WO2018227665A1 - Methods for extracting and purifying nostoc sphaeroides kutzing phycobiliprotein, and purified phycocyanin - Google Patents

Abstract

Provided is a method for purifying a Nostoc sphaeroides Kutzing phycocyanin, the method comprising the following steps: (1) selecting an anion exchange column for being passed through ; (2) formulating a Tris-HCl buffer solution, i.e., buffer A, and a Tris-HCl-NaCl buffer solution, i.e., buffer B; (3) dissolving a coarsely extracted Nostoc sphaeroides Kutzing phycobiliprotein powder with buffer A, and subjecting same to centrifugation and filtration; (4) washing a pump, and balancing the ion exchange column with the buffer A buffer solution; (5) directly loading the Nostoc sphaeroides Kutzing phycobiliprotein dissolved in buffer A and prepared in step (3), carrying out gradient elution with buffer B, and collecting an eluent; (6) cleaning the ion exchange column with a salt solution, and then reversely rinsing same with a base solution; and (7) subjecting the eluent to ultrafiltration concentration and dialysis to obtain a purified Nostoc sphaeroides Kutzing phycocyanin aqueous solution.

Description

Method for extracting and purifying phycobiliprotein of genus genus and phycocyanin Technical field

The present invention relates to a method for efficiently extracting and purifying phycobiliproteins and purified phycocyanin.

Background technique

Nostoc sphaeroids Kutzing, the scientific name of the genus Saccharomyces cerevisiae, belongs to the genus Nostocaceae (Nostoc.), which belongs to the genus of the genus. It is called the celestial rice and the celestial genus. It is also known as the water fungus, the wormwood, and the earth. Novoca commune vauch is a precious and edible nitrogen-and-nitrogen algae that is traditionally exported in China. It is a multicellular filamentous plant with a simple cell structure, a single filament of undivided individuals composed of round spherical cells, a filamentous body in the shape of a bead, a group of colloids, spheres or other irregular shapes. , blue-green or yellow-brown, visible to the naked eye, with nitrogen-fixing capacity. "Compendium of Materia Medica", "Pharmaceutical Test", "National Herbal Medicine Compilation", etc., said that the effect of Ge Xianmi is for eyesight, qi, enthusiasm, gastrointestinal, bonfire, long-term food Eliminate fatigue, convergence, cure night blindness, burns and other effects.

Phycobiliprotein is a light-harvesting pigment protein widely found in phycobilisomes (PBS) with red algae, blue-green algae and cryptophyta, including phycocyanin (PC) and phycoerythrimide (PE). ), phycoerythrocyanin (PEC) and allococyanin (APC). Gexian rice is rich in biliary protein and is superior to other algae. The phycocyanin is a kind of fluorochrome protein with very high added value. It can be used as a soluble antigen, an antibody detection diagnostic reagent, a fluorescent probe, a fluorescent label, etc. for immunoassay, and the higher the purity, the higher the price.

The purification of phycobiliprotein usually requires first purification, such as salting out, isoelectric point or crystallization, to remove the heteroprotein, and then purified by column chromatography, including hydroxy limestone adsorption chromatography, cellulose series ion exchange layer. Analytical, affinity chromatography and size exclusion chromatography.

In the phycobiliprotein crude powder, the amount of heteroprotein is extremely high. In order to isolate the phycocyanin from the commercial application standard, the phycobiliprotein separation and purification method has been reported, and the operation is complicated, resulting in a production cost that is too high to be prepared in large quantities to meet potential market demands. Therefore, it is necessary to develop a simple and effective method for preparing and purifying phycocyanin in large quantities, and provide a technical basis for the industrial production of phycocyanin.

Summary of the invention

It is an object of the present invention to provide a method for efficiently extracting and purifying phycocyanin and purified phycocyanin.

In order to achieve the above object, the present invention provides a method for extracting phycobiliprotein of genus, comprising the steps of: (a) adding dry powder of cemetery to water; (b) adding liquid nitrogen, continuously stirring, and volatilizing liquid nitrogen Completely; (c) centrifugal filtration to obtain a gentamicin extract; (d) freeze-drying to obtain a crude extract of genomic protein.

In the step (a), the water may be pure water, deionized water, distilled water or the like.

In the step (a), the mass ratio of the dry powder of the genus and the water is 1:40 to 1:70; preferably, 1:50, or 1:60.

In the step (b), the liquid nitrogen is added in an amount of 0.5% to 1% of the total mass of the dried starch and water.

In the step (b), the stirring time is from 4 h to 10 h; preferably, 6 h.

In the step (c), the centrifugal rotation speed is 5000 rpm-8000 rpm; the centrifugation time is 20 min-30 min; and the centrifugal filtration can be performed by a disc centrifuge.

