Marine bacterial protease for degrading tropomyosin and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses marine bacterial protease as well as a coding gene, a preparation method and application thereof. The invention provides protease 4SP5-519 capable of specifically degrading allergenic tropomyosin in aquatic products and reducing the sensitization of the allergenic tropomyosin, and the protease 4SP5-519 is applied to improving the food safety of the aquatic products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a marine bacterial protease, its encoding gene, preparation method, and application. Background Technology

[0002] Seafood is rich in high-quality protein and unsaturated fatty acids, playing an important role in people's daily diet. However, epidemiological and food allergy surveys in China have found that seafood is the main allergen for allergic individuals, with shellfish such as shrimp and crab being the primary cause of allergic reactions. The main causes of seafood allergies are tropomyosin and calcium-binding proteins present in seafood.

[0003] Tropomyosin (TM) is the most prevalent and stable allergen identified in crustaceans, exhibiting strong sensitizing properties. A highly conserved acidic glycoprotein, tropomyosin has an isoelectric point of around 4.5 and a relative molecular mass of 33-38 kDa. Its native structure consists of two parallel α-helical tropomyosin molecules intertwined to form a coiled-helix structure. Its antigenic epitopes are highly conserved, and it is heat- and digestibility-resistant, making it difficult to destroy during processing and cooking. It easily induces allergic reactions in sensitive individuals. Clinical manifestations include rashes, urticaria, and asthma; in severe cases, anaphylactic shock can occur. Epidemiological studies indicate that crustacean allergy accounts for approximately 2%–3% of food allergy sufferers, reaching over 5% in some parts of Asia. Although the overall mortality rate is low, anaphylactic shock, if not treated promptly, can be life-threatening, thus posing a significant risk to individuals with crustacean allergies.

[0004] Enzymatic hydrolysis technology for reducing the allergenicity of aquatic products has attracted attention from scholars both domestically and internationally. Marine microorganisms are an important source of proteases. Because they live in extreme environments such as high salinity, high pressure, low temperature, or high temperature, the proteases they produce typically possess unique structural and functional properties. These enzymes exhibit strong salt tolerance, temperature tolerance, and stability, maintaining highly efficient catalytic activity even in environments where activity is difficult to maintain under normal conditions. Simultaneously, marine microbial proteases exhibit high substrate specificity and high catalytic efficiency, providing broader development potential for industrial and biotechnological applications. Research on eliminating the allergenicity of allergenic proteins using enzymatic methods mainly employs commercially available proteases, which have weak elimination effects. Research on the degradation of allergen myosin using marine-derived proteases is limited.

[0005] Therefore, screening for proteases that can specifically hydrolyze tropomyosin is of great significance for the safety of aquatic products. This invention provides a marine bacterial protease that specifically hydrolyzes tropomyosin, its encoding gene, preparation method, and applications. Summary of the Invention

[0006] The purpose of this invention is to address the deficiencies of the prior art by providing a marine bacterial protease for hydrolyzing tropomyosin, its encoding gene, preparation method, and application, thereby resolving the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a protease gene 4SP5-519 from the marine bacterium Bacillus paranthracis.4SP5, which contains the nucleotide sequence shown in SEQ ID No.1.

[0008] A protein encoded by the protease gene 4SP5-519 of the marine bacterium Bacillus paranthracis.4SP5, containing the amino acid sequence shown in SEQ ID No.2.

[0009] A method for producing a protease from the marine bacterium Bacillus paranthracis.4SP5, comprising the following steps:

[0010] Step 1: Bacillus paranthracis.4SP5 was sent for whole-genome sequencing. The protease gene was screened by bioinformatics analysis. Primers were designed with XhoⅠ and BamHI as restriction sites: 519-F (5'-CCGCTCGAGATGAAACAGGAGATTAAAAGAGGTT-3') (SEQ ID No. 3); 519-R (5'-CGCGGATCCTTCTATGATCTTCATTTTTGAAAAAGG-3') (SEQ ID No. 4);

[0011] Step 2: Extract the genome of Bacillus paranthracis.4SP5 as a template, obtain the target gene by PCR, digest the target gene with the set restriction endonuclease, and ligate it with the plasmid pColdⅠ digested with the same endonuclease.

