A process for large-scale extraction of microbial genomic DNA

Abstract

This invention discloses a process for large-scale extraction of microbial genomic DNA, comprising the following steps: cell harvesting and cell disruption pretreatment, neutral protease pre-digestion, SDS-based strong lysis and enzyme termination, primary clarification, arginine precipitation to capture DNA, precipitate redissolution, anion exchange chromatography purification, and concentration and productization. The neutral protease pre-digestion is performed in an SDS / EDTA-free environment, with calcium salt added first to compete with EDTA to maintain enzyme activity; lysis and digestion are performed continuously in the same reaction vessel; arginine precipitation and anion exchange chromatography are combined to form a chloroform-free purification process; and extracellular DNA in the fermentation broth supernatant can be captured simultaneously. This invention uses neutral protease instead of expensive proteinase K, and arginine precipitation and chromatography combination instead of chloroform extraction, offering advantages such as low cost, safety and environmental friendliness, high purity, intact fragments, simultaneous capture of extracellular DNA, and ease of industrial scale-up.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a process for large-scale extraction of high-purity genomic DNA from microorganisms (including bacteria, yeast, and filamentous fungi). Background Technology

[0002] Genomic DNA extraction is a fundamental step in molecular biology, genetic engineering, diagnostic reagents, and biopharmaceuticals. With the rapid development of synthetic biology, gene therapy, and the vaccine industry, the industrial demand for large-scale, high-purity, and high-molecular-weight microbial genomic DNA is becoming increasingly urgent.

[0003] Existing large-scale DNA extraction methods mainly face the following mutually restrictive technical challenges: First, there is the high cost associated with using proteinase K. Proteinase K is currently the most commonly used proteolytic enzyme in DNA extraction, but its production cost is high, accounting for 40% to 60% of the reagent cost. Literature reports that Nagarse (a subtilisin, a neutral protease) can be a cheaper alternative to proteinase K, performing similarly in digesting leukocytes and bacterial DNA, at only 5% to 10% of the price. However, this research is limited to small-scale laboratory operations (Mori et al., J.Clin. Lab. Anal. 2000; 14:97-100), and does not address the process integration issues for industrial-scale applications, especially the maintenance of the activity of neutral proteases, which are highly sensitive to EDTA in the reaction system.

[0004] Secondly, there are safety and environmental issues associated with using controlled reagents such as chloroform. While traditional chloroform / isoamyl alcohol extraction yields good purification results, chloroform is a strictly controlled precursor chemical, posing challenges in large-scale production due to procurement difficulties, operational safety hazards, and high costs associated with hazardous waste disposal. Although some studies have attempted to replace organic solvents with non-toxic reagents, such as the method for precipitating proteins using saturated saline solution disclosed in patent CN102140451A, these methods generally suffer from unstable purification results and limited applicability.

[0005] Third, the purification process is cumbersome and difficult to scale up linearly. Conventional purification methods (such as alcohol precipitation and column purification) face challenges when scaled up to industrial scale, including large processing volumes, demanding equipment requirements, severe DNA shearing damage, and substandard purity due to polysaccharide / protein co-precipitation. While the DNA separation and purification method reported in patent CN104152436B can be completed quickly, it relies on consumables such as purification columns, making it unsuitable for continuous large-scale production. Furthermore, many existing technologies require multiple transfers of lysis buffer between different containers, increasing the risk of DNA shearing degradation and operational complexity.

[0006] Fourth, there is a lack of simultaneous capture strategies for microbial extracellular DNA. Free DNA present in the fermentation broth supernatant (released from the lysis of dead bacteria) is discarded as waste in conventional processes. Patent CN202410432611.5 discloses a method for separating and extracting intracellular and extracellular DNA from microbial colony biofilms, but this technology is designed for biofilms on solid culture media, requires scraping operations, is not suitable for liquid fermentation systems, and is not integrated with large-scale industrial DNA extraction processes.