The invention also provides a method for purifying phycocyanin, which comprises the following steps: (1) using an anion exchange column to pass the column; (2) disposing Tris-HCl buffer Buffer A, Tris-HCl-NaCl buffer Buffer B, filtration; (3) Puxian A bilirubin crude powder is dissolved with Buffer A, centrifuged, filtered; (4) wash the pump, balance the ion exchange column with Buffer A buffer; (5) step (3) The prepared phycobiliprotein dissolved in Buffer A is directly loaded, Buffer B gradient elutes, and the eluate is collected; (6) the ion exchange column is washed with a salt solution, and then washed back with an alkali solution; (7) the step The eluate of (5) is subjected to ultrafiltration concentration and dialysis to obtain a purified aqueous solution of phycocyanin; (8) The aqueous solution of phycocyanin obtained in the step (7) is stored at 0 to 10 ° C.

In the step (1), preferably, 20% of the ethanol water in the anion exchange column may be replaced with deionized water before the column is passed, and the purpose is to store the anion exchange column with 20% ethanol water, which should be used before use. It is replaced.

In the step (1), the anion exchange column is an Agarosix FF-DEAE anion exchange column; the medium of the anion exchange column is composed of an agarose gel having a particle diameter of 50 to 150 μm and a degree of crosslinking of 6%; preferably , consisting of a crosslinked agarose gel with a particle size of 90 μm and a cross-linking degree of 6%; the agarose gel relies on a secondary chain such as a hydrogen bond between sugar chains to maintain a network structure, a network structure The density depends on the concentration of agarose. In general, the structure of the agarose gel is stable and can be used under many conditions (such as water, salt solution in the range of pH 4-9); the agarose gel starts to melt above 40 °C, can not be autoclaved, available chemistry Sterilization treatment. Cross-linked agarose gel is a commonly used chromatographic matrix in biological separation. It uses different functional groups to modify the hydroxyl groups on the surface to prepare commercial fillers such as hydrophobic chromatography, ion exchange chromatography and affinity chromatography.

In the step (2), the Tris-HCl buffer has a pH of 6 to 6.8 and an electric conductivity of 4 to 8 μs/cm; preferably, the pH is 6.5 to 6.8, and the conductivity is 6 to 8 μs/cm; further preferably The pH was 6.6 and 6.8, and the electrical conductivity was 6 μs/cm and 8 μs/cm, respectively.

In step (2), the pH of the Tris-HCl-NaCl buffer is 5 to 6.8, and the conductivity is 4 to 8.5 μs/cm; preferably, the pH is 6.5 to 6.8, and the conductivity is 6 to 8 μs/cm; Further preferably, the pH is 6.5, 6.8, and the electrical conductivity is 6 μs/cm and 8 μs/cm, respectively.

In the step (2), further preferably, when the pH of the Tris-HCl buffer is 6, the conductivity is 4 μs/cm, the pH of the Tris-HCl-NaCl buffer is 6, and the conductivity is 4 μs/cm; or, Tris- When the pH of the HCl buffer is 6.5, the conductivity is 6 μs/cm, the pH of the Tris-HCl-NaCl buffer is 6.5, the conductivity is 6 μs/cm, or the conductivity of the Tris-HCl buffer is 6.8, the conductivity is 8 μs. At /cm, the Tris-HCl-NaCl buffer had a pH of 6.8 and an electrical conductivity of 8 μs/cm.

In the step (2), the method for preparing the Tris-HCl buffer is to consult the amount of Tris-HCl at different pH values, add water, and finally adjust the accurate pH value with HCl; preparation of the Tris-HCl-NaCl buffer Method for reviewing different pH values The amount of Tris-HCl, water configuration, and finally adjust the exact pH value with HCl, add NaCl, and check the conductivity until the target is met.

In the step (2), the filtration means that the solid impurities are removed by filtration under reduced pressure with a 0.2 μm filter membrane, and the pressure is reduced by a vacuum pump under reduced pressure.

In the step (3), the crude extract of the genus Phytophthora bilirubin may be prepared according to the above method; or the crude extract powder of the genus Phytophthora glutamate is composed of dry powder of pure sage and pure water (liquid to liquid ratio 1 : 60) Extraction for 6 h, filtration, and lyophilization.

In the step (3), the amount of the crude extract of the phycocyanin protein and the PB buffer is 5 g: 1 L to 15 g: 1 L; preferably, 10 g: 1 L.

In the step (3), the centrifugal rotation speed may be, but not limited to, 5000 to 10000 r/min, such as 8000 r/min.

In the step (3), the centrifugation time is 20 min to 40 min; preferably, 30 min.

In the step (3), the filtration is preferably carried out by using a 0.2 μm filter.

In the step (4), the pump is washed with deionized water for 1 to 1.5 column volumes; preferably, 1 column volume is washed with deionized water.

