WO2005098014A1 - Microbe method for producing valienamine and validamine - Google Patents

Abstract

microbe method for producing valienamine and validamine The present invention relates to a microbe method for producing valienamine and validamine, the used microbe is Stenotropgomonas maltrophilia CCTCC No.M204024, by fermenting the microbe to, or by the microbe cells or enzymes to disassemble the substrate validamycin or validoxylamine to produce valienamine and validamine, which are two kinds of cyclitol substrate, weight/volume compositions of the medium are: validamycine 0.5% ~20.0%, (NH4)2S04 0.5%~10.0%, KCl 0.5%~5.0%, Na2HPO4 12H2O 0.1%~10.0%, NaH2PO4?2H2O 0.1%~5.0%, MgSO4 0.01~1.0%, formulating with tap water, the culture conditions are : fermenting temperature 20~40°C, initial pH 6.0~8.0, culturing duration 1h~180h, the fermenting product are subjected to ion exchange, chromatography to separate and purify to provide valienamine and validamine.

Description

 TECHNICAL FIELD

 The present invention relates to a novel oligotrophomonas maltophilia screened from soil

{Stenotrophomonas maltrophilia), also involves the use of this new strain to break down potent mycin (also known as Jinggangmycin) or potent mycotylamine (also known as pinamycin or pinam hydroxylamine) to produce potent myelenamine and potent mycotoxamine Amine method. Background technique

 The research on the effective method of manufacturing mycolylamine and effective mycylamine has been over 30 years. The Japanese Kameda et al. Have used the bacterial cells of denitrifying Pseudomonas to degrade the effective mycin A or the effective myridine A. Then, effective mynomycin and effective myramine are obtained by separation, but it cannot be grown on a medium using only effective mycins as the sole carbon source; and the ability to decompose effective mycetin is very weak, and it is not suitable for effective mycotoxins. Mass production of enamines and effective myramine.

A Japanese strain, Kameda, and the like, isolated a strain capable of decomposing effective mycin into effective myenylamine and effective myramine in the soil. Avobacterium saccharophiluni IF013948 (Public License: JP57-54593) ₀

 Japanese Kameida and others used the enzyme-producing microorganism Cytophaga heparina) Cytophaga heparina) IFO12017 (ATCC13125) and IFO14087 strains to degrade potent mycin or potent mymidine amine to produce potent myelenamine and potent mycolamine [Patent Bulletin (B2): Hei 2-26957].

Japanese Kameida and others found that microorganisms of the genus Agrobacteriund or Aeromonas {Aeromonas ^ and their variants can effectively decompose effective mycin or effective mylidylamine to produce effective myenylamine and effective myramine. For example, the soil microorganism Ugrobacterium includes 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, And Agrobacterium tumefaciens IFO 3058 strains, etc .; Aeromonas microorganisms, Aeromonas hydrophila subsp. Anaerogenes IFO 13282, subsp. Hydrophila IFO 13286, subsp. Proteolytica IFO 13287, aeromonas punctata subsp. Cavice IFO 13288, and aeromonas salmonicida subsp. salmonicida lFO 12659, etc. [Patent Gazette (B2): Hei 6-69380].

 The raw material used in the present invention is a widely used agricultural antibiotic, effective mycin, namely Jinggangmycin, which is a highly efficient and safe agricultural antibiotic, does not pollute the environment, and is harmless to humans and animals. A safe, pollution-free pesticide with a large area and the lowest cost per acre is an important species 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 And β-D-glucose and other structural components.

 The effective mycoglycoside bond is hydrolyzed to be decomposed into the effective mymidine amine. There are two kinds of effective mymidine amine. There are two kinds of effective mymidine amine. validamine).

 The effective mylenylamine in the present invention is named valienamine in English, and its structural formula is shown in Figure 1 (Chem. Rev. 2003, 103: 1955-1977).

 Figure 1 Chemical structure of effective valienamine

Effective mycenylamine, also known as gangliomycin, (1S, 2S, 3S, 4R) -1-amino-5- (hydroxymethyl) cyclohex-5-ene-2, 3, 4-triol; The molecular formula is C ₇ H ₁₃ N0 _{4. The} important groups are: a primary amino group (-N¾), a carbon-carbon double bond (C = C), a methylol group (-CH ₂ OH), and three hydroxyl groups (- OH). The hydrochloride (C ₇ H ₁₃ NO ₄ · HC1) [is + 68.6 ° (1N-HC1), the melting point of pentaacetate (C ₁₇ H ₂₃ N0 ₉ ) is 95 ° C, [«is +30.2 ° (CHC1 ₃ ), color reaction with ninhydrin. Since effective myrcenamine contains a primary amino group (_NH ₂ ), first-order dissociation occurs in aqueous solution.

 The effective mycolamine referred to in the present invention, the English name is validamine, and its structural formula is shown in Figure 2 (Chem.

Rev. 2003, 103: 1955-1977).

