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

Microbe method for producing valienamine and validamine Download PDF

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Publication number
WO2005098014A1
WO2005098014A1 PCT/CN2005/000267 CN2005000267W WO2005098014A1 WO 2005098014 A1 WO2005098014 A1 WO 2005098014A1 CN 2005000267 W CN2005000267 W CN 2005000267W WO 2005098014 A1 WO2005098014 A1 WO 2005098014A1
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effective
culture
fermentation
substrate
preparation
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PCT/CN2005/000267
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French (fr)
Chinese (zh)
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Yuguo Zheng
Xiaolong Chen
Yaping Xue
Yuanshan Wang
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Zhejiang University Of Technology
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00Preparation of nitrogen-containing organic compounds
    • C12P13/001Amines; Imines

Definitions

  • 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.
  • potent mycin also known as Jinggangmycin
  • potent mycotylamine also known as pinamycin or pinam hydroxylamine
  • 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.
  • Avobacterium saccharophiluni IF013948 Public License: JP57-54593
  • 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.
  • 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 ⁇ -D-glucose and other structural components.
  • the effective mycoglycoside bond is hydrolyzed to be decomposed into the effective mymidine amine.
  • effective mymidine amine There are two kinds of effective mymidine amine.
  • 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).
  • 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 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.
  • Effective mycotylamine also known as Jinggangmycin, has the molecular formula C 7 H 15 N0 4 , one primary amino group (-N3 ⁇ 4), 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, [o3 ⁇ 4 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.
  • 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.
  • 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)]
  • PseMifowcwas denitrificans are Aegeroides sp Aerobic bacteria, capable of denitrification.
  • Agrobacterium short bacillus, large zj, 1.5 ⁇ 0 ⁇ 3 ⁇ m X 0.6 ⁇ 1 ⁇ 0 ⁇ m, 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 pastesperm, 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; top1 ⁇ 2): 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 ⁇ m X 1.0-3.0 ⁇ m. 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.
  • 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.
  • 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 ⁇ m x 1.3 to 1.4 ⁇ m. 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 3 ⁇ 48, 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.
  • 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
  • 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.
  • 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 ⁇ 12 ⁇ 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:
  • 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.
  • the reaction can be carried out under standing , But under the conditions of stirring, ventilation, shaking is better.
  • the pH value is adjusted and controlled with inorganic or organic acids and alkalis.
  • 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.
  • the effective mylidinylamine is decomposed to generate valienamine and glutamine is validamine o
  • 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.
  • the pH value is changed with inorganic
  • the acid or organic acid and alkali are adjusted and controlled to obtain a fermentation product.
  • 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.
  • 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.
  • 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.
  • the reaction can be performed under standing, but under stirring, ventilation, and shaking conditions.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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 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).
  • the microbial strain of the present invention iStenotrophomonas maltrophilia) CCTCC No. M 204024 is suitable for the production of effective myramide and effective myramine.
  • the noodle embodiment is intended to illustrate rather than limit the scope of the invention.
  • 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.
  • Example 3 400 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 3 400 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.
  • 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%, Na3 ⁇ 4P0 4 ⁇ 23 ⁇ 40: 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 - 123 ⁇ 40: 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.
  • Example 4 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 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.
  • Validamycin media formulations 2.0%, (NH 4) 2 S0 4: 1.0%, KC1: 0.5%, Na 2 HP0 4 - 123 ⁇ 40: 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.
  • Example 5 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.
  • Example 6 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 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.
  • 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.
  • Example 7 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.
  • Example 8 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 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 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 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 6L of the above-mentioned fermentation broth was collected, and separated and purified. The procedures were
  • Effective formula of culture medium 2.0%, (H 4 ) 2 S0 4 : 1.2%, KC1: 4.0%, Na 2 HP0 4 ⁇ 123 ⁇ 40: 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.
  • Example 9 480 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.
  • Example 10 580 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 10 580 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.
  • Example 11 580 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 11 580 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.
  • 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.
  • Example 12 200 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 12 200 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 12 200 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 12 200 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.
  • 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 ⁇ 2 ⁇ 2 0: 0.8%, MgS0 4 : 0.01%, adjust pH to 8.0 with NaOH solution.
  • Example 13 100 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 13 100 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 13 100 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 13 100 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.
  • Effective formula of culture medium 2.0%, (NH 4 ) 2 S0 4 : 1.2%, KC1: 0.5%, Na 2 HP0 4 ⁇ 12 ⁇ 2 0: 1.0%, NaH 2 P0 4 ⁇ 2 ⁇ 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.
  • Example 14 100 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 14 100 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 14 100 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 14 100 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.
  • Effective formula of culture medium 2.0%, (H 4 ) 2 S0 4 : 1.2%, KC1: 0.5%, Na 2 HP0 4 ⁇ 12 ⁇ 2 0: 1.0%, NaH 2 P0 4 ⁇ 2 ⁇ 2 0: 0.1%, MgS0 4 : 0.01%, pH was adjusted to 7.0 with NaOH.

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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

有效霉烯胺和有效霉胺的微生物制备方法 技术领域  TECHNICAL FIELD
本发明涉及从土壤中筛选到的新的嗜麦芽寡养单胞菌 The present invention relates to a novel oligotrophomonas maltophilia screened from soil
{Stenotrophomonas maltrophilia), 还涉及利用该新菌株分解有效霉素(又称 井冈霉素)或有效霉亚基胺(又称井闪霉亚基胺或井闪羟胺)生产有效霉烯 胺和有效霉胺的方法。 背景技术 {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
有关有效霉烯胺和有效霉胺的制造方法的研究已经有 30多年的历史, 日本人龟田等人曾经采用过脱硝假单孢菌的菌体降解有效霉素 A或有效霉 亚基胺 A, 再进行分离得到有效霉烯胺和有效霉胺, 可是不能在仅以有效霉 素类物质为唯一碳源的培养基上生长; 而且对有效霉素的分解能力很弱, 不 适用于有效霉烯胺和有效霉胺的大量生产。  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.