[0007] The four technical challenges mentioned above are interconnected: cost reduction cannot come at the expense of purity; replacing chloroform cannot introduce more complex operating procedures; and simplifying operations cannot result in reduced yield. Therefore, developing a process that simultaneously addresses these challenges, is suitable for large-scale continuous production, and can yield high-purity, high-molecular-weight DNA has significant industrial value. Summary of the Invention

[0008] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a process for large-scale extraction of microbial genomic DNA.

[0009] A process for large-scale extraction of microbial genomic DNA includes the following steps performed sequentially: (1) Cell harvesting and cell disruption pretreatment: Wet cells were collected from the microbial fermentation broth by continuous flow centrifugation and cell disruption was performed by enzymatic cell disruption in a buffer solution containing EDTA. (2) Enzyme activation and pre-digestion: In the system containing EDTA obtained in step (1), before adding SDS, first add calcium salt to the final concentration to exceed the initial EDTA concentration by 5-15 mM, so that calcium ions can fully compete for EDTA, and then add neutral protease and stir at a suitable temperature to carry out the pre-digestion reaction. (3) In situ lysis and enzyme termination: After the reaction in step (2) is completed, without intermediate separation, add SDS and EDTA to the same reaction vessel in sequence to the working concentration, heat to the appropriate temperature, and continue stirring until lysis is complete; (4) Primary clarification and impurity removal: Cool the lysate, add potassium acetate solution to precipitate proteins and polysaccharides, and obtain a clear supernatant by continuous flow centrifugation or deep filtration; (5) Arginine precipitation to capture DNA: Add neutral salt to the clear supernatant to a final concentration of 0.5-1.0 M, dissolve and then slowly add arginine salt to a final concentration of 0.3-0.8 M. Let stand at low temperature to form a DNA-arginine complex flocculent precipitate, and collect the precipitate by centrifugation or filtration. (6) Resolution of precipitate: Resuspend the precipitate in a buffer solution containing neutral salt and stir to dissolve; (7) Anion exchange chromatography purification: After filtering the heavy solution, the sample is loaded onto a strong anion exchange chromatography column, washed sequentially with binding buffer and washing buffer, eluted with elution buffer, and the eluent is collected; (8) Concentration and productization: The eluent is desalted and concentrated by a tangential flow ultrafiltration system, replaced with the final product buffer, and sterilized by filtration to obtain the DNA product; Steps (2) to (3) are carried out continuously in the same reaction vessel, and no intermediate transfer or centrifugation is required between steps (2) and (3).

[0010] Preferably, when the supernatant of the fermentation broth contains free DNA, the supernatant is collected after centrifugation of the fermentation broth and combined with the clarified supernatant obtained in step (4) for arginine precipitation in step (5) to achieve simultaneous capture of intracellular DNA and extracellular DNA.

[0011] Preferably, the arginine salt is selected from at least one of arginine hydrochloride, arginine sulfate, or arginine acetate.

[0012] Preferably, the neutral salt in step (5) is sodium chloride, and the final concentration added is 0.5-1.0 M; the final concentration added of the arginine salt is 0.3-0.8 M.

[0013] Preferably, the packing material of the strong anion exchange chromatography column in step (7) is agarose gel based on quaternary ammonium groups or polystyrene-divinylphenyl packing material.

[0014] Compared with the prior art, the present invention has the following beneficial effects: First, it significantly reduces material costs, making it suitable for large-scale production. Replacing expensive proteinase K with neutral proteases has been proven in the literature; neutral proteases (such as Nagarse) are far less expensive than proteinase K while achieving comparable results. This invention solves the problem of activity inhibition of neutral proteases in EDTA-containing pre-digestion buffers through a calcium salt competitive EDTA strategy and integrates it into industrial-scale processes. Simultaneously, using arginine precipitation as a pre-capture step before chromatography significantly reduces the average purification cost per gram of DNA produced.

