CN1273606C - Microbe method for preparing enamine and amine from valinemia - Google Patents
Microbe method for preparing enamine and amine from valinemia Download PDFInfo
- Publication number
- CN1273606C CN1273606C CN 200410017516 CN200410017516A CN1273606C CN 1273606 C CN1273606 C CN 1273606C CN 200410017516 CN200410017516 CN 200410017516 CN 200410017516 A CN200410017516 A CN 200410017516A CN 1273606 C CN1273606 C CN 1273606C
- Authority
- CN
- China
- Prior art keywords
- validamycin
- preparation
- thalline
- effective
- validamine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 150000002081 enamines Chemical class 0.000 title claims abstract description 55
- 150000001412 amines Chemical class 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 25
- 208000008766 Valinemia Diseases 0.000 title 1
- 229930195482 Validamycin Natural products 0.000 claims abstract description 68
- 239000000758 substrate Substances 0.000 claims abstract description 29
- 244000005700 microbiome Species 0.000 claims abstract description 17
- 239000008399 tap water Substances 0.000 claims abstract description 14
- 235000020679 tap water Nutrition 0.000 claims abstract description 14
- 241000122971 Stenotrophomonas Species 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 5
- 238000000926 separation method Methods 0.000 claims abstract description 3
- JARYYMUOCXVXNK-IMTORBKUSA-N validamycin Chemical compound N([C@H]1C[C@@H]([C@H]([C@H](O)[C@H]1O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)CO)[C@H]1C=C(CO)[C@H](O)[C@H](O)[C@H]1O JARYYMUOCXVXNK-IMTORBKUSA-N 0.000 claims abstract 13
- GSQYAWMREAXBHF-UOYQFSTFSA-N validamine Chemical compound N[C@H]1C[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GSQYAWMREAXBHF-UOYQFSTFSA-N 0.000 claims description 49
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 48
- GSQYAWMREAXBHF-UHFFFAOYSA-N Validamine Natural products NC1CC(CO)C(O)C(O)C1O GSQYAWMREAXBHF-UHFFFAOYSA-N 0.000 claims description 48
- 230000001580 bacterial effect Effects 0.000 claims description 30
- 238000002360 preparation method Methods 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 25
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- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 5
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- 238000004809 thin layer chromatography Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 abstract description 2
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- 235000011130 ammonium sulphate Nutrition 0.000 abstract description 2
- 238000005342 ion exchange Methods 0.000 abstract description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 abstract 2
- 241000108056 Monas Species 0.000 abstract 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract 2
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- 239000001301 oxygen Substances 0.000 abstract 2
- LUMNWCHHXDUKFI-UHFFFAOYSA-N 5-bicyclo[2.2.1]hept-2-enylmethanol Chemical class C1C2C(CO)CC1C=C2 LUMNWCHHXDUKFI-UHFFFAOYSA-N 0.000 abstract 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 abstract 1
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- 235000019341 magnesium sulphate Nutrition 0.000 abstract 1
- JARYYMUOCXVXNK-CSLFJTBJSA-N validamycin A Chemical compound N([C@H]1C[C@@H]([C@H]([C@H](O)[C@H]1O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)CO)[C@H]1C=C(CO)[C@@H](O)[C@H](O)[C@H]1O JARYYMUOCXVXNK-CSLFJTBJSA-N 0.000 description 60
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- XPHOBMULWMGEBA-UHFFFAOYSA-N Valienamine Natural products NC1C=C(CO)C(O)C(O)C1O XPHOBMULWMGEBA-UHFFFAOYSA-N 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- XPHOBMULWMGEBA-VZFHVOOUSA-N valienamine Chemical compound N[C@H]1C=C(CO)[C@@H](O)[C@H](O)[C@H]1O XPHOBMULWMGEBA-VZFHVOOUSA-N 0.000 description 7
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- 239000004202 carbamide Substances 0.000 description 1
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- 230000001413 cellular effect Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 239000013530 defoamer Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 235000013882 gravy Nutrition 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 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
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 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
- 239000001053 orange pigment Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- PGMYKACGEOXYJE-UHFFFAOYSA-N pentyl acetate Chemical compound CCCCCOC(C)=O PGMYKACGEOXYJE-UHFFFAOYSA-N 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 238000013138 pruning Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000001052 yellow pigment Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
Abstract
The present invention relates to a method for preparing microorganisms of effective mildew enamine and effective mildew amine. The referred microorganisms are pantropic malt oligoclonal oxygen monas (Stenotrophomonas maltrophilia), CCTCC No. M 204024 of the oligoclonal oxygen monas (Stenotrophomonas). The microorganisms are utilized for fermentation and decomposition, or the cells or the enzymes, etc. of the microorganisms are utilized for decomposing validamycin or effective mildew imine of substrates so as to produce two types of substances in the cyclol class of the effective mildew enamine and effective mildew amine. A culture medium comprises 0.5% to 20.0% of validamycin, 0.5% to 10.0% of (NH4)2SO4, 10.5% to 5.0% of KCl, 0.1% to 10.0% of Na2HPO4-12H2O, 0.1% to 5.0% of NaH2PO4-2H2O and 0.01% to 1.0% of MgSO4 by weight/volume, which are prepared by tap water. The present invention has the culture conditions that the fermentation temperature is from 20 DEG C to 40 DEG C, an initial pH is from 6.0 to 8.0, and the culture time is from 1h to 180h; and the effective mildew enamine and the effectively mildew amine are obtained by the separation and the purification of an ion exchange method and a chromatography method.
