CN1273606C - Microbe method for preparing enamine and amine from valinemia - Google Patents

Abstract

The present invention relates to a method for preparing microorganisms of effective mildew enamine and effective mildew amine. The referred microorganisms are pantropic malt oligoclonal oxygen monas (Stenotrophomonas maltrophilia), CCTCC No. M 204024 of the oligoclonal oxygen monas (Stenotrophomonas). The microorganisms are utilized for fermentation and decomposition, or the cells or the enzymes, etc. of the microorganisms are utilized for decomposing validamycin or effective mildew imine of substrates so as to produce two types of substances in the cyclol class of the effective mildew enamine and effective mildew amine. A culture medium comprises 0.5% to 20.0% of validamycin, 0.5% to 10.0% of (NH4)2SO4, 10.5% to 5.0% of KCl, 0.1% to 10.0% of Na2HPO4-12H2O, 0.1% to 5.0% of NaH2PO4-2H2O and 0.01% to 1.0% of MgSO4 by weight/volume, which are prepared by tap water. The present invention has the culture conditions that the fermentation temperature is from 20 DEG C to 40 DEG C, an initial pH is from 6.0 to 8.0, and the culture time is from 1h to 180h; and the effective mildew enamine and the effectively mildew amine are obtained by the separation and the purification of an ion exchange method and a chromatography method.

Description

Microbial preparation method of effective mycamine and effective mycamine

Technical field

The present invention relates to new stenotrophomonas maltrophilia (Stenotrophomonas maltrophilia) screened from soil, and also relates to using the new bacterial strain to decompose effective mycomycin (also known as Jinggangmycin) or effective mycoylidene amine (also known as Jinggang A method for producing effective mycylamine and effective mycylamine.

Background technique

There have been more than 30 years of research on the production methods of effective mycosylamine and effective mycosylamine. Japanese Kameda et al. once used the bacteria of Pseudomonas denitrification to degrade effective mycomycin A or effective mycoylidene amine A , and then separated to obtain effective mycamine and effective mycamine, but they cannot grow on a medium with only effective mycin as the sole carbon source; and the decomposition ability of effective mycin is very weak, so it is not suitable for effective myc Mass production of enamines and potent mycoamines.

The Japanese Kameda et al. isolated a strain of Flavobacterium saccharophilum IFO13948 (Flavobacterium saccharophilum) IFO13948 (public patent: JP57-54593) that can decompose effective mycin into effective mycylamine and effective mycamine in soil.

Japanese Kameda et al. used Cytophaga heparina IFO12017 (ATCC13125) and IFO14087 strains to degrade effective mycomycin or effective mycoylidene amine to produce effective mycylamine and effective mycamine [Patent Bulletin (B2): Ping 2-26957].

Japanese Kameda et al. discovered that microorganisms of the genus Agrobacterium or Aeromonas and their mutant strains can effectively decompose effective mycosylidene or effective mycoylidene amine to produce effective mycosyramine and effective mycosylamine . For example, Agrobacterium microorganisms include Agrobacterum Radiobacter IFO 12664 (ATCC 4718) strain, IFO 13258 (ATCC 13332) strain, IFO 13259 (ATCC 13333) strain, IFO 13532 (ATCC 19358) strain, IFO 13533 (ATCC 25235) strain strains, and Agrobacterium tumefaciens IFO 3058 strains, etc.; Aeromonas (Aeromonas) microorganisms, including Aeromonas hydrophila subsp. 13288, and aeromonas salmonicida subsp.salmonicida IFO 12659, etc. [Patent Bulletin (B2): Flat 6-69380].

The raw material used in the present invention is a widely used agricultural antibiotic - validamycin, i.e. Jinggangmycin, which is an efficient and safe agricultural antibiotic that does not pollute the environment and is harmless to humans and animals. Safe and pollution-free pesticides with a wide area and the lowest cost per mu are an important product in the pesticide industry. Effective mycin is an aminoglycoside agricultural antibiotic, the main components are A, B, C, D, E, F, etc. It can be found from the structure of effective mycin that effective mycin is composed of effective mycylamine, effective mycamine And β-D-glucose and other structural components.

The glycosidic bonds of validamycin are hydrolyzed and decomposed into effective mycoylidene amines. There are two types of effective mycoylidene amines, A and B, and effective mycoylidene amines are decomposed to produce effective mycoylidene amine (valienamine) and effective mycoylidene ( validamine).

The effective mycylamine involved in the present invention is called valienamine in English. Its structural formula is shown in Figure 1 (Chem. Rev. 2003, 103: 1955-1977).

Figure 1 The chemical structure of effective valienamine

Effective mycylamine, also known as Jinggangamine or Jinggangmycylamine, (1S, 2S, 3S, 4R)-1-amino-5-(hydroxymethyl)cyclohex-5-ene-2,3,4-tri Alcohol; molecular formula is C ₇ H ₁₃ NO ₄ , important groups are: a primary amino group (-NH ₂ ), a carbon-carbon double bond (C=C), a hydroxymethyl group (-CH ₂ OH), three a hydroxyl group (-OH). The [α] _D ²³ of its hydrochloride (C ₇ H ₁₃ NO ₄ ·HCl) is +68.6° (1N-HCl), the melting point of pentaacetate (C ₁₇ H ₂₃ NO ₉ ) is 95°C, [α ] _D ²³ is +30.2° (CHCl ₃ ), and reacts with ninhydrin. Since effective mycylamine contains a primary amino group (-NH ₂ ), it undergoes first-order dissociation in aqueous solution.

