A method for improving gene transformation efficiency of a microorganism polysaccharide-producing strain

Abstract

The application discloses a method for improving gene transformation efficiency of a microorganism polysaccharide-producing strain, and the method comprises the following steps: treating the microorganism polysaccharide-producing strain by using a polysaccharide-degrading enzyme, removing the wrapping of the polysaccharide on the strain cell, obtaining the cell, and preparing a competent cell; then, an exogenous plasmid is transformed into the treated competent cell by using an electric shock; wherein the polysaccharide-degrading enzyme can degrade the extracellular polysaccharide produced in the culture process of the microorganism polysaccharide-producing strain. The application effectively improves the success rate and the transformation efficiency of plasmid transformation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for improving the gene transformation efficiency of microbial polysaccharide-producing strains. In particular, it relates to the rapid separation of bacterial cells and extracellular polysaccharides through the action of degrading enzymes, which facilitates the preparation of competent cells of such polysaccharide-producing strains and significantly improves transformation efficiency. Background Technology

[0002] Microbial polysaccharides can be produced by many bacteria and fungi in nature. Based on their location within microbial cells, they can be classified into extracellular polysaccharides, cell wall polysaccharides, and extracellular polysaccharides. Extracellular polysaccharides are mainly composed of homopolysaccharides or heteropolysaccharides. Extracellular polysaccharides primarily play a protective role for bacteria, participating in cell membrane integrity, capturing nutrients, allowing adhesion to surfaces, and enabling bacteria to resist extreme environmental conditions, protecting them from toxic compounds. Simultaneously, due to their functional structures and diverse biological activities, as well as their unique biodegradability and biocompatibility, extracellular polysaccharides have attracted significant attention and are widely used in industry, agriculture, food, and medicine. The global annual production value of microbial polysaccharides reaches as high as US$50 billion.

[0003] With the development of synthetic biology, it has become possible to enhance and reconstruct microbial polysaccharide synthesis pathways and modify polysaccharide structures to further improve their performance through genetic engineering. However, microbial polysaccharide-producing strains are mainly selected from nature and are usually non-model strains, lacking relatively mature genetic engineering methods, especially plasmids and transformation methods. For plasmid manipulation of non-model strains, conjugation transfer and electroporation transformation are commonly used to introduce exogenous plasmids into cells. Conjugation transfer is cumbersome and time-consuming, which is extremely unfavorable for large-scale molecular modification. In contrast, electroporation transformation is more convenient, faster, and widely used. However, the efficiency of electroporation transformation is greatly affected by the competent state, especially since microbial polysaccharide-producing strains secrete extracellular sugar copolymers during cultivation. These extracellular polysaccharide macromolecules form a mucus layer surrounding the cell, and some components, acting as capsules, bind to the cell wall. Conventional separation methods cannot separate polysaccharides from bacterial cells. Dilution separation not only increases the number of steps and poses a risk of contamination, but also makes it extremely difficult to obtain bacterial cells completely free of polysaccharide coatings, resulting in significant challenges in preparing competent cells of such microbial polysaccharide-producing strains. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for improving the conversion efficiency of microbial polysaccharide-producing strains, addressing the shortcomings of the prior art.

[0005] To address the aforementioned technical problems, this invention discloses a method for improving the transformation efficiency of microbial polysaccharide-producing strains. By adding polysaccharide-degrading enzymes, the preparation method of competent cells for polysaccharide-producing strains is improved, thereby increasing the success rate and efficiency of plasmid transformation.

[0006] Specifically, the present invention provides a method for improving the transformation efficiency of microbial polysaccharide-producing strains, characterized by comprising the following steps:

[0007] (1) Preparation of crude enzyme solution of polysaccharide degrading enzyme;

[0008] (2) Use polysaccharide-degrading enzymes to treat microbial polysaccharide-producing strains, remove the polysaccharide from the bacterial cells of the microbial polysaccharide-producing strains, obtain bacterial cells, and then prepare competent cells;

[0009] (3) The exogenous plasmid was transferred into competent cells treated in step (2) by electroporation.

[0010] The polysaccharide-degrading enzyme is a polysaccharide-degrading enzyme capable of degrading the extracellular polysaccharides produced during the cultivation of the microbial polysaccharide-producing strain.