Description
Technical field
The new germ oligotrophy unit cell that the present invention relates to screen from soil (Stenotrophomonas maltrophilia) also relates to this new bacterial strain of utilization and decomposes the method that validamycin (claim not only jingganmycin) or effective mould ylidene amines (but also claiming mould ylidene amines in well ridge or well ridge azanol) are produced effective mildew enamine and validamine.
Background technology
About the research of the manufacture 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) IFO13948 (open special permission: JP57-54593) in soil.
Japanese tortoise fields etc. adopt Cytophaga microbes producing cellulase (Cytophaga heparina) IFO12017 (ATCC13125) and IFO14087 strain 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 effectively 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 (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 (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].
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, having become at present wide, mu minimum safety, the public nuisance-free agricultural chemicals of usefulness cost of China's usable floor area, is 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.
The validamycin glycosidic link is decomposed into effective mould ylidene amines through hydrolysis, and effective mould ylidene amines has two kinds of A, B, and effective mould ylidene amines through decomposing, generates effective mildew enamine (valienamine) and validamine (validamine) again.
The effective mildew enamine that the present invention relates to, English valienamine by name.Its structural formula such as Fig. 1 (Chem.Rev.2003,103:1955-1977).
The chemical structure of Fig. 1 effective mildew enamine (valienamine)
Effective mildew enamine claims well ridge amine or valienamine again, (1S, 2S, 3S, 4R)-and 1-amino-5-(methylol) hexamethylene-5-alkene-2,3, the 4-trivalent alcohol; 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.Because effective mildew enamine contains a primary amino (NH
2), one-level takes place in the aqueous solution dissociate.
The validamine that the present invention relates to, English validamine by name, its structural formula such as Fig. 2 (Chem.Rev.2003,103:1955-1977).
The chemical structure of Fig. 2 validamine (validamine)
Validamine claims the well ridge mould amine again, and molecular formula is C
7H
15NO
4, a primary amino (NH
2), a methylol (CH
2OH), three hydroxyl (OH), its hydrochloride (C
7H
13NO
4HCl) [α]
D 23Be+57.4 °, fusing point is 229 ℃-232 ℃, [α] (1N-HCl)
D 23Be+60.6 °, meet the triketohydrindene hydrate color reaction.Validamine also contains a primary amino (NH
2), one-level takes place in the aqueous solution dissociate.
Cyclic alcohol such as effective mildew enamine, validamine class material is the core texture of glycosidase inhibitor, also is a kind of stronger glycosidase inhibitor simultaneously.
Glycosylase is the pruning enzyme of oligonucleotide chain in the glycoprotein biosynthesizing, and is very important to the formation of the oligonucleotide chain in the glycoprotein.The composition of sugar chain and structure are the recognition sites of the special biological function of glycoprotein, influence Protein Folding, solubleness, modification, antigenicity and biological activity etc.Glycosidase activity to glycoprotein biosynthesizing play a part very crucial, research glycosidase inhibitor, be with a wide range of applications.
Because the biological activity of effective mildew enamine and validamine, people are to its interest and day sharp increase.These years, be one of active research field, especially in Japan and German, and in China, the research of this respect almost is blank.
Summary of the invention
Task of the present invention is the autotelic new microorganism strains that validamycin or effective mould ylidene amines is decomposed into effective mildew enamine and validamine that screens from soil; Next provides a kind of cell of this new microbe fermentation decomposition or this new microbe or method that enzyme decomposes microbiotic validamycin, effective mould ylidene amines production effective mildew enamine and validamine utilized.
Microorganism provided by the invention is oligotrophy zygosaccharomyces (Stenotrophomonas), be germ oligotrophy unit cell (Stenotrophomonas maltrophilia), this bacterial strain has been preserved in Chinese typical culture collection center on March 15th, 2004, be called for short CCTCC, deposit number is CCTCC No.M 204024.
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 | Flat 6-69380 |
| (ATCC 25235) | |
| Agrobacterium tumefaciens IFO 3058 | Flat 6-69380 |
| Aeromonas hydrophila subsp. anaerogenes IFO 13282 | Flat 6-69380 |
| Aeromonas hydrophila subsp. hydrophila IFO 13286 | Flat 6-69380 |
| Aeromonas hydrophila subsp. proteolytica IFO13287 | Flat 6-69380 |
| Aeromonas punctata subsp.cavice IFO 13288 | Flat 6-69380 |
| Aeromonas salmonicida subsp. salmonicida IFO 12659 | Flat 6-69380 |
It is as follows that each belongs to physiological and biochemical property:
Rhodopseudomonas (Pseudomonas): denitrifying pseudomonas (Pseudomonasdenitrificans) is the Gram-negative sporeless bacterium, polar flagella motion, oxidized form metabolism, common oxidase positive, chemoorganotrophy.Aerobic bacteria can carry out denitrification.
Soil Pseudomonas (Agrobacterium): bacillus pumilis, size are 1.5～0.3 μ m * 0.6～1.0 μ m, single paired arrangement, and rare peritricha, the Gram-reaction feminine gender does not produce gemma, the bacterium colony projection, full edge is smooth, and non-pigment is to ecru.In containing sugar culture-medium, produce a large amount of polysaccharide mucus, fixing atmospheric nitrogen not, most kinds can be utilized inorganic nitrogen, the radiation edaphic bacillus can produce 3-ketone group lactose from lactose, weak or the nothing of the ability of decomposing protein, can extensively utilize carbohydrate, organic acid salt and amino acid to be carbon source, can not utilize Mierocrystalline cellulose, starch, agarose, semi-lactosi and other carbohydrates.