The effective mycoamine involved in the present invention is called validamine in English, and its structural formula is shown in Figure 2 (Chem. Rev. 2003, 103: 1955-1977).

Figure 2 The chemical structure of validamine

Effective mycoamine, also known as Jinggang mycoamine, has a molecular formula of C ₇ H ₁₅ NO ₄ , one primary amino group (-NH ₂ ), one hydroxymethyl group (-CH ₂ OH), three hydroxyl groups (-OH), and its hydrochloric acid The [α] _D ²³ of the salt (C ₇ H ₁₃ NO ₄ ·HCl) is +57.4°, the melting point is 229°C-232°C, the [α] _D ²³ of (1N-HCl) is +60.6°, when ninhydrin Color reaction. Effective mycoamine also contains a primary amino group (-NH ₂ ), which undergoes first-order dissociation in aqueous solution.

Effective mycylamine, effective mycamine and other cyclic alcohols are the core structure of glycosidase inhibitors, and they are also a strong glycosidase inhibitor.

Glycosidases are trimming enzymes of oligosaccharide chains in glycoprotein biosynthesis, and are extremely important for the formation of oligosaccharide chains in glycoproteins. The composition and structure of sugar chains are the recognition sites for specific biological functions of glycoproteins, which affect protein folding, solubility, modification, antigenicity, and biological activity. Glycosidase activity plays a key role in the biosynthesis of glycoproteins, and research on glycosidase inhibitors has broad application prospects.

Due to the biological activity of mycamine and effective mycamine, people's interest in it is increasing day by day. These years, it is one of the active research fields, especially in Japan and Germany, but in our country, the research in this area is almost blank.

Contents of the invention

The task of the present invention is to purposefully screen effective mycoylidene or effective mycoylidene into effective mycylamine and effective mycamine strains from soil; Or the method for producing effective mycylamine and effective mycylamine by decomposing antibiotics validamycin and valid mycoylidene with cells or enzymes of the new microorganism.

The microorganism provided by the present invention is Stenotrophomonas (Stenotrophomonas), is Stenotrophomonas maltrophilia (Stenotrophomonas maltrophilia), and this bacterial strain has been preserved in China Type Culture Collection Center on March 15, 2004, is called for short CCTCC , the deposit number is CCTCC No.M 204024.

According to literature and patent search result, the patent strains that have reported that can degrade validamycin include Pseudomonas (Pseudomonas, 1 species, Pseudomonas denitrificans, Pseudomonas denitrificans), Flavobacterium (Flavobacterium, 3 1 species), Cytophaga (1 species, 3 strains), Agrobacterium (2 species, of which Agrobacterium radiobacter has 5 strains, Agrobacterium tumefaciens, 1 species), Aeromonas (Aeromonas , 5 species). The specific results are shown in Table 1.

Table 1 Summary of strains of validamycin-degrading patent [Patent Gazette (B2)] bacteria patent Pseudomonas denitrificans Flat 2-26957 (1982), Flat 2-2598 Flavobacterium saccharophilum IFO13984 Flat 2-26957, Flat 2-2598 Flavobacterium heparinum Flat 2-26957 Flavobacterium keratolyticus Flat 2-26957 Cytophaga heparina IFO 12017 Flat 2-26957 Cytophaga heparina ATCC 13125 Flat 2-26957 Cytophaga heparina IFO 14087 Flat 2-26957 Flavobacterium saccharophilum n.sp. Flat 2-2598 Flavobacterium saccharophilum FERM-P Flat 2-2598 Flavobacterium saccharophilumNO.5707 Flat 2-2598 Agrobacterium Radiobacter IFO 12664 (ATCC 4718) Flat 6-69380 Agrobacterium Radiobacter IFO 13258 (ATCC 13332) Flat 6-69380 Agrobacterium Radiobacter IFO 13259 (ATCC 13333) Flat 6-69380 Agrobacterium Radiobacter IFO 13532 (ATCC 19358) Flat 6-69380 Agrobacterium Radiobacter IFO 13533 Flat 6-69380 (ATCC 25235) Agrobacterium tumefaciens IFO 3058 Flat 6-69380 Aeromonas hydrophila subsp. anaerogenes IFO 13282 Flat 6-69380 Aeromonas hydrophila subsp.hydrophila IFO 13286 Flat 6-69380 Aeromonas hydrophila subsp. proteolytica IFO13287 Flat 6-69380 Aeromonas punctata subsp.cavice IFO13288 Flat 6-69380 Aeromonas salmonicida subsp.salmonicida IFO 12659 Flat 6-69380

The physiological and biochemical characteristics of each genus are as follows:

Pseudomonas (Pseudomonas): denitrifying Pseudomonas (Pseudomonasdenitrificans) is a Gram-negative non-bacillus, polar flagellate movement, oxidative metabolism, usually positive for oxidase, organic chemical energy nutrition. Aerobic bacteria capable of denitrification.

Agrobacterium: short bacillus, 1.5-0.3μm×0.6-1.0μm in size, arranged in single pairs, sparsely hairy, Gram-negative, no spores, raised colonies, smooth entire edges, no Pigment to light grayish yellow. Produces a large amount of polysaccharide mucus in sugar-containing medium, does not fix atmospheric nitrogen, and most species can use inorganic nitrogen. Agrobacterium actinum can produce 3-keto-lactose from lactose, with weak or no ability to decompose protein, and can widely utilize carbohydrates, Organic acid salts and amino acids are carbon sources, and cellulose, starch, agarose, galactose and other sugars cannot be used.