日本人龟田等在土壤中分离到一株能将有效霉素分解为有效霉烯胺和 有效霉胺的菌株嗜糖黄杆菌 avobacterium saccharophiluni) IF013948 (公 开特许: JP57-54593 ) 0 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
日本人龟田等采用噬细胞菌属产酶微生物 ί Cytophaga heparina ) IFO12017 (ATCC13125 ) 和 IFO14087株降解有效霉素或有效霉亚基胺生产 有效霉烯胺和有效霉胺 [特许公报(B2) : 平 2-26957]。  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].
日本人龟田等发现了土壤菌属 Agrobacteriund 或者气单胞菌属 {Aeromonas^ 的微生物以及它们的变异株能有效分解有效霉素或有效霉亚 基胺生产有效霉烯胺和有效霉胺。 比如土壤菌属 Ugrobacterium 微生物, 有 Agrobacterum Radiobacter IFO 12664 (ATCC 4718)株, IFO 13258 (ATCC 13332)株, IFO 13259 (ATCC 13333)株, IFO 13532 (ATCC 19358)株, IFO 13533 (ATCC 25235)株, 及 Agrobacterium tumefaciens IFO 3058株等; 气单胞菌 属 {Aeromonas )微生物, Aeromonas hydrophila subsp. Anaerogenes IFO 13282,同亚种 subsp.hydrophila IFO 13286, subsp .proteolytica IFO 13287, aeromonas punctata subsp. cavice IFO 13288,以及 aeromonas salmonicida subsp.salmonicida lFO 12659等 [特许公报 (B2): 平 6-69380]。 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].
本发明所用到的原料是一种广泛应用的农用抗生素——有效霉素, 即井 冈霉素, 它是一种高效、 安全的农用抗生素, 不污染环境, 对人畜无害, 目 前已经成为我国使用面积较广、 亩用成本最低的安全、 无公害农药, 是农药 工业的一个重要品种。 有效霉素是氨基糖苷类农用抗生素, 主要组分有 A、 B、 C、 D、 E、 F 等, 从有效霉素的结构中可以发现, 有效霉素是由有效霉 烯胺、 有效霉胺和 β -D-葡萄糖等结构组成。  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.
有效霉素糖苷键经过水解, 分解为有效霉亚基胺, 有效霉亚基胺有八、 B 两种, 有效霉亚基胺再经过分解, 生成有效霉烯胺 (valienamine)和有效霉 胺 (validamine)。  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).
本发明涉及的有效霉烯胺, 英文名为 valienamine 其结构式如图 1 (Chem. Rev. 2003, 103:1955-1977)。  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 imgf000003_0001
Figure imgf000003_0001
图 1 有效霉烯胺 (valienamine) 的化学结构  Figure 1 Chemical structure of effective valienamine
有效霉烯胺, 又称井冈霉烯胺, (1S, 2S, 3S, 4R)-1-氨基 -5- (羟甲基) 环己 -5-烯 -2, 3, 4-三元醇; 分子式为 C7H13N04, 重要的基团有: 一个伯氨 基(-N¾) , 一个碳碳双键 (C=C) , 一个羟甲基 (-CH2OH) , 三个羟基 (-OH) 。 其盐酸盐(C7H13NO4 · HC1) 的 [ 为 +68.6° (1N-HC1), 五乙酸 盐 (C17H23N09)的熔点为 95°C, [« 为 +30.2° (CHC13), 遇茚三酮显色反应。 由于有效霉烯胺含有一个伯氨基 (_NH2) , 在水溶液中发生一级解离。 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.
本发明涉及的有效霉胺,英文名为 validamine,其结构式如图 2(Chem. 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) 。 Rev. 2003, 103: 1955-1977).
Figure imgf000003_0002
Figure imgf000003_0002
有效霉胺 (validamine) 的化学结构 有效霉胺,又称井冈霉胺,分子式为 C7H15N04,一个伯氨基(-N¾), 一个羟甲基 (-CH2OH), 三个羟基 (-OH), 其盐酸盐 (C7H13N04 · HC1)的 [« 为 +57.4。 , 熔点为 229°C-232°C, ( 1N-HC1)的 [o¾3为 +60.6° , 遇茚三酮显色 反应。 有效霉胺也含有一个伯氨基(-NH2) , 在水溶液中发生一级解离。 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.
有效霉烯胺、 有效霉胺等环醇类物质是糖苷酶抑制剂的核心结构, 同 时也是一种较强的糖苷酶抑制剂。  Cycloalcohols, such as effective mycolylamine and effective mycoamine, are the core structure of glycosidase inhibitors, and they are also strong glycosidase inhibitors.
糖苷酶是糖蛋白生物合成中寡糖链的修剪酶, 对糖蛋白中的寡糖链的 形成极为重要。糖链的组成与结构是糖蛋白特异生物功能的识别部位, 影响 蛋白质的折叠、 溶解度、 修饰、 抗原性及生物活性等。 糖苷酶活性对糖蛋白 生物合成起着十分关键的作用, 研究糖苷酶抑制剂, 具有广泛的应用前景。  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.
由于有效霉烯胺和有效霉胺的生物活性, 人们对它的兴趣与日剧增。 这些年, 是活跃的研究领域之一, 尤其在日本、 韩国和德国, 而在我国, 这 方面的研究几乎是空白。 发明内容  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
本发明的任务是有目的的从土壤中筛选到将有效霉素或有效霉亚基胺 分解为有效霉烯胺和有效霉胺的新的微生物菌株;其次是提供一种利用该新 微生物发酵分解或该新微生物的细胞或酶分解抗生素有效霉素、有效霉亚基 胺生产有效霉烯胺和有效霉胺的方法。  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.
