Separation and purification method of porphyridium phycoerythrin

Abstract

The invention relates to the technical field of phycoerythrin extraction, and discloses a porphyridium phycoerythrin separation and purification method which comprises the following steps: extracting porphyridium, carrying out fractional precipitation, and carrying out separation and purification. The method comprises the following steps: firstly, simply extracting porphyridium in an extraction manner; then carrying out fractional precipitation in an ammonium sulfate fractional precipitation manner; secondly, separating and purifying through column chromatography; finally, the extracted phycoerythrin is subjected to purity analysis and method optimization, and phycoerythrin with high purity is successfully separated from porphyridium through the porphyridium separation and purification step. The method has the advantages of simplicity and convenience in operation, good purification effect and the like, and provides a reliable basis for subsequent research. Meanwhile, a purity detection result shows that the purified phycoerythrin has relatively high purity and is suitable for further biological research and application.

Description

Technical Field

[0001] The invention relates to the technical field of phycoerythrin extraction, in particular to a method for separating and purifying phycoerythrin from Porphyridium cruentum. Background Art

[0002] Porphyridium is a microalgae found in seawater, freshwater and moist soil. Most of its cells are spherical, bright blood red or dark purple, with an axial star-shaped or irregular shaped chromatin and a sheathless nucleus. Porphyridium can produce abundant phycobiliproteins, which have potential application value in biochemistry, medical research and other fields. Among them, phycoerythrin is a new type of fluorescent labeling agent separated and purified from red algae (including Porphyridium), which has unique fluorescent properties and biological activity.

[0003] The extraction of phycoerythrin is an important biotechnological process, which can effectively separate and purify phycoerythrin from algae cells. During the extraction process, the extraction method and environment will affect the purity of the extracted phycoerythrin. Therefore, in the process of extracting phycoerythrin, it is necessary to select appropriate methods and parameters for optimization and adjustment according to the specific situation. By optimizing lighting conditions, regulating temperature, optimizing nutritional conditions, physical crushing and extraction, and other physical methods, the yield of phycoerythrin can be effectively improved. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for separating and purifying Porphyridium phycoerythrin.

[0005] To achieve the above object, the present invention provides the following technical solutions: A method for separating and purifying phycoerythrin from Porphyridium cruzi, which comprises extracting Porphyridium cruzi, and then separating and purifying the phycoerythrin from Porphyridium cruzi by graded precipitation;

[0006] Firstly, the Porphyridium cruentum was simply extracted by leaching;

[0007] Then, fractional precipitation is carried out by ammonium sulfate fractional precipitation;

[0008] Secondly, separation and purification are carried out by column chromatography;

[0009] Finally, the purity of the extracted phycoerythrin was analyzed and the method was optimized.

[0010] Preferably,

[0011] The specific steps of extraction are:

[0012] Pretreatment of Porphyridium cruentum: washing, grinding, drying and sieving the harvested microalgae to obtain microalgae powder;

[0013] Stirring treatment: placing the obtained microalgae powder in a stirrer, adding 8 to 10 times of buffer (such as tris(hydroxymethyl)aminomethane buffer, pH 6.5 to 7) and stirring evenly, standing for 60 to 120 minutes, and then adding complex enzymes (such as protease, glycosidase, carboxylase, etc.) and organic solvents (such as ethanol or styrene, concentration of 40% to 55%) and stirring again, the stirring speed is 50 to 80 r / min;

[0014] Extraction: After the obtained mixed solution is allowed to stand for 10 to 30 minutes, it is heated to 35 to 40°C for 30 to 35 minutes, and then enzymatically hydrolyzed for 20 to 30 minutes to obtain an enzyme-inactivated extract;

[0015] Solid-liquid separation and drying: The enzyme-inactivated extract is centrifuged to obtain the supernatant, and the supernatant is vacuum freeze-dried (temperature is -50°C to -30°C, time is 40 to 50 minutes) to obtain a microalgae protein extract.

[0016] Preferably,

[0017] Ammonium sulfate graded precipitation: solid ammonium sulfate is added to the supernatant to different saturations, such as 0%, 20%, 40%, 60% and 80%;

[0018] Let it stand overnight, then perform high-speed refrigerated centrifugation to separate the precipitate;

[0019] The precipitate was dissolved in a small amount of distilled water and placed in a dialysis bag for dialysis. First, it was dialyzed with 4°C distilled water for 24 h, and then balanced with 0.01 mol / L sodium phosphate buffer, pH 7.0, for 24 h. After dialysis, the supernatant was centrifuged and used as the loading sample.