Aeromonas: cells are straight, rod-shaped with round ends, often move with unipolar hairs, ferment glucose, fructose, maltose and trehalose, decompose carbohydrates into acids, some produce gas, hydrolyze starch and Dextrin, hydrolyzes casein, liquefies gelatin, produces DNase, arginine deaminase, a few species do not move. Gram-negative, respiratory and fermentative metabolic types, positive for oxidase and catalase, facultative anaerobes.

Cytophaga: does not form fruiting bodies, the cells are rod-shaped, often form very long cell chains, aerobic, can utilize carbohydrates, require a large amount of CO ₂ in the metabolic process, can completely decompose cellulose and hydrolyze chitin Polysaccharides such as gelatin and agar can slide on the interface.

Flavobacterium (Flavobacterium): straight rod shape, round end, size 0.5μm×1.0～3.0μm. Cells do not contain PHB salts, do not form endospores, are Gram negative, do not motile, do not slide or swim, are strictly aerobic, grow on solid media, produce typical yellow or orange pigments, some strains do not Pigment, colony translucent, round, raised or slightly raised, smooth and shiny, entire, positive for contact enzyme, oxidase, phosphatase, indigestible agar, organic chemical nutrition. No gas is produced in low-concentration peptone medium.

Based on the above results, the new bacterial strain screened by the present invention does not belong to any of the above-mentioned patented strains.

The new strain is characterized by:

Colony morphology: smooth, shiny, with neat edges, white, gray or light yellow, and the diameter of the colony after culturing for 18h-36h is about 1mm-2mm; the lawn on the slope is white and sticky.

Cell morphology: no PHB particles are formed in the cells, no movement, rod-shaped, and the average size of the bacteria is: 0.4-0.5μm×1.3-1.4μm. Gram-negative, non-spore-forming.

Physiological and biochemical characteristics: liquefied gelatin, positive yolk reaction, positive lipase (TW 80) reaction, negative oxidase, positive catalase, negative indole reaction, negative methyl red, negative VP reaction, produces H ₂ S, does not oxidize Decompose glucose, produce oxidative acid, use malonate, use citrate, do not reduce nitrate, cannot denitrify, cannot use starch, urease reaction is positive, hydrolyze casein, cannot decompose lactose to produce 3-ketolactose.

Need growth factors, produce lysine decarboxylase, hydrolyze chitin; use asparagine as carbon and nitrogen source.

According to the above bacteriological characteristics, this new bacterial strain was identified as a strain belonging to the genus Stenotrophomonas and was Stenotrophomonas maltrophilia.

The medium used for the strain CCTCC No.M 204024 of the present invention contains nutrients that can be utilized by the above-mentioned strains, either liquid or solid, but liquid medium is more suitable for mass industrial production. The medium is properly mixed with carbon sources, nitrogen sources, inorganic salts, etc. that can be used by the above-mentioned microorganisms. As carbon sources: validamycin, glucose, lactose, maltose, dextrin, starch, glycerin, mannitol, behenitol, oils (soybean oil, lard, etc.); as nitrogen sources: gravy, yeast extract , dry yeast, soybean flour, corn steep liquor, peptone, urea, ammonia salt (ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.), peptides (dipeptide, tripeptide, etc.); inorganic salts include: Na, K, Ca, Mg, Fe, Mn, Zn, Co, Ni and other metal salts and organic acid salts such as phosphate and acetate; in addition: amino acids (such as glutamic acid, aspartic acid, Lysine, glycine, methionine, etc.), vitamins (V _B1 , V _B2 , V _B12 , V _C , _VE , niacin, etc.), nucleic acids (such as purine, pyrimidine and their derivatives, etc.) . Adjust the pH value of the culture medium with inorganic or organic acids and alkalis, and add an appropriate amount of defoaming agents, such as foam enemies.

The culture methods that can be used include: static culture, shaking culture, or ventilation and stirring culture, etc., and deep ventilation and stirring culture should be used for mass production of effective mycamine and effective mycylamine.

Another important feature of the present invention is to use the new microbial fermentation or the new microbial cells or their enzymes to decompose the substrate validamycin or effective mycoylidene amine, an intermediate product of validycin, to produce effective mycylamine and effective mycylamine .

New microorganism Stenotrophomonas (Stenotrophomonas) maltophilia Stenotrophomonas (Stenotrophomonas maltrophilia) of the present invention, CCTCC No.M 204024, and the culture medium containing carbon source, nitrogen source, inorganic salt, substrate is The antibiotic validamycin is fermented, and then the effective mycylamine and effective mycylamine are separated from the decomposed fermentation liquid and purified.

The composition of the medium of the present invention is (w/v, %): validamycin: 0.5% to 20%, (NH ₄ ) ₂ SO ₄ : 0.5% to 10%, KCl: 0.5% to 5.0%, Na ₂ HPO ₄ ·12H ₂ O: 0.1%-10.0%, NaH ₂ PO ₄ ·2H ₂ O: 0.1%-5.0%, MgSO ₄ : 0.01%-1.0%, prepared with tap water, pH 6.0-8.0.

