Use of ursolic acid as affinity resin ligand and its application in isolation and purification of phycocyanin

Abstract

The application relates to an application of ursolic acid as an affinity resin ligand and separation and purification of phycocyanin, and belongs to the field of biochemistry and development and utilization of marine product resources. The application is that ursolic acid is used as a ligand of an affinity resin for separating and purifying phycocyanin, and is covalently connected with the amino group of an amino resin through an amide bond to form an affinity resin. The application also provides a preparation method of the affinity resin. The affinity resin prepared by using the method can separate and purify a crude extract of phycocyanin to an analytical pure grade in one step.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochemistry and marine product resource development and utilization, and particularly relates to ursolic acid as an affinity resin ligand and its application in the separation and purification of phycocyanin. BACKGROUND

[0002] Affinity chromatography is a method with high selectivity and high efficiency, which can separate specific proteins with high purity from complex mixtures. In particular, for biological molecules with long separation process, low concentration, and many impurities, it is difficult to separate by conventional methods, and affinity chromatography has incomparable advantages. Small molecules and metal ions are the most widely studied fields, with low price and wide source, and have become the fastest developing and most widely used affinity materials. At present, there are various affinity filler commercial reagents on the market, which provide convenience for the wide application of affinity purification. When used, only a specific ligand small molecule needs to be connected to form a specific affinity resin for a certain type of protein.

[0003] The structure-based affinity ligand design through virtual screening and molecular docking research has become an important step in establishing affinity chromatography method. Through virtual screening, small molecule ligands that can specifically bind to the target protein can be screened from a large number of small molecule databases. Through computer simulation of molecular docking, the complex process of protein and small molecule docking can be visualized, which is convenient for research.

[0004] Phycocyanin is a light-harvesting pigment protein found in cyanobacteria and some cryptophytes. It is composed of phycobilins and apo-proteins. The maximum absorption peak of phycocyanin is at 620 nm. Phycocyanin has antioxidant, anti-inflammatory, anti-obesity and anti-cancer activities, and can also mediate photodynamic therapy of tumors, and can be used as a photosensitizer.

[0005] The commercial value of phycocyanin is directly proportional to its purity; phycocyanin with a purity of 0.7 is generally considered to be food grade, a purity of 3.0 to 3.9 is equivalent to reaction grade, and a purity of >4.0 is considered to be analytical grade. The higher the purity of phycocyanin, the higher its commercial value. Since phycocyanin and allophycocyanin coexist in spirulina, the separation of phycocyanin and allophycocyanin is a key step in the purification of phycocyanin. In order to obtain analytical grade phycocyanin, most purification methods need to be combined in multiple steps, such as ammonium sulfate precipitation combined with ion exchange chromatography, hydroxyapatite column chromatography, etc. However, these methods are tedious and have low yield of phycocyanin, which is not suitable for large-scale production. Therefore, in order to avoid time-consuming multi-step operation steps, it is necessary to find a more efficient and economical method for the purification of phycocyanin. SUMMARY

[0006] The technical problem solved by the present application is to provide an application of ursolic acid as an affinity resin ligand and its use in the separation and purification of phycocyanin, the present application screens small molecule ligands that specifically bind to phycocyanin, prepares an affinity resin, and establishes a column chromatography process for purifying phycocyanin using the same. The phycocyanin prepared by the method has a purity of 4 or more, meeting the standard of analytical grade.

[0007] The present application is realized by the following technical solutions:

[0008] The application of ursolic acid as an affinity resin ligand, wherein the application is that ursolic acid is used as a ligand of an affinity resin for the separation and purification of phycocyanin.

[0009] An affinity resin for purifying phycocyanin, wherein the affinity resin is covalently connected by an amide bond between the carboxyl group of ursolic acid and the amino group of an amino resin.

[0010] The present application also provides a preparation method of the affinity resin, wherein small molecule ursolic acid is dissolved in methanol with a concentration of not more than 10 mM, the amino resin washed with methanol is mixed with the ursolic acid solution at a volume ratio of 1:2, 1-ethyl(3-dimethylaminopropyl)-3-carbodiimide hydrochloride coupling agent powder is added to the mixed solution, the solution is shaken in the dark overnight, centrifuged to remove the supernatant, the precipitate is washed with methanol and then with pure water, and then the affinity resin is obtained by alternately washing with acid and alkali solutions and preserving with a 20% ethanol solution.

