CN100371451C - Preparation of effective mycoenamine with microbial lytic acrose and its derivative - Google Patents
Preparation of effective mycoenamine with microbial lytic acrose and its derivative Download PDFInfo
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- CN100371451C CN100371451C CNB2005100606385A CN200510060638A CN100371451C CN 100371451 C CN100371451 C CN 100371451C CN B2005100606385 A CNB2005100606385 A CN B2005100606385A CN 200510060638 A CN200510060638 A CN 200510060638A CN 100371451 C CN100371451 C CN 100371451C
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- Prior art keywords
- acarbose
- derivative
- thalline
- effective mildew
- klebsiella
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- HCPOCMMGKBZWSJ-UHFFFAOYSA-N ethyl 3-hydrazinyl-3-oxopropanoate Chemical compound CCOC(=O)CC(=O)NN HCPOCMMGKBZWSJ-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 235000013882 gravy Nutrition 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 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
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 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
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening 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
- 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
- 239000007787 solid Substances 0.000 description 1
- 239000011973 solid acid Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- YCJYNBLLJHFIIW-MBABXGOBSA-N validoxylamine A Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)C[C@@H]1N[C@@H]1[C@H](O)[C@@H](O)[C@H](O)C(CO)=C1 YCJYNBLLJHFIIW-MBABXGOBSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 150000008495 β-glucosides Chemical class 0.000 description 1
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention discloses a new microorganism capable of preparing validamycin enamine by cracking acarbose and/or derivatives thereof, which belongs to the klebsiella oxytoca (CCTCC No. M 205091) of the klebsiella. The present invention also discloses a method by using the microorganism to crack acarbose and/or derivatives thereof to prepare validamycin enamine, namely that the microorganism (CCTCC No. M 205091) directly ferments and decomposes substrate acarbose and/or derivatives thereof by the culture of a culture medium, or mycelia are obtained by the culture of the culture medium; cells are used for the catalytic decomposition of the substrate acarbose and/or derivatives thereof to produce the validamycin enamine.
Description
Technical field
The present invention relates to a kind of from soil screening and separating to new microorganism, also relate to the method for utilizing this new microbiological degradation acarbose (acarbose) and acarbose derivative (acarbosederivatives) thereof to prepare effective mildew enamine (valienamine).
Background technology
The effective mildew enamine that the present invention relates to, English valienamine by name, its structural formula is as follows: (Chem.Rev.2003,103:1955-1977).
Effective mildew enamine claims valienamine again, its chemical name: (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.
Effective mildew enamine is a kind of stronger glycosidase inhibitor, also is simultaneously the core texture of a lot of other glycosidase inhibitors, can be used for synthetic effective Valiolamine (valiolamine) and voglibose (voglibose).
The method of producing effective mildew enamine mainly contains following several:
(1) with validamycin (validamycins) [jingganmycin (Jinggangmycins)], effective mould ylidene amines [the mould ylidene amines in well ridge (validoxylamine)] and derivative thereof are the feedstock production effective mildew enamine.Validamycin is a kind of efficient, safe agricultural antibiotic, and is 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, 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.
1. microbial lytic: major microorganisms has Flavobacterium saccharophilum, Pseudomonas denitrificans etc.,, decomposing validamycin and obtain effective mildew enamine by technology such as fermentation, cell catalysis as substrate with validamycin, carry out separation and purification through methods such as ion-exchange, chromatographies again, is one of method of present suitability for industrialized production effective mildew enamine.
2. NBS (N-Bromosuccinimide) reagent cracking process preparation, utilize NBS reagent, C-N key in effective mould ylidene amines or derivatives thereof can rupture, obtain effective mildew enamine, also can generate some cyclohexanone analog materials in addition, the lysate composition is comparatively complicated, can separate by methods such as ion-exchanges to obtain effective mildew enamine, but not have the report of suitability for industrialized production at present.