With new bacterial strain stenotrophomonas maltophilia (Stenotrophomonas maltrophilia) CCTCC No.M 20402) of the present invention, produce effective mycylamine and effective mycylamine, its method has:

(1) The culture medium prepared above is sterilized, inserted into the cultivated strain CCTCC No.M 204024, inoculated on a slant or with seed liquid, and cultivated. The concentration of effective mycin in the culture medium is 0.5% to 20.0%. , 1.0% to 10.0% is the best; usually the temperature is 20°C to 40°C, preferably 28°C to 35°C, the initial pH is 6.0 to 8.0, and it is better to be close to neutral, that is, the initial pH value is 6.8 to 7.2 is better, and the culture time is preferably 100h to 180h, among which 150h is the best. In addition, the reaction can be carried out under static conditions, but it is better to carry out under the conditions of stirring, ventilation and shaking. During the fermentation process, the pH value is adjusted and controlled with inorganic or organic acids and alkalis. In this method, validamycin in the medium composition is not only a carbon source utilized by microorganisms, but also a substrate decomposed by microorganisms; the substrate validamycin is decomposed into effective mycoylidene amines, and effective mycoylidene amines include A and B Two kinds of effective mycoylidene amines are then decomposed to produce effective mycosyramine (valienamine) and effective mycosamine (validamine).

(2) The culture medium prepared above is sterilized, inserted into the cultured strain CCTCC No.M204024, inoculated with a slant or seed liquid, and cultivated bacteria. ℃～35℃ is better, the initial pH is 6.0～8.0, and it is better to be close to neutral, that is, the initial pH value is preferably 6.5～7.5; during the cultivation process, the substrate for microbial decomposition—effectivemycin or effective The mycosylidene solution can be added when the bacteria begin to grow, or after the bacteria have grown for a period of time, the substrate effective mycoylidene or effective mycosyramine in the culture can be added The concentration is 1.0%～50.0%, and the feeding speed can be adjusted according to the concentration of effective mycoylidene or effective mycoylidene amine. The culture time is preferably 100h～180h, among which 150h is the best. In addition, the reaction can be carried out in static Place it down, but it is better to carry out under the conditions of stirring, ventilating and shaking; during the fermentation process, the change of pH value is adjusted and controlled by inorganic acid or organic acid and alkali to obtain the fermentation product.

(3) The culture medium prepared above is sterilized, inserted into the cultivated strain CCTCC No.M204024, inoculated with a slant or seed liquid, and cultivated bacteria. ℃～35℃ is better, the initial pH is 6.0～8.0, it is better to be close to neutral, that is, the initial pH value is preferably 6.5～7.5; cultivate the bacteria to the logarithmic growth phase, and perform centrifugation to obtain the bacteria; The obtained thalline is added to the substrate effective mycoylidene or effective mycoylidene amine solution, the substrate effective mycoylidene or effective mycoylidene amine is decomposed by the bacterium, and the concentration of effective mycin or effective mycoylidene amine is 1.0 % to 20.0%, the reaction time is preferably 1h to 100h, and 70h is the best. In addition, the reaction can be carried out under static conditions, but it is better to be carried out under stirring, ventilating, and shaking conditions; during the fermentation process, the pH value The change of the fermentation product is adjusted and controlled by inorganic acid or organic acid and alkali.

(4) The culture medium prepared above is sterilized, inserted into the cultured strain CCTCC No.M204024, inoculated with a slant or seed solution, and cultivated bacteria. ℃～35℃ is better, the initial pH is 6.0～8.0, it is better to be close to neutral, that is, the initial pH value is preferably 6.5～7.5; the bacteria are cultivated to the logarithmic growth phase, centrifuged to obtain the bacteria, and then The bacteria are crushed, and the lyase is extracted; the extracted lyase is added to the substrate effective mycoylidene or effective mycoylidene amine solution, and the enzyme reaction is used to decompose the substrate effectivemycin or effective mycoylidene amine, and the effective mycoylidene The concentration of mycosine or effective mycoylidene amine solution is 1.0% to 20.0%, and the enzyme reaction time is preferably 1h to 100h, among which 10h is the best. In addition, the reaction can be carried out under static conditions, but the enzyme reaction time can be carried out under stirring, ventilation, and shaking. It is better to carry out under conditions; during the fermentation process, the change of pH value is adjusted and controlled by inorganic acid or organic acid and alkali to obtain the fermentation product.

Separating and purifying the decomposed fermentation broth containing effective mycylamine and effective mycylamine, the specific method is:

When extracting the target product from the fermentation broth, a conventional fermentation product extraction method can be used. Such as: filtration, centrifugation, precipitation, crystallization, recrystallization, concentration, drying, freeze-drying, adsorption, ion exchange, chromatography, etc. In addition, effective mycylamine and effective mycylamine are alkaline substances that are easily soluble in water and difficult to dissolve in general solvents, so the separation and purification methods of water-soluble alkaline substances can be used, such as ion exchange resin, activated carbon, porous polymer , Sephadex, ion exchanger, etc. after adsorption and desorption or chromatography.

The complete extraction process can be divided into three stages: pretreatment, mid-term purification and refinement: (1) pretreatment stage, the goal of this stage is to extract from complex liquid The target product was isolated, solid particles were removed, and concentrated. (2) In the mid-term purification stage, non-specific and low-resolution purification techniques are generally used first, followed by high-resolution chromatography and other techniques to remove impurities with similar physical and chemical properties and product properties. (3) Refining stage, this is the final processing procedure, the purpose is to further remove impurities and purify the product, the method depends on the application purpose of the product, the most commonly used method is crystallization, combined with drying, to obtain the desired purity and a certain shape The product.