[0015] Secondly, it eliminates the need for controlled reagents such as chloroform. This invention, through a combination of selective arginine precipitation and subsequent anion exchange chromatography, completely avoids the use of chloroform / phenol, making it safe and environmentally friendly. Arginine precipitation utilizes the electrostatic interaction between the arginine guanidine group and the DNA phosphate backbone under high-salt conditions, causing selective precipitation of DNA while retaining most proteins, polysaccharides, and other impurities in the supernatant, thus avoiding the polysaccharide co-precipitation problem associated with traditional alcohol precipitation.

[0016] Third, it can simultaneously capture intracellular and extracellular DNA. This invention is the first to integrate an extracellular DNA capture strategy into a large-scale industrial DNA extraction process. By combining the supernatant after centrifugation of the fermentation broth with the clarified supernatant of the lysis buffer for arginine precipitation, free DNA that is discarded as waste in conventional processes can be recovered simultaneously, thereby increasing the total DNA yield.

[0017] Fourth, the "one-pot" continuous operation. Existing technologies typically require multiple transfers and centrifugations at each step, increasing operation time and labor costs, and easily causing DNA loss and cross-contamination. This invention completes the entire process from cell disruption to lysis continuously in the same reaction vessel. Through a specific timing design of "calcium salt preferentially competing for EDTA → neutral protease pre-digestion → SDS in-situ lysis and enzyme termination," it solves the two key problems of neutral protease activity inhibition and enzyme termination without intermediate transfers, reducing transfer steps and the risk of DNA shearing and degradation, making it suitable for industrial automated production.

[0018] Fifth, high purity and relatively intact fragments. The combination of arginine precipitation and anion exchange chromatography forms a complementary multi-stage purification chain: arginine precipitation, as a crude capture step, removes most of the polysaccharides and proteins, protecting the subsequent chromatography column; anion exchange chromatography, as a purification step, achieves pharmaceutical-grade purity. Both are indispensable—omitting arginine precipitation leads to packing blockage and increased column pressure when the crude lysis buffer is directly loaded (see Comparative Example 2); omitting anion exchange chromatography results in substandard purity of the arginine precipitation product (see Comparative Example 3). The synergistic effect of both yields a high-purity (A260 / A280 ≥ 1.80, A260 / A230 ≥ 2.0) final product containing high molecular weight (>50 kb).

[0019] In particular, this invention, through a specific time sequence design of "preferential competition for calcium salts → pre-digestion with neutral protease → in-situ cleavage with SDS" and a complementary functional combination of "crude purification by arginine precipitation → fine purification by anion exchange," for the first time simultaneously resolves the long-standing contradictions in cost, safety, scalability, and yield in the same process flow in existing technologies. Comparative Examples 2 and 3, and synergistic effect verification experiments, demonstrate that the combination of arginine precipitation and anion exchange chromatography produces a synergistic effect of "1 + 1 > 2," which is something that those skilled in the art could not have foreseen using any single technical means. Attached Figure Description

[0020] Figure 1 This is a flowchart of the process of the present invention; Figure 2 These are agarose gel electrophoresis images of purified DNA samples from Examples 1, 1 Comparative Examples, and 3 of this invention. Detailed Implementation

[0021] To more clearly illustrate the present invention, the invention will be further described in detail below with reference to preferred embodiments and comparative examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention. Any modifications, equivalent substitutions, improvements, etc., made within the concept and principles of the present invention should be included within the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All names of materials used herein are commercially available conventional products, the specific sources and models of which are described in the examples, but should not be construed as being limited to these specific brands and models.

[0023] Example 1: Extraction of genomic DNA from Saccharomyces cerevisiae fermentation broth 1. Harvesting of microorganisms The fermentation broth (OD) of Saccharomyces cerevisiae cultured to the logarithmic growth phase was used to... 600 =8.0, volume 100 L), pumped into a disc centrifuge at a flow rate of 2 L / min (speed 10,000 rpm), collect wet bacterial cells, weigh approximately 0.9 kg.

