CN1325655C - Microbial validamycin cracking process of producing validamycin anamine and validamycin amine - Google Patents
Microbial validamycin cracking process of producing validamycin anamine and validamycin amine Download PDFInfo
- Publication number
- CN1325655C CN1325655C CNB2005100613637A CN200510061363A CN1325655C CN 1325655 C CN1325655 C CN 1325655C CN B2005100613637 A CNB2005100613637 A CN B2005100613637A CN 200510061363 A CN200510061363 A CN 200510061363A CN 1325655 C CN1325655 C CN 1325655C
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- Prior art keywords
- validamycin
- pseudomonas
- substrate
- thalline
- validamine
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- -1 validamycin amine Chemical class 0.000 title abstract description 9
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- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical class CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- DLRVVLDZNNYCBX-ABXHMFFYSA-N melibiose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-ABXHMFFYSA-N 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- PGMYKACGEOXYJE-UHFFFAOYSA-N pentyl acetate Chemical compound CCCCCOC(C)=O PGMYKACGEOXYJE-UHFFFAOYSA-N 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 229960002429 proline Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 229960001153 serine Drugs 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229960001866 silicon dioxide Drugs 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- TYFQFVWCELRYAO-UHFFFAOYSA-L suberate(2-) Chemical compound [O-]C(=O)CCCCCCC([O-])=O TYFQFVWCELRYAO-UHFFFAOYSA-L 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 229940070710 valerate Drugs 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses a new microbe capable of cracking validamycin into validamycin enamine and validamycin amine, which belongs to the pseudomonas fluorescence (CCTCC No. M205098) of the pseudomonas. The present invention also discloses a method by using the microbe to crack the validamycin to prepare validamycin enamine and validamycin amine. The pseudomonas fluorescence (CCTCC No. M205098) is cultured by a seed culture medium, and then is transferred to a fermentation medium containing substrate validamycin for fermentation or substrate decomposition, or is cultured by a seed culture medium, then is separated to obtain strain; the obtained strain is added to solution containing substrate validamycin for reaction, the strain is used for decomposing substrates, and the decomposing solution is separated and purified to obtain the validamycin enamine and the validamycin amine.
Description
Technical field
The new microorganism fluorescent pseudomonas (Pseudomonas fluorescence) that the present invention relates to screen from soil also relates to the method that this new bacterial strain cracking validamycin (claiming jingganmycin again) of utilization is produced effective mildew enamine and validamine.
Background technology
Effective mildew enamine that the present invention relates to (valienamine) and validamine (validamine) structural formula following (Chem.Rev.2003,103:1955～1977):
Effective mildew enamine claims valienamine again, and molecular formula is C
7H
13NO
4, important group has: a primary amino (NH
2), a carbon-carbon double bond (C=C), a methylol (CH
2OH), three hydroxyls (OH).Its hydrochloride (C
7H
13NO
4HCl) [α]
D 23Be+68.6 ° (1N-HCl), pentacetate (C
17H
23NO
9) fusing point be 95 ℃, [α]
D 23Be+30.2 ° of (CHCl
3), meet the triketohydrindene hydrate color reaction.
Validamine claims the well ridge mould amine again, and molecular formula is C
7H
15NO
4, important group has: a primary amino (NH
2), a methylol (CH
2OH), three hydroxyl (OH), its hydrochloride (C
7H
13NO
4HCl) [α]
D 23Be+57.4 ° (1N-HCl) that fusing point is 229 ℃-232 ℃, [α]
D 23Be+60.6 ° of (H
2O), meet the triketohydrindene hydrate color reaction.
Effective mildew enamine and validamine are two kinds of stronger glycosidase inhibitors, also are simultaneously the core textures of a lot of other glycosidase inhibitors, can be used for synthetic effective Valiolamine (valiolamine) and voglibose (voglibose).
About the research of the production method of effective mildew enamine and validamine has had the history in more than 30 year, people such as Japanese tortoise field once adopted thalline degraded Validacin (Takeda) or the effective mould ylidene amines A of denitration pseudomonas, separate obtaining effective mildew enamine and validamine again, can not on the substratum that only is sole carbon source, grow with validamycin class material; And very weak to the capacity of decomposition of validamycin, be not suitable for the mass production of effective mildew enamine and validamine.
