CN101899410B - Streptomyces parvus and application thereof for preparing daptomycin - Google Patents
Streptomyces parvus and application thereof for preparing daptomycin Download PDFInfo
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- 241000933218 Streptomyces parvus Species 0.000 title claims abstract description 31
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 title claims abstract description 24
- 108010013198 Daptomycin Proteins 0.000 title claims abstract description 23
- 229960005484 daptomycin Drugs 0.000 title claims abstract description 23
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a streptomyces parvus and application thereof for preparing daptomycin. The strain is reserved in the CCTCC and has the reservation number of CCTCC NO: M2010136, and the reservation data is June 4, 2010. The method for preparing the daptomycin is as follows: a, taking streptomyces parvus CCTCC NO: M2010136 as a fermentation strain; b, strain cultivation: inoculating slope lawn into a shake flask seeding tank to cultivate to obtain shake flask seeding liquid; inoculating the shake flask seeding liquid into the seeding tank to be cultivated; inoculating the seeding tank culture solution into fermentation tank culture medium to be cultivated, and collecting fermentation liquor; carrying out feed supplement in the fermentation and cultivation process; and c, extracting a fermentation product. The fermentation technology provided by the invention has stable production capability, high fermentation unit and few fermentation byproducts by the pilot plant test and the experiment in a 10-ton fermentation tank, greatly lowers post-extracting difficulty and is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of novel microorganism and uses thereof and application, relate in particular to a kind of streptomyces parvus and the application in the preparation daptomycin thereof.
Background technology
Daptomycin is one type of first product that is called cyclic ester peptide class new antibiotic family.It is in the middle of streptomyces parvus (Streptomyces parvus) fermented liquid, to extract to obtain.It not only has novel chemical structure, and its binding mode is also with arbitrary to have got permission microbiotic different.Daptomycin can destroy the bacterial cell membrane function in many aspects, kills gram positive organism thus rapidly.The daptomycin decapacitation acts on outside most of clinical relevant gram positive organisms, the more important thing is external still to have a strong active to being resistance isolated strains such as X-1497 (methicillin), vancomyein and Linezolid.
Daptomycin is a tunning, and the ferment filtrate through fermentation culture obtains can finally obtain daptomycin through a series of purifying process such as chromatography, decolouring, crystallization and recrystallization etc. again.General daptomycin produces bacterium, and throughput is unstable, and output is lower; Fermentation byproduct is more, and impurity is more, causes back extraction work comparatively complicated; Increase the purification difficulty of postorder widely, be difficult to obtain highly purified final product, thus can't industrialization production.
Summary of the invention
The objective of the invention is to the problems referred to above, provide a kind of fermentation unit high, can stably manufactured, output is many and by product is few daptomycin produces bacterium.
A kind of streptomyces parvus SW0702, classification called after streptomyces parvus (Streptomyces parvus SW0702), by China's typical culture collection center preservation, preserving number is: CCTCC NO:M2010136, preservation date is: on June 4th, 2010.
The colony characteristics of the bacterial strain of streptomyces parvus CCTCC NO:M2010136: substrate mycelium physically well develops, and the elongated branch of mycelia, diameter are 0.8～0.9 μ m; Aerial hyphae well-grown, fibrillae of spores and are given birth on aerial mycelium, and fibrillae of spores is straight, gentle bent, spore ovalize or oval, and smooth surface, diameter is 0.65～0.95 * 0.95～2.6 μ m, ripe spore chain spore number is more than 20.Sucrose nitrate salt agar: the gas silk is light yellow to pink.
Description according to microbial morphology and external related data; Various cultural characteristics and morphological specificity in conjunction with the bacterial strain of streptomyces parvus CCTCC NO:M2010136; The bacterial strain of this streptomyces parvus CCTCC NO:M2010136 should belong to streptomyces parvus and belong to, and names the SW0702 into Streptomyces parvus.
Another object of the present invention provides the application of this bacterial classification in the preparation daptomycin.