(2) be the feedstock production effective mildew enamine with acarbose and/or its derivative
Acarbose is used for the treatment of a kind of medicine of diabetes, and from the structure of acarbose as can be seen, acarbose mainly is made up of maltose and effective mildew enamine.Korea S HUR YUL, the patent WO 2004108657 of et al. application and WO 2004000782 have reported with trifluoroacetic acid (TFA) or solid acid hydrolysis acarbose and/or its derivative and have prepared effective mildew enamine.Acarbose and/or its derivative are that feedstock production effective mildew enamine reaction formula is as follows:
(3) chemical synthesis preparation since separation in 1972 obtains effective mildew enamine, the method for many chemosynthesis effective mildew enamines occurred, and still the major part by chemosynthesis is a racemic modification, and yield is lower.It is one of method of present industrial production effective mildew enamine.
(4) extract from streptomyces hygroscopicus (Streptomyces hygroscopicus) fermented liquid, streptomyces hygroscopicus can generate a spot of precursor substance effective mildew enamine in the process of the synthetic validamycin of fermentation.Therefore, the method by ion-exchange can obtain effective mildew enamine by extraction separation from the fermented liquid of validamycin.But the concentration of effective mildew enamine is very low in the fermented liquid, and it is bigger to extract difficulty, does not have industrial report.
At present, the method for suitability for industrialized production effective mildew enamine mainly is: microbiological deterioration validamycin method; With glucose is that starting raw material carries out chemical synthesis.
Till now, domestic and foreign literature does not prepare the report in this field of effective mildew enamine about microbiological degradation acarbose and derivative thereof.
Summary of the invention
The invention provides a kind of new microorganism strains that from soil, screens acarbose and/or its derivative cracking production effective mildew enamine; Next provides a kind of method of utilizing this new microbe cracking acarbose and/or its derivative to prepare effective mildew enamine.
New microorganism strains provided by the invention belongs to the acid-producing Klebsiella bacterium (Klebsiella oxytoca) of Klebsiella (Klebsiella), oneself is preserved in Chinese typical culture collection center this bacterial strain on August 18th, 2005, be called for short CCTCC, deposit number is CCTCC No.M205091.
The new strain characteristics that the present invention screens from soil is as follows:
Colonial morphology: cultivated 2 days for 30 ℃, rounded at the bacterium colony that beef extract-peptone is dull and stereotyped to be cultivated, 1 millimeter of diameter, projecting shape is a lens-shaped, neat in edge is smooth.
Cellular form: the straight-bar bacterium, size 0.3～0.5 μ m * 0.8～2.0 μ m has the folder film, single, paired or catenation.
Physiological and biochemical property: the new bacterial strain that from soil, screens, be Gram-negative, bacillus, aerobic, do not move edwardsiella hoshinae, lecithinase feminine gender, hydrolyzed starch not, arginine decarboxylase feminine gender, phenylalanine deaminase feminine gender, esterase (Tween 80) positive, the nitrate reduction positive, the nitrite reduction is positive, the denitrification positive, clark and Lubsreaction feminine gender, VP reacting positive, casein hydrolysis feminine gender, the tyrosine hydrolysis positive does not produce 3-ketone group lactose.Oxidase negative, the catalase positive, the urase positive, the lysine decarboxylase positive, beta-glucoside enzyme positive, the beta-galactosidase enzymes positive, the ornithine decarboxylase feminine gender, the arginine dihydrolase feminine gender, lipase feminine gender, N-acetyl-beta-glucosidase feminine gender, β-gluconic acid enzyme feminine gender, α-Fructus Hordei Germinatus glucosidase feminine gender, phenol red reaction negative, edwardsiella hoshinae.Arabitol, semi-lactosi hydrochlorate 5-ketone group-Sunmorl N 60S, N.F,USP MANNITOL, maltose, glucose, sucrose, L-arabinose, D-arabitol, trehalose, rhamnosyl, inositol, Pentitol, cellobiose, sorbyl alcohol, propanedioic acid can be utilized, α-Pu Taotang can not be utilized.Comprehensive The above results, this bacterial strain is acid-producing Klebsiella bacterium (Klebsiella oxytoca).
