Production method and application of gelatin degradation active protein

Abstract

The invention relates to a production method of gelatin degradation active protein, which comprises the following steps: fermenting lysine bacillus GX9210 to produce the gelatin degradation active protein, the lysine bacillus GX9210 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20242440. Compared with the prior art, the method has the advantages that the gelatin degradation active protein is prepared by fermenting the safe and non-pathogenic bacteria, so that the safety is improved. In addition, the production method is efficient and simple, and the produced gelatin degradation active protein has good activity and has obvious inhibition capability on phytopathogen. The gelatin degradation active protein is creatively applied to preparation of the bacteriostatic agent capable of inhibiting phytopathogen, and a brand new choice and thought are provided for prevention and treatment of plant diseases.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a method for producing a gelatin-degrading active protein and an application of the protein as an antibacterial agent for plant pathogens. Background Art

[0002] Gelatin degradation active protein refers to a protein that can specifically degrade natural collagen and gelatin (partially denatured product of collagen), including collagenase, gelatinase, hyaluronidase, etc., with a molecular weight of about 68-130kDa. Gelatin degradation active protein is widely used in many fields such as pharmaceutical industry, food processing, and environmental pollutant treatment. In the pharmaceutical industry, gelatin degradation active protein plays an important role in the remodeling of extracellular matrix and tissue repair. In food processing, gelatin degradation active protein can improve the mechanical properties and water resistance of gelatin film. By mixing with other natural polymer materials, it improves its water vapor permeability, oxygen barrier, moisture retention and other properties. It is widely used as a microencapsulation agent and the production of biodegradable packaging materials. In addition, it is also used as a food additive in cheese manufacturing, hypoallergenic baby food, meat tenderization, etc. In the field of environmental pollutant treatment, gelatin degradation active protein is often used to treat waste pollutants such as chrome scraps, leather, hair, etc. in traditional aquatic products, slaughterhouses, and leather industries to reduce environmental pollution.

[0003] Gelatin-degrading active proteins can be derived from microorganisms, plants, and animals, among which microbial fermentation is one of the most efficient production and preparation methods for gelatin-degrading active proteins. At present, gelatin-degrading active proteins represented by collagenase are mainly obtained by fermenting strains from Clostridium and Vibrio, among which Clostridium histolyticum is the main one. However, Clostridium and Vibrio are both pathogenic bacteria, and toxins are produced during the fermentation process, which poses certain safety hazards to the environment and human health, limiting the application scenarios of microbial gelatin-degrading active proteins. In addition, if toxic substances in the fermentation products are removed by various methods, the target protein will be lost, resulting in a decrease in yield. Summary of the invention

[0004] Based on this, one object of the present invention is to provide a method for producing a gelatin-degrading active protein, comprising obtaining the gelatin-degrading active protein by fermenting a new lysinibacillus sp. GX9210. Another object of the present invention is to provide an application of the gelatin-degrading active protein in the preparation of an antibacterial agent.

[0005] The specific technical solution is:

[0006] A method for producing a gelatin-degrading active protein comprises the following steps: producing the gelatin-degrading active protein by fermenting Bacillus lysinicus GX9210; the Bacillus lysinicus GX9210 is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242440.

[0007] Compared with the prior art, the present invention uses a safe and non-pathogenic bacterium to ferment and prepare gelatin-degrading active protein, which improves safety. In addition, the production method is efficient and simple, and the produced gelatin-degrading active protein has good activity and has obvious inhibitory ability against plant pathogens when used as an antibacterial agent. The present invention pioneered the use of gelatin-degrading active protein to prepare antibacterial agents for the inhibition of plant pathogens, providing a new choice and idea for the prevention and control of plant diseases.

[0008] Furthermore, the method further comprises: subjecting the lysinic Bacillus GX9210 to shake flask fermentation for 24-48 hours to obtain fermentation broth, and then dialysis purification of the supernatant of the fermentation broth to obtain a gelatin-degrading active protein solution.

[0009] Furthermore, the conditions of the shake flask fermentation include an inoculation amount of 1-5%, a fermentation temperature of 35-40°C, and an oscillation speed of 180-250rpm; the dialysis method includes dialysis purification of the supernatant of the fermentation broth using a dialysis bag with a pore size of 10-50KD.

[0010] Furthermore, the culture medium used in the shake flask fermentation includes 2-8 parts of yeast powder, 5-13 parts of tryptone, 5-10 parts of NaCl, and 1000 parts of water, with a pH of 6.5-8.0.

[0011] Furthermore, the method also includes freeze-drying the obtained gelatin-degrading active protein solution to obtain gelatin-degrading active protein freeze-dried powder.

