A method for isolating intracellular water-soluble proteins from Haematococcus pluvialis

Abstract

This invention belongs to the technical field of separation and extraction of bioactive components, specifically relating to a method for separating intracellular water-soluble proteins from *Hydrocotyle erythrorhizon*, comprising the following steps: Step 1, preparation of extraction medium: preparing a copolymer solution from an amphiphilic temperature-responsive block copolymer; Step 2, mixing and permeation: mixing *Hydrocotyle erythrorhizon* algal powder with the copolymer solution prepared in Step 1 to form a mixed slurry, and stirring for 30 to 180 minutes; Step 3, thermally triggered separation: heating to 32°C to 45°C, maintaining the temperature and allowing it to stand for 10 to 60 minutes to allow the system to separate into phases, and collecting the condensed phase rich in water-soluble proteins; Step 4, protein recovery: dialysis and drying the condensed phase collected in Step 3 to obtain the protein product. This invention, by employing a temperature-responsive block copolymer, integrates the gentle disruption of cell walls, selective capture, and thermally triggered preliminary separation of intracellular water-soluble proteins from *Hydrocotyle erythrorhizon* into a continuous aqueous process, which is beneficial for maintaining protein activity.

Description

Technical Field

[0001] This invention belongs to the field of separation and extraction of bioactive components, specifically relating to a method for separating intracellular water-soluble proteins from Haematococcus pluvialis. Background Technology

[0002] Haematococcus pluvialis, as an important source of the high-value natural product astaxanthin, has seen relatively advanced industrial development. However, the development and utilization of another important class of active components in algae—water-soluble proteins—faces significant bottlenecks. These proteins (including phycoerythrin, etc.) possess unique fluorescent properties and antioxidant, anti-inflammatory, and other biological activities, showing great potential in high-end foods, cosmetics, and biomarkers.

[0003] Existing processes for extracting water-soluble proteins from *Haemaphysalis* typically involve two separate and inefficient stages: cell disruption and protein separation. First, to release intracellular proteins, the tough cell wall of *Haemaphysalis* must be disrupted. Commonly used physical disruption methods, such as high-pressure homogenization, sonication, or repeated freeze-thaw cycles, while effective, can easily cause denaturation and inactivation of water-soluble proteins sensitive to shear and heat due to intense mechanical forces or ice crystals. Chemical disruption methods, such as treatment with dilute alkalis or surfactants, easily introduce foreign chemicals that may alter protein structure and are difficult to remove subsequently. Enzymatic hydrolysis offers relatively mild conditions but is costly, and enzyme formulations for the complex cell wall of *Haemaphysalis* are complex and lack versatility. These disruption methods, while pursuing high disruption rates, often fail to simultaneously maintain protein activity and only address the issue of "release."

[0004] Secondly, selectively separating the target water-soluble protein from the complex homogenate containing a large amount of cell debris, chlorophyll, lipids, and nucleic acids after the release of cell contents is another technical challenge. Conventional centrifugation and filtration can only remove solid impurities and are ineffective against soluble impurities. Ammonium sulfate precipitation or organic solvent precipitation are commonly used methods for crude protein separation, but the former introduces a large amount of inorganic salts that require time-consuming dialysis for removal, while the latter directly faces the risk of protein denaturation caused by organic solvents, and the steps are cumbersome, making it difficult to achieve both high yield and activity. Existing technologies separate cell disruption and preliminary separation into multiple steps, resulting in a long process, low efficiency, and the protein is constantly exposed to an environment that can easily lead to its inactivation throughout the process.

[0005] In recent years, temperature-responsive polymers have attracted attention in the field of bioseparation due to their unique reversible phase transition properties of "dissolving at low temperatures and precipitating at high temperatures." Some studies have attempted to use them for the precipitation and separation of proteins, but these applications are mostly for protein solutions with relatively simple compositions, such as pre-purified proteins or cell lysate supernatants. Their mode of action typically involves adding the polymer after the protein is in a homogeneous solution, and then precipitating the protein by changing the temperature. Essentially, this is still a separation step and does not solve the core challenge of gently and efficiently releasing and capturing proteins in one step from intact cells. More importantly, ordinary temperature-responsive polymers tend to form dense aggregates during phase transitions, which may embed or entrain impurities, and their interactions with proteins lack selectivity.

