in that: The following steps are involved: Step 1: Prepare sample eluent: Step 1.1, mixing and incubating the cross-linked carbonic anhydrase obtained by the preparation method according to any one of claims 1 to 3 with a salvianolic acid solution to obtain an incubation solution; Step 1.2, centrifuging the incubation solution to obtain a precipitate, and washing the precipitate to obtain a washed precipitate; Step 1.3, adding a desorption solution to the washed precipitate for desorption, oscillation, centrifugation, and collecting the supernatant as the sample eluent; Step 2: Prepare the negative control group eluate: Step 2.1, placing the cross-linked carbonic anhydrase obtained by the preparation method according to any one of claims 1 to 3 in an oil bath and heating it to inactivate it, mixing the inactivated cross-linked carbonic anhydrase with a salvianolic acid solution and incubating them to obtain a negative control group incubation solution; Step 2.2: Repeat steps 1.2 and 1.3 for the negative control group incubation solution, and collect the supernatant as the negative control group eluate; Step 3: Structural identification: Step 3.1, performing HPLC-MS analysis on the sample eluate and the negative control group eluate, respectively, and comparing the peak areas of each peak in the total ion current graphs of the two groups of samples; Step 3.2, if the difference in peak area in step 3.1 is greater than 10%, further comparing the peak area of ​​the extracted ion map corresponding to the mass-to-charge ratio shown in one of the primary mass spectra; Step 3.3: If the peak area difference in step 3.2 is greater than 10%, further UPLC-QTOF analysis is performed to confirm the structure.

8. A carbonic anhydrase inhibitor obtained according to the method of claim 7, characterized in that The carbonic anhydrase inhibitor includes one or more of lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B and salvianolic acid C.

9. Use of the carbonic anhydrase inhibitor according to claim 8 in the preparation of a medicament for treating and / or preventing vascular diseases, altitude sickness, eye diseases or osteoporosis.

10. The use according to claim 9, characterized in that: The eye disease includes ocular hypertension.

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