本发明提供的微生物是寡氧单胞菌属 C ^^c^o w^),为嗜麦芽寡养
Figure imgf000004_0001
maltrophilia), 该菌株己于 2004年 3月 15日保藏 于中国典型培养物保藏中心, 简称 CCTCC , 保藏编号为 CCTCC No. M 204024。
The microorganism provided by the present invention is Oligomonas spp. (C ^^ c ^ ow ^), which is maltotrophophilic
Figure imgf000004_0001
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.
根据文献及专利查阅结果, 己^ ¾道的能降解有效霉素的专利菌种包括 叚单胞菌属 ^Pseudomonas , 1个中, Pseudomonas denitrificans, 反石肖化叚单 胞菌)、 黄杆菌属 iFlavobacterium, 3个种)、 嗜纤维菌属 i Cytophaga 1个 种, 3 个菌株)、 土壤菌属 (Agrobacterium , 2 个禾中, 其中 Agrohacterium radiobacter ^ 5个菌株, Agrobacterium tumefaciens , 1个禾中)、 气单胞菌属 {Aeromonas, 5个种)。 具体结果见附表 1。 降解有效霉素专利 [特许公报 (B2 ) ]菌种汇总 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)]
Figure imgf000005_0001
各属生理生化特征如下:
Figure imgf000005_0001
The physiological and biochemical characteristics of each genus are as follows:
jg^. |¾¾fj¾ (Pseudomonas): 反石肖化假单胞菌 (PseMifowcwas denitrificans) 为享兰氏阴性无芽孢杆菌, 极生鞭毛运动, 氧化型代谢, 通常氧化酶阳性, 有机化能营养。 好氧菌, 能进行反硝化作用。  jg ^. | ¾¾fj¾ (Pseudomonas): PseMifowcwas denitrificans are Aegeroides sp Aerobic bacteria, capable of denitrification.
± (Agrobacterium):短小杆菌,大 zj、为 1.5〜0·3 μ m X 0.6~1·0 μ m, 单个成对排列, 稀周毛, 革兰氏反应阴性, 不产生芽孢, 菌落凸起, 全缘光 滑,无色素至浅灰黄色。在含糖培养基中产生大量多糖黏液,不固定大气氮, 多数种可利用无机氮,放射土壤杆菌能从 ¥L糖产生 3-酮基乳糖,分解蛋白质 的能力弱或无, 可广泛利用碳水化合物、有机酸盐和氨基酸为碳源, 不能利 用纤维素、 淀粉、 琼脂糖、 半乳糖及其他糖类。 ± (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.
气单胞菌属 era ): 细胞挺直, 具圆端杆状, 常以单极毛运动, 发酵葡萄糖、 果糖、 麦芽糖和海藻糖, 碳水化合物分解成酸, 有的产气, 水 解淀粉和糊精, 水解酪蛋白, 液化明胶, 产生 DNA酶, 精氨酸脱氨酶, 少 数种不运动。革兰氏反应阴性, 有呼吸和发酵两种代谢类型, 氧化酶和过氧 化氢酶阳性, 兼性厌氧菌。  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.
嗜纤维菌属(C3;top½ ): 不形成子实体, 细胞杆状, 往往形成很长的 细胞链, 好氧, 能利用碳水化合物, 代谢过程中需要大量 co2, 能彻底分解 纤维素、 水解几丁质和琼脂等多糖类物质, 可以在界面上滑行。 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.
黄杆菌属 Flavobacterium): 直杆状, 端圆,大小 0.5 μ m X 1.0-3.0 μ m。 细胞内不含 PHB盐, 不形成内生孢子, 革兰氏阴性, 不运动, 无滑动或泳 动, 严格好氧, 在固体培养基上生长, 产生典型的黄色或橙色色素, 有些菌 株不产生色素, 菌落半透明, 圆形, 隆起或微隆起, 光滑且又光泽, 全缘, 接触酶、 氧化酶、 磷酸酶均阳性, 不消化琼脂, 有机化能营养。 在低浓度蛋 白胨培养基中不产气。  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.
综合以上结果,本发明筛选到的新的菌株不属于上述专利菌种的任何一 种。  Based on the above results, the new strains screened by the present invention do not belong to any of the aforementioned patent strains.
该新的菌株特征为:  The new strain is characterized by:
菌落形态: 光滑, 有光泽, 边缘整齐, 白、 灰或淡黄色, 培养 181!〜 36h 的菌落直径 lmm〜2mm左右; 斜面菌苔白色, 粘稠。  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.
细胞形态: 细胞内不形成 PHB颗粒, 不运动, 杆状, 菌体平均大小为: 0.4〜0.5 μ πι Χ 1.3~1.4 μ ηι。 革兰氏阴性, 无芽孢。  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.
生理生化特征: 液化明胶, 卵黄反应阳性, 脂酶 (土温 80) 反应阳性, 氧化酶阴性, 接触酶阳性, 吲哚反应阴性, 甲基红阴性, V-P反应阴性, 产 生¾8, 不氧化分解葡萄糖, 氧化型产酸, 利用丙二酸盐, 利用柠檬酸盐, 不还原硝酸盐, 不能反硝化, 不能利用淀粉, 脲酶反应阳性, 水解酪蛋白, 不能分解乳糖产生 3-酮基乳糖。 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.
需生长因子, 产赖氨酸脱羧酶,水解几丁质; 以天门冬酰胺作碳、氮源。 根据以上细菌学特征, 这个新的菌株被鉴定属于寡氧单胞菌属 ( Stenotrophomomas ) 的菌株, 为嗜麦芽寡养单胞菌 {Stenotrophomonas maltrophilid)。  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).
