A method for producing high-purity SOD by cell culture

Abstract

The present application relates to the technical field of natural product separation, in particular to a preparation method for producing high-purity SOD by cell culture method. The present application takes sterile suspension cells of Portulaca oleracea as raw material, adopts a two-step culture process in stages: in the first stage, the cells are rapidly proliferated in a suitable environment to increase biomass, and in the second stage, the endogenous antioxidant metabolic pathway in the plant cells is activated in situ by mild saline-alkaline, weak light and trace metal ion stress, so that SOD is highly expressed. In combination with low-temperature mild cell wall breaking, double-gradient salting-out impurity removal and gel chromatography deep refining process, pharmaceutical-grade high-purity SOD is finally obtained. The present application is completely free of microorganisms, exogenous inducing bacteria, agricultural residues and seasonal restrictions, and has very high batch stability, which completely solves the technical pain points of traditional field extraction, such as many impurities, low purity, poor stability, low yield of conventional cell culture, etc. The process is green and controllable, and is suitable for standardized factory production.

Description

Technical Field

[0001] This invention relates to the field of natural product separation technology, and in particular to an industrial production method for preparing high-purity superoxide dismutase based on two-step culture of sterile suspended cells, induction by combined physicochemical stress, and fine purification. Background Technology

[0002] Superoxide dismutase (SOD) is a core functional antioxidant enzyme in living organisms that specifically scavenge superoxide anion free radicals. It is widely used in high-end cosmetics, pharmaceutical anti-inflammatory agents, functional foods, bio-preservation, and skin repair. However, existing SOD production processes have significant technical defects.

[0003] Traditional plant field extraction methods rely on field cultivation and are greatly affected by seasons, climate, pests and diseases, soil heavy metals, and pesticide residues. The natural plant's SOD content is extremely low, and it contains complex impurities such as pigments, polysaccharides, and miscellaneous proteins. The purity of the finished product from conventional processes is generally below 85%, which cannot meet the access requirements for pharmaceutical-grade and high-end cosmetic raw materials.

[0004] Microbial fermentation: The product contains a large amount of bacterial protein, metabolic byproducts, and endotoxins, resulting in high purification costs; moreover, the enzyme-protein structure has poor adaptability and weak storage stability.

[0005] The shortcomings of existing plant cell culture technologies: Most of the currently disclosed plant cell culture SOD processes use single constant culture conditions and lack targeted enzyme production induction mechanisms. The cells only express SOD in a basic manner, resulting in extremely low yield and poor activity, making them unsuitable for industrialization. A small number of stress-induced technologies are simple and lack stepwise regulatory logic, failing to balance "biomass accumulation" and "enrichment of highly active enzymes," resulting in poor overall technical performance and no substantial industrialization value. Summary of the Invention

[0006] In view of this, the purpose of this invention is to develop a high-purity SOD mass production process using pure plant cells, without microbial involvement, and with complete aseptic control, in order to overcome the following existing technological bottlenecks:

[0007] Firstly, traditional plant-derived SOD has many impurities, low purity, and uncontrollable safety.

[0008] Secondly, conventional suspension cell culture does not induce enzyme production in a targeted manner, resulting in extremely low SOD expression levels.

[0009] Thirdly, a single stress method cannot achieve simultaneous high expression of two subtypes of SOD;

[0010] Fourthly, conventional purification processes suffer from high losses and insufficient purity, making it difficult to achieve the high purity level required for pharmaceutical applications.

[0011] Fifthly, existing processes suffer from significant batch-to-batch variations and cannot be standardized for continuous production.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] This invention provides a method for increasing the yield, enzyme activity and / or purity of SOD during the production process, using purslane to prepare SOD, including a step of stress-induced purslane cells;

[0014] The conditions for stress-induced induction include at least one of the following:

[0015] (i) Reduce carbon source supply;

[0016] (ii) Increase the salinity of the culture medium;

[0017] (iii) Adding metal ions or increasing the concentration of metal ions;

[0018] (iv) Reduce light exposure.