Japanese tortoise field etc. is separated to the bacterial strain that a strain can be decomposed into validamycin effective mildew enamine and validamine and has a liking for sugared Flavobacterium (Flavobacterium saccharophilum) IFO 13948 (open special permission: JP57-54593) in soil.
Japanese tortoise fields etc. adopt Cytophaga microbes producing cellulase (Cytophaga heparina) IFO 12017 (ATCC 13125) and IFO 14087 strains degraded validamycin or effective mould ylidene amines to produce effective mildew enamine and validamine [special permission communique (B2): flat 2-26957].
The microorganism of soil Pseudomonas (Agrobacterium) or Aeromonas (Aeromonas) has been found in Japanese tortoise field etc. and their variant can decompose validamycin or effective mould ylidene amines is produced effective mildew enamine and validamine.Such as soil Pseudomonas (Agrobacterium) microorganism, Agrobacterum Radiobacter IFO 12664 (ATCC 4718) strain is arranged, IFO 13258 (ATCC13332) strain, IFO 13259 (ATCC 13333) strain, IFO 13532 (ATCC 19358) strain, IFO13533 (ATCC 25235) strain, and Agrobacterium tumefaciens IFO 3058 strains etc.; Aeromonas (Aeromonas) microorganism, Aeromonas hydrophila subsp.Anaerogenes IFO 13282 is arranged, with subspecies subsp.hydrophila IFO 13286, subsp.proteolytica IFO 13287, Aeromonas punctata subsp.cavice IFO 13288, and Aeromonas salmonicida subsp.salmonicida IFO 12659 etc. [special permission communique (B2): flat 6-69380].
Consult the result according to document and patent, the patent bacterial classification of the validamycin of having reported of degrading comprises Rhodopseudomonas (Pseudomonas, 1 kind, Pseudomonas denitrificans, denitrifying pseudomonas), Flavobacterium (Flavobacterium, 3 kinds), have a liking for Cellulomonas (Cytophaga1 kind, 3 bacterial strains), soil Pseudomonas (Agrobacterium, 2 kinds, wherein Agrobacteriumradiobacter has 5 bacterial strains, Agrobacterium tumefaciens, a kind), Aeromonas (Aeromonas, 5 kinds).Concrete outcome sees attached list 1.
Table 1 degraded validamycin patent [special permission communique (B2)] bacterial classification gathers
| Bacterial classification | Patent |
| ?Pseudomonas?denitrificans | Flat 2-26957 (1982), flat 2-2598 |
| ?Flavobacterium?saccharophilum?IFO?13984 | Flat 2-26957, flat 2-2598 |
| ?Flavobacterium?heparinum | Flat 2-26957 |
| ?Flavobacterium?keratolyticus | Flat 2-26957 |
| ?Cytophaga?heparina?IFO?12017 | Flat 2-26957 |
| ?Cytophaga?heparina?ATCC?13125 | Flat 2-26957 |
| ?Cytophaga?heparina?IFO?14087 | Flat 2-26957 |
| ?Flavobacterium?saccharophilum?n.sp. | Flat 2-2598 |
| ?Flavobacterium?saccharophilum?FERM-P | Flat 2-2598 |
| ?Flavobacterium?saccharophilum?NO.5707 | Flat 2-2598 |
| ?Agrobacterium?Radiobacter?IFO?12664(ATCC?4718) | Flat 6-69380 |
| ?Agrobacterium?Radiobacter?IFO?13258(ATCC?13332) | Flat 6-69380 |
| ?Agrobacterium?Radiobacter?IFO?13259(ATCC?13333) | Flat 6-69380 |
| ?Agrobacterium?Radiobacter?IFO?13532(ATCC?19358) | Flat 6-69380 |
| ?Agrobacterium?Radiobacter?IFO?13533(ATCC?25235) | Flat 6-69380 |
| ?Agrobacterium?tumefaciens?IFO?3058 | Flat 6-69380 |
| ?Aeromonas?hydrophila?subsp.anaerogenes?IFO?13282 | Flat 6-69380 |
| ?Aeromonas?hydrophila?subsp.hydrophila?IFO?13286 | Flat 6-69380 |
| ?Aeromonas?hydrophila?subsp.proteolytica?IFO?13287 | Flat 6-69380 |
| ?Aeromonas?punctata?subsp.cavice?IFO?13288 | Flat 6-69380 |
| ?Aeromonas?salmonicida?subsp.salmonicida?IFO?12659 | Flat 6-69380 |
In addition, in April, 2004, people such as the Zhejiang Polytechnical University abundant state of Zheng have screened germ oligotrophy unit cell (Stenotrophomonas maltrophilia) (CN 1563397), and this bacterium can be used for the production of effective mildew enamine and validamine.2004, Japan also reported the production technology (JP 3586684 B1) of utilizing Paenibacillus to prepare validamine and effective mildew enamine.
