A bleomycin producing strain and its application

Abstract

The present invention belongs to the technical field of microbial fermentation, and specifically relates to a bleomycin production strain and its application. The present invention obtains a strain with high resistance to bleomycin by mutagenesis. Streptomyces mobaraensis BL‑G2‑202402, deposit number: CCTCC NO: M 20241482, and add bleomycin skeleton amino acids to the fermentation medium formula to improve the bleomycin fermentation titer and affect the ratio of the main components A2 and B2; add substances that increase cell permeability and substances that increase the oxygen carrying capacity of the fermentation liquid to the bleomycin fermentation medium at the same time, and finally determine that adding 0.5 g / L of chlorhexidine and 2.5‑10 g / L of oxygenator can significantly improve the bleomycin titer; replace part of the nitrogen source in the culture medium with dried bacterial residues, and find that freeze-dried bacterial residues can replace 10‑40% of corn steep liquor powder as the bleomycin fermentation nitrogen source, and the fermentation titer is increased. The above research provides a reference for pilot production in fermentation tanks.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a bleomycin production strain and application thereof. Background Art

[0002] Bleomycin (Blm) is a water-soluble glycopeptide antibiotic that was first discovered by Umezawa et al. in 1966 from Streptomyces verticillium. Streptomyces verticillus ) fermentation broth. According to the different Rf values ​​in paper chromatography, bleomycin can be divided into group A and group B, and the main difference between the homologues lies in the different C-terminal amine structures on the glycoside ligand side chain. At present, the imported bleomycin injection drug BLENOXANE used in clinical practice ® The main components are bleomycin A2 (60%) and B2 (30%) (structure as Figure 1 Bleomycin does not cause significant bone marrow suppression during chemotherapy, so it can be used in combination with other antibiotics to treat malignant germ cell cancer, head and neck cancer, skin cancer, Hodgkin lymphoma and squamous cell carcinoma. For example, the combination of bleomycin, etoposide and cisplatin can treat 90% of testicular cancer.

[0003] Given the complexity of bleomycin structure and the difficulty of chemical synthesis, biological fermentation is still the main industrial production method for bleomycin A2 and B2. Domestic research on bleomycin mainly focuses on the optimization of fermentation conditions and the identification of new analogs. There are few companies in China that have the qualifications to produce bleomycin drugs, and there is a great demand for raw materials in the market.

[0004] Bleomycin fermentation is a process that consumes a lot of oxygen. Dissolved oxygen is crucial to the success and yield of bleomycin production. In addition, the ratio of A2 and B2 needs to be adjusted through the fermentation process conditions and fermentation formula to achieve its optimal ratio close to 2:1, which saves a lot of cost for the subsequent purification process.

[0005] Bleomycin mainly exists in the fermentation liquid. The conventional collection method is to first adjust the pH of the bleomycin fermentation liquid to 6.0 with acetic acid, then separate the solid and liquid by centrifugation or plate and frame filtration, collect the liquid, and then enrich, recover and purify bleomycin by resin adsorption to obtain bleomycin. However, the bacterial cells after solid-liquid separation can only be treated as solid waste. How to turn waste into treasure and realize "green production" is also a problem that we urgently need to solve. Summary of the invention

[0006] In order to solve the above problems, the present invention uses a strain of Streptomyces mobara preserved in the strain bank of the applicant ( Streptomyces mobaraensis)BL-G2-202402 was taken as the research object. On the basis of the original research, bleomycin backbone amino acids were added to its fermentation medium formula to investigate the effect of the addition of backbone amino acids on the bleomycin fermentation titer and the ratio of the main components A2 and B2. At the same time, previous studies found that the bleomycin production process has a particularly high requirement for oxygen. In order to make the existing equipment meet the production needs of bleomycin, substances that increase cell permeability and substances that increase the oxygen carrying capacity of the fermentation liquid were added to the bleomycin fermentation medium at the same time. The effects of these substances on the bleomycin fermentation level and the ratio of the main components A2 and B2 were investigated, providing a reference for the pilot production of fermenters.

