Lysobacter strain and application thereof

Abstract

The application discloses a lysobacter strain and application thereof, the strain is a new species in the lysobacter, and the preservation number is CGMCC No.1.19230.The strain D1-1-M9 obtained by the application is a new species different from known species of the lysobacter genus, and can produce extracellular protease.The extracellular protease can maintain relatively high protease activity in a medium-temperature or low-temperature environment, so has very wide development and application prospects in medicine, food and industrial production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus lysinus and its applications, and more specifically to the study of the enzymatic properties of the extracellular proteases of the Bacillus lysinus strain. Background Technology

[0002] The genus *Lysobacter* was first proposed by Canadian scholars Christensen and Cook in 1978. Taxonomically, it belongs to the kingdom Procaryotes, phylum Proteobacteria, class Gammaproteobacteria, order Xanthomonadales, and family Xanthomonadaceae. The main characteristics of *Lysobacter* include: Gram-negative, aerobic corynoid bacteria; the main respiratory quinone is ubiquinone Q-8; the main polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE); the genome size is 2.5–6.7 Mb, and the G+C percentage is 61.6%–71.6%.

[0003] Lysobacteria are widely distributed in terrestrial soils, especially in the rhizosphere soils of crops, and most lysobacteria exhibit broad-spectrum antagonistic effects against plant pathogens. Lysobacteria have also been found in freshwater, seawater, and Antarctic coastal sediments, indicating their strong adaptability to extreme environments. New species of lysobacteria have also been discovered in animal and plant organisms and their related environments, such as tomato stems, sponge samples, Antarctic penguin feathers, giant panda enclosures, Pacific shrimp intestines, python respiratory tracts, human meibomian gland secretions, Oriental white stork feces, and cow dung deposits.

[0004] Lysobacter has high metabolic activity and can secrete a variety of active substances such as antibiotics and extracellular hydrolases. These active substances have good antagonistic activity against a variety of plant pathogens, nematodes, and bacteria. Therefore, it can be used as a new type of biocontrol bacteria with broad-spectrum antagonistic activity against bacteria, fungi, and nematodes.

[0005] Lysobacterium can secrete a variety of macromolecular active substances, such as cell membrane-bound phosphatases, nucleases produced after the exponential growth phase, cellulases with β-1,4-glucanase and chitosanase activities, keratinases with high keratinase decomposition activity, endopeptidases (hydrolases) that specifically recognize the carboxyl groups of lysine residues, and chitinases, β-1,3-glucanases, and proteases that degrade the cell walls of microorganisms such as nematodes. Lysobacterium can utilize these extracellular enzymes to antagonize the growth of plant pathogenic microorganisms, thereby achieving the purpose of controlling crop diseases.

[0006] Proteolytic activity is one of the most noteworthy common characteristics of the *Lysobacterium* genus, and it can be used as a hemostatic agent after wound cleaning. In the food industry, proteases play an important role in the processing of meat, dairy products, and beverages. Bacterial proteases are important detergent enzymes, accounting for 90% of global enzyme sales. Existing experimental data show that proteases produced by *Lysobacterium* have antagonistic activity against various pathogens, and researchers have used protease inhibitor experiments to confirm the correlation between proteases and the formation and release of spores and endospores.

[0007] Bacterial proteases are characterized by short production cycles, low costs, and ease of management and control. Furthermore, most microbially synthesized proteases are extracellular enzymes, making purification and preparation relatively easy and significantly reducing production costs. Microorganisms are increasingly becoming the main source of industrial enzyme preparations. Obtaining superior strains is crucial for protease production; therefore, isolating and identifying novel strains is an important step in obtaining high-yielding protease strains. With the deepening research into Bacillus lysate resources and their natural products, it is believed that more and more potent natural products will be discovered, providing abundant lead compounds for the development of biocontrol and biomedicine. Summary of the Invention

[0008] The main objective of this invention is to provide a Lysobacter sp.D1-1-M9 strain.

[0009] The Lysobacterium strain D1-1-M9 provided by this invention was isolated from the soil of Longmu Lake in Ngari Prefecture, Tibet Autonomous Region. The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.19230, dated November 8, 2023. Bacterial identification revealed that this strain is a novel species within the genus *Lysobacter*. The 16S rRNA gene sequence of this strain is shown in SEQ ID NO. 1.