[0020] Preferably,

[0021] The specific steps of column chromatography separation and purification are:

[0022] Hydroxyapatite (HA) column chromatography:

[0023] Equilibrate the column: pre-equilibrate the HA chromatography column with appropriate PBS buffer;

[0024] Loading: Load the dialyzed sample onto the HA chromatography column;

[0025] Elution: Elution was performed using PBS buffer with increasing ionic strength, and the eluate was collected in portions;

[0026] Purity test: Purity is assessed by measuring the absorbance of the eluate at a specific wavelength.

[0027] Preferably,

[0028] Purity testing and property analysis:

[0029] Use a spectrophotometer to measure the absorbance of the purified phycoerythrin at a specific wavelength and calculate the purity;

[0030] Perform polyacrylamide gradient gel electrophoresis and observe the electrophoresis pattern to determine whether the purified phycoerythrin is a single component;

[0031] The maximum absorption peak and room temperature fluorescence emission peak of the purified phycoerythrin were determined.

[0032] Preferably,

[0033] The purified phycoerythrin was subjected to UV absorption spectrum, fluorescence spectrum, fluorescence quantum yield, Stokes shift property determination, and stability test under different temperature and pH conditions;

[0034] Stability tests include antioxidant activity, photostability, and thermodynamic stability tests;

[0035] After testing, the optimal separation and purification method was determined and the living environment for improving the phycoerythrin of Porphyridium cruentum was evaluated.

[0036] Preferably,

[0037] Antioxidant activity test: DPPH free radical scavenging ability assay, by comparing the DPPH free radical scavenging rates of phycoerythrin solutions with different concentrations to evaluate their antioxidant activity;

[0038] Light stability test: External light irradiation experiment, by comparing the absorption value preservation rate under different irradiation time to evaluate the light stability of phycoerythrin;

[0039] Thermodynamic stability test: Circular dichroism spectroscopy test, the thermodynamic stability of phycoerythrin is evaluated by comparing the circular dichroism spectra under different conditions.

[0040] Preferably,

[0041] By evaluating the content, performance and stability of phycoerythrin in Porphyridium cruzi, the temperature, light and humidity of the growth and preservation environment of Porphyridium cruzi were evaluated, so as to select a better growth and preservation environment.

[0042] Compared with the prior art, the present invention provides a method for separating and purifying phycoerythrin from Porphyridium cruentum, which has the following beneficial effects:

[0043] 1. The method for separating and purifying phycoerythrin from Porphyridium cruentum successfully separates phycoerythrin with high purity from Porphyridium cruentum through the separation and purification steps of Porphyridium cruentum. The method has the advantages of simple operation and good purification effect, and provides a reliable basis for subsequent research. At the same time, the purity test results show that the purified phycoerythrin has high purity and is suitable for further biological research and application.

[0044] 2. The method for separating and purifying phycoerythrin from Porphyridium cruzi is to extract and purify the phycoerythrin from Porphyridium cruzi multiple times by means of leaching, graded precipitation and column chromatography separation and purification. In a progressive manner, the phycoerythrin from Porphyridium cruzi can be better extracted, and the extraction purity of the phycoerythrin from Porphyridium cruzi can be improved.

[0045] 3. The method for separating and purifying phycoerythrin from Porphyridium cruentum is to extract phycoerythrin from Porphyridium cruentum by testing and evaluating the phycoerythrin from Porphyridium cruentum, and to measure the ultraviolet absorption spectrum, fluorescence spectrum, fluorescence quantum yield, and Stokes shift properties of the purified phycoerythrin, as well as to conduct stability tests under different temperature and pH conditions; the stability tests include antioxidant activity, photostability, and thermodynamic stability tests; after the tests, the optimal separation and purification method is determined and the improvement of the living environment of phycoerythrin from Porphyridium cruentum is evaluated. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a schematic diagram of the process of the present invention;

[0047] Figure 2 This is a schematic diagram of the purity of phycoerythrin after fractional precipitation according to the present invention. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] See also Figure 1-Figure 2 , a method for separating and purifying phycoerythrin from Porphyridium cruentum, which comprises extracting the phycoerythrin from Porphyridium cruentum, and then separating and purifying the phycoerythrin after graded precipitation;

[0050] Firstly, the Porphyridium cruentum was simply extracted by leaching;

[0051] Then, fractional precipitation is carried out by ammonium sulfate fractional precipitation;

[0052] Secondly, separation and purification are carried out by column chromatography;

[0053] Finally, the purity of the extracted phycoerythrin was analyzed and the method was optimized.

