Novel method for extracting high-purity R-phycoerythrin in laver
阅读说明：本技术 一种提取紫菜中高纯度r-藻红蛋白的新方法 (Novel method for extracting high-purity R-phycoerythrin in laver ) 是由 王全富 侯艳华 王一帆 于 2020-06-05 设计创作，主要内容包括：本发明公开了一种提取高纯度藻红蛋白的方法。该方法将紫菜反复冻融使细胞破碎,经离心、盐析后透析,得到上清液粗蛋白。利用新型绿色的低共熔溶剂,并加入磷酸盐构成双水相体系,萃取得到较高纯度的藻红蛋白。本发明提取得到的藻红蛋白纯度高,所应用的低共熔溶剂双水相体系绿色环保,生物相容性好,且该提取方法成本低,易规模化、工业化,在提取高纯度藻红蛋白、保护环境等方面有应用前景。(The invention discloses a method for extracting high-purity phycoerythrin. The method comprises repeatedly freezing and thawing thallus Porphyrae to break cells, centrifuging, salting out, and dialyzing to obtain supernatant crude protein. A novel green eutectic solvent is utilized, phosphate is added to form a two-aqueous-phase system, and the phycoerythrin with higher purity is obtained through extraction. The phycoerythrin extracted by the invention has high purity, the applied eutectic solvent aqueous two-phase system is green and environment-friendly, the biocompatibility is good, and the extraction method has low cost, is easy to realize large-scale and industrialization, and has application prospects in the aspects of extracting high-purity phycoerythrin, protecting the environment and the like.)
1. A method for extracting phycoerythrin from high-purity laver is characterized in that: separating and purifying R-phycoerythrin from a crude extract containing the R-phycoerythrin in the laver by adopting a eutectic solvent (DES) -aqueous two-phase method, and specifically performing the following steps: (1) freezing the crude extract laver obtained by ammonium sulfate salting-out method at-20 deg.C overnight, thawing at room temperature, sieving to remove a large amount of algae residue, centrifuging the extractive solution at 7000-8000 rpm for 15-30 min, salting out with ammonium sulfate to obtain R-phycoerythrin precipitate, and dissolving the precipitate with 10 mM phosphate buffer;
(2) and establishing a eutectic solvent (DES) -aqueous two-phase system to obtain the high-purity R-phycoerythrin.
2. The method of claim 1, wherein the hydrogen bond acceptor of the eutectic solvent is choline chloride, the hydrogen bond donor is urea, the synthesis temperature is 80-90 ℃, and the synthesis time is 2-3 h.
3. The method for extracting high-purity phycoerythrin from laver as claimed in claim 1, wherein the aqueous two-phase system has a phase temperature of 20-25 ℃.
4. The aqueous two-phase system according to claim 4, wherein the choline chloride-urea and the dipotassium hydrogen phosphate solution form an aqueous two-phase system for purifying R-phycoerythrin.
5. The aqueous two-phase system according to claim 4, wherein the phycoerythrin extracted has a higher extraction rate and a higher purity, 92.60% and 3.825% respectively, relative to the crude protein, and the purity reaches the pharmaceutical grade level, which indicates the high efficiency of the protein purification of the present invention.
Technical Field
The invention belongs to the technical field of protein extraction and purification, and particularly relates to a method for preparing high-purity phycoerythrin from laver by a eutectic solvent-aqueous two-phase method.
Background
Phycoerythrin (PE) is a kind of phycobiliprotein, and is isolated and purified from red algae. Phycoerythrin has excellent light absorbing performance and high quantum yield, and may be combined with biotin and various monoclonal antibodies conveniently, so that it may be used widely as natural pigment in food, cosmetics, dye and other industries, as well as fluorescent reagent for clinical medicine diagnosis, immunochemistry, bioengineering and other fields. Has important theoretical and practical significance for the extraction and research of phycoerythrin.
Currently, the separation and purification of phycoerythrin mainly includes ammonium sulfate precipitation combined with agarose gel chromatography (Indian J. Geo-Mar. Sci. 2013, 42, 21-28), hydroxyapatite column chromatography (J. Biotechnol. 2003, 101, 289-293), polyacrylamide gel electrophoresis (J. chromatogr. B. 2000, 739, 117-123), and the like. These conventional methods have the disadvantages of excessive steps, complicated operation, solvent consumption, high kinetic energy consumption and limited purity of the extracted protein, so that additional multi-step chromatography is required to achieve the purity target, and the industrial large-scale production of some proteins is extremely disadvantageous. Proteins are easily denatured under acidic, alkaline or heat conditions due to their instability. Thus, there is a need for a more efficient, stable, economical and scalable way to obtain a product that is both high purity and high yield and is biologically active.