Summary of the invention
The invention provides a kind of new microorganism strains that validamycin can be cracked into effective mildew enamine and validamine that from soil, screens; Next provides a kind of method of utilizing this new microbial lytic validamycin to produce effective mildew enamine and validamine.
New microorganism strains provided by the invention belongs to the fluorescent pseudomonas (Pseudomornas fluorescence) of Pseudomonas (Pseudomonas), this bacterial strain has been preserved in Chinese typical culture collection center on September 2nd, 2005, be called for short CCTCC, deposit number is CCTCC No.M205098.
The new strain characteristic that the present invention screens from soil is as follows:
Cellular form: shaft-like, size 0.7～0.8 μ m * 2.0～2.8 μ m, amphitrichous.
Colonial morphology: aerobic, on the beef extract-peptone flat board, can secrete water-soluble green pigment;
Physiological and biochemical property: oxidase positive, the catalase positive.Can utilize N-acetylglucosamine, ribose, sucrose, methylene-succinic acid, malonate, acetate, DL-lactic acid salt, 5-ketone group-gluconate, N.F,USP MANNITOL, D-glucose, D-sorbyl alcohol, L-arabinose, propionic salt, caprate, valerate, Citrate trianion, 2-ketone group-gluconate, 3-hydroxyl-butyrates, 4-hydroxy-benzoic acid salt, can be nitrogenous source with Histidine, L-Serine and L-proline(Pro), can not utilize inositol, suberate, 3-hydroxy-benzoic acid salt, glycogen, D-melibiose, L-Fucose etc.
According to above bacterial characteristics, this new bacterial strain is belonged to the Pseudomonas fluorescens (Pseudomonas fluorescens) of Rhodopseudomonas (Pseudomonas) by evaluation, and this microorganism has the ability that the cracking validamycin is produced effective mildew enamine and validamine.
The new bacterial strain Pseudomonas fluorescens (Pseudomonas fluorescens) that the present invention screens does not belong to any of the above-mentioned bacterial classification of publication.
The used raw material of the present invention is a kind of agricultural antibiotic of widespread use a---validamycin, it is jingganmycin, it is a kind of efficient, safe agricultural antibiotic, free from environmental pollution, to the person poultry harmless, become at present that China's usable floor area is wide, mu is minimum with cost, safety, public nuisance-free agricultural chemicals, be an important kind of pesticide industry.Validamycin is the aminoglycoside agricultural antibiotic, and main ingredient has A, B, C, D, E, F etc., can find from the structure of validamycin, and validamycin is made up of effective mildew enamine, validamine and β-structures such as D-glucose.
New microorganism of the present invention is screened from soil and obtains, this new bacterial strain Pseudomonas fluorescens (Pseudomonas fluorescens), CCTCC No.M 205098 all can grow under 20 ℃～40 ℃ culture temperature, the suitableeest growth temperature is 25 ℃～37 ℃, culture condition: pH value 6.0～8.0, the suitableeest growth pH value are neutral or approaching neutral.This new bacterial classification has the ability that the cracking validamycin prepares effective mildew enamine and validamine.
