CN106167815A - The Preparation method and use of it dimension streptozotocin derivative - Google Patents
The Preparation method and use of it dimension streptozotocin derivative Download PDFInfo
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- 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
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- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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Abstract
The invention belongs to biological technical field, be specifically related to the Preparation method and use of sky dimension streptozotocin derivative, including 1) prepare red saccharopolyspora seed liquor；2) sky dimension streptozotocin derivative is prepared.The conversion ratio that the method for the present invention prepares sky dimension streptozotocin derivative is high, economically feasible.Sky dimension streptozotocin derivative prepared by the present invention is for preparing preventing and treating agriculture and forestry injurious insect or evil demodicid mite medicine, and this medicine has the effect of excellence when preventing and treating agriculture and forestry injurious insect or evil demodicid mite.
Description
Technical field
The invention belongs to biological technical field, be specifically related to 4 "-O-glucosyl tenvermectins A/B and profit thereof
The method prepared with microorganism conversion and the application in preparation preventing and treating agriculture and forestry injurious insect and evil demodicid mite medicine.
Background technology
It dimension rhzomorph (Tenvermectin) is novel ten hexa-atomic macrolide antibiotics, has wide spectrum, efficient, low
Poison, dosage is little, use the features such as safer, wherein sky dimension rhzomorph A/B chemical structural formula as follows:
Through determination of activity, it is compared with avilamycin, ivermectin and mibemycin, to aphid, demodicid mite, Lepidoptera class evil
Worm and the insect of other damage to crops and livestock intestinal tract parasite all have more preferable prevention effect, are that a kind of prospect is the best
New generation of environment protection insecticide.
It is, by microbial cell, complicated substrate is carried out structural modification that microorganism converts, and namely utilizes microorganism generation
Certain or certain a series of enzyme produced during thanking carries out catalytic reaction to substrate specific part (group), makes substrate molecule
Structure is changed to similar another kind of compound.Microorganism converts possesses reaction orientation, and product is the most single, and side reaction is few,
The advantages such as biomass accumulation is fast, transformation time is short, invertase expression efficiency high, and reaction condition is gentle, safety and environmental protection, are natural work
Property compound carry out structural modification a kind of convenient means.Convert sky dimension rhzomorph A/B with microorganism, obtain new, structure class
As reactive compound, to Expansion development utilize a new generation sky dimension rhzomorph Macrocyclolactone lactone kind medicine significant.
Summary of the invention
The present invention provides 4, and " Preparation method and use of-O-glucosyl tenvermectins A/B, the present invention passes through
Microorganism converts sky dimension rhzomorph and obtains a kind of new sky dimension streptozotocin derivative, has good Insecticiding-miticiding effect, has exploitation
Become the potentiality of a new generation's environment-friendly pesticide.
Offer a kind of 4 is provided " preparation method of-O-glucosyl tenvermectins A/B,
Comprise the steps:
1) red saccharopolyspora seed liquor is prepared: take the S. erythraea strain that preserving number is ATCC 11635
Saccharopolyspora erythraea, streak inoculation is on YMS solid medium, in the constant incubator of 20-28 DEG C
Cultivate 5-7 days, by the strain transfer after activation in fluid medium, be placed in 20-28 DEG C, the horizontal shaker of 250rpm is cultivated
40 hours, obtain seed liquor；
2) "-O-glucosyl tenvermectins A/B: seed liquor is transferred in converting in culture medium, cultivate for preparing 4
The conversion of substrate sky dimension rhzomorph A/B that after 24 hours, addition is dissolved with organic solvent, at 20-28 DEG C, converts under conditions of 250rpm
After cultivating 150 hours, obtain fermentation liquid；After using extractant extractive fermentation liquid and reclaiming extractant, purifies and separates obtains 4 "-O-
Glucosyl tenvermectins A/B sterling；Described 4 " chemical constitution of-O-glucosyl tenvermectins A/B
It is characterized as:
" preparation method of-O-glucosyl tenvermectins A/B, the method is by fermentation in the 4 of present invention offer
Cultivate red saccharopolyspora Saccharopolyspora erythraea (ATCC 11635), then add the end in certain time
Thing sky dimension rhzomorph A/B (tenvermectins A/B) continues to cultivate 150h afterwards, eventually passes the extraction hexa-atomic macro ring of isolated ten
Lactone compound 4 "-O-glucosyl tenvermectins A/B.