Chemical structure of validamine Effective mycotylamine, also known as Jinggangmycin, has the molecular formula C ₇ H ₁₅ N0 ₄ , one primary amino group (-N¾), one methylol group (-CH ₂ OH), three hydroxyl groups (-OH), and its hydrochloride (C ₇ H ₁₃ N0 ₄ · HC1) [«is +57.4. , Melting point is 229 ° C-232 ° C, [o¾ ³ of (1N-HC1) is + 60.6 °, and it reacts with ninhydrin to develop color. Effective myramine also contains a primary amino group (-NH ₂ ), which undergoes first-order dissociation in aqueous solution.

 Cycloalcohols, such as effective mycolylamine and effective mycoamine, are the core structure of glycosidase inhibitors, and they are also strong glycosidase inhibitors.

 Glycosidases are trimming enzymes for 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 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 glycoprotein biosynthesis. Research on glycosidase inhibitors has broad application prospects.

 Due to the biological activity of effective myramine and effective myramine, people's interest in it has been increasing day by day. In recent years, it is one of the active research fields, especially in Japan, South Korea, and Germany, but in our country, the research in this area is almost blank. Summary of the invention

 The task of the present invention is to purposefully screen from the soil to a new microbial strain that decomposes effective mycotoxin or effective myclidene into effective mycenylamine and effective mycamine; secondly, to provide a new microorganism to decompose by effective fermentation Or the method of decomposing antibiotic effective omycin and effective myco subunit amine by the cell or enzyme of the new microorganism.

The microorganism provided by the present invention is Oligomonas spp. (C ^^ c ^ ow ^), which is maltotrophophilic

maltrophilia), which was deposited at the China Type Culture Collection on March 15, 2004, referred to as CCTCC, with the accession number CCTCC No. M 204024.

According to the literature and patent inspection results, the patented strains that can degrade the effective mycotoxin include Pseudomonas ^ Pseudomonas, 1 of which is Pseudomonas denitrificans, Flavomyces iFlavobacterium (3 species), i Cytophaga (1 species, 3 strains), Agrobacterium (2 species of grasses, of which Agrohacterium radiobacter ^ 5 strains, Agrobacterium tumefaciens, 1 species), Aeromonas {Aeromonas, 5 species). The specific results are shown in Schedule 1. Patent for Degradation of Effective Mycin [Patent Gazette (B2)]

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

 jg ^. | ¾¾fj¾ (Pseudomonas): PseMifowcwas denitrificans are Aegeroides sp Aerobic bacteria, capable of denitrification.

± (Agrobacterium): short bacillus, large zj, 1.5 ~ 0 · 3 μm X 0.6 ~ 1 · 0 μm, arranged in pairs in a single pair, thin peripheral hair, negative Gram reaction, no spores, colony raised Full light Smooth, non-pigmented to light grayish yellow. A large amount of polysaccharide slime is produced in the sugar-containing medium, and atmospheric nitrogen is not fixed. Most species can use inorganic nitrogen. Agrobacterium radians can produce 3-ketolactose from ¥ L sugar, and the ability to break down proteins is weak or non-existent. Carbon water can be widely used Compounds, organic acid salts, and amino acids are carbon sources, and cellulose, starch, agarose, galactose, and other sugars cannot be used.

 Aeromonas era): Cells are straight, with rounded rods, often moving with unipolar hairs, fermenting glucose, fructose, maltose and trehalose, carbohydrates are broken down into acids, some produce gas, hydrolyzed starch and paste Sperm, hydrolyzed casein, liquefied gelatin, produces DNase, arginine deaminase, a few species do not move. Gram reaction is negative, there are two types of metabolism of breathing and fermentation, oxidase and catalase positive, facultative anaerobic bacteria.

Cellophyllum (C3; top½): does not form fruiting bodies, cells are rod-shaped, often form very long cell chains, aerobic, can utilize carbohydrates, a large amount of co ₂ is required in the metabolic process, can completely decompose cellulose, and hydrolyze Polysaccharides such as chitin and agar can glide across the interface.

 Flavobacterium): Straight rod-shaped, rounded end, size 0.5 μm X 1.0-3.0 μm. Cells do not contain PHB salt, do not form endospores, Gram-negative, do not move, have no sliding or swimming, strictly aerobic, grow on solid media, produce typical yellow or orange pigments, and some strains do not Pigments, colonies are translucent, round, raised or slightly raised, smooth and lustrous, whole, positive for contact enzymes, oxidases, and phosphatases, do not digest agar, and organically nutrition. It does not produce gas in low-concentration egg white culture medium.

 Based on the above results, the new strains screened by the present invention do not belong to any of the aforementioned patent strains.

 The new strain is characterized by:

 Colony form: smooth, shiny, neat edges, white, gray or light yellow, culture 181! The colony diameter of ~ 36h is about lmm ~ 2mm; the bevel moss is white and sticky.

 Cell morphology: PHB particles are not formed in the cells, do not move, and are rod-shaped. The average cell size is: 0.4 to 0.5 μm x 1.3 to 1.4 μm. Gram-negative and spore-free.

Physiological and biochemical characteristics: Liquefied gelatin, positive yolk reaction, positive lipase (soil temperature 80), negative oxidase, positive contact enzyme, negative indole reaction, negative methyl red, negative VP reaction, produces ¾8, does not decompose glucose oxidatively Oxidative acid production, using malonate, citrate, does not reduce nitrate, cannot denitrify, cannot use starch, positive urease reaction, hydrolyzes casein, Inability to break down lactose to produce 3-ketolactose.