[0011] The polysaccharide-degrading enzyme is a polysaccharide-degrading enzyme capable of degrading the extracellular polysaccharides produced during the cultivation of the microbial polysaccharide-producing strain. This invention utilizes a degrading enzyme capable of degrading the extracellular polysaccharides produced during the cultivation of the microbial strain, facilitating subsequent separation of the bacterial gel by centrifugation.

[0012] According to the concept of this invention, by treating the strain with an enzyme that can degrade the extracellular polysaccharides produced during the cultivation of the corresponding microbial strain, the conversion efficiency of the microbial polysaccharide-producing strain can be improved.

[0013] In a specific aspect of this invention, a method is provided to improve the transformation efficiency of *Sphingomonas* strains. The sphingomonas gum family comprises a series of extracellular polysaccharides with the same tetrasaccharide repeating unit backbone structure produced by *Sphingomonas* fermentation, including gellan gum, velan gum, rhamnose gum, Dettol gum, S88, and S7. The degradative enzyme gelR gene used has the nucleotide sequence shown in SEQ ID NO.1:

[0014]

[0015] The sphingomonas strains, i.e., microbial polysaccharide-producing strains, include, but are not limited to, any one of the strains with accession numbers ATCC31555, Sphingomonas sp. CGMCC No. 2428, Sphingomonas sp. HT-1CCTCC NO: M2012062, ATCC 31461, ATCC 31961, CGMCC No. 6833, and ATCC 53159, all of which are publicly disclosed strains.

[0016] Specifically, enzyme solutions can be prepared using genetic engineering methods to reduce costs. In one embodiment of the present invention, a polysaccharide-degrading enzyme solution is prepared by the following method: the gene of the degrading enzyme is ligated into the expression vector pET-28a and transformed into competent E. coli BL21(DE3) cells to obtain recombinant cells expressing the degrading enzyme, and cultured at 32-37°C until OD200. 600 After the concentration reaches between 0.4 and 0.8, add IPTG as an inducer to a final concentration of 0.1-1M. Continue culturing at 24-28℃ for 16-24 hours, then collect the bacterial cells, resuspend them several times with PBS buffer, and then sonicate them. The supernatant after centrifugation is filtered through a membrane to obtain the crude enzyme solution, which is then stored at -80℃.

[0017] The method for treating microbial polysaccharide-producing strains with degrading enzymes is as follows: The microbial polysaccharide-producing strains are inoculated into an oligotrophic medium and cultured in a shaker at 25-35℃ until the absorbance OD reaches a certain level. 600 When the pH value is between 0.2 and 0.6, add 1-5% (v / v) crude enzyme solution. Enzyme activity: 3-10 U / mL. Crude enzyme activity is defined as the amount of enzyme required to produce 1 μmol / mL of reducing sugar per minute under optimal reaction conditions. Continue culturing for 10-60 min, centrifuge to collect the cells, wash several times with pre-cooled glycerol, and resuspend to OD. 600 Store at -80°C for temperatures between 2 and 5%. Preferably, use 5-15% glycerol by volume.

[0018] The oligotrophic culture medium contains 0.1-0.5M sucrose.

[0019] In one specific embodiment, the oligotrophic culture medium is composed of: 3M sucrose, 5g / L fish meal peptone, and 5g / L NaCl.

[0020] The electric shock conditions used are 2.0KV-5KV, pulse time is 2-10ms, and electric shocks are performed 1-5 times.

[0021] According to the method for improving the transformation efficiency of microbial polysaccharide-producing strains, the number of transformants obtained on the plate after electroporation transformation in step (3) is approximately 30 to 200.

[0022] Beneficial Effects: This invention addresses the difficulties in preparing competent cells and the low transformation efficiency of microbial polysaccharide-producing strains by providing a simple and easy-to-implement rapid transformation method for these strains. Based on conventional electroporation transformation, this method is clear in its approach, simple to operate, and yields good transformation results, laying the foundation for genetic manipulation of microbial polysaccharide-producing strains. The approach of this invention can also be applied to the treatment of strains that ferment to produce high molecular weight polymers, showing great promise for future applications. Attached Figure Description

[0023] Figure 1 SDS-PAGE electrophoresis image of crude polysaccharide-degrading enzyme solution;

[0024] Figure 2 This is a colony PCR verification image;

[0025] Figure 3 Comparison chart showing the separation effect of crude enzyme solution with added polysaccharide hydrolase and bacterial gel. Detailed Implementation

[0026] The strains and plasmids involved in the following examples:

[0027] Escherichia coli BL21(DE3) competent cells were used for protein expression and were purchased from Anhui General Biosystems Co., Ltd.