[0010] Studies have found that the *Lystomum lysate* strain D1-1-M9 can produce extracellular proteases. Furthermore, it can utilize D-trehalose, D-cellobiose, D-raffinose, β-methyl-D-glucoside, N-acetyl-D-glucosamine, N-acetyl-β-D-mannitol, N-acetyl-D-glucosamine, α-D-glucose, D-fructose, D-galactose, 3-methyl-D-glucose, D-fucose, L-fucose, L-rhamnose, inositol, glycerol, D-glucose-6-PO4, D-fructose-6-PO4, D-aspartic acid, and gelatin. L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, pectin, D-galactonic acid, L-galactonolactone, D-gluconic acid, D-glucuronic acid, L-lactic acid, citric acid, α-ketoglutarate, L-malic acid, Tween 40, α-hydroxy-butyric acid, β-hydroxy-D, L-butyric acid, α-keto-butyric acid, acetoacetic acid, propionic acid, and acetic acid are used as the sole carbon source, exhibiting broad substrate spectrum.

[0011] Physiological experiments showed that strain D1-1-M9 can grow at temperatures of 10-35℃, pH values ​​of 6.5-10, and NaCl concentrations of 1.0%-4.0% (w / v). The strain exhibits optimal growth at 25℃, pH 7.0, and a NaCl concentration of 1.0%.

[0012] Optimized culture revealed that the optimal protease production capacity of D1-1-M9 was achieved with a NaCl concentration of 1.0% and the addition of 10% glucose to the 2216 medium. Enzymatic studies showed that the highest protease activity produced by Bacillus lysinensis D1-1-M9 was observed at 40℃ and pH 7. Relatively high protease activity was maintained under mesophilic and hypothermic conditions, but it was not heat-resistant. (The text also mentions the presence of Mg metal ions, but this seems unrelated to the preceding information and is likely a separate, incomplete thought.) 2+ and Fe 3+ (5.0 mmol / L) could inhibit its activity, and the relative activity after inhibition was only 52.3% and 30.6% of the control. This strain has broad application prospects in the study of microbial extracellular protease production.

[0013] In addition, the present invention also discloses the fermentation broth and liquid inoculum prepared from the above-mentioned Bacillus lysinus strain, as well as a method for preparing the same, the method comprising:

[0014] 1) Strain activation: Pick a strain and streak it onto 2216 solid medium, place it in a constant temperature incubator, and incubate at 25℃ for 24 hours;

[0015] 2) Preparation of seed culture: Pick activated bacterial cells and inoculate them into a large test tube containing seed culture medium, seal the tube with a rubber stopper, place it on a shaker at 25℃ and shake at 160 rpm for 16-24 h to obtain seed culture.

[0016] 3) Liquid fermentation: Then, the seed liquid and seed culture medium are inoculated at a volume ratio of 5-10% for expansion culture. The culture conditions are 25℃ and 160rpm. The culture obtained after 48h of culture is the fermentation broth. The fermentation broth is centrifuged at 12000rpm for 10min, and the bacterial cells are collected to obtain liquid inoculum.

[0017] The seed culture medium is 2216 liquid culture medium, and the formula of 2216 liquid culture medium is as follows: 5.0g peptone, 1.0g yeast extract, 0.1g citric acid triiron, 19.45g sodium chloride, 8.8g magnesium chloride, 3.24g sodium sulfite, 1.3g calcium chloride, 0.55g potassium chloride, 0.16g sodium bicarbonate, 0.08g potassium bromide, 34.0mg strontium chloride, 22.0mg boric acid, 4.0mg sodium silicate, 2.4mg sodium fluoride, 1.6mg ammonium nitrate, and 8.0mg disodium hydrogen phosphate, with distilled water added to a final volume of 1000mL, and sterilized at 121℃ for 20min; the formula of 2216 solid culture medium is based on 2216 liquid culture medium with the addition of 15.0g agar.

[0018] In addition, experiments have shown that the Bacillus lysinus strain of the present invention, or the fermentation broth and liquid inoculum obtained using this strain, can produce extracellular proteases.

[0019] Furthermore, the optimal culture conditions for the production of extracellular proteases were determined: the D1-1-M9 protease synthesis capacity was optimal when the NaCl concentration in the 2216 liquid medium was 1.0% and 10% glucose was added.