 Requires growth factors, produces lysine decarboxylase, hydrolyzes chitin; uses asparagine as carbon and nitrogen source. Based on the above bacteriological characteristics, this new strain was identified as a strain belonging to the genus Stenotrophomomas (Stenotrophomonas maltrophilid).

The medium used for the strain CCTCC No. M 204024 of the present invention contains some nutrients that can be utilized by the above-mentioned strains, both liquid and solid. However, a liquid medium is more suitable for large-scale industrial production. The medium is reasonably formulated with a carbon source, a nitrogen source, an inorganic salt, and the like that can be used by the microorganisms. The carbon sources are: effective mycin, glucose, lactose, maltose, dextrin, starch, glycerol, mannitol, sorbitol, lipids (soy oil, lard, etc.); as nitrogen sources are-gravy, yeast Cream, dry yeast, soybean meal, corn pulp, peptone, urea, ammonia salts (ammonium sulfate, ammonium chloride, ammonium nitrate, amine acetate, etc.), peptides (dipeptide, tripeptide, etc.); inorganic salts are: Na , K, Ca, Mg, Fe, Mn, Zn, Co, NTi and other metal salts and phosphates, acetates and other organic acid salts; can also be added-amino acids (such as glutamic acid, aspartic acid , Lysine, glycine, methionine, etc.), microbiotin (V _B1 , V _B2 , V _{B 1 2} , V _c V _E , niacin, etc.), nucleic acids (such as purines, pyrimidines and their derivatives Wait). Inorganic or organic acids and alkalis can be used to adjust the pH value of the culture medium, and an appropriate amount of antifoaming agent such as foaming enemy can also be added.

 The culture methods that can be used are: static culture, shaking culture, or aeration and agitation culture. When a large amount of effective mycol and effective myramine are produced, deep aeration and agitation culture should be used.

 Another important feature of the present invention is to use the new microbial fermentation or the new microbial cell or its enzymatic decomposition substrate, effective mycotoxin or an effective intermediate of effective mycomycin, to produce effective mycenylamine and effective mycotylamine .

 The novel microbial oligooxymonas iSt otrophomoncis (M Stenotrophornonas mcdtrophilia), CCTCC No. M 204024, and a medium containing a carbon source, a nitrogen source, and an inorganic salt, and the substrate is an antibiotic effective mold And fermentation, and then decomposing the fermentation broth to separate effective melamine and effective myramine, and then purify.

The composition of the culture medium of the present invention is (w / v,%): effective mycin: 0.5% to 20%, (NH ₄ ) 2SO4: 0.5% to 10%, KC1: 0.5% to 5.0%, Na ₂ HPO ₄ · _{12Η 2 0: 0.1% ~ 10.0} %, NaH 2 PO 4 «2H 2 0: 0.1% ~5.0%, MgSQ 4: 0.01% ~ 1.0%, formulated in tap water, pH value 6.0 to 8.0. Tear the invention of the new

maltrophilia) CCTCC No. M 20402) produces menomynamine and effective myramine by the following methods:

 1) The above-prepared culture medium is sterilized, inserted into the cultured strain CCTCC No. M 204024, slanted or inoculated with seed solution, and cultured, usually selected at a temperature of 20 ° C ~ 40 ° C, at 28 ° C ~ 35 ° C is better, the initial pH is 6.0 ~ 8.0, it is better to be near neutral, the culture time is 100 h to 180 h, and the best is about 150 h. In addition, the reaction can be carried out under standing , But under the conditions of stirring, ventilation, shaking is better. During the fermentation process, the pH value is adjusted and controlled with inorganic or organic acids and alkalis. In this method, the effective mycin in the medium composition is both a carbon source utilized by the microorganism and a substrate decomposed by the microorganism; the substrate effective mycin is decomposed into an effective mycoimine amine. Two types, the effective mylidinylamine is decomposed to generate valienamine and glutamine is validamine o

 (2) The above-prepared culture medium is sterilized, inserted into the cultured strain CCTCC No. M 204024, inoculated with bevel or seed solution, and cultured, and the culture conditions: Generally, the temperature is selected at 20 ° C ~ 40 ° C It is better to use 28 ° C ~ 35 ° C, the initial pH is 6.0 ~ 8.0, and it is better to be near neutral; in the course of culture, add microbially-decomposed substrate-effective mycin or effective mymidine solution, It can be added at the beginning of the growth of the bacteria, or it can be added after the growth of the bacteria for a period of time. The concentration of the effective mycotoxin or effective mymidine in the substrate is 1.0% ~ 50.0%, the flow acceleration can be adjusted according to the concentration of effective mycin or effective mymidine, the culture time is 100 1! ~ 180 h is better, of which about 160 h is the best. The ifc external reaction can be carried out under static conditions, but it is better to carry out under stirring, aeration, and shaking conditions. During the fermentation process, the pH value is changed with inorganic The acid or organic acid and alkali are adjusted and controlled to obtain a fermentation product.