S. erythraea strain is in fawn or lark on YMS solid medium, and spore is spherical, oval,
Smooth surface.Red saccharopolyspora be have mycelia and spore differentiation gram positive bacteria G (+), can produce by surface thorniness
The molecular short spore chain of spore.Producing sporogenic mode is to be started to curl up by nutrition aerial hyphae end, and then mycelia can produce
Raw barrier film, is divided into mycelia several sections, the most gradually adds thick skirt wall-forming, eventually forms circular spores release.This bacterium is purchased from the " U.S.
Standard biological product collecting center ", preserving number is: ATCC 11635.
As preferred technical scheme, described step 1) the preparation method of fluid medium be: take glucose 10g, ferment
Female extract 2g, beef extract 2g and peptone 3g, after being settled to 1000ml with distilled water, regulate pH with the NaOH of 1mol/l
To 7.0, use every bottle of subpackage 40ml of 250ml triangular flask, at 121 DEG C, sterilizing 20min under the conditions of 0.15MPa.
As preferred technical scheme, described step 2) conversion culture medium include M-1 culture medium, the M-that carbon-nitrogen ratio is different
2 culture medium, M-3 culture medium and M-4 culture medium, wherein,
The component of M-1 culture medium is: glucose 5g, brown sugar 10g, tryptone 5g, yeast extract 2.5g, ethylenediamine tetraacetic
Sodium acetate 0.036g, glycine betaine 1.2g and sodium propionate 0.11g；
The component of M-2 culture medium is: cerelose 44g, Carnis Bovis seu Bubali cream 5.0g, peptone 6.0g, yeast extract 4g, cheese
Fried soybean cake powder 11.0g, NaCl 1.5g and K of peptone 3.0g, heat2HPO40.5g；
The component of M-3 culture medium is: glucose 50g, soybean cake powder 5g, yeast extract 5g, peptone 4g, NaCl 5g,
K2HPO42g and KH2PO41g；
The component of M-4 culture medium is: cerelose 50g, NaCl 5g, (NH4)2SO42g、CaCO36g and Semen sojae atricolor powder
30g；
After above component distilled water is settled to 1000ml, regulate pH to 7.0-7.2,250ml tri-with the NaOH of 1mol/l
Every bottle of subpackage 40ml of angle bottle, at 121 DEG C, sterilizing 20min under the conditions of 0.15MPa.
The invention still further relates to, by changing conversion nutrient media components, improve ten hexa-atomic macrolides compound 4 "-O-
The method of the conversion ratio of glucosyl tenvermectins A/B.Seed liquor is transferred in above-mentioned four kinds of different conversion culture medium
In (M-1 culture medium, M-2 culture medium, M-3 culture medium and M-4 culture medium), these four converts the carbon source of culture medium all with glucose
Under based on, the variation of nitrogen source, and possess the trace element of necessity, in the case of other conversion conditions are the most constant, compare
Dimension rhzomorph the A/B "-O-glucosyl tenvermectins A/ that is converted into 4 by microorganism in sky under the conditions of four groups of different culture medias
The height of the conversion ratio of B.Result shows that the conversion ratio converting culture medium M-1 is minimum, and (carbon source is single to convert culture medium M-4
Glucose, carbon-nitrogen ratio is about 1.5:1) conversion ratio the highest (details is shown in embodiment), therefore when converting culture medium carbon source choosing
For glucose, when carbon-nitrogen ratio is about 1.5:1, beneficially sky dimension rhzomorph A/B microorganism is converted into 4 "-O-glucosyl
tenvermectins A/B。
As preferred technical scheme, described step 2) organic solvent include the one in methanol, ethanol, DMSO or several
Kind.