[0012] Furthermore, the present invention also provides the use of gelatin-degrading active protein in the preparation of antibacterial agent. The gelatin-degrading active protein is produced by the production method provided by the present invention.

[0013] Furthermore, the concentration of gelatin degradation active protein in the antibacterial agent is 1-10 mg / mL.

[0014] Furthermore, the antibacterial agent can inhibit plant pathogens.

[0015] Furthermore, the plant pathogens include plant pathogenic bacteria and / or plant pathogenic fungi.

[0016] Furthermore, the plant pathogenic bacteria include Ralstonia solanacearum, Xanthomonas solani, Pantoea anacardiae, and Pantoea sternbergii; the plant pathogenic fungi include Fusarium moniliforme, Neodiscus polytrichosporium, Verticillium nigromatis, and Pythium glomerulosa.

[0017] The lysinibacillus sp. GX9210 described in the present invention has been deposited in the China Center for Type Culture Collection (abbreviated as: CCTCC; address: China Center for Type Culture Collection, Wuhan University, Wuhan City, Hubei Province, China) on November 6, 2024, with a deposit date of November 6, 2024 and a deposit number of CCTCC NO: M20242440. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The colony morphology of the lysinibacillus GX9210 described in the present invention after being cultured on LB agar solid medium for 24 hours;

[0019] Figure 2 The electrophoresis diagram of the 16S rDNA PCR amplification product of the lysinic Bacillus GX9210 of the present invention; the left lane is the nucleic acid molecular weight standard band; the right lane is the 16S rDNA amplification product of the lysinic Bacillus GX9210;

[0020] Figure 3 A phylogenetic tree constructed from 16S rDNA sequences of the lysinic Bacillus GX9210 of the present invention and similar bacterial species;

[0021] Figure 4 The SDS-PAGE map of the gelatin-degrading active protein produced by the lysinic Bacillus GX9210 of the present invention, the left lane is a protein molecular weight standard, and the right lane is a protein band of the gelatin-degrading active protein solution prepared in step S403;

[0022] Figure 5 It is a total ion current diagram of LC-MS / MS mass spectrometry identification of gelatin-degrading active protein produced by lysinibacillus GX9210 of the present invention;

[0023] Figure 6 The gelatin degradation activity of the gelatin-degrading active protein produced by the lysinic Bacillus GX9210 of the present invention on gelatin; negative control: sterile water; positive control: commercial collagenase IV, concentration 0.5 mg / mL; gelatin-degrading active protein: 0.5 mg / mL protein dilution prepared in step S505 of the present invention;

[0024] Figure 7The experimental results of the inhibitory effect of the gelatin degradation active protein produced by the lysinic Bacillus GX9210 of the present invention on plant pathogenic bacteria; negative control: sterile water; antibacterial agent: the antibacterial agent containing 2 mg / mL gelatin degradation active protein described in step S602 of the present invention; positive control: streptomycin sulfate, concentration 0.2 mg / mL;

[0025] Figure 8 These are the experimental results of the inhibitory effect of the gelatin-degrading active protein produced by the lysinic Bacillus GX9210 of the present invention on plant pathogenic fungi; negative control: sterile water; antibacterial agent: the antibacterial agent containing 2 mg / mL gelatin-degrading active protein described in step S702 of the present invention; the positive control for Neodiscussora is 0.5 mg / mL mancozeb; the positive control for Pythium glomerulosa is 0.5 mg / mL carbendazim; the positive control for Fusarium moniliforme is 0.5 mg / mL carbendazim; the positive control for Verticillium nigromatis is 10 mg / mL carbendazim.

[0026] Fig. 9 Schematic diagram of the comparison result between the microbial collagenase (No: A0A1E4R6K6) and the amino acid sequence of SEQ ID NO: 4-21 obtained in step S503; the sequence in the figure is the sequence of the microbial collagenase (No: A0A1E4R6K6) in the Uniprot database, and the gray shadow marks are the peptides compared with SEQ ID NO: 4-22. DETAILED DESCRIPTION

[0027] The present invention hopes to use safer strains to ferment and produce gelatin-degrading active proteins, making the produced proteins safer and more widely used, while avoiding additional toxin removal steps and improving yields. Therefore, the present invention collects samples from various natural environments, isolates strains, screens non-pathogenic bacteria or probiotics, and investigates their potential in preparing gelatin-degrading active proteins.

[0028] After screening, investigation and verification, the present invention finally obtains a strain of Lysinibacillus sp. GX9210 (Lysinibacillus sp. GX9210) that is easy to culture and non-pathogenic and can ferment to produce gelatin-degrading active protein. After optimizing the fermentation product purification method, the present invention finally formed a production method of gelatin-degrading active protein using Lysinibacillus sp. GX9210 for fermentation, combined with dialysis and freeze-drying technology. Using this production method, more than 20 mg of gelatin-degrading active protein freeze-dried powder can be obtained per 100 mL of fermentation broth. The production method provided by the present invention has high yield, simple process, non-toxic and safe, and can be applied in more application scenarios.