In the step (4), the equilibration process uses 4 to 6 column volumes of Tris-HCl buffer; preferably, 5 column volumes of Tris-HCl buffer Buffer A are used.

In the step (5), the Tris-HCl buffer has a pH of 6 to 6.8 and an electric conductivity of 4 to 8 μs/cm; preferably, the pH is 6.5 to 6.8, and the conductivity is 6 to 8 μs/cm; further preferably The pH was 6.6 and 6.8, and the electrical conductivity was 6 μs/cm and 8 μs/cm, respectively.

In the step (5), the pH of the Tris-HCl-NaCl buffer is 6 to 6.8, and the conductivity is 4 to 8 μs/cm; preferably, the pH is 6.5 to 6.8, and the conductivity is 6 to 8 μs/cm; Preferably, the pH is 6.5, 6.8, and the electrical conductivity is 6 μs/cm and 8 μs/cm, respectively.

In the step (5), it is further preferred that the Tris-HCl buffer has a pH of 6, the conductivity is 4 μs/cm, the Tris-HCl-NaCl buffer has a pH of 6, and the conductivity is 4 μs/cm; or, Tris-HCl When the pH of the buffer was 6.8 and the conductivity was 8 μs/cm, the pH of the Tris-HCl-NaCl buffer was 6.8, and the conductivity was 8 μs/cm.

In the step (5), the loading amount is 2-4 column volumes; preferably, 2, 2.5, 3, 4 column volumes.

In the step (5), the collected eluate is 4 to 5 column volumes of 45 to 100% of an eluent.

In the step (6), the salt solution is preferably NaCl or KCl, and the salt solution has a concentration of 0.02 to 0.1 M; preferably, 0.05 M.

In the step (6), the salt solution is used in an amount of from 1 to 2 column volumes.

In the step (6), the alkali solution is preferably NaOH or KOH, and the concentration of the alkali solution is 0.1 to 0.5 M; preferably, 0.1 M.

In the step (6), the alkali solution is used in an amount of from 3 to 4 column volumes.

In the step (7), the ultrafiltration membrane has a pore diameter of from 1000 D to 8000 D; preferably, it is 3000 D.

In step (7), the dialysis can be carried out by any conventional dialysis method.

In the step (8), the temperature at which the aqueous solution is stored is preferably 4 °C.

Specifically, the method for purifying phycocyanin of the present invention comprises the steps of: dissolving the crude extract of phycobiliprotein of genus Phytophthora with a Tris-HCl buffer Buffer A having a pH of 6 to 6.8 and an electric conductivity of 4 to 8 μs/cm. Centrifuge at 8000 r/min for 30 min, filter with 20 μm filter; 6% cross-linked agarose gel medium column filled with deionized water, wash the pump with 1 column volume of deionized water, 5 column volume buffer The liquid Buffer A is equilibrated, and 4 column volumes are loaded. The gradient eluent of Tris-HCl-NaCl buffer Buffer B with a pH of 6 to 6.8 and a conductivity of 4 to 8 μs/cm is collected for 4 to 5 column volumes. The 3000D ultrafiltration membrane was concentrated by ultrafiltration, dialyzed, and stored in an aqueous solution at 4 °C.

Compared with the prior art, the beneficial effects of the present invention are:

i) The anion exchange column selected in the present invention, such as the Agarosix FF-DEAE anion exchange column, significantly improves the purification efficiency, eliminates the need for ammonium sulfate precipitation in the existing purification method, and greatly increases the concentration and purity of phycocyanin. The method is simple, the effect is obvious, and the processing capacity is large, which provides a basis for industrial application.

Ii) The loading conditions selected by the invention have been proved by experiments to be large, and the number of protein samples that can be purified at one time is simple, the purification method is simple, the cost is low, the purification efficiency of phycoerythrin is greatly improved, and the basis for industrialized large production is provided.

Iii) The present invention can remove most of the impurities in the phycobiliprotein crude powder, so that the extraction rate of phycocyanin is close to 100%, and the amount of loss is small.

Iv) The present invention can purify the purity of phycocyanin to over 90% or even higher.

v) The purified phycocyanin of the invention has high added value and can be applied to the fields of cosmetics, food, health food and biomedicine, and has broad development prospects.

DRAWINGS

Figure 1 shows a gel filtration chromatogram of the purified phycocyanin in Example 7. Gel filtration chromatography peaks at different times depending on the molecular weight of the protein, and the purity of each test sample is calculated by the ratio of the peak area to the total area. The relative molecular weight of phycocyanin was estimated to be 50 KD-54 KD based on the peak position.

Figure 2 shows the ultraviolet spectrum (1. phycocyanin; 2. phycocyanin standard) of the purified phycocyanin and phycocyanin reference substance in Example 7. By comparison, the peak positions of the two samples are basically the same, and there are similar conjugate structures, which are similar samples.