[0028] Sphingomonas sp. CGMCC No. 2428 was isolated and preserved in our laboratory.

[0029] Sphingomonas sp. ATCC 31461 (Sphingomonas sp. ATCC 31961).

[0030] Sphingosomalidone (CGMCC No. 6833).

[0031] The plasmid pBBR1MCS-2 is a commercially available plasmid.

[0032] The culture media involved in the following examples:

[0033] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar powder 2%

[0034] Oligonutrient liquid culture medium: 3M sucrose, 5g / L fish meal peptone, 5g / L NaCl.

[0035] SOC liquid culture medium: peptone 20 g / L, yeast extract 5 g / L, NaCl 0.5 g / L, glucose 20 mM, MgSO4 10 mM, KCl 2.5 mM.

[0036] Electroshock buffer: 10% glycerol.

[0037] The function of polysaccharide-degrading enzymes is to degrade extracellular polysaccharides to maintain bacterial metabolism when the carbon source in the culture medium is insufficient. In this invention, we utilize the property of gelR to degrade sphingosine mucin. By adding crude gelR enzyme solution during the culture of Sphingosine monocytogenes, cell recovery is facilitated and high-quality competent cells are prepared, thereby significantly improving the transformation efficiency of bacteria.

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0039] Example 1: Construction of a heterologous expression system for sphingosine gum degrading enzyme.

[0040] The heterologous expression system of sphingosine gum degrading enzyme was obtained through the following steps:

[0041] (1) The degradative enzyme gene gelR sequence was obtained by PCR from the genome of Sphingomonas CGMCC No.2428 using primers (F: 5′-ATGCTTACCATGCCGGAC-3′, R: 5′-GGAATTGCACCACGTCTG-3′). The gelR gene was positively expressed between BamHⅠ and SacⅠ in the MCS region of the pET-28a plasmid to obtain the plasmid pET-28a-gelR.

[0042] (2) Add 100 μg of plasmid pET-28a-gelR to BL21(DE3) competent cells under sterile conditions, mix by pipetting and aspiration, and then place on ice for about 30 min.

[0043] (3) Quickly place it in a 42℃ water bath and heat shock for 90 seconds. After the time is up, quickly remove it and place it on ice for 3-5 minutes.

[0044] (4) Add 600 μL of LB medium to the system in step (3), mix well, and incubate at 37°C with shaking for 1 h;

[0045] (5) Spread the culture medium from step 4 onto LB solid medium plates containing a final concentration of 40 μg / mL kanamycin and incubate at 37°C for 12 h to obtain positive single colonies.

[0046] Example 2: Preparation of crude sphingosine gum degrading enzyme solution.

[0047] The crude enzyme solution of sphingosine gum degrading enzyme was prepared by the following method.

[0048] (1) Pick a positive single colony from Example 1 and inoculate it into 10 ml of LB liquid medium containing a final concentration of 40 μg / mL kanamycin, and incubate at 37°C for 6 h;

[0049] (2) Take 1 mL of the culture medium from step (1) and inoculate it into a 1 L shake flask containing 200 mL of LB liquid culture medium. Incubate at 37 °C and 200 rpm until OD. 600 It is 0.6;

[0050] (3) Add IPTG to the culture system of step (2) to a final concentration of 0.2M, and continue to culture at 25℃ and 200rpm for 20h;

[0051] (4) Centrifuge the fermentation broth from step (3) at 4°C and 8000×g for 10 min to collect the cells;

[0052] (5) The bacterial cells collected in step (4) were resuspended twice in pre-cooled PBS buffer (pH=7.0), then sonicated and centrifuged at 12000×g at 4℃ to obtain the supernatant sample.

[0053] (6) Take 10 μL of the supernatant sample obtained in step (5), heat it to denature, and then perform Western blotting analysis. The results are as follows: Figure 1 It can be seen that there is protein at 71kD in the supernatant, and its size is consistent with that of the polysaccharide degrading enzyme gelR.