As preferred technical scheme, described step 2) extractant include ethyl acetate, n-butyl alcohol, isobutyl acetate,
One or more in ether, petroleum ether, dichloromethane or chloroform.
As preferred technical scheme, described step 2) purification include that reversed-phase high-performance liquid chromatography chromatographs.
As preferred technical scheme, described step 2) purification also include silica gel column chromatography.
A kind of 4 " purposes of-O-glucosyl tenvermectins A/B, 4 "-O-glucosyl tenvermectins
A/B is used for preparing preventing and treating agriculture and forestry injurious insect or evil demodicid mite medicine.
As preferred technical scheme, described agriculture and forestry injurious insect includes that Lepidoptera diamond-back moth section, Lepidoptera Noctuidae, Lepidoptera are withered
One or more in Ye E section, Lepidoptera snout moths larva section, coleoptera Elateridae, Tylenchida Aphelenchoidea；Described harmful demodicid mite includes leaf
Mite pest.
As preferred technical scheme, described 4 "-O-glucosyl tenvermectins A/B includes single 4 "-O-
Glucosyl tenvermectin A, 4 single "-O-glucosyl tenvermectin B or both mixture.
" preparation method of-O-glucosyl tenvermectins A/B, can prepare conversion ratio high to the 4 of the present invention
4″-O-glucosyl tenvermectins A/B.These are 4 years old, and "-O-glucosyl tenvermectins A/B is in preventing and treating agricultural evil
There is when worm or evil demodicid mite the effect of excellence.
Detailed description of the invention
It is explained the present invention below, it should be understood that example is for illustrating rather than with instantiation
Limitation of the present invention, the scope of the present invention is determined according to claims with core content.
Embodiment 1
1) take the red saccharopolyspora strain that preserving number is ATCC 11635, streak inoculation on YMS solid medium,
The constant incubator of 28 DEG C is cultivated 7 days, by the strain transfer after activation in fluid medium, is placed in 28 DEG C, 250rpm's
Horizontal shaker is cultivated 40 hours, obtains seed liquor, with 5% inoculum concentration, seed liquor is transferred in converting in culture medium, cultivate 24
After hour, every bottle converts the conversion of substrate sky dimension rhzomorph A/B mixture adding the dissolving of 500U methanol in culture medium, at 28 DEG C,
Continue under conditions of 250rpm to convert to cultivate 150 hours, obtain fermentation liquid.
2) fermentation culture obtains 3L fermentation liquid as stated above, by fermentation liquid 6L industrial alcohol soaked overnight, filters
Ethanol extract.Gained extracting solution removes ethanol phase at 50 DEG C of concentrating under reduced pressure, then extracts three respectively by equal-volume ethyl acetate
Secondary acetic acid ethyl acetate extract, extract is concentrated to dryness under 50 DEG C of reduced pressure, obtains 5g oily mater.
Silicagel column on the oily mater of gained (particle diameter 100-200 mesh) is carried out column chromatography, with chloroform: methanol=100:0-
60:40 (V/V) carries out gradient elution, identifies that (TLC) detects by thin layer, obtains 2 components.By component 2 through gel
(Sephadex LH-20) column chromatography obtains component 2-1, then use semi-preparative column chromatograph be further purified obtain pure
4 "-O-glucosyl tenvermectin A and 4 "-O-glucosyl tenvermectin B.
Wherein thin layer authentication method: sample after conversion and blank point are on silica gel g thin-layer plate, at chloroform: methanol
(9:1) developing solvent condition is launched, and takes out and dry after running through, and observes, then develops the color with concentrated sulphuric acid, inspect under first uviol lamp 254nm
Conversion results.