Figure 3 is a graph showing the fluorescence spectrum (1. phycocyanin; 2. phycocyanin standard) of the purified phycocyanin and phycocyanin reference substance in Example 7. By comparison, the peak positions of the two samples are the same, indicating that they are homogeneous samples.

Figure 4 shows a three-component SGS-PAGE analysis diagram (1: Marker (Mr is 14.3 kD, 20.1 kD, 29.0 kD, 44.3 kD, 66.4 kD, 97.2 kD from bottom to top, respectively); 2: prepared according to the above method of the present invention Phycobiliprotein crude powder; 3: phycoerythrin; 4: Example 7 purified phycocyanin) analysis of phycocyanin subunit molecular weight of about 17kD according to electrophoresis data, 18kD, 19kD.

detailed description

The present invention will be further described in detail in conjunction with the following specific embodiments and drawings. The processes, conditions, experimental methods, and the like of the present invention are generally known in the art and common general knowledge, except for the contents specifically mentioned below, and the present invention is not particularly limited. Specific techniques or conditions not specified in the examples are carried out according to the techniques or conditions described in the literature in the art, or in accordance with the product specifications. If the reagents or instruments used do not indicate the manufacturer, they are regular products that can be purchased through regular channels.

The method for detecting the concentration of phycocyanin of the present invention is Coomassie Brilliant Blue G250 staining method, and the absorbance value at a wavelength of 595 nm is measured, and a standard curve is drawn and calculated according to the method described in Li Hesheng's Principles and Techniques of Plant Physiology and Biochemistry Experiment, and the algae are purchased. The phycocyanin standard curve measured by the cyanoprotein standard was OD ₅₉₅ = 0.5919 c + 0.0062, R ² = 0.9975; the concentration was calculated as: concentration c = (OD _{595 -} 0.0062) ÷ 0.5919.

The purity detection of the phycocyanin of the present invention is detected by an ultraviolet absorption spectrometer, and the purity is calculated as:

Purity = A ₆₂₀ /A ₂₈₀ ; purity % = A ₆₂₀ / (A ₆₂₀ + A ₂₈₀ ) × 100%.

Example 1 Laboratory primordia purification of phycocyanin using a DEAE-Cellulose 52 anion exchange column

50.01 g, 50.03 g, and 49.99 g of dried cemetery powder were separately added to 2500 mL of pure water, stirred uniformly, and then added with 20 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 9.96 g, 10.02 g, and 9.98 g of crude extract of phycobiliprotein, respectively, with a pH of 7.0 and conductivity. 3.6 μs/cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; column was passed through a DEAE-52 anion exchange column: 1) The pump was washed with 1 column volume of deionized water. 2) Balance with 5 column volumes of buffer Buffer A; 3) Load 2 column volumes. A Tris-HCl-NaCl eluate with a pH of 7.0 and a conductivity of 3.6 μs/cm was collected from 45 to 100%. The results are shown in Table 1 below:

Table 1

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	0.93	4.3	81
the second time	0.93	4.3	81
the third time	0.93	4.3	81

As seen from the above Table 1, the average concentration of phycocyanin obtained by three purifications was 0.93 g/mL, and the average purity was 4.3 (81%). After three repeated experiments, the experiment was very reproducible. However, the purity and concentration of purified phycocyanin are low. It can be seen that the purification of phycocyanin by DEAE-Cellulose 52 anion exchange column is not efficient and the effect is poor.

Example 2 Purification of phycocyanin by laboratory small test using DEAE-Cpato anion exchange column

Add 50.01g, 50.03g, 49.99g dry powder of Gexianmi to 2500mL of pure water, stir evenly and add 20mL liquid Nitrogen is stirred vigorously while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 10.01 g, 10.03 g, and 9.91 g of crude extract of phycobiliprotein, respectively, with a pH of 7.0 and conductivity. 3.6 μs/cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; column was passed through a DEAE-Cpato anion exchange column: 1) The pump was washed with 1 column volume of deionized water. 2) Balance with 5 column volumes of buffer Buffer A; 3) Load 2 column volumes. A Tris-HCl-NaCl eluent with a pH of 7.0 and a conductivity of 3.6 μs/cm was collected from 45 to 100%. The results are shown in Table 2 below:

Table 2

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	1.03	4.9	83
the second time	1.03	4.9	83
the third time	1.03	4.9	83

As can be seen from Table 2 above, the three purifications gave a mean phycocyanin concentration of 1.03 g/mL and a mean purity of 4.9 (83%). After three repeated experiments, the experiment was very reproducible. Similarly, the purity and concentration of purified phycocyanin are low, and it can be seen that the purification of phycocyanin by DEAE-Cpato column is not efficient and the effect is not good.