With the development of green chemistry, green solvents are more and more valued by people. The Eutectic solvent (Deep Eutectic Solvents, DESs) is a green solvent, consisting of hydrogen bond acceptor (quaternary ammonium salt) and hydrogen bond donor (acid amide, carboxylic acid and polyol) in appropriate molar ratios (Am. chem. Soc. 2004, 126, 9142-. The aqueous two-phase system contains a large amount of water, and the extraction is close to the physiological environment of biological substances, so most of biomacromolecules still keep the biological activity after being separated by the aqueous two-phase technology (biochem. Eng. J. 2009, 46, 306-.
In view of the above discussion, the method combines the eutectic solvent and the aqueous two-phase system, adopts the eutectic solvent/salt aqueous two-phase system to extract phycoerythrin in laver on the basis of the traditional ammonium sulfate salting-out, and has application prospect in the aspect of preparing high-purity phycoerythrin.
Disclosure of Invention
The invention aims to provide a novel method for extracting high-purity phycoerythrin from laver, which simplifies the purification process of the phycoerythrin, has high product purity, is green and environment-friendly and is easy to amplify.
In order to achieve the purpose, the invention adopts the following technical scheme:
(1) preparing crude protein: centrifuging thallus Porphyrae frozen at-20 deg.C overnight, collecting supernatant, salting out, and adding ammonium sulfate to reach 35% saturation degree. Centrifuging, collecting precipitate, and dialyzing in phosphate buffer solution with pH =6.8 to obtain crude phycoerythrin extractive solution.
(2) Synthesis of eutectic solvent: choline chloride and urea are used as raw materials, and are stirred for 2-3 hours at the temperature of 80-90 ℃ to obtain clear transparent liquid, namely the eutectic solvent.
(3) Establishing a two-water-phase system: adding 300-400 μ L of K with pH of 8.0 to 0.3-0.5 g of eutectic solvent2HPO4Standing the salt solution in water bath at 25 deg.C to obtain aqueous two-phase system.
(4) Adding the crude protein into aqueous two phase, oscillating, placing in 20-25 deg.C water bath, absorbing upper phase after phase formation is stable, measuring absorbance at 280 nm, 565 nm, 618 nm and 650 nm with ultraviolet spectrophotometer, and calculating purity and extraction rate.
Purity:
the extraction rate is as follows:
concentration:
wherein C istAnd CbThe concentrations of phycoerythrin in the DES-rich upper phase and the salt-rich lower phase, VtAnd VbThe volumes of the upper and lower phases are indicated separately.
(5) The technical advantages are as follows:
1. the phycoerythrin extracted by the invention has higher purity and yield.
2. The aqueous two-phase system has mild separation conditions, and the whole operation can be carried out at normal temperature and normal pressure.
3. The invention has less separation steps and more economical separation process.
4. The invention can amplify each parameter in proportion, can be operated continuously, and is easy to realize industrialized mass production.
Drawings
FIG. 1a shows the purity and extraction rate of phycoerythrin at different amounts of eutectic solvent.
FIG. 1b shows the purity and extraction rate of phycoerythrin at different crude protein addition levels.
FIG. 1c shows the purity and extraction rate of phycoerythrin at different extraction times.
FIG. 2 is a gel electrophoresis diagram of phycoerythrin.
FIG. 3a is a UV-Vis spectrum of phycoerythrin in pure water and in the low eutectic solvent double water phase in the examples of the present invention.
FIG. 3b is a graph showing fluorescence spectra of phycoerythrin in pure water and in a low eutectic solvent double aqueous phase in an example of the present invention.
Detailed Description
The invention will be further illustrated and described by means of specific embodiments in conjunction with the accompanying drawings.
Example 1:
(1) adding 250 mL of sterilized water into 5g of dry laver, freezing at-20 ℃ overnight, centrifuging, taking supernatant, salting out, and adding ammonium sulfate until 35% saturation.
(2) Centrifuging, collecting precipitate, dialyzing in phosphate buffer solution with pH =6.8, and removing ammonium sulfate to obtain crude phycoerythrin extractive solution.