According to above bacterial characteristics, this new bacterial strain is belonged to the bacterial strain of Klebsiella (Klebsiella) by evaluation, this bacterial strain is acid-producing Klebsiella bacterium (Klebsiella oxytoca), and this microorganism strains has the ability of cracking acarbose and/or its derivative generation effective mildew enamine.
Being used for the present invention is acarbose and/or its derivative as substrate, and the structural formula of acarbose such as figure below have connected a trisaccharide maltose on the amino of effective mildew enamine.
Acarbose derivative is meant the compound that connects a monose, disaccharide, the trisaccharide except that trisaccharide maltose, tetrose on the amino of effective mildew enamine, these materials are present in the fermented liquid of producing acarbose, can carry out extraction separation by methods such as ion-exchange, chromatographies.
New microorganism of the present invention belongs to the acid-producing Klebsiella bacterium (Klebsiella oxytoca) of Klebsiella (Klebsiella), CCTCC No.M 205091.Being used for bacterial classification CCTCC No.M 205091 of the present invention can be by method cracking acarbose and/or its derivative of fermentation.The usually used substratum of culturing micro-organisms is except containing acarbose and/or its derivative, can also contain the nutritive substance that some can be utilized by above-mentioned bacterial strains.The carbon source that can add on a small quantity has: glucose, lactose, maltose, dextrin, starch, glycerine 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.) etc.; Inorganic salts has: metallic salts such as Na, K, Ca, Mg, phosphoric acid salt or acetate etc.Rationally allocate carbon source, nitrogenous source and inorganic salt etc. that mentioned microorganism can utilize in the substratum into.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 stir culture.
The present invention's fermentative medium formula (ratio of weight/volume) preferably is as follows: acarbose and/or acarbose derivative: 1.0%～5.0%, and (NH
4)
2SO
4: 0.5%～5.0%, KCl:0.1%～1.0%, Na
2HPO
412H
2O:0.1%～2.0%, MgSO
4: 0.02%～0.1%, with the tap water preparation, regulate pH value 6.0～8.0 with hydrochloric acid or the NaOH solution of 1.0mol.
Fermentating controling condition: select 28 ℃～35 ℃ of temperature usually, initial pH is 6.0～8.0, and during shake flask fermentation, shaking speed is: 100～300r/min, ventilation during ferment tank: 1.0～10.0vvm, fermentation time 72h～192h.
Another key character of the present invention is to utilize this new microorganism cells catalytic decomposition acarbose and/or its derivative to produce effective mildew enamine, this method as follows:
Microorganism CCTCC No.M 205091 (Klebsiella oxytoca ZJB-051), cultivate logarithmic phase through above-mentioned substratum and control condition, then with medium centrifugal, obtain thalline, thalline added in acarbose and/or its derivative solution react, acarbose and/or its derivative solution concentration are (ratio of weight/volume): 1.0%～10.0%; 28 ℃～35 ℃ of temperature of reaction, reaction times 4h～72h carries out under wave and culture or airy condition.
From the reaction solution of microbial fermentation solution or cell catalysis, extract effective mildew enamine, can adopt filtration, method such as centrifugal to carry out solid-liquid separation earlier, again supernatant liquor is carried out ion-exchange with macroporous ion exchange resin, use the ammonia soln wash-out, collect the elutriant concentrating under reduced pressure, concentrated solution carries out chromatography with the strong basicity chromatographic resin, and water is collected the part that contains effective mildew enamine as eluent.Method with thin-layer chromatography and gas phase analysis is analyzed the effective mildew enamine of producing, 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 energy cracking acarbose of the present invention and/or its derivative to prepare effective mildew enamine.