Use thin-layer chromatography and gas phase analysis to analyze the effective mycamine and effective mycamine produced by fermentation. The operation process and experimental conditions are in accordance with the literature [J.Antibiot, 1984, 37:1301～1305] and patent [EP0063456] method, and the results obtained are completely consistent with them. It can be seen that effective mycocin and effective mycoylidene can be cracked into effective mycylamine and effective mycylamine by using the microbial strain Stenotrophomonas maltrophilia CCTCC No.M 204024 of the present invention.

The microbial strain Stenotrophomonas maltrophilia (Stenotrophomonas maltrophilia) CCTCC No.M 204024 of the present invention can not only grow on the culture medium with effective mycins as the sole carbon source, but also has a strong ability to decompose effective mycins ; Utilize CCTCC No.M 204024 to produce effective mycylamine and effective mycylamine. Under optimal process conditions, the decomposition rate of effectivemycin can reach 70% to 90%, and the molar conversion of effectivemycin A into effective mycylamine The rate reaches 40%-80% (that is, 1 mole of effectivemycin A can be converted into 0.40-0.80 moles of effective mycylamine), and the molar conversion rate of effective mycoylidene into effective mycylamine reaches 35%-75%. %, obtain effective mycamine simultaneously; Microbial strain Stenotrophomonas maltrophilia (Stenotrophomonas maltrophilia) CCTCC No.M 204024 of the present invention is suitable for the production of effective mycamine and effective mycamine.

Specific implementation plan

The following examples are intended to illustrate but not limit the scope of the invention.

Example 1:

Medium formula (weight/volume percentage, the same below): effectivemycin: 1.0%, (NH ₄ ) ₂ SO ₄ : 1.0%, KCl: 0.5%, Na ₂ HPO ₄ ·12H ₂ O: 1.0%, NaH ₂ PO ₄ ·2H ₂ O: 0.1%, MgSO ₄ : 0.01%, prepared with tap water, and adjusted the pH to 6.0 with HCl solution.

Take 100mL of the above-mentioned culture medium, divide it equally into two 250mL Erlenmeyer flasks, and sterilize. Insert the slant strain CCTCC No.M 204024, cultivate the bacteria, shake the table at a speed of 150r/min, and cultivate it at 28°C for 72 hours as a seed solution for later use.

Take 2L of the above culture medium, divide it into 20 500mL shake flasks, and sterilize. The seed liquid was inserted, the inoculum amount was 2% (v/v), and culture was carried out, the culture temperature was 20° C., the culture time was 160 h, and the shaker speed was 200 r/min.

Collect 1.8L of the above-mentioned fermentation broth, centrifuge to remove the bacteria, and the supernatant is adsorbed on the column (1L macroporous weakly acidic resin D113, NH ₄ ⁺ ), washed with distilled water, and then eluted with 0.5mol/L ammonia water to reduce Concentrate under pressure, then put the concentrated solution on column chromatography (500mL strong base chromatography resin Dowex 1×2, OH ^- ), elute with distilled water, collect the eluate in sections, collect the first flow out containing effective mycamine and the latter The part of the effective mycylamine that flows out is analyzed with the method of thin-layer chromatography and gas phase analysis, then carries out vacuum freeze-drying, obtains 0.31 gram of effective mycylamine and 0.74 gram of effective mycylamine.

Example 2:

Medium formula effectivemycin: 17.0%, (NH ₄ ) ₂ SO ₄ : 8.0%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 1.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1%, MgSO ₄ : 0.01%, prepared with tap water, and adjusted the pH to 7.0 with NaOH solution.

Take 500mL of culture medium, evenly distribute it in five 500mL Erlenmeyer flasks, and sterilize. Insert the slant strain CCTCC No.M 204024 for cultivation, the cultivation temperature is 28°C, the cultivation time is 150h, the reaction is carried out under the conditions of stirring, ventilation and shaking, and the speed of the shaker is 200r/min.

400 mL of the above-mentioned fermentation broth was collected, separated and purified, and the steps were the same as in Example 1 to obtain 0.56 g of available mycylamine and 0.85 g of available mycylamine.

Example 3:

Fermentation medium formula Effectivemycin: 8.0%, (NH ₄ ) ₂ SO ₄ : 6%, KCl: 0.1%, Na ₂ HPO ₄ 12H ₂ O: 1.0%, NaH ₂ PO ₄ 2H ₂ O: 0.16% , MgSO ₄ : 0.02%, prepared with tap water, and adjusted the pH to 7.5 with NaOH solution.

Seed medium formula Effectivemycin: 1.0%, (NH ₄ ) ₂ SO ₄ : 1%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 1.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1% , MgSO ₄ : 0.5%, prepared with tap water, and adjusted the pH to 7.5 with NaOH solution.

Take 500mL seed culture medium, divide it into five 500mL Erlenmeyer flasks, and sterilize. Insert the slant strain CCTCC No.M 204024, cultivate the bacteria, shake the table at a speed of 200r/min, and cultivate it on a shaker at 30°C for 72 hours as a seed solution for later use.