(3) Synthesis of eutectic solvent: choline chloride and urea are used as raw materials, and are stirred for 2-3 hours at the temperature of 80-90 ℃ to obtain clear transparent liquid, namely the eutectic solvent.
(4) Establishing a two-water-phase system: adding 300-400 μ L of K with pH of 8.0 to 0.30-0.50 g of eutectic solvent2HPO4And (4) putting the salt solution into a water bath at the temperature of 20-25 ℃ to obtain a double water phase system.
(5) Optimizing phycoerythrin extraction conditions: adding 300-400 μ L of K with pH of 8.0 to 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55 g DES2HPO4Adding crude protein into salt solution, shaking, placing in 25 deg.C water bath, absorbing upper phase after phase formation is stable, and calculating purity and extraction rate, the result is shown in FIG. 1 a. When the DES amount is 0.35g, the purity and the extraction rate are highest; adding 0.035, 0.040, 0.045, 0.050, 0.055, 0.060 mg algae into DES-salt aqueous two-phase systemOscillating the hemoglobin, placing in 20-25 deg.C water bath, absorbing the upper phase after phase formation is stable, and calculating purity and extraction rate, the result is shown in FIG. 1 b. When the protein addition amount is 0.04 mg, the purity and the extraction rate are highest. Establishing 7 eutectic solvent aqueous two-phase systems, adding crude protein extract, shaking, standing in 25 deg.C water bath for 0, 10, 20, 30, 40, 50, and 60 min, and calculating purity and extraction rate, the result is shown in FIG. 1 c. When the time is 20-30min, the purity and extraction rate are highest, respectively 3.825 and 92.60%.
(6) The high purity protein obtained under the optimal conditions was verified by SDS-PAGE, and the results are shown in FIG. 2. The molecular weight is about 18.0, 21.0 and 30.0 kDa, and the subunits are alpha subunit, beta subunit and gamma subunit.
(7) Ultraviolet-visible spectrum and fluorescence spectrum are adopted to research the conformation of phycoerythrin before and after extraction. The UV-Vis spectra curves of R-phycoerythrin in pure water and DES phases are similar in shape, and the maximum absorption peaks are still present at 495, 540 and 565 nm (FIG. 3 a). In addition, in both the water and DES phases, the R-phycoerythrin excitation wavelength was 495 nm and the emission wavelength was 570 nm (FIG. 3 b). These results indicate that there is no interaction between R-phycoerythrin and DESS, nor formation of chemical bonds.
Example 2:
(1) adding 250 mL of sterilized water into 5g of dry laver, freezing at-20 ℃ overnight, centrifuging, taking supernatant, salting out, and adding ammonium sulfate until 35% saturation.
(2) Centrifuging, collecting precipitate, dialyzing in phosphate buffer solution with pH =6.8, and removing ammonium sulfate to obtain crude phycoerythrin extractive solution.
(3) Synthesis of eutectic solvent: choline chloride and urea are used as raw materials, and are stirred for 2-3 hours at the temperature of 80-90 ℃ to obtain clear transparent liquid, namely the eutectic solvent.
(4) Establishing a two-water-phase system: adding 300-400 μ L of K with pH of 8.0 to 0.30-0.50 g of eutectic solvent2HPO4And (5) putting the salt solution into a water bath at 25 ℃ to obtain a two-water-phase system.
(5) Optimization of phycoerythrin extraction conditions
To the ratio of 0.25, 0.30, 0.35,0.40, 0.45, 0.50, 0.55 g DES was added with 300-2HPO4Adding crude protein into salt solution, shaking, placing in water bath at 25 deg.C, absorbing upper phase after phase formation is stable, and calculating purity and extraction rate. When the DES amount is 0.35g, the purity and the extraction rate are highest; adding 0.035, 0.040, 0.045, 0.050, 0.055 and 0.060 mg phycoerythrin into DES-salt aqueous two-phase system, oscillating, placing in 20-25 deg.C water bath, absorbing upper phase after phase stabilization, calculating purity and extraction rate, when the protein addition is 0.04 mg, the purity and extraction rate are highest. Establishing 7 eutectic solvent aqueous two-phase systems, adding crude protein extract, oscillating, standing in 25 deg.C water bath for 0, 10, 20, 30, 40, 50, and 60 min, and calculating purity and extraction rate, wherein when the time is 20-30min, the purity and extraction rate are highest, respectively 3.775 and 93.20%.