CN113292648A - Method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae and phycobiliprotein - Google Patents
Method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae and phycobiliprotein Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae, which comprises the following steps: inoculating a nitrogen-fixing blue algae strain into a BG11 culture medium, performing illumination culture, centrifuging to collect algae liquid, performing secondary centrifugation, and removing supernatant to obtain an algae body; then, the algae body is resuspended in PBS buffer solution to obtain PBS algae body weight suspension; then repeatedly freezing and thawing PBS algae heavy suspension, centrifuging to obtain crude phycobiliprotein extract; then, adding the crude phycobiliprotein extract into a two-aqueous-phase system, and primarily purifying to obtain a phycobiliprotein extract; and then, adopting a hydroxyapatite column, adopting different ion gradients to perform isocratic elution on the phycobiliprotein extracting solution, and then dialyzing to remove salt to obtain the microalgae phycobiliprotein. The invention also discloses the phycobiliprotein prepared by the method. The invention adopts the plasma intensity elution process with different concentrations, can simultaneously realize the separation and purification of high-purity phycoerythrin and phycocyanin, and has high utilization rate of raw materials.
Description
Technical Field
The invention belongs to the technical field of biochemical separation, and particularly relates to a method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae and the phycobiliprotein.
Background
Algae, a type of microorganism capable of photosynthesis, belongs to one of protists and can be classified into unicellular microalgae and macroalgae. Phycobilisomes or chromogens mainly exist in blue algae and red algae, are main light-capturing antenna compounds thereof, generally consist of a plurality of phycobiliproteins and connexins, and the phycobiliproteins are formed by covalently connecting phycobiliproteins or chromogens and corresponding apoproteins through thioether bonds. The structures of different phycobiliproteins are basically similar, apoproteins generally consist of alpha subunits and beta subunits, a small number of apoproteins contain gamma subunits, the alpha subunits and the beta subunits form a trimer or a hexamer, and the gamma subunits are mostly present in the hexamer to play a role in stabilizing the structure. The fluorescence characteristics of phycobiliproteins are mainly determined by the type, location and amount of phycobiliproteins bound to apoproteins. Phycobiliproteins can be classified into Phycocyanin (PC), Phycoerythrin (PE), and Allophycocyanin (APC) according to their absorption spectrum properties. In general, spectroscopic purity of phycobiliproteins (A)max/A280) The higher the price, the more finely divided it can be in food grade (A) according to its puritymax/A280>0.7), pharmaceutical grade (A)max/A280>3.0) and reagent grade (A)max/A280>4.0). Phycobiliprotein has the functions of oxidation resistance, inflammation resistance, aging resistance, cancer resistance, immunofluorescence and the like, is widely used as natural pigments (foods, skin care products, dyes and the like), medical and health products and fluorescent reagents in molecular biology research, and has huge potential market demands.
Nitrogen-fixing blue algae is a low-grade prokaryotic plant. It can fix nitrogen, and fix molecular nitrogen in air into nitrogen compound; can also carry out photosynthesisThe carbon dioxide is changed into carbon compound and oxygen is released, and energy and reducing agent are provided for self nitrogen fixation. Chlorophyll a contained in the cells can perform oxygen-producing photosynthesis; its heteromorphic cell (heterocyst) is the site for nitrogen fixation; the cell can secrete Extracellular Polysaccharide (EPS), which is polyanionic macromolecule capable of interacting with cation and is capable of reacting with Na+Can affect the change in external environmental pH.
The acquisition of intracellular active substances such as phycobiliprotein is mainly divided into two processes of extraction and purification. In terms of extraction, the effect of cell disruption is achieved mainly through physical treatment, and cell disruption solution, namely crude phycobiliprotein extract, is collected through centrifugation. The cell disruption methods commonly used at present include an extraction method, a glass bead grinding method, a liquid nitrogen grinding method, a homogenizer treatment method and an ultrasonic disruption method. For example: in the invention patent CN 104292316B, the R-type phycoerythrin in the asparagus is extracted by adopting a method of crushing and leaching, the method is long in time consumption, and only the leaching step needs 24-48 h. In chinese patent application CN 109206504A, phycocyanin in spirulina is extracted by a method combining mixing grinding and ultrasonic disruption, which is complex, and is not feasible in large-scale industrial production of high-purity phycobiliprotein due to the limitation of ultrasonic disruptor.
