A method for rapid isolation and purification of rubisco from microalgae

Abstract

The application discloses a method for rapidly separating and purifying Rubisco from microalgae. The method comprises the following steps: filtering supernatant of a crude protein extract solution after breaking microalgae; combining the supernatant of the crude protein extract solution with anion exchange magnetic beads; separating the magnetic beads and eluting the magnetic beads with elution buffer containing 40-550 mM NaCl in sequence to obtain Rubisco of the microalgae. Experiments prove that the method for rapidly separating and purifying Rubisco can separate and purify Rubisco from Chlamydomonas reinhardtii CC-125 strain, is simple to operate, needs only one-step directional enrichment, is short in time consumption, and the overall process is less than 3 hours, and more importantly, the obtained Rubisco is high in purity and few in impure proteins. Therefore, the method provided by the application can rapidly separate and purify Rubisco and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for rapidly isolating and purifying Rubisco from microalgae. Background Technology

[0002] Plants, algae, and certain bacteria convert solar energy into more stable energy sources within their bodies through photosynthesis. Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) is a key enzyme in photosynthesis that catalyzes CO2 fixation and is one of the most abundant proteins in nature. Rubisco not only determines the photosynthetic rate of plants but has also received widespread attention in recent years as a novel food protein resource.

[0003] Rubisco comprises 30-60% of plant-soluble proteins and can be directly extracted from biomass such as plant leaves, algae, and duckweed. Its production process generates significantly less carbon emissions than that of animal protein sources. Furthermore, Rubisco possesses excellent food-grade properties, such as a balanced amino acid composition, rich in essential amino acids, high protein digestibility, good thermal stability, neutral flavor, light color, and excellent gelling properties. Therefore, Rubisco holds significant application potential in the food industry and functional protein fields.

[0004] However, due to the significant differences in cell structure among different photosynthetic organisms, existing Rubisco separation and purification techniques mainly rely on multi-step column chromatography processes, including ammonium sulfate precipitation, gel filtration chromatography, and ion exchange chromatography. These methods have the following shortcomings: (1) The purification process is cumbersome and time-consuming, usually requiring 2-3 days; (2) It heavily relies on expensive chromatography equipment, requiring the use of protein purification systems such as AKTA; (3) Sample loss is severe, with multiple transfers causing protein loss and decreased enzyme activity; (4) It is difficult to prepare on a large scale; (5) The sample has a high content of impurities and proteins, affecting purification efficiency. Therefore, it is of great significance to develop a simple, low-cost, fast purification method for Rubisco that is suitable for crude microalgae extraction systems. Summary of the Invention

[0005] The purpose of this invention is to rapidly isolate and purify Rubisco from microalgae under conventional experimental conditions without the need for a complex chromatography system.

[0006] This invention first protects a method for rapidly isolating and purifying Rubisco from microalgae, which may include the following steps: (1) After crushing the microalgae, filter to obtain the supernatant of the crude protein extract; (2) Combine the supernatant of the crude protein extract obtained in step (1) with anion exchange magnetic beads; (3) After completing step (2), separate the magnetic beads and elute them sequentially with a gradient of elution buffer containing 40-550 mM NaCl to obtain Rubisco of microalgae.

[0007] In the above method, step (1) of breaking microalgae can specifically involve uniformly mixing microalgae and lysis buffer, and then deeply breaking them.

[0008] The solute and concentration of the lysis buffer may specifically be 10-20 mM (e.g., 10-15 mM, 15-20 mM, 10 mM, 15 mM or 20 mM) NaHCO3, 10-20 mM (e.g., 10-15 mM, 15-20 mM, 10 mM, 15 mM or 20 mM) MgCl2 and 0.5-1.0 mM (e.g., 0.5-0.8 mM, 0.8-1.0 mM, 0.5 mM, 0.8 mM or 1.0 mM) PMSF, and the solvent may be pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.6-8.0, pH 7.0, pH 7.6 or pH 8.0) or 20-30 mM (e.g., 20-25 mM, 25-30 mM, 20 mM, 25 mM or 30 mM) Tris-HCl buffer.

[0009] In the above method, the amount of lysis buffer used is determined according to the mass (specifically, wet weight) of the microalgae. The mass (wet weight) to volume ratio of the microalgae and the lysis buffer is 1g:10-15mL (e.g., 1g:10-13mL, 1g:13-15mL, 1g:10mL, 1g:13mL or 1g:15mL).

[0010] In the above method, step (1) of breaking down microalgae further includes a homogenization step. Specifically, the microalgae are homogenized and broken down, tissue fragments are removed, and then the mixture is homogenized with lysis buffer before being deeply broken down.

[0011] The homogenization and crushing can be achieved by mechanical stirring.