The substratum that is used for bacterial classification CCTCC No.M 205098 of the present invention is that some contain and can be utilized nutritive substance by above-mentioned bacterial strains, as carbon source, nitrogenous source, inorganic salt etc., as carbon source except validamycin, can also have: glucose, lactose, maltose, dextrin, starch, glycerine, N.F,USP MANNITOL, sorbyl alcohol, lipid (soya-bean oil, lard etc.); As having of nitrogenous source: gravy, yeast extract paste, dry yeast, soyflour, corn steep liquor, peptone, urea, ammonia salt (ammonium sulfate, ammonium chloride, ammonium nitrate, Ammoniom-Acetate etc.), peptide class (dipeptides, tripeptides etc.); Inorganic salts has: metallic salt and organic acid salts such as phosphoric acid salt, acetate such as Na, K, Ca, Mg, Fe, Mn, Zn, Co, Ni; In addition, the material that also can add on a small quantity has: amino acids (as L-glutamic acid, aspartic acid, Methionin, glycine, methionine(Met) etc.), little element (V that gives birth to
B1, V
B2, V
B12, V
C, V
E, nicotinic acid etc.), nucleic acid class (as purine, pyrimidine and derivative thereof etc.).PH value with mineral acid or organic acid, bases are regulated substratum also can add appropriate amount of defoamer, opposes as bubble etc.Liquid culture, solid culture all can, but during a large amount of suitability for industrialized production, liquid nutrient medium is comparatively suitable.Can adopt training method to have: to leave standstill cultivation, wave and culture or ventilation stir culture etc., the appropriate to the occasion employing deep ventilation of mass production effective mildew enamine and validamine stir culture.
The Pseudomonas fluorescens (Pseudomonas fluorescens) of new microorganism Rhodopseudomonas (Pseudomonas) of the present invention, CCTCC No.M 205098, substratum with carbonaceous sources, nitrogenous source, inorganic salt, substrate is the microbiotic validamycin, ferment, then decomposing and fermenting liquid is separated effective mildew enamine and validamine, and purify.
Seed culture medium bulking value of the present invention such as down (g/L): glucose 10～40, peptone 5～20, yeast extract paste 1～10, MgSO
40.1～1, the tap water preparation.
Main component is a validamycin in the fermention medium, other components are the carbon source that can be utilized by new microorganism of the present invention, nitrogenous source, inorganic salt etc., fermention medium of the present invention form each bulking value such as down (g/L): validamycin 5～50, glucose 1～20, peptone 1～10, yeast powder 1～10, MgSO
40.1～1, prepare with tap water.
Decompose the substrate validamycin with new bacterial strain fluorescent pseudomonas CCTCC No.M 205098 of the present invention and produce effective mildew enamine and validamine, its method has:
(1) utilize this new microbial fermentation to decompose the substrate validamycin and prepare effective mildew enamine and validamine: bacterial classification CCTCC No.M 205098 is through overactivation, insert the seed culture medium cultivation of above-mentioned sterilization and make seed liquor, again seed liquor is linked into cultivation and fermentation in the fermention medium after above-mentioned preparation is sterilized.
The control condition of seed culture is as follows: 25 ℃～37 ℃ of culture temperature, and pH6.0～8.0, incubation time 24h～48h, the pH value, is carried out to good under ventilate stirring or oscillating condition for well near neutral;
The control condition of fermentation culture: 25 ℃～37 ℃ of temperature, pH is 6.0～8.0, and fermentation time is 72h～192h, and the pH value, is carried out to good under ventilate stirring or oscillating condition for well near neutral.Validamycin during substratum is formed in this method is the carbon source that microorganism utilizes, again by the substrate of microbial lytic; The substrate validamycin is cracked into effective mould ylidene amines, and effective mould ylidene amines passes through cracking again, generates effective mildew enamine (valienamine) and validamine (validamine).
(2) utilize this new microorganism cells catalytic pyrolysis substrate validamycin to prepare effective mildew enamine and validamine: bacterial classification CCTCC No.M 205098 is through overactivation, insert in the seed culture medium of sterilizing and cultivate, 25 ℃～37 ℃ of culture temperature, pH6.0～8.0, the pH value near neutral for well, it is centrifugal to the logarithmic growth after date to cultivate thalline, collects thalline; The thalline that obtains is added in the substrate validamycin solution, utilize cellular lysate substrate validamycin to produce effective mildew enamine and validamine; Validamycin concentration in the solution, with the ratio of bulking value represent (kg/L, %): 1.0%～10.0%; 25 ℃～37 ℃ of temperature of reaction, reaction times 4h～96h carries out to good under the condition that stirs or vibrate of ventilating.
The pH value is regulated control with mineral acid or organic acid, bases in cracking validamycin process.