[0029] Furthermore, since various bacteria, especially non-pathogenic bacteria and probiotics are often reported to produce a variety of antibacterial substances including antibacterial proteins, small molecule antibacterial peptides, and volatile antibacterial substances, they can be effectively applied in the fields of agriculture, animal husbandry, and medicine, and although gelatin-degrading active proteins can also be secreted and produced by bacteria, they have never been tried to be used as an antibacterial substance in the agricultural field. Therefore, the present invention explores the antibacterial activity of the gelatin-degrading active protein secreted by Bacillus lysine GX9210. The present invention finds that when the produced gelatin-degrading active protein is used as an antibacterial agent to inhibit plant pathogens, it has a good antibacterial effect on plant pathogens. The present invention uses gelatin-degrading active proteins to inhibit plant pathogens, providing a new idea and option for the prevention and control of plant diseases.

[0030] The following, in conjunction with the accompanying drawings and specific examples, introduces the separation and purification of Bacillus lysinii GX9210, the cultivation of Bacillus lysinii GX9210, the identification of the strain of Bacillus lysinii GX9210, the production of gelatin-degrading active protein, the identification of gelatin-degrading active protein, the experiment of the inhibitory effect of gelatin-degrading active protein on plant pathogenic bacteria, and the experiment of the inhibitory effect of gelatin-degrading active protein on plant pathogenic fungi, to further explain in detail the production method of the gelatin-degrading active protein of the present invention and the application of the gelatin-degrading active protein in the field of inhibiting plant pathogenic bacteria.

[0031] Isolation and Purification of Lysinibacillus GX9210

[0032] S101 dilutes the soil: weigh 10 g of soil sample, the soil sample comes from the soil of diseased tomato roots in Tianyang District, Baise City, Guangxi; then, in an ultra-clean workbench, add 90 mL of sterile water to the soil sample, place it on an oscillator and oscillate for 60 min, so that the soil sample is evenly dispersed in the diluent to form a soil suspension; after the soil is dispersed, draw 100 μL of the soil suspension into 900 μL of sterile water and shake to obtain a 10-fold dilution, and then perform a 10-fold gradient dilution in sequence to obtain 10-10 6 The whole process was carried out in an ultra-clean workbench.

[0033] S102 strain culture: Take 100 μL of 10-10 6 The gradient dilutions of 100 times were spread on culture plates containing LB solid medium, and then the culture plates were cultured at 37°C for 24 hours until single colonies grew on the culture plates.

[0034] Screening, isolation and purification of S103 strain: After the cultivation, single colonies were picked one by one from the appropriate dilution gradient plates according to their growth conditions, and streaked on culture plates containing LB solid culture medium. The culture plates were then placed at 37°C and cultured for 24 hours until single colonies grew on the culture plates. The single colonies obtained at this time were the isolated and purified lysinibacillus GX9210.

[0035] S104 strain preservation: Pick the isolated and purified lysine Bacillus GX9210 monoclone from the culture dish containing LB solid culture medium, inoculate it into a 500mL conical flask containing 200ml LB liquid culture medium, and culture it for 20 hours at a constant temperature of 37°C and shaking at 200rpm to obtain seed liquid. The seed liquid and 25% sterile glycerol aqueous solution are placed in a tube at a volume ratio of 1:4 and mixed to obtain glycerol bacteria containing 20% ​​glycerol. After marking on the tube wall, store it at -20°C for a long time to complete the strain preservation.

[0036] The preparation method of LB solid culture medium is as follows: weigh 10g of tryptone, 5g of yeast powder, 7g of NaCl, and 15g of agar powder, stir and dissolve each component in distilled water in a conical flask, adjust to pH 7.0-7.5 and make up to 1L, seal the conical flask with sterilization paper, place it in a sterilizer for 121°C, 101KPa high-pressure steam sterilization for 20min, pour the sterilized culture medium into the culture dish in the clean bench before solidification, pour about 20mL of culture medium into each culture dish, and after the culture medium is cooled and solidified, a culture dish containing LB solid culture medium is obtained.

[0037] The preparation method of LB liquid culture medium is as follows: weigh 10 g of tryptone, 5 g of yeast extract powder, and 7 g of sodium chloride, stir and dissolve in distilled water, adjust the pH to 7.0-7.5 and make the volume to 1000 mL, dispense into conical flasks, and sterilize at 121°C, 101 KPa high-pressure steam for 20 min.