[0012] The removal of tissue fragments can be achieved by filtering the homogenate obtained by breaking up the homogenate.

[0013] The aforementioned deep disruption can be performed in a high-pressure cell disruptor at a pressure of 800-1200 bar.

[0014] The crude protein extract of microalgae is obtained after any of the above-mentioned deep crushing processes.

[0015] In the above method, in step (1), the filtration can be the process of filtering the crude protein extract of microalgae through gauze.

[0016] In the above method, in step (2), the amount of anion exchange magnetic beads added can be 5-25% of the volume of the supernatant of the crude protein extract (e.g., 5-10%, 10-15%, 15-20%, 20-25%, 5%, 10%, 15%, 20% or 25%).

[0017] Any of the anion exchange magnetic beads mentioned above can be DEAE magnetic beads.

[0018] In the above method, in step (2), the binding is 2-8℃ (such as 2-4℃, 4-8℃, 2℃, 4℃ or 8℃) for 30-90min (such as 30-60min, 60-90min, 30min, 60min or 90min).

[0019] In the above method, in step (3), the elution buffer containing 40-550mM NaCl can specifically be Washbuffer 1, Wash buffer 2, Wash buffer 3 and Wash buffer 4.

[0020] The solute and concentration of Wash buffer 1 are 40-60mM (e.g., 40-50mM, 50-60mM, 40mM, 50mM or 60mM) NaCl, 10-20mM (e.g., 10-15mM, 15-20mM, 10mM, 15mM or 20mM) NaHCO3 and 10-20mM (e.g., 10-15mM, 15-20mM, 10mM, 15mM or 20mM) MgCl2, and the solvent is pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.6-8.0, pH 7.0, pH 7.6 or pH 8.0) or 20-30mM (e.g., 20-25mM, 25-30mM, 20mM, 25mM or 30mM) Tris-HCl buffer.

[0021] The solute and concentration of Wash buffer 2 are 90-110 mM (e.g., 90-100 mM, 100-110 mM, 90 mM, 100 mM or 110 mM) NaCl, 10-20 mM (e.g., 10-15 mM, 15-20 mM, 10 mM, 15 mM or 20 mM) NaHCO3 and 10-20 mM (e.g., 10-15 mM, 15-20 mM, 10 mM, 15 mM or 20 mM) MgCl2, and the solvent is pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.6-8.0, pH 7.0, pH 7.6 or pH 8.0) or 20-30 mM (e.g., 20-25 mM, 25-30 mM, 20 mM, 25 mM or 30 mM) Tris-HCl buffer.

[0022] The solute and concentration of Wash buffer 3 are 240-260mM (e.g., 240-250mM, 250-260mM, 240mM, 250mM or 260mM) NaCl, 10-20mM (e.g., 10-15mM, 15-20mM, 10mM, 15mM or 20mM) NaHCO3 and 10-20mM (e.g., 10-15mM, 15-20mM, 10mM, 15mM or 20mM) MgCl2, and the solvent is pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.6-8.0, pH 7.0, pH 7.6 or pH 8.0) or 20-30mM (e.g., 20-25mM, 25-30mM, 20mM, 25mM or 30mM) Tris-HCl buffer.

[0023] The solute and concentration of Wash buffer 4 are 450-550mM (e.g., 450-500mM, 500-550mM, 450mM, 500mM or 550mM) NaCl, 10-20mM (e.g., 10-15mM, 15-20mM, 10mM, 15mM or 20mM) NaHCO3 and 10-20mM (e.g., 10-15mM, 15-20mM, 10mM, 15mM or 20mM) MgCl2, and the solvent is pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.6-8.0, pH 7.0, pH 7.6 or pH 8.0) or 20-30mM (e.g., 20-25mM, 25-30mM, 20mM, 25mM or 30mM) Tris-HCl buffer.

[0024] In any of the methods described above, the elution time of Wash buffer 1, Wash buffer 2, Wash buffer 3 and Wash buffer 4 is 5-15 min (e.g. 5-10 min, 10-15 min, 5 min, 10 min or 15 min).

[0025] In any of the methods described above, the elution volumes of Wash buffer 1 and Wash buffer 2 can be 25-35 mL (e.g., 25-30 mL, 30-35 mL, 25 mL, 30 mL, or 35 mL).

[0026] In any of the methods described above, the elution volumes of Wash buffer 3 and Wash buffer 4 can be 5-15 mL (e.g., 5-10 mL, 10-15 mL, 5 mL, 10 mL or 15 mL).

[0027] Any of the microalgae mentioned above may be from the genus Chlamydomonas (Chlamydomonas Microalgae.

[0028] Any of the above-mentioned Chlamydomonas species ( Chlamydomonas The microalgae that can be specifically Chlamydomonas reinhardtii ( ) Chlamydomonas reinhardii ).