[0024] 2. One-pot cell wall breaking and pyrolysis In a 100 L jacketed reactor, lysozyme buffer (containing 1 M sorbitol, 25 mM EDTA, and 20 mM Tris-HCl, pH 7.5) was added at a wet cell to buffer ratio of 1:2 (w / v) to bring the total volume to approximately 18 L. Lysozyme was added to a final concentration of 0.5 mg / mL, and β-mercaptoethanol was added to a final concentration of 15 mM. The reactor was incubated at 37 °C with stirring (150 rpm) for 2 hours. Microscopic examination revealed a protoplast formation rate of approximately 95%.

[0025] Then, without centrifugation, add sterile CaCl2 solution (1 M) directly to the jar to a final concentration of 35 mM. Add neutral protease (food-grade neutral protease (high-activity concentrated type), product number: FDG-2230, enzyme activity ≥110,000 U / mg) to a concentration of 0.2 mg / mL. Adjust the pH to 7.5 with 1 M Tris base, raise the temperature to 50°C, and continue incubation with stirring for 1 hour.

[0026] Subsequently, SDS (20% stock solution) was added sequentially to a final concentration of 1% (w / v), and EDTA (0.5 M stock solution, pH 8.0) was added to a final concentration of 50 mM. The temperature was raised to 65°C, and stirring was continued for 30 minutes until the lysis solution became a clear, viscous liquid.

[0027] 3. Initial Clarification The lysis buffer was cooled to 4°C, and 5 M potassium acetate solution was added to a final concentration of 0.5 M. The mixture was stirred in an ice bath for 30 minutes. The solution was then centrifuged using a continuous flow centrifuge (15,000 rpm) to obtain approximately 23 L of clear supernatant.

[0028] 4. Arginine precipitation captures DNA Slowly add solid NaCl to the supernatant to a final concentration of 0.8 M, stirring to dissolve. Then slowly add arginine hydrochloride (arginine·HCl) solution (4 M, pH 7.0) to a final concentration of 0.5 M, stirring continuously. Let stand at 4 °C for 2 hours to form a white flocculent precipitate. Collect the precipitate by continuous flow centrifugation (8,000 rpm), discarding the supernatant.

[0029] 5. Precipitation and dissolution The wet precipitate was resuspended in 10 L of 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl, stirred at room temperature for 1 hour until completely dissolved, and centrifuged (10,000 rpm, 30 minutes) to remove insoluble matter.

[0030] 6. Anion exchange chromatography After filtering the above solution through a 0.45 μm filter membrane, the solution was loaded at a flow rate of 5 mL / min onto a chromatography column (20 cm in diameter, approximately 7 cm in height) packed with strong anion exchange packing material (Q Sepharose Fast Flow, 2 L). Five column volumes were equilibrated with binding buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0). After loading the solution, the column was washed with binding buffer until the baseline stabilized. Then, three column volumes were washed with washing buffer (20 mM Tris-HCl, 0.8 M NaCl, pH 8.0). Finally, the column was eluted with a linear gradient using elution buffer (20 mM Tris-HCl, 1.5 M NaCl, pH 8.0), and approximately 1.5 L of the eluent corresponding to the UV absorption peak (A260) was collected.

[0031] 7. Ultrafiltration Concentration and Finished Product The collected eluent was concentrated to 200 mL using a tangential flow ultrafiltration system (30 kDa membrane pack, 0.5 m² area), and simultaneously washed and desalted with a 5-fold volume of final product buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Finally, the eluent was filtered through a 0.22 μm filter membrane for sterilization, and the filtrate was lyophilized into powder (approximately 0.51 g) and then aliquoted for storage.

[0032] 8. Quality Inspection The final product was tested for the following: Concentration (A260 method): 1.85 mg / mL (after reconstitution); Purity: A260 / A280 = 1.81, A260 / A230 = 2.04; Integrity: Agarose gel electrophoresis (1% agarose, DL5000 marker) showed a gradient distribution of DNA from large to small (see lane 1 in Figure 2), and the presence of continuous high-molecular-weight DNA fragments (>50 kb). The calculated DNA yield was approximately 0.568 mg / g wet bacterial cells.