Take 8L of fermentation medium, divide it into 80 shake flasks of 500mL, and sterilize. Insert the seed solution, the inoculation amount is 5% (v/v), and cultivate, the cultivation temperature is 30°C, the cultivation time is 180h, the reaction is carried out under stirring, ventilating and shaking conditions, and the shaking table speed is 200r/min.

7.5 L of the above-mentioned fermented liquid was collected, separated and purified, and the steps were the same as in Example 1 to obtain 3.19 g of available mycylamine and 7.77 g of available mycylamine.

Example 4:

Medium formulation Effectivemycin: 2.0%, (NH ₄ ) ₂ SO ₄ : 1.0%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 2.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1%, MgSO ₄ : 0.1%, prepared with tap water, and adjusted the pH to 7.0 with NaOH solution.

Take 500mL of culture medium, evenly distribute it in five 500mL Erlenmeyer flasks, and sterilize. Insert the slant strain CCTCC No.M 204024, cultivate the bacteria, shake the table at a speed of 150r/min, and cultivate it at 28°C for 72 hours as a seed solution for later use.

Take 8L of culture medium, divide it into 80 shake flasks of 500mL, and sterilize. Insert the seed solution, the inoculum amount is 5% (v/v), and cultivate. The culture temperature is 28° C., and the culture time is 100 h. The reaction is carried out under stirring, ventilating, and shaking conditions, and the shaking table speed is 200 r/min.

7.5 liters of the above-mentioned fermented liquid were collected, separated and purified, and the steps were the same as in Example 1 to obtain 2.11 g of available mycylamine and 5.63 g of available mycylamine.

Example 5:

Medium formulation Effectivemycin: 1.5%, (NH ₄ ) ₂ SO ₄ : 0.75%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 2.0%, NaH ₂ PO ₄ 2H ₂ O: 1.0%, MgSO ₄ : 0.01%, prepared with tap water, natural pH.

Take 200mL of the above-mentioned culture medium, evenly distribute it in four 250mL Erlenmeyer flasks, and sterilize. Insert the slant strain CCTCC No.M 204024, cultivate the bacteria, shake the table at a speed of 150r/min, and cultivate it at 28°C for 72 hours as a seed solution for later use.

Add 5L of the above-mentioned culture medium into a 10L fermenter, sterilize, insert 200mL of seed liquid, and carry out fermentation.

After inoculation, start to feed 1L of the substrate effective mycin solution with a concentration of 10%, the flow rate is 0.2mL/min; the culture temperature is 30°C, the culture time is about 160h, the ventilation rate is 2.5L/min, and the stirring speed is 200r/min .

6L of the above-mentioned fermentation broth was collected, separated and purified, and the steps were the same as in Example 1 to obtain 3.67 g of available mycylamine and 6.53 g of available mycylamine.

Embodiment 6:

Medium formula Effectivemycin: 1.0%, (NH ₄ ) ₂ SO ₄ : 0.7%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 0.1%, NaH ₂ PO ₄ 2H ₂ O: 4.0%, MgSO ₄ : 0.01%, prepared with tap water, natural pH.

Take 500mL of the above-mentioned culture medium, evenly distribute it in five 500mL Erlenmeyer flasks, and sterilize. Insert the slant strain CCTCC No.M 204024, cultivate the bacteria, shake the table at a speed of 150r/min, and cultivate it at 28°C for 72 hours as a seed solution for later use.

Add 5L of the above-mentioned culture medium into a 10L fermenter, sterilize, insert 200mL of seed liquid, and carry out fermentation.

When the bacteria grow to the logarithmic phase, 3L of the substrate effectivemycin solution with a concentration of 2% is fed, and the flow rate is 0.5mL/min; the culture temperature is 35°C, the culture time is about 180h, and the ventilation rate is 2.5L/min. The stirring speed is 200r/min.

8 L of the above-mentioned fermented liquid was collected, separated and purified, and the steps were the same as in Example 1 to obtain 3.08 g of available mycylamine and 5.82 g of available mycylamine.

Embodiment 7:

Medium formula Effectivemycin: 1.0%, (NH ₄ ) ₂ SO ₄ : 0.7%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 6.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1%, MgSO ₄ : 0.7%, prepared with tap water, natural pH.

Take 500mL of culture medium, evenly distribute it in five 500mL Erlenmeyer flasks, and sterilize. Insert the slant strain CCTCC No.M 204024, cultivate the bacteria, shake the table at a speed of 150r/min, and cultivate it at 28°C for 72 hours as a seed solution for later use.

6 L of the above-mentioned medium was added to a 10 L fermenter as a fermentation broth, and sterilized. Insert 500mL of seed solution.

When the bacteria grow to the logarithmic phase, add 100mL of effective mycoylidene amine solution with a concentration of 50%, and the flow rate is 0.05mL/min; culture conditions: temperature is 30°C, culture time is 120h, ventilation volume is 3.5L/min , stirring speed 200r/min.

6L of the above-mentioned fermentation liquid was collected, separated and purified, and the steps were the same as in Example 1 to obtain 3.26 g of available mycylamine and 7.01 g of available mycylamine.

Embodiment 8:

Medium formula Effectivemycin: 2.0%, (NH ₄ ) ₂ SO ₄ : 1.2%, KCl: 4.0%, Na ₂ HPO ₄ 12H ₂ O: 4.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1%, MgSO ₄ : 0.01%, prepared with tap water, adjusted to pH 6.5 with HCl.