The bacterial strain of streptomyces parvus CCTCC NO:M2010136 can be applicable to the fermentative prepn daptomycin.This preparation method comprises the following steps:
A. fermentation strain adopts the bacterial strain of streptomyces parvus CCTCC NO:M2010136;
B. strain culturing: by the inclined-plane eggplant bottle of ordinary method preparation, take the inclined-plane lawn insert shake-flask seed cultivate shake-flask seed liquid; Shake-flask seed liquid is inoculated in the shake flask fermentation substratum or in the seeding tank cultivates; Seed tank culture liquid is inoculated in fermentation tank culture medium cultivates, collect fermented liquid; Preferably in the fermentation culture process, carry out feed supplement.
C. tunning extracts: chromatography, decolouring, crystallization and recrystallization.
Wherein said step b is: the inclined-plane by the ordinary method preparation takes 0.5 * 1.0cm
2The inclined-plane lawn insert the shake-flask seed substratum, 150～250rpm cultivated 16～32 hours, must shake-flask seed liquid; Shake-flask seed liquid is inoculated in seeding tank by the inoculum size of seed tank culture matrix long-pending 0.1～5%, and 80～120rpm cultivates 16～32h; Seed tank culture liquid is inoculated in fermentation tank culture medium by the amount of fermention medium volume 5～15%, 120～200rpm, fermentation culture 130～240 hours is collected fermented liquid; Wherein culture temperature is 28～32 ℃.
Wherein the feed supplement among the step b is very important in the whole process of fermentation.Several respects such as concrete supplying technics is dense from pH, bacterium according to the fermented liquid practical situation, carbon nitrogen source consumption, dissolved oxygen and mycelia microscopy situation consider, feed supplement can adopt even flow to add or the mode of fed-batch.
The wherein feed supplement in the fermentation culture process also is that the fermentor tank feed supplement is looked different concrete fermentation situation and had multiplely, mainly contains following several kinds of diverse ways,
1, treat that mycelial growth is good after, begin to mend capric acid and Witconol 2301 with 1: 1 mixture of volume ratio, adjust the feed supplement amount according to mycelia microscopy situation and fermentation unit situation, the amount of being mended is 0.1ml/L～0.6ml/L fermented liquid .h;
Perhaps
2, treat that mycelial growth is good after, begin stream and add and mend 8% capric acid ammonia, flow velocity 1.0～3.0ml/L/h, dissolved oxygen is not less than 10% during the control fermentation.Specifically be exactly that (this moment, fermented liquid was than thickness cultivating certain hour; Mycelial growth is vigorous, and bacterium is dense more than 20%) beginning feed supplement stream adds capric acid ammonia, when capric acid ammonia add a certain amount of after mycelia begin self-dissolving; The dense decline of bacterium; Nutrient solution is thinning, and dissolved oxygen rises, and at this moment can suitably reduce rotating speed, air flow.
Perhaps
3, pH is raised to for the first time at 7.0～8.5 o'clock and begins and mends during the fermentation; Mended for capric acid and Witconol 2301 with 1: 1 mixture of volume ratio (for the description of easy postorder; Abbreviate capric acid and Witconol 2301 as precursor with 1: 1 mixture of volume ratio), the amount of being mended is 0.03～0.2ml/L fermented liquid .h; Proceed to about 35～45 hours in fermentation until finish, the amount of being mended is 0.10～0.55ml/L fermented liquid .h;
In order to improve fermentation unit further, can also in the feed supplement operation, increase the feed supplement of rape oil, be specially: when fermenting to 70～78 hours; The benefit that temporarily stops precursor being gone into, and disposable benefit is gone into rape oil for the first time, and benefit amount by volume is 0.5～1.7% of a fermentor tank volume; Air flow quantity suitably reduces; Reduce 10～20% approximately, finish the back in the rape oil feed supplement first time and restarted to mend precursor again in 4～8 hours, the feed supplement amount is 0.10～0.55ml/L fermented liquid .h; After for the first time mending rape oil 24～40 hours, suspend and mend precursor, change and mend rape oil (for the second time), disposable benefit is gone into, and feed supplement amount by volume is about 0.5～1.5% of a fermentor tank volume, and air flow quantity continues suitably to reduce, and reduces 10～20% approximately.Rape oil feed supplement for the second time finishes back 4～8 hours, recovers the benefit of precursor and goes into.