Aeromonas (Aeromonas): cell is straight and upright, the tool nose circle is shaft-like, often with the motion of one pole hair, glucose fermentation, fructose, maltose and trehalose, carbohydrate breakdown becomes acid, the aerogenesis that has, hydrolyzed starch and dextrin, caseinhydrolysate, liquefy gelatin, produce the DNA enzyme, the arginine desaminase, the minority kind is not moved.The Gram-reaction feminine gender has and breathes and the two kinds of metabolic types that ferment oxydase and hydrogen peroxide enzyme positive, facultative anaerobe.
Have a liking for Cellulomonas (Cytophaga): do not form sporophore, cell is shaft-like, often forms very long cell chains, and is aerobic, can utilize carbohydrate, needs a large amount of CO in the metabolic process
2, thoroughly polysaccharose substances such as decomposition of cellulose, hydrolysis chitin and agar can slide on the interface.
Flavobacterium (Flavobacterium): direct rod shape, end circle, big or small 0.5 μ m * 1.0～3.0 μ m.Do not contain PHB salt in the cell, do not form statospore, Gram-negative, do not move, fricton-tight or swimming, strict aerobic, grow on solid medium, produce typical yellow or orange pigment, some bacterial strain is chromogenesis not, bacterium colony is translucent, circle, protuberance or little protuberance, smooth and gloss again, full edge, catalase, oxydase, Phosphoric acid esterase are all positive, indigestion agar, chemoorganotrophy.Aerogenesis not in the lower concentration protein culture medium.
Based on the above results, the new bacterial strain that screens of the present invention does not belong to any of above-mentioned patent bacterial classification.
This new strain characteristics is:
Colonial morphology: smooth, glossy, neat in edge, white, ash or faint yellow is cultivated about colony diameter 1mm～2mm of 18h～36h; Inclined-plane lawn white, thickness.
Cellular form: do not form the PHB particle in the cell, do not move, shaft-like, the thalline mean size is: 0.4～0.5 μ m * 1.3～1.4 μ m.Gram-negative, no gemma.
Physiological and biochemical property: liquefy gelatin, the egg yolk reaction positive, lipase (soil temperature 80) reacting positive, oxidase negative, the catalase positive, the indole reaction feminine gender, the methyl red feminine gender, the V-P reaction negative produces H
2S, oxygenolysis glucose not, oxidized form produces acid, utilizes malonate, utilizes Citrate trianion, does not reduce nitrate, can not denitrification, can not utilize starch, the urase reacting positive, caseinhydrolysate, can not reduce lactose produces 3-ketone group lactose.
Need somatomedin, produce lysine decarboxylase, the hydrolysis chitin; Make carbon, nitrogenous source with asparagine.
According to above bacterial characteristics, this new bacterial strain is belonged to the bacterial strain of oligotrophy zygosaccharomyces (Stenotrophomonas) by evaluation, is germ oligotrophy unit cell (Stenotrophomonasmaltrophilia).
The substratum that is used for bacterial classification CCTCC No.M 204024 of the present invention is that some contain and can be utilized nutritive substance by above-mentioned bacterial strains, liquid, solid all can, but during a large amount of suitability for industrialized production, liquid nutrient medium is comparatively suitable.Rationally allocate carbon source that mentioned microorganism can utilize, nitrogenous source, inorganic salt etc. in the substratum into.As having of carbon source: validamycin, glucose, lactose, maltose, dextrin, starch, glycerine, N.F,USP MANNITOL, mountain plough 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; Can be added with in addition: 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.
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 validamine and effective mildew enamine stir culture.
Another key character of the present invention is that the effective mould ylidene amines of intermediate product that utilizes this new microbial fermentation or this new microorganism cells or its enzyme to decompose substrate validamycin or validamycin is produced effective mildew enamine and validamine.
New microorganism oligotrophy zygosaccharomyces (Stenotrophomonas) germ oligotrophy unit cell (Stenotrophomonas maltrophilia) of the present invention, CCTCC No.M 204024, substratum with carbonaceous sources, nitrogenous source, inorganic salt, substrate is the microbiotic validamycin, ferment, then decomposing and fermenting liquid is separated validamine and effective mildew enamine, and purify.
Substratum of the present invention consist of (w/v, %): validamycin: 0.5%～20%, (NH
4)
2SO
4: 0.5%～10%, KCl:0.5%～5.0%, Na
2HPO
412H
2O:0.1%～10.0%, NaH
2PO
42H
2O:0.1%～5.0%, MgSO
4: 0.01%～1.0%, with tap water preparation, pH value 6.0～8.0.
With new bacterial strain germ oligotrophy unit cell (Stenotrophomonasmaltrophilia) CCTCC No.M 20402 of the present invention) to produce and imitate Valienamine and validamine, its method has:
(1) substratum of above-mentioned preparation inserts the bacterial classification CCTCC No.M 204024 after cultivating through sterilization, and inclined-plane or with the seed liquor inoculation is cultivated, and the concentration of validamycin is 0.5%～20.0% in the substratum, is good with 1.0%～10.0%; Usually select temperature at 20 ℃～40 ℃, with 28 ℃～35 ℃ better, initial pH is 6.0～8.0, with better near neutrality, be initial pH value with 6.8～7.2 for well, incubation time is that 100h～180h is better, is best with 150h wherein, reaction can be carried out under leaving standstill in addition, but carries out to good under stirring, ventilation, oscillating condition.During the fermentation, the pH value is regulated control with mineral acid or organic acid, bases.Validamycin during substratum is formed in this method is the carbon source that microorganism utilizes, again by the substrate of microbiological degradation; The substrate validamycin is broken down into effective mould ylidene amines, and effective mould ylidene amines has two kinds of A, B, and effective mould ylidene amines through decomposing, generates effective mildew enamine (valienamine) and validamine (validamine) again.