用于本发明菌种 CCTCC No. M 204024的培养基是一些含有可以被上述 菌株利用营养物质, 液体、 固体均可, 但是大量工业化生产时, 液体培养基 较为合适。 培养基中合理配入上述微生物能利用的碳源、 氮源、 无机盐等。 作为碳源的有: 有效霉素、 葡萄糖、 乳糖、 麦芽糖、 糊精、 淀粉、 甘油、 甘 露醇、 山犁醇、 ^脂类 (豆油、 猪油等); 作为氮源的有 - 肉汁、 酵母膏、 干酵母、大豆粉、 玉米浆、 蛋白胨、尿素、氨盐(硫酸铵、 氯化铵、硝酸铵、 醋酸胺等), 肽类 (二肽、 三肽等); 无机盐类有: Na、 K、 Ca、 Mg、 Fe、 Mn、 Zn、 Co、 NTi等金属盐类和磷酸盐、 醋酸盐等有机酸盐类; 另可加有- 氨基酸类 (如谷氣酸、 天冬氨酸、 赖氨酸、 甘氨酸、 甲硫氨酸等)、 微生素 (VB1、 VB2、 VB 1 2、 Vc VE、 烟酸等)、 核酸类 (如嘌呤、 嘧啶及其衍生物 等)。用无机酸或有机酸、碱类调节培养基的 pH值,还可力 0入适量的消泡剂, 如泡敌等。 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.
可采用培养方式有: 静置培养、 摇动培养、 或通风搅拌培养等, 大量生 产有效霉胺和有效霉烯胺时宜采用深层通风搅拌培养。  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.
本发明的另一个重要特征是利用该新的微生物发酵或该新的微生物细 胞或其酶分解底物有效霉素或有效霉素的中间产物有效霉亚基胺生产有效 霉烯胺和有效霉胺。  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 .
本发明的新的微生物寡氧单胞菌属 iSt otrophomoncis 嗜麦芽寡养单 M Stenotrophornonas mcdtrophilia), CCTCC No. M 204024, 与含碳源、 氮 源、 无机盐的培养基, 底物为抗生素有效霉素, 进行发酵, 然后对分解发酵 液进行分离有效霄胺和有效霉烯胺, 并进行提纯。  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.
本发明的培养基组成为(w/v, %) :有效霉素: 0.5%〜20%, (NH4) 2SO4: 0.5%〜10%, KC1: 0.5%~5.0%, Na2HPO4 ·12Η20: 0.1%~10.0%, NaH2PO4 «2H20: 0.1%〜5.0%, MgSQ4: 0.01%~1.0%, 以自来水配制, pH值 6.0~8.0。 泪本发明的新
Figure imgf000008_0001
maltrophilia) CCTCC No. M 20402)生产效霉烯胺和有效霉胺, 其方法有:
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
Figure imgf000008_0001
maltrophilia) CCTCC No. M 20402) produces menomynamine and effective myramine by the following methods:
1 )上述配制的培养基经过灭菌, 接入经培养后的菌种 CCTCC No. M 204024, 斜面或用种子液接种, 进行培养, 通常选择温度在 20°C〜40°C, 以 28°C~35 °C较好, 初始 pH为 6.0~8.0, 以接近中性较好, 培养时间为 100 h〜 180 h较好,其中以 150 h左右为最好,此外反应可在静置下进行,但在搅拌、 通风、 振荡条件下进行为佳。 在发酵过程中, pH值用无机酸或有机酸、 碱 类调节控制。 该方法中培养基组成中的有效霉素既是微生物利用的碳源, 又 是被微生物分解的底物; 底物有效霉素被分解为有效霉亚基胺, 有效霉亚基 胺有 A、 B两种, 有效霉亚基胺再经过分解, 生成有效霉烯胺 (valienamine) 禾口有欢霉胺 (validamine) o  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 ) 上述配制的培养基经过灭菌, 接入经培养后菌种 CCTCC No. M 204024, 用斜面或种子液接种, 培养菌体, 培养条件: 通常选择温度在 20 °C〜40°C, 以 28°C〜35 °C较好, 初始 pH为 6.0~8.0, 以接近中性较好; 在培 养的 程中流加微生物分解的底物——有效霉素或有效霉亚基胺溶液,可以 在菌体刚开始生长的时候流加, 也可以在菌体生长了一段时间后再进行流 加,流加入培养物中的底物有效霉素或有效霉亚基胺的浓度为 1.0%~50.0%, 流加 速度可以根据有效霉素或有效霉亚基胺的浓度进行调节,培养时间为 100 1!〜 180 h较好, 其中以 160 h左右为最好, ifc外反应可在静置下进行, 但在撳拌、 通风、 振荡条件下进行为佳; 在发酵过程中, pH值的变化用无 机酸或有机酸、 碱类调节控制, 制得发酵产物。  (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 ) 上述配制的培养基经过灭菌, 接入经培养后菌种 CCTCC No. M 204024 , 用斜面或种子液接种, 培养菌体, 培养条件: 通常选择温度在 20 °C〜4(TC, 以 28°C~35 °C较好, 初始 pH为 6.0〜8.0, 以接近中性较好; 培养 菌体 IJ对数生长期, 进行离心分离, 得到菌体; 1每得到的菌体加入底物有效 霉素或底物有效霉亚基胺溶液中,利用菌体分解底物有效霉素或有效霉亚基 胺, ^效霉素或有效霉亚基胺的浓度为 1.0%~20.0%, 反应时间为 lh ~ 100h 较好,其中以 70 h左右为最好,此外反应可在静置下进行,但在搅拌、通风、 振荡条件下进行为佳; 在发酵过程中, pH值的变化用无机酸或有机酸、 碱 类调节控制, 制得发酵产物。 [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) 上述配制的培养基经过灭菌, 接入经培养后菌种 CCTCC No. M 204024, 用斜面或种子液接种, 培养菌体, 培养条件: 通常选择温度在 20 °C〜40°C, 以 28°C〜35°C较好, 初始 pH为 6.0〜8.0, 以接近中性较好; 培养 菌体到对数生长期, 进行离心分离, 得到菌体, 然后将菌体破碎, 提取出裂 解酶; 将提取的裂解酶加入到底物有效霉素或底物有效霉亚基胺溶液中, 利 用酶反应分解底物有效霉素或有效霉亚基胺,有效霉素或有效霉亚基胺溶液 的浓度为 1.0%〜20.0%,酶反应时间为 lh〜 100hi较好,其中以 10 h左右为最 好, 此外反应可在静置下进行, 但在搅拌、 通风、 振荡条件下进行为佳; 在 发酵过程中, pH值的变化用无机酸或有机酸、 碱类调节控制, 制得发酵产 物。  (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.