[0038] Cultivation of Lysinibacillus GX9210

[0039] S201 strain activation: Use a sterile inoculation stick to dip the glycerol bacteria obtained from S104, streak on LB solid medium, and place in a constant temperature incubator at 37°C for 24 hours. After 24 hours of incubation, the colony morphology of Lysinibacillus GX9210 is as follows: Figure 1 As shown, the colonies are round or oval, light yellow, with neat edges and a moist and smooth surface.

[0040] Preparation of S202 seed solution: Use a sterile inoculation stick to pick up a single colony of Bacillus lysinicus GX9210 and inoculate it into 100 mL of LB liquid culture medium, and culture it at a constant temperature of 37°C and a shaking speed of 220 rpm for 20 hours to obtain seed solution.

[0041] Identification of Lysinibacillus GX9210

[0042] S301 Extraction of genomic DNA: Use Omega Bacterial DNA Kit (D3350-01) kit to extract genomic DNA. First, take 2 ml of the seed solution obtained in step S202 into a sterile 2 ml centrifuge tube, centrifuge at 12000 rpm for 2 min, discard the supernatant and retain the precipitate; then, add 100 μL 1×TE Buffer to the precipitate, vortex to mix, add 10 μL lysozyme to mix, and warm at 37°C for 10 min; add 100 μL BTL Buffer and 20 μL proteinase K, mix, warm at 55°C for 1 h, and oscillate and mix three times in between; add 5 μL RNase A enzyme, mix, stand at room temperature for 5 min, centrifuge at 10000 rpm for 2 min, take 200 μL supernatant and transfer it to a new sterile 1.5 ml centrifuge tube; add 200 μL BTL Buffer, mix well, and place in a 65℃ warm bath for 10min; add 200μL anhydrous ethanol, vortex mix well, transfer all the samples to the adsorption column, centrifuge at 10000rpm for 2min, discard the supernatant and the adsorption column, and place the adsorption column in a new collection tube; add 500μL HBC Buffer to the adsorption column, centrifuge at 10000rpm for 2min, and discard the supernatant; add 700μL DNA Wash Buffer to the adsorption column, centrifuge at 10000rpm for 2min, discard the supernatant, and repeat twice; put the empty adsorption column back into the collection tube, centrifuge at 10000rpm for 2min; add 30μL~50μL Elution Buffer (preheated at 65℃) to the adsorption column to dissolve the DNA precipitate, and obtain genomic DNA, which is stored at -20℃ for later use.

[0043] S302 PCR amplification of 16S rDNA sequence: Using the genomic DNA obtained in step S301 as a template, PCR amplification was performed using Eubac27F and Eubac1492R as primers. The PCR reaction system (50 μL) included: 0.25 μL TaKaRa LA Taq (5 U / μl), 2.5 μL 10× LA Taq Buffer II (Mg 2+Plus), 4μl dNTPs Mixture (2.5mM), 1μL genomic DNA, 0.5μL upstream primer Eubac27F (10μM), 0.5μL downstream primer Eubac1492R (10μM), 16.25μL ddH2O. The sequence of the upstream primer Eubac27F is: 5'-agagtttgat cctggctcag-3' (as shown in SEQ ID NO: 1); the sequence of the downstream primer Eubac1492R is: 5'-ggttaccttg ttacgactt-3' (as shown in SEQ ID NO: 2).

[0044] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The process of denaturation, annealing, and extension was repeated 35 times; extension was performed at 72°C for another 10 min, and the PCR amplification product was stored at 4°C.

[0045] S303 PCR product electrophoresis: Take 5 μL of the PCR amplification product obtained in step S302 and perform electrophoresis on a 1.2% agarose gel at 120V for 25 minutes. The electrophoresis results are as follows: Figure 2 As shown, the band of the PCR amplification product is single and bright, with a length of about 1500 bp.

[0046] S304 16S rDNA sequence determination: 30 μL of the PCR amplification product obtained in step S302 was sent to Guangzhou Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results showed that the 16S rDNA length of the lysinic Bacillus GX9210 of the present invention was 1409 bp, and the specific sequence was shown in SEQ ID NO: 3.

[0047] S305 strain identification: The 16S rDNA sequence of Bacillus lysinicus GX9210 obtained by sequencing in step S304 was input into NCBI and BLAST comparison was performed. The strain GX9210 was molecularly identified based on its homology. Sequences with high homology to SEQ ID NO: 3 were downloaded, and a molecular evolution tree was constructed using MEGA7.0 software. The results are as follows Figure 3As shown, according to the results of 16srDNA homology comparison, the 16S rDNA sequence of the aminicidal Bacillus GX9210 provided by the present invention has a sequence similarity of 97% with that of Lysinibacillus fusiformis strain PLT 16 (ON365834.1). Therefore, it can be seen that the strain obtained by the present invention is classified as a new strain of the genus Lysinibacillus, and the present invention names the strain as Lysinibacillus sp. GX9210.