For separation and purification, the method can be generally divided into two processes, wherein the first step is pretreatment, and the substances such as heteroproteins, polysaccharides and the like with large differences in molecular weight or charge characteristics are removed by adopting a method of ammonium sulfate fractional precipitation (salting-out) and isoelectric point precipitation; the second step is separation and purification in the true sense, and according to the hydrophobic property difference, the molecular weight difference and the charge characteristic difference of the phycobiliprotein, corresponding hydroxyapatite column chromatography, agarose gel column chromatography and anion exchange column chromatography are selected for separation and purification. Further, a system of optimizing eluent can be selected to simultaneously separate and purify a plurality of phycobiliproteins, or a plurality of column chromatography combination treatments are adopted to obtain a phycobiliprotein purification solution with higher purity. For example: in patent CN106478810B, salting-out and column chromatography are combined to separate and purify reagent-level phycocyanin and reagent-level phycocyanin, the method needs to perform dialysis treatment on a sample after salting-out, and then two-time column chromatography is combined to separate and purify phycobiliprotein, so that the purification process is complex and consumes long time; in the Chinese patent application CN 106220728A, DEAE-Sepharose centrifugal exchange column purification is adopted, then the purification is further carried out through Sephacryl S-300HR column, and finally the high-purity phycobiliprotein is obtained through hydroxyapatite column chromatography and Superdex 75 column purification in sequence.
In fact, in the aspect of separation and purification of phycobiliprotein, the research of related technologies in China is late, the research foundation is weak, the problems of complex separation and purification process, high cost and low yield exist, the development and utilization force and depth are very small at present, most of the research is only limited to the development of simple food-grade products, and few reports exist on the separation and purification process of reagent grade. The complicated separation and purification process limits the application of phycobiliprotein. Therefore, it is necessary to develop a method which is simple in operation, high in purity and suitable for large-scale preparation, separation and purification of phycobiliproteins, and provide a technical basis for realizing industrial production of phycobiliproteins from marine microalgae.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provides a method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae, which is simple to operate, high in purity and suitable for large-scale production. The second purpose of the invention is to provide a phycobiliprotein.
In order to achieve the purpose, the invention adopts the following technical scheme:
as a first aspect of the invention, a method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae comprises the following steps:
step one, inoculating a nitrogen-fixing blue algae strain into a BG11 culture medium, performing illumination culture, centrifuging to collect algae liquid, performing secondary centrifugation, and removing a supernatant to obtain an algae body; suspending the algae in PBS buffer solution to obtain PBS algae suspension;
step two, repeatedly freezing and thawing the PBS algae heavy suspension in the step one to obtain algae cell crushing liquid, and centrifuging to obtain crude phycobiliprotein extract;
step three, adding the crude phycobiliprotein extracting solution obtained in the step two into a double aqueous phase system, and taking an upper phase after balancing to obtain a primarily purified phycobiliprotein extracting solution;
purifying the phycobiliprotein extracting solution obtained in the fourth step and the third step by adopting a hydroxyapatite column to obtain a phycobiliprotein purifying solution, and then dialyzing and desalting to obtain microalgae phycobiliprotein, wherein the hydroxyapatite column is subjected to isocratic elution by adopting different ion gradients.
According to the invention, the hydroxyapatite column of the fourth step adopts phosphate buffer solution with the ionic strength of 0.01-0.03M sodium chloride and the pH value of 7.2 to perform isocratic elution to collect red phycoerythrin, and adopts phosphate buffer solution with the ionic strength of 0.04-0.05M and the pH value of 7.2 to perform isocratic elution to collect blue phycocyanin.