[0029] Any of the above-mentioned Chlamydomonas reinhardtii ( Chlamydomonas reinhardii It can be the Chlamydomonas reinhardtii CC-125 strain.

[0030] Experiments have demonstrated that the method provided by this invention can isolate and purify Rubisco from the Chlamydomonas reinhardtii CC-125 strain. It is not only simple to operate, requiring only one step of targeted enrichment, but also quick, with the entire process taking ≤3 hours. More importantly, it yields Rubisco with high purity and very few contaminating proteins. Therefore, the method provided by this invention can rapidly isolate and purify Rubisco and has significant application value. Attached Figure Description

[0031] Figure 1 This is the supernatant of the crude protein extract of Chlamydomonas reinhardtii in step 1 of Example 1.

[0032] Figure 2 The centrifuge tubes and beakers after step 3 in Example 1 are shown.

[0033] Figure 3 The centrifuge tubes and beakers after completing steps (1), (2), (3) and (4) in step 4 of Example 1.

[0034] Figure 4 SDS-PAGE analysis of samples Wc, S, FT, W1, W2, W3, and W4 in step 5 of Example 1. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] The solutes and their concentrations in the lysis buffer were 10 mM NaHCO3, 10 mM MgCl2 and 1 mM PMSF (JSENB, CAS No.: 329-98-6), and the solvent was pH 7.6, 25 mM Tris-HCl buffer.

[0038] The *Chlamydomonas reinhardtii* CC-125 strain used in this embodiment can be obtained from the Freshwater Algae Culture Collection, Chinese Academy of Sciences (FACHB, address: No. 7, East Lake South Road, Wuchang District, Wuhan, telephone: 027-68780871), where its accession number is FACHB-2218. In the following text, the *Chlamydomonas reinhardtii* CC-125 strain will be referred to as *Chlamydomonas reinhardtii*.

[0039] Example 1: Rapid isolation and purification of Rubisco from Chlamydomonas reinhardtii The anion exchange magnetic beads are specifically DEAE magnetic beads (BeaverBeads, product code: 70809-5).

[0040] 1. Collect *Chlamydomonas reinhardtii* cultured for three weeks, homogenize using mechanical stirring to obtain a homogenate; then filter the homogenate (to remove tissue fragments) and collect the filtrate. Next, mix the filtrate with lysis buffer and transfer to a high-pressure cell disruptor for deep disruption at 800-1200 bar to obtain a crude protein extract of *Chlamydomonas reinhardtii* (this sample is designated Wc sample). Filter the crude protein extract through three layers of gauze to obtain the supernatant of the crude protein extract (this sample is designated S sample).

[0041] The amount of lysis buffer used depends on the mass of Chlamydomonas reinhardtii (specifically, wet weight). The mass-to-volume ratio of Chlamydomonas reinhardtii to lysis buffer is 1g:10-15mL (in actual operation, it is 1g:10mL).

[0042] The supernatant of the crude protein extract of Chlamydomonas reinhardtii is shown below. Figure 1 .

[0043] 2. After completing step 1, mix 25 mL of the supernatant of Chlamydomonas reinhardtii crude protein extract and 5 mL of DEAE magnetic beads (in actual operation, the amount of DEAE magnetic beads added is 5-25% of the volume of the supernatant of Chlamydomonas reinhardtii crude protein extract) in a 50 mL centrifuge tube and incubate at 2-8℃ for 30-90 min (in actual operation, incubate at 4℃ for 1 h).

[0044] 3. After completing step 2, take the centrifuge tube and separate the magnetic beads using an external magnetic field. Pour the liquid that has not bound to the magnetic beads into a beaker (this sample is then referred to as the FT sample). The purpose of this step is to remove unbound proteins.

[0045] See details of the centrifuge tubes and beakers after completing step 3. Figure 2 .

[0046] 4. Gradient salt elution Take the magnetic beads separated in step 3 and elute them sequentially with elution buffers of different salt concentrations (i.e., Wash buffer 1, Wash buffer 2, Wash buffer 3, and Wash buffer 4) (the purpose is to remove non-specifically bound proteins). Details are as follows: (1) Take the magnetic beads separated in step 3, wash with 30 mL Wash buffer 1 for 10 min, and then pour the liquid into a beaker (at this time, the sample is recorded as sample W1).

[0047] Wash buffer 1 contains 50 mM NaCl, 10 mM NaHCO3, and 10 mM MgCl2 as solutes and their concentrations, and 25 mM Tris-HCl buffer at pH 7.6 as the solvent.

[0048] (2) Take the magnetic beads from step (1) and wash them with 30 mL Wash buffer 2 for 10 min. Then pour the liquid into a beaker (at this time, the sample is recorded as sample W2).