[0020] Advantages of this invention:

[0021] The Bacillus lysinus D1-1-M9 of this invention was isolated from the soil on the shore of Longmu Co Salt Lake in Ngari Prefecture, Tibet Autonomous Region. The environment was not polluted, and it has no ecotoxicity, high safety and strong stability.

[0022] The Lysobacter D1-1-M9 of this invention was identified as a new species of the genus Lysobacter after 16S rRNA gene sequence analysis, genomic analysis, phylogenetic analysis and multiple taxonomic analyses.

[0023] The Bacillus lysinicus D1-1-M9 of this invention is simple to cultivate, has a short cycle, strong stability, broad substrate spectrum, and can produce extracellular proteases. The produced extracellular proteases can maintain relatively high protease activity under medium and low temperature environments, so it has very broad development and application prospects in medicine, food and industrial production. Attached Figure Description

[0024] Figure 1Morphological image of strain D1-1-M9 under an electron transmission microscope (left image) and the transparent ring formed on a milk plate (right image);

[0025] Figure 2 Phylogenetic tree of 16S rRNA gene of strain D1-1-M9;

[0026] Figure 3 Effect of NaCl concentration on protease synthesis in strain D1-1-M9;

[0027] Figure 4 The effect of carbon source on protease synthesis in strain D1-1-M9;

[0028] Figure 5 Relative protease activities of strain D1-1-M9 at different temperatures;

[0029] Figure 6 Relative protease activities of strain D1-1-M9 under different pH conditions;

[0030] Figure 7 Relative protease activities of strain D1-1-M9 under different metal ions. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0032] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0034] Example 1: Isolation and Identification of Bacillus lysinus D1-1-M9 strain

[0035] a. Soil sample collection: Soil samples were collected from the shore of Longmu Lake in Ngari Prefecture, Tibet Autonomous Region, and brought back to the laboratory at 4°C. The bacterial strains were isolated using the plate dilution coating method.

[0036] b. The culture medium for isolating the strain was 2216 solid medium: 5.0g peptone, 1.0g yeast extract, 0.1g citric acid triiron, 19.45g sodium chloride, 8.8g magnesium chloride, 3.24g sodium sulfite, 1.3g calcium chloride, 0.55g potassium chloride, 0.16g sodium bicarbonate, 0.08g potassium bromide, 34.0mg strontium chloride, 22mg boric acid, 4.0mg sodium silicate, 2.4mg sodium fluoride, 1.6mg ammonium nitrate, 8.0mg disodium hydrogen phosphate, and 15g agar. Distilled water was added to bring the volume to 1000mL, pH 7.2-7.4, and sterilized at 121℃ for 20min.

[0037] c. Separation using the plate dilution method: Thoroughly mix the collected soil sample, weigh 10g, and place it in an Erlenmeyer flask containing an appropriate amount of glass beads and 90mL of sterile water. Place the flask on a shaker, shake for 20min, let stand for 5min, and then dilute sequentially using a 10-fold serial dilution method. Take 10g of the diluted sample... -4 10 -5 10 -6 200 μL of each soil dilution was evenly spread onto 2216 agar plates. Three plates were spread for each dilution, and the plates were incubated upside down in a 20°C incubator for 14 days. Single colonies with different morphologies were selected for further streaking purification. The purified strains were then transferred to 2216 slant agar and stored at 4°C for later use.

[0038] d. Identification of the strain: The purified strain D1-1-M9 was streaked onto a 2216 plate. Colony morphology was observed: colonies were round, yellowish-creamy, smooth, opaque, and with regular edges. Cell morphology was observed under an electron transmission microscope; the cells were rod-shaped. Figure 1 (Left figure in the image). PCR amplicon sequencing was performed using universal 16S rRNA primers 27F and 1492R, and the resulting sequence is shown in SEQ ID NO.1. Comparison with 16S rRNA sequences of known strains in the Genbank database revealed that strain D1-1-M9 showed the highest similarity (97.80%) to the 16S rRNA gene sequence of *Lysobacter erysipheiresistens* RS-LYSO-3. Phenotypic and physicochemical characteristics of the strain were analyzed according to Bergey's Manual of Bacteriological Identification and multiple taxonomic methods, combined with phylogenetic analysis of the 16S rRNA gene sequence. Figure 2 Genome sequencing analysis confirmed that strain D1-1-M9 is a new species of Lysobacter sp. nov. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.19230 on November 8, 2023.