[3] The above-prepared culture medium is sterilized, inserted into the cultured strain CCTCC No. M 204024, inoculated with a slant or seed solution, and cultured, and the culture conditions: Generally, the temperature is selected at 20 ° C ~ 4 (TC It is better to use 28 ° C ~ 35 ° C, the initial pH is 6.0 ~ 8.0, and it is better to be close to neutral; the IJ logarithmic growth phase of the cultured cells is centrifuged to obtain the cells; 1 added to each of the cells obtained In the substrate effective mycin or substrate effective mymidine solution, the substrate is used to decompose the substrate effective mycin or effective mymidine. The reaction time is preferably from lh to 100h, of which about 70h is the best. In addition, the reaction can be performed under standing, but it is better to perform under stirring, ventilation, and shaking conditions; during the fermentation process, the pH value changes Use of inorganic or organic acids, bases Similar control, to obtain fermentation products.

 (4) The above-prepared culture medium is sterilized, inserted into the cultured strain CCTCC No. M 204024, inoculated with bevel or seed solution, and cultured, and the culture conditions: Generally, the temperature is selected at 20 ° C ~ 40 ° C It is better to use 28 ° C ~ 35 ° C, the initial pH is 6.0 ~ 8.0, and it is better to be close to neutral; the bacterial cells are cultured to the logarithmic growth phase, and centrifuged to obtain bacterial cells, and then the bacterial cells are broken and extracted The lyase is extracted; the extracted lyase is added to the solution of substrate effective mycin or substrate effective mydiimine, and the substrate is used to decompose the substrate effectivemycin or effective mydiiamine, effective mycin or effective mydii The concentration of the amine solution is 1.0% ~ 20.0%, and the reaction time of the enzyme is preferably 1h ~ 100hi. Among them, about 10h is the best. In addition, the reaction can be performed under standing, but under stirring, ventilation, and shaking conditions. In the fermentation process, the change in pH value is adjusted and controlled with inorganic or organic acids and alkalis to obtain a fermentation product.

 Isolation and purification of the decomposed fermentation broth containing effective mycenylamine and effective mycelamine are as follows: 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, layering, etc. In addition, effective mycenylamine and effective myramine are alkaline substances that are easily soluble in water and difficult to dissolve in common solvents. For this reason, separation and purification methods of water-soluble alkaline substances can be used, such as ion exchange resins, activated carbon, and porous polymers. Substances, dextran gels, ion exchangers, etc. are subjected to adsorption and desorption or chromatography.

 The complete extraction process can be divided into three stages: pretreatment, intermediate purification and purification: (1) The pretreatment stage, the goal of this stage is to minimize the number of operation steps and design as simple as possible, from complex materials The target product was separated from the solution, solid particles were removed, and concentrated. (2) In the intermediate purification stage, generally: ^ using non-specific and low-resolution purification technology, followed by high-resolution chromatography and other technologies ^: mainly remove impurities with similar physical and chemical properties and physical and chemical 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 and combined with f drying to obtain the required purity and a certain degree. Shaped products.

The thin-layer chromatography and gas-phase analysis were used to analyze the effective mycolylamine and effective myramine produced by fermentation. The operation process and experimental conditions were based on the literature μ ^^ / ^^, 1984, 37: 1301 ~ 1305] and patents [ Ep0063456] method, the results obtained are completely consistent with them. It can be seen that the use of the microbial strain of the present invention iStenotrophomonas maltropMia) CCTCC No. M 204024 can cleave potent mycin and potent mycotylamine into potent myramine and potent myramine.

 The microbial strain Stenotrophomo maltrophilia CCTCC No. M 204024 of the present invention can not only grow on a culture medium with an effective mycotoxin as the sole carbon source, but also has the ability to decompose the effective mycin Strong; Use CCTCC No. M 204024 to produce effective mycenylamine and effective mycelylamine. Under the best process conditions, the effective mycotoxin decomposition rate reaches 70% ~ 90%. The conversion rate is 40% ~ 80% (that is, 1 mol of effective mycin A can be converted to 0.40-0.80 mol of effective mycenenamine). 35% ~ 75%, at the same time effective mycol is obtained; the microbial strain of the present invention iStenotrophomonas maltrophilia) CCTCC No. M 204024 is suitable for the production of effective myramide and effective myramine. detailed description

 The noodle embodiment is intended to illustrate rather than limit the scope of the invention.

 Example 1:

： Media formula (weight / volume percentage, the same below) = effective mycin: 1.0%, (N) ₂ S0 _4: 1.0%, KC1: 0.5%, Na ₂ HP0 ₄ · 12Η ₂ 0 ·· 1.0%, " aH ₂ P0 ₄ · 2Η ₂ 0: 0.1%, MgS0 ₄ : 0.01%, prepared with tap water, and adjusted the pH to 6.0 with HC1 solution.

 Take 100mL of the above medium, aliquot it in two 250mL Erlenmeyer flasks, and sterilize. Introduce the slant strain CCTCC No. M 204024, culture the bacteria, rotate the shaker at 150r / min, and cultivate at 28 ° C for 72 hours as seed liquid for future use.