[0006] Therefore, a method for isolating intracellular water-soluble proteins from Haematococcus pluvialis is needed to address the problems existing in the current technology. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, a method for isolating intracellular water-soluble proteins from Haematococcus pluvialis is provided.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for isolating intracellular water-soluble proteins from Haematococcus pluvialis, the method comprising the following steps:

[0010] Step 1: Preparation of extraction medium: The amphiphilic temperature-responsive block copolymer is dissolved in a buffer solution with a pH of 6.5 to 7.5 to prepare a copolymer solution with a concentration of 1.0 to 10 g / L; the amphiphilic temperature-responsive block copolymer is polyethylene glycol-block-poly(N-isopropylacrylamide), which includes hydrophilic polyethylene glycol segments and temperature-sensitive poly(N-isopropylacrylamide) segments, which are covalently linked together;

[0011] Step 2, Mixing and Infiltration: Mix the Haematococcus pluvialis powder with the copolymer solution obtained in Step 1 to form a mixed slurry, and stir at a temperature of 10°C to 25°C for 30 to 180 minutes;

[0012] Step 3, thermally triggered separation: The mixed system after step 2 is heated to 32°C to 45°C and kept at this temperature for 10 to 60 minutes to allow the system to separate into phases. The condensed phase rich in water-soluble proteins is then collected.

[0013] Step 4, Protein Recovery: The condensed phase collected in Step 3 is dialyzed and dried to obtain water-soluble protein products from Haematococcus pluvialis.

[0014] The preparation method of the amphiphilic temperature-responsive block copolymer includes the following steps:

[0015] Step 1, Preparation of initiator: Methoxy polyethylene glycol is dissolved in an anhydrous solvent and reacted with an acyl halide reagent under an inert atmosphere and ice bath cooling. After purification, a polyethylene glycol macromolecular initiator with a terminal haloacyl group is obtained.

[0016] Step 2, chain extension polymerization: The macromolecular initiator, monomer N-isopropylacrylamide, catalyst and organic ligand obtained in step 1 are placed in a reaction vessel, vacuumed and replaced with inert gas, and then a dehydrating solvent is added. The reaction is stirred at 60°C to 80°C for 6 to 24 hours.

[0017] Step 3, purification and drying: After the reaction in step 2 is completed, the mixture is precipitated, filtered and washed, and then vacuum dried at 30°C to 40°C to obtain the amphiphilic temperature-responsive block copolymer.

[0018] In the first step, the number average molecular weight of the methoxy polyethylene glycol is 2000, the acyl halide reagent is 2-bromoisobutyryl bromide, and the molar ratio of the methoxy polyethylene glycol to 2-bromoisobutyryl bromide is 1:1.5 to 1:2.5.

[0019] In the second step, the molar ratio of the monomer N-isopropylacrylamide to the macromolecular initiator is 50:1 to 200:1; the molar ratio of the monomer N-isopropylacrylamide, the catalyst, and the organic ligand is (50-200):1:(1-2).

[0020] The catalyst is cuprous bromide, and the organic ligand is pentamethyldiethylenetriamine.

[0021] In the second step, the functional monomer acrylic acid was also added to the reaction vessel, with the molar ratio of N-isopropylacrylamide to acrylic acid being 30:1 to 10:1.

[0022] Existing technologies typically treat cell disruption and protein separation as two separate processes. Cell disruption inevitably poses a potential threat to protein activity and only releases the contents. Separation often relies on salting out or organic solvent precipitation. These methods introduce new impurities or denaturation risks, and are characterized by numerous steps and lengthy processes, resulting in overall low efficiency and impaired protein activity. To address this problem, this invention first designs and employs a polyethylene glycol-block-poly(N-isopropylacrylamide) amphiphilic temperature-responsive block copolymer to simultaneously address the challenges of cell disruption and separation complexity.

[0023] This copolymer is not a simple mixture or homopolymer. Its polyethylene glycol segment, acting as a flexible hydrophilic chain, not only ensures good solubility of the entire molecule in a low-temperature aqueous phase but also allows for gentle interactions with the biomembrane structure. Through osmosis and hydration, it gently disturbs the cell wall structure of *Haemaphysalis*, promoting the release of intracellular substances while minimizing mechanical damage to protein structures. The poly(N-isopropylacrylamide) segment imparts a unique temperature-responsive behavior to the entire molecule. Below its phase transition point, this segment is in a hydrophilic extended state, causing the copolymer to dissolve; when the temperature rises above the phase transition point, the segment rapidly transitions to a hydrophobic collapsed state, becoming the physical cross-linking point driving molecular aggregation. Dissolving this copolymer in a near-neutral buffer solution to prepare the extraction medium aims to allow the hydrophilic segment to assist in contacting and penetrating the algal cell wall under mild conditions, while simultaneously preparing a smart "switch" for subsequent capture and separation.

[0024] In step one, the buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution; a reducing agent with a concentration of 0.1 g / L to 1.0 g / L, namely dithiothreitol, is also added to the copolymer solution.

[0025] In step two, the mass-to-volume ratio of *Rhodophyta globulinii* powder to copolymer solution in the mixed slurry is from 1 g to 30 ml to 1 g to 100 ml, based on dry weight.