[0048] Production of gelatin-degradable active protein

[0049] S401 Fermentation of gelatin-degrading active protein: prepare a culture bottle containing 100 mL of LB liquid culture medium, inoculate the seed solution obtained in step S202 into the LB liquid culture medium at an inoculation rate of 2%, and then perform shake flask fermentation at a constant temperature of 37°C and a shaking speed of 220 rpm for 32 hours to obtain fermentation liquid.

[0050] S402 Preparation of fermentation broth supernatant: The fermentation broth obtained in step S401 is centrifuged at 8000 rpm for 10 minutes at 4°C, the supernatant is collected and filtered using a 0.22 μm polyethersulfone (PES) filter membrane, and the filtrate obtained is the supernatant of the fermentation broth.

[0051] S403 dialysis: put the supernatant obtained in step S402 into a dialysis bag with a pore size of 20KD, pierce the bag mouth, and put it into 20mM Tris-HCl buffer. The volume of the dialysate is 10 times the volume of the supernatant in the dialysis bag. Perform dialysis at 4°C with continuous magnetic stirring. Replace the buffer every 3-4 hours, and a total of 3 changes are required to remove small molecules and obtain a gelatin-degrading active protein solution.

[0052] S404 Preparation of gelatin degradation active protein freeze-dried powder: freeze-dry the gelatin degradation active protein solution obtained in step S403 using a conventional freeze dryer to obtain gelatin degradation active protein freeze-dried powder. More than 20 mg of microbial collagenase freeze-dried powder can be prepared per 100 mL of the fermentation liquid obtained in S401.

[0053] Identification of gelatin-degrading active proteins

[0054] S501 Sample pretreatment: The gelatin degradation active protein solution obtained in S403 was subjected to SDS-PAGE gel electrophoresis analysis, with a separation gel concentration of 12%, a constant voltage of 120V, and an electrophoresis time of about 1 hour. Figure 4As shown, a single protein band between molecular weight 100KD and 140KD is the target band. The target band is cut with a clean surgical blade and entrusted to Shanghai Zhongke New Life Biotechnology Co., Ltd. for LC-MS / MS mass spectrometry identification. The specific experimental parameters are as described in steps S502 and S503.

[0055] S502 Enzymatic hydrolysis of the test sample: The protein strip cut from the SDS-PAGE electrophoresis gel in step S501 is used as the test sample. After the test sample is subjected to conventional reduction and alkylation treatment, trypsin with a mass of one-fiftieth of the test sample is added and enzymatic hydrolysis is carried out at 37°C for 20 hours. The enzymatic hydrolysis product is desalted by solid phase extraction and freeze-dried, redissolved in a 0.1% formic acid aqueous solution, and stored at 20°C for use.

[0056] S503 mass spectrometry analysis: Liquid A is 0.1% formic acid in water, and liquid B is 0.1% formic acid in acetonitrile (84% acetonitrile). After the chromatographic column is equilibrated with 95% liquid A, the sample is loaded onto the Trap column by an automatic sampler. After chromatographic separation, the sample is analyzed by mass spectrometry using a mass spectrometer. The detection method is positive ion, the parent ion scanning range is 300-1800m / z, the primary mass spectrometry resolution is 70,000at 200m / z, the AGC (Automatic gain control) target is 1e6, the Maximum IT is 50ms, and the dynamic exclusion time (Dynamic exclusion) is 30.0s. The mass-to-charge ratio of peptides and peptide fragments was collected according to the following method: 20 fragment spectra (MS2 scan) were collected after each full scan (fμLl scan), MS2 Activation Type was HCD, Isolation window was 2m / z, secondary mass spectrometry resolution was 17,500at 200m / z, Normalized CollisionEnergy was 27eV, Underfill was 0.1%. The total ion current obtained by mass spectrometry analysis is shown in Figure 5 As shown, the amino acid sequence of the polypeptide of the gelatin-degrading active protein obtained in step S403 after trypsinization was obtained by analysis using Mascot 2.2.2 software. The specific sequence is shown in SEQ ID NO: 4-22.

[0057] S504 Data analysis: Compare the amino acid sequences of SEQ ID NO: 4-22 obtained in step S503 with the sequences in the Uniprot protein database. Fig. 9As shown, it was found that the protein was consistent with the molecular weight (122.82 kDa) of a microbial collagenase in a comparison (No: A0A1E4R6K6; microbial collagenase OS=Lysinibacillus fusiformis OX=28031GN=SAMN02787113_00501PE=3SV=1). It was preliminarily inferred that the protein was a type of microbial collagenase and should therefore have gelatin degradation activity.