Example 3 Purification of phycocyanin by laboratory mini-test using DEAE-Sephadex Fast Flow anion exchange column

50.01 g, 50.03 g, and 49.99 g of dried cemetery powder were separately added to 2500 mL of pure water, stirred uniformly, and then added with 20 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 10.06 g, 9.97 g, and 10.01 g of the crude extract powder of genus phycobiliprotein, respectively, with a pH of 7.0 and conductivity. 3.6 μs/cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; column was passed through a DEAE-Sephadex Fast Flow anion exchange column: 1) Deionized with 1 column volume Washing pump; 2) equilibrating with 5 column volumes of Buffer A; 3) loading 1 column volume, collecting 45-100% pH-7.0, and conducting 3.6 μs/cm Tris-HCl-NaCl elution Liquid, the results are shown in Table 3 below:

table 3

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	1.00	5.6	85
the second time	1.00	5.6	85
the third time	1.00	5.6	85

As can be seen from Table 3 above, the three purifications gave a mean phycocyanin concentration of 1.00 g/mL and an average purity of 5.6 (85%). After three repeated experiments, the experiment was very reproducible. However, the purity and concentration of purified phycocyanin are not high. It can be seen that the purification of phycocyanin by DEAE-Sephadex Fast Flow column is slightly higher than that of the above two anion exchange columns, but the efficiency is still not high and the effect is poor.

Example 4 Laboratory Auxiliary Purification of Phycocyanin Using Agarosix FF-DEAE Anion Exchange Column

50.01 g, 50.03 g, and 49.99 g of dried cemetery powder were separately added to 2500 mL of pure water, stirred uniformly, and then added with 20 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 9.98 g, 10.01 g, and 10.01 g of crude extract of genomic protein of genus Phytophthora, respectively, with a pH of 7.0 and conductivity. 3.6 μs/cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; column was passed through an Agarosix FF-DEAE anion exchange column: 1) Washed with 1 column volume of deionized water Pump; 2) equilibrate with 5 column volumes of buffer Buffer A; 3) load 2 column volumes. A Tris-HCl-NaCl eluate with a pH of 7.0 and a conductivity of 3.6 μs/cm was collected from 45 to 100%. The results are shown in Table 4 below:

Table 4

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	2.21	10.2	91.7
the second time	2.21	10.2	91.7
the third time	2.21	10.2	91.7

As can be seen from Table 4 above, the average concentration of phycocyanin obtained by three purifications was 2.21 g/mL, and the average purity was 10.2 (91.7%). After three repeated experiments, the experiment was very reproducible. In addition, the purity and concentration of phycocyanin purified by Agarosix FF-DEAE anion exchange column were significantly improved. It can be seen that the purification method of phycocyanin is suitable for Agarosix FF-DEAE anion exchange column.

Example 5 Purification of phycocyanin by laboratory test using the method of the present invention

60.05 g, 60.01 g, and 60.02 g of dried cemetery powder were separately added to 3000 mL of pure water, stirred uniformly, and then 25 mL of liquid nitrogen was added, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 12.480 g, 12.51 g, and 12.50 g of the crude extract of the phycobiliprotein, respectively. The pH is 6.0, and the conductivity is 4.0 μs. /cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; 2) Balance with 5 column volumes of buffer Buffer A; 3) Load 2.5 column volumes. The Tris-HCl-NaCl eluate with a pH of 6.0 to 100% and a conductivity of 4 μs/cm was collected. The results are shown in Table 5 below:

table 5

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	2.61	12.0	92.3
the second time	2.61	12.0	92.3
the third time	2.61	12.0	92.3

As can be seen from Table 5 above, the three purifications gave a mean phycocyanin concentration of 2.61 g/mL and an average purity of 12.0 (92.3%). After three repeated experiments, the experiment was very reproducible. In addition, the purity and concentration of phycocyanin purified by the Agarosix FF-DEAE anion exchange column were significantly improved. It can be seen that the Agarosix FF-DEAE anion exchange column is suitable for the purification of phycocyanin.

Example 6 Purification of phycocyanin by laboratory test using the method of the present invention

75.11g, 75.08g, and 75.10g of dried cemetery powder were separately added to 3750mL of pure water, stirred uniformly, and then added with 30mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 15.10 g, 15.02 g, and 15.08 g of the crude extract of the phycobiliprotein, respectively, with a pH of 6.5 and a conductivity of 6.0 μs. /cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; 2) Balance with 5 column volumes of buffer Buffer A; 3) Load 3 column volumes. A Tris-HCl-NaCl eluate with a pH of 6.5 and a conductivity of 6.0 μs/cm was collected from 45 to 100%. The results are shown in Table 6 below:

Table 6

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	3.13	14.8	93.6
the second time	3.13	14.8	93.6
the third time	3.13	14.8	93.6

As can be seen from the above Table 6, the average concentration of phycocyanin obtained by three purifications was 3.13 g/mL, and the average purity was 14.8 (93.6%). After three repeated experiments, the experiment was very reproducible. In addition, the purity and concentration of phycocyanin purified by the Agarosix FF-DEAE anion exchange column were significantly improved. It can be seen that the Agarosix FF-DEAE anion exchange column is suitable for the purification of phycocyanin.