After fermentor tank is mended rape oil for the second time 24 hours, can also suspend the benefit of precursor once more and go into, and mend a rape oil (for the third time) again, benefit amount by volume is 0.5～1.2% of a fermentor tank volume, recovers the benefit of precursor afterwards again and goes into.In whole fermentor tank feed supplement process, total benefit amount by volume of rape oil is 1.5～4.4% of a fermentor tank volume.
Perhaps
4, treat that mycelial growth begins to mend precursor after good, according to mycelia microscopy situation and fermentation unit situation adjustment feed supplement amount, mycelial growth is sturdy; Amount is many, and fermentation unit progressively produces and begins and rises, and the amount of being mended is 0.1ml/L～0.4ml/L fermented liquid .h; When the reducing sugar in the substratum is reduced to below 1%; Begin to mend glucose, to keep the concentration of reducing sugar, concentration maintains about 1%.
In the feed supplement process, oleic acid formicester and capric acid are by 1: 1 volume mixture, through being used for feed supplement after the membrane filtration degerming.Because capric acid has very strong corrodibility, the pipeline quality that feed supplement is used necessarily will be got well.Prevent that the capric acid feed supplement is undesired, cause fermentation unusual.In addition, temperature is lower than below 20 ℃, and capric acid is easy to solidify, and causes the feed supplement line clogging, therefore will be incubated pipeline, keeps pipeline unobstructed.
The invention also discloses substratum: volume ratio meter by weight, the carbon source in the shake-flask culture process is 1.0～9.5%, and is preferred 1.5～9.2%, most preferably is 8.0～9.0%; Nitrogenous source is 0.4～3.8%, and is preferred 1.0～3.5%, most preferably is 2.0～2.8%; Inorganic salt are 0.2～2.5%, and are preferred 0.4～2.0%, most preferably 0.5～1.3%; All the other are water;
Carbon source in the seed tank culture process is 1.0～9.5%, and is preferred 1.3～9.2%, most preferably is 7.0～9.0%; Nitrogenous source is 0.8～3.8%, and is preferred 1.5～3.3%, most preferably is 2.0～3.0%; Inorganic salt are 0.2～2.5%, and are preferred 0.4～2.0%, most preferably 0.5～1.3%; All the other are water;
Wherein carbon source is selected from one or more in maltodextrin, starch, amylum hydrolysate of the sugar, molasses, dextrin, glycerine, glucose, SANMALT-S, wort, potato leaching powder, potato dextrin, the rolled oats; In preferred maltodextrin, amylum hydrolysate of the sugar, molasses, glycerine, glucose, wort, potato dextrin, the rolled oats one or more; One or more in maltodextrin, amylum hydrolysate of the sugar, molasses, glycerine, glucose, the potato dextrin most preferably.
Wherein nitrogenous source is selected from soybean cake powder, steeping water, zein, peptone, vegetable protein peptone, pancreatin Sunlover 10, meat peptone, urea, ammonium sulfate iron, yeast powder, YE, gives birth in bean powder, nitrate salt, the monosodium glutamate one or more; Preferred soybean cake powder, zein, peptone, vegetable protein peptone, pancreatin Sunlover 10, urea, ammonium sulfate iron, yeast powder, give birth in bean powder, nitrate salt, the monosodium glutamate one or more; Most preferably give birth to bean powder, pancreatin Sunlover 10, yeast powder three's mixing, perhaps vegetable protein peptone, both mixing of pancreatin Sunlover 10.
Wherein inorganic salt are selected from K
2HPO
4, NaCl, Fe (NH
4)
2(SO
4)
2.6H
2O, NaNO
3, KNO
3, MgSO
4.7H
2O, FeSO
4.7H
2O, KH
2PO
4In one or more; Preferred K
2HPO
4, NaCl, Fe (NH
4)
2(SO
4)
2.6H
2O, NaNO
3, KNO
3In one or more; Fe (NH most preferably
4)
2(SO
4)
2.6H
2O, KNO
3In one or both.