[0019] In some specific embodiments of the present invention, the above method is:

[0020] The reduction in carbon source supply includes adjusting the carbon source content of the culture medium to 18 g / L, 19 g / L, 20 g / L, 21 g / L or 22 g / L;

[0021] The carbon source includes sucrose;

[0022] Increasing the salinity of the culture medium includes adjusting the salt content of the culture medium to 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, or 1.2 g / L;

[0023] The salt includes sodium chloride;

[0024] The addition of metal ions or increase of metal ion concentration includes increasing the Cu content in the culture medium. 2+ Increasing the concentration by 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or 10 nM, Mn 2+ The concentration was increased by 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM or 0.5 μM;

[0025] The reduction in light intensity includes reducing the light intensity to 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the normal light intensity.

[0026] The standard illumination is 4500 lx.

[0027] In some specific embodiments of the present invention, the method for preparing the purslane cells described above includes:

[0028] Young purslane leaves were soaked in ethanol for disinfection, rinsed, and then inoculated into callus induction medium and cultured in the dark to obtain callus tissue.

[0029] The callus tissue was cultured and subcultured 2 or 3 times to obtain purslane cells.

[0030] In some specific embodiments of the present invention, the above method is:

[0031] The concentration of the ethanol includes 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

[0032] The soaking time is 25 s, 26 s, 27 s, 28 s, 29 s, 30 s, 31 s, 32 s, 33 s, 34 s or 35 s;

[0033] The callus induction medium includes MS medium, 0.7 mg / L, 0.71 mg / L, 0.72 mg / L, 0.73 mg / L, 0.74 mg / L, 0.75 mg / L, 0.76 mg / L, 0.77 mg / L, 0.78 mg / L, 0.79 mg / L, 0.8 mg / L, 0.81 mg / L, 0.82 mg / L, 0.83 mg / L, 0.84 mg / L, 0.85 mg / L, 0.86 mg / L, 0.87 mg / L, 0.88 mg / L, 0.89 mg / L or 0.9 mg / L of 2,4-D, and 0.15 mg / L, 0.16 mg / L, 0.17 mg / L, 0.18 mg / L, 0.19 mg / L, 0.20 mg / L, 0.21 mg / L, and 0.22 mg / L of 2,4-D. NAA at concentrations of 0.23 mg / L, 0.24 mg / L, or 0.25 mg / L;

[0034] The conditions for the dark culture include a temperature of 24°C, 25°C, or 26°C, and a time of 18 days, 19 days, 20 days, 21 days, or 22 days.

[0035] This invention also provides a method for preparing SOD, comprising:

[0036] S1: Suspension culture of purslane cells for 10, 11, 12, 13 or 14 days;

[0037] S2: The purslane cells are subjected to stress induction for 5, 6, 7, or 8 days, wherein the stress induction conditions include at least one of the following:

[0038] (i) Reduce carbon source supply;

[0039] (ii) Increase the salinity of the culture medium;

[0040] (iii) Adding metal ions or increasing the concentration of metal ions;

[0041] (iv) Reduce light exposure;

[0042] S3: Collect the purslane cells and isolate SOD.

[0043] In some specific embodiments of the present invention, the culture medium for suspension culture in the above preparation method includes MS medium.

[0044] In some specific embodiments of the present invention, the MS culture medium prepared by the above method comprises ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, potassium iodide, boric acid, zinc manganate sulfate, sodium molybdate, copper sulfate, cobalt chloride, ferrous sulfate, disodium EDTA, inositol, glycine, vitamin B1, vitamin B6, niacin, and sucrose.

[0045] In some specific embodiments of the present invention, the method for preparing the purslane cells in the above preparation method includes:

[0046] Young purslane leaves were soaked in ethanol for disinfection, rinsed, and then inoculated into callus induction medium and cultured in the dark to obtain callus tissue.

[0047] The callus tissue was cultured and subcultured 2 or 3 times to obtain purslane cells.