[0007] One of the technical solutions provided by the present invention is a strain of Streptomyces mobaraensis, specifically Streptomyces mobaraensis BL-G2-202402, the strain was deposited in the China Center for Type Culture Collection on July 4, 2024; address: Wuhan University, Wuhan, China; Postal Code: 430072; Deposit Number: CCTCC NO: M 20241482.

[0008] The second technical solution provided by the present invention is the one described in the technical solution Streptomyces mobaraensis Application of BL-G2-202402, especially in the production of bleomycin;

[0009] Furthermore, using Streptomyces mobaraensis The method for producing bleomycin by BL-G2-202402 is as follows:

[0010] The seed liquid is inoculated into the fermentation medium at an inoculation rate of 10-20% (v:v), the shaking speed is 250-270rpm, the culture temperature is 28-29℃, and the culture period is 8-10d;

[0011] Fermentation medium: glucose 8-12 g / L, corn starch 30-50 g / L, sucrose 20-30 g / L, soybean cake powder 30-40 g / L, corn steep liquor powder 15-20 g / L, zinc sulfate 1-1.5 g / L, copper sulfate 0.02-0.05 g / L, pH 7.0;

[0012] Furthermore, the fermentation medium is also supplemented with 1 g / L of L-tryptophan, 1 g / L of DL-methionine, and 1 g / L of L-cysteine;

[0013] Furthermore, the fermentation medium is further added with 0.5 g / L of chlorhexidine and 2.5-10 g / L of oxygenant; the oxygenant is any one of liquid paraffin, n-dodecane or n-hexadecane; preferably, the fermentation medium is further added with 0.5 g / L of chlorhexidine and 5 g / L of n-hexadecane;

[0014] Further, 10-40% (w / w) of the corn steep liquor powder in the fermentation medium is replaced by Streptomyces mobaraensis Dried mushroom residue of BL-G2-202402;

[0015] Furthermore, the preparation method of the dried mushroom residue is as follows:

[0016] Will Streptomyces mobaraensis The bleomycin fermentation liquid of BL-G2-202402 was centrifuged and the bacterial sludge was collected. The bacterial sludge was repeatedly washed with water and the bacterial bodies were collected and dried. After drying, the dried bacterial residues were collected and crushed for standby use.

[0017] Furthermore, the drying method is high temperature drying or freeze drying;

[0018] Furthermore, the high temperature drying condition is 60-80°C for 2-3 h;

[0019] Furthermore, the freeze-drying process conditions are:

[0020] ① Pre-freezing: Pre-freeze at -40℃ for 3 hours and then start vacuuming;

[0021] ②Sublimation: divided into three stages, the first stage, the rapid heating stage, the material temperature rises from -40℃ to -30℃, and the heating rate is controlled at 10℃ / hour; the second stage, the temperature maintenance stage, the material temperature is maintained between -30℃ and -25℃, and the maintenance time is 10 hours; the third stage, the rapid heating stage, the material temperature rises from -25℃ to 20℃, and the heating rate is controlled at 5℃ / hour (automatic heating is turned on);

[0022] ③ Collect the freeze-dried mushroom residue and crush it for later use.

[0023] Beneficial effects:

[0024] 1. The present invention obtains a strain having high resistance to bleomycin by mutagenesis Streptomyces mobaraensis BL-G2-202402 was then applied to the production of bleomycin. Streptomyces mobaraensis BL-G2-202402 can grow normally on a plate containing 500 mg / L bleomycin; adding 100-300 mg / L bleomycin to the fermentation system has no obvious effect on its growth and metabolism.

[0025] 2. The present invention adds bleomycin backbone amino acids to the fermentation medium formula and optimizes their combination. It is found that when 1 g / L of L-tryptophan, 1 g / L of DL-methionine, and 1 g / L of L-cysteine ​​are added to the medium, the total titer can reach 309.21 mg / L and the ratio of A2:B2 can reach 1.77:1.

[0026] 3. The present invention simultaneously adds a substance that increases cell permeability and a substance that increases the oxygen carrying capacity of the fermentation liquid to the bleomycin fermentation medium, and investigates the effects of these substances on the bleomycin fermentation level and the ratio of the main components A2 and B2. It is finally determined that the addition of 0.5 g / L of chlorhexidine and 2.5-10 g / L of oxygenant can improve the bleomycin titer; in particular, when 0.5 g / L of chlorhexidine and 5 g / L of n-hexadecane are added to the culture medium, the bleomycin titer can reach 385.96 mg / L.