[0026] In step two, intermittent ultrasonic treatment is performed while stirring, with an ultrasonic power of 50 watts to 300 watts, an ultrasonic time of 1 minute to 3 minutes per session, an interval of 2 minutes, and a total ultrasonic time of no more than 15 minutes.

[0027] In step four, the dialysate used in the dialysis is a phosphate buffer solution with a concentration of 0.002 mol / L, and the dialysis time is 12 to 48 hours, during which the dialysate is changed 3 to 6 times.

[0028] However, simply possessing responsive copolymers is insufficient. The next challenge is to enable them to effectively bind to target proteins under mild conditions and achieve selective enrichment in complex mixtures. Ordinary temperature-sensitive polymers often lack affinity for specific proteins and tend to indiscriminately aggregate impurities. Therefore, the extraction process of this invention is designed with two distinct temperature stages: "mixing and permeation" and "thermally triggered separation."

[0029] At low temperatures ranging from 10°C to 25°C, the copolymer molecules completely dissolve and extend. At this point, the polyethylene glycol segments and the carboxyl groups provided by the optionally introduced acrylic acid units can bind affinityily to the water-soluble proteins slowly released from algal cells through steric stabilization, hydrogen bonding, or weak electrostatic interactions. This binding process occurs in an aqueous phase under near-physiological conditions, avoiding the damage to the protein structure caused by a harsh chemical environment. Simultaneously, the presence of the copolymer molecules may have a mild permeability and loosening effect on the cell wall, synergistically promoting protein release in conjunction with low-intensity sonication, rather than relying on forceful destruction.

[0030] Once the target protein is effectively captured by copolymer molecules at low temperatures, the key to simplifying the process lies in how to easily separate it from the mixed slurry containing numerous impurities such as cell debris, pigments, and nucleic acids. This invention utilizes the inherent temperature-responsive characteristics of copolymers to achieve separation through a simple heating operation. Heating the system to the range of 32°C to 45°C causes a hydrophilic-hydrophobic transition in the poly(N-isopropylacrylamide) segments of the copolymer molecules. This transition drives the entire copolymer molecule, along with the water-soluble protein bound to it, to aggregate and form a dense condensate phase. This condensate phase can be easily separated from the supernatant phase, which is rich in insoluble algal residue and most hydrophilic impurities, by gravity or low-speed centrifugation. The ingenuity of this step lies in the fact that the driving force for separation is a physical temperature change, eliminating the need to add large amounts of salt to alter ionic strength or introduce organic solvents that may denature the protein. This simplifies the operation, reduces secondary damage to protein activity, and improves the selectivity of the separation.

[0031] In this invention, the polyethylene glycol-block-poly(N-isopropylacrylamide) copolymer plays multiple roles: it is a mild permeation aid, an affinity trap for the target protein, and a trigger for thermally driven phase separation. The entire process begins with mixing the copolymer solution with algal powder, and by adjusting only the external parameter of temperature, permeation binding and phase separation are completed sequentially. Finally, the collected protein-rich condensed phase is dialyzed and dried to obtain the product. This method avoids repeated transfer and processing of materials between multiple steps, shortens the total process time, reduces the risk of target protein inactivation during lengthy processes, and provides a new approach to obtaining highly active and relatively pure water-soluble protein products from *Haematococcus pluvialis*.

[0032] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0033] 1. This invention employs a self-designed amphiphilic temperature-responsive block copolymer of polyethylene glycol-block copolymer-poly(N-isopropylacrylamide) as the extraction medium. The polyethylene glycol segments of this copolymer exhibit good hydrophilicity and biocompatibility, while the poly(N-isopropylacrylamide) segments possess temperature-responsive characteristics. At lower temperatures, the entire copolymer molecule exhibits an extended hydrophilic conformation and dissolves in the aqueous phase. When the temperature rises above its phase transition range, the poly(N-isopropylacrylamide) segments rapidly transition to a hydrophobic state, driving the entire molecule to aggregate.

[0034] 2. The method of this invention achieves a gentle and efficient extraction of intracellular water-soluble proteins from *Hylocereus pluvialis*. In the low-temperature extraction stage, a polyethylene glycol-block-poly(N-isopropylacrylamide) copolymer dissolved in a buffer solution is mixed with *Hylocereus pluvialis* powder. Its amphiphilic structure allows it to interact with the *Hylocereus pluvialis* cell wall, and, with the synergy of gentle ultrasound, promotes the release of intracellular substances. The polyethylene glycol segments and carboxyl groups extended by the copolymer molecules in the low-temperature aqueous phase can selectively bind to the released water-soluble proteins through non-covalent forces such as hydrogen bonding and electrostatic interactions. This process is carried out under near-neutral, gentle conditions, avoiding damage to the protein structure caused by strong acids, strong bases, or violent mechanical shearing, thus preserving the protein's natural activity.