[0048] In some specific embodiments of the present invention, the above preparation method is as follows:

[0049] The concentration of the ethanol includes 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

[0050] The soaking time is 25 s, 26 s, 27 s, 28 s, 29 s, 30 s, 31 s, 32 s, 33 s, 34 s or 35 s;

[0051] The callus induction medium includes MS medium, 0.7 mg / L, 0.71 mg / L, 0.72 mg / L, 0.73 mg / L, 0.74 mg / L, 0.75 mg / L, 0.76 mg / L, 0.77 mg / L, 0.78 mg / L, 0.79 mg / L, 0.8 mg / L, 0.81 mg / L, 0.82 mg / L, 0.83 mg / L, 0.84 mg / L, 0.85 mg / L, 0.86 mg / L, 0.87 mg / L, 0.88 mg / L, 0.89 mg / L or 0.9 mg / L of 2,4-D, and 0.15 mg / L, 0.16 mg / L, 0.17 mg / L, 0.18 mg / L, 0.19 mg / L, 0.20 mg / L, 0.21 mg / L, and 0.22 mg / L of 2,4-D. NAA at concentrations of 0.23 mg / L, 0.24 mg / L, or 0.25 mg / L;

[0052] The conditions for the dark culture include a temperature of 24°C, 25°C, or 26°C, and a time of 18 days, 19 days, 20 days, 21 days, or 22 days.

[0053] In some specific embodiments of the present invention, the above preparation method is as follows:

[0054] The reduction in carbon source supply includes adjusting the carbon source content of the culture medium to 18 g / L, 19 g / L, 20 g / L, 21 g / L or 22 g / L;

[0055] The carbon source includes sucrose;

[0056] Increasing the salinity of the culture medium includes adjusting the salt content of the culture medium to 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, or 1.2 g / L;

[0057] The salt includes sodium chloride;

[0058] The addition of metal ions or increase of metal ion concentration includes increasing the Cu content in the culture medium. 2+ Increasing the concentration by 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or 10 nM, Mn 2+ The concentration was increased by 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM or 0.5 μM;

[0059] The reduction in light intensity includes reducing the light intensity to 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of the normal light intensity.

[0060] In some specific embodiments of the present invention, the above preparation method is as follows:

[0061] The collection of purslane cells includes centrifugation to collect purslane cells;

[0062] After centrifugation and collection, a resuspension step is also included;

[0063] The centrifugation conditions include a temperature of 4°C and a centrifugation speed of 5500~6500 rpm;

[0064] The resuspension includes resuspension using phosphate buffer at pH 6.8, 6.9, 7.0, 7.1, or 7.2 at 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C.

[0065] The separation includes the steps of cell wall disruption, impurity removal, and purification;

[0066] The cell wall disruption process involves intermittently using ultrasound to disrupt the collected purslane cells to obtain a crude enzyme solution.

[0067] The impurity removal process includes centrifuging the crude enzyme solution, taking the supernatant, and obtaining a clear and transparent crude enzyme solution.

[0068] The purification process includes gradient stepwise salting out, dialysis, and chromatography, including:

[0069] a) The clear and transparent crude enzyme solution is subjected to low salt precipitation, with ammonium sulfate saturation of 32%, 33%, 34%, 35%, 36%, 37%, or 38%;

[0070] b) The clear and transparent crude enzyme solution after low salt precipitation is subjected to high salt precipitation, with ammonium sulfate saturation of 58%, 59%, 60%, 61%, 62%, 63%, or 64%;

[0071] c) Dialyze using a 10 kDa molecular weight cutoff dialysis bag at 2°C, 3°C, 4°C, 5°C, or 6°C;

[0072] d) SOD was collected by Sephadex G-75 dextran gel chromatography.