Semi-preparative column chromatographic condition: flowing phase: methanol-acetonitrile-water (45:45:10)；Chromatographic column C18,9.4*250mm；Inspection
Survey wavelength: 244nm；Flow velocity: 1.5ml/min；Column temperature: 24.4 DEG C；Sample size is: 60ul.Collect retention time be 16min and
The peak of 19.7min obtains 4 "-O-glucosyl tenvermectin A and 4 "-O-glucosyl tenvermectin B.Sample
Product and the configuration of standard substance: weigh appropriate amount of sample and standard substance, using flowing is solvent mutually, is configured to solution.
3) 4 " Structural Identification of-O-glucosyl tenvermectins A and B
By the Spectrum Analysis such as 1D and 2D NMR, MS " structure of-O-glucosyl tenvermectins A/B that determines 4
As follows:
4 " physicochemical property of-O-glucosyl tenvermectin A is as follows:
Character: white powder material
Dissolubility: be easily soluble in chloroform, acetone, methanol, water insoluble
Molecular formula: C51H78O19
ESI-MS m/z:993.23[M-H]-
UVλmax(EtOH) nm (log ε): 245 (4.67), 239 (4.63)
IR vmaxcm-1: 3387,2930,2875,1721,1646,1450,1383,1340,1305,1270,1170,
1062,989
4 " physicochemical property of-O-glucosyl tenvermectin B is as follows:
Character: white powder material
Dissolubility: be easily soluble in chloroform, acetone, methanol, water insoluble
Molecular formula: C52H80O19
ESI-MS m/z:1007.22[M-H]-
UVλmax(EtOH) nm (log ε): 245 (4.50), 239 (4.46)
IR vmaxcm-1: 3369,2928,2873,1716,1637,1452,1382,1340,1304,1268,1168,
1118,1064,985
1H NMR(CDCl3, 400MHz) and13C NMR(CDCl3, 100MHz) and it is shown in Table 1.
"-O-glucosyl tenvermectins A and B is at CDCl for table 143In nuclear magnetic data
(hydrogen is composed, 400MHz；Carbon is composed, 100MHz)
Embodiment 2
Taking the red saccharopolyspora kind that preserving number is ATCC 11635, streak inoculation is on YMS solid medium, at 28 DEG C
Constant incubator in cultivate 7 days, will activation after strain transfer in fluid medium, be placed in 28 DEG C, the level of 250rpm
Shaking table is cultivated 40 hours, obtains seed liquor, with 5% inoculum concentration, seed liquor is transferred in converting in culture medium (M-1), cultivate 24
After hour, every bottle converts the conversion of substrate sky dimension rhzomorph A/B mixture adding methanol dissolving in culture medium, at 28 DEG C, 250rpm's
Under the conditions of continue convert cultivate 150 hours, ethyl acetate extractive fermentation liquid, detect to obtain 4 "-O-glucosyl by HPLC
Tenvermectins A and B conversion ratio are respectively 5.77% and 7.69%.
Embodiment 3
Taking the red saccharopolyspora kind that preserving number is ATCC 11635, streak inoculation is on YMS solid medium, at 28 DEG C
Constant incubator in cultivate 7 days, will activation after strain transfer in fluid medium, be placed in 28 DEG C, the level of 250rpm
Shaking table is cultivated 40 hours, obtains seed liquor, with 5% inoculum concentration, seed liquor is transferred in converting in culture medium (M-2), cultivate 24
After hour, every bottle converts the conversion of substrate sky dimension rhzomorph A/B mixture adding ethanol dissolving in culture medium, at 28 DEG C, 250rpm's
Under the conditions of continue convert cultivate 150 hours, ether extractive fermentation liquid, detect to obtain 4 "-O-glucosyl by HPLC
Tenvermectins A and B conversion ratio are respectively 4.20% and 11.15%.