[0058] The present invention further detects the degradation activity of the protein on gelatin, and the specific steps are as follows:

[0059] S505 Dissolving the lyophilized powder of gelatin-degrading active protein: Weigh the lyophilized powder of gelatin-degrading active protein prepared in S404, dissolve it in sterile water, and dilute it to 0.5 mg / L with sterile water to prepare a 0.5 mg / L protein diluent.

[0060] S506 gelatin degradation experiment: holes with a diameter of 5 mm were made at equal distances on the gelatin solid plate, and 50 μL of 0.5 mg / mL protein dilution was applied to the holes. Sterile water was used as a negative control, and a commercially available collagenase IV (enzyme activity ≥ 125 CDU / mg soild, where CDU is collagen digestion units) aqueous solution with a concentration of 0.5 mg / mL was used as a positive control. Three technical replicates were performed for each group. The gelatin solid plate was then placed at a constant temperature of 30°C for 48 hours to measure the degradation zone diameter.

[0061] The results are as follows Figure 6 As shown, the protein dilution with a concentration of 0.5 mg / mL produced by the lysinic Bacillus GX9210 of the present invention can produce a degradation zone of 24.0±0.89 mm on a gelatin solid plate, which is equivalent to the positive control group with a degradation zone diameter of 24.3±1.03 mm. The above experiment proves that the gelatin-degrading active protein freeze-dried powder obtained by S404 of the present invention has gelatin-degrading activity.

[0062] The preparation method of the gelatin solid plate is as follows: weigh 20 g of gelatin, 5 g of tryptone, 0.1 g of NaCl, 0.5 g of KH2PO4, 0.2 g of MgSO4·7H2O, and 15 g of agar powder into a conical flask, add 1000 mL of distilled water, seal it with sterilization paper, place it in a sterilizer and sterilize it with 121°C and 101 KPa high-pressure steam for 20 minutes, pour the sterilized culture medium into a culture dish in a clean bench before solidification, pour about 20 mL of culture medium into each culture dish, and after the culture medium is cooled and solidified, a solid plate containing gelatin is obtained.

[0063] Experimental study on the inhibitory effect of antibacterial agent containing gelatin degradation active protein on plant pathogenic bacteria

[0064] Activation of S601 plant pathogenic bacteria: Dip the glycerol strains of common plant pathogenic bacteria: Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, Pantoea ananatis, and Pantoea stewartii, and streak them on solid culture medium plates. After single colonies grow, pick out single colonies and inoculate them into liquid culture medium to prepare seed solutions of the four plant pathogenic bacteria.

[0065] Ralstonia solanacearum was purchased from Guangdong Microbiological Culture Collection Center, GDMCC No: 1.1561, and the culture medium was LB + glucose medium; Bacillus subtilis was purchased from Beijing Biobo Biotechnology Co., Ltd., with the product number: bio-099491, and the culture medium was NA medium; Pantoea ananas was purchased from Guangdong Microbiological Culture Collection Center, GDMCC No: 1.1601, and the culture medium was LB medium; Pantoea stewartii was purchased from China Industrial Microbiological Culture Collection Administration Center, CICC No: 10978, and the culture medium was LB medium.

[0066] Identification of the antibacterial activity of S602: The four plant pathogenic bacteria in S601 were used as indicator bacteria, and the OD values ​​of the seed solutions of the four plant pathogenic bacteria prepared in S601 were measured with sterile water. 600 Adjust to 1.0 and add 100 μL of the adjusted OD 600 The bacterial solution was evenly coated on the corresponding solid culture medium. Then, three holes with a diameter of 5 mm were made at equal distances on the solid culture medium, and the gelatin degradation active protein freeze-dried powder prepared in step S404 was dissolved with sterile water, and diluted to 2 mg / mL with sterile water to prepare an antibacterial agent. 50 μL of the antibacterial agent was dotted in the hole, and sterile water was used as a negative control and 0.2 mg / mL streptomycin sulfate was used as a positive control. After culturing at a constant temperature of 30°C for 48 hours, the diameter of the antibacterial zone was measured.

[0067] The results are as follows Figure 7 As shown in the figures: the gelatin-degrading active protein produced by lysinibacillus GX9210 of the present invention has good antibacterial effect on a variety of plant pathogenic bacteria, among which it has antibacterial activity against Ralstonia solanacearum, White blight, Pantoea steuneri and Pantoea ananas, and the diameters of the inhibition zones are 14.83±0.75mm, 23.00±0.63mm, 11.30±1.03mm and 11.3±0.52mm, respectively. Therefore, the gelatin-degrading active protein needs to be developed as an antagonist of plant pathogenic bacteria.