[0054] (7) The supernatant sample obtained in step (5) is filtered through a 0.22 μm water filter to remove bacteria and obtain crude polysaccharide degrading enzyme solution.

[0055] Example 3: Preparation of competent cells of the velan gum producing strain

[0056] Competent cells of the flavonoid-producing strain were prepared using the following method:

[0057] (1) Streak Sphingomonas NX-3, which was stored in glycerol tubes at -80℃, on LB plates and incubate at 34℃ for 20h.

[0058] (2) Pick a single colony from step (1) and place it in 10 mL of oligotrophic medium. Incubate at 34°C for 12 h to obtain a seed culture.

[0059] (3) Take 1 mL of the seed culture from step (2) and inoculate it into a 500 mL shake flask containing 100 mL of oligotrophic medium. Incubate at 34 °C until OD reaches 100 mL. 600 It ranges from 0.4 to 0.6;

[0060] (4) Add 1 mL of the crude sphingosine gum degrading enzyme solution obtained in Example 2 to the culture system in step (3) and continue to culture for 30 min;

[0061] (5) After incubating the culture medium from step (4) on ice for 30 min, centrifuge at 8000×g for 10 min at 4℃ to collect competent cells;

[0062] (6) Resuspend the bacterial cells collected in step (5) with electroporation buffer, centrifuge at 8000×g for 10 min at 4℃, remove the supernatant, and repeat this step twice.

[0063] (7) Resuspend the bacterial cells obtained by centrifugation in step (6) with electroporation buffer and adjust the concentration of competent cells to OD. 600 Aliquot between 2 and 5 μL (100 μL per tube) and store at -80 °C.

[0064] Example 4 Electroporation Conversion of Vilan Gum Producing Bacteria

[0065] (1) Take about 100 ng of pBBR1MCS-2 plasmid and add it to 100 μL of competent cells of the vilan gum producing strain prepared in Example 3 on ice. Mix the plasmid and competent cells thoroughly by gently aspirating and spitting with a pipette and incubate on ice for 10 min.

[0066] (2) Transfer the mixed system in step (1) to a pre-cooled electrostatic cup (1 mm) at 0℃ and electrostatically shock it (voltage 3KV / cm, time constant = 4ms) 3 times.

[0067] (3) Immediately after the electric shock, add 700 μL of Soc medium to the electric transfer cup in step (2), mix well, and transfer to a sterile 1.5 mL centrifuge tube. Incubate at 34 °C with shaking for 1 h.

[0068] (4) The culture medium from step (3) was spread on LB solid medium plates containing a final concentration of 40 μg / mL kanamycin and incubated at 34°C for 48 h.

[0069] (5) 150-200 transducers were obtained on the plate after the electric shock in step (4);

[0070] (6) Randomly select the grown single clones and perform colony PCR using primers (F: 5′-CAGGATGAGGATCGTTTC-3′, R: 5′-CTTGGTCGGTCATTTCGAAC-3′) at both ends of the cannabidiol resistance gene on plasmid pBBR1MCS-2, followed by agarose gel electrophoresis. Figure 2 The results of PCR gel electrophoresis identification of the kanamycin resistance gene of the transformed strain in Example 4 of this invention show that the amplified band size is 848 bp, which is consistent with the size of the kanamycin resistance gene on pBBR1MCS-2.

[0071] Example 5: Preparation of competent strains for gellan gum production

[0072] (1) Streak Sphingomonas ATCC 31961 stored in glycerol tubes at -80℃ on LB plates and incubate at 30℃ for 20h.

[0073] (2) Pick a single colony from step 1 and place it in 10 mL of oligotrophic medium. Incubate at 30°C for 12 h to obtain seed culture.

[0074] (3) Take 1 mL of the seed culture from step (2) and inoculate it into a 500 mL shake flask containing 100 mL of oligotrophic medium. Incubate at 28 °C until OD. 600 It ranges from 0.4 to 0.6;

[0075] (4) Add 2 mL of the crude sphingosine gum degrading enzyme solution obtained in Example 2 to the culture system in step (3) and continue to culture for 45 min;

[0076] (5) After incubating the culture medium from step (4) on ice for 30 min, centrifuge at 8000×g for 10 min at 4℃ to collect competent cells;

[0077] (6) Resuspend the bacterial cells collected in step (5) with electroporation buffer, centrifuge at 8000×g for 10 min at 4℃, remove the supernatant, and repeat this step twice.