Further, the eluent is phosphate buffer solution with pH 7.2, and the elution speed is 18-20 mL/h.
And step four, balancing the phycobiliprotein extracting solution obtained in the step three by using phosphate buffer solution with the volume of 3-5 times of the column volume of 7.0 of 0.01M, pH to balance a hydroxyapatite chromatography column, after balancing, loading the pretreated phycobiliprotein extracting solution to the balanced hydroxyapatite column, wherein the loading amount is 2.5-3.0mL, and purifying to obtain the phycobiliprotein purifying solution.
According to the invention, the hydroxyapatite column type is an HA (1.8cm multiplied by 4.0cm) chromatographic column, and the loading amount is 2.5-3.0 mL.
According to the invention, the fourth step also comprises the steps of observing the color of the phycobiliprotein purification solution, collecting the red component to obtain phycoerythrin, and collecting the blue component to obtain phycocyanin.
According to the invention, the illumination conditions of the illumination culture in the step one are 50-75 mu mol/m2/s, the light source is an LED lamp, the light-dark cycle is 24/0h, and preferably 100-200mM sodium citrate is externally added.
According to the invention, the PBS buffer of step one has a pH of 6.8-7.5 and a concentration of 20 mM.
Preferably, the PBS buffer has a pH of 7.2.
According to the invention, the times of freezing and thawing in the step two are 4-6, the freezing and thawing temperature is-20 ℃, and the thawing is complete at 0-4 ℃.
Preferably, the number of times of freeze thawing in the second step is 5, and the thawing is completed at 4 ℃.
According to the present invention, the aqueous two-phase system in the third step is a PEG/inorganic salt system, and the molecular weight of PEG is 1000-4000.
Preferably, the inorganic salt is ammonium sulfate and the PEG has a molecular weight of 2500.
Further, the volume ratio of the PEG/inorganic salt aqueous two-phase system is 2/l, the phase separation treatment condition is 4 ℃, the mixture is kept standing for 1h in a dark place, an organic phase of a layer is taken, and the upper layer is centrifuged and collected to obtain the phycobiliprotein extracting solution.
As a second aspect of the invention, the phycobiliprotein is prepared by the method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae.
The method for separating and purifying the high-purity phycobiliprotein from the nitrogen-fixing blue algae has the beneficial effects that:
(1) the invention provides a nitrogen-fixing blue algae as a raw material for extracting phycobiliprotein, which has higher content of phycobiliprotein, more advanced culture process and capability of obviously improving the yield.
(2) The traditional extraction process needs to prepare algae powder firstly, relates to freeze-drying, and brings high cost due to high energy consumption.
(3) Different from the traditional extraction process, the PBS algal body heavy suspension is crushed by adopting a repeated freeze-thaw method, so that the high energy consumption and high equipment cost caused by a large homogenizer can be avoided, the problem of non-scalability of an ultrasonic crushing method can be solved, and the production cost is obviously reduced.
(4) Compared with ammonium sulfate fractional precipitation, the double aqueous phase pretreatment method adopted by the invention has the advantages of simple operation, low cost and more obvious effect of removing foreign protein and polysaccharide impurities.
(5) The invention adopts the plasma intensity elution process with different concentrations, can simultaneously realize the separation and purification of high-purity phycoerythrin and phycocyanin, and has high utilization rate of raw materials.
Drawings
FIG. 1 is OD of example 1750Results plot, wherein BG11(100) is BG11 medium supplemented with a nitrogen source, supplemented with 100mM sodium citrate; BG11(500) is BG11 medium supplemented with nitrogen source and added with 500mM sodium citrate; BG110(100) is BG11 medium without nitrogen source and added with 100mM sodium citrate; BG110(500) is BG11 medium with nitrogen source removed and 500mM sodium citrate added.
FIG. 2 is a graph showing the results of the phycobiliprotein content of example 1 (ninth day).
FIG. 3 shows the results of a full wavelength scan of the ultrasonication of example 2.