[0068] The preparation method of LB+glucose solid medium is as follows: weigh 10 g of tryptone, 5 g of yeast powder, 5 g of glucose, 7 g of NaCl, and 15 g of agar powder, add distilled water and stir, adjust the pH to 7.0-7.5, and make up to 1000 mL.

[0069] The preparation method of LB+glucose liquid culture medium is as follows: weigh 10 g of tryptone, 5 g of yeast powder, 5 g of glucose, and 7 g of NaCl, add distilled water and stir, adjust the pH to 7.0-7.5, and make up to 1000 mL.

[0070] The preparation method of NA solid culture medium is as follows: 1 g yeast powder, 5 g tryptone, 3 g beef extract, 10 g sucrose, 15 g agar powder, add distilled water and stir, adjust the pH to 7.0, and make up to 1000 mL.

[0071] The preparation method of NA liquid culture medium is as follows: 1 g of yeast powder, 5 g of tryptone, 3 g of beef extract, and 10 g of sucrose, add distilled water and stir, adjust the pH to 7.0, and make up to 1000 mL.

[0072] After the above culture medium is prepared, seal it with sterilization paper and place it in a sterilizer for sterilization at 121°C and 101KPa high-pressure steam for 20 minutes. When the temperature of the sterilizer drops below 70°C and the pressure returns to 0KPa, take it out for use.

[0073] Experimental study on the inhibitory effect of antibacterial agent containing gelatin degradation active protein on plant pathogenic fungi

[0074] Activation of S701 plant pathogenic fungi: Use a sterilized inoculation loop to scrape the slant strains of four common plant pathogenic fungi, Fusarium tandemis, Fusarium neoformans, Verticillium discoideum, and Pythium myriotylum, and inoculate them into PDA solid culture medium, place it in a 30°C biochemical incubator, culture Fusarium tandemis for 5 days, Fusarium neoformans for 5 days, Verticillium discoideum for 3 days, and Pythium myriotylum for 7 days, until they are full of hyphae, to obtain fungal cakes of the four plant pathogenic fungi.

[0075] Fusarium moniliforme was purchased from Wuhan Huizao Biotechnology Co., Ltd. with the product number: HZB120142; Neodiscus polytrichosporium was purchased from Wuhan Huizao Biotechnology Co., Ltd. with the product number: HZB177234; Verticillium nigromatum was purchased from Wuhan Huizao Biotechnology Co., Ltd. with the product number: HZB120427; Pythium glomerulosa was purchased from Wuhan Huizao Biotechnology Co., Ltd. with the product number: HZB208579.

[0076] Identification of antibacterial activity of S702: Using the four plant pathogenic fungi in S701 as indicator bacteria, take a round plant pathogenic fungus cake with a diameter of 8 mm and place it in the center of a new PDA solid culture medium, and make three holes with a diameter of 5 mm at equal distances on the periphery of the cake. Use sterile water to dissolve the gelatin degradation active protein freeze-dried powder prepared in step S404 and dilute it to 2 mg / mL with sterile water to prepare an antibacterial agent. 50 μL of antibacterial agent was dotted in the hole, and sterile water was used as a negative control, and carbendazim / mancozeb manganese was used as a positive control. Among them, the positive control for Neodiscussops multilocularis was 0.5 mg / mL mancozeb; the positive control for Pythium spp. was 0.5 mg / mL carbendazim; the positive control for Fusarium moniliformis was 0.5 mg / mL carbendazim; the positive control for Verticillium nigromatis was 10 mg / mL carbendazim. Then the PDA solid medium with the bacterial cake was placed in a 30°C biochemical incubator, and Fusarium moniliformis was cultured for 5 days, Neodiscus polytrichosporium was cultured for 5 days, Verticillium nigromatum was cultured for 3 days, and Pythium glomerulosa was cultured for 7 days. Then the colony diameter was measured and the fungal inhibition rate was calculated. Inhibition rate = (mycelium radius of pathogens in the negative control group - mycelium radius of pathogens in the antibacterial agent experimental group) / mycelium radius of pathogens in the negative control group × 100%. Among them, when the colony is irregular in shape, the shortest distance from the center of the colony to the colony boundary is recorded as the colony diameter.

[0077] The results are as follows Figure 8 As shown in the figure, the gelatin degradation active protein produced by lysinibacillus GX9210 of the present invention has an antibacterial rate of 66.57%, 61.90%, 61.50% and 55.26% against Pythium glomerulosa, Neodiscus polytrichosporium, Verticillium nigromatum and Fusarium moniliforme respectively. That is, the gelatin degradation active protein produced by lysinibacillus GX9210 has a good antibacterial effect on a variety of plant pathogenic fungi, and can be used to inhibit the growth of a variety of plant pathogenic fungi. The microbial collagenase needs to be developed as an antagonist of plant pathogenic fungi.