Take 1000mL of culture medium, evenly distribute it in ten 500mL Erlenmeyer flasks, and sterilize. Insert the slant strain CCTCC No.M 204024, cultivate the cells to the logarithmic growth phase, and then perform centrifugation to obtain the cells, and add these cells to 500mL of the substrate activemycin solution with a concentration of 5%. The bacteria decompose effective mycin; reaction conditions: temperature 30°C, initial pH 6.5, culture time about 100h, shaker speed 150r/min.

480 mL of the above-mentioned reaction solution was collected, separated and purified, and the steps were the same as in Example 1 to obtain 0.45 g of available mycylamine and 1.21 g of available mycylamine.

Embodiment 9:

Medium formula Effectivemycin: 2.0%, (NH ₄ ) ₂ SO ₄ : 1.2%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 1.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1%, MgSO ₄ : 0.01%, prepared with tap water, adjusted to pH 7.5 with NaOH solution.

Take 1000mL culture medium, divide it into ten 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCTCC No.M 204024, cultivate the bacteria to the logarithmic growth phase, and then perform centrifugation to obtain the bacteria. The bacteria are added to 500mL of substrate effective mycosylamine solution with a concentration of 15%, and the effective mycosylamine is decomposed by the bacteria; culture conditions: temperature 32°C, initial pH 7.5, culture time about 40h, shaker The speed is 200r/min.

580 mL of the above reaction solution was collected, separated and purified, and the steps were the same as in Example 1 to obtain 1.03 g of available mycylamine and 2.77 g of available mycylamine.

Example 10:

Medium formula Effectivemycin: 2.0%, (NH ₄ ) ₂ SO ₄ : 1.2%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 3.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1%, MgSO ₄ : 0.01%, prepared with tap water, adjusted to pH 7.5 with NaOH solution.

Take 1000mL culture medium, divide it evenly into ten 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCTCC No.M 204024, cultivate the bacteria to the logarithmic growth phase, and then perform centrifugation to obtain the bacteria. The cells were added to 500mL of substrate effective mycosylamine solution with a concentration of 3%, and the effective mycosylamines were decomposed by the cells; culture conditions: temperature 34°C, initial pH 7.5, culture time about 20h, shaker speed 150r/min.

580 mL of the above reaction solution was collected, separated and purified, and the steps were the same as in Example 1 to obtain 0.53 g of available mycylamine and 1.06 g of available mycylamine.

Example 11:

Medium formula Effectivemycin: 2.0%, (NH ₄ ) ₂ SO ₄ : 1.2%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 0.2%, NaH ₂ PO ₄ 2H ₂ O: 1.5%, MgSO ₄ : 0.01%, adjust the pH to 6.0 with HCl solution.

Take 2000mL of the above-mentioned culture medium, divide it into 20 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCTCC No.M 204024, cultivate the bacteria to the logarithmic growth phase, and then perform centrifugation to obtain the bacteria. The bacteria are broken up by ultrasonic waves, and then centrifuged, salted out, dialysis, and chromatography are carried out to extract the lytic enzyme, which is added to 200mL of 8% substrate activemycin solution to decompose the effective enzyme by enzymatic reaction. Mycin produces effective mycamine and effective mycylamine. Culture conditions: temperature is 25°C, initial pH is 6.0, culture time is about 10 hours, and the reaction can be carried out under static condition.

200 mL of the above-mentioned reaction solution was collected, separated and purified, and the steps were the same as in Example 1 to obtain 0.15 g of available mycylamine and 0.34 g of available mycylamine.

Example 12:

Medium formula Effectivemycin: 2.0%, (NH ₄ ) ₂ SO ₄ : 1.2%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 1.5%, NaH ₂ PO ₄ 2H ₂ O: 0.8%, MgSO ₄ : 0.01%, adjust the pH to 8.0 with NaOH solution.

Take 2000mL culture medium, divide it evenly into 20 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCTCC No.M 204024, cultivate the bacteria to the logarithmic growth phase, and then perform centrifugation to obtain the bacteria. Break up the bacteria, then carry out centrifugation, salting out, dialysis, and chromatography to extract the lyase, add the above-mentioned extracted lyase to 100mL of the substrate effective mycin solution with a concentration of 20%, and use the enzyme reaction to decompose the effective Mycin. Reaction conditions: the temperature is 32°C, the initial pH is 8.0, the incubation time is about 80 hours, and the reaction can be carried out under static conditions.

100 mL of the above reaction solution was collected, separated and purified, and the steps were the same as in Example 1 to obtain 0.21 g of available mycylamine and 0.39 g of available mycylamine.

Example 13:

Medium formula Effectivemycin: 2.0%, (NH ₄ ) ₂ SO ₄ : 1.2%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 1.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1%, MgSO ₄ : 0.01%, adjust the pH to 7.8 with NaOH.

Take 2000mL culture medium, divide it evenly into 20 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCTCC No.M 204024, cultivate the bacteria to the logarithmic growth phase, and then perform centrifugation to obtain the bacteria. Break up the bacteria, then carry out centrifugation, salting out, dialysis, and chromatography to extract the lyase, add the above-mentioned extracted lyase to 100mL of substrate-effective mycolidene amine solution with a concentration of 10%, and use the enzyme reaction Decompose effective mycoylidene to produce effective mycamine and effective mycylamine. Reaction conditions: temperature 30°C, initial pH 7.0, incubation time about 30 hours, shaker speed 100r/min.