FIG. 4 shows the results of a full-wavelength scan of the disruption solution of example 2 by the repeated freeze-thaw method.
FIG. 5 shows the results of a full wavelength scan of the PEG 5000/ammonium sulfate purified solution of example 3.
FIG. 6 shows the results of full-wavelength scanning of the PEG 2500/ammonium sulfate purification solution of example 3.
FIG. 7 is a graph showing the test results of example 4. Wherein, a is a repeated freeze-thawing cell disruption solution (the solution is dark red due to the existence of impure proteins), b is a PEG 2500/ammonium sulfate two-aqueous-phase system purification solution (the impure proteins are removed, the solution is mauve), c is high-purity phycoerythrin (the main component is phycoerythrin, the solution is fluorescent red) obtained by hydroxyapatite column chromatography, and d is high-purity phycocyanin (the main component is phycocyanin, the solution is fluorescent blue) obtained by hydroxyapatite column chromatography.
Detailed Description
The present invention will be further illustrated by reference to the following specific examples. The specific techniques or conditions not mentioned in the examples are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The reagent or the apparatus is not indicated to the manufacturer, and is a conventional product available from a normal distributor.
OD for growth of nitrogen-fixing cyanobacteria in the following examples750(A750) Characterization, absorbance of phycocyanin and phycoerythrin is measured by ultravioletVisible spectrophotometer detection, calculation of phycocyanin purity according to formula P1＝A620/A280The purity of phycoerythrin is calculated according to the formula P3 ═ A565/A280The calculation of phycocyanin concentration is based on the formula [ PC]＝(A620-0.7×A650) /7.38, calculation of phycoerythrin concentration according to the formula [ PE]＝(A565-2.8×[PC]-1.34×[APC])/12.7([APC](a650-0.19 × a620)/5.65), wherein a280、A565、A620、A650、A750Absorbance at wavelengths 280, 565, 620, 650, 750nm, respectively.
Example 1
(1) Collecting fresh nitrogen-fixing blue algae (Nostoc commune) algae liquid, centrifuging to collect algae body, and performing OD (origin density) treatment7500.25 of BG11 culture medium (BG11) supplemented with a nitrogen source and BG11 culture medium (BG110) without a nitrogen source were inoculated into a fresh BG11 culture medium, and 100mM and 500mM of sodium citrate were added to the control group and the experimental group, respectively;
(2) placing the experimental group and the control group in the step (1) under the conditions that the illumination intensity is 50 mu mol/m2/s, the light source is an LED lamp, and the light-dark cycle is 24/0h for culturing;
(3) measuring the OD of the experimental group and the control group in the step (2) at regular intervals750And phycobiliprotein content. The chlorophyll a content is measured by soaking and extracting with 95% acetone, and measuring the specific absorbance value.
The experimental data for example 1 are shown in fig. 1 and 2.
The results show that: the nitrogen BG11 culture medium grows better than the nitrogen-deficient BG11 culture medium, and the nitrogen-fixing blue algae yield is highest in an experimental group (BG11(100)) which adds 100mM sodium citrate in the nitrogen BG11 culture.
And (4) conclusion: the biomass yield and the phycobiliprotein content can be effectively improved by adding sodium citrate from an external source.
Note: the fresh nitrogen-fixing cyanobacteria liquid used in the following examples 2 to 4 was obtained by the irradiation culture of nitrogen-fixing cyanobacteria (Nostoc commune) in BG11(100) medium in example 1.