[0039] The sequence of SEQ ID NO.1 is as follows:

[0040]

[0041] Studies of this strain revealed that strain D1-1-M9 can utilize D-trehalose, D-cellobiose, D-raffinose, β-methyl-D-glucoside, N-acetyl-D-glucosamine, N-acetyl-β-D-mannitol, N-acetyl-D-glucosamine, α-D-glucose, D-fructose, D-galactose, 3-methyl-D-glucose, D-fucose, L-fucose, L-rhamnose, inositol, glycerol, D-glucose-6-PO4, and D-fructose-6-PO4. 4. D-Aspartic acid, gelatin, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, pectin, D-galactonic acid, L-galactonolactone, D-gluconic acid, D-glucuronic acid, L-lactic acid, citric acid, α-keto-glutaric acid, L-malic acid, Tween 40, α-hydroxy-butyric acid, β-hydroxy-D, L-butyric acid, α-keto-butyric acid, acetoacetic acid, propionic acid, and acetic acid are used as the sole carbon source, exhibiting broad substrate spectrum. Furthermore, according to physiological experiments, strain D1-1-M9 can grow under conditions of 10-35℃, pH 6.5-10, and NaCl concentration of 1.0-4.0% (w / v); the optimal growth conditions for strain D1-1-M9 are 25℃, pH 7.0, and 1.0% NaCl.

[0042] Furthermore, strain D1-1-M9 was able to form a clear zone on milk plates. Figure 1 (See right figure) This illustrates that it can produce extracellular proteases. We first optimized the culture conditions for the synthesis of proteases by the Bacillus lysinus strain D1-1-M9, and then verified some enzymatic properties of the produced proteases through further experiments.

[0043] Example 2: Optimization of culture conditions for protease synthesis by Bacillus lysinic strain D1-1-M9

[0044] 1) Effect of NaCl concentration on D1-1-M9 synthetic protease

[0045] NaCl was added to 2216 liquid medium to achieve final salt concentrations of 1.0%, 2.0%, 3.0%, and 4.0%. The culture was scaled up using seed culture and fermentation medium (2216 liquid medium) at an inoculum ratio of 5–10% (v / v). The culture conditions were 25°C, 160 rpm, and 48 h. The resulting culture was the fermentation broth. Cells were collected using the above method to obtain crude enzyme solution, and the enzyme activity of the crude enzyme solution was determined. Results are as follows: Figure 3As shown, the D1-1-M9 protein synthesizes the best protease when the NaCl concentration is 1.0%.

[0046] 2) The effect of carbon source on protease yield

[0047] 10% glucose, sucrose, lactose, maltose, soluble starch, and corn flour were added to 2216 liquid medium as supplementary carbon sources. The culture was scaled up by inoculating the seed culture and fermentation medium (2216 liquid medium) at a volume ratio of 5–10% under the following conditions: 25℃, 160 rpm, and culturing for 48 h. The resulting culture was the fermentation broth. Cells were collected using the above method to obtain crude enzyme solution, and the enzyme activity of the crude enzyme solution was determined. Results are as follows: Figure 4 As shown, the D1-1-M9 protein synthesizes the best protease when supplemented with 10% glucose.

[0048] Example 3 Enzymatic characteristics of Bacillus lysinensis strain D1-1-M9

[0049] I. Construction of the tyrosine standard curve for Bacillus lysinus strain D1-1-M9

[0050] a. Take 18 test tubes and divide them into six groups of 3. Add standard tyrosine, deionized water, 0.4 mol / L Na2CO3 and Folin-Ciocalteu reagent to each group and mix well.

[0051] b. Place in a 40℃ water bath for 20 minutes, then remove and allow to cool to room temperature. Perform colorimetric determination using a UV spectrophotometer at a wavelength of 680 nm to measure the OD. 680 The blank control was a solution without added tyrosine; a standard curve of the tyrosine solution was plotted based on the experimental results.