[0073] The present invention has the following beneficial effects:

[0074] In existing technologies, there is no complete technical solution for preparing high-purity SOD (SOD) with a purity of over 97% through a two-step suspension cell culture coupled with a gradient purification process of "first proliferation, then rational stress-induced enzyme production". This invention develops a high-purity SOD mass production process using pure plant cells, without microbial involvement, and with complete aseptic control, breaking through the bottlenecks of existing technologies. Specifically, it includes:

[0075] This invention belongs to a pure plant cell system, with no microorganisms involved throughout the entire process: it abandons exogenous biological interventions such as strain induction and fermentation symbiosis, and relies entirely on endogenous metabolic regulation of plant cells, resulting in a clean system with a high level of safety.

[0076] This invention features a unique two-step differentiated culture system: strictly distinguishing between the "cell proliferation phase" and the "enzyme enrichment induction phase," first ensuring high biomass, and then directionally activating antioxidant pathways through rational stress, resulting in SOD production that is more than 8 times higher than that of conventional cell culture;

[0077] This invention implements a multi-repetitive rationalized stress dual-subtype simultaneous production increase: through the synergistic mild stress of weak light, slight salt and alkali, and trace metal ions, Cu / Zn-SOD and Mn-SOD are simultaneously induced to accumulate efficiently, and the antioxidant activity of the products is comprehensively superior to that of a single subtype of SOD.

[0078] This invention employs a low-temperature gradient fractional purification process: through two-stage salting out, dialysis for impurity removal, and gel chromatography for precise impurity removal, the finished product has a stable purity of ≥97%, reaching the industry's top pharmaceutical-grade standard.

[0079] This invention enables year-round sterile factory production: it is not limited by season, environment, or climate, has no pesticide residues or heavy metal contamination, and has extremely strong batch consistency, making it suitable for large-scale standardized mass production. Detailed Implementation

[0080] This invention discloses a method for producing high-purity SOD using cell culture. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0081] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0082] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0083] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0084] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0085] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0086] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0087] This invention employs sterile plant suspension cell culture and pure physicochemical stress induction to produce high-purity, high-stability superoxide dismutase (SOD). In short, it uses sterile purslane suspension cells as raw material and employs a two-stage, phased culture process: the first stage involves rapid cell biomass proliferation in a suitable environment; the second stage uses mild salinity, low light, and trace metal ions as synergistic stress to activate endogenous antioxidant metabolic pathways in plant cells, achieving simultaneous and efficient expression of Cu / Zn-SOD and Mn-SOD dual subtypes. Combined with low-temperature gentle cell disruption, dual-gradient salting out for impurity removal, and gel chromatography for deep purification, the final product is pharmaceutical-grade high-purity SOD with a purity ≥97% and an enzyme activity ≥6400 U / mg. This invention is entirely free of microorganisms, exogenous induced bacteria, pesticide residues, and seasonal limitations, exhibiting extremely high batch stability. It completely solves the technical pain points of traditional field extraction, such as high impurities, low purity, poor stability, and low yield from conventional cell culture. The process is green, controllable, and suitable for standardized industrial mass production.

[0088] In its specific implementation, the present invention includes the following steps:

[0089] 1. Induction of aseptic callus from purslane

[0090] Select healthy young purslane leaves, and soak them in 75% ethanol for 25-35 seconds, disinfect them with 0.1% mercuric chloride for 5-7 minutes, and rinse them with sterile pure water 4-6 times.

[0091] Inoculate onto callus induction medium: MS + 2,4-D 0.7~0.9 mg / L + NAA 0.15~0.25 mg / L, sucrose 28~32 g / L, pH 5.7~5.9;

[0092] Incubate in the dark at 24-26℃ for 18-22 days to obtain loose, pale yellow, and uniformly growing sterile callus tissue.

[0093] 2. Establishment of stable suspension cell lines

[0094] Select high-quality callus tissue, transfer it into liquid MS suspension medium, and culture it in a constant temperature shaker at 24~26℃ and 100~120 rpm.

[0095] After 2-3 consecutive subcultures, a sterile purslane suspension cell system with fine particles, good dispersibility, and stable growth was obtained through screening.