将含有效霉烯胺和有效霉胺的分解发酵液进行分离、纯化,具体做法是: 从发酵液提取目标产物时, 可采用常规的发酵产物提取法。 如: 过滤、 离心、 沉淀、 结晶、 重结晶、 浓缩、 干燥、 冷冻干燥, 吸附、 离子交换、 层 析等。此外,有效霉烯胺和有效霉胺易溶于水、难溶于一般溶剂的碱性物质, 因面可以采用水溶性碱性物质的分离、 精制方法, 例如离子交换树脂、 活性 炭、 多孔高聚物, 葡聚糖凝胶、 离子交换剂等进行吸附后解吸或层析。  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.
完整的提取过程可分为预处理、 中期纯化和精制三个阶段: (1 )预处理 阶段, 这一阶段的目标是以尽可能少的操作歩骤和尽可能简单的设计, 从复 杂的料液中分离出目标产物、 除去固体颗粒、 浓缩。 (2) 中期纯化阶段, 一 般:^采用非特异性和低分辨率的纯化技术,随后再采用高分辨率的层析法等 技^:主要是除去理化性质和产物理化性质相似的的杂质。 (3 )精制阶段, 这 是最后的加工程序, 目的是进一步除去杂质, 纯化产物, 其方法取决于产品 的应用目的, 最常用的方法是结晶法, 并结合 f燥, 获得所需纯度和一定形 状的产品。  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.
用薄层层析和气相分析的方法对发酵生产的有效霉烯胺和有效霉胺进 行分析, 操作过程和实验条件按文献 μ^^/^^, 1984, 37: 1301〜1305]和专 利 [ΕΡ0063456]的方法进行, 所得到的结果与它们完全一致。 由此可见, 利 用本发明的微生物菌株嗜麦芽寡养单胞菌 iStenotrophomonas maltropMia) CCTCC No. M 204024可以将有效霉素、 有 ¾¾霉亚基胺裂解为有效霉烯胺和 有效霉胺。 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.
本发明的微生物菌株嗜麦芽寡养单胞菌( Stenotrophomo画 maltrophilia) CCTCC No. M 204024, 不仅能在以有效霉素类物质为唯一碳源的培养基上 生长, 而且对有效霉素的分解能力较强; 利用 CCTCC No. M 204024生产有 效霉烯胺和有效霉胺, 在较佳工艺条件下, 有效霉素的分解率达到 70%〜90%, 有效霉素 A转化为有效霉烯胺的嫁尔转化率达到 40%〜80% (即 1摩^的有效霉素 A能转化为 0.40 -0.80摩 的有效霉烯胺),有效霉亚基胺 转化为有效霉烯胺的摩尔转化率达到 35%〜75%, 同时得到有效霉胺; 本发 明的微生物菌株嗜麦芽寡养单胞菌 iStenotrophomonas maltrophilia) CCTCC No. M 204024适用于有效霉烯胺和有效霉胺的生产。 具体实施方式  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
丁面实施例旨在举例说明而不是限制本发明的范围。  The noodle embodiment is intended to illustrate rather than limit the scope of the invention.
实施例 1:  Example 1:
:培养基配方(重量 /体积百分比, 以下同) = 有效霉素: 1.0%,(N )2S04 : 1.0% , KC1: 0.5%, Na2HP04 · 12Η20·· 1.0%, "aH2P04 ·2Η20: 0.1%, MgS04: 0.01%, 用自来水配制, 用 HC1溶液将 pH调到 6.0。 : 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.
取 lOOmL上述培养基, 平均分装在 2个 250mL三角瓶中, 灭菌。 接入 斜面菌种 CCTCC No. M 204024, 培养菌体, 摇床转速 150r/min, 在 28°C摇 床培赛 72小时作为种子液, 备用。  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.
取 2L上述培养基, 平均分装入 20个 50 OmL摇瓶中, 灭菌。 接入种子 液, 接种量为 2% (v/v) , 进行培养, 培养温度为 20°C, 培养时间为 160 h, 摇床转速 200r/min。  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.
收集上述发酵液 1.8L, 离心分离除去菌体, 上清液上柱吸附 (1L大孔 弱酸'性树脂 D113, NH4+), 用蒸馏水洗后, 再用 0.5mol/L的氨水洗脱, 减压 浓缩, 再将浓缩液上柱层析 (500mL强碱层析树脂 Dowex 1 X 2, OH ), 用 蒸馏冰洗脱, 分段收集洗出液, 收集先流出来的含有效霉胺和后流出来的有 效霉錄胺的部分, 用薄层层析方法和气相分析的方法进行分析, 然后进行真 空冷 干燥, 得到 0.31克有效霉胺和 0.74克有效霉烯胺。 实施例 2: 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:
培养基配方 有效霉素: 17.0%, (NH4)2S04 : 8.0% , KC1 : 0.5% , Na2HP04 - 12H20: 1.0%, NaH2P04 · 2H20: 0.1%, MgS04: 0.01%, 用自 来水配制, 用 NaOH溶液将 pH调到 7.0。 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.
取 500mL培养基, 平均分装在 5个 500mL三角瓶中, 灭菌。 接入斜面 菌种 CCTCC No. M 204024,进行培养,培养温度为 28°C,培养时间为 150 h, 反应在搅拌、 通风、 振荡条件下进行, 摇床转速 200r/min。  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.
收集上述发酵液 400mL, 进行分离、 纯化, 步骤同实施例 1, 得到 0.56 克有效霉胺和 0.85克有效霉烯胺。 实施例 3 :  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:
发酵培养基配方 有效霉素: 8.0% , (NH4)2S04: 6%, KC1: 0.1%, Na2HP04 · 12H20: 1.0%, Na¾P04 · 2¾0: 0.16%, MgS04: 0.02%, 用自 来水配制, 用 NaOH溶液将 pH调到 7.5。 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.