Example 2
(1) Taking fresh solidPutting the blue algae solution into a centrifuge tube, and diluting the algae solution to OD with water according to a proportion7502.0; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10 min; resuspending the alga body in an equal volume of 20mM PBS buffer solution, wherein the pH value is 7.2, and obtaining nitrogen-fixing blue alga PBS resuspension;
(2) repeatedly freezing and thawing the nitrogen-fixing blue algae PBS resuspension in the step (1) for 5 times at-20 ℃, completely thawing at 4 ℃ to obtain microalgae cell freeze-thaw method crushing liquid, and centrifuging at 10000rpm for 10min for later use;
(3) treating the nitrogen-fixing blue algae PBS resuspension in the step (1) by adopting an ultrasonic disruption method as a control example, wherein the ultrasonic disruption conditions are 150W, 7s/5s and 18min x 2, obtaining a microalgae cell ultrasonic disruption method disruption solution, and centrifuging at 10000rpm for 10min for later use;
(4) and (3) measuring the characteristic absorbance values of the two crushing liquids by using an ultraviolet-spectrophotometer, and scanning the two crushing liquids by using a multifunctional microplate reader at the full wavelength of 240-700 nm.
The experimental data for example 2 are shown in table 1, fig. 3 and fig. 4.
The results show that: as can be seen from Table 1, the Phycoerythrin (PE) and Phycocyanin (PC) in the freeze-thaw disruption extract have higher spectroscopic purity; as can be seen from the comparison of FIG. 3 and FIG. 4, the absorption peaks of the hetero-proteins in the extract solution obtained by the freeze-thaw crushing method are fewer, and the absorption peaks of phycoerythrin (565nm) and phycocyanin (620nm) are relatively more obvious.
And (4) conclusion: the extraction process can be simplified and the equipment cost can be reduced by repeated freeze thawing extraction.
TABLE 1 spectroscopic purity of phycobiliprotein extract
Example 3
(1) Taking fresh nitrogen-fixing blue algae liquid in a centrifuge tube, and diluting the algae liquid to OD by using water according to a proportion7502.0; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized water once, and centrifuging at 4000rpm for 10 min; resuspending the algal cells inIn an equal volume of 20mM PBS buffer solution, the pH value is 7.2, and nitrogen-fixing blue algae PBS resuspension is obtained;
(2) repeatedly freezing and thawing the nitrogen-fixing blue algae PBS resuspension in the step (1) for 5 times at-20 ℃, completely thawing at 4 ℃ to obtain microalgae cell freeze-thaw method crushing liquid, and centrifuging at 10000rpm for 10min for later use;
(3) adding the freeze-thaw method crushing liquid in the step (2) into a PEG 2500/ammonium sulfate aqueous two-phase system, wherein the volume ratio is 2:1 (20% w/w and 10% w/w), standing for 1h in darkness at 4 ℃, taking an upper organic phase, and centrifuging for 10min at 10000rpm for later use;
(4) adding the freeze-thaw method crushing liquid in the step (2) into a PEG 5000/ammonium sulfate aqueous two-phase system, wherein the volume ratio is 2:1 (20% w/w and 10% w/w), standing for 1h in darkness at 4 ℃, taking an upper organic phase, and centrifuging for 10min at 10000rpm for later use;
(5) and (3) measuring the characteristic absorbance values of the two crushing liquids by using an ultraviolet-spectrophotometer, and scanning the two crushing liquids by using a multifunctional microplate reader at the full wavelength of 240-700 nm.
The experimental data for example 3 are shown in table 2, fig. 5 and fig. 6.
The experimental results are as follows: as can be seen from Table 2, the Phycoerythrin (PE) and Phycocyanin (PC) of the PEG 2500/ammonium sulfate purified solution have higher spectroscopic purity; as can be seen from the comparison of FIG. 5 and FIG. 6, the absorption peaks of hetero-proteins in the PEG 2500/ammonium sulfate purified solution are less, and the absorption peaks of phycoerythrin (565nm) and phycocyanin (620nm) are relatively more obvious.
And (4) conclusion: through the purification of a PEG/inorganic salt double aqueous phase system, the purity can be greatly improved, and the pollution of protein and polysaccharide can be effectively removed.