[0054] The specific steps of extraction are:

[0055] Pretreatment of Porphyridium cruentum: washing, grinding, drying and sieving the harvested microalgae to obtain microalgae powder;

[0056] Stirring treatment: placing the obtained microalgae powder in a stirrer, adding 8 to 10 times of buffer (such as tris(hydroxymethyl)aminomethane buffer, pH 6.5 to 7) and stirring evenly, standing for 60 to 120 minutes, and then adding complex enzymes (such as protease, glycosidase, carboxylase, etc.) and organic solvents (such as ethanol or styrene, concentration of 40% to 55%) and stirring again, the stirring speed is 50 to 80 r / min;

[0057] Extraction: After the obtained mixed solution is allowed to stand for 10 to 30 minutes, it is heated to 35 to 40°C for 30 to 35 minutes, and then enzymatically hydrolyzed for 20 to 30 minutes to obtain an enzyme-inactivated extract;

[0058] Solid-liquid separation and drying: The enzyme-inactivated extract is centrifuged to obtain the supernatant, and the supernatant is vacuum freeze-dried (temperature is -50°C to -30°C, time is 40 to 50 minutes) to obtain a microalgae protein extract.

[0059] Ammonium sulfate graded precipitation: solid ammonium sulfate is added to the supernatant to different saturations, such as 0%, 20%, 40%, 60% and 80%;

[0060] Let it stand overnight, then perform high-speed refrigerated centrifugation to separate the precipitate;

[0061] The precipitate was dissolved in a small amount of distilled water and placed in a dialysis bag for dialysis. First, it was dialyzed with 4°C distilled water for 24 h, and then balanced with 0.01 mol / L sodium phosphate buffer, pH 7.0, for 24 h. After dialysis, the supernatant was centrifuged and used as the loading sample.

[0062] The specific steps of column chromatography separation and purification are:

[0063] Hydroxyapatite (HA) column chromatography:

[0064] Equilibrate the column: pre-equilibrate the HA chromatography column with appropriate PBS buffer;

[0065] Loading: Load the dialyzed sample onto the HA chromatography column;

[0066] Elution: Elution was performed using PBS buffer with increasing ionic strength, and the eluate was collected in portions;

[0067] Purity test: Purity is assessed by measuring the absorbance of the eluate at a specific wavelength.

[0068] Purity testing and property analysis:

[0069] Use a spectrophotometer to measure the absorbance of the purified phycoerythrin at a specific wavelength and calculate the purity;

[0070] Perform polyacrylamide gradient gel electrophoresis and observe the electrophoresis pattern to determine whether the purified phycoerythrin is a single component;

[0071] The maximum absorption peak and room temperature fluorescence emission peak of the purified phycoerythrin were determined.

[0072] The purified phycoerythrin was subjected to UV absorption spectrum, fluorescence spectrum, fluorescence quantum yield, Stokes shift property determination, and stability test under different temperature and pH conditions;

[0073] Stability tests include antioxidant activity, photostability, and thermodynamic stability tests;

[0074] After testing, the optimal separation and purification method was determined and the living environment for improving the phycoerythrin of Porphyridium cruentum was evaluated.

[0075] Antioxidant activity test: DPPH free radical scavenging ability assay, by comparing the DPPH free radical scavenging rates of phycoerythrin solutions with different concentrations to evaluate their antioxidant activity;

[0076] Light stability test: External light irradiation experiment, by comparing the absorption value preservation rate under different irradiation time to evaluate the light stability of phycoerythrin;

[0077] Thermodynamic stability test: Circular dichroism spectroscopy test, the thermodynamic stability of phycoerythrin is evaluated by comparing the circular dichroism spectra under different conditions.

[0078] By evaluating the content, performance and stability of phycoerythrin in Porphyridium cruzi, the temperature, light and humidity of the growth and preservation environment of Porphyridium cruzi were evaluated, so as to select a better growth and preservation environment.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for separating and purifying Porphyridium phycoerythrin, characterized in that: The method is a separation and purification method that extracts Porphyridium cruentum, and then separates and purifies it after graded precipitation. Firstly, the Porphyridium cruentum was simply extracted by leaching; Then, fractional precipitation is carried out by ammonium sulfate fractional precipitation; Secondly, separation and purification are carried out by column chromatography; Finally, the purity of the extracted phycoerythrin was analyzed and the method was optimized.