[0035] 3. This invention achieves rapid preliminary separation of the target protein from the complex matrix through simple temperature control, a significant advantage over traditional multi-step processes. After low-temperature extraction, only the mixture needs to be heated. At this point, the poly(N-isopropylacrylamide) segments in the copolymer molecules undergo a hydrophilic-hydrophobic transition, causing the entire copolymer and its bound water-soluble proteins to aggregate, forming a protein-rich condensed phase. This condensed phase is physically separated from the protein-poor aqueous phase containing impurities such as cell debris, pigments, and lipids. This thermally triggered phase separation process does not require the addition of large amounts of salt or organic precipitants, is simple in procedure, and effectively reduces the co-precipitation of impurity proteins and non-protein impurities, thus improving the selectivity of the separation.

[0036] 4. This invention integrates three typically independent unit operations—cell disruption, target component extraction, and primary separation—into a single, continuous process. Traditional methods require cell disruption, centrifugation to obtain a crude extract, and subsequent separation and purification of the crude extract, resulting in a lengthy process. This method utilizes the multifunctionality of polyethylene glycol-block-poly(N-isopropylacrylamide) copolymers, enabling them to simultaneously perform osmosis-assisted processes, protein capture, and phase separation-driven processes. The entire process can be completed in a buffer system through steps such as mixing, temperature-controlled stirring, temperature-induced phase separation, and collection. This avoids frequent transfer and processing of materials between different steps, simplifying the operation, shortening the time, and reducing the risk of target protein inactivation during lengthy processes, thus providing a higher-quality primary product for subsequent higher-level purification. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows:

[0039] Methoxylated polyethylene glycol: Shanghai Tuoyang Biotechnology Co., Ltd., with a number average molecular weight of 2000.

[0040] 2-Bromoisobutyryl bromide: Shanghai Lianmai Biotechnology Co., Ltd., CAS No. 20769-85-1.

[0041] Anhydrous dichloromethane: Tianjin Yirenda Chemical Co., Ltd., purity 99.5%.

[0042] N-Isopropylacrylamide, Hubei Xinyuhong Biomedical Technology Co., Ltd., 99% purity.

[0043] Cuprous bromide: Shanghai Huzhen Industrial Co., Ltd., brand: Amresco, purity 99.99%.

[0044] Pentamethyldiethylenetriamine: Shanghai Boyun New Materials Co., Ltd., purity 99%, CAS No. 3030-47-5.

[0045] Anhydrous N,N-dimethylformamide: Shanghai Xinshengnuo Chemical Co., Ltd., purity 99.99%.

[0046] Dithiothreitol: Hubei Jianchu Biomedical Co., Ltd., CAS No. 3483-12-3, purity 98%.

[0047] Haematococcus pluvialis powder: Yunnan Aierfa Biotechnology Co., Ltd.

[0048] The technical solution of this application is as follows:

[0049] A method for isolating intracellular water-soluble proteins from Haematococcus pluvialis, the method comprising the following steps:

[0050] Step 1: Preparation of extraction medium: The amphiphilic temperature-responsive block copolymer is dissolved in a buffer solution with a pH of 6.5 to 7.5 to prepare a copolymer solution with a concentration of 1.0 to 10 g / L; the amphiphilic temperature-responsive block copolymer is polyethylene glycol-block-poly(N-isopropylacrylamide), which includes hydrophilic polyethylene glycol segments and temperature-sensitive poly(N-isopropylacrylamide) segments, which are covalently linked together;

[0051] Step 2, Mixing and Infiltration: Mix the Haematococcus pluvialis powder with the copolymer solution obtained in Step 1 to form a mixed slurry, and stir at a temperature of 10°C to 25°C for 30 to 180 minutes;

[0052] Step 3, thermally triggered separation: The mixed system after step 2 is heated to 32°C to 45°C and kept at this temperature for 10 to 60 minutes to allow the system to separate into phases. The condensed phase rich in water-soluble proteins is then collected.

[0053] Step 4, Protein Recovery: The condensed phase collected in Step 3 is dialyzed and dried to obtain water-soluble protein products from Haematococcus pluvialis.

[0054] In step one, the buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution;

[0055] A reducing agent, namely dithiothreitol, is also added to the copolymer solution at a concentration of 0.1 g / L to 1.0 g / L.

[0056] In step two, the mass-to-volume ratio of *Rhodophyta globulinii* powder to copolymer solution in the mixed slurry is from 1 g to 30 ml to 1 g to 100 ml, based on dry weight.

[0057] In step two, intermittent ultrasonic treatment is performed while stirring, with an ultrasonic power of 50 watts to 300 watts, an ultrasonic time of 1 minute to 3 minutes per session, an interval of 2 minutes, and a total ultrasonic time of no more than 15 minutes.