(2) substratum of above-mentioned preparation is through sterilization, access is bacterial classification CCTCC No.M204024 after cultivating, with inclined-plane or seed liquor inoculation, cultivate thalline, culture condition: select temperature at 20 ℃～40 ℃ usually, with 28 ℃～35 ℃ better, initial pH is 6.0～8.0, with near neutral better, promptly initial pH value with 6.5～7.5 for well; Stream adds substrate---validamycin or effective mould ylidene amines solution of microbiological degradation in the process of cultivating, can when just having begun to grow, thalline add by stream, also can be at thalli growth carry out stream for some time of after again and add, the substrate validamycin in the stream adding culture or the concentration of effective mould ylidene amines are 1.0%～50.0%, the speed that stream adds can be regulated according to the concentration of validamycin or effective mould ylidene amines, incubation time is that 100h～180h is better, be best wherein with 150h, reaction can be carried out under leaving standstill in addition, but is stirring, ventilate, carry out under the oscillating condition to good; During the fermentation, the variation of pH value is regulated control with mineral acid or organic acid, bases, makes tunning.
(3) substratum of above-mentioned preparation is through sterilization, access is bacterial classification CCTCC No.M204024 after cultivating, with inclined-plane or seed liquor inoculation, cultivate thalline, culture condition: select temperature at 20 ℃～40 ℃ usually, with 28 ℃～35 ℃ better, initial pH is 6.0～8.0, with near neutral better, promptly initial pH value with 6.5～7.5 for well; Cultivate thalline to logarithmic phase, carry out centrifugation, obtain thalline; The thalline that obtains is added in substrate validamycin or the effective mould ylidene amines solution of substrate, utilize thalline to decompose substrate validamycin or effective mould ylidene amines, the concentration of validamycin or effective mould ylidene amines is 1.0%～20.0%, reaction times is that 1h～100h is better, be best wherein with 70h, reaction can be carried out under leaving standstill in addition, but carries out under stirring, ventilation, oscillating condition to good; During the fermentation, the variation of pH value is regulated control with mineral acid or organic acid, bases, makes tunning.
(4) substratum of above-mentioned preparation is through sterilization, access is bacterial classification CCTCC No.M204024 after cultivating, with inclined-plane or seed liquor inoculation, cultivate thalline, culture condition: select temperature at 20 ℃～40 ℃ usually, with 28 ℃～35 ℃ better, initial pH is 6.0～8.0, with near neutral better, promptly initial pH value with 6.5～7.5 for well; Cultivate thalline to logarithmic phase, carry out centrifugation, obtain thalline,, extract lyase then with bacterial cell disruption; The lyase that extracts is joined in substrate validamycin or the effective mould ylidene amines solution of substrate, utilize enzyme reaction to decompose substrate validamycin or effective mould ylidene amines, the concentration of validamycin or effective mould ylidene amines solution is 1.0%～20.0%, the enzyme reaction time is that 1h～100h is better, be best wherein with 10h, reaction can be carried out under leaving standstill in addition, but carries out under stirring, ventilation, oscillating condition to good; During the fermentation, the variation of pH value is regulated control with mineral acid or organic acid, bases, makes tunning.
The decomposing and fermenting liquid that will contain effective Valienamine and validamine separates, purifying, and specific practice is:
During from the broth extraction target product, can adopt conventional tunning extraction method.As: filter, centrifugal, precipitation, crystallization, recrystallization, concentrate, dry, lyophilize, absorption, ion-exchange, chromatography etc.In addition, the alkaline matter that effective mildew enamine and validamine be soluble in water, be insoluble in common solvent, 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 or chromatography.
Complete leaching process can be divided into pre-treatment, mid-term purifying and refining three phases: (1) pretreatment stage, the target in this stage is with the least possible operation steps and as far as possible simply design, isolates target product from the feed liquid of complexity, remove solid particulate, concentrates.(2) mid-term purification phase, the general purification technique that adopts non-specific and low resolution earlier, adopt again subsequently technology such as high-resolution chromatography mainly be remove physico-chemical property similar with the product physico-chemical property impurity.(3) refining stage, this is last work program, purpose is further to remove impurity, purified product, its method depends on the application purpose of product, the most frequently used method is a crystallization process, and in conjunction with dry, obtains the product of required purity and definite shape.
Method with thin-layer chromatography and gas phase analysis is analyzed the effective mildew enamine and the validamine of fermentative production, operating process and experiment condition are pressed document [J.Antibiot, 1984,37:1301～1305] and the method for patent [EP0063456] carry out, resulting result and they are in full accord.This shows, utilize microorganism strains germ oligotrophy unit cell of the present invention (Stenotrophomonasmaltrophilia) CCTCC No.M 204024 validamycin, effective mould ylidene amines can be cracked into effective mildew enamine and validamine.