Table 2 experimental data for example 3
Example 4
(1) Taking fresh nitrogen-fixing blue algae liquid in a centrifuge tube, and diluting the algae liquid to OD by using water according to a proportion7502.0; centrifuging at 4000rpm for 10min, and discarding the supernatant; washing the precipitate with deionized waterCentrifuging at 4000rpm for 10 min; resuspending the alga body in an equal volume of 20mM PBS buffer solution, wherein the pH value is 7.2, and obtaining nitrogen-fixing blue alga PBS resuspension;
(2) repeatedly freezing and thawing the nitrogen-fixing blue algae PBS resuspension in the step (1) for 5 times at-20 ℃, completely thawing at 4 ℃ to obtain microalgae cell freeze-thaw method crushing liquid, and centrifuging at 10000rpm for 10min for later use;
(3) adding the freeze-thaw method crushing liquid in the step (2) into a PEG 2500/ammonium sulfate aqueous two-phase system, wherein the volume ratio is 2:1 (20% w/w and 10% w/w), standing for 1h in darkness at 4 ℃, taking an upper organic phase, and centrifuging for 10min at 10000rpm for later use;
(4) and (3) balancing the hydroxyapatite chromatography column by using phosphate buffer solution with the volume of 3-5 times of that of 7.0 times of 0.01M, pH, after balancing, loading the pretreated phycobiliprotein extracting solution to the well-balanced hydroxyapatite column, and loading the phycobiliprotein purifying solution in the step (3) to 2.5 mL.
(5) And (3) collecting red protein purification solution 1 (phycoerythrin) by adopting a phosphate buffer solution with the ionic strength of 0.02M (sodium chloride) and the pH value of 7.2 and collecting blue protein purification solution 2 (phycocyanin) by adopting a phosphate buffer solution with the ionic strength of 0.04M (sodium chloride) and the pH value of 7.2 aiming at the phycobiliprotein adsorbed in the step (4).
(5) And (3) measuring the characteristic absorbance values of the two crushing liquids by using an ultraviolet-spectrophotometer, and photographing the high-purity phycoerythrin and the high-purity phycocyanin obtained after purification.
The experimental data for example 4 are shown in table 3 and fig. 7. In fig. 7, a is a repeated freeze-thawing cell disruption solution (the solution is dark red due to the presence of impure proteins), b is a PEG 2500/ammonium sulfate two-aqueous phase system purification solution (the impure proteins are removed, the solution is purple red), c is high-purity phycoerythrin (the main component is phycoerythrin, the solution is fluorescent red) obtained by hydroxyapatite column chromatography, and d is high-purity phycocyanin (the main component is phycocyanin, the solution is fluorescent blue) obtained by hydroxyapatite column chromatography.
Table 3 experimental data for example 4
The experimental results are as follows: the phycocyanin purity of the phycocyanin prepared by the method of the embodiment 4 is more than 4(4.025), the phycoerythrin purity is more than 4(4.807), and the reagent grade requirements can be met.
And (4) conclusion: the separation and purification of high-purity phycocyanin and phycoerythrin can be realized through hydroxyapatite column chromatography.
In conclusion, the invention takes fresh nitrogen-fixing microalgae as raw material for culture, and can obviously improve the biomass yield and the phycobiliprotein content by adding sodium citrate from an external source; the extraction process can be simplified and the equipment cost can be reduced by repeated freeze thawing extraction; further, the PEG/inorganic salt double aqueous phase system is used for purification, so that the purity can be greatly improved, and the protein and polysaccharide pollution can be effectively removed; finally, separation and purification of high-purity phycocyanin and phycoerythrin can be realized through hydroxyapatite column chromatography. Compared with the traditional extraction and separation purification process, the method has the advantages of high phycobiliprotein content, simple extraction process, low cost and high raw material utilization rate, and is suitable for large-scale industrial production of phycobiliprotein.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (10)
1. A method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae is characterized by comprising the following steps:
step one, inoculating a nitrogen-fixing blue algae strain into a BG11 culture medium, performing illumination culture, centrifuging to collect algae liquid, performing secondary centrifugation, and removing a supernatant to obtain an algae body; suspending the algae in PBS buffer solution to obtain PBS algae suspension;
step two, repeatedly freezing and thawing the PBS algae heavy suspension in the step one to obtain algae cell crushing liquid, and centrifuging to obtain crude phycobiliprotein extract;
step three, adding the crude phycobiliprotein extracting solution obtained in the step two into a double aqueous phase system, and taking an upper phase after balancing to obtain a primarily purified phycobiliprotein extracting solution;
purifying the phycobiliprotein extracting solution obtained in the fourth step and the third step by adopting a hydroxyapatite column to obtain a phycobiliprotein purifying solution, and then dialyzing and desalting to obtain microalgae phycobiliprotein, wherein the hydroxyapatite column is subjected to isocratic elution by adopting different ion gradients.