种子培养基配方 有效霉素: 1.0%, (NH4)2S04: 1%, KC1: 0.5%, Na2HP04 - 12¾0: 1.0%, NaH2P04 · 2H20: 0.1%, MgS04: 0.5%, 用自来 水配制, 用 NaOH溶液将 pH调到 7.5。 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.
取 500mL种子培养基, 平均分装在 5个 500mL三角瓶中, 灭菌。 接入 斜面菌种 CCTCC No. M 204024, 培养菌体, 摇床转速 200r/min, 在 30°C摇 床培养 72小时作为种子液, 备用。  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.
取 8L发酵培养基, 平均分装在 80个 500mL的摇瓶中, 灭菌。 接入种 子液,接种量为 5% (v/v), 进行培养,培养温度为 30°C, 培养时间为 180 h, 反应在搅拌、 通风、 振荡条件下进行, 摇床转速 200r/min。  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.
收集上述发酵液 7.5L, 进行分离、 纯化, 步骤同实施例 1, 得到 3.19 克有效霉胺和 7.77克有效霉烯胺。 实施例 4:  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:
培养基配方 有效霉素: 2.0%, (NH4)2S04: 1.0% , KC1 : 0.5% , Na2HP04 - 12¾0: 2.0%, NaH2P04 · 2H20: 0.1%, MgS04: 0.1%, 用自来 水配制, 用 NaOH溶液将 pH调到 7.0。 取 500mL培养基, 平均分装在 5个 500mL三角瓶中, 灭菌。 接入斜面 菌种 CCTCC No. M 204024, 培养菌体, 摇床转速 150r/min, 在 28°C摇床培 养 72小时作为种子液, 备用。 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.
取 8L培养基, 平均分装在 80个 500mL的摇瓶中, 灭菌。 接入种子液, 接种量为 5% ( v/v), 进行培养, 培养温度为 28°C, 培养时间为 100 h, 反应 在搅拌、 通风、 振荡条件下进行, 摇床转速 200r/min。  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.
收集上述发酵液 7.5升, 进行分离、 纯化, 步骤同实施例 1, 得到 2.11 克有效霉胺和 5.63克有效霉烯胺。 实施例 5 :  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:
培养基配方 有效霉素: 1.5%, (N )2S04 : 0.75% , KC1 : 0.5% , Na2HP04 · 12H20: 2.0%, NaH2P04 · 2H20: 1.0%, MgS04: 0.01%, 用自 来水配制, 自然 pH。 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.
取 200mL上述培养基, 平均分装在 4个 250mL三角瓶中, 灭菌。 接入 斜面菌种 CCTCC No. M 204024, 培养菌体, 摇床转速 150r/min, 在 28°C摇 床培养 72小时作为种子液, 备用。  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.
在 10L发酵罐中加入上述培养基 5L, 灭菌, 接入种子液 200mL, 进行 发酵。  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.
在接种后, 开始流加浓度为 10%的底物有效霉素溶液 1L, 流加速度 0.2mL/min; 培养温度 30°C, 培养时间为 160 h左右, 通气量 2.5L/min, 搅 拌速度 200r/min。  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.
收集上述发酵液 6L, 进行分离、 纯化, 步骤同实施例 1, 得到 3.67克 有效霉胺和 6.53克有效霉烯胺。 实施例 6:  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:
培养基配方 有效霉素: 1.0%, (NH4)2S04 : 0.7% , KC1 : 0.5% , Na2HP04 - 12H20: 0.1%, NaH2P04 - 2H20: 4.0%, MgS04: 0.01%, 用自 来水配制, 自然 pH。 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.
取 500mL上述培养基, 平均分装在 5个 500mL三角瓶中, 灭菌。 接入 斜面菌种 CCTCC No. M 204024, 培养菌体, 摇床转速 150r/min, 在 28°C摇 床培养 72小时作为种子液, 备用。 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.
在 10L发酵罐中加入上述培养基 5L, 灭菌, 接入种子液 200mL, 进行 发酵。  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.
在菌体生长到对数期时进行流加浓度为 2%的底物有效霉素溶液 3L, 流 加速度 0.5mL/min;培养温度 35°C,培养时间为 180 h左右,通气量 2.5L/min, 搅拌速度 200r/min。  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.
收集上述发酵液 8L, 进行分离、 纯化, 步骤同实施例 1, 得到 3.08克 有效霉胺和 5.82克有效霉烯胺。 实施例 7:  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:
培养基配方 有效霉素: 1.0%, (NH4)2S04 : 0.7% , KC1 : 0.5% , Na2HP04 · 12H20: 6.0%, NaH2P04 · 2H20: 0.1%, MgS04: 0.7%, 用自来 水配制, 自然 pH。 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.
取 500mL培养基, 平均分装在 5个 500mL三角瓶中, 灭菌。 接入斜面 菌种 CCTCC No. M 204024, 培养菌体, 摇床转速 150r/min, 在 28°C摇床培 养 72小时作为种子液, 备用。  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.
在 10L发酵罐中加入上述培养基 6L作为发酵液, 灭菌。 接入种子液 500mL o  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
在菌体生长到对数期时流加浓度为 50%的有效霉亚基胺溶液 lOOmL,流 加速度 0.05 mL/min; 培养条件: 温度为 30°C, 培养时间为 120 h, 通气量 3.5L/min, 搅拌速度 200r/min。  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.
收集上述发酵液 6L, 进行分离、 纯化, 步骤同实施例 1, 得到 3.26克 有效霉胺和 7.01克有效霉烯胺。 实施例 8:  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:
培养基配方 有效霉素: 2.0%, ( H4)2S04: 1.2% , KC1 : 4.0% , Na2HP04 · 12¾0: 4.0%, NaH2P04 · 2H20: 0.1%, MgS04: 0.01%, 用自 来水配制, 用 HC1调节 pH为 6.5。 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.