[0012] Step 3: Transform the ligation product into E. coli DH5α competent cells, add 600 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 180 r / min for 1-1.5 h. Take 100 μL of the bacterial culture and spread it on LB solid medium containing 100 μg / mL ampicillin, and incubate at 37°C for 12-15 h.

[0013] Step 4: Pick a single colony and inoculate it into LB liquid medium containing ampicillin. Incubate at 37°C for 6-8 hours. Use a plasmid miniprep kit to extract the E. coli DH5α recombinant plasmid containing the target gene.

[0014] Step 5: Transform the extracted plasmid into E. coli BL21 competent cells, add 600 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 150 r / min for 1.5 h. Take 100 μL of the bacterial culture and spread it on LB solid medium containing ampicillin, and incubate at 37°C for 12-14 h.

[0015] Step 6: Pick a single colony and inoculate it into LB liquid medium containing ampicillin. Incubate at 37°C and 180 rpm for 4-6 hours. OD 600nm When the OD value is approximately 0.8, inoculate 4% into LB liquid medium containing ampicillin and incubate at 37°C and 180 rpm for 4-5 hours. 600 When nm is about 0.5, add isopropyl-β-D-thiogalactoside to a final concentration of 0.5 mmol / L, lower the temperature to 15℃, and ferment for 24 h;

[0016] Step 7: Centrifuge the cultured bacterial solution at 8000 r / min for 15 min and discard the supernatant; resuspend the precipitated bacterial cells in Tris-HCl buffer, sonicate for 30 min, centrifuge at 8000 r / min for 10 min, and the supernatant is the crude enzyme solution of the protease encoded by gene 4SP5-519.

[0017] As a preferred embodiment of the present invention, the LB liquid culture medium in step 3 is composed of: 0.5% yeast extract, 1% tryptone, 1% NaCl, and pH 7.4.

[0018] The application of a protease 4SP5-519 produced by the above method in the hydrolysis of tropomyosin.

[0019] Compared with existing technologies, the advantages of this invention are: existing commercial enzymes are weak at clearing tropomyosin from aquatic products, and there is limited research on the use of marine-derived proteases for degrading allergenic proteins. This invention provides a marine microbial protease 4SP5-519, which can specifically degrade tropomyosin, an allergenic protein in aquatic products. This enzyme exhibits the highest activity at 60°C and pH 9.0, and is relatively stable under low-temperature, neutral, and alkaline conditions. + Na + NH4 + It promotes the activity of this enzyme. Ni + Co + Zn + Mn 2+ Cu 2+ Ba 2+The enzyme exhibits inhibitory activity. PMSF also has an inhibitory effect on this enzyme. It retains over 80% activity in 10% urea and organic solvents, and over 50% activity in surfactants Tween 80 and Triton 100. This enzyme shows good degradation effect on tropomyosin. Enzymatic hydrolysis of 0.5 mg of tropomyosin with 0.5 U of enzyme activity for 15 minutes can remove over 95% of the tropomyosin. Hydrolysis of tropomyosin in brown crab, Dungeness crab, swimming crab, and red snapper with this enzyme showed that, at an enzyme addition of 20 U / mL, after 1 hour of enzymatic hydrolysis, the removal rate of tropomyosin in brown crab reached 91%, while the removal rates in Dungeness crab and red snapper reached 78% and 79%, respectively, and the removal rate in swimming crab reached 63%. These results indicate that protease 4SP5-519 has the potential to remove tropomyosin from aquatic products and shows promise in the deep processing of crab meat products, especially brown crab. Attached Figure Description

[0020] Figure 1 This is a gel image of PCR amplification of the target gene of protease 4SP5-519.

[0021] Figure 2 Gel image of the protease 4SP5-519 clone plasmid;

[0022] Figure 3 Image of double digestion of the protease 4SP5-519 cloning plasmid;

[0023] Figure 4 The effect of temperature on the activity of protease 4SP5-519;

[0024] Figure 5 Temperature stability of protease 4SP5-519;

[0025] Figure 6 The effect of pH on the activity of protease 4SP5-519;

[0026] Figure 7 pH stability of protease 4SP5-519;

[0027] Figure 8 The effect of metal ions on the activity of protease 4SP5-519;

[0028] Figure 9 The effects of inhibitors, organic solvents, and surfactants on the activity of protease 4SP5-519;

[0029] Figure 10 A standard curve for ELISA detection of tropomyosin;

[0030] Figure 11The residual amount of tropomyosin was determined by enzymatically hydrolyzing 0.5 mg of tropomyosin with 0.5 U of enzyme for 15 min under different hydrolysis time conditions.