[0027] 4. The present invention replaces part of the nitrogen source in the culture medium with dried fungus residue, and finds that freeze-dried fungus residue can replace 10-40% of corn steep liquor powder as the nitrogen source for bleomycin fermentation. The fermentation titer increases slightly, with the maximum increase reaching 7.87%, and the ratio of A2 to B2 does not change significantly. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the chemical structure of bleomycin

[0029] X in the picture - Indicates an acid radical ion, such as X in the bleomycin sulfate molecule - is sulfate ion, X in the bleomycin hydrochloride molecule - For chloride ion. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0031] Bleomycin fermentation broth processing and HPLC detection method used in the present invention:

[0032] Sample processing: After the fermentation of bleomycin, the pH value of the culture medium is neutral or weakly alkaline, while bleomycin is relatively stable under weakly acidic conditions. To improve the stability of bleomycin in the fermentation broth, before the bleomycin concentration determination, the pH of the fermentation broth was adjusted to about 6.0 with 10% acetic acid solution, 10 mL of the fermentation broth was centrifuged (12000 r / min, 10 min), 1 mL of the supernatant was aspirated, and it was filtered with a 0.22 μm water microporous filter membrane before liquid chromatography determination.

[0033] Chromatographic analysis conditions: The aqueous phase preparation method is to take 7.53 g of sodium hexane sulfonate and 3.72 g of disodium ethylenediaminetetraacetate and dissolve them in 1 L of ultrapure water, add 4.6 mL of glacial acetic acid (chromatographic grade), and adjust the pH to 4.3 with ammonia water. The organic phase preparation method is methanol: acetonitrile = 7:3; LC Column 250×4.6 mm, 5 μm C18 chromatographic column is used, the column temperature is 40℃, the flow rate is 1.0 mL / min, the ultraviolet detection wavelength is 294 nm, and the liquid chromatography gradient elution program is shown in Table 1.

[0034] Table 1 Liquid chromatography gradient elution program

[0035]

[0036] Calculation formula:

[0037]

[0038] Where:

[0039] A 供 ---The sum of the peak areas of bleomycin A2 and bleomycin B2 in the chromatogram of the test solution;

[0040] A 对 ---The sum of the peak areas of bleomycin A2 and bleomycin B2 in the chromatogram of the reference solution;

[0041] C 对 ---Concentration of reference solution, mg / ml;

[0042] P----purity of bleomycin sulfate reference substance, %;

[0043] 0.94----Conversion factor; the ratio of the molecular weight of bleomycin (about 1418) to the molecular weight of bleomycin sulfate (about 1515);

[0044] 1000----Unit conversion factor.

[0045] Remark:

[0046] 1. The bleomycin reference substance used in this application is bleomycin sulfate, with a purity of 97% (A2:B2 ratio: 2.23:1).

[0047] 2. Treatment of bleomycin reference substance: Prepare 1000 mg / L bleomycin sulfate with sterile saline and filter it with a 0.22-micron microporous membrane for sterilization.

[0048] The present invention is further explained below by means of specific examples.

[0049] Example 1 Comparison of resistance between bleomycin producing strain BL-G2-202402 and original strain BL-G1-1

[0050] The strain BL-G2-202402 is a strain of Streptomyces BL-G1-1 preserved in our laboratory. It was induced by ARTP normal temperature plasma technology and combined with resistance screening to obtain a strain with high resistance to bleomycin. The original strain and the resistant strain were inoculated in solid culture medium and liquid fermentation medium containing different concentrations of bleomycin, respectively, and the growth and colony size of the strains on the solid culture medium containing different concentrations of bleomycin were observed; the titers of the two strains on the fermentation medium with different concentrations of bleomycin were measured to judge the difference in the feedback ability of the resistant strain to its own metabolites, so as to predict its potential high-yield capacity.

[0051] 1. Culture medium

[0052] Slant culture medium: glucose 4 g / L, yeast powder 4 g / L, maltose powder 10 g / L, calcium carbonate 2 g / L, agar 18 g / L.