Wherein, in the fermentation culture process, volume ratio meter by weight, carbon source is 1.5～15%, and is preferred 2.5～13%, most preferably 5～11%; Nitrogenous source is 0.5～7.0%, and is preferred 0.7～5%, most preferably 0.7～3%; Inorganic salt are 0.1～2.0%, and are preferred 0.3～1.6%, most preferably 0.5～1.3%; Amino acid 0～1.5%, preferred 0.2～1.2%, most preferably 0.3～0.9%; All the other are water;
Substratum material in the fermentation culture process is selected as follows:
Carbon source is selected from one or more in maltodextrin, starch, Zulkovsky starch, amylum hydrolysate of the sugar, cane molasses, beet sirup, dextrin, glycerine, rape oil, glucose, SANMALT-S, wort, potato leaching powder, potato dextrin, the rolled oats; In preferred maltodextrin, Zulkovsky starch, amylum hydrolysate of the sugar, cane molasses, glycerine, rape oil, glucose, wort, potato dextrin, the rolled oats one or more, most preferably one or more in maltodextrin, Zulkovsky starch, cane molasses, glucose, rape oil, the potato dextrin.
Nitrogenous source is selected from one or more in soybean cake powder, analysis for soybean powder, steeping water, zein, peptone, vegetable protein peptone, pancreatin Sunlover 10, meat peptone, urea, ferrous ammonium sulphate, yeast powder, YE, living bean powder, nitrate salt, the monosodium glutamate; Preferred soybean cake powder, analysis for soybean powder, zein, peptone, vegetable protein peptone, pancreatin Sunlover 10, urea, ferrous ammonium sulphate, yeast powder, give birth in bean powder, nitrate salt, the monosodium glutamate one or more; Most preferably be and select analysis for soybean powder, KNO simultaneously
3, three kinds of materials of Sodium Glutamate select peptone, urea, ferrous ammonium sulphate, four kinds of materials of yeast powder simultaneously for use.
Inorganic salt are selected from K
2HPO
4, NaCl, Fe (NH
4)
2(SO
4)
2.6H
2O, NaNO
3, KNO
3, MgSO
4.7H
2O, FeSO
4.7H
2O, CH
3COONa, CaCO
3In one or more; Preferred Fe (NH
4)
2(SO
4)
2.6H
2O, NaNO
3, KNO
3, MgSO
4.7H
2O, FeSO
4.7H
2O, CH
3COONa, CaCO
3In one or more; Fe (NH most preferably
4)
2(SO
4)
2.6H
2O, NaNO
3, KNO
3, CH
3COONa, CaCO
3In one or more.
Through fermentation accomplish put jar after, tunning is extracted, through resin absorption and desorb, carry out can obtaining purity behind chromatography and the purifying again and reach the daptomycin more than 98%.
The disclosed streptomyces parvus CCTCC of the present invention NO.M2010136 is that the daptomycin of a kind of high unit of being used at present to produce produces bacterium, and leavening property is good, can be used for scale operation.Fermenting process is the important step that daptomycin is produced, and its fermentation level is directly related with the technology quality, and zymotechnique provided by the invention makes an experiment its stable production capacity through shaking in bottle and 10 tons of fermentor tanks.Through detecting, the daptomycin unit for preparing with bacterial classification provided by the invention and fermentation culture method can up to 3100 μ g/ml or more than, thereby for follow-up suitability for industrialized production good basis is provided.Fermentation byproduct of the present invention is less relatively simultaneously, and has reduced the difficulty of back extraction, and the technology of extraction and purifying is simple relatively, has improved yield and purity greatly, on suitability for industrialized production, has more advantage.
Bacterial strain preservation situation: be preserved in Wuhan China typical culture collection center (being called for short CCTCC); Deposit number is CCTCCNO:M2010136; Classification called after streptomyces parvus SW0702 (Streptomyces parvusSW0702), preservation date is on June 4th, 2010.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but can not method related in the scheme and technical parameter be interpreted as
Limitation of the present invention.
Embodiment 1: the cultural characteristic of streptomyces parvus CCTCC NO:M2010136
The substratum that the inoculation of streptomyces parvus CCTCC NO:M2010136 is used to observe morphological specificity in Gause I, glucose asparagine etc.Cultivate 5,8,10 days regular hours for 28 ℃, observe the line description of going forward side by side.