[0058] In step four, the dialysate used in the dialysis is a phosphate buffer solution with a concentration of 0.002 mol / L, and the dialysis time is 12 to 48 hours, during which the dialysate is changed 3 to 6 times.

[0059] The preparation method of the amphiphilic temperature-responsive block copolymer includes the following steps:

[0060] Step 1, Preparation of initiator: Methoxy polyethylene glycol is dissolved in an anhydrous solvent and reacted with an acyl halide reagent under an inert atmosphere and ice bath cooling. After purification, a polyethylene glycol macromolecular initiator with a terminal haloacyl group is obtained.

[0061] Step 2, chain extension polymerization: The macromolecular initiator, monomer N-isopropylacrylamide, catalyst and organic ligand obtained in step 1 are placed in a reaction vessel, vacuumed and replaced with inert gas, and then a dehydrating solvent is added. The reaction is stirred at 60°C to 80°C for 6 to 24 hours.

[0062] Step 3, purification and drying: After the reaction in step 2 is completed, the mixture is precipitated, filtered and washed, and then vacuum dried at 30°C to 40°C to obtain the amphiphilic temperature-responsive block copolymer.

[0063] In the first step, the number average molecular weight of the methoxy polyethylene glycol is 2000, the acyl halide reagent is 2-bromoisobutyryl bromide, and the molar ratio of the methoxy polyethylene glycol to 2-bromoisobutyryl bromide is 1:1.5 to 1:2.5.

[0064] In the second step, the molar ratio of the monomer N-isopropylacrylamide to the macromolecular initiator is 50:1 to 200:1;

[0065] The molar ratio of the monomer N-isopropylacrylamide, the catalyst, and the organic ligand is (50-200):1:(1-2).

[0066] The catalyst is cuprous bromide, and the organic ligand is pentamethyldiethylenetriamine.

[0067] In the second step, the functional monomer acrylic acid was also added to the reaction vessel, with the molar ratio of N-isopropylacrylamide to acrylic acid being 30:1 to 10:1.

[0068] This invention integrates the gentle disruption, selective capture, and thermally triggered preliminary separation of intracellular water-soluble proteins from Haematococcus pluvialis into a continuous aqueous process by employing temperature-responsive block copolymers, which simplifies the process and helps maintain protein activity.

[0069] The present invention will be described in detail below through examples and comparative examples, but the scope of protection of the present invention is not limited to these examples. Unless otherwise specified, the chemical reagents and raw materials used in the following examples and comparative examples are all conventional commercially available products.

[0070] Example 1

[0071] This embodiment first prepares the desired polyethylene glycol-block-poly(N-isopropylacrylamide) amphiphilic temperature-responsive block copolymer. 30 g of methoxy polyethylene glycol with a number-average molecular weight of 2000 was dissolved in 150 mL of anhydrous dichloromethane. Under a nitrogen atmosphere and ice-water bath cooling, 2-bromoisobutyryl bromide was added dropwise at a molar ratio of methoxy polyethylene glycol to 2-bromoisobutyryl bromide of 1:2.5. After the addition was complete, the reaction was continued at approximately 0°C for 8 hours. After the reaction was complete, the byproduct salt was removed by filtration, and the filtrate was concentrated by rotary evaporation and then poured into a large amount of pre-cooled anhydrous diethyl ether to precipitate. The white solid was collected by filtration, washed three times with cold diethyl ether, and finally dried under vacuum at 35°C for 12 hours to obtain a polyethylene glycol macromolecular initiator with 2-bromoisobutyryl groups at the end.

[0072] Subsequently, chain extension polymerization was carried out. Five grams of the aforementioned macromolecular initiator, the monomer N-isopropylacrylamide (molar ratio to macromolecular initiator: 200:1), the catalyst cuprous bromide, and the organic ligand pentamethyldiethylenetriamine (molar ratio: 200:1:2) were placed together in a dry Schlenk flask. Anhydrous N,N-dimethylformamide was added as a dehydrating solvent, and the mixture was evacuated and purged with nitrogen three times to remove oxygen. The flask was then placed in an 80°C oil bath, and the reaction was continuously stirred for 24 hours.

[0073] After the reaction was completed, the reaction solution was exposed to air to terminate the reaction, and then it was added dropwise to a large amount of deionized water to precipitate the copolymer. The precipitate was collected by filtration and washed several times with deionized water and methanol in sequence. Finally, it was dried under vacuum at 40°C to constant weight to obtain the target copolymer.