Effective mildew enamine and validamine are alkaline matter soluble in water, thereby can adopt separation, the process for purification of water-soluble alkaline material, for example ion exchange resin, gac, porous polymer, dextrane gel, ion-exchanger etc. adsorb back desorb, chromatography etc.
Sepn process is as follows: it is centrifugal or remove by filter thalline to contain the fermented liquid of effective mildew enamine and validamine or cell catalysis reaction solution, again supernatant liquor or filtrate are carried out ion-exchange with macroporous ion exchange resin, use the ammonia soln wash-out, collect elutriant, concentrating under reduced pressure, concentrated solution carries out chromatography with the strong basicity chromatographic resin, and water is collected the part that only contains effective mildew enamine or validamine as eluent.Lyophilize obtains finished product.Method with thin-layer chromatography and gas phase analysis is analyzed effective mildew enamine and the validamine produced, operating process and experiment condition are pressed document J.Antibiot, 1984, the method for 37:1301～1305 and patent documentation EP0063456 is carried out, and resulting result and they are in full accord.This shows, utilize microorganism of the present invention to produce effective mildew enamine and validamine by the cracking validamycin.
Specific embodiments
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Embodiment 1
Seed culture medium (g/L): glucose 20, peptone 10, yeast extract paste 5, MgSO
40.2, the tap water preparation, it is standby to sterilize.
Fermention medium (g/L): validamycin 15, glucose 5, peptone 10, yeast powder 10, MgSO
40.2 with the tap water preparation, it is standby to sterilize.
The preparation of seed liquor: get the 500mL seed culture medium, average mark is contained in 10 250mL triangular flasks, sterilization.Insert inclined-plane CCTCC No.M 205098 and cultivate, shaking speed 200r/min cultivates 36h as seed liquor at 30 ℃ of shaking tables.
Fermentation: the 10L fermention medium of in the 15L fermentor tank, packing into, sterilization.Then, insert seed liquor and ferment inoculum size 5% (promptly inserting the 0.5L seed liquor), 30 ℃ of leavening temperatures, pH7.0, ventilation 3.5vvm, fermentation time 96h.
The separation of product: collect above-mentioned fermented liquid 10L, thalline is removed in centrifugation, supernatant liquor upper prop (D113 resin, NH
4 +), carry out ion-exchange, behind distilled water wash, use the ammoniacal liquor wash-out of 0.5mol/L again, concentrating under reduced pressure, (Dowex 1 * 2, OH with column chromatography on the concentrated solution again
-), use the distilled water wash-out, analyze (silica-gel plate G: Haiyang Chemical Plant, Qingdao with the thin-layer chromatography method; The chromatography agent, n-propyl alcohol/acetic acid/water=4: 1: 1; The colour developing of 0.5% triketohydrindene hydrate, effective mildew enamine R
f=0.45, the R of validamine
f=0.39), the Fractional Collections elutriant is collected the part that only contains effective mildew enamine or validamine, carries out vacuum lyophilization then.Obtain 7.5 gram effective mildew enamines and 4.1 gram validamines.
Embodiment 2
Seed culture medium (g/L): glucose 10, peptone 20, yeast extract paste 1, MgSO
40.1, the tap water preparation, it is standby to sterilize.
Fermention medium (g/L): validamycin 20, glucose 5, peptone 10, yeast powder 10, MgSO
40.2 with the tap water preparation, it is standby to sterilize.
The preparation of seed liquor: get the 150mL seed culture medium, average mark is contained in 3 250mL triangular flasks, sterilization.Insert inclined-plane CCTCC No.M 205098 and cultivate, shaking speed 200r/min cultivates 48h as seed liquor at 25 ℃ of shaking tables.
Fermentation: in the triangular flask of 20 500mL, every bottled 100mL fermention medium of going into, sterilization, insert seed liquor then and ferment, inoculum size 5% (being that every bottle graft is gone into the 5mL seed liquor), 28 ℃ of leavening temperatures, pH7.0, shaking speed 200r/min, fermentation time 144h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step is with embodiment 1.Obtain 1.4 gram effective mildew enamines and 0.9 gram validamine.
Embodiment 3
Seed culture medium (g/L): glucose 40, peptone 5, yeast extract paste 10, MgSO
41, the tap water preparation, it is standby to sterilize.