At present, the production technology comparative maturity of China's acarbose, a lot of producers are all producing this medicine.In producing the fermented liquid of acarbose, except containing acarbose, also contain acarbose derivative, in existing acarbose production technique, extract acarbose after, other components all abandon need not.Microorganism CCTCC No.M 205091 among the present invention not only can decompose acarbose, can also decompose the derivative of acarbose.Therefore, production cost is low, and economic benefit is huge, is suitable for suitability for industrialized production, has a good application prospect.
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
Fermentative medium formula (ratio of weight/volume, as follows): acarbose 2.0%, (NH
4)
2SO
41.5%, KCl 0.5%, Na
2HPO
412H
2O 1.0%, MgSO
40.02%, with the tap water preparation, pH is adjusted to 6.0, and is standby.
Get the above-mentioned substratum of 150mL, average mark is contained in 3 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h as seed liquor at 30 ℃ 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 5% (v/v), cultivates, and culture temperature is 28 ℃, and shaking speed 200r/min, fermentation time are 120h.
Collect above-mentioned fermented liquid 1.8L, 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, R
f=0.45), the Fractional Collections elutant, the part of collecting the effective mildew enamine that flows out after containing is carried out vacuum lyophilization then, obtains 2.8 gram effective mildew enamines.
Embodiment 2
Fermentative medium formula: acarbose 1.0%, (NH
4)
2SO
40.5%, KCl 0.1%, Na
2HPO
412H
2O 0.1%, MgSO
40.02%, with the tap water preparation, pH is adjusted to 7.0, and is standby.
Get the above-mentioned seed culture medium of 150mL, average mark is contained in 3 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 150r/min cultivates 48h 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 5% (v/v), cultivates, and culture temperature is 30 ℃, and shaking speed 200r/min, fermentation time are 72h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 1.1 gram effective mildew enamines with embodiment 1.
Embodiment 3
Fermentative medium formula: acarbose 2.5%, (NH
4)
2SO
42.0%, KCl 1.0%, Na
2HPO
412H
2O 2.0%, MgSO
40.1%, with the tap water preparation, pH is adjusted to 8.0, and is standby.
Get the above-mentioned substratum of 150mL, average mark is contained in 3 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h as seed liquor at 32 ℃ 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 5% (v/v), cultivates, and culture temperature is 32 ℃, shaking speed 200r/min.Fermentation time is 144h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 3.0 gram effective mildew enamines with embodiment 1.
Embodiment 4
Fermentative medium formula: acarbose 5.0%, (NH
4)
2SO
45.0%, KCl 0.5%, Na
2HPO
412H
2O 0.5%, MgSO
40.05%, with the tap water preparation, pH is adjusted to 7.2, and is standby.
Get the above-mentioned substratum of 150mL, average mark is contained in 3 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h 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 5% (v/v), cultivates, and culture temperature is 35 ℃, shaking speed 200r/min.Fermentation time is 192h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 3.4 gram effective mildew enamines with embodiment 1.
Embodiment 5
Fermentative medium formula: acarbose 2.0%, (NH
4)
2SO
41.5%, KCl 0.5%, Na
2HPO
412H
2O 0.5%, MgSO
40.02%, with the tap water preparation, pH regulator is 6.8, and is standby.
Get the above-mentioned seed culture medium of 500mL, average mark is contained in 10 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h as seed liquor at 32 ℃ of shaking tables, and is standby.
Get the above-mentioned substratum of 10L, the 15L fermentor tank of packing into, sterilization.Insert seed liquor, inoculum size is 5% (v/v), ferments, and culture temperature is 30 ℃, rotating speed 200r/min, and ventilation 4.0vvm, fermentation time are 120h.
Collect above-mentioned fermented liquid 10L, separate, purifying, step obtains 8.5 gram effective mildew enamines with embodiment 1.
Embodiment 6
Culture medium prescription: acarbose derivative (connect a monose on the amino of effective mildew enamine, or tetrose, the mixture of two kinds of derivatives) 5.0%, (NH
4)
2SO
45.0%, KCl 0.5%, Na
2HPO
412H
2O 1.0%, MgSO
40.1%, with the tap water preparation, pH is adjusted to 7.0, and is standby.