[0074] Next, the water-soluble proteins from *Rhodotorula purpureus* were extracted. 1.0 g of *Rhodotorula purpureus* powder (dry weight) was weighed and added to 100 mL of the above copolymer solution with a concentration of 10 g / L. This solution was prepared by dissolving the copolymer in a pH 7.5 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution and contained 1.0 g / L dithiothreitol as a reducing agent. The mixed slurry was placed in a 10°C water bath and subjected to intermittent ultrasonic treatment at 300 watts, with a 3-minute ultrasonic treatment followed by a 2-minute interval, for a total ultrasonic treatment time of 15 minutes, accompanied by gentle mechanical stirring for 180 minutes.

[0075] The mixture was then transferred to a 40°C water bath and allowed to stand for 60 minutes, during which clear phase separation was observed. The lower protein-rich condensed phase was collected and placed in a dialysis bag with a molecular weight cutoff of 3500. Dialysis was performed with 0.002 mol / L phosphate buffer at 4°C for 48 hours, with the dialysate changed 6 times during this period. Finally, the solution in the dialysis bag was freeze-dried to obtain the water-soluble protein product from *Hydrocotyle vulgaris*.

[0076] Example 2

[0077] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0078] Methoxylated polyethylene glycol with a number average molecular weight of 2000 was reacted with 2-bromoisobutyryl bromide at a molar ratio of 1:1.5 for 6 hours. In the chain extension polymerization step, the molar ratio of monomer N-isopropylacrylamide to macromolecular initiator was 50:1, and the molar ratio of monomer, catalyst cuprous bromide, and organic ligand pentamethyldiethylenetriamine was 50:1:1. The polymerization reaction was carried out at 60°C for 6 hours. For protein extraction, 1.0 g of algal powder was added to 30 mL of a 1.0 g / L copolymer solution (pH 6.5, containing 0.1 g / L dithiothreitol). The solution was sonicated at 25°C with 50 W for 1 minute, followed by 2-minute intervals for a total of 5 minutes, while stirring for 30 minutes. The solution was then allowed to stand at 45°C for 10 minutes for phase separation. The collected condensed phase was dialyzed against the same buffer for 12 hours, with three buffer changes, and finally lyophilized to obtain the product.

[0079] Example 3

[0080] In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows:

[0081] This embodiment introduces functional monomers into the copolymer preparation. First, the molar ratio of methoxy polyethylene glycol to 2-bromoisobutyryl bromide was set to 1:2.0, and the reaction was carried out for 7 hours. During chain extension polymerization, in addition to N-isopropylacrylamide (molar ratio to macromolecular initiator 125:1), acrylic acid was also added, with a molar ratio of N-isopropylacrylamide to acrylic acid of 10:1. The molar ratio of monomer, cuprous bromide, and pentamethyldiethylenetriamine was 125:1:1.5. Polymerization was carried out at 70°C for 15 hours. During the extraction stage, 1.0 g of algal powder was added to 65 mL of a copolymer solution with a concentration of 5.5 g / L (pH 7.0, containing 0.55 g / L dithiothreitol). The solution was ultrasonicated at 175 W for 2 minutes, with 2-minute intervals, for a total of 10 minutes, while simultaneously stirring for 105 minutes. Subsequently, the solution was allowed to stand at 35°C for 35 minutes for phase separation. The condensed phase was dialyzed for 30 hours, the medium was changed 4 times, and then it was freeze-dried.

[0082] Comparative Example 1

[0083] In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows:

[0084] In the copolymer solution of step one, the amphiphilic temperature-responsive block copolymer was not added at all, and the extraction operation was carried out using only a sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer (containing an equal amount of dithiothreitol) with the same pH and volume.

[0085] Comparative Example 2

[0086] In this comparative example, the similarities with Example 2 will not be repeated, and the differences are as follows:

[0087] In step two, the mixed slurry is directly placed at 45℃ (i.e., the separation temperature) for stirring and extraction, which eliminates the low-temperature permeation step, while the other conditions remain unchanged.

[0088] Comparative Example 3

[0089] In this comparative example, the similarities with Example 3 will not be repeated, and the differences are as follows:

[0090] The amphiphilic temperature-responsive block copolymer used was replaced with a common, non-block poly(N-isopropylacrylamide) homopolymer with a number-average molecular weight similar to that of the poly(N-isopropylacrylamide) segments in the copolymer prepared in Example 3.

[0091] Comparative Example 4

[0092] In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows:

[0093] The copolymers used are different. In the preparation of this copolymer, no acrylic functional monomers were added in the second-step chain extension polymerization; it consists only of polyethylene glycol and poly(N-isopropylacrylamide) segments.

[0094] Comparative Example 5

[0095] In this comparative example, the similarities with Example 2 will not be repeated, and the differences are as follows:

[0096] After the thermally triggered separation in step three, the condensed phase is not collected. Instead, the entire mixed system is centrifuged at high speed, and all the supernatant is collected, followed by the addition of ammonium sulfate to the supernatant to 70% saturation for salting out. The precipitate is then collected and dialyzed and freeze-dried.