2. The method for separating and purifying phycoerythrin from Porphyridium cruentum according to claim 1, characterized in that: The specific steps of extraction are: Pretreatment of Porphyridium cruentum: washing, grinding, drying and sieving the harvested microalgae to obtain microalgae powder; Stirring treatment: placing the obtained microalgae powder in a stirrer, adding 8 to 10 times of buffer (such as tris(hydroxymethyl)aminomethane buffer, pH 6.5 to 7) and stirring evenly, standing for 60 to 120 minutes, and then adding complex enzymes (such as protease, glycosidase, carboxylase, etc.) and organic solvents (such as ethanol or styrene, concentration of 40% to 55%) and stirring again, the stirring speed is 50 to 80 r / min; Extraction: After the obtained mixed solution is allowed to stand for 10 to 30 minutes, it is heated to 35 to 40°C for 30 to 35 minutes, and then enzymatically hydrolyzed for 20 to 30 minutes to obtain an enzyme-inactivated extract; Solid-liquid separation and drying: The enzyme-inactivated extract is centrifuged to obtain the supernatant, and the supernatant is vacuum freeze-dried (temperature is -50°C to -30°C, time is 40 to 50 minutes) to obtain a microalgae protein extract.

3. The method for separating and purifying Porphyridium phycoerythrin according to claim 1, characterized in that: Ammonium sulfate graded precipitation: solid ammonium sulfate is added to the supernatant to different saturations, such as 0%, 20%, 40%, 60% and 80%; Let it stand overnight, then perform high-speed refrigerated centrifugation to separate the precipitate; The precipitate was dissolved in a small amount of distilled water and placed in a dialysis bag for dialysis. First, it was dialyzed with 4°C distilled water for 24 h, and then balanced with 0.01 mol / L sodium phosphate buffer, pH 7.0, for 24 h. After dialysis, the supernatant was centrifuged and used as the loading sample.

4. The method for separating and purifying phycoerythrin from Porphyridium cruentum according to claim 1, characterized in that: The specific steps of column chromatography separation and purification are: Hydroxyapatite (HA) column chromatography: Equilibrate the column: pre-equilibrate the HA chromatography column with appropriate PBS buffer; Loading: Load the dialyzed sample onto the HA chromatography column; Elution: Elution was performed using PBS buffer with increasing ionic strength, and the eluate was collected in portions; Purity test: Purity is assessed by measuring the absorbance of the eluate at a specific wavelength.

5. The method for separating and purifying phycoerythrin from Porphyridium cruentum according to claim 1, characterized in that: Purity testing and property analysis: Use a spectrophotometer to measure the absorbance of the purified phycoerythrin at a specific wavelength and calculate the purity; Perform polyacrylamide gradient gel electrophoresis and observe the electrophoresis pattern to determine whether the purified phycoerythrin is a single component; The maximum absorption peak and room temperature fluorescence emission peak of the purified phycoerythrin were determined.

6. The method for separating and purifying Porphyridium phycoerythrin according to claim 1, characterized in that: The purified phycoerythrin was subjected to UV absorption spectrum, fluorescence spectrum, fluorescence quantum yield, Stokes shift property determination, and stability test under different temperature and pH conditions; Stability tests include antioxidant activity, photostability, and thermodynamic stability tests; After testing, the optimal separation and purification method was determined and the living environment for improving the phycoerythrin of Porphyridium cruentum was evaluated.

7. The method for separating and purifying phycoerythrin from Porphyridium cruentum according to claim 1, characterized in that: Antioxidant activity test: DPPH free radical scavenging ability assay, by comparing the DPPH free radical scavenging rates of phycoerythrin solutions with different concentrations to evaluate their antioxidant activity; Light stability test: External light irradiation experiment, by comparing the absorption value preservation rate under different irradiation time to evaluate the light stability of phycoerythrin; Thermodynamic stability test: Circular dichroism spectroscopy test, the thermodynamic stability of phycoerythrin is evaluated by comparing the circular dichroism spectra under different conditions.

8. The method for separating and purifying Porphyridium phycoerythrin according to claim 1, characterized in that: By evaluating the content, performance and stability of phycoerythrin in Porphyridium cruzi, the temperature, light and humidity of the growth and preservation environment of Porphyridium cruzi were evaluated, so as to select a better growth and preservation environment.

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