[0096] 3. Two-step differential suspension culture

[0097] Step 1: Rapid cell proliferation culture (0~12 days)

[0098] A high-nutrient culture system was used: MS complete nutrition, 30 g / L sucrose, normal light, and no stress.

[0099] Objective: To rapidly accumulate cell biomass and ensure total yield in the later stages.

[0100] Step 2: Induction culture for enzyme production under rational stress (12-18 days)

[0101] The culture system was adjusted to low nutrition + multiple compound stresses to induce SOD overexpression:

[0102] 1) Reduce sucrose to 18-22 g / L, moderate carbon source stress;

[0103] 2) Add 0.8~1.2 g / L NaCl to the culture medium to induce micro-salt and alkali stress;

[0104] 3) Add trace amounts of Cu 2+ Mn 2+ Complex metal ions activate enzyme precursors;

[0105] 4) Culture under low light stress, with light intensity reduced to 30%–40% of normal light intensity;

[0106] After 6 days of continuous induction culture, endogenous dual-subtype SOD was enriched in large quantities.

[0107] 4. Aseptic cell collection and low-temperature gentle cell disruption

[0108] After the induction culture was completed, the cell pellet was collected by low-temperature centrifugation at 4℃ and 5500~6500 rpm.

[0109] Resuspended in a low-temperature phosphate buffer solution with a pH of 6.8–7.2;

[0110] The cell wall is gently broken down by intermittent ultrasonic treatment at 4℃ to avoid denaturation and inactivation of SOD protein caused by high temperature.

[0111] 5. Pretreatment of crude enzyme solution to remove impurities

[0112] Centrifuge the cell wall disruption mixture at low temperature (10,000-12,000 rpm) for 15-20 min to thoroughly remove cell debris and insoluble fiber, obtaining a clear and transparent crude enzyme solution.

[0113] 6. Gradient stepwise salting-out purification

[0114] 1) Low salting-out stage: Ammonium sulfate saturation 32%~38%, precipitation removes macromolecular polysaccharides, pigments, and impurities;

[0115] 2) High salt precipitation stage: Ammonium sulfate saturation 58%~64%, accurately enriching SOD active components;

[0116] Stepwise salting out can remove impurities to the maximum extent, reduce enzyme activity loss, and significantly improve the purity of the finished product.

[0117] 7. Dialysis desalination and deep purification

[0118] A 10 kDa molecular weight cutoff dialysis bag was used for low-temperature dialysis at 4℃ for 10-14 h to completely remove salts and small molecule impurities.

[0119] The sample was then subjected to Sephadex G-75 dextran gel chromatography to collect the single symmetrical SOD activity elution peak and remove trace amounts of residual proteins.

[0120] 8. Low-temperature concentration and vacuum freeze-drying

[0121] After ultrafiltration and low-temperature concentration, vacuum freeze-drying is performed to obtain a pure white, highly active, and high-purity SOD powder product.

[0122] Core technical specifications of the product:

[0123] 1. The purity of the finished product's SOD is ≥97%;

[0124] 2. Total enzyme activity ≥ 6400 U / mg;

[0125] 3. Contains both Cu / Zn-SOD and Mn-SOD dual active isoforms;

[0126] 4. Enzyme activity retention rate ≥82% after 30 min in a 90℃ water bath;

[0127] 5. Good stability over a wide acid-base range of pH 3.0 to 11.0;

[0128] 6. Store at room temperature in a sealed, light-protected container for 18 months; enzyme activity retention ≥92%.

[0129] 7. Sterile, free of pesticide residues, free of heavy metals, and free of pyrogens, meeting the standards for food, cosmetics, and pharmaceutical raw materials.

[0130] The present invention will be further illustrated below with reference to the embodiments.

[0131] Example 1

[0132] Sterile purslane suspension cells were first cultured for 12 days to proliferate, then induced for 6 days by a combined stress of micro-salt alkali + weak light + trace metal ions; followed by low-temperature cell disruption, two-step gradient salting out, and gel chromatography purification. The specific steps are as follows.