Microorganism strains germ oligotrophy unit cell of the present invention (Stenotrophomonasmaltrophilia) CCTCC No.M 204024, can not only on the substratum that with validamycin class material is sole carbon source, grow, and stronger to the capacity of decomposition of validamycin; Utilize CCTCC No.M 204024 to produce effective mildew enamine and validamine, under the preferred process condition, the rate of decomposition of validamycin reaches 70%～90%, the molar yield that Validacin (Takeda) is converted into effective mildew enamine reaches 40%～80% (promptly 1 mole Validacin (Takeda) can be converted into 0.40～0.80 mole effective mildew enamine), the molar yield that effective mould ylidene amines is converted into effective mildew enamine reaches 35%～75%, obtains validamine simultaneously; Microorganism strains germ oligotrophy unit cell of the present invention (Stenotrophomonasmaltrophilia) CCTCC No.M 204024 is applicable to the production of effective mildew enamine and validamine.
Specific embodiments
Following embodiment is intended to illustrate rather than limit the scope of the invention.
Embodiment 1:
Culture medium prescription (weight/volume percent, as follows): validamycin: 1.0%, (NH
4)
2SO
4: 1.0%, KCl:0.5%, Na
2HPO
412H
2O:1.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, with the tap water preparation, pH is transferred to 6.0 with HCl solution.
Get the above-mentioned substratum of 100mL, average mark is contained in 2 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 204024, cultivate thalline, shaking speed 150r/min cultivates 72 hours as seed liquor at 28 ℃ of shaking tables, and is standby.
Get the above-mentioned substratum of 2L, average mark 20 500mL that pack into shake in the bottle, sterilization.Insert seed liquor, inoculum size is 2% (v/v), cultivates, and culture temperature is 20 ℃, and incubation time is 160h, shaking speed 200r/min.
Collect above-mentioned fermented liquid 1.8L, thalline is removed in centrifugation, the absorption of supernatant liquor upper prop (1L macropore weakly acidic resin D113, NH
4 +), after the distillation washing, use the ammoniacal liquor wash-out of 0.5mol/L again, concentrating under reduced pressure is again with column chromatography on the concentrated solution (500mL highly basic chromatographic resin Dowex 1 * 2, OH
-), use the distilled water wash-out, the Fractional Collections elutant, collect flow out earlier contain validamine and after the part of the effective mildew enamine that flows out, method with thin-layer chromatography method and gas phase analysis is analyzed, carry out vacuum lyophilization then, obtain 0.31 gram validamine and 0.74 gram effective mildew enamine.
Embodiment 2:
The culture medium prescription validamycin: 17.0%, (NH
4)
2SO
4: 8.0%, KCl:0.5%, Na
2HPO
412H
2O:1.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, with the tap water preparation, pH is transferred to 7.0 with NaOH solution.
Get the 500mL substratum, average mark is contained in 5 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 204024, cultivate, culture temperature is 28 ℃, and incubation time is 150h, is reflected under stirring, ventilation, the oscillating condition to carry out shaking speed 200r/min.
Collect above-mentioned fermented liquid 400mL, separate, purifying, step obtains 0.56 gram validamine and 0.85 gram effective mildew enamine with embodiment 1.
Embodiment 3:
The fermentative medium formula validamycin: 8.0%, (NH
4)
2SO
4: 6%, KCl:0.1%, Na
2HPO
412H
2O:1.0%, NaH
2PO
42H
2O:0.16%, MgSO
4: 0.02%, with the tap water preparation, pH is transferred to 7.5 with NaOH solution.
Seed culture based formulas validamycin: 1.0%, (NH
4)
2SO
4: 1%, KCl:0.5%, Na
2HPO
412H
2O:1.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.5%, with the tap water preparation, pH is transferred to 7.5 with NaOH solution.
Get the 500mL seed culture medium, average mark is contained in 5 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 204024, cultivate thalline, shaking speed 200r/min cultivates 72 hours as seed liquor at 30 ℃ of shaking tables, and is standby.
Get the 8L fermention medium, average mark is contained in shaking in the bottle of 80 500mL, sterilization.Insert seed liquor, inoculum size is 5% (v/v), cultivates, and culture temperature is 30 ℃, and incubation time is 180h, is reflected under stirring, ventilation, the oscillating condition to carry out shaking speed 200r/min.
Collect above-mentioned fermented liquid 7.5L, separate, purifying, step obtains 3.19 gram validamines and 7.77 gram effective mildew enamines with embodiment 1.
Embodiment 4:
The culture medium prescription validamycin: 2.0%, (NH
4)
2SO
4: 1.0%, KCl:0.5%, Na
2HPO
412H
2O:2.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.1%, with the tap water preparation, pH is transferred to 7.0 with NaOH solution.
Get the 500mL substratum, average mark is contained in 5 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 204024, cultivate thalline, shaking speed 150r/min cultivates 72 hours as seed liquor at 28 ℃ of shaking tables, and is standby.
Get the 8L substratum, average mark is contained in shaking in the bottle of 80 500mL, sterilization.Insert seed liquor, inoculum size is 5% (v/v), cultivates, and culture temperature is 28 ℃, and incubation time is 100h, is reflected under stirring, ventilation, the oscillating condition to carry out shaking speed 200r/min.
Collect 7.5 liters of above-mentioned fermented liquids, separate, purifying, step obtains 2.11 gram validamines and 5.63 gram effective mildew enamines with embodiment 1.
Embodiment 5:
The culture medium prescription validamycin: 1.5%, (NH
4)
2SO
4: 0.75%, KCl:0.5%, Na
2HPO
412H
2O:2.0%, NaH
2PO
42H
2O:1.0%, MgSO
4: 0.01%, with tap water preparation, natural pH.
Get the above-mentioned substratum of 200mL, average mark is contained in 4 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 204024, cultivate thalline, shaking speed 150r/min cultivates 72 hours as seed liquor at 28 ℃ of shaking tables, and is standby.