[0097] Comparative Example 6

[0098] In this comparative example, the similarities with Example 3 will not be repeated, and the differences are as follows:

[0099] In the mixing and permeation process of step two, no ultrasonic treatment is used; only mechanical stirring is performed for the same amount of time.

[0100] Performance Test Results and Analysis

[0101] To evaluate the extraction efficiency, the products obtained in the examples and comparative examples were tested using the following methods: the total protein concentration in the extracted products was determined using the Coomassie Brilliant Blue method, and the protein extraction rate relative to the raw algal powder was calculated. The characteristic peak area of ​​phycoerythrin in the products was analyzed by high-performance liquid chromatography (HPLC), and the relative purity of phycoerythrin in the total protein was calculated. Simultaneously, the molecular weight distribution of the protein products was observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All tests were performed in triplicate, and the results were averaged. The specific test results are shown in Table 1.

[0102] As shown in Table 1, all three embodiments of the present invention achieved good extraction results. The total protein extraction rate exceeded 65%, the relative purity of phycoerythrin was close to or exceeded 50%, and electrophoresis showed clear target protein bands with few non-target impurity proteins. This verifies the overall effectiveness of the present technical solution, namely, that by utilizing polyethylene glycol-block-poly(N-isopropylacrylamide) as a specific block copolymer, efficient extraction and preliminary purification of proteins can be achieved under mild conditions.

[0103] Table 1 Analysis of Test Results

[0104] serial number Total protein extraction rate (%) Phycoerythrin relative purity (%) Electrophoretic band main band clarity Example 1 68.5 52.3 Clear, with few background impurities Example 2 65.2 49.8 Clear, with few background impurities Example 3 71.8 58.1 Very clear, clean background Comparative Example 1 22.4 18.7 Diffusion, multiple stray bands Comparative Example 2 45.6 35.2 The main band is weak, and the background is dark. Comparative Example 3 58.1 41.5 The bands are blurry and have a trailing effect. Comparative Example 4 66.7 50.5 Clear Comparative Example 5 61.0 46.9 Multiple miscellaneous straps, the main strap is still visible. Comparative Example 6 63.1 48.2 Clear

[0105] The results of Comparative Example 1 are the most intuitive. Without using the copolymer, the extraction rate and purity decreased significantly with only buffer and ultrasound. The electrophoresis pattern showed a diffuse appearance, which indicates that the copolymer is not a simple auxiliary agent in this method, but a core substance for achieving selective extraction and separation. Its indispensable role has been confirmed.

[0106] Comparative Example 2 used high-temperature extraction, and its extraction effect was significantly worse than that of Example 2, which was carried out at low temperature. This shows that the "low-temperature penetration binding" step designed in this invention is crucial for the effective and gentle binding of copolymers and proteins, and direct high temperature is not conducive to the binding process.

[0107] Comparative Example 3 used a conventional poly(N-isopropylacrylamide) homopolymer instead of a block copolymer, and its performance indicators were all lower than those of the corresponding examples. This is mainly because the poly(N-isopropylacrylamide) homopolymer lacks hydrophilic polyethylene glycol segments, resulting in poor amphiphilicity and dispersibility in the aqueous phase. Its interaction with the cell wall and its affinity for and protein capture ability may be inferior to those of the block copolymer, leading to a decrease in its efficiency and selectivity.

[0108] The comparison between Comparative Example 4 and Example 1 demonstrates the potential advantages of introducing acrylic functional monomers. Although the extraction rate of Comparative Example 4 was not significantly different from that of Example 1, its phycoerythrin purity was noticeably improved. This suggests that the carboxyl groups introduced into the copolymer segments may enhance the selective binding ability to specific target proteins (such as phycoerythrin) through additional electrostatic interactions.

[0109] Comparative Example 5 simulated the traditional salting-out process, and its extraction rate and purity were both lower than those of Example 2, which directly used thermally triggered phase separation. Furthermore, electrophoresis showed more impurity bands. This demonstrates that the thermally triggered separation step of the present invention, compared to traditional ammonium sulfate salting-out, simplifies the operation while achieving better primary separation selectivity and reducing the co-precipitation of impurities.

[0110] Comparative Example 6 omitted ultrasonic treatment, and its extraction rate and purity were slightly lower than those of the complete Example 3. This shows that intermittent ultrasound, as an auxiliary physical cell disruption method, has a synergistic effect with the chemical permeation of the copolymer and can more effectively promote the release of intracellular proteins. However, it is not an irreplaceable core step, and mechanical stirring itself can also achieve most of the functions.