Embodiment 4
Taking the red saccharopolyspora kind that preserving number is ATCC 11635, streak inoculation is on YMS solid medium, at 28 DEG C
Constant incubator in cultivate 7 days, will activation after strain transfer in fluid medium, be placed in 28 DEG C, the level of 250rpm
Shaking table is cultivated 40 hours, obtains seed liquor, with 5% inoculum concentration, seed liquor is transferred in converting in culture medium (M-3), cultivate 24
After hour, every bottle converts the conversion of substrate sky dimension rhzomorph A/B mixture adding DMSO dissolving in culture medium, at 28 DEG C, 250rpm's
Under the conditions of continue convert cultivate 150 hours, petroleum ether extraction fermentation liquid, detect to obtain 4 "-O-glucosyl by HPLC
Tenvermectins A and B conversion ratio are respectively 7.78% and 14.24%.
Embodiment 5
Taking the red saccharopolyspora kind that preserving number is ATCC 11635, streak inoculation is on YMS solid medium, at 28 DEG C
Constant incubator in cultivate 7 days, will activation after strain transfer in fluid medium, be placed in 28 DEG C, the level of 250rpm
Shaking table is cultivated 40 hours, obtains seed liquor, with 5% inoculum concentration, seed liquor is transferred in converting in culture medium (M-4), cultivate 24
After hour, every bottle converts the conversion of substrate sky dimension rhzomorph A/B mixture adding methanol dissolving in culture medium, at 28 DEG C, 250rpm's
Under the conditions of continue convert cultivate 150 hours, dichloromethane extractive fermentation liquid, detect to obtain 4 "-O-glucosyl by HPLC
Tenvermectins A and B conversion ratio are respectively 9.74% and 19.8%.
Embodiment 6
4 "-O-glucosyl tenvermectins A and the B biological activity to Tetranychus cinnabarinus
Reagent agent: 98% (w/w) 4 "-O-glucosyl tenvermectins A, B, 98% (w/w)
Tenvermectin A: weigh 1g 98%4 respectively "-O-glucosyl tenvermectins A and B adds in beaker, and
Add 93g methanol and 6g surfactant NPE, make the preparation that concentration is 10000mg/L, dilute with water
To become concentration be 0.005,0.01,0.02,0.04, the medicinal liquid of 0.08mg/L is for examination.
For examination biology: Tetranychus cinnabarinus (Tetranychus cinnabarinus): under the conditions of in the controlled environment chamber [(26 ±
1) DEG C, RH (70 ± 5) %, H/D14], it is inoculated on Semen Viciae fabae Seedling cultivation.
Experimental technique: employing leaf dish leaching worm immersion method: selection indoor feeding, the one-tenth acarid that physiological status is consistent.Choose life
Long consistent Broad Bean Leaves, makes diameter 2cm leaf dish with card punch, and blade back is placed on the absorbent cotton at plastic ware center, often upward
3 leaf butterflies of ware, choose with small size brush pen and are connected into demodicid mite and are inoculated on leaf dish, 30, every leaf dish, and add suitable quantity of water, are put in (26 ± 1) DEG C,
Intensity of illumination 3000～4500lx, 14h/d, in the culturing room of RH 50%～75%.Under stereomicroscope, demodicid mite is checked into after 2h
Number, on every ware leaf dish, the quantity of demodicid mite is not less than 20.The mass concentration 0.005 for preparing, 0.01,0.02,0.04,0.08mg/L
Medicament be put in beaker, clamp blade with tweezers from low concentration to high concentration, soak medicine successively, the leaching medicine time is 5s, comparison steaming
Distilled water processes female one-tenth demodicid mite, and each mass concentration is a process, often processes and is repeated 3 times.Treat that the medicament on blade dries, will process
Leaf dish be placed in the phjytotron of (26 ± 1) DEG C and 14h photoperiod and cultivate 24h, and in culture dish, add a small amount of water moisturizing.Leaching
After medicine, acarid is the most active, within after process 5-8 hour, begins to activity of slowing down, and after 12-24 hour, polypide is static.Death determination mark
Accurate: touching demodicid mite body with brush pen during inspection, complete motionless person is judged to death.With Tetranychus cinnabarinus for examination worm, to 4 "-O-glucosyl
The toxicity of tenvermectins A and B is determined, and with tenvermectin A for comparison medicament, compares 4 "-
The activity of O-glucosyl tenvermectins A and B and tenvermectin A.