[0033] Example 2: Extraction of genomic DNA from Escherichia coli fermentation broth This embodiment is basically the same as Example 1, except that the microorganism is Escherichia coli (Gram-negative bacteria), the cell wall breaking step is changed to lysozyme treatment, and unlike Example 1, the amount of calcium salt added is calculated based on the initial EDTA concentration.

[0034] Microbial harvest The fermentation broth of E. coli cultured to the logarithmic growth phase (OD) 600 = 5.0, volume 50 L) Continuous flow centrifugation was used to collect wet bacterial cells, approximately 0.32 kg.

[0035] One-pot cell wall breaking and pyrolysis In a 50 L jacketed reaction vessel, lysozyme buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0) was added at a wet cell to buffer ratio of 1:2. Lysozyme was added to a final concentration of 1 mg / mL, and the mixture was incubated at 37°C with stirring for 90 minutes. CaCl2 was then added to a final concentration of 20 mM, followed by neutral protease to a concentration of 0.2 mg / mL, and the mixture was incubated at 50°C for 60 minutes. SDS (1%) and EDTA (50 mM) were then added sequentially, and the mixture was lysed at 65°C for 30 minutes. Subsequent steps were the same as steps 3-8 in Example 1. Approximately 0.29 g of DNA product was obtained, with a purity of A260 / A280 = 1.86 and A260 / A230 = 2.05.

[0036] Example 3: Simultaneous capture of intracellular and extracellular DNA in fermentation broth This embodiment is basically the same as Embodiment 1, except that the supernatant after centrifugation of the fermentation broth is collected additionally in the cell harvesting step.

[0037] Fermentation broth separation The yeast fermentation broth (100 L) was pumped into a disc centrifuge at a flow rate of 2 L / min, and the wet cells (about 0.9 kg) and the centrifugation supernatant (about 97 L, containing free DNA released by the lysis of dead bacteria during fermentation) were collected separately.

[0038] Extracellular DNA capture The supernatant obtained by centrifugation was combined with the clarified supernatant obtained in step 3 of Example 1 (total volume approximately 120 L), and arginine was precipitated together: NaCl was added to a final volume of 0.8 M, arginine hydrochloride was added to a final volume of 0.5 M, the mixture was allowed to stand at 4°C for 2 hours, and the precipitate was collected by centrifugation. Subsequent steps were the same as steps 5-7 of Example 1.

[0039] result Approximately 0.54 g of finished DNA was obtained, an increase of about 5.9% compared to Example 1 (intracytoplasmic DNA only, approximately 0.51 g), confirming successful extracellular DNA capture. Purity A260 / A280 = 1.87, A260 / A230 = 2.02.

[0040] Comparative Example 1: Using proteinase K instead of neutral protease (comparison of calcium salt addition strategies) Same as in Example 1, except that the neutral protease is replaced with proteinase K (0.1 mg / mL) of equal activity units, and no calcium salt is added (because proteinase K is not metal ion dependent). The remaining steps are exactly the same.

[0041] The results showed that the DNA yields of the two groups were roughly equivalent (0.55 g for proteinase K and 0.51 g for neutral proteinase) and their purity (A260 / A280 both > 1.80) was also roughly equivalent. However, the unit cost of proteinase K was approximately 15 times that of neutral proteinase, increasing the total material cost by about 55%. This comparative study demonstrates that neutral proteinase can significantly reduce costs without sacrificing yield.

[0042] Comparative Example 2: Omitting the arginine precipitation step (to verify the synergistic relationship between arginine precipitation and chromatography) Same as in Example 1, but omitting the arginine precipitation step (step 4), the clear supernatant obtained in step 3 (containing 0.5 M potassium acetate) was directly loaded onto the anion exchange chromatography column (diluted 7 times to reduce conductivity due to excessive salt content). Other chromatography parameters were the same as in Example 1.

[0043] The results showed that due to the excessively large volume of supernatant (approximately 140 L), the chromatography time was extended to over 10 hours; severe blockage of the packing material occurred during sample loading, and the column pressure increased from the initial 0.2 MPa to 0.8 MPa (exceeding the operating limit), making purification impossible. This comparative example confirms that arginine precipitation, as a pre-capture step before chromatography, plays an irreplaceable role in protecting the chromatography column and reducing the sample loading volume.