2. The method for separating and purifying phycobiliprotein from nitrogen-fixing cyanobacteria as claimed in claim 1, wherein the hydroxyapatite column in step four is prepared by performing isocratic elution with phosphate buffer solution of pH 7.2 having ionic strength of 0.01-0.03M sodium chloride to collect phycoerythrin in red, and performing isocratic elution with phosphate buffer solution of pH 7.2 having ionic strength of 0.04-0.05M to collect phycocyanin in blue.
3. The method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing cyanobacteria as claimed in claim 2, wherein the elution rate is 18-20 mL/h.
4. The method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing cyanobacteria as claimed in claim 1, wherein in the fourth step, the phycobiliprotein extract in the third step is subjected to equilibrium chromatography on hydroxyapatite by using 0.01M, pH phosphate buffer solution with 7.0 and 3-5 times column volume, after equilibrium, the pretreated phycobiliprotein extract is loaded on the well-balanced hydroxyapatite column, the loading amount is 2.5-3.0mL, and the phycobiliprotein purified solution is obtained after purification.
5. The method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing cyanobacteria as claimed in claim 1, wherein the fourth step further comprises observing the color of the phycobiliprotein purification solution, collecting the red component to obtain phycoerythrin, and collecting the blue component to obtain phycocyanin.
6. The method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing cyanobacteria as claimed in claim 1, wherein the illumination condition of the illumination culture in the first step is 50-75 μmol/m2/s, the light source is an LED lamp, the light-dark cycle is 24/0h, and preferably 100-200mM sodium citrate is externally added.
7. The method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing cyanobacteria as claimed in claim 1, wherein the PBS buffer solution in the first step has pH of 6.8-7.5 and concentration of 20 mM.
8. The method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing cyanobacteria as claimed in claim 1, wherein the number of times of freeze thawing in the second step is 4-6, the freeze thawing temperature is-20 ℃, and the complete thawing is performed at 0-4 ℃.
9. The method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing cyanobacteria as claimed in claim 1, wherein the aqueous two phase system in the third step is PEG/inorganic salt system, and the molecular weight of PEG is 1000-4000.
10. The phycobiliprotein is prepared by the method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae according to any one of claims 1 to 9.
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| CN116239674A (en) * | 2022-12-30 | 2023-06-09 | 广东湛江海洋医药研究院 | Method for refining phycobiliprotein from dilute solution of phycobiliprotein and flocculation formula |
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| CN112851777A (en) * | 2021-04-16 | 2021-05-28 | 华东理工大学 | Method for efficiently separating and purifying marine microalgae phycobiliprotein and phycobiliprotein |
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| CN114878289A (en) * | 2022-07-12 | 2022-08-09 | 北京大学 | Preparation method of low-temperature-resistant and low-salt-resistant cyanobacteria phycobilisome sample |
| CN114878289B (en) * | 2022-07-12 | 2022-09-27 | 北京大学 | Preparation method of low-temperature-resistant and low-salt-resistant cyanobacteria phycobilisome sample |
| CN116239674A (en) * | 2022-12-30 | 2023-06-09 | 广东湛江海洋医药研究院 | Method for refining phycobiliprotein from dilute solution of phycobiliprotein and flocculation formula |
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