The colony characteristics (see table 1) of the bacterial strain of table 1, streptomyces parvus CCTCC NO:M2010136 on various substratum
| Substratum | Growing state | Aerial hyphae | Substrate mycelium | Soluble pigment |
| Gause I agar | Luxuriant | White to pink | Brown | Grey black |
| Glucose asparagine agar | Luxuriant | White | Colourless | Colourless |
| Sucrose is examined formula agar | Luxuriant | White to pink | Pale red | Colourless |
| The inorganic salt Starch Agar | Luxuriant | Ash is red | Ash | Ash |
| Calcium malate agar | Poor | Brown | Colourless | Colourless |
| Oatmeal agar | Luxuriant | Light pink | Colourless | Colourless |
| Potato ball | Moderate | White | Colourless | Colourless |
| Glucose yeast cream agar | Luxuriant | White | Colourless | Colourless |
| Tyrosine agar | Luxuriant | White | Colourless | Colourless |
| The tryptone yeast extract medium | Poor | Colourless | Colourless | Colourless |
| The yeast extract paste malt extract agar | Poor | Colourless | Colourless | Colourless |
| Glycerine asparagine agar | Poor | Colourless | Colourless | Light brown |
Embodiment 2: physiological and biochemical property and utilization of carbon source
Physiology and biochemistry character adopts the standard method in the streptomyces parvus classification.
The biochemical reactions of streptomyces parvus CCTCC NO:M2010136 is: this bacterium gelatine liquefication is slow, the cadmium yellow pigment.Milk does not solidify, and peptonizes rapidly.The starch hydrolysis.Mierocrystalline cellulose is not grown.A little less than the nitrate reduction, type of generation melanochrome not.Utilize Biolog automatic microbe identification systems to investigate the metabolism situation of streptomyces parvus to 95 kinds of different carbon sources: with inoculation in the BUG+B plate culture medium; 37 ℃ of constant temperature culture 16h; With aseptic cotton carrier the thalline on the flat board is washed; (GN/GP-IF+T) mixes with inoculation liquid, processes bacteria suspension, is adjusted to 20%T with turbidometer.Bacteria suspension is added in each hole of Biolog GP2 micropore every hole 150uL respectively with the electronic liquid fillers in 8 holes.The micropore identification plate is placed in 37 ℃ of incubators, after cultivating 6h, 24h and 48h, is placed on the Biolog readout instrument respectively and reads the result.
Analyze the metabolism fingerprint through the Biolog readout instrument, streptomyces parvus CCTCC NO:M2010136 can utilize wherein 22 kinds of carbon sources more by force, and 73 kinds of utilization of carbon source abilities are more weak maybe can not utilize to other.System provides the 48h qualification result and sees table 2.
Table 2 streptomyces parvus CCTCC NO:M2010136 is to the ability of utilizing of 95 kinds of carbon sources on the Biolog GP2 plate
Explain :+, carbon source can be utilized;-, can not utilize carbon source; B: a little less than utilizing the carbon source ability
Embodiment 3: the production fermentation culture of daptomycin
1. seed spawn culture and preservation
Solid medium: glucose 0.5g, yeast extract paste 0.3g, wort 0.3g, CaCO30.3g, agar 1.8g adds water 100ml, pH7.0
Cultivate: inoculation was cultivated 7～10 days for 30 ℃ on culture medium slant.
After solid culture finished, it is subsequent use that 4～10 ℃ of refrigerations are placed on the inclined-plane.
2. shake-flask seed is cultivated
Substratum: glycerine 20g, Zulkovsky starch 80g, yeast powder 6g gives birth to bean powder 20g, pancreatin Sunlover 10 20g, Fe (NH
4)
2(SO
4)
2.6H
2O 10g adds water 2000ml, and pH 7.0
Inoculum size: take inclined-plane lawn piece and insert in the 2L shake-flask seed substratum
Cultivate: 30 ℃, 24h
Shaking speed: 250rpm
Grow mycelia, having obvious wall cling phenomenon and bacterium dense is about 11%, with 2L shake-flask seed nutrient solution, inserts in the following seeding tank.
3. seeding tank seed culture
Substratum: glycerine 4kg, yeast powder 1.2kg, soybean cake powder 1.6kg, NaNO
30.4kg, skimmer 0.2kg, pH7.0 loading amount: dress substratum 400L in the 1000L seeding tank, 121 ℃ of sterilization 30min.
Inoculum size: 2L shake-flask seed liquid
Cultivate: 30 ℃, 28h
4. fermentation culture
Substratum: Zulkovsky starch 100kg, glucose 50kg, saltpetre 30kg, analysis for soybean powder 