[0044] Comparative Example 3: Isopropanol precipitation was used instead of arginine precipitation (to verify the selectivity advantage of arginine precipitation). Same as in Example 1, but with the arginine precipitation step replaced by conventional isopropanol precipitation: 0.6 times the volume of pre-cooled isopropanol was added to the supernatant, and the mixture was allowed to stand at -20 °C for 2 hours. The precipitate was then collected by centrifugation (10,000 rpm, 30 minutes). The precipitate was washed twice with 70% ethanol and then redissolved according to step 5 of Example 1.

[0045] The results showed that the precipitate obtained from isopropanol precipitation was muddy but not viscous, and the solution was turbid after redissolution. The test results were: A260 / A280 = 1.67 (standard requirement ≥ 1.8), A260 / A230 = 1.34 (standard requirement ≥ 2.0), indicating the presence of protein and polysaccharide contamination. Agarose gel electrophoresis showed lane 3 (see...). Figure 2 The presence of a significant protein-DNA complex indicates co-precipitation of DNA and protein, and the fragments do not increase uniformly from large to small. This comparative example confirms that arginine precipitation has better selectivity than conventional alcohol precipitation, effectively avoiding the problem of protein-polysaccharide co-precipitation.

[0046] Comparative Example 4: Omitting the calcium salt competing EDTA step (verifying the necessity of the "add calcium salt first" strategy) Same as Example 1, except that in step 2, CaCl2 solution is not added; instead, neutral protease is added directly. The remaining steps are the same.

[0047] The results showed that the activity of neutral protease (metalloproteinase) was significantly inhibited due to the presence of 25 mM EDTA in the pre-digestion buffer, resulting in poor pre-digestion. The final DNA yield was only 0.097 g (approximately 19% of that in Example 1), with impurities mainly consisting of protein, indicating that the neutral protease did not maintain protease activity. This comparative example confirms that calcium salt competition for EDTA is a necessary technique for maintaining neutral protease activity.

[0048] Comparative Example 5: Change the order of adding ingredients (add neutral protease first, then calcium salt). Same as Example 1, except that in step 2, CaCl2 is added 30 minutes after the addition of neutral protease. The rest of the steps are the same.

[0049] The results showed that because EDTA initially inhibited the activity of the neutral protease, the enzyme activity could not be fully restored even with the subsequent addition of calcium salt. The final DNA yield was 0.147 g (approximately 32% of that in Example 1), with a purity of A260 / A280 = 1.79 and A260 / A230 = 2.02. This comparative example confirms that the specific order of adding the calcium salt first, followed by the neutral protease, is crucial for ensuring enzyme activity.

[0050] Synergistic effect verification experiment To verify the synergistic effect of arginine precipitation and anion exchange chromatography, the following comparison was designed: Experiment A (Anion Exchange Chromatography Only): The clarified supernatant from step 3 of Example 1 was directly loaded onto the chromatography column. Result: The packing material became clogged, the column pressure was >0.8 MPa, and purification could not be completed.

[0051] Experiment B (arginine precipitation only): Steps 4-5 of Example 1 were performed, but steps 6-7 were omitted, and the heavy solution was used as the final product. Results: A260 / A280 = 1.65, indicating the presence of protein and polysaccharide residues (A260 / A230 = 1.4).

[0052] Experiment C (the combination of this invention): Arginine precipitation + anion exchange chromatography. Results: A260 / A280 = 1.81, A260 / A230 = 2.04, and the column pressure did not increase significantly after 10 consecutive batches of the packing material.

[0053] The results showed that Experiment A yielded no effective product due to its inability to purify; Experiment B's purity was insufficient; and Experiment C achieved both high purity and operable process stability. The actual effect of Experiment C functionally surpassed the simple sum of the effects of Experiments A and B (one being unable to purify, the other insufficiently pure), constituting the "superior combined technical effect compared to the sum of the effects of each individual technical feature" as described in the Patent Examination Guidelines. This demonstrates a non-obvious synergistic effect between arginine precipitation and anion exchange chromatography.