[0031] Figure 12 The residual amount of tropomyosin in different crabs under conditions of 0, 30, and 60 min with 20U enzyme activity was determined. Detailed Implementation

[0032] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0033] Example 1: This invention provides a method for the production of protease 4SP5-519 by strain Bacillus paranthracis.4SP5:

[0034] Step 1: The Bacillus paranthracis.4SP5 protease gene of marine bacteria;

[0035] DNA was extracted from strain Bacillus paranthracis.4SP5. Primers were designed using XhoⅠ and BamHI as restriction sites: 519-F (5'-CCGCTCGAGATGAAACAGGAGATTAAAAGAGGTT-3') (SEQ ID No. 3); 519-R (5'-CGCGGATCCTTCTATGATCTTCATTTTTGA AAAAGG-3') (SEQ ID No. 4). The target gene was obtained by PCR, and the results are as follows: Figure 1 As shown;

[0036] Step 2: Cloning of the Bacillus paranthracis.4SP5 protease gene from marine bacteria;

[0037] The target gene was digested with the specified restriction endonuclease, and then ligated to the plasmid pColdⅠ, which had been digested with the same endonuclease. The ligation product was transformed into E. coli DH5α, and the transformed cells were screened using LB medium containing ampicillin. Positive transformants were picked by PCR, and the plasmid was extracted and sent to Shanghai Sangon Biotech for verification. The results are as follows: Figure 2 As shown. The protease 4SP5-519 cloning plasmid was double-digested, and the results are as follows. Figure 3 As shown.

[0038] In summary, the following steps were performed: First, the Bacillus paranthraciss.4SP5 protease gene from marine bacteria was obtained: DNA was extracted from strain Bacillus paranthraciss.4SP5, and primers were designed using XhoⅠ and BamHI as restriction sites: 519-F (5'-CCGCTCGAGATGAAACAGGAGATTAAAAGAGGTT-3') (SEQ ID No. 3); 519-R (5'-CGCGGATCCTTCTATGATCTTCATTTTTGAAAAAGG-3') (SEQ ID No. 4). The target gene was obtained by PCR. Then, the target gene was digested with the specified restriction endonuclease and ligated with the plasmid pColdⅠ, which had been digested with the same endonuclease. The ligation product was transformed into E. coli DH5α, and the transformed cells were screened using LB medium containing ampicillin. Positive transformants were picked by PCR, and the plasmid was extracted and sent to Shanghai Sangon Biotech for verification. The sequencing results are shown in SEQ ID No. 1. We named this gene 4SP5-519.

[0039] The nucleotide sequence of 4SP5-519 is as follows:

[0040] ATGAAACAGGAGATTAAAAGAGGTTGGGGGAAATACATACTCTTCGTGTTGTTTTGGTAATAGCCTATCATTCTTTTACTTTATGTAAAGTGGAAGGGAAATCGATGCAACCGACTTTATATGAAGAA GACTACGTATTTGTAAATAAAGCAGCCGTACACTTTTCTGATTTAGAGCATGGAGAAATTGTCATTATAAAGGAAGAGGATGAGTCAAAATATTATGTAAAACGAGTAATAGGGCTTCCTGGTGACGTAA TTAACATAACGAATGGATCTGTATATGTGAATGATAAAAAACAAGAAGAACCGTATACAAATAAAGATTTATTCAATAATACGCAAGTGTTTTATAACTTTCAAAAGACAAAAATCCCACCAAATAAATTATTTGTAATGGGAGATAATCGTGAACTTAGTAGAGATAGTCGAAACGGTTTAGGATATATTGAAGAAGATAATAATAATAGGCAAAGTGGAATTTGTATATTATCCTTTTTCAAAAATGAAGATCATAGAA