[0053] Seed culture medium: glucose 2.5 g / L, malt extract powder 10 g / L, yeast extract powder 2 g / L, corn steep liquor powder 0.5 g / L, pH 7.0.

[0054] Fermentation medium: glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, pH 7.0.

[0055] 2. Experimental Methods

[0056] 2.1 Comparison of bleomycin resistance culture

[0057] The bleomycin-producing bacteria BL-G1-1 and BL-G2-202402 were inoculated on solid culture media containing 100-700 mg / L bleomycin (same slant) and cultured at 28°C for 7 days. The growth and colony size of the strains on the solid culture media containing different concentrations of bleomycin were observed. The specific results are shown in Table 2.

[0058] Table 2. Effect of adding bleomycin to bleomycin solid medium on bleomycin yield See the table below

[0059]

[0060] Note: What is added is bleomycin sulfate, and the above added amount is the amount converted into bleomycin.

[0061] As shown in Table 2, the resistance of the screened resistant strains to bleomycin is significantly higher than that of the original strains, which has the potential to increase the bleomycin production.

[0062] 2.2 Shake flask fermentation method

[0063] Inoculate the bleomycin-producing bacteria BL-G1-1 and BL-G2-202402 into 60 mL / 250 mL eggplant flask slant medium, culture at 28°C for 7 days, and set aside. Use a sterile inoculation needle to scrape 1 cm 2 The slant seeds were inoculated into the seed culture medium bottle with a liquid volume of 30mL / 250mL. The culture was carried out at 28°C and 270rpm for about 48h. The seed liquid was viscous and the amount of mycelium was large and the mycelium was long and network-like under the microscope. At this time, the seed liquid could be transferred to the fermentation bottle.

[0064] The volume of the fermentation bottle liquid is 30mL / 250mL, the inoculation amount is 10%, the shaker speed is 270rpm, the culture temperature is 28℃, the culture period is 9d, and the fermentation liquid is gray in color when the growth is normal. At the same time, microscopic examination shows that the mycelium is slender, reticular, darkly stained, and free of foreign bacteria.

[0065] The seed liquid of the above two strains was transplanted into the fermentation medium added with different concentrations of bleomycin, and the titer of the fermentation liquid was measured after cultivation according to the above control parameters to infer whether the resistant strains can resist the feedback inhibition of their own metabolites. The specific results are shown in Table 3.

[0066] Table 3. Effect of adding bleomycin to bleomycin liquid medium on bleomycin production

[0067]

[0068] Note: The bleomycin added to the fermentation broth is bleomycin sulfate, and the above-mentioned amount is converted into bleomycin; the fermentation titer of strain BL-G1-1 / strain BL-G2-202402 is the titer after deducting the added bleomycin sulfate.

[0069] It can be seen from Table 3 that adding 100 mg / L bleomycin to the fermentation broth of strain BL-G1-1 has no effect on its growth and metabolism, while adding 200 mg / L bleomycin has a significant effect on the growth and metabolism of strain BL-G1-1; adding 100-300 mg / L bleomycin to strain BL-G2-202402 has no significant effect on its growth and metabolism, but when the addition amount reaches 400 mg / L, it has a significant effect on its growth and metabolism.

[0070] Example 2 Effect of adding bleomycin backbone amino acids to the fermentation medium on the potency of bleomycin fermentation broth and the ratio of A2 to B2

[0071] The bleomycin molecule contains a typical hybrid peptide-polyketide structure and a disaccharide unit, in which the peptide-polyketide backbone molecule is composed of 9 amino acid molecules (serine, asparagine, histidine, threonine, cysteine, alanine, β-alanine), 1 acetic acid molecule and two molecules of S-adenosylmethionine (AdoMet).

[0072] This example is used to determine the effects of L-tryptophan and DL-methionine, and their combination with other amino acids, on increasing the potency of bleomycin and improving the ratio of A2 to B2.

[0073] 1. Culture medium

[0074] Slant culture medium (g / L): glucose 4 g / L, yeast powder 4 g / L, maltose powder 10 g / L, calcium carbonate 2 g / L, agar 18 g / L.