Add above-mentioned substratum 5L in the 10L fermentor tank, sterilization inserts seed liquor 200mL, ferments.
After inoculation, beginning stream, to add concentration be 10% substrate validamycin solution 1L, flow acceleration 0.2mL/min; 30 ℃ of culture temperature, incubation time are about 160h, air flow 2.5L/min, stirring velocity 200r/min.
Collect above-mentioned fermented liquid 6L, separate, purifying, step obtains 3.67 gram validamines and 6.53 gram effective mildew enamines with embodiment 1.
Embodiment 6:
The culture medium prescription validamycin: 1.0%, (NH
4)
2SO
4: 0.7%, KCl:0.5%, Na
2HPO
412H
2O:0.1%, NaH
2PO
42H
2O:4.0%, MgSO
4: 0.01%, with tap water preparation, natural pH.
Get the above-mentioned substratum of 500mL, average mark is contained in 5 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 204024, cultivate thalline, shaking speed 150r/min cultivates 72 hours as seed liquor at 28 ℃ of shaking tables, and is standby.
Add above-mentioned substratum 5L in the 10L fermentor tank, sterilization inserts seed liquor 200mL, ferments.
Carrying out stream during to logarithmic phase at thalli growth, to add concentration be 2% substrate validamycin solution 3L, flow acceleration 0.5mL/min; 35 ℃ of culture temperature, incubation time are about 180h, air flow 2.5L/min, stirring velocity 200r/min.
Collect above-mentioned fermented liquid 8L, separate, purifying, step obtains 3.08 gram validamines and 5.82 gram effective mildew enamines with embodiment 1.
Embodiment 7:
The culture medium prescription validamycin: 1.0%, (NH
4)
2SO
4: 0.7%, KCl:0.5%, Na
2HPO
412H
2O:6.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.7%, with tap water preparation, natural pH.
Get the 500mL substratum, average mark is contained in 5 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 204024, cultivate thalline, shaking speed 150r/min cultivates 72 hours as seed liquor at 28 ℃ of shaking tables, and is standby.
In the 10L fermentor tank, add above-mentioned substratum 6L as fermented liquid, sterilization.Insert seed liquor 500mL.
Thalli growth during to logarithmic phase stream to add concentration be 50% effective mould ylidene amines solution 100mL, flow acceleration 0.05mL/min; Culture condition: temperature is 30 ℃, and incubation time is 120h, air flow 3.5L/min, stirring velocity 200r/min.
Collect above-mentioned fermented liquid 6L, separate, purifying, step obtains 3.26 gram validamines and 7.01 gram effective mildew enamines with embodiment 1.
Embodiment 8:
The culture medium prescription validamycin: 2.0%, (NH
4)
2SO
4: 1.2%, KCl:4.0%, Na
2HPO
412H
2O:4.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, with the tap water preparation, regulating pH with HCl is 6.5.
Get the 1000mL substratum, average mark is contained in 10 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 204024, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, it is in 5% the substrate validamycin solution that these thalline are joined 500mL concentration, utilizes thalline decomposition validamycin; Reaction conditions: 30 ℃ of temperature, initial pH is 6.5, incubation time is about 100h, shaking speed 150r/min.
Collect above-mentioned reaction solution 480mL, separate, purifying, step obtains 0.45 gram validamine and 1.21 gram effective mildew enamines with embodiment 1.
Embodiment 9:
The culture medium prescription validamycin: 2.0%, (NH
4)
2SO
4: 1.2%, KCl:0.5%, Na
2HPO
412H
2O:1.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, with the tap water preparation, regulating pH with NaOH solution is 7.5.
Get the 1000mL substratum, average mark is contained in 10 500mL triangular flasks, sterilization, insert slant strains CCTCC No.M 204024, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, it is in 15% the effective mould ylidene amines solution of substrate that these thalline are joined 500mL concentration, utilizes thalline to decompose effective mould ylidene amines; Culture condition: 32 ℃ of temperature, initial pH is 7.5, incubation time is about 40h, shaking speed 200r/min.
Collect above-mentioned reaction solution 580mL, separate, purifying, step obtains 1.03 gram validamines and 2.77 gram effective mildew enamines with embodiment 1.
Embodiment 10:
The culture medium prescription validamycin: 2.0%, (NH
4)
2SO
4: 1.2%, KCl:0.5%, Na
2HPO
412H
2O:3.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, with the tap water preparation, regulating pH with NaOH solution is 7.5.
Get the 1000mL substratum, average mark is contained in 10 500mL triangular flasks, sterilization, insert slant strains CCTCC No.M 204024, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, it is in 3% the effective mould ylidene amines solution of substrate that thalline is joined 500mL concentration, utilizes thalline to decompose effective mould ylidene amines; Culture condition: 34 ℃ of temperature, initial pH is 7.5, incubation time is about 20h, shaking speed 150r/min.
Collect above-mentioned reaction solution 580mL, separate, purifying, step obtains 0.53 gram validamine and 1.06 gram effective mildew enamines with embodiment 1.
Embodiment 11:
The culture medium prescription validamycin: 2.0%, (NH
4)
2SO
4: 1.2%, KCl:0.5%, Na
2HPO
412H
2O:0.2%, NaH
2PO
42H
2O:1.5%, MgSO
4: 0.01%, regulating pH with HCl solution is 6.0.