Get the above-mentioned substratum of 150mL, average mark is contained in 3 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h as seed liquor at 30 ℃ 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 5% (v/v), cultivates, and culture temperature is 35 ℃, and shaking speed 200r/min, fermentation time are 192h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 2.1 gram effective mildew enamines with embodiment 1.
Embodiment 7
Culture medium prescription: acarbose and acarbose derivative thereof (connect a monose on the amino of effective mildew enamine, or trisaccharide, the mixture of two kinds of derivatives) 2.5%, (NH
4)
2SO
42.0%, KCl 1.0%, Na
2HPO
412H
2O 2.0%, MgSO
40.5%, with the tap water preparation, pH is adjusted to 8.0, and is standby.
Get the above-mentioned substratum of 150mL, average mark is contained in 3 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h as seed liquor at 30 ℃ 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 5% (v/v), cultivates, and culture temperature is 32 ℃, and shaking speed 200r/min, fermentation time are 152h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 1.5 gram effective mildew enamines with embodiment 1.
Embodiment 8
Culture medium prescription: acarbose derivative (connect a monose on the amino of effective mildew enamine, or disaccharide, the mixture of two kinds of derivatives) 1.0%, (NH
4)
2SO
40.5%, KCl 0.1%, Na
2HPO
412H
2O 0.1%, MgSO
40.02%, with the tap water preparation, pH is adjusted to 6.0, and is standby.
Get the above-mentioned substratum of 150mL, average mark is contained in 3 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h as seed liquor at 30 ℃ 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 5% (v/v), cultivates, and culture temperature is 28 ℃, and shaking speed 200r/min, fermentation time are 72h.
Collect above-mentioned fermented liquid 1.8L, separate, purifying, step obtains 1.1 gram effective mildew enamines with embodiment 1.
Embodiment 9
Culture medium prescription: acarbose derivative (connect a trisaccharide on the amino of effective mildew enamine, or tetrose, the mixture of two kinds of derivatives) 2.5%, (NH
4)
2SO
41.5%, KCl 0.5%, Na
2HPO
412H
2O 0.5%, MgSO
40.03%, with the tap water preparation, pH is adjusted to 7.0, and is standby.
Get the above-mentioned seed culture medium of 500mL, average mark is contained in 10 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h as seed liquor at 30 ℃ of shaking tables, and is standby.
Get the above-mentioned ferment tank substratum of 10L, the 15L fermentor tank of packing into, sterilization.Insert seed liquor, inoculum size is 5% (v/v), ferments, and culture temperature is 30 ℃, ventilation 4.0vvm, and rotating speed 200r/min, fermentation time are 144h.
Collect above-mentioned fermented liquid 10L, separate, purifying, step obtains 5.6 gram effective mildew enamines with embodiment 1.
Embodiment 10
Culture medium prescription: acarbose and/or acarbose derivative: 1.0%～5.0%, (NH
4)
2SO
4: 0.5%～5.0%, KCl:0.1%～1.0%, Na
2HPO
412H
2O:0.1%～2.0%, MgSO
4: 0.02%～0.1%, with the tap water preparation, be hydrochloric acid or the NaOH solution adjusting pH value 6.0～8.0 of 1.0mol/L with concentration.
Get the above-mentioned substratum of 150mL, average mark is contained in 3 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205091, cultivate thalline, shaking speed 200r/min cultivates 48h as seed liquor at 35 ℃ 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 5% (v/v), cultivates, and culture temperature is 30 ℃, and shaking speed 200r/min cultivates logarithmic phase, and medium centrifugal obtains thalline.
It is in 10.0% the acarbose solution that the above-mentioned thalline that obtains is joined 500mL concentration, utilizes thalline to decompose acarbose; Reaction conditions: 35 ℃ of temperature, shaking speed 100r/min, the reaction times is about 72h.Collect above-mentioned reaction solution 500mL, separate, purifying, step obtains 1.7 gram effective mildew enamines with embodiment 1.