[0054] The above results indicate that the effect of the specific combination (arginine precipitation + chromatography) claimed in this invention (high purity, operability) is not a simple superposition of the effect of arginine precipitation (poor purity) and the effect of chromatography (inability to operate alone), but rather a non-obvious improvement that solves the fatal defects that exist when each is used alone through the synergistic effect of the two.

[0055] Although the present invention has been described in detail above with reference to specific embodiments, those skilled in the art will readily understand from this disclosure that various changes or modifications can be made to the present invention without departing from the principles and spirit defined by the claims. Therefore, the detailed description of the above embodiments is for illustrative purposes only and is not intended to limit the present invention; the scope of protection of the present invention should be determined by the content of the claims.

Claims

1. A process for large-scale extraction of microbial genomic DNA, characterized in that, The following steps are performed sequentially: (1) Cell harvesting and cell disruption pretreatment: Wet cells were collected from the microbial fermentation broth by continuous flow centrifugation and cell disruption was performed by enzymatic cell disruption in a buffer containing EDTA. (2) Enzyme activation and pre-digestion: In the system containing EDTA obtained in step (1), before adding SDS, first add calcium salt to the final concentration to exceed the initial EDTA concentration by 5-15 mM, so that calcium ions can fully compete for EDTA, and then add neutral protease and stir at a suitable temperature to carry out the pre-digestion reaction. (3) In situ lysis and enzyme termination: After the reaction in step (2) is completed, without intermediate separation, add SDS and EDTA to the same reaction vessel in sequence to the working concentration, heat to the appropriate temperature, and continue stirring until lysis is complete; (4) Primary clarification and impurity removal: Cool the lysate, add potassium acetate solution to precipitate proteins and polysaccharides, and obtain a clear supernatant by continuous flow centrifugation or deep filtration; (5) Arginine precipitation to capture DNA: Add neutral salt to the clear supernatant to a final concentration of 0.5-1.0 M, dissolve, and then slowly add arginine salt to a final concentration of 0.3-0.8 M. The arginine salt is selected from any one of arginine hydrochloride, arginine sulfate or arginine acetate. Let stand at low temperature to form a flocculent precipitate of DNA-arginine complex. Collect the precipitate by centrifugation or filtration. (6) Resolution of precipitate: Resuspend the precipitate in a buffer solution containing neutral salt and stir to dissolve; (7) Anion exchange chromatography purification: After filtering the heavy solution, the sample is loaded onto a strong anion exchange chromatography column, washed sequentially with binding buffer and washing buffer, eluted with elution buffer, and the eluent is collected; (8) Concentration and productization: The eluent is desalted and concentrated by a tangential flow ultrafiltration system, replaced with the final product buffer, and sterilized by filtration to obtain the DNA product; Steps (2) to (3) are carried out continuously in the same reaction vessel, and no intermediate transfer or centrifugation is required between steps (2) and (3).

2. The process for large-scale extraction of microbial genomic DNA according to claim 1, characterized in that, It also includes the step of simultaneously capturing extracellular DNA: after centrifuging the fermentation broth, the supernatant is collected and combined with the clarified supernatant obtained in step (4) to carry out the arginine precipitation in step (5).

3. The process for large-scale extraction of microbial genomic DNA according to claim 1, characterized in that, The arginine salt is selected from at least one of arginine hydrochloride, arginine sulfate, or arginine acetate.

4. The process for large-scale extraction of microbial genomic DNA according to claim 1, characterized in that, The neutral salt mentioned in step (5) is sodium chloride, and the final concentration added is 0.5-1.0 M; the final concentration added of the arginine salt is 0.3-0.8 M.

5. The process for large-scale extraction of microbial genomic DNA according to claim 1, characterized in that, The packing material of the strong anion exchange chromatography column in step (7) is agarose gel based on quaternary ammonium groups or polystyrene-divinylphenyl packing material.

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