[0111] Test results show that the use of block copolymers, the temperature program for low-temperature binding and thermally triggered separation, and the introduction of functional monomers in this invention together constitute a complete method for the gentle, efficient, and selective separation of water-soluble proteins from Haematococcus pluvialis.

[0112] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for separating intracellular water-soluble proteins from Haematococcus pluvialis, characterized in that, The method includes the following steps: Step 1: Preparation of the extraction medium: The amphiphilic temperature-responsive block copolymer is dissolved in a buffer solution with a pH of 6.5 to 7.5 to prepare a copolymer solution with a concentration of 1.0 to 10 g / L; the amphiphilic temperature-responsive block copolymer is polyethylene glycol-block-poly(N-isopropylacrylamide), which includes hydrophilic polyethylene glycol segments and temperature-sensitive poly(N-isopropylacrylamide) segments, which are covalently linked; acrylic acid is added during the preparation of the amphiphilic temperature-responsive block copolymer, and the molar ratio of N-isopropylacrylamide to acrylic acid is 10:1; Step 2, Mixing and Infiltration: Mix the Haematococcus pluvialis powder with the copolymer solution obtained in Step 1 to form a mixed slurry, and stir at a temperature of 10°C to 17°C for 30 to 180 minutes; Step 3, thermally triggered separation: The mixed system after step 2 is heated to 32°C to 45°C and kept at this temperature for 10 to 60 minutes to allow the system to separate into phases. The condensed phase rich in water-soluble proteins is then collected. Step 4, Protein Recovery: The condensed phase collected in Step 3 is dialyzed and dried to obtain water-soluble protein products from Haematococcus pluvialis.

2. The method for separating intracellular water-soluble proteins from *Rhodochophora* according to claim 1, characterized in that, In step one, the buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution; a reducing agent with a concentration of 0.1 g / L to 1.0 g / L, namely dithiothreitol, is also added to the copolymer solution.

3. The method for separating intracellular water-soluble proteins from *Rhodochophora* according to claim 1, characterized in that, In step two, the mass-to-volume ratio of *Rhodophyta globulinii* powder to copolymer solution in the mixed slurry is from 1 g to 30 ml to 1 g to 100 ml, based on dry weight.

4. The method for separating intracellular water-soluble proteins from *Rhodochophora* according to claim 1, characterized in that, In step two, intermittent ultrasonic treatment is performed while stirring, with an ultrasonic power of 50 watts to 300 watts, an ultrasonic time of 1 minute to 3 minutes per session, an interval of 2 minutes, and a total ultrasonic time of no more than 15 minutes.

5. The method for separating intracellular water-soluble proteins from *Rhodochophora* according to claim 1, characterized in that, In step four, the dialysate used in the dialysis is a phosphate buffer solution with a concentration of 0.002 mol / L, and the dialysis time is 12 to 48 hours, during which the dialysate is changed 3 to 6 times.

6. The method for separating intracellular water-soluble proteins from *Rhodochophora* according to claim 1, characterized in that, The preparation method of the amphiphilic temperature-responsive block copolymer includes the following steps: Step 1, Preparation of initiator: Methoxy polyethylene glycol is dissolved in an anhydrous solvent and reacted with an acyl halide reagent under an inert atmosphere and ice bath cooling. After purification, a polyethylene glycol macromolecular initiator with a terminal haloacyl group is obtained. Step 2, chain extension polymerization: The macromolecular initiator, monomer N-isopropylacrylamide, acrylic acid, catalyst and organic ligand obtained in step 1 are placed in a reaction vessel, vacuumed and replaced with inert gas, and then a dehydrating solvent is added. The reaction is stirred at 60°C to 80°C for 6 to 24 hours. Step 3, purification and drying: After the reaction in step 2 is completed, the mixture is precipitated, filtered and washed, and then vacuum dried at 30°C to 40°C to obtain the amphiphilic temperature-responsive block copolymer.

7. The method for separating intracellular water-soluble proteins from *Rhodochophora* according to claim 6, characterized in that, In the first step, the number average molecular weight of the methoxy polyethylene glycol is 2000, the acyl halide reagent is 2-bromoisobutyryl bromide, and the molar ratio of the methoxy polyethylene glycol to 2-bromoisobutyryl bromide is 1:1.5 to 1:2.

5.

8. The method for separating intracellular water-soluble proteins from *Rhodochophora* according to claim 6, characterized in that, In the second step, the molar ratio of the monomer N-isopropylacrylamide to the macromolecular initiator is 50:1 to 200:1; the molar ratio of the monomer N-isopropylacrylamide, the catalyst, and the organic ligand is (50-200):1:(1-2).

9. The method for separating intracellular water-soluble proteins from *Rhodochophora* according to claim 6, characterized in that, The catalyst is cuprous bromide, and the organic ligand is pentamethyldiethylenetriamine.