Fermention medium (g/L): validamycin 30, glucose 5, peptone 10, yeast powder 10, MgSO
40.2 with the tap water preparation, it is standby to sterilize.
The preparation of seed liquor: get the 150mL seed culture medium, average mark is contained in 3 250mL triangular flasks, sterilization.Insert inclined-plane CCTCC No.M 205098 and cultivate, shaking speed 200r/min cultivates 24h as seed liquor at 37 ℃ of shaking tables.
Fermentation: in the triangular flask of 20 500mL, every bottled 100mL fermention medium of going into, sterilization, insert seed liquor then and ferment, inoculum size 5% (being that every bottle graft is gone into the 5mL seed liquor), 35 ℃ of leavening temperatures, pH7.2, shaking speed 200r/min, fermentation time 144h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 1.4 gram effective mildew enamines and 1.1 gram validamines with embodiment 1.
Embodiment 4
Seed culture medium (g/L): glucose 25, peptone 15, yeast extract paste 5, MgSO
40.4, the tap water preparation, it is standby to sterilize.
The preparation of seed liquor is with embodiment 1.
Fermention medium (g/L): validamycin 50, glucose 1, peptone 10, yeast powder 1, MgSO
41, with the tap water preparation, it is standby to sterilize.
Fermentation: in the triangular flask of 20 500mL, every bottled 100mL fermention medium of going into, sterilization inserts seed liquor then and ferments, inoculum size 5% (promptly inserting the 5mL seed liquor), 25 ℃ of leavening temperatures, pH6.0, shaking speed 200r/min, fermentation time 192h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 1.5 gram effective mildew enamines and 1.2 gram validamines with embodiment 1.
Embodiment 5
Seed culture medium (g/L): glucose 30, peptone 8, yeast extract paste 6, MgSO
40.5, the tap water preparation, it is standby to sterilize.
The preparation of seed liquor is with embodiment 1.
Fermention medium (g/L): validamycin 5, glucose 20, peptone 1, yeast powder 10, MgSO
40.1 with the tap water preparation, it is standby to sterilize.
Fermentation: in the triangular flask of 20 500mL, every bottled 100mL fermention medium of going into, sterilization inserts seed liquor then and ferments, inoculum size 5% (promptly inserting the 5mL seed liquor), 37 ℃ of leavening temperatures, pH8.0, shaking speed 200r/min, fermentation time 72h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 0.5 gram effective mildew enamine and 0.3 gram validamine with embodiment 1.
Embodiment 6
Seed culture medium (g/L): glucose 20, peptone 10, yeast extract paste 5, MgSO
40.2, the tap water preparation, it is standby to sterilize.
The preparation of thalline: get the 500mL seed culture medium, average mark is contained in 10 250mL triangular flasks, sterilization.Insert inclined-plane CCTCC No.M 205098 and cultivate, shaking speed 200r/min cultivates thalline to logarithmic phase at 30 ℃ of shaking tables, and is centrifugal, collects thalline.
Cell catalysis reaction: the thalline that collection is obtained joins 100mL and contains in 5.0% the validamycin aqueous solution 30 ℃ of temperature of reaction, reaction times 48h.
Collect above-mentioned fermented liquid 100mL, separate, purifying, step obtains 0.25 gram effective mildew enamine and 0.2 gram validamine with embodiment 1.
Embodiment 7
Seed culture medium (g/L): glucose 10, peptone 20, yeast extract paste 10, MgSO
40.1, the tap water preparation, it is standby to sterilize.
The preparation of thalline is with embodiment 6.
The cell catalysis reaction: the thalline that collection is obtained joins in the validamycin aqueous solution of 100mL 1.0% 37 ℃ of temperature of reaction, reaction times 4h.
Collect above-mentioned fermented liquid 100mL, separate, purifying, step obtains 0.08 gram effective mildew enamine and 0.07 gram validamine with embodiment 1.
Embodiment 8
Seed culture medium (g/L): glucose 40, peptone 5, yeast extract paste 1, MgSO
41, the tap water preparation, it is standby to sterilize.
The preparation of thalline is with embodiment 6.
The cell catalysis reaction: the thalline that collection is obtained joins in the validamycin aqueous solution of 100mL 10.0% 25 ℃ of temperature of reaction, reaction times 96h.