[0011] The present application also provides an application of the affinity resin, wherein the application is the separation and purification of phycocyanin using the affinity resin.

[0012] As one of the preferred embodiments, the method comprises the following specific steps:

[0013] In the first step, a crude phycocyanin solution is prepared, 20 mM sodium dihydrogen phosphate-sodium hydrogen phosphate combined buffer with a pH of 7.0 is flowed through the affinity resin chromatography column, two or more column volumes are balanced, the crude phycocyanin solution is added to the affinity resin chromatography column, the combined buffer is first used to flush and wash away the impure proteins that cannot be combined, then 20 mM acetic acid-sodium acetate elution buffer containing 50 mM NaCl with a pH of 4.0 is added, the elution is performed at a flow rate of 1 ml / min, the eluate is collected according to the color change to blue or the formation of an absorption peak at 620 nm, and the purity of the phycocyanin is determined by calculating the ratio of the 620 nm and 280 nm absorbance values of the phycocyanin collection solution;

[0014] Second step, regeneration of affinity resin, after collecting the phycocyanin elution peak, the affinity resin is washed with binding buffer until the effluent pH is neutral, the resin is washed with binding buffer containing 1M NaCl to remove the bound impurities, and the washing is continued until the effluent conductivity is stable, and then the next phycocyanin crude extract purification can be carried out;

[0015] Third step, complete washing of the affinity resin; before long-term preservation of the affinity resin, the resin is washed with 3M potassium thiocyanate solution at a flow rate of 1ml / min to completely wash the bound proteins, and the washing is continued until the protein detection baseline (280nm absorbance value) is stable, then the resin is washed with pure water for two to three column volumes, washed with 20% ethanol, and preserved at 4℃.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The present application provides an affinity resin ligand and an affinity resin for separating and purifying phycocyanin, and the affinity resin and separation and purification method prepared by the method of the present application can separate and purify the phycocyanin crude extract to an analytical pure level through only one step of chromatography. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Visualization of the docking force of ursolic acid and phycocyanin;

[0019] Figure 2 Visualization of the docking force of ursolic acid and allophycocyanin;

[0020] Figure 3 Chromatogram of eluting phycocyanin with pH 7.0 binding buffer and 50mM pH 4.0 (acetic acid-sodium acetate) elution buffer. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are further illustrated by the following examples, but the protection scope of the present application is not limited to the following examples.

[0022] Ursolic acid (Ursolic Acid, CAS: 77-52-1) or English name: (3beta)-3-hydroxyurs-12-en-28-oic acid.

[0023] Example 1: Screening method of affinity ligand: screening of affinity resin ligand, which is a small molecule that can specifically bind to phycocyanin and allophycocyanin and has the largest difference in docking binding energy with phycocyanin and allophycocyanin through computer simulation virtual screening. The computer virtual screening technology is used to perform preliminary screening from the ZINC 15 database. The screening criteria are: 3D / Standard / In-Stock / Mid / 0, a total of 663041 small molecules. The obtained small molecules are downloaded to establish a molecular library. Further screening of the database. The polymer three-dimensional structure of Spirulina platensis phycocyanin (1HA7) and allophycocyanin (4F0U) is obtained from the RCSB protein database (www.rcsb.org). The AutoDock Tools1.5.7 software is used to remove ligands and water molecules. By retaining the alpha, beta subunit and phycobilin as a monomer, two protein monomer models are obtained, and the docking site is determined. The SailVina-AutodockVina software is used for virtual docking of the two monomer proteins with the small molecule database, and the obtained binding energy scores are sorted. The screening obtains ursolic acid that can specifically bind to phycocyanin and allophycocyanin, and has the largest difference in docking binding energy with phycocyanin and allophycocyanin, and is more likely to distinguish phycocyanin and allophycocyanin and other impurities. Finally, the PyMOL software is used to visualize the docking mode, as shown in Figure 1 、 Figure 2