Conclusion: experimental result is shown in Table 2, tenvermectin A is 0.0084mg/L to the LC50 of Tetranychus cinnabarinus, after conversion
"-O-glucosyl tenvermectins A/B is respectively 0.0156mg/L, 0.0113mg/ to the LC50 of Tetranychus cinnabarinus to product 4
L, converts afterproduct 4 "-O-glucosyl tenvermectins A/B and the tenvermectin A activity nothing to Tetranychus cinnabarinus
Significant difference.
Table the 2:4 "-O-glucosyl tenvermectins A/B activity to Tetranychus cinnabarinus
Embodiment 7
4 "-O-glucosyl tenvermectins A and the B determination of activity to Bursaphelenchus xylophilus
"-O-glucosyl tenvermectins A/B is from above-described embodiment, tenvermectin A for test medicine: 4
From Haizheng Medicine Stock Co., Ltd., Zhejiang Prov.
For examination biology: with Bursaphelenchus xylophilus for examination worm
Test method: use immersion method.Every kind of medicament set up 5 mass concentrations 1,2,5,10,20mg/L, every concentration repeats
3 times, 24h statistical experiment result.
Result of the test: various medicaments are shown in Table 3 to the virulence of Bursaphelenchus xylophilus.The tenvermectin A LC50 to Bursaphelenchus xylophilus
" LC50 of Bursaphelenchus xylophilus is respectively by-O-glucosyl tenvermectins A/B for 2.4391mg/L, to convert afterproduct 4
6.7984mg/L, 5.7980mg/L, convert the afterproduct 4 "-O-glucosyl tenvermectins A/B work to Bursaphelenchus xylophilus
Property, compared with tenvermectin A, there was no significant difference.
Table the 3:4 "-O-glucosyl tenvermectins A/B activity to Bursaphelenchus xylophilus
Embodiment 8
4 "-O-glucosyl tenvermectins A and the B determination of activity to diamondback moth
"-O-glucosyl tenvermectins A/B is from above-described embodiment, tenvermectin A for test medicine: 4
From Haizheng Medicine Stock Co., Ltd., Zhejiang Prov.
For examination biology: with diamondback moth for examination worm
Test method: use medicine embrane method.5 mass concentrations 25,50,100,200 and 250mg/L set up by every kind of medicament, often
Concentration is repeated 3 times, 24h statistical experiment result.
Result of the test: various medicaments are shown in Table 4 to the virulence of diamondback moth.Tenvermectin A to diamondback moth at 250mg/L
Corrected mortality be 79.62%, convert afterproduct 4 " diamondback moth is existed by-O-glucosyl tenvermectins A/B
The corrected mortality of 250mg/L is respectively 70.21%, 74.87%, converts afterproduct 4 "-O-glucosyl
Diamondback moth activity compared with tenvermectin A, be there was no significant difference by tenvermectins A/B.
Table the 4:4 "-O-glucosyl tenvermectins A/B activity to diamondback moth
Embodiment 9
4 "-O-glucosyl tenvermectins A and the B determination of activity to bamboo snout moth's larva
"-O-glucosyl tenvermectins A/B is from above-described embodiment, tenvermectin A for test medicine: 4
From Haizheng Medicine Stock Co., Ltd., Zhejiang Prov.
For examination biology: with bamboo snout moth's larva for examination worm
Test method: use infusion process.Every kind of medicament sets up 5 mass concentrations 1,2,5,10 and 20mg/L, every concentration weight
Multiple 3 times, 24h statistical experiment result.
Result of the test: various medicaments are shown in Table 5 to the virulence of bamboo snout moth's larva.Tenvermectin A to bamboo snout moth's larva in the correction of 20mg/L
Mortality rate is 77.52%, converts afterproduct 4 "-O-glucosyl tenvermectins A/B to bamboo snout moth's larva in the correction of 20mg/L
Mortality rate is respectively 68.56%, and 70.62%, "-O-glucosyl tenvermectins A/B is to bamboo snout moth's larva to convert afterproduct 4
Activity, compared with tenvermectin A, there was no significant difference.