[0133] 1. Induction of aseptic callus from purslane

[0134] Select healthy young purslane leaves, and soak them in 75% ethanol for 25 seconds, disinfect them with 0.1% mercuric chloride for 5-7 minutes, and rinse them with sterile pure water 4-6 times.

[0135] Inoculate onto callus induction medium: MS + 2,4-D 0.7~0.9 mg / L + NAA 0.15~0.25 mg / L, sucrose 28~32 g / L, pH 5.7~5.9;

[0136] Incubate in the dark at 24-26℃ for 18-22 days to obtain loose, pale yellow, and uniformly growing sterile callus tissue.

[0137] 2. Establishment of stable suspension cell lines

[0138] Select high-quality callus tissue, transfer it into liquid MS suspension medium, and culture it in a constant temperature shaker at 24~26℃ and 100~120 rpm.

[0139] After 2-3 consecutive subcultures, a sterile purslane suspension cell system with fine particles, good dispersibility, and stable growth was obtained through screening.

[0140] The MS suspension medium formulation is as follows (1 L):

[0141] Ammonium nitrate (NH4NO3) 1650 mg;

[0142] Potassium nitrate (KNO3) 1900 mg;

[0143] Calcium chloride (CaCl2·2H2O) 440 mg;

[0144] Magnesium sulfate MgSO4·7H2O 370 mg;

[0145] Potassium dihydrogen phosphate (KH₂PO₄) 170 mg;

[0146] Potassium iodide (KI) 0.83 mg;

[0147] Boric acid (H3BO3) 6.2 mg;

[0148] Manganese sulfate (MnSO4·4H2O) 22.3 mg;

[0149] Zinc sulfate ZnSO4·7H2O 8.6 mg;

[0150] Sodium molybdate Na₂MoO₄·2H₂O 0.25 mg;

[0151] Copper sulfate CuSO4·5H2O 0.025 mg;

[0152] Cobalt chloride (CoCl₂·6H₂O) 0.025 mg;

[0153] Ferrous sulfate (FeSO4·7H2O) 27.8 mg;

[0154] Disodium ethylenediaminetetraacetate (Na2-EDTA·2H2O) 37.3 mg;

[0155] Inositol 100 mg;

[0156] Glycine 2 mg;

[0157] Thiamine B1 hydrochloride 0.1 mg;

[0158] Pyridoxine B6 hydrochloride 0.5 mg;

[0159] Niacin 0.5 mg;

[0160] 30 g of sucrose;

[0161] Autoclave at 121℃ for 20 minutes.

[0162] 3. Two-step differential suspension culture

[0163] Step 1: Rapid cell proliferation culture (0~12 days)

[0164] A suitable high-nutrient culture system was used: MS complete nutrition (same as the MS suspension medium above), sucrose 30 g / L, normal light (4500 lx), and no stress.

[0165] Step 2: Induction culture for enzyme production under rational stress (12-18 days)

[0166] 1) Reduce sucrose to 18-22 g / L, moderate carbon source stress;

[0167] 2) Add 0.8~1.2 g / L NaCl to the culture medium to induce micro-salt and alkali stress;

[0168] 3) Add trace amounts of Cu 2+ Mn 2+ Complex metal ions (causing Cu) 2+ Concentration increased by 5 nM, Mn 2+ Increasing the concentration by 0.1 μM activates the enzyme precursor;

[0169] 4) Culture under low light stress, with light intensity reduced to 30%–40% of normal light intensity;

[0170] After 6 days of continuous induction culture, endogenous dual-subtype SOD was enriched in large quantities.

[0171] 4. Aseptic cell collection and low-temperature gentle cell disruption

[0172] After the induction culture was completed, the cell pellet was collected by low-temperature centrifugation at 4℃ and 5500~6500 rpm.