Get the above-mentioned substratum of 2000mL, average mark is contained in 20 500mL triangular flasks, and sterilization inserts slant strains CCTCC No.M 204024, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, with ultrasonic wave with bacterial cell disruption, carry out again centrifugal, saltout, dialysis, chromatography, extract lyase, it is in 8% the substrate validamycin solution, to utilize enzyme reaction to decompose validamycin and produce validamine and effective mildew enamine that the lyase that said extracted is gone out adds 200mL concentration.Culture condition: 25 ℃ of temperature, initial pH is 6.0, and incubation time is about 10h, and reaction can be carried out under leaving standstill.
Collect above-mentioned reaction solution 200mL, separate, purifying, step obtains 0.15 gram validamine and 0.34 gram effective mildew enamine with embodiment 1.
Embodiment 12:
The culture medium prescription validamycin: 2.0%, (NH
4)
2SO
4: 1.2%, KCl:0.5%, Na
2HPO
412H
2O:1.5%, NaH
2PO
42H
2O:0.8%, MgSO
4: 0.01%, regulating pH with NaOH solution is 8.0.
Get the 2000mL substratum, average mark is contained in 20 500mL triangular flasks, sterilization, insert slant strains CCTCC No.M 204024, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, with ultrasonic wave with bacterial cell disruption, carry out again centrifugal, saltout, dialysis, chromatography, extract lyase, the lyase that said extracted is gone out, add 100mL concentration and be in 20% the substrate validamycin solution, utilize enzyme reaction to decompose validamycin.Reaction conditions: 32 ℃ of temperature, initial pH is 8.0, and incubation time is about 80h, and reaction can be carried out under leaving standstill.
Collect above-mentioned reaction solution 100mL, separate, purifying, step obtains 0.21 gram validamine and 0.39 gram effective mildew enamine with embodiment 1.
Embodiment 13:
The culture medium prescription validamycin: 2.0%, (NH
4)
2SO
4: 1.2%, KCl:0.5%, Na
2HPO
412H
2O:1.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, regulating pH with NaOH is 7.8.
Get the 2000mL substratum, average mark is contained in 20 500mL triangular flasks, sterilization, insert slant strains CCTCC No.M 204024, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, with ultrasonic wave with bacterial cell disruption, carry out again centrifugal, saltout, dialysis, chromatography, extract lyase, the lyase that said extracted is gone out, add 100mL concentration and be in 10% the effective mould ylidene amines solution of substrate, utilize enzyme reaction to decompose effective mould ylidene amines and produce validamine and effective mildew enamine.Reaction conditions: 30 ℃ of temperature, initial pH is 7.0, incubation time is about 30h, shaking speed 100r/min.
Collect above-mentioned reaction solution 100mL, separate, purifying, step obtains 0.10 gram validamine and 0.31 gram effective mildew enamine with embodiment 1.
Embodiment 14:
The culture medium prescription validamycin: 2.0%, (NH
4)
2SO
4: 1.2%, KCl:0.5%, Na
2HPO
412H
2O:1.0%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, regulating pH with NaOH is 7.0.
Get the above-mentioned substratum of 2000mL, average mark is contained in 20 500mL triangular flasks, sterilization, insert slant strains CCTCC No.M 204024, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, with ultrasonic wave with bacterial cell disruption, carry out again centrifugal, saltout, dialysis, chromatography, extract lyase, the lyase that said extracted is gone out, join 100mL concentration and be in 18% the effective mould ylidene amines solution of substrate, utilize enzyme reaction to decompose effective mould ylidene amines.Reaction conditions: 30 ℃ of temperature, initial pH is 7.0, incubation time is about 50h, shaking speed 100r/min.
Collect above-mentioned reaction solution 100mL, separate, purifying, step obtains 0.33 gram validamine and 0.59 gram effective mildew enamine with embodiment 1.
Claims (12)
1. the microbe preparation method of effective mildew enamine and validamine, it is characterized in that described microorganism is oligotrophy zygosaccharomyces (Stenotrophomonas) germ oligotrophy unit cell (Stenotrophomonas maltrophilia), CCTCC No.M 204024, the substratum of germ oligotrophy unit cell and carbonaceous sources, nitrogenous source, inorganic salt, substrate is the microbiotic validamycin, ferment, decomposing and fermenting liquid is separated validamine and effective mildew enamine, and purify.
2. preparation method according to claim 1 is characterized in that described substratum composition, its weight/volume: validamycin 0.5%～20.0%, (NH
4)
2SO
40.5%～10.0%, KCl0.5%～5.0%, Na
2HPO
412H
2O 0.1%～10.0%, NaH
2PO
42H
2O 0.1%～5.0%, MgSO
40.01%～1.0%, with the tap water preparation, regulate pH value 6.0～8.0.
3. preparation method according to claim 1, the separation, the purification step that it is characterized in that described fermented liquid are: reaction solution is centrifugal, remove thalline, the absorption of supernatant liquor upper prop, after the distillation washing, use the ammoniacal liquor wash-out of 0.5mol/L again, concentrating under reduced pressure, with column chromatography on the concentrated solution, use the distilled water wash-out again, the Fractional Collections elutant, analyze with the thin-layer chromatography method, collect flow out earlier contain validamine and after the part of the effective mildew enamine that flows out, carry out vacuum lyophilization respectively, obtain validamine and effective mildew enamine.
4. preparation method according to claim 1 and 2, it is characterized in that the fermention medium after inserting CCTCC No.M 204024 ferments, the concentration 0.5%～20.0% of validamycin in the substratum, culture condition: 20 ℃～40 ℃ of temperature, initial pH is 6.0～8.0, and incubation time is 100h～180h.
5. preparation method according to claim 4, the concentration that it is characterized in that validamycin in the fermention medium is 1.0%～10.0%, 28 ℃～35 ℃ of leavening temperatures, initial pH is 6.8～7.2, incubation time is 150h.