Table the 5:4 "-O-glucosyl tenvermectins A/B activity to bamboo snout moth's larva
The present invention 4 " preparation method of-O-glucosyl tenvermectins A/B, can prepare that conversion ratio is high 4 "-
O-glucosyl tenvermectins A/B.These are 4 years old, and "-O-glucosyl tenvermectins A/B is at preventing and treating agriculture and forestry injurious insect
Or there is during evil demodicid mite the effect of excellence.
The present invention 4, and " purposes of-O-glucosyl tenvermectins A/B is retouched by concrete example
Stating, those skilled in the art can use for reference present invention, the link such as suitable feed change, process conditions realize corresponding its
Its purpose, its relevant change is all without departing from present disclosure, and all similar replacements and change are for people in the art
It is apparent from for Yuan, is considered as being included within the scope of the present invention.
Embodiment described above is the present invention preferably scheme, and the present invention not makees any pro forma restriction,
Other variant and remodeling is also had on the premise of without departing from the technical scheme described in claim.
Claims (10)
1.4 " preparation method of-O-glucosyl tenvermectins A/B, it is characterised in that comprise the steps:
1) red saccharopolyspora seed liquor is prepared: take the S. erythraea strain that preserving number is ATCC 11635
Saccharopolyspora erythraea, streak inoculation is on YMS solid medium, in the constant incubator of 20-28 DEG C
Cultivate 5-7 days, by the strain transfer after activation in fluid medium, be placed in 20-28 DEG C, the horizontal shaker of 250rpm is cultivated
40 hours, obtain seed liquor；
2) 4 are prepared "-O-glucosyl tenvermectins A/B: seed liquor is transferred in converting in culture medium, cultivate 24 little
The conversion of substrate sky dimension rhzomorph A/B that time after, addition is dissolved with organic solvent, at 20-28 DEG C, converts under conditions of 250rpm and cultivates
After 150 hours, obtain fermentation liquid；4 are obtained through chromatographic isolation after using extractant extractive fermentation liquid and reclaiming extractant "-O-
Glucosyl tenvermectins A/B sterling；Described 4 " chemical constitution of-O-glucosyl tenvermectins A/B
It is characterized as:
The most according to claim 14 " preparation method of-O-glucosyl tenvermectinsA/B, it is characterised in that
Described step 1) in the preparation method of fluid medium be: take glucose 10g, yeast extract 2g, beef extract 2g and egg
White peptone 3g, after being settled to 1000ml with distilled water, regulates pH to 7.0 with the NaOH of 1mol/l, uses 250ml triangular flask every bottle point
Dress 40ml, at 121 DEG C, sterilizing 20min under the conditions of 0.15MPa.
The most according to claim 14 " preparation method of-O-glucosyl tenvermectinsA/B, it is characterised in that
Described step 2) conversion culture medium include that M-1 culture medium that carbon-nitrogen ratio is different, M-2 culture medium, M-3 culture medium and M-4 cultivate
Base, wherein,
The component of M-1 culture medium is: glucose 5g, brown sugar 10g, tryptone 5g, yeast extract 2.5g, ethylenediaminetetraacetic acid
Sodium 0.036g, glycine betaine 1.2g and sodium propionate 0.11g；
The component of M-2 culture medium is: cerelose 44g, Carnis Bovis seu Bubali cream 5.0g, peptone 6.0g, yeast extract 4g, casein
Fried soybean cake powder 11.0g, NaCl 1.5g and K of peptone 3.0g, heat2HPO40.5g；
The component of M-3 culture medium is: glucose 50g, soybean cake powder 5g, yeast extract 5g, peptone 4g, NaCl 5g,
K2HPO42g and KH2PO41g；
The component of M-4 culture medium is: cerelose 50g, NaCl 5g, (NH4)2SO4 2g、CaCO36g and Semen sojae atricolor powder 30g；
After above component distilled water is settled to 1000ml, regulate pH to 7.0-7.2,250ml triangular flask with the NaOH of 1mol/l
Every bottle of subpackage 40ml, at 121 DEG C, sterilizing 20min under the conditions of 0.15MPa.