[0173] Resuspended in a low-temperature phosphate buffer solution with a pH of 6.8–7.2;

[0174] The cell wall is gently broken down by intermittent ultrasonic treatment at 4℃ to avoid denaturation and inactivation of SOD protein caused by high temperature.

[0175] The parameters for intermittent ultrasonic gentle cell disruption are as follows:

[0176] Time: Ultrasound on for 2 seconds, intermittent off for 6 seconds, single cycle for 8 seconds;

[0177] Power: 120~180W;

[0178] Ice bath temperature: ≤30℃;

[0179] Equipment: 650W probe crusher.

[0180] 5. Pretreatment of crude enzyme solution to remove impurities

[0181] Centrifuge the cell wall disruption mixture at low temperature (10,000-12,000 rpm) for 15-20 min to thoroughly remove cell debris and insoluble fiber, obtaining a clear and transparent crude enzyme solution.

[0182] 6. Gradient stepwise salting-out purification

[0183] 1) Low salting-out stage: Ammonium sulfate saturation 32%~38%, precipitation removes macromolecular polysaccharides, pigments, and impurities;

[0184] 2) High salt precipitation stage: Ammonium sulfate saturation of 58%~64% accurately enriches SOD active components.

[0185] 7. Dialysis desalination and deep purification

[0186] A 10 kDa molecular weight cutoff dialysis bag was used for low-temperature dialysis at 4℃ for 10-14 h to completely remove salts and small molecule impurities.

[0187] The sample was then subjected to Sephadex G-75 dextran gel chromatography to collect the single symmetrical SOD activity elution peak and remove trace amounts of residual proteins.

[0188] Finished product testing: SOD purity 97.1%, total enzyme activity 6480 U / mg, 90℃ heat residue 82.6%, batch stability, and extremely low impurities.

[0189] Comparative Example 1: Conventional constant culture cell group

[0190] The entire process was conducted using a single culture medium (the same MS suspension medium as described above) and without stress induction culture; all other extraction and purification processes were completely consistent.

[0191] Results: The SOD enzyme activity was only 860 U / mg and the purity was 83.5%, which were far lower than those of the present invention.

[0192] Comparative Example 2: Traditional Field Plant Extracts

[0193] The conventional extraction process for the same variety of purslane during field harvesting involves the following steps:

[0194] 1. Raw material pretreatment: Wash the above-ground parts of fresh purslane with clean water, drain the surface water, and chop it; keep it on ice throughout the process and avoid placing it at room temperature.

[0195] 2. Add materials and homogenize: Add pre-cooled buffer solution at a material-to-liquid ratio of 1:10 (g:mL); homogenize the tissue at high speed for 2 minutes under ice bath conditions, and homogenize intermittently to prevent temperature rise.

[0196] 3. Low-temperature extraction: Transfer the homogenate to an Erlenmeyer flask and extract by low-speed shaking on a shaker at 4 ℃ for 60 min.

[0197] 4. Centrifuge to obtain crude enzyme supernatant: Centrifuge at 4℃, 10000 r / min for 15 min; carefully aspirate the supernatant, which is the crude SOD extract, and discard the precipitate.

[0198] The finished product has a purity of 78.9% and an enzyme activity of 2150 U / mg. It contains pigment impurities and has poor stability.

[0199] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle 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 increasing the yield, enzyme activity, and / or purity of SOD during production, characterized in that, The preparation of SOD using purslane includes a step of stress-induced purslane cell preparation; The conditions for stress-induced induction include at least one of the following: (i) Reduce carbon source supply; (ii) Increase the salinity of the culture medium; (iii) Adding metal ions or increasing the concentration of metal ions; (iv) Reduce light exposure.

2. The method as described in claim 1, characterized in that, include: The reduction in carbon source supply includes adjusting the carbon source content of the culture medium to 18-22 g / L; The carbon source includes sucrose; Increasing the salinity of the culture medium includes adjusting the salt content of the culture medium to 0.8~1.2 g / L; The salt includes sodium chloride; The addition of metal ions or increase of metal ion concentration includes increasing the Cu content in the culture medium. 2+ Concentration increased by 5~10 nM, Mn 2+ Concentration increased by 0.1~0.5 μM; The reduction in light intensity includes reducing the light intensity to 30% to 40% of that under normal lighting conditions.