100 mL of the above reaction solution was collected, separated and purified, and the steps were the same as in Example 1 to obtain 0.10 g of available mycylamine and 0.31 g of available mycylamine.

Example 14:

Medium formula Effectivemycin: 2.0%, (NH ₄ ) ₂ SO ₄ : 1.2%, KCl: 0.5%, Na ₂ HPO ₄ 12H ₂ O: 1.0%, NaH ₂ PO ₄ 2H ₂ O: 0.1%, MgSO ₄ : 0.01%, adjust pH to 7.0 with NaOH.

Take 2000mL of the above-mentioned culture medium, divide it into 20 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCTCC No.M 204024, cultivate the bacteria to the logarithmic growth phase, and then perform centrifugation to obtain the bacteria. The bacterium is broken up by ultrasonic waves, and then centrifuged, salted out, dialysis, and chromatography are carried out to extract the lyase, and the above-mentioned extracted lyase is added to 100 mL of substrate-effective mycolidene amine solution with a concentration of 18%, and the The enzymatic reaction breaks down effective mycoylidene amines. Reaction conditions: temperature 30°C, initial pH 7.0, incubation time about 50 hours, shaker speed 100r/min.

100 mL of the above reaction solution was collected, separated and purified, and the steps were the same as in Example 1 to obtain 0.33 g of available mycylamine and 0.59 g of available mycylamine.

Claims (12)

1. a microbial preparation method of effective mycylamine and effective mycylamine is characterized in that described microorganism is Stenotrophomonas (Stenotrophomonas) stenotrophomonas maltrophilia (Stenotrophomonas maltrophilia), CCTCC No.M 204024, Stenotrophomonas maltophilia and a medium containing carbon source, nitrogen source, and inorganic salt, the substrate is the antibiotic validamycin, and the fermentation is carried out, and the effective mycylamine and effective mycylamine are separated from the decomposed fermentation liquid, and Purify. 2. The preparation method according to claim 1, characterized in that the composition of the culture medium is weight/volume: validamycin 0.5% to 20.0%, (NH ₄ ) ₂ SO ₄ 0.5% to 10.0%, KCl0 .5%～5.0%, Na ₂ HPO ₄ 12H ₂ O 0.1%～10.0%, NaH ₂ PO ₄ 2H ₂ O 0.1%～5.0%, MgSO ₄ 0.01%～1.0%, prepare with tap water, adjust the pH value 6.0～8.0. 3. The preparation method according to claim 1, characterized in that the separation and purification steps of the fermented liquid are: centrifugation of the reaction solution, removal of the thallus, adsorption of the upper column on the supernatant, washing with distilled water, and then using 0.5 mol/L ammonia water, concentrated under reduced pressure, and then put the concentrated solution on column chromatography, eluted with distilled water, collected the eluate in sections, and analyzed it by thin layer chromatography, collected the effective mold containing The part of the amine and the effective mycylamine that flows out afterward are subjected to vacuum freeze-drying respectively to obtain the effective mycylamine and the effective mycylamine. 4. The preparation method according to claim 1 or 2, characterized in that the fermentation medium after accessing CCTCC No.M 204024 is fermented, the concentration of effectivemycin in the medium is 0.5%～20.0%, and the culture conditions are: The temperature is 20°C-40°C, the initial pH is 6.0-8.0, and the culture time is 100h-180h. 5. The preparation method according to claim 4, characterized in that the concentration of effectivemycin in the fermentation medium is 1.0% to 10.0%, the fermentation temperature is 28°C to 35°C, the initial pH is 6.8 to 7.2, and the culture time is 150h . 6. The preparation method according to claim 1, characterized in that after the bacterial strain CCTCC No.M 204024 is inserted into the culture medium, the substrate validamycin is added during the cultivation process, and it begins when the thalline just begins to grow. Fed-batch feeding, culture conditions: the temperature is 20°C-40°C, the initial pH is 6.0-8.0, and the culture time is 100h-180h. 7. The preparation method according to claim 6, characterized in that the substrate activemycin is added after the logarithmic phase of the growth of the bacteria, the culture conditions are: 28°C-35°C, the initial pH is 6.5-7.5, and the culture Time 150h. 8. The preparation method according to claim 1, characterized in that after inserting the strain CCTCC No.M 204024 in the culture medium and cultivating the thalline to the logarithmic growth phase, then performing centrifugation to obtain the thalline, and the thalline The cells are added to the substrate validamycin solution, and the substrate effective mycin is decomposed by the cells. The culture conditions: the temperature is 20°C-40°C, the initial pH is 6.0-8.0, and the culture time is 1h-100h. 9. The preparation method according to claim 8, characterized in that the bacteria are added to the substrate validamycin solution, culture conditions: temperature 28°C-35°C, initial pH 6.5-7.5, culture time 70h. 10. The preparation method according to claim 1, characterized in that after inserting the strain CCTCC No.M 204024 in the culture medium, the thallus is cultivated to the logarithmic growth phase, and then centrifuged to obtain the thallus, and then ultrasonic Break up the bacteria, extract the lyase, add the lyase to the validamycin solution, culture conditions: temperature 20°C-40°C, initial pH 6.0-8.0, culture time 1h-100h. 11. The preparation method according to claim 10, characterized in that the lyase is added to the substrate validamycin solution, culture conditions: temperature 28°C-35°C, initial pH 6.5-7.5, culture time 10h. 12. The preparation method according to any one of claims 1 or 6-11, characterized in that the substrate is effective mycolide.