取 lOOOmL培养基, 平均分装在 10个 500mL三角瓶中, 灭菌。 接入斜 面菌种 CCTCC No. M 204024,培养菌体到对数生长期,然后进行离心分离, 得到菌体, 将这些菌体加入到 500mL浓度为 5%的底物有效霉素溶液中, 利 用菌体分解有效霉素; 反应条件: 温度 30°C, 初始 pH为 6.5, 培养时间为 100 h左右, 摇床转速 150r/min。 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.
收集上述反应液 480mL, 进行分离、 纯化, 步骤同实施例 1, 得到 0.45 克有效霉胺和 1.21克有效霉烯胺。 实施例 9:  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:
培养基配方 有效霉素: 2.0%, (NH4)2S04 : 1.2% , KC1 : 0.5% , Na2HP04 · 12H20: 1.0%, NaH2P04 - 2H20: 0.1%, MgS04: 0.01%, 用自 来水配制, 用 NaOH溶液调节 pH为 7.5。 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.
取 lOOOmL培养基, 平均分装在 10个 500mL三角瓶中, 灭菌, 接入斜 面菌种 CCTCC No. M 204024,培养菌体到对数生长期,然后进行离心分离, 得到菌体, 将这些菌体加入到 5O0mL浓度为 15%的底物有效霉亚基胺溶液 中, 利用菌体分解有效霉亚基胺; 培养条件: 温度 32°C, 初始 pH为 7.5, 培养时间为 40 h左右, 摇床转速 200r/min。  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.
收集上述反应液 580mL, 进行分离、 纯化, 步骤同实施例 1, 得到 1.03 克有效霉胺和 2.77克有效霉烯胺。 实施例 10:  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:
培养基配方 有效霉素: 2.0%, (NH4)2S04: 1.2% , KC1 : 0.5% , Na2HP04 - 12H20: 3.0%, NaH2P04 · 2H20: 0.1%, MgS04: 0.01%, 用自 来水配制, 用 NaOH溶液调节 pH为 7.5。 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.
取 lOOOmL培养基, 平均分装在 10个 500mL三角瓶中, 灭菌, 接入斜 面菌种 CCTCC No. M 204024, ±咅养菌体到对数生长期,然后进行离心分离, 得到菌体, 将菌体加入到 500mL浓度为 3%的底物有效霉亚基胺溶液中, 利 用菌体分解有效霉亚基胺; 培养条件: 温度 34°C, 初始 pH为 7.5, 培养时 间为 20 h左右, 摇床转速 150r/min。  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.
收集上述反应液 580mL, 进行分离、 纯化, 步骤同实施例 1, 得到 0.53 克有效霉胺和 1.06克有效霉烯胺。 实施例 11 : 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:
培养基配方 有效霉素: 2.0%, (NH4)2S04: 1.2% , KC1 : 0.5%, Na2HP04 - 12H20: 0.2%, NaH2P04 · 2H20: 1.5%, MgS04: 0.01%, 用 HC1 溶液调节 pH为 6.0。 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.
取 2000mL上述培养基, 平均分装在 20个 500mL三角瓶中, 灭菌, 接 入斜面菌种 CCTCC No. M 204024, 培养菌体到对数生长期, 然后进行离心 分离, 得到菌体, 用超声波将菌体破碎, 再进行离心、 盐析、 透析、 层析, 提取出裂解酶,将上述提取出的裂解酶加入 200mL浓度为 8%的底物有效霉 素溶液中,利用酶反应分解有效霉素生产有效霉胺和有效霉烯胺。培养条件: 温度 25°C, 初始 pH为 6.0, 培养时间为 10 h左右, 反应可在静置下进行。  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.
收集上述反应液 200mL, 进行分离、 纯化, 步骤同实施例 1, 得到 0.15 克有效霉胺和 0.34克有效霉烯胺。 实施例 12:  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:
培养基配方 有效霉素: 2.0%, (NH4)2S04: 1.2% , KC1 : 0.5% , Na2HP04 - 12H20: 1.5%, NaH2P04 · 2Η20: 0.8%, MgS04: 0.01%,用 NaOH 溶液调节 pH为 8.0。 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.
取 2000mL培养基, 平均分装在 20个 500mL三角瓶中, 灭菌, 接入斜 面菌种 CCTCC No. M 204024, 培养菌体到对数生长期,然后进行离心分离, 得到菌体, 用超声波将菌体破碎, 再进行离心、 盐析、 透析、 层析, 提取出 裂解酶, 将上述提取出的裂解酶, 加入 lOOmL浓度为 20%的底物有效霉素 溶液中, 利用酶反应分解有效霉素。 反应条件: 温度 32°C, 初始 pH为 8.0, 培养时间为 80 h左右, 反应可在静置下进行。  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.
收集上述反应液 lOOmL, 进行分离、 纯化, 步骤同实施例 1, 得到 0.21 克有效霉胺和 0.39克有效霉烯胺。 实施例 13 :  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:
培养基配方 有效霉素: 2.0%, (NH4)2S04 : 1.2% , KC1 : 0.5% , Na2HP04 · 12Η20: 1.0%, NaH2P04 · 2Η20: 0.1%, MgS04: 0.01%,用 NaOH 调节 pH为 7.8。 取 2000mL培养基, 平均分装在 20个 500mL三角瓶中, 灭菌, 接入斜 面菌种 CCTCC No. M 204024, 培养菌体到对数生长期,然后进行离心分离, 得到菌体, 用超声波将菌体破碎, 再进行离心、 盐析、 透析、 层析, 提取出 裂解酶, 将上述提取出的裂解酶, 加入 lOOmL浓度为 10%的底物有效霉亚 基胺溶液中, 利用酶反应分角有效霉亚基胺生产有效霉胺和有效霉烯胺。反 应条件:温度 30°C,初始 pH为 7.0,培养时间为 30 h左右,摇床转速 100r/min。 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.