[0078] The preparation method of PDA solid culture medium is as follows: weigh 200g potato, 20g tryptone, 3g NaCl, 1.5g KH2PO4, and 15g agar powder into a conical flask, dissolve with distilled water and make up to 1000mL of sterile water, seal with sterilization paper, place in a sterilizer for 121°C, 101KPa high-pressure steam sterilization for 20min, pour the sterilized culture medium into the culture dish in the clean bench before solidification, pour about 20mL of culture medium into each culture dish, and obtain PDA solid culture medium after the culture medium is cooled and solidified.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] 1. A new method for producing gelatin-degrading active protein is provided, which uses a new strain of Lysinibacillus sp. GX9210 to ferment gelatin-degrading active protein. Since Lysinibacillus sp. GX9210 is not a pathogenic bacterium and is non-pathogenic to soil, plants and humans, compared with Clostridium and Vibrio, the use of Lysinibacillus sp. GX9210 for gelatin-degrading active protein fermentation is safer and has a wider range of application scenarios.

[0081] 2. 100 mL of fermentation liquid of Bacillus lysinic acid GX9210 of the present invention can produce more than 20 mg of gelatin degradation active protein, and the fermentation conditions are not harsh, and only the culture medium and culture conditions used can obtain a higher yield of microbial collagenase. At the same time, Bacillus lysinic acid GX9210 is a non-toxin-producing bacterium, so the gelatin degradation active protein produced by fermentation does not require a toxin removal step, further improving the yield. The strain provided by the present invention is a fermentation strain with great potential in large-scale production of safe microbial source gelatin degradation active protein.

[0082] 3. The purification step in the production process of gelatin-degrading active protein of the present invention is simple, and only requires dialysis with a dialysis bag with a pore size of 20KD and then freeze-drying to obtain gelatin-degrading active protein powder.

[0083] 4. The gelatin-degrading active protein produced by the present invention has high activity and has a significant degradation effect on gelatin.

[0084] 5. The gelatin-degrading active protein produced by the present invention has an antibacterial effect on a variety of plant pathogenic bacteria and pathogenic fungi, providing a new choice for gelatin-degrading active protein in the field of inhibiting plant pathogens.

[0085] 6. The present invention innovatively applies gelatin-degrading active protein in the field of inhibiting plant pathogens, providing a new option and idea for the prevention and treatment of plant diseases.

[0086] The experimental methods in the following examples of the present invention that do not specify specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The various common chemical reagents used in the examples are all commercially available products. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0087] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for producing a gelatin-degradable active protein, characterized in that: The method comprises the following steps: using lysinibacillus GX9210 to ferment and produce gelatin-degrading active protein; the lysinibacillus GX9210 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20242440.

2. The method for producing gelatin-degrading active protein according to claim 1, characterized in that: include: The lysinic Bacillus GX9210 is subjected to shake flask fermentation for 24-48 hours to obtain a fermentation broth, and then the supernatant of the fermentation broth is dialyzed and purified to obtain a gelatin-degrading active protein solution.

3. The method for producing a gelatin-degrading active protein according to claim 2, characterized in that: The shake flask fermentation includes the following conditions: inoculation amount of 1-5%, fermentation temperature of 35-40°C, and shaking speed of 180-250rpm; the dialysis method includes using a dialysis bag with a pore size of 10-50KD to dialyze and purify the supernatant of the fermentation liquid.

4. The method for producing a gelatin-degrading active protein according to claim 2, wherein: The culture medium used in the shake flask fermentation includes 2-8 parts of yeast powder, 5-13 parts of tryptone, 5-10 parts of NaCl, and 1000 parts of water, with a pH of 6.5-8.

0.

5. The method for producing gelatin-degrading active protein according to claim 2, characterized in that: The method also includes freeze-drying the obtained gelatin degradation active protein solution to obtain gelatin degradation active protein freeze-dried powder.

6. The application of gelatin degradation active protein in the preparation of antibacterial agent, characterized in that: The gelatin-degrading active protein is produced according to the production method according to any one of claims 1-5.

7. The use according to claim 6, characterized in that: The concentration of gelatin degradation active protein in the antibacterial agent is 1-50 mg / mL.

8. The use according to claim 7, characterized in that: The antibacterial agent can inhibit plant pathogens.

9. The use according to claim 8, characterized in that: The plant pathogens include plant pathogenic bacteria and / or plant pathogenic fungi.

10. The use according to claim 9, characterized in that: The plant pathogenic bacteria include Ralstonia solanacearum, Xanthomonas solani, Pantoea ananas, and Pantoea steieryanus; the plant pathogenic fungi include Fusarium moniliforme, Neodiscus polytrichosporium, Verticillium nigromatis, and Pythium glomerulosa.

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