Example 7 Purification of phycocyanin by laboratory test using the method of the present invention

100.10g, 99.98g, and 100.01g of dried cemetery powder were separately added to 5000 mL of pure water, stirred uniformly, and then added with 40 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 20.03 g, 20.01 g, and 20.05 g of the crude extract of the phycobiliprotein, respectively, with a pH of 6.8 and an electric conductivity of 8 μs/ Cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; ) equilibrate with 5 column volumes of buffer Buffer A; 3) load 4 column volumes. A Tris-HCl-NaCl eluent with a pH of 6.8 and a conductivity of 8 μs/cm was collected from 45 to 100%. The results are shown in Table 7 below:

Table 7

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	4.50	18.4	94.8

the second time	4.50	18.4	94.8
the third time	4.50	18.4	94.8

As can be seen from the above Table 7, the average concentration of phycocyanin obtained by three purifications was 4.50 g/mL, and the average purity was 18.4 (94.8%). After three repeated experiments verified. It was found that when the loading conditions were as follows: Buffer A was dissolved in Tris-HCl buffer Buffer A with a pH of 6.8 and a conductivity of 8 μs/cm, the volume of the four columns was up to 4, and the highest concentration of phycocyanin was obtained, 4.50. g/mL; at the same time, elution conditions of pH 6.8, conductivity of 8μs / cm Tris-HCl-NaCl buffer Buffer B gradient elution can obtain a purity of 18.4 phycocyanin, greatly improving the purification efficiency.

The gel filtration chromatogram of the purified phycocyanin in Example 7 is shown in FIG. Gel filtration chromatography peaks at different times depending on the molecular weight of the protein, and the purity of each test sample is calculated by the ratio of the peak area to the total area. The peak position is about 11 mL. According to the standard map, the molecular weight of about 9 mL is about 67 KD, the molecular weight of 12 mL is about 43 KD, and the relative molecular weight of phycocyanin is estimated to be 50 KD-54 KD.

The ultraviolet spectrum of the purified phycocyanin and phycocyanin reference substance in Example 7 is shown in Fig. 2. By comparison, the peak positions of the two samples were substantially coincident, and the absorption peak was 610 nm, which had the same conjugated structure and was judged to be a homogeneous sample.

The fluorescence spectrum of the purified phycocyanin and phycocyanin reference substance in Example 7 is shown in Fig. 3 (1. phycocyanin; 2. phycocyanin standard). By comparison, the characteristic peak positions of the two samples are basically the same (450 nm and 660 nm), indicating that they are homogeneous samples.

Example 8 Purification of phycocyanin by laboratory test using the method of the present invention

50.02 g, 50.01 g, and 49.99 g of dried cemetery powder were separately added to 2500 mL of pure water, stirred uniformly, and then added with 20 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and freeze-dried to obtain 9.97 g, 9.99 g, and 9.91 g of the crude extract of the phycobiliprotein, respectively. The pH is 7.5 and the conductivity is 3.9 μs. /cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; the column was passed through an Agarosix FF-DEAE anion exchange column: 1) The pump was washed with 1 column volume of deionized water; 2) Balance with 5 column volumes of buffer Buffer A; 3) Load 2 column volumes. A Tris-HCl-NaCl eluate with a pH of 7.5 and a conductivity of 3.9 μs/cm was collected from 45 to 100%. The results are shown in Table 8 below:

Table 8

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	2.01	4.7	82.5
the second time	2.01	4.7	82.5
the third time	2.01	4.7	82.5

As can be seen from the above Table 8, the average concentration of phycocyanin obtained by three purifications was 2.01 g/mL, and the average purity was 4.7 (82.5%). After three repeated experiments, the experiment was very reproducible. When the pH of the buffer exceeds 6.8, the purification effect is remarkably lowered.