[0078] (7) Resuspend the bacterial cells obtained by centrifugation in step (6) with electroporation buffer and adjust the concentration of competent cells to OD. 600 Aliquot between 2 and 5 μL (100 μL per tube) and store at -80 °C.

[0079] Example 6 Electrostatic Conversion of Gel-Producing Bacteria

[0080] (1) Take about 100 ng of pBBR1MCS-2 plasmid and add it to 100 μL of competent cells prepared in Example 5 on ice. Mix the plasmid and competent cells thoroughly by gently aspirating and spitting with a pipette, and incubate on ice for 10 min.

[0081] (2) Transfer the mixed system in step (1) to a pre-cooled electrostatic cup (1 mm) at 0℃ and electrostatically discharge it (voltage 3KV / cm, time constant = 4ms) once.

[0082] (3) Immediately after the electric shock, add 700 μL of Soc medium to the electric transfer cup in step (2), mix well, and transfer to a sterile 1.5 mL centrifuge tube. Incubate at 30 °C with shaking for 2 h.

[0083] (4) The culture medium from step (3) was spread on LB solid medium plates containing a final concentration of 40 μg / mL kanamycin and incubated at 30°C for 48 h.

[0084] (5) After step (4), 80-120 transducers are obtained on the plate after the electric shock;

[0085] (6) Randomly selected single clones were subjected to colony PCR using primers (F: 5′-CAGGATGAGGATCGTTTC-3′, R: 5′-CTTGGTCGGTCATTTCGAAC-3′) at both ends of the kanamycin resistance gene on plasmid pBBR1MCS-2. The amplified band size was verified by agarose gel electrophoresis. The amplified band size was 848bp, which was consistent with the size of the kanamycin resistance gene on pBBR1MCS-2.

[0086] Example 7: Preparation of competent cells of rhamnogum production strain

[0087] (1) Streak Sphingomonas CGMCC No.6833, which was stored in glycerol tubes at -80℃, on LB plates and incubate at 28℃ for 20h.

[0088] (2) Pick a single colony from step (1) and place it in 10 mL of oligotrophic medium. Incubate at 28°C for 12 h to obtain a seed culture.

[0089] (3) Take 1 mL of the seed culture from step (2) and inoculate it into a 500 mL shake flask containing 100 mL of oligotrophic medium. Incubate at 28 °C until OD. 600 It ranges from 0.4 to 0.6;

[0090] (4) Add 4 mL of the crude sphingosine gum degrading enzyme solution obtained in Example 2 to the culture system in step (3) and continue to culture for 60 min;

[0091] (5) After incubating the culture medium from step (4) on ice for 30 min, centrifuge at 8000×g for 10 min at 4℃ to collect competent cells;

[0092] (6) Resuspend the bacterial cells collected in step (5) with electroporation buffer, centrifuge at 8000×g for 10 min at 4℃, remove the supernatant, and repeat this step twice.

[0093] (7) Resuspend the bacterial cells obtained by centrifugation in step (6) with electroporation buffer and adjust the concentration of competent cells to OD. 600 Aliquot between 2 and 5 μL (100 μL per tube) and store at -80 °C.

[0094] Example 8 Electroporation Conversion of Rhamnol Production Bacteria

[0095] (1) Take about 100 ng of pBBR1MCS-2 plasmid and add it to 100 μL of competent cells prepared in Example 7 on ice. Mix the plasmid and competent cells thoroughly by gently aspirating and spitting with a pipette, and incubate on ice for 10 min.

[0096] (2) Transfer the mixed system in step (1) to a pre-cooled electrostatic cup (1 mm) at 0℃ and electrostatically discharge it (voltage 3KV / cm, time constant = 4ms) twice.

[0097] (3) Immediately after the electric shock, add 700 μL of Soc medium to the electric transfer cup in step (2), mix well, and transfer to a sterile 1.5 mL centrifuge tube. Incubate at 28 °C with shaking for 1 h.

[0098] (4) The culture medium from step (3) was spread on LB solid medium plates containing a final concentration of 40 μg / mL kanamycin and incubated at 28°C for 48 h.