6. preparation method according to claim 1, after it is characterized in that in substratum, inserting bacterial classification CCTCC No.M 204024, stream adds the substrate validamycin in culturing process, beginning stream when thalline has just begun to grow adds, culture condition: temperature is at 20 ℃～40 ℃, initial pH is 6.0～8.0, and incubation time is 100h～180h.
7. preparation method according to claim 6 is characterized in that carrying out the current adding substrate validamycin, culture condition again after the thalli growth logarithmic phase: 28 ℃～35 ℃, initial pH is 6.5～7.5, incubation time 150h.
8. preparation method according to claim 1, it is characterized in that in substratum, inserting bacterial classification CCTCC No.M 204024 backs and cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, thalline is joined in the substrate validamycin solution, utilize thalline to decompose the substrate validamycin, culture condition: temperature is at 20 ℃～40 ℃, initial pH is 6.0～8.0, and incubation time is 1h～100h.
9. preparation method according to claim 8 is characterized in that thalline being joined in the substrate validamycin solution culture condition: 28 ℃～35 ℃ of temperature, and initial pH is 6.5～7.5, incubation time is 70h.
10. preparation method according to claim 1, it is characterized in that in substratum, inserting bacterial classification CCTCC No.M 204024 backs and cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, again with supersonic method with bacterial cell disruption, extract lyase, lyase is joined in the validamycin solution, culture condition: 20 ℃～40 ℃ of temperature, initial pH is 6.0～8.0, incubation time is 1h～100h.
11. preparation method according to claim 10 is characterized in that lyase being joined in the substrate validamycin solution culture condition: 28 ℃～35 ℃ of temperature, initial pH is 6.5～7.5, incubation time is 10h.
12., it is characterized in that substrate is effective mould ylidene amines according to the described preparation method in one of claim 1 or 6～11.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410017516 CN1273606C (en) | 2004-04-05 | 2004-04-05 | Microbe method for preparing enamine and amine from valinemia |
| PCT/CN2005/000267 WO2005098014A1 (en) | 2004-04-05 | 2005-03-07 | Microbe method for producing valienamine and validamine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410017516 CN1273606C (en) | 2004-04-05 | 2004-04-05 | Microbe method for preparing enamine and amine from valinemia |
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| Publication Number | Publication Date |
|---|---|
| CN1563397A CN1563397A (en) | 2005-01-12 |
| CN1273606C true CN1273606C (en) | 2006-09-06 |
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ID=34479006
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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| Country | Link |
|---|---|
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| WO (1) | WO2005098014A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245325B (en) * | 2007-02-12 | 2010-12-08 | 青岛大学 | Stenotrophomonas maltophilia of strain producing novel alkali proteinase |
| CN102329751A (en) * | 2011-09-21 | 2012-01-25 | 江南大学 | Stenotrophomonas maltophilia for generating keratinase and application of stenotrophomonas maltophilia |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100371451C (en) * | 2005-09-06 | 2008-02-27 | 浙江工业大学 | Preparation of effective mycoenamine with microbial lytic acrose and its derivative |
| CN1935778B (en) * | 2005-09-21 | 2011-04-27 | 上海来益生物药物研究开发中心有限责任公司 | Method for separating and purifying valienamine and validamine |
| CN100362108C (en) * | 2005-11-01 | 2008-01-16 | 浙江工业大学 | Microbial process of producing validamycin anamine and validamycin amine |
| CN1325655C (en) * | 2005-11-01 | 2007-07-11 | 浙江工业大学 | Microbial validamycin cracking process of producing validamycin anamine and validamycin amine |
| CN101050447B (en) * | 2006-04-07 | 2010-10-13 | 上海来益生物药物研究开发中心有限责任公司 | Gene engineering bacterium of beta - glucosaccharase, and application |
| CN100460501C (en) * | 2006-09-01 | 2009-02-11 | 江苏新天地生物肥料工程中心有限公司 | Organism preparation method of agricultural amino acid and fertilizer product |
| CN104823977B (en) * | 2015-04-24 | 2017-06-06 | 浙江省桐庐汇丰生物科技有限公司 | A kind of bactericidal composition of validoxylamine A with amino-oligosaccharide and its application |
| CN111254091B (en) * | 2020-01-20 | 2022-04-19 | 浙江工业大学 | Stenotrophomonas maltophilia GYH and application thereof in degradation of chlorinated hydrocarbon pollutants |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| JPH0669380B2 (en) * | 1985-10-08 | 1994-09-07 | 武田薬品工業株式会社 | Method for producing valienamine and validamine |
-
2004
- 2004-04-05 CN CN 200410017516 patent/CN1273606C/en not_active Expired - Lifetime
-
2005
- 2005-03-07 WO PCT/CN2005/000267 patent/WO2005098014A1/en active Application Filing
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245325B (en) * | 2007-02-12 | 2010-12-08 | 青岛大学 | Stenotrophomonas maltophilia of strain producing novel alkali proteinase |
| CN102329751A (en) * | 2011-09-21 | 2012-01-25 | 江南大学 | Stenotrophomonas maltophilia for generating keratinase and application of stenotrophomonas maltophilia |
| CN102329751B (en) * | 2011-09-21 | 2013-07-24 | 江南大学 | Stenotrophomonas maltophilia for generating keratinase and application of stenotrophomonas maltophilia |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1563397A (en) | 2005-01-12 |
| WO2005098014A1 (en) | 2005-10-20 |
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