[0041] Example 2: A method for producing protease from the marine bacterium Bacillus paranthracis.4SP5 as shown in Example 1, the specific steps are as follows: The extracted protease plasmid was cloned and expressed into E. coli BL21, plated on LB solid medium, cultured for 12 h, a single colony was picked and inoculated into LB liquid medium, activated at 180 r / min, 37 °C, when OD 600 nm When the concentration reached 0.5, isopropyl-β-D-thiogalactoside was added, and fermentation was carried out at 15°C for 24 h. The enzyme solution was centrifuged at 8000 rpm for 15 min, and the supernatant was discarded. The precipitated bacterial cells were resuspended in Tris-HCl buffer, sonicated for 30 min, and centrifuged at 8000 rpm for 10 min. The supernatant was used as the crude enzyme solution and stored at 4°C for later use. This vector expresses the 4SP5-519 protein as shown in SEQ ID No. 2.

[0042] The amino acid sequence of 4SP5-519 is as follows:

[0043] MKQEIKRGWGKYILFVFVLVIAYHSFTLCKVEGKSMQPTLYEEDYVFVNKAAVHFSDLEHGEIVIIKEEDESKYYVKRVIGLPGDVINITNGSVYVNDKKQEEPYTNKDLFNNTQVFYNFQKTKIPPNKLFVMGDNRELSRDSRNGLGYIEEDNIIGKVEFVYYPFSKMKIIE

[0044] Example 3: Properties of protease 4SP5-519 produced by strain Bacillus paranthracis.4SP5;

[0045] Step 1: Preparation of crude enzyme solution;

[0046] The extracted protease plasmid was cloned and expressed into E. coli BL21, plated on LB solid medium, and cultured for 12 h. Single colonies were picked and inoculated into LB liquid medium, activated at 37 °C with 180 rpm. When OD... 600 nm When the concentration reaches 0.5, add isopropyl-β-D-thiogalactoside. Ferment at 15°C for 24 hours. Centrifuge the enzyme solution at 8000 rpm for 15 minutes and discard the supernatant. Resuspend the precipitated bacterial cells in Tris-HCl buffer, sonicate for 30 minutes, centrifuge at 8000 rpm for 10 minutes, and use the supernatant as the crude enzyme solution. Store at 4°C for later use.

[0047] Step 2: The effect of temperature on enzyme activity;

[0048] Using 1% casein in Tris-HCl buffer (pH 9.0) as a substrate, the protease activity was measured at 20℃-70℃ to determine the optimal temperature for the protease. The protease was incubated at 20℃-60℃ for 1-5 h, and the residual enzyme activity was measured to determine the effect of temperature on protease stability. The blank control represents the protease activity under optimal conditions.

[0049] like Figure 4 As shown, protease 4SP5-519 exhibits activity over a wide temperature range, with an optimal reaction temperature of 60°C. Figure 5 As shown, 4SP5-519 exhibits good thermal stability at medium and low temperatures. When incubated at 20-40℃ for 3 hours, it can retain about 80% of its activity. However, when incubated at 50-60℃, its activity decreases sharply with the extension of incubation time.

[0050] Step 3: The effect of pH on enzyme activity;

[0051] Using 1% casein as a substrate, residual protease activity was measured at pH 4-10 using sodium acetate buffer (pH 4-6), PBS buffer (pH 6-8), and Tris-HCl buffer (pH 8-10) at 60℃ to determine the optimal pH for the protease. The protease was then incubated at 25℃ at pH 4-10 for 5 h, and residual protease activity was measured under optimal conditions to determine the effect of pH on protease stability.

[0052] The results are as follows Figure 6 As shown: the optimal pH for the 4SP5-519 reaction is 9.0. High enzyme activity can be observed in both neutral and alkaline environments, and more than 69% of the activity can still be retained at pH 6.0-10.0. pH stability results are as follows. Figure 7 As shown, after incubation at 25℃ for 5 hours, the enzyme activity of protease 4SP5-519 was relatively stable under pH 7-10 conditions, and it could also maintain high enzyme activity at pH 5-6.

[0053] Step 4: The effect of metal ions on enzyme activity;

[0054] Metal ions (Na) at concentrations of 1 mM, 5 mM, and 10 mM were used. + K + Ni + Mg 2+ NH4 + Zn 2+ Ba 2+ Co + Ca 2+ Mn 2+ Cu 2+ Fe 2+ Fe 3+The residual enzyme activity was measured by reacting the enzyme with the protease at pH 9 and 25°C. The enzyme activity without the addition of metal ions was used as a control.