[0024] Example 2: Preparation method of affinity resin: Take 5ml Bio-rad Affi-gel 102 amino agarose resin (containing 75μmol amino) in a 50ml centrifuge tube, wash with 10ml analytical pure methanol for 5 times, and remove the supernatant. Add 10ml 10mM ursolic acid solution (containing ursolic acid 100μmol) and 0.0144g 1-ethyl(3-dimethylaminopropyl)-3-carbodiimide hydrochloride (75μmol). Use glass slow stirring, avoid light, 22℃ shaking bed 150rpm shaking, overnight reaction 16h. After the reaction is completed, it is placed in a gravity column, washed with methanol for 3 times, and the unbound ursolic acid is washed away, and then washed with 0.1M pH 4.0 acetate buffer containing 0.5M NaCl and pH 8.0 Tris-HCl buffer alternately for three times. Then wash the ursolic acid affinity resin with pure water, and store it in 20% ethanol solution at 4℃ in the dark.

[0025] ​Example 3C - Preparation of crude phycocyanin: The frozen Arthrospira platensis paste was thawed at room temperature. The paste was centrifuged at 10,000 rpm for 20 min at low temperature using a Xiangyi H2050 centrifuge. The supernatant was removed and the paste was collected. The paste was mixed with 10 ml of 10 mM phosphate buffer at pH 7.0 at a ratio of 1 g of paste to 10 ml of buffer. The mixture was repeatedly frozen and thawed twice for 2 h each time between -20 °C and room temperature, and then was stored at -20 °C overnight. The next day, the mixture was stored at 4 °C for 24 h. The mixture was centrifuged at 10,000 rpm for 20 min at low temperature. The supernatant was collected and was crude phycocyanin with a purity of 1.28 and a content of 3.2 mg / ml. The crude phycocyanin was stored at 4 °C.

[0026] Example 4 - Purification of crude phycocyanin by gravity column chromatography: 5 mL of ursolic acid affinity resin was loaded into a gravity flow plastic chromatography column. The column was first washed with 40 ml of water until the liquid level was 0.5 cm above the affinity resin. Then, 20 mL of 20 mM NaH2P04-Na2HP04 buffer at pH 7.0 (hereinafter referred to as "A solution") was added to wash and balance the column. 10 ml of the A solution and 5 ml of crude phycocyanin at a concentration of 2 mg / ml were added. Then, 30 ml of the A solution was added to wash and balance the column. Next, 40 mL of 20 mM acetic acid-NaOAc buffer at pH 4.0 was added to elute the C-phycocyanin. When blue solution flowed out, it was collected in a 10 mL centrifuge tube. When the eluate was no longer blue, the affinity resin was washed with 20 mL of the A solution containing 1 M NaCl, and then was washed with 80 ml of water for the next round of purification. If the column was not used immediately, it was washed with 40 mL of 20% ethanol, the liquid level was lowered to 2 cm above the affinity resin, and the outlet was closed. The column was stored in a refrigerator at 4 °C.

[0027] The phycocyanin collection solution was concentrated using an Amicon Ultra-15 centrifugal concentration tube (molecular weight cut-off of 3 kDa) from Merck. The purity of the phycocyanin in the collection solution was measured to be 4.0, which met the purity requirement for analytical grade phycocyanin.