[0049] Wash buffer 2 contains 100 mM NaCl, 10 mM NaHCO3, and 10 mM MgCl2 as solutes and their concentrations, and 25 mM Tris-HCl buffer at pH 7.6 as the solvent.

[0050] (3) Take the magnetic beads from step (2) and wash them with 15 mL Wash buffer 3 for 10 min. Then pour the liquid into a beaker (at this time, the sample is recorded as sample W3).

[0051] Wash buffer 3 contains 250 mM NaCl, 10 mM NaHCO3, and 10 mM MgCl2 as solutes and their concentrations, and 25 mM Tris-HCl buffer at pH 7.6 as the solvent.

[0052] (4) Take the magnetic beads from step (3) and wash them with 15 mL Wash buffer 4 for 10 min. Then pour the liquid into a beaker (at this time, the sample is recorded as sample W4).

[0053] Wash buffer 4 contains 500 mM NaCl, 10 mM NaHCO3, and 10 mM MgCl2 as solutes and their concentrations, and 25 mM Tris-HCl buffer at pH 7.6 as the solvent.

[0054] For details of the centrifuge tubes and beakers after completing steps (1), (2), (3), and (4), please refer to [link to documentation]. Figure 3 .

[0055] 5. Detection of elution components Samples Wc, S, FT, W1, W2, W3, and W4 were analyzed by 15% denaturing polyacrylamide gel electrophoresis (SDS-PAGE) combined with Coomassie brilliant blue staining.

[0056] Test results are shown Figure 4 (M represents the molecular weight standard of the protein marker (kDa), and the red arrows indicate the positions of the Rubisco large subunit (approximately 55 kDa) and small subunit (approximately 15 kDa). The results show that both the Rubisco large and small subunit bands are clearly visible in sample W3; the Rubisco large subunit is purer in sample W4.

[0057] Therefore, the above method can rapidly isolate Rubisco from Chlamydomonas reinhardtii, and Rubisco has high purity and very few impurities.

[0058] In summary, the method provided in this application can isolate and purify Rubisco from microalgae and has the following advantages: simple operation, low cost, only one-step directional enrichment, no need for chromatography column separation and complex equipment; short processing time: the whole process is ≤3 hours; the native conformation is intact and the Rubisco activity is well maintained; and it is easy to scale up for production.

[0059] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for rapidly isolating and purifying Rubisco from Chlamydomonas reinhardtii, comprising the following steps: (1) After crushing Chlamydomonas reinhardtii, filter to obtain the supernatant of crude protein extract; (2) Combine the supernatant of the crude protein extract obtained in step (1) with DEAE magnetic beads; (3) After completing step (2), separate the magnetic beads and elute them sequentially with Wash buffer 1, Wash buffer 2 and Wash buffer 3. Collect the eluent of Wash buffer 3 to obtain Rubisco of Chlamydomonas reinhardtii. In step (1), the specific process of breaking Chlamydomonas reinhardtii involves breaking the Chlamydomonas reinhardtii homogenate, removing tissue fragments, mixing it evenly with lysis buffer, and then deeply breaking it. The solutes and their concentrations in the lysis buffer are 10-20 mM NaHCO3, 10-20 mM MgCl2 and 0.5-1.0 mM MMSF, and the solvent is a pH 7.0-8.0, 20-30 mM Tris-HCl buffer. In step (3), the solute and concentration of Wash buffer 1 are 50 mM NaCl, 10 mM NaHCO3 and 10 mM MgCl2, and the solvent is pH 7.6, 25 mM Tris-HCl buffer; the solute and concentration of Wash buffer 2 are 100 mM NaCl, 10 mM NaHCO3 and 10 mM MgCl2, and the solvent is pH 7.6, 25 mM Tris-HCl buffer; the solute and concentration of Wash buffer 3 are 250 mM NaCl, 10 mM NaHCO3 and 10 mM MgCl2, and the solvent is pH 7.6, 25 mM Tris-HCl buffer. The elution time for Wash buffer 1, Wash buffer 2 and Wash buffer 3 is 5-15 minutes; The elution volume of Wash buffer 1 and Wash buffer 2 is 25-35 mL; The elution volume of Wash buffer 3 is 5-15 mL.

2. The method according to claim 1, characterized in that: The mass-to-volume ratio of Chlamydomonas reinhardtii to lysis buffer is 1 g: 10-15 mL.

3. The method according to claim 1, characterized in that: In step (2), the amount of DEAE magnetic beads added is 5-25% of the volume of the supernatant of the crude protein extract.

4. The method according to claim 1, characterized in that: In step (2), the bonding is performed at 2-8℃ for 30-90 minutes.

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