 Take 2L of the above-mentioned culture medium, divide it into 20 50-mL shake flasks on average, and sterilize. The seed solution was introduced, and the inoculation amount was 2% (v / v). The culture was performed at a temperature of 20 ° C, a cultivation time of 160 h, and a shaker speed of 200 r / min.

Collect 1.8L of the above fermentation broth, centrifuge to remove the bacteria, and adsorb the supernatant on the column (1L macroporous weakly acidic resin D113, NH ₄ +), wash with distilled water, and then elute with 0.5mol / L ammonia water. The solution was concentrated under reduced pressure, and the concentrated solution was subjected to column chromatography (500 mL strong base chromatography resin Dowex 1 X 2, OH), eluting with distilled ice, and the eluate was collected in stages. A portion of the effective myclopamine flowing out afterwards was analyzed by thin-layer chromatography and gas phase analysis method, and then subjected to vacuum cold drying to obtain 0.31 g of effective myramine and 0.74 g of effective myramide. Example 2:

Validamycin media _{formulations: 17.0%, (NH4) 2} S0 4: 8.0%, KC1: 0.5%, Na 2 HP0 4 - 12H 2 0: 1.0%, NaH 2 P0 4 · 2H 2 0: 0.1%, MgS0 ₄ : 0.01%, prepared with tap water, and adjusted to pH 7.0 with NaOH solution.

 Take 500mL culture medium, aliquot into 5 500mL Erlenmeyer flasks, and sterilize. The slant strain CCTCC No. M 204024 was introduced and cultured. The culture temperature was 28 ° C and the culture time was 150 h. The reaction was carried out under stirring, ventilation and shaking conditions, and the speed of the shaker was 200 r / min.

 400 mL of the above fermentation broth was collected, and separated and purified. The steps were the same as those in Example 1 to obtain 0.56 g of effective myramine and 0.85 g of effective myramine. Example 3:

Fermentation medium formula effective mycin: 8.0%, (NH ₄ ) ₂ S0 ₄ : 6%, KC1: 0.1%, Na ₂ HP0 ₄ · 12H ₂ 0: 1.0%, Na¾P0 ₄ · 2¾0: 0.16%, MgS0 ₄ : 0.02%, prepared with tap water, and adjusted to pH 7.5 with NaOH solution.

Formulation validamycin seed _{medium: 1.0%, (NH 4)} 2 S0 4: 1%, KC1: 0.5%, Na 2 HP0 4 - 12¾0: 1.0%, NaH 2 P0 4 · 2H 2 0: 0.1%, MgS0 ₄ : 0.5%, prepared with tap water, and adjusted to pH 7.5 with NaOH solution.

 Take 500mL seed medium, aliquot into 5 500mL Erlenmeyer flasks, and sterilize. Introduce the slant strain CCTCC No. M 204024, culture the bacterial cells, shake at 200r / min in a shaker, and culture in a shaker at 30 ° C for 72 hours as the seed solution for future use.

 Take 8L of fermentation medium, aliquot it into 80 500mL shake flasks, and sterilize. The seed solution was introduced, and the inoculation amount was 5% (v / v). The culture was performed at a temperature of 30 ° C and a cultivation time of 180 h. The reaction was performed under stirring, ventilation, and shaking conditions, and the speed of the shaker was 200 r / min.

 7.5 L of the above fermentation broth was collected, and separated and purified, and the steps were the same as those in Example 1 to obtain 3.19 g of effective myramine and 7.77 g of effective myramine. Example 4:

Validamycin media _{formulations: 2.0%, (NH 4)} 2 S0 4: 1.0%, KC1: 0.5%, Na 2 HP0 4 - 12¾0: 2.0%, NaH 2 P0 4 · 2H 2 0: 0.1%, MgS0 4 : 0.1%, prepared with tap water, and adjusted to pH 7.0 with NaOH solution. Take 500mL culture medium, aliquot it into 5 500mL Erlenmeyer flasks, and sterilize. Introduce the slant strain CCTCC No. M 204024, culture the bacteria, rotate the shaker at 150 r / min, and culture in a shaker at 28 ° C for 72 hours as the seed solution for future use.

 Take 8L of medium and aliquot it into 80 500mL shake flasks and sterilize. The seed solution was introduced, the inoculation amount was 5% (v / v), and the culture was performed at a cultivation temperature of 28 ° C and a cultivation time of 100 h. The reaction was performed under stirring, ventilation, and shaking conditions, and the shaking speed was 200 r / min.

 7.5 liters of the above-mentioned fermentation broth was collected, and separated and purified. The steps were the same as in Example 1 to obtain 2.11 g of effective myramine and 5.63 g of effective myramine. Example 5:

Effective formula of culture medium: 1.5%, (N) ₂ S0 _4: 0.75%, KC1: 0.5%, Na ₂ HP0 ₄ · 12H ₂ 0: 2.0%, NaH ₂ P0 ₄ · 2H ₂ 0: 1.0%, MgS0 ₄ : 0.01%, formulated with tap water, natural pH.

 Take 200mL of the above medium, aliquot it into 4 250mL Erlenmeyer flasks, and sterilize. Introduce the slant strain CCTCC No. M 204024, culture the bacteria, rotate the shaker at 150r / min, and culture in a shaker at 28 ° C for 72 hours as the seed solution, and reserve it.