Collect above-mentioned fermented liquid 100mL, separate, purifying, step obtains 0.63 gram effective mildew enamine and 0.41 gram validamine with embodiment 1.
Claims (6)
1. method of utilizing microorganism fluorescent pseudomonas cracking validamycin to prepare effective mildew enamine and validamine, it is characterized in that described microorganism belongs to the fluorescent pseudomonas of Pseudomonas (Pseudomonas) (Pseudomonas fluorescence), CCTCC No.M 205098; Described fluorescent pseudomonas (Pseudomonas fluorescence), CCTCC No.M 205098, after cultivating, seed culture medium inserts the decomposition substrate that ferments in the fermention medium that contains the substrate validamycin, or seed culture medium cultivation back separation obtains thalline, the thalline that obtains added in the solution that contains validamycin carry out the reaction decomposes substrate, then decomposed solution is separated, purified, obtain effective mildew enamine and validamine.
2. method according to claim 1 is characterized in that described seed culture medium, the ratio of the bulking value that each is formed, and promptly g/L is: glucose 10～40, peptone 5～20, yeast extract paste 1～10, MgS0
40.1～1, the tap water preparation.
3. method according to claim 1 is characterized in that described fermention medium, the ratio of the bulking value that each is formed, and promptly g/L is: validamycin 5～50, glucose 1～20, peptone 1～10, yeast powder 1～10, MgSO
40.1～1, prepare with tap water.
4. according to claim 1 or 2 or 3 described methods, it is characterized in that in seed culture medium, inserting microorganism CCTCC No.M 205098 and cultivate, 25 ℃～37 ℃ of culture temperature, pH6.0～8.0, incubation time 24h～48h obtains seed liquor; Seed liquor inserts in the fermention medium that contains the substrate validamycin ferments, and its fermentation condition is: 25 ℃～37 ℃ of leavening temperatures, and pH6.0～8.0, fermentation time is 72h～192h.
5. method according to claim 1 and 2 is characterized in that inserting microorganism CCTCC No.M 205098 and cultivates 25 ℃～37 ℃ of culture temperature in seed culture medium, pH6.0～8.0, cultivate thalline to logarithmic phase, centrifugation then obtains thalline; With the thalline that separation obtains, add in the aqueous solution that contains the substrate validamycin and react, utilize thalline to decompose substrate, 25 ℃～37 ℃ of temperature of reaction, reaction times 4h～96h.
6. method according to claim 5, the concentration that it is characterized in that the substrate validamycin aqueous solution is 1.0%～10.0%, 25 ℃～37 ℃ of temperature of reaction, reaction times 4h～96h.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5754593A (en) * | 1980-09-16 | 1982-04-01 | Takeda Chem Ind Ltd | Preparation of valienamine |
| JPS586684B2 (en) * | 1978-05-23 | 1983-02-05 | 日立造船株式会社 | Energy saver for contact type sulfuric acid production equipment |
| JPS58152496A (en) * | 1982-03-04 | 1983-09-10 | Takeda Chem Ind Ltd | Production of valienamine and validamine |
| JPS62181793A (en) * | 1985-10-08 | 1987-08-10 | Takeda Chem Ind Ltd | Production of valineamine and validamine |
| JP3586684B1 (en) * | 2003-10-28 | 2004-11-10 | 合同酒精株式会社 | Novel microorganisms that convert validamycin to valienamin and validamin |
| CN1563397A (en) * | 2004-04-05 | 2005-01-12 | 浙江工业大学 | Microbe method for preparing enamine and amine from valinemia |
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2005
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPS586684B2 (en) * | 1978-05-23 | 1983-02-05 | 日立造船株式会社 | Energy saver for contact type sulfuric acid production equipment |
| JPS5754593A (en) * | 1980-09-16 | 1982-04-01 | Takeda Chem Ind Ltd | Preparation of valienamine |
| JPS58152496A (en) * | 1982-03-04 | 1983-09-10 | Takeda Chem Ind Ltd | Production of valienamine and validamine |
| JPS62181793A (en) * | 1985-10-08 | 1987-08-10 | Takeda Chem Ind Ltd | Production of valineamine and validamine |
| JP3586684B1 (en) * | 2003-10-28 | 2004-11-10 | 合同酒精株式会社 | Novel microorganisms that convert validamycin to valienamin and validamin |
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