[0052] II. Method for determining the protease activity of Bacillus lysinus strain D1-1-M9

[0053] a. Inoculate a single colony of strain D1-1-M9 into 2216 liquid medium and incubate at 25°C and 160 rpm for 48 h;

[0054] b. Inoculate the bacterial culture into the fermentation medium and incubate at 25°C and 160 rpm for 48 hours;

[0055] c. Adjust the bacterial concentration to OD 680 The value is 0.500. Centrifuge at 12000 rpm for 10 min, and the supernatant is the crude enzyme solution.

[0056] d. Determine protease activity using the Folin-Ciocalteu method. Before the assay, preheat the crude enzyme solution and casein solution in a 40°C water bath for 5 min. For the experimental group, take 1 mL of crude enzyme solution and add it to 1 mL of 10 g / L casein solution, incubate in a 40°C water bath for 10 min; immediately add 3 mL of 0.4 mol / L trichloroacetic acid solution to terminate the reaction.

[0057] e. For the control group, take 1 mL of crude enzyme solution, add 3 mL of 0.4 mol / L trichloroacetic acid solution, incubate at 40℃ for 10 min to inactivate the protease, then add 1 mL of 10 g / L casein solution, let stand for 10 min, and centrifuge at 12000 rpm for 10 min.

[0058] f. Take 1 mL of the supernatant from the experimental group and the control group respectively, add 5.0 mL of 0.4 mol / L Na2CO3 solution and 1 mL of Folin reagent working solution, and develop color in a water bath at 40℃ for 20 min;

[0059] g. Measure the OD of the sample at 680 nm using a UV-Vis spectrophotometer. 680 Value. Using the experimental group OD 680 Value minus control group OD 680 It is worthwhile to obtain the net OD value.

[0060] The formula for calculating enzyme activity is:

[0061]

[0062] In the formula: X is the enzyme activity of the sample (U / mL); 5 represents 1 mL of 5 mL reaction solution to be measured, i.e., 5 times; A is the tyrosine content corresponding to the standard curve obtained from the net OD value; n is the dilution factor of the enzyme solution; 10 represents 1 min of the 10 min reaction time to be calculated, i.e., 1 / 10.

[0063] III. Stability test of protease activity of Bacillus lysinus strain D1-1-M9

[0064] 1) Thermal stability of proteases

[0065] Enzyme solution and casein solution were reacted in the optimal pH solution at 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃ for 60 min, respectively. Enzyme activity was measured to determine the optimal reaction temperature. After the reaction was complete, the system was immediately placed in an ice bath, and enzyme activity was measured again to determine the enzyme's heat resistance. The results are as follows: Figure 5As shown, the protease activity of *Lysobacterium lysate* D1-1-M9 increases slowly between 20-40℃, reaching its maximum at 40℃. At 20℃ and 30℃, the relative protease activities are 86.9% and 91.26%, respectively. As the temperature continues to rise from 40℃, the protease activity of *Lysobacterium lysate* D1-1-M9 begins to decrease rapidly. At 50℃ and 60℃, the relative protease activity decreases to 62.5% and 10.21%, respectively; at 70℃, the relative protease activity is only 0.82%. Therefore, the optimal temperature for the protease produced by *Lysobacterium lysate* D1-1-M9 is 40℃. It can maintain relatively high protease activity under medium and low temperature environments but is not resistant to high temperatures.

[0066] 2) Optimal reaction pH of proteases

[0067] A 1 mg / mL casein substrate solution was prepared using 50 mmol / L Tris-HCl with a pH range of 1.5-9.0. 10 μL of the enzyme solution and 100 μL of substrate solutions at different pH values ​​were reacted at 30 °C for 60 min. The reaction was then terminated by adding trichloroacetic acid. Enzyme activity was measured to determine the enzyme's pH stability. The results are as follows: Figure 6 As shown, the protease produced by Lysobacterium D1-1-M9 exhibits a gradual increase in activity with increasing pH from 3.0 to 7.0, reaching its maximum relative activity at pH 7.0. Under alkaline conditions, the relative activity decreases relatively slowly, and once the pH reaches 10, the protease activity essentially remains unchanged, maintaining a relative activity of around 30%.