 5L of the above-mentioned culture medium was added to a 10L fermentation tank, sterilized, and 200 mL of seed solution was inserted into the fermenter.

 After inoculation, start to add 1L of 10% substrate effective mycin solution with a flow acceleration of 0.2mL / min; incubation temperature 30 ° C, incubation time is about 160 h, aeration 2.5L / min, stirring speed 200r / min.

 6L of the above fermentation broth was collected, and separated and purified, and the steps were the same as those in Example 1 to obtain 3.67 g of effective myramine and 6.53 g of effective myramine. Example 6:

Validamycin media _{formulations: 1.0%, (NH 4)} 2 S0 4: 0.7%, KC1: 0.5%, Na 2 HP0 4 - 12H 2 0: 0.1%, NaH 2 P0 4 - 2H 2 0: 4.0%, MgS0 ₄ : 0.01%, formulated with tap water, natural pH.

Take 500mL of the above medium, aliquot it into 5 500mL Erlenmeyer flasks, and sterilize. Access to the slant strain CCTCC No. M 204024, culture the bacteria, shake at 150r / min, shake at 28 ° C The bed was cultured for 72 hours as a seed solution and used as a spare.

 5L of the above-mentioned culture medium was added to a 10L fermentation tank, sterilized, and 200 mL of a seed solution was inserted to perform fermentation.

 When the growth of the bacteria reached the logarithmic phase, 3 L of a substrate effectivemycin solution with a concentration of 2% was added, and the flow acceleration was 0.5 mL / min; the culture temperature was 35 ° C, the culture time was about 180 h, and the ventilation volume was 2.5 L / min, stirring speed is 200r / min.

 8L of the above-mentioned fermentation broth was collected, and separated and purified. The steps were the same as in Example 1 to obtain 3.08 g of effective myramine and 5.82 g of effective myramine. Example 7:

Effective formula of culture medium: 1.0%, (NH ₄ ) ₂ S0 ₄ : 0.7%, KC1: 0.5%, Na ₂ HP0 ₄ · 12H ₂ 0: 6.0%, NaH ₂ P0 ₄ · 2H ₂ 0: 0.1%, MgS0 ₄ : 0.7%, formulated with tap water, natural pH.

 Take 500mL culture medium, aliquot into 5 500mL Erlenmeyer flasks, and sterilize. The slanted strain CCTCC No. M 204024 was inserted, and the bacterial cells were cultured at a shaking speed of 150 r / min. The seeds were cultured in a shaker at 28 ° C for 72 hours as a seed solution and used as a spare.

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

 When the growth of the bacteria reached the logarithmic phase, 100 mL of an effective mymidine solution with a concentration of 50% was added, and the flow acceleration was 0.05 mL / min; Culture conditions: The temperature was 30 ° C, the culture time was 120 h, and the ventilation volume was 3.5L. / min, stirring speed is 200r / min.

 6L of the above-mentioned fermentation broth was collected, and separated and purified. The procedures were the same as those in Example 1 to obtain 3.26 g of effective myramine and 7.01 g of effective myramine. Example 8:

Effective formula of culture medium: 2.0%, (H ₄ ) ₂ S0 ₄ : 1.2%, KC1: 4.0%, Na ₂ HP0 ₄ · 12¾0: 4.0%, NaH ₂ P0 ₄ · 2H ₂ 0: 0.1%, MgS0 ₄ : 0.01%, prepared with tap water, and adjusted to pH 6.5 with HC1.

Take 1000mL culture medium, aliquot it into 10 500mL Erlenmeyer flasks, and sterilize. Introduce the slant strain CCCCC No. M 204024, culture the bacteria to the logarithmic growth phase, and then perform centrifugation. The bacterial cells were obtained, and these cells were added to 500 mL of a 5% substrate effective mycin solution, and the effective cells were decomposed. The reaction conditions were: temperature 30 ° C, initial pH 6.5, and incubation time 100 h. Left and right, the speed of the shaker is 150r / min.

 480 mL of the above reaction solution was collected, and separated and purified. The steps were the same as those in Example 1 to obtain 0.45 g of effective myramine and 1.21 g of effective myramine. Example 9:

Validamycin media _{formulations: 2.0%, (NH 4)} 2 S0 4: 1.2%, KC1: 0.5%, Na 2 HP0 4 · 12H 2 0: 1.0%, NaH 2 P0 4 - 2H 2 0: 0.1%, MgS0 ₄ : 0.01%, prepared with tap water, and adjusted to pH 7.5 with NaOH solution.

 Take 1000mL culture medium, aliquot it into 10 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCTCC No. M 204024, culture the bacteria to the logarithmic growth phase, and then centrifuge to obtain the bacteria. The cells were added to 5OmL of a 15% substrate effective mold subunit amine solution, and the cells were used to decompose the effective mold subunit amines. Culture conditions: The temperature was 32 ° C, the initial pH was 7.5, and the culture time was about 40 h. Shaker speed is 200r / min.