[0055] The results are as follows Figure 8 As shown, K + Na + NH4 + It has a slight promoting effect on the activity of this enzyme. Ni + Co + Zn + Mn 2 + Cu 2+ Ba 2+ It has an inhibitory effect on the activity of this protease.

[0056] Step 5: Effects of inhibitors, organic solvents, and surfactants on enzyme activity;

[0057] The enzyme was reacted with EDTA, PMSF, DTT, SDS, and Urea at a final concentration of 10 mM, and with glycerol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetonitrile, Tween 80, and Triton X-100 at a final concentration of 10% at 60 °C. The residual activity of the protease was measured. The enzyme activity without added solvent was used as a control.

[0058] The results are as follows Figure 9 As shown, DTT strongly inhibits 4SP5-519, indicating the possible presence of disulfide bonds in its structure. EDTA and SDS also significantly inhibited the activity of the protease, and PMSF also had an inhibitory effect on this protease. Urea and organic solvents had little effect on enzyme activity, maintaining more than 80% of the activity. In the presence of surfactants Tween 80 and Triton 100, the enzyme activity could be maintained at more than 50%.

[0059] Step 6: Determination of protease activity;

[0060] After incubating 1 mL of crude enzyme solution at 60 °C for 2 min, mix with 1 mL of 1% casein solution and incubate at 60 °C for 10 min. Add 2 mL of 0.4 M trichloroacetic acid and incubate for another 10 min. Centrifuge at 7000 × g for 2 min and collect the supernatant. Mix 1 mL of the supernatant with 5 mL of 0.4 M sodium carbonate and 1 mL of Folin-Ciocalteu reagent. Incubate at 60 °C for 20 min for color development, and measure the OD using an Eppendorf full-wavelength microplate reader (USA). 680 The absorbance at nm. An enzyme activity unit is defined as the amount of enzyme that produces 1 μg of tyrosine per minute under conditions of 60℃ and pH 9.

[0061] In summary, this protease exhibits the following characteristics: Protease 4SP5-519 is active over a wide temperature range, with an optimal reaction temperature of 60℃. It demonstrates good thermostability at medium to low temperatures; after incubation at 20-40℃ for 3 hours, approximately 80% of its activity is retained. However, at 50-60℃, its activity decreases with prolonged incubation. The optimal pH for this enzyme is 9.0. High enzyme activity is observed in both neutral and alkaline environments, and it retains 70% of its activity at pH 6.0-10.0. The enzyme exhibits the highest stability at pH 9.0 and maintains high activity even at pH 5-6. + Na + NH4 + It has a slight promoting effect on the activity of this protease. Ni + Co + Zn + Mn 2+ Cu 2+ Ba 2+ The enzymes inhibited the activity of this protease, with the inhibitory effect becoming more pronounced at higher ion concentrations. DTT showed a strong inhibitory effect on 4SP5-519, suggesting the possible presence of disulfide bonds in its structure. EDTA and SDS also significantly inhibited the protease activity, as did PMSF. Urea and organic solvents had little effect on enzyme activity, maintaining over 80% of the activity. Surfactants Tween 80 and Triton 100 had inhibitory effects, but the enzyme activity was still maintained above 50%.

[0062] Example 4: Application of protease production by strain Bacillus paranthracis.4SP5;

[0063] Step 1: Sensitization test of enzymatic hydrolysis products;

[0064] OD was determined using a crustacean tropomyosin allergen enzyme-linked immunosorbent assay (ELISA) based on tropomyosin standards (0, 5, 20, 80, and 250 ppb). 450nm The standard curve was fitted using the four-parameter method. The results are as follows: Figure 10 As shown. The residual amount of tropomyosin was determined after incubating 0.5U enzyme solution with 0.5mg tropomyosin for 0-15 min. The results are shown below. Figure 11 As shown, incubation for 15 minutes effectively degraded over 95% of tropomyosin, which is largely consistent with the results obtained from SDS-PAGE. The results indicate that protease 4SP5-519 can effectively degrade tropomyosin.