收集上述反应液 lOOmL, 进行分离、 纯化, 步骤同实施例 1, 得到 0.10 克有效霉胺和 0.31克有效霉烯胺。 实施例 14:  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:
培养基配方 有效霉素: 2.0%, ( H4)2S04 : 1.2% , KC1 : 0.5% , Na2HP04 · 12Η20: 1.0%, NaH2P04 · 2Η20: 0.1%, MgS04: 0.01%,用 NaOH 调节 pH为 7.0。 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.
取 2000mL上述培养基, 平均分装在 20个 500mL三角瓶中, 灭菌, 接 入斜面菌种 CCTCC No. M 204024, 培养菌体到对数生长期, 然后进行离心 分离, 得到菌体, 用超声波将菌体破碎, 再进行离心、 盐析、 透析、 层析, 提取出裂解酶, 将上述提取出的裂解酶, 加入到 lOOmL浓度为 18%的底物 有效霉亚基胺溶液中, 利用酶反应分解有效霉亚基胺。 反应条件: 温度 30 °C, 初始 pH为 7.0, 培养时间为 50 h左右, 摇床转速 100r/min。  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.
收集上述反应液 100mL, 进行分离、 纯化, 步骤同实施例 1, 得到 0.33 克有效霉胺和 0.59克有效霉烯胺。  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

权 利 要 求 书 Claim
1.一种有效霉烯胺和有效霉胺的微生物制备方法,其特征是所述的微生 物是寡氧单胞菌属 Stmotrophomonas 嗜麦芽寡养单胞菌, CCTCC No. M 204024, 嗜麦芽寡养单胞菌与含碳源、 氮源、 无机盐的培养基, 底物为抗生 素有效霉素, 进行发酵, 对分解发酵液进行分离有效霉胺和有效霉烯胺, 并 进行提纯。 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.
2.根据权利要求 1所述的制备方法,其特征是所述的培养基重量 /体积的 组成为: 有效霉素 0.5%~20.0%, (NH4)2SO4 0.5%~10.0%, KC1 0.5%~5.0%, Na2HP04 · 12H20 0.1%〜10.0%, NaH2P04 · 2Η20 0.1%〜5·0%, MgS04 0.01%~1.0%, 用自来水配讳 lj, 调节 pH值 6.0〜8.0。 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.
3.根据权利要求 1所述的制备方法, 其特征是所述的发酵液的分离、提 纯步骤为: 反应液离心, 除去菌体, 上清液上柱吸附, 用蒸馏水洗后, 再用 0.5mol/L的氨水洗脱, 减压浓缩, 再将浓缩液上柱层析, 用蒸馏水洗脱, 分 段收集洗出液, 用薄层层析方法进行分析, 收集先流出来的含有效霉胺和后 流出来的有效霉烯胺的部分, 分别进行真空冷冻干燥, 得到有效霉胺和有效 霉烯胺。  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.
4.根据权利要求 1或 2所述的制备方法,其特征是在接入 CCTCC No. M 204024后的发酵培养基进行发酵, 培养基中有效霉素的浓度 0.5%〜20.0°/。, 培养条件: 温度 20°C〜40°C, 初始 pH为 6·0〜8.0, 培养时间为 100 1!〜 180 h, 在静置下进行发酵。  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.
5.根据权利要求 4所述的制备方法,其特征是发酵培养基中有效霉素的 浓度为 1.0%~10.0%,发酵温度 28°C~35°C, 初始 pH为 6.8~7.2,培养时间为 150 h, 在搅拌、 通风、 振荡条件下进行发酵。  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.
6.根据权利要求 1 所述的制备方法, 其特征是在培养基中接入菌种 CCTCC No. M 204024后, 在培养过程中流加入底物有效霉素, 在菌体刚开 始生长的时候开始流加, 培养条件:温度在 20°C〜40°C,初始 pH为 6.0〜8.0, 培养时间为 100 h〜180 h, 在静置下进行发酵。  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.
7.根据权利要求 6所述的制备方法,其特征是在菌体生长对数期后再进 行流加底物有效霉素, 培养条件: 28°C~35 °C, 初始 pH为 6.5〜7.5 , 培养时 间 150 h, 在搅拌、 通风、 振荡条件下进行发酵。 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.
8.根据权利要求 1 所述的制备方法, 其特征是在培养基中接入菌种 CCTCC No. M 204024后并培养菌体到对数生长期, 然后进行离心分离, 得 到菌体, 将菌体加入 ¾1底物有效霉素溶液中, 利用菌体分解底物有效霉素, 培养条件: 温度在 20°C〜40°C, 初始 pH为 6.0〜8.0, 培养时间为 1 1!〜 100 h, 在静置下进行发酵。  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.
9.根据权利要求 8所述的制备方法,其特征是将菌体加入到底物有效霉 素溶液中, 培养条件: 温度 28°C~35°C, 初始 pH为 6.5〜7.5, 培养时间为 70 h, 在搅拌、 通风、 振荡条件下进行发酵。  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. 根据权利要求 1所述的制备方法, 其特征是在培养基中接入菌种 CCTCC No. M 204024后培养菌体到对数生长期, 然后进行离心分离, 得到 菌体, 再用超声波法 4每菌体破碎, 提取出裂解酶, 将裂解酶加入到有效霉素 溶液中, 培养条件: 温度 20°C〜40°C, 初始 pH为 6.0〜8.0, 培养时间为 1 1!〜 100h, 在静置下进行。  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. 根据权利要求 10所述的制备方法,其特征是将裂解酶加入到底物 有效霉素溶液中, 培养条件: 温度 28°C〜35°C, 初始 pH为 6.5〜7.5, 培养时 间为 10 h, 在搅拌、 通风、 振荡条件下进行。  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. 根据权利要求 1或 6〜11所述的制备方法,其特征是底物可以为有 效霉亚基胺。  12. The preparation method according to claim 1 or 6 to 11, characterized in that the substrate can be an effective mold subunit amine.
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