Example 9 Purification of phycocyanin by laboratory test using the method of the present invention

50.01 g, 49.99 g, and 49.98 g of dried cemetery powder were separately added to 2500 mL of pure water, stirred uniformly, and then added with 20 mL of liquid nitrogen, and vigorously stirred while adding. After the liquid nitrogen is completely evaporated, stirring is continued, the ice is completely dissolved, centrifugally filtered, and lyophilized to obtain 10.01 g, 9.95 g, and 9.99 g of the crude extract of the phycobiliprotein, respectively, with a pH of 9.3 and an electric conductivity of 9 μs/ Cm Tris-HCl buffer Buffer A was dissolved, centrifuged at 8000 r/min for 30 min, filtered through a 0.2 μm filter; washed with 1 column volume of deionized water, 5 column volume buffer Buffer A equilibrated, loaded 4 For the column volume, collect 45-100% Tris-HCl-NaCl eluent with a pH of 9.3 and a conductivity of 9 μs/cm. The results are shown in Table 9 below:

Table 9

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	1.99	1.9	65.5
the second time	1.98	1.9	65.5
the third time	2.00	1.9	65.5

As seen from the above Table 9, the average concentration of phycocyanin obtained by three purifications was 1.99 g/mL, and the average purity was 1.9 (65.5%). After three repeated experiments, the experiment was very reproducible. However, its concentration and purity are significantly lower than other examples, indicating that the pH range of the loading and elution is not suitable. The optimal loading condition is that the pH of the Tris-HCl buffer Buffer A is 6-6.8, and the conductivity is 6 to 8 μs/cm, the elution condition is Tris-HCl-NaCl buffer Buffer B has a pH of 6 to 6.8 and an electric conductivity of 6 to 8 μs/cm.

Comparative example 1

10.06g, 9.99g, 10.03g of crude extract of genus phycobiliprotein, according to the literature "Tian Panpan, stepwise salting out method combined with aqueous two-phase extraction and purification of genomic phycocyanin", using step by step The phycocyanin was purified by NH ₄ SO ₄ salting out method combined with aqueous two-phase extraction. The test results are shown in Table 10 below:

Table 10

As can be seen from the above Table 10, after three repeated experiments, the average phycocyanin concentration was 1.04 g/mL, and the average purity was 6.11 (85.9%), and the concentration and purity were significantly lower than those in the examples.

The stepwise NH ₄ SO ₄ salting out method and the two-aqueous phase extraction method selected in Comparative Example 1 are complicated in operation steps, and the experimental period is long, which increases the cost of actual operation. The method of the invention can complete the purification process by one-time chromatography, and the purity and concentration of the phycocyanin purified by the method of Comparative Example 1 are far lower than the method of the invention, further verifying that the method of the invention has strong operability, simple operation and use cost. Low, the obtained phycocyanin concentration and purity are high.

Comparative example 2

Purification of algae by a one-step Macro-Prep Methyl hydrophobic chromatography method using 10.01 g, 10.03 g, and 9.98 g of crude phycobiliproteins from the genus Blue protein, the test results are shown in Table 11 below:

Table 11

	Phycocyanin concentration (g/mL)	purity	purity(%)
the first time	0.93	7.23	87.8
the second time	0.93	7.23	87.8
the third time	0.93	7.23	87.8

As can be seen from the above Table 11, after three repeated experiments, the average phycocyanin concentration was 0.93 g/mL, and the average purity was 7.23 (87.8%), and the concentration and purity were significantly lower than those of Examples 5-7.

The one-step Macro-Prep Methyl hydrophobic chromatography used in Comparative Example 2, although single-operated, has low purity and low recovery, and it is difficult to achieve the desired purity of phycocyanin. The method of the invention can complete the purification process by one DEAE chromatography, and can obtain the high-purity and high-concentration phycocyanin, and proves that the method of the invention has strong operability, simple operation and low use cost.

Comparative example 3

10.01g, 10.03g, 10.02g of crude extract of genus phycobiliprotein, according to the method described in the literature "Zhang Yunyun, isolation, purification and properties of cyanobacteria chlorophyll", using NH ₄ SO ₄ salting out, SephadexG The phycocyanin was purified by the -100 chromatography separation and purification method, and the test results are shown in Table 12 below:

Table 12

As can be seen from the above Table 12, after three repeated experiments, the average concentration of phycocyanin was 0.76 g/mL, and the average purity was 7.07 (87.6%), and the concentration and purity were significantly lower than those of Examples 5-7.

The phycocyanin was purified by the NH ₄ SO ₄ salting out-Sephadex G-100 chromatography method selected in Comparative Example 3, and the operation was relatively simple, and the purification was carried out in two steps, but the obtained phycocyanin was low in purity, only 88%. The method of the invention can complete the purification process only by one DEAE chromatography, and can obtain the high-purity and high-concentration phycocyanin, and proves that the method of the invention has strong operability, simple operation and low use cost.