The most according to claim 14 " preparation method of-O-glucosyl tenvermectinsA/B, it is characterised in that
Described step 2) organic solvent include one or more in methanol, ethanol, DMSO.
The most according to claim 14 " preparation method of-O-glucosyl tenvermectinsA/B, it is characterised in that
Described step 2) extractant include ethyl acetate, n-butyl alcohol, isobutyl acetate, ether, petroleum ether, dichloromethane or three chloromethanes
One or more in alkane.
The most according to claim 14 " preparation method of-O-glucosyl tenvermectins A/B, its feature exists
In, described step 2) purification include that reversed-phase high-performance liquid chromatography chromatographs.
The most according to claim 64 " preparation method of-O-glucosyl tenvermectins A/B, its feature exists
In, described step 2) purification also include silica gel column chromatography.
8. an according to claim 14 " purposes of-O-glucosyl tenvermectins A/B, its feature exists
In, 4 "-O-glucosyl tenvermectins A/B is used for preparing preventing and treating agriculture and forestry injurious insect or evil demodicid mite medicine.
The most according to claim 84 " purposes of-O-glucosyl tenvermectins A/B, it is characterised in that institute
State agriculture and forestry injurious insect and include that Lepidoptera diamond-back moth section, Lepidoptera Noctuidae, Lepidoptera Lasiocampidae, Lepidoptera snout moths larva section, coleoptera are kowtowed
One or more in first section, Tylenchida Aphelenchoidea；Described harmful demodicid mite includes spider mite kind insect.
The most according to claim 84 " purposes of-O-glucosyl tenvermectins A/B, it is characterised in that institute
State 4 "-O-glucosyl tenvermectins A/B includes single 4 "-O-glucosyl tenvermectinA, single
4 "-O-glucosyl tenvermectin B or both mixture.
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| CN113461757A (en) * | 2021-06-25 | 2021-10-01 | 湖州师范学院 | Preparation method and application of novel sixteen-membered macrolide |
| CN113817008A (en) * | 2021-07-15 | 2021-12-21 | 湖州师范学院 | Preparation method and application of novel succinyl sixteen-membered macrolide |
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| CN108660101A (en) * | 2017-04-01 | 2018-10-16 | 浙江海正药业股份有限公司 | Express recombinant microorganism, the preparation method and its usage of day dimension rhzomorph B |
| CN108660101B (en) * | 2017-04-01 | 2021-07-09 | 浙江海正药业股份有限公司 | Recombinant microorganism expressing ivermectin B, preparation method and application thereof |
| WO2019228260A1 (en) * | 2018-05-28 | 2019-12-05 | 浙江海正药业股份有限公司 | Crystal form of tenvermectin b, preparation method therefor, and use thereof |
| AU2019276480B2 (en) * | 2018-05-28 | 2022-01-20 | Shenzhen Tenver Biopharm Co., Ltd. | Crystal form of tenvermectin B, preparation method therefor, and use thereof |
| US11912717B2 (en) | 2018-05-28 | 2024-02-27 | Shenzhen Tenver Biopharm Co., Ltd. | Crystal form of tenvermectin B, preparation method therefor, and use thereof |
| CN113461757A (en) * | 2021-06-25 | 2021-10-01 | 湖州师范学院 | Preparation method and application of novel sixteen-membered macrolide |
| CN113461757B (en) * | 2021-06-25 | 2023-08-22 | 湖州师范学院 | Preparation method and application of novel sixteen-membered macrolide |
| CN113817008A (en) * | 2021-07-15 | 2021-12-21 | 湖州师范学院 | Preparation method and application of novel succinyl sixteen-membered macrolide |
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Application publication date: 20161130 |