 580 mL of the above reaction solution was collected, and separated and purified. The steps were the same as those in Example 1 to obtain 1.03 g of effective myramine and 2.77 g of effective myramine. Example 10:

Validamycin media _{formulations: 2.0%, (NH 4)} 2 S0 4: 1.2%, KC1: 0.5%, Na 2 HP0 4 - 12H 2 0: 3.0%, NaH 2 P0 4 · 2H 2 0: 0.1%, MgS0 ₄ : 0.01%, prepared with tap water, and adjusted to pH 7.5 with NaOH solution.

 Take 1000mL culture medium, aliquot it into 10 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCTCC No. M 204024, and maintain the bacteria to the logarithmic growth phase, and then centrifuge to obtain the bacteria. Add the bacterial cells to 500 mL of a substrate effective 3% amine solution, and use the bacterial cells to decompose the effective fulminine; Culture conditions: temperature 34 ° C, initial pH 7.5, culture time 20 hours , Shaker speed is 150r / min.

580 mL of the above reaction solution was collected, and separated and purified, and the steps were the same as those in Example 1 to obtain 0.53 g of effective myramine and 1.06 g of effective myramine. Example 11:

Validamycin media _{formulations: 2.0%, (NH 4)} 2 S0 4: 1.2%, KC1: 0.5%, Na 2 HP0 4 - 12H 2 0: 0.2%, NaH 2 P0 4 · 2H 2 0: 1.5%, MgS0 ₄ : 0.01%, adjust pH to 6.0 with HC1 solution.

 Take 2000mL of the above medium, aliquot it into 20 500mL triangle flasks, sterilize, insert the slant strain CCTCC No. M 204024, culture the bacteria to the logarithmic growth phase, and then centrifuge to obtain the bacteria. The ultrasonic wave was used to break the bacterial cells, followed by centrifugation, salting out, dialysis, and chromatography to extract the lyase, and the above-mentioned extracted lyase was added to 200 mL of a substrate effectivemycin solution with a concentration of 8%. Mycetin produces potent myramine and potent myramine. Culture conditions: The temperature is 25 ° C, the initial pH is 6.0, and the culture time is about 10 h. The reaction can be performed under standing.

 200 mL of the above reaction solution was collected, and separated and purified. The steps were the same as those in Example 1 to obtain 0.15 g of effective myramine and 0.34 g of effective myramine. Example 12:

Validamycin media _{formulations: 2.0%, (NH 4)} 2 S0 4: 1.2%, KC1: 0.5%, Na 2 HP0 4 - 12H 2 0: 1.5%, NaH 2 P0 4 · 2Η 2 0: 0.8%, MgS0 ₄ : 0.01%, adjust pH to 8.0 with NaOH solution.

 Take 2000mL culture medium, aliquot it into 20 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCCCC No. M 204024, culture the bacteria to the logarithmic growth phase, and then centrifuge to obtain the bacteria, using ultrasound The bacterial cells are broken, and then centrifuged, salted out, dialyzed, and chromatographed to extract the lyase, and the extracted lyase is added to 100 mL of a 20% substrate effectivemycin solution, which is effectively decomposed by an enzyme reaction. Mycin. Reaction conditions: The temperature is 32 ° C, the initial pH is 8.0, and the incubation time is about 80 h. The reaction can be performed under standing.

 100 mL of the above reaction solution was collected, and separated and purified. The procedures were the same as those in Example 1 to obtain 0.21 g of effective myramine and 0.39 g of effective myramine. Example 13:

Effective formula of culture medium: 2.0%, (NH ₄ ) ₂ S0 ₄ : 1.2%, KC1: 0.5%, Na ₂ HP0 ₄ · 12Η ₂ 0: 1.0%, NaH ₂ P0 ₄ · 2Η ₂ 0: 0.1%, MgS0 ₄ : 0.01%, pH was adjusted to 7.8 with NaOH. Take 2000mL culture medium, aliquot it into 20 500mL Erlenmeyer flasks, sterilize, insert the slant strain CCCCC No. M 204024, culture the bacteria to the logarithmic growth phase, and then centrifuge to obtain the bacteria, using ultrasound The bacterial cells were crushed, and then centrifuged, salted out, dialysis, and chromatography were performed to extract the lyase. The lyase extracted above was added to 100 mL of a 10% substrate effective mold subunit amine solution, and the enzyme reaction was used. Fragmentation of effective myco subunit amines produces effective myco amines and effective mycenyl amines. Reaction conditions: the temperature is 30 ° C, the initial pH is 7.0, the culture time is about 30 h, and the rotation speed of the shaker is 100 r / min.

 100 mL of the above reaction solution was collected, and separated and purified. The steps were the same as those in Example 1 to obtain 0.10 g of effective myramine and 0.31 g of effective myramine. Example 14:

Effective formula of culture medium: 2.0%, (H ₄ ) ₂ S0 ₄ : 1.2%, KC1: 0.5%, Na ₂ HP0 ₄ · 12Η ₂ 0: 1.0%, NaH ₂ P0 ₄ · 2Η ₂ 0: 0.1%, MgS0 ₄ : 0.01%, pH was adjusted to 7.0 with NaOH.