3. The method as described in claim 1, characterized in that, The method for preparing the purslane cells includes: Young purslane leaves were soaked in ethanol for disinfection, rinsed, and then inoculated into callus induction medium and cultured in the dark to obtain callus tissue. The callus tissue was cultured and subcultured 2-3 times to obtain purslane cells.

4. The method as described in claim 3, characterized in that, include: The concentration of the ethanol is 70% to 80%; The soaking time is 25-35 seconds; The callus induction medium includes MS medium, 0.7-0.9 mg / L 2,4-D and 0.15-0.25 mg / L NAA; The conditions for the dark culture include a temperature of 24-26°C and a time of 18-22 days.

5. A method for preparing SOD, characterized in that, include: S1: Suspension culture of purslane cells for 10-14 days; S2: The purslane cells are subjected to stress induction for 5-8 days, wherein the stress induction conditions include at least one of the following: (i) Reduce carbon source supply; (ii) Increase the salinity of the culture medium; (iii) Adding metal ions or increasing the concentration of metal ions; (iv) Reduce light exposure; S3: Collect the purslane cells and isolate SOD.

6. The preparation method according to claim 5, characterized in that, The culture medium for suspension culture includes MS medium.

7. The preparation method according to claim 5, characterized in that, The method for preparing the purslane cells includes: Young purslane leaves were soaked in ethanol for disinfection, rinsed, and then inoculated into callus induction medium and cultured in the dark to obtain callus tissue. The callus tissue was cultured and subcultured 2-3 times to obtain purslane cells.

8. The preparation method according to claim 7, characterized in that, include: The concentration of the ethanol is 70% to 80%; The soaking time is 25-35 seconds; The callus induction medium includes MS medium, 0.7-0.9 mg / L 2,4-D and 0.15-0.25 mg / L NAA; The conditions for the dark culture include a temperature of 24-26°C and a time of 18-22 days.

9. The preparation method according to claim 5, characterized in that, include: The reduction in carbon source supply includes adjusting the carbon source content of the culture medium to 18-22 g / L; The carbon source includes sucrose; Increasing the salinity of the culture medium includes adjusting the salt content of the culture medium to 0.8~1.2 g / L; The salt includes sodium chloride; The addition of metal ions or increase of metal ion concentration includes increasing the Cu content in the culture medium. 2+ Concentration increased by 5~10 nM, Mn 2+ Concentration increased by 0.1~0.5 μM; The reduction in light intensity includes reducing the light intensity to 30% to 40%.

10. The preparation method according to claim 5, characterized in that, include: The collection of purslane cells includes centrifugation to collect purslane cells; After centrifugation and collection, a resuspension step is also included; The centrifugation conditions include a temperature of 4°C and a centrifugation speed of 5500~6500 rpm; The resuspension includes resuspension using phosphate buffer solution at pH 6.8-7.2 and a temperature of 4°C to 20°C. The separation includes the steps of cell wall disruption, impurity removal, and purification; The cell wall disruption process involves intermittently using ultrasound to disrupt the collected purslane cells to obtain a crude enzyme solution. The impurity removal process includes centrifuging the crude enzyme solution, taking the supernatant, and obtaining a clear and transparent crude enzyme solution. The purification process includes gradient stepwise salting out, dialysis, and chromatography, including: a) The clear and transparent crude enzyme solution was subjected to low salt precipitation, with an ammonium sulfate saturation of 32%~38%; b) The clear, transparent crude enzyme solution after low salt precipitation is subjected to high salt precipitation, with an ammonium sulfate saturation of 58% to 64%; c) Dialyze using a 10 kDa molecular weight cutoff dialysis bag at 4°C; d) SOD was collected by Sephadex G-75 dextran gel chromatography.