Embodiment 11
The preparation of thalline is with embodiment 10;
It is in 5.0% acarbose derivative (connecting a monose on the amino of effective mildew enamine, or disaccharide, or tetrose, the mixture of the three kinds of derivatives) solution that the thalline that obtains is joined 500mL concentration, utilizes thalline to decompose acarbose; Reaction conditions: 28 ℃ of temperature, shaking speed 100r/min, the reaction times is about 36h.Collect above-mentioned reaction solution 500mL, separate, purifying, step obtains 0.8 gram effective mildew enamine with embodiment 1.
Embodiment 12
The preparation of thalline is with embodiment 10;
It is in 1.0% the acarbose solution that the thalline that obtains is joined 500mL concentration, utilizes thalline to decompose acarbose; Reaction conditions: 32 ℃ of temperature, shaking speed 100r/min, the reaction times is about 4h.Collect above-mentioned reaction solution 500mL, separate, purifying, step obtains 0.5 gram effective mildew enamine with embodiment 1.
Embodiment 13
The preparation of thalline is with embodiment 10;
It is in 2.0% the acarbose and acarbose derivative (connect a disaccharide on the amino of effective mildew enamine, or connect tetrose, the mixture of two kinds of derivatives) solution thereof that the thalline that obtains is joined 500mL concentration, utilizes thalline to decompose acarbose; Reaction conditions: 32 ℃ of temperature, shaking speed 100r/min, the reaction times is about 48h.Collect above-mentioned reaction solution 500mL, separate, purifying, step obtains 1.3 gram effective mildew enamines with embodiment 1.
Claims (4)
1. method of utilizing microbial lytic acarbose and/or its derivative to prepare effective mildew enamine, it is characterized in that: described microorganism belongs to the acid-producing Klebsiella bacterium (Klebsiella oxytoca) of Klebsiella (Klebsiella), CCTCC No.M 205091; The substratum of described acid-producing Klebsiella bacterium and carbonaceous sources, nitrogenous source, inorganic salt, substrate is acarbose and/or its derivative, cultivate, ferment, decompose substrate acarbose and/or its derivative, then the effective mildew enamine in the decomposed solution is separated purification; Described acarbose derivative is meant the compound that connects monose, disaccharide, the trisaccharide except that trisaccharide maltose or a tetrose on the amino of effective mildew enamine.
2. method according to claim 1, it is characterized in that inserting microorganism CCTCC No.M 205091 and ferment in substratum, decompose substrate acarbose and/or its derivative, leavening temperature is 28 ℃～35 ℃, initial pH is 6.0～8.0, fermentation time 72h～192h.
3. method according to claim 1, it is characterized in that inserting in substratum microorganism CCTCC No.M 205091 cultivates, cultivate thalline to logarithmic phase, centrifugation obtains thalline, thalline joined in substrate acarbose and/or its derivative solution carry out cell catalysis, utilize thalline to decompose substrate.
4. method according to claim 3 is characterized in that the specific concentration of the weight/volume of acarbose and/or its derivative solution is: 1.0%～10.0%; 28 ℃～35 ℃ of temperature of reaction, reaction times 4h～72h.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004000782A1 (en) * | 2002-06-25 | 2003-12-31 | B T Gin., Inc. | Preparation method of valienamine from acarbose and/or acarbose derivatives using trifluoroacetic acid |
| WO2004108657A1 (en) * | 2003-06-11 | 2004-12-16 | B T Gin., Inc. | Preparation method of valienamine using solid catalysts |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004000782A1 (en) * | 2002-06-25 | 2003-12-31 | B T Gin., Inc. | Preparation method of valienamine from acarbose and/or acarbose derivatives using trifluoroacetic acid |
| WO2004108657A1 (en) * | 2003-06-11 | 2004-12-16 | B T Gin., Inc. | Preparation method of valienamine using solid catalysts |
| 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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