 Take 2000mL of the above medium, aliquot it into 20 500mL triangle flasks, sterilize, insert the slant strain CCTCC No. M 204024, culture the bacteria to the logarithmic growth phase, and then centrifuge to obtain the bacteria. The bacterial cells were broken by ultrasound, and then centrifuged, salted out, dialyzed, and chromatographed to extract the lysing enzyme. The above-mentioned extracted lysing enzyme was added to 100 mL of a 18% substrate effective mold subunit amine solution. The enzymatic reaction breaks down the effective mold subunit amine. Reaction conditions: The temperature is 30 ° C, the initial pH is 7.0, the incubation time is about 50 h, and the speed of the shaker is 100 r / min.

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

Claims

Claim

A method for preparing an effective mycolylamine and an effective mycobacterium, characterized in that the microorganisms are Stomotrophomonas oligotrophomonas, CCTCC No. M 204024, Maltotrophophilic The bacterium and the medium containing a carbon source, a nitrogen source, and an inorganic salt are fermented with a substrate of effective antibiotic mycotoxin, and the effective fermentation of the decomposed fermentation broth is separated and purified, and then purified.

The preparation method according to claim 1, characterized in that the composition of the weight / volume of the culture medium is: 0.5% to 20.0% of effective mycin, 0.5% to 10.0% of (NH4) ₂ SO ₄ and KC1 0.5 % ~ 5.0%, Na ₂ HP0 ₄ · 12H ₂ 0 0.1% ~ 10.0%, NaH ₂ P0 ₄ · 2Η ₂ 0 0.1% ~ 5 · 0%, MgS0 ₄ 0.01% ~ 1.0%, use tab water to match lj, adjust pH value is 6.0 ~ 8.0.

 The preparation method according to claim 1, characterized in that the steps of separating and purifying the fermentation broth are: centrifuging the reaction liquid, removing bacterial cells, adsorbing the supernatant on a column, washing with distilled water, and then using 0.5 mol / L ammonia water was eluted, concentrated under reduced pressure, and then the concentrated solution was subjected to column chromatography, eluting with distilled water, and the eluate was collected in stages, and analyzed by thin layer chromatography. The amine and the effective mycolylamine flowing out afterwards are respectively vacuum freeze-dried to obtain the effective mycolylamine and the effective mycolylamine.

 The preparation method according to claim 1 or 2, characterized in that fermentation is performed in a fermentation medium after accessing CCTCC No. M 204024, and a concentration of effective mycin in the medium is 0.5% to 20.0 ° /. , Culture conditions: Temperature 20 ° C ~ 40 ° C, initial pH is 6.0 · 8.0, and culture time is 100 1! ~ 180 h. Fermentation was performed under standing.

 The preparation method according to claim 4, characterized in that the concentration of effective mycin 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 For 150 h, fermentation was performed under stirring, aeration, and shaking conditions.

 The preparation method according to claim 1, characterized in that after inserting the strain CCTCC No. M 204024 into the culture medium, the substrate effective mycotoxin is added during the culture process, which starts when the bacteria body just starts to grow. Feeding, culture conditions: the temperature is 20 ° C ~ 40 ° C, the initial pH is 6.0 ~ 8.0, the culture time is 100 h ~ 180 h, and the fermentation is performed under standing.

The preparation method according to claim 6, characterized in that, after the logarithmic growth period of the bacterial cells, the substrate is added with the effective mycin, and the culture conditions are: 28 ° C ~ 35 ° C, and the initial pH is 6.5 ~ 7.5 When training After 150 h, fermentation was performed under stirring, aeration, and shaking conditions.

 The preparation method according to claim 1, characterized in that after cultivating the strain CCTCC No. M 204024 in the medium and culturing the bacterial cells to the logarithmic growth phase, and then centrifuging to obtain the bacterial cells, the bacterial cells are obtained. The substrate is added to a substrate effective omycin solution of ¾1, and the substrate is used to decompose the substrate effective omycin. The culture conditions are: the temperature is 20 ° C ~ 40 ° C, the initial pH is 6.0 ~ 8.0, and the culture time is 11! ~ 100 h. Fermentation was performed at rest.

 The preparation method according to claim 8, characterized in that the bacterial cells are added to the substrate effective mycin solution, and the culture conditions are: temperature 28 ° C ~ 35 ° C, initial pH is 6.5 ~ 7.5, culture time is 70 h. Fermentation is performed under stirring, aeration, and shaking conditions.

 10. The preparation method according to claim 1, characterized in that the bacterial cells are cultured to logarithmic growth phase after being introduced into the culture medium CCTCC No. M 204024, and then centrifuged to obtain the bacterial cells, and then ultrasonic waves are used. Method 4: Each bacterial cell is broken, the lyase is extracted, and the lyase is added to the effective mycin solution. The culture conditions are: temperature 20 ° C ~ 40 ° C, initial pH is 6.0 ~ 8.0, and the culture time is 11! ~ 100h, performed under standing.

 11. The preparation method according to claim 10, characterized in that the lyase is added to the substrate effective mycin solution, and the culture conditions are: temperature 28 ° C ~ 35 ° C, initial pH is 6.5 ~ 7.5, and culture time is 10 h, under stirring, ventilation and shaking conditions.

 12. The preparation method according to claim 1 or 6 to 11, characterized in that the substrate can be an effective mold subunit amine.