[0028] Example 5 Affinity resin column purification process of crude phycocyanin: 5 ml of ursolic acid affinity resin was loaded into an XK16 / 20 empty column of GE Healthcare, and the column was connected to an AKTA explorer 10S protein fast purification process development system of GE Healthcare. The absorbance values at three wavelengths of 280 nm, 620 nm and 650 nm were detected. Two to three column volumes of pure water were first used to flush at a flow rate of 1 ml / min, then two to three column volumes of 20 mM pH 7.0 sodium dihydrogen phosphate-sodium hydrogen phosphate buffer (hereinafter referred to as A solution) were used to flush to pH equilibrium, and 5 ml of 2 mg / ml C-phycocyanin crude extract filtered by a 0.22 μm filter was added. The A solution was continuously used to flush to wash away the impurities that could not be combined, and when the protein detection baseline (absorbance value at 280 nm) was stable, 20 mM pH 4.0 acetic acid-sodium acetate elution buffer containing 50 mM NaCl was used to elute at a flow rate of 1 ml / min. According to the color change to blue or the formation of an absorption peak at 620 nm, the eluate was collected. The ratio of the absorbance values at 620 nm and 280 nm of the collected solution containing phycocyanin was calculated to determine the purity of phycocyanin. After collecting the phycocyanin elution peak, the affinity resin was flushed with the binding buffer until the effluent pH was neutral. The resin was washed with the binding buffer containing 1 M NaCl to wash away the combined impurities, and the flushing was continued until the effluent conductivity was stable, and the next phycocyanin purification could be carried out. If not used temporarily, the resin was flushed with two to three column volumes of pure water and then with 20% ethanol, and stored at 4°C. The purity of C-phycocyanin in the collected solution was measured to be 4.53, which exceeded the purity requirement of analytical grade phycocyanin. The chromatogram at 280 nm during the elution process is shown in Figure 6. Figure 3

[0029] Before long-term storage of the affinity resin, the resin was washed with a 3M potassium thiocyanate solution at a flow rate of 1 ml / min to completely wash away the proteins combined on the resin. The flushing was continued until the protein detection baseline (absorbance value at 280 nm) was stable, and then the resin was flushed with two to three column volumes of pure water and then with 20% ethanol, and stored at 4°C.​

Claims

1. Use of ursolic acid as a ligand for affinity resins, characterized in that, The application is that ursolic acid is used as a ligand for separating and purifying phycocyanin affinity resin.

2. An affinity resin for purifying phycocyanin, characterized in that, The affinity resin is obtained by covalently connecting the carboxyl of ursolic acid and the amino of an amino resin through an amide bond.

3. Process for the preparation of the affinity resin according to claim 2, characterized in that, A small molecule ursolic acid is dissolved in methanol with a concentration not exceeding 10 mM, the amino resin washed with methanol is mixed with the ursolic acid solution at a volume ratio of 1:2, 1-ethyl(3-dimethylaminopropyl)-3-carbodiimide hydrochloride coupling agent powder is added to the mixed solution, and the mixture is shaken in the dark overnight, centrifuged to remove the supernatant, and the precipitate is washed with methanol multiple times, then washed with pure water, and then washed with acid and alkali solutions multiple times to obtain the affinity resin, which is stored at 4°C with 20% ethanol solution.

4. Use of the affinity resin according to claim 2, characterized in that, The application is that the affinity resin of claim 2 is used for separating and purifying phycocyanin.

5. Use according to claim 4, characterized in that, The application method comprises the following specific steps: preparing a phycocyanin crude extract, flowing through an affinity resin chromatographic column with a 20 mM sodium dihydrogen phosphate-sodium hydrogen phosphate binding buffer at pH 7.0, equilibrating two or more column volumes, adding the phycocyanin crude extract to the affinity resin chromatographic column, first washing with the binding buffer to wash away the impure proteins that cannot be combined, then adding an elution buffer containing 50 mM NaCl and 20 mM acetic acid-sodium acetate at pH 4.0, eluting at a flow rate of 1 ml / min, collecting the eluate according to the color change to blue or the formation of an absorption peak at 620 nm, calculating the ratio of the 620 nm and 280 nm absorbance values of the phycocyanin collection to determine the purity of the phycocyanin.

6. Use according to claim 5, characterized in that, After collecting the phycocyanin elution peak, the affinity resin is washed with the binding buffer until the effluent pH is neutral, the resin is washed with the binding buffer containing 1 M NaCl to wash away the combined impure proteins, and the washing is continued until the effluent conductivity is stable, and the next phycocyanin crude extract purification can be performed.

7. Use according to claim 5, characterized in that, Before long-term storage of the affinity resin, the resin is washed with a 3 M potassium thiocyanate solution at a flow rate of 1 ml / min to completely wash away the proteins combined on the resin, and then the resin is washed with pure water for two to three column volumes and then with 20% ethanol, and stored at 4°C.

Images