[0068] 3) Metal ion tolerance of proteases

[0069] 10 μL of enzyme solution was reacted with casein solution at the optimal pH and temperature for 60 min, and then trichloroacetic acid was added to terminate the reaction. Enzyme activity was measured and set as 100%. FeCl3·6H2O, ZnCl2, CaCl2·2H2O, MnCl2, and MgCl2·6H2O were added to this reaction system at concentrations of 0.2, 1.0, 5.0, and 10 mmol / L, respectively. Samples were processed and measured according to standard sample processing methods. The results are as follows: Figure 7 As shown. The enzyme activity of the control group (without added metal ions) was set to 100%. Compared with the control group, the Mn content in the reaction system was [missing information]. 2+ At a concentration of 5.0 mmol / L, the enzyme activity was 115.2% of the control group. 2+ and Zn 2+ At a concentration of 5.0 mmol / L, the relative protease activities were 102.3% and 98.3% of the control group, respectively, indicating that Ca... 2+ and Zn 2+ It has virtually no effect on the relative activity of the protease. However, the Mg in the reaction system... 2+and Fe 3+ At a concentration of 5.0 mmol / L, it can inhibit relative activity, with its relative activity being only 52.3% and 30.6% of the control.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art,

[0071] Of course, based on the technical content disclosed in this specification, substitutions or modifications can be made.

[0072] Other implementation methods can be easily derived from this invention; therefore, any changes and modifications made based on the principles of this invention are not permitted.

[0073] All of the above should be included within the scope of the patent application of this invention.

Claims

1. A strain of Bacillus lysinensis, characterized in that, The strain is lysobacterium ( Lysobacter sp. D1-1-M9 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.19230.

2. The lysozyme strain according to claim 1, characterized in that, The nucleotide sequence of the 16S rRNA gene of the lysozyme strain is shown in SEQ ID NO.

1.

3. The lysozyme strain according to claim 1, characterized in that, The lysozyme strain can grow under conditions of 10-35℃, pH 6.5-10, and NaCl concentration of 1.0%-4.0% w / v.

4. The lysozyme strain according to claim 3, characterized in that, The optimal growth conditions for the lysozyme strain are 25°C, pH 7.0, and NaCl concentration of 1.0% w / v.

5. The fermentation broth and liquid inoculum prepared from the Bacillus lysinus strain according to claim 1.

6. A method for preparing the fermentation broth and liquid inoculant according to claim 5, characterized in that, The method includes: 1) Strain activation: Pick a strain and streak it onto 2216 solid medium, place it in a constant temperature incubator, and incubate at 25℃ for 24 hours; 2) Preparation of seed culture: Pick activated bacterial cells and inoculate them into a large test tube containing seed culture medium, seal the tube with a rubber stopper, place it on a shaker at 25℃ and shake at 160 rpm for 16-24 h to obtain seed culture. 3) Liquid fermentation: Then, the seed liquid and seed culture medium are inoculated at a volume ratio of 5-10% for expansion culture. The culture conditions are 25℃ and 160rpm. The culture obtained after 48h of culture is the fermentation broth. The fermentation broth is centrifuged at 12000rpm for 10min, and the bacterial cells are collected to obtain liquid inoculum.

7. The method according to claim 6, characterized in that, The seed culture medium is 2216 liquid culture medium, and the formulation of 2216 liquid culture medium is as follows: 5.0 g peptone, 1.0 g yeast extract, 0.1 g citric acid triiron, 19.45 g sodium chloride, 8.8 g magnesium chloride, 3.24 g sodium sulfite, 1.3 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium bicarbonate, 0.08 g potassium bromide, 34.0 mg strontium chloride, 22.0 mg boric acid, 4.0 mg sodium silicate, 2.4 mg sodium fluoride, 1.6 mg ammonium nitrate, and 8.0 mg disodium hydrogen phosphate, with distilled water added to a final volume of 1000 mL, and sterilized at 121°C for 20 min; the formulation of 2216 solid culture medium is based on the 2216 liquid culture medium with the addition of 15.0 g agar.

8. The use of the Bacillus lysinus strain of claim 1 or the fermentation broth and liquid inoculum of claim 5 in the production of extracellular proteases.

9. The application according to claim 8, characterized in that, The lysozyme strain can directly synthesize extracellular proteases during the culture process. The optimal culture conditions for the lysozyme strain to synthesize extracellular proteases are: a NaCl concentration of 1.0% w / v in 2216 liquid medium, and the addition of 10% glucose.

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