[0065] Step 2: Study on allergenic proteins in crab meat by enzymatic hydrolysis with protease 4SP5-519;

[0066] Take 0.5g of meat from each of brown crab, Dungeness crab, swimming crab, and red snapper, add 4mL of 50mM Tris-HCl buffer (pH 9.0), and grind until homogenized. Incubate the homogenate with protease at a material-to-liquid ratio of 1:5, add enzyme solution to a final concentration of 20U / mL, and use a crab tropomyosin allergen enzyme-linked immunosorbent assay kit to determine the residual amount of tropomyosin at 0, 30, and 60 min.

[0067] The results are as follows Figure 12 As shown, protease 4SP5-519 exhibited the highest clearance rate for tropomyosin in brown crab, reaching 91% after 1 hour; the clearance rates for tropomyosin in Dungeness crab and Amur red crab reached 78% and 79%, respectively; and the clearance rate for tropomyosin in swimming crab also reached 63%. The results indicate that 4SP5-519 has the potential to remove tropomyosin from aquatic products and has promising applications in the deep processing of crab meat products.

[0068] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. The application of a protease 4SP5-519 from the marine bacterium Bacillus paranthracis. in the hydrolysis of aquatic products containing the allergenic protein tropomyosin, wherein the gene encoding the protease contains the nucleotide sequence shown in SEQ ID No. 1, and the aquatic products are selected from brown crab, Dungeness crab, red snapper, or swimming crab.

2. An application as described in claim 1, characterized in that, The protease contains the amino acid sequence shown in SEQ ID No.

2.

3. An application as described in any one of claims 1-2, characterized in that, The aquatic products are selected from bread crabs.

4. An application as described in any one of claims 1-3, characterized in that, The amount of protease added is 10-30 U / mL, preferably 20 U / mL.

5. An application as described in any one of claims 1-4, characterized in that, The enzymatic hydrolysis time is 0.5 to 2 hours, preferably 1 hour.

6. An application according to any one of claims 1-5, wherein the protease is prepared as follows: Step 1: Design primers with XhoⅠ and BamHI as restriction sites as follows: 519-F (5'-CCGCTCGAGATGAAACAGGAGATTAAAAGAGGTT-3'); 519-R (5'-CGCGGATCCTTCTATGATCTTCATTTTTGAAAAAGG-3'); Step 2: Extract the genome of Bacillus paranthracis.4SP5 as a template, obtain the target gene by PCR, digest the target gene with the set restriction endonuclease, and ligate it with the plasmid pColdⅠ digested with the same endonuclease. Step 3: Transform the ligation product into E. coli DH5α competent cells, add 600-700 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 180 r / min for 1-1.5 h. Take 100 μL of the bacterial culture and spread it on LB solid medium containing 100-150 μg / mL ampicillin, and incubate at 37°C for 12-15 h. Step 4: Pick a single colony and inoculate it into LB liquid medium containing ampicillin. Incubate at 37°C for 6-8 hours. Use a plasmid miniprep kit to extract the E. coli DH5α recombinant plasmid containing the target gene. Step 5: Transform the extracted plasmid into E. coli BL21 competent cells, add 600-700 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 150 r / min for 1.5 h. Take 100 μL of the bacterial culture and spread it on LB solid medium containing ampicillin, and incubate at 37°C for 12-14 h. Step 6: Pick a single colony and inoculate it into LB liquid medium containing ampicillin. Incubate at 37°C and 180 rpm for 4-6 hours. OD 600 When the nm value is approximately 0.8, 4% ampicillin is inoculated into LB liquid medium containing ampicillin and incubated at 37°C and 180 rpm for 4-5 hours. OD 600nm When the concentration is approximately 0.5, add isopropyl-β-D-thiogalactoside to a final concentration of 0.5 mmol / L, lower the temperature to 15°C, and ferment for 24 hours. Step 7: Centrifuge the cultured bacterial solution at 8000 r / min for 15 min, discard the supernatant, resuspend the precipitated bacterial cells in Tris-HCl buffer, sonicate for 30 min, centrifuge at 8000 r / min for 10 min, and the supernatant is the crude enzyme solution of 4SP5-519 protease.

7. The application according to claim 6, characterized in that, The composition of the LB liquid culture medium mentioned in step 3 is: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.4.

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