[0075] Seed culture medium: glucose 2.5 g / L, malt extract powder 10 g / L, yeast extract powder 2 g / L, corn steep liquor powder 0.5 g / L, pH 7.0.

[0076] Fermentation medium (w / v):

[0077] Control 0: glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, pH 7.0.

[0078] Control 1: glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, pH 7.0.

[0079] Formula 1: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-serine 1 g / L, pH 7.0.

[0080] Formula 2: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-asparagine 1 g / L, pH 7.0.

[0081] Formula 3: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-histidine 1 g / L, pH 7.0.

[0082] Formula 4: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-threonine 1 g / L, pH 7.0.

[0083] Formula 5: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, pH 7.0.

[0084] Formula six: glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-alanine 1 g / L, pH 7.0.

[0085] Formula 7: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, β-alanine 1 g / L, pH 7.0.

[0086] 2. Shake flask fermentation method

[0087] Inoculate the bleomycin-producing bacteria BL-G2-202402 on a 60 mL / 250 mL eggplant flask slant medium at 28°C for 7 days and set aside. Scrape 1 cm 2 The slant seeds were inoculated into the seed culture medium bottle with a liquid volume of 30mL / 250mL. The culture was carried out at 28°C and 270rpm for about 48h. The seed liquid was viscous and the amount of mycelium was large and the mycelium was long and network-like under the microscope. At this time, the seed liquid could be transferred to the fermentation bottle.

[0088] The seed liquid was inoculated into 7 kinds of fermentation medium bottles containing different structural amino acids, among which the culture medium without amino acids and without structural amino acids were used as controls, respectively. The liquid volume in the fermentation bottle was 30mL / 250mL, the inoculation amount was 10%, the shaker speed was 270rpm, the culture temperature was 28℃, the culture period was 9d, and the fermentation liquid was gray in color when it grew normally. At the same time, microscopic examination showed that the mycelium was slender, reticular, darkly stained, and free of foreign bacteria.

[0089] 3. Experimental results

[0090] After the fermentation was completed, the contents of bleomycin A2 and B2 in the fermentation broth were determined. The results are shown in Table 4:

[0091] Table 4 Effect of amino acid combination on bleomycin fermentation level and ratio of A2 and B2

[0092]

[0093] From the above results, it can be seen that by adding 1 g / L of L-tryptophan, 1 g / L of DL-methionine and 1 g / L of L-cysteine ​​to the basic formula, not only the fermentation level of bleomycin was greatly improved, but also the ratio of A2 and B2 was significantly improved.

[0094] Example 3 Effect of oxygenator and surfactant on bleomycin fermentation level

[0095] Previous studies have found that adding a low concentration of chlorhexidine solution has no negative effect on the growth and metabolism of bleomycin-producing bacteria, but adding chlorhexidine can improve the permeability of the cell membrane and enhance the level of cell metabolism; while adding oxygenators can increase the dissolved oxygen level in the fermentation broth, enhance the metabolic capacity of microbial cells, and thus improve the fermentation level of bleomycin. In this study, oxygenators and surfactant chlorhexidine were added to the bleomycin fermentation medium to investigate their effects on bleomycin production.

[0096] 1. Culture medium

[0097] Slant culture medium (g / L): glucose 4 g / L, yeast powder 4 g / L, maltose powder 10 g / L, calcium carbonate 2 g / L, agar 18 g / L.

[0098] Seed culture medium (g / L): glucose 2.5 g / L, malt extract powder 10 g / L, yeast extract powder 2 g / L, corn steep liquor powder 0.5 g / L, pH 7.0.

[0099] Fermentation medium formulation

[0100] Control 0: glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, pH 7.0.

[0101] Control 1: glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, pH 7.0.

[0102] Control 2: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 1 g / L, pH 7.0.

[0103] Formula 1: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, liquid paraffin 2.5 g / L, pH 7.0.

[0104] Formula 2: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, n-dodecane 2.5 g / L, pH 7.0.

[0105] Formula 3: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, n-hexadecane 2.5 g / L, pH 7.0.

[0106] Formula 4: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, liquid paraffin 5 g / L, pH 7.0.

[0107] Formula 5: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, n-dodecane 5 g / L, pH 7.0.