[0099] (5) 30-60 transducers are obtained on the plate after the electric shock in step (4);

[0100] (6) Randomly selected single clones were subjected to colony PCR using primers (F: 5′-CAGGATGAGGATCGTTTC-3′, R: 5′-CTTGGTCGGTCATTTCGAAC-3′) at both ends of the kanamycin resistance gene on plasmid pBBR1MCS-2. The amplified band size was verified by agarose gel electrophoresis. The amplified band size was 848bp, which was consistent with the size of the kanamycin resistance gene on pBBR1MCS-2.

[0101] Comparative Example 1: Mycelial Gel Separation After Adding Degrading Enzyme

[0102] This invention provides a comparison chart of cell acquisition effects by centrifugation with and without polysaccharide-degrading enzyme in Example 3. Figure 3 The figures show centrifugation of fermentation broth with added polysaccharide-degrading enzyme and centrifugation of the original fermentation broth, respectively. The comparison clearly shows that this invention achieves mycelial separation by adding polysaccharide-degrading enzyme, facilitating the recovery of microbial cells, eliminating interference from extracellular polysaccharides in the preparation of competent cells, and thus improving the gene transformation efficiency of polysaccharide-producing strains.

[0103] Based on conventional electroporation transformation methods, this invention presents a gene transformation method suitable for microbial polysaccharide-producing strains. By adding polysaccharide-degrading enzymes to remove the polysaccharide coating from the bacterial cells, rapid separation of the bacterial gel is achieved. This improves the preparation method of competent cells for this type of strain, significantly increasing the success rate and efficiency of gene transformation. It provides an effective method for the genetic engineering of microbial polysaccharide-producing strains and has significant practical value.

[0104] The embodiments of the present invention described above are intended to explain the relevant content of the present invention. Any modifications, deletions, additions, or other operations based on the present invention will be included within the protection scope of the present invention. The concept of the present invention can also be applied to the treatment of strains that produce high molecular weight polymers through fermentation, and its application prospects are very broad.

Claims

1. A method for improving the gene transformation efficiency of microbial polysaccharide-producing strains, characterized in that, Includes the following steps: (1) Preparation of enzyme solution for polysaccharide degrading enzyme; (2) Treat the polysaccharide-producing strain with polysaccharide-degrading enzymes to remove the polysaccharide coating on the strain cells, obtain cell bodies, and prepare competent cells; wherein, the polysaccharide-producing strain is inoculated into an oligotrophic medium containing 0.1-0.5 M sucrose and cultured in a shaker at 25-35 °C until the absorbance OD reaches a certain level. 600 When the enzyme concentration is between 0.2 and 0.6, add 1-5% v / v of the enzyme solution obtained in step (1). The enzyme activity of the solution is 3-10 U / mL. Continue culturing for 10-60 min, centrifuge to collect the cells, wash several times with pre-cooled glycerol and resuspend to OD. 600 Store at -80 °C between 2 and 5 °C; (3) The exogenous plasmid was transformed into competent cells after step (2) by electroporation. The electroporation conditions were 2.0KV-5KV, the pulse time was 2-10 ms, and the electroporation was performed 1-5 times. The polysaccharide-degrading enzyme is a polysaccharide-degrading enzyme capable of degrading the extracellular polysaccharides produced during the culture of the microbial polysaccharide-producing strain; the nucleotide sequence of the polysaccharide-degrading enzyme is shown in SEQ ID NO. 1; the accession number of the microbial polysaccharide-producing strain is CGMCC No. 6833.

2. The method according to claim 1, characterized in that, In step (1), the polysaccharide-degrading enzyme solution was prepared as follows: the gene for the polysaccharide-degrading enzyme was ligated into the expression vector pET-28a and transformed into E. coli competent cells BL21(DE3) to obtain recombinant cells expressing the polysaccharide-degrading enzyme, which were then cultured at 32-37 °C until OD200. 600 Once the value is between 0.4 and 0.8, add the inducing agent IPTG and continue culturing at 24-28°C for 16-24 h. Collect the bacterial cells, resuspend them several times with PBS buffer, and then sonicate to disrupt them. Centrifuge the supernatant and filter it through a membrane to obtain the crude enzyme solution, which is then stored at -80°C.

3. The method according to claim 2, characterized in that, The final concentration of the added inducing agent IPTG is 0.1-1M.

4. The method according to claim 1, characterized in that, The glycerol is 5-15% by volume.

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