As can be seen from Examples 1 to 9 and Comparative Examples 1 to 3, (1) the filler of the Agarosix FF-DEAE anion exchange column selected for the present invention is a 6% cross-linking degree agarose gel, which exhibits high load in purification. The amount (120mg/mL) and high stability (operating temperature 4～40°C, operating pressure ≦3Bar), the performance is better. The experiment proves that the method of the invention is suitable for using the resin for purifying phycocyanin, and the effect is obviously superior to other DEAE anion exchange columns (including DEAE-52 cellulose column and DEAE-Sephadex Fast Flow column); and the loss of phycoblue protein is small. The sample loading is large and the purity is high, which may be because the ion exchange column ligand of the present invention binds to cytoprotein, and does not bind to other impurity proteins, thereby increasing the adsorption amount of phycocyanin; (2) the pH value in the buffer When 6 to 6.8 (preferably 6.5 to 6.8), the purity of the phycocyanin purified by the method of the present invention is significantly higher than that of the comparative example because the salt ion inclusion in the Tris-HCl system is only in the suitable pH range. The negatively charged part of the phycocyanin is exposed, allowing phycocyanin to adsorb as much as possible on the Agarosix FF-DEAE column, while other heteroproteins are not adsorbed and are excluded from the column during loading equilibrium; Tris - HCl buffer system is relatively difficult to stabilize phycocyanin (3) The present invention can be isolated by a step Agarosix FF-DEAE to obtain high purity, high yield (large sample loading, high phycoerythrin concentration) phycocyanin, Greatly simplified Test steps.

In summary, the method of the present invention can extract phycocyanin having a purified concentration of 4.50 g/mL or more and a purity of 18.4 (purity of 94.8%) or more, and the method of the present invention has a large sample loading amount. The method is simple and the cycle time is short; at the same time, the purity of the purified phycocyanin is increased from 6.11 (purity 85.9%) reported in the literature to 18.4 (94.8%), which significantly improves the purity of phycocyanin. A large span has been achieved in the field. Therefore, the method of the invention has obvious advantages and innovations compared to other extraction processes.

The protection of the present invention is not limited to the above embodiment. Variations and advantages that may be conceived by those skilled in the art are intended to be included within the scope of the invention and the scope of the appended claims.

Claims (11)

1. A method for purifying phycocyanin, characterized in that the method comprises the following steps: (1) using an anion exchange column to pass the column; (2) configuring a Tris-HCl buffer Buffer A, Tris-HCl-NaCl Buffer B; (3) Crude powder of Phytophthora glutamate is dissolved in Buffer A, centrifuged, filtered; (4) Wash the pump, equilibrate the ion exchange column with Buffer A buffer; (5) Step (3) The prepared phycobiliprotein prepared by dissolving in Buffer A is directly loaded, the Buffer B gradient is eluted, and the eluate is collected; (6) the ion exchange column is washed with a salt solution, and then backwashed with an alkali solution; (7) The eluate of the step (5) is subjected to ultrafiltration concentration and dialysis to obtain a purified aqueous solution of phycocyanin;

   Wherein, the pH of the Tris-HCl buffer is 6-6.8, and the pH of the Tris-HCl-NaCl buffer is 6-6.8.
2. The method according to claim 1, wherein the Tris-HCl buffer has a conductivity of 4 to 8 μs/cm, and the Tris-HCl-NaCl buffer has a conductivity of 4 to 8 μs/cm.
3. The method according to claim 1, wherein the pH of the Tris-HCl buffer is 6.5 to 6.8, and the pH of the Tris-HCl-NaCl buffer is 6.5 to 6.8.
4. The method according to claim 1, wherein the Tris-HCl buffer has a pH of 6, the conductivity is 4 μs/cm, the pH of the Tris-HCl-NaCl buffer is 6, and the conductivity is 4 μs/cm; Or, when the pH of the Tris-HCl buffer is 6.5, the conductivity is 6 μs/cm, the pH of the Tris-HCl-NaCl buffer is 6.5, the conductivity is 6 μs/cm, or the conductivity of the Tris-HCl buffer is 6.8. At a rate of 8 μs/cm, the Tris-HCl-NaCl buffer had a pH of 6.8 and an electrical conductivity of 8 μs/cm.
5. The method of claim 1 wherein in step (1), the anion exchange column is an Agarosix FF-DEAE anion exchange column.
6. The method according to claim 1, wherein in the step (1), the medium of the anion exchange column is an agarose gel having a particle diameter of 50 to 150 μm and a degree of crosslinking of 6%.
7. The method of claim 1 wherein in step (1), 20% of the ethanol water in the ion exchange column medium is replaced with deionized water prior to passing the column.
8. The method according to claim 1, wherein in the step (3), the amount of the crude extract of the genus Phytophthora bilirubin and the Tris-HCl buffer is in a ratio of 5 g:1 L to 15 g:1 L.
9. The method according to claim 1, wherein in the step (4), the ultrafiltration membrane has a pore diameter of from 1000 D to 8000 D.
10. The method according to claim 1, wherein in the step (6), the concentration of the NaCl or KCl is 0.02 to 0.1 M; and the concentration of the NaOH or KOH is 0.1 to 0.5 M.
11. A genus Phytocyanin obtained by the method according to any one of claims 1 to 10.

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