[0108] Formula six: glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, n-hexadecane 5 g / L, pH 7.0.

[0109] Formula 7: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, liquid paraffin 10 g / L, pH 7.0.

[0110] Formula 8: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, n-dodecane 10 g / L, pH 7.0.

[0111] Formula 9: Glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, n-hexadecane 10 g / L, pH 7.0.

[0112] 2. Shake flask fermentation method

[0113] Inoculate the bleomycin-producing bacteria BL-G2-202402 into a 60 mL / 250 mL eggplant flask slant medium at 28°C for 7 days and set aside. Scrape 1 cm 2 The slant seeds were inoculated into the seed culture medium bottle with a liquid volume of 30mL / 250mL. The culture was carried out at 28°C and 270rpm for about 48h. The seed liquid was viscous and the amount of mycelium was large and the mycelium was long and network-like under the microscope. At this time, the seed liquid could be transferred to the fermentation bottle.

[0114] The above seed liquid was inoculated into the above 11 different fermentation culture medium bottles containing different concentrations of chloramphenicol and combinations of chloramphenicol and oxygenator, among which the culture medium without chloramphenicol and oxygenator was used as control. The liquid volume in the fermentation bottle was 30mL / 250mL, the inoculation amount was 10%, the shaking speed was 270rpm, the culture temperature was 28℃, the culture period was 9d, and the fermentation liquid was gray in color when the growth was normal. At the same time, microscopic examination showed that the mycelium was slender, reticular, darkly stained, and free of foreign bacteria.

[0115] 3. Experimental results

[0116] After the fermentation was completed, the contents of bleomycin A2 and B2 in the fermentation broth were determined. The results are shown in Table 5 below:

[0117] Table 5. Effect of the combination of chlorhexidine and oxygenator on the fermentation level of bleomycin

[0118]

[0119] From the results shown in Table 5, it can be seen that adding 0.5 g / L of chlorhexidine can improve the fermentation level of bleomycin; the combined addition of 0.5 g / L chlorhexidine and 2.5-10 g / L concentration of liquid paraffin, n-dodecane or n-hexadecane can improve the fermentation level of bleomycin, and at the same time, the ratio of A2 and B2 did not change significantly; the best combination was comprehensively determined to be 0.5 g / L chlorhexidine + 5 g / L n-hexadecane.

[0120] Example 4 Application of Bleomycin Fermentation Residue as a Substitute for Nitrogen Source

[0121] 1 Culture medium

[0122] Slant culture medium (g / L): glucose 4 g / L, yeast powder 4 g / L, maltose powder 10 g / L, calcium carbonate 2 g / L, agar 18 g / L.

[0123] Seed culture medium (g / L): glucose 2.5 g / L, malt extract powder 10 g / L, yeast extract powder 2 g / L, corn steep liquor powder 0.5 g / L, pH 7.0.

[0124] Fermentation medium: glucose 10 g / L, corn starch 45 g / L, sucrose 25 g / L, soybean cake powder 35 g / L, corn steep liquor powder 17.5 g / L, zinc sulfate 1.22 g / L, copper sulfate 0.05 g / L, L-tryptophan 1 g / L, DL-methionine 1 g / L, L-cysteine ​​1 g / L, Sanisol 0.5 g / L, n-hexadecane 5 g / L, pH 7.0.

[0125] 2 Experimental methods

[0126] 2.1. Preparation of dried bleomycin cells

[0127] The pH of the fermentation liquid after the fermentation of the six bleomycins in the formula of Example 3 was adjusted to 6.0 with glacial acetic acid, and solid-liquid separation was performed by high-speed centrifugation. The bacterial sludge after centrifugation was repeatedly washed three times with 3 times the weight of deionized water. The specific operation was as follows: the bacterial sludge after centrifugation was collected, 3 times the weight of deionized water was added, stirred evenly, and then centrifuged. This was repeated three times to collect the bacterial sludge.

[0128] The fungus mud was dried by high temperature drying and low temperature drying, respectively. The high temperature drying was carried out by drying at 60℃ for 2.5 hours; the low temperature drying was carried out by freeze drying. The process parameters of the drying process were as follows: ① Pre-freezing: vacuum was started after pre-freezing at -40℃ for 3 hours; ② Sublimation: divided into three stages, the first stage, the rapid heating stage, the material temperature was raised from -40℃ to -30℃, and the heating rate was controlled at 10℃ / hour; the second stage, the temperature maintenance stage, the material temperature was maintained between -30℃ and -25℃, and the maintenance time was 10 hours; the third stage, the rapid heating stage, the material temperature was raised from -25℃ to 20℃, and the heating rate was controlled at 5℃ / hour (automatic heating was turned on). ③ After freeze drying, the dried fungus residue was collected.

[0129] The mushroom residues were dried by different drying methods and crushed separately for later use.

[0130] 2.2. Results of the bleomycin residue replacement test

[0131] After partially replacing the nitrogen source (soybean cake powder / corn steep liquor powder) in the fermentation medium with high and low temperature dried fungus residues, bleomycin fermentation was carried out using the same shake flask fermentation method as in Example 3. The results are shown in Tables 6 and 7.

[0132] Table 6. Experiment on high temperature drying of fungus residue as a nitrogen source

[0133]

[0134] Conclusion: High-temperature dried fungus residue cannot replace soybean cake powder as a nitrogen source for bleomycin fermentation; high-temperature dried fungus residue can replace 10-40% corn steep liquor powder as a nitrogen source for bleomycin fermentation, the fermentation titer increases slightly, and the ratio of A2 to B2 does not change significantly.

[0135] Table 7. Experiment on low temperature drying of fungus residue as a nitrogen source

[0136]

[0137] Conclusion: Low-temperature dried mushroom residue can replace 10-20% soybean cake powder as the nitrogen source for bleomycin fermentation; at the same time, low-temperature dried mushroom residue can also replace 10-40% corn steep liquor powder as the nitrogen source for bleomycin fermentation, and the fermentation titer increases slightly, with the maximum increase of 7.87%, and the ratio of A2 to B2 does not change significantly.

[0138] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A strain of Streptomyces mobaraensis, characterized in that: Specific strains Streptomyces mobaraensis BL-G2-202402, accession number: CCTCC NO: M 20241482.

2. The strain according to claim 1 Streptomyces mobaraensis Application of BL-G2-202402 in the production of bleomycin.

3. The use according to claim 2, characterized in that The method for producing bleomycin is as follows: Streptomyces mobaraensis The BL-G2-202402 seed liquid was inoculated into the fermentation medium at an inoculation rate of 10-20%, the shaking speed was 250-270 rpm, the culture temperature was 28-29°C, and the culture period was 8-10 days.

4. The use according to claim 3, characterized in that The fermentation medium is as follows: glucose 8-12 g / L, corn starch 30-50 g / L, sucrose 20-30 g / L, soybean cake powder 30-40 g / L, corn steep liquor powder 15-20 g / L, zinc sulfate 1-1.5 g / L, copper sulfate 0.02-0.05 g / L, pH 7.

0.

5. The use according to claim 4, characterized in that The fermentation medium is also supplemented with 1 g / L of L-tryptophan, 1 g / L of DL-methionine, and 1 g / L of L-cysteine.

6. The use according to claim 5, characterized in that The fermentation medium is further added with 0.5 g / L of chlorhexidine and 2.5-10 g / L of an oxygenator; the oxygenator is any one of liquid paraffin, n-dodecane or n-hexadecane.

7. The use according to claim 6, characterized in that The fermentation medium is added with 0.5 g / L of chlorhexidine and 5 g / L of n-hexadecane.

8. The use according to claim 7, characterized in that 10-40% of the corn steep liquor in the fermentation medium is replaced by Streptomyces mobaraensis Dried mushroom residue of BL-G2-202402.

9. The use according to claim 8, characterized in that The preparation method of the dried fungus residue is as follows: Will Streptomyces mobaraensis The bleomycin fermentation liquid of BL-G2-202402 was centrifuged and the bacterial sludge was collected. After the bacterial sludge was repeatedly washed with water, the bacterial bodies were collected and dried. After drying, the dried bacterial residues were collected and crushed for later use.

10. The use according to claim 9, characterized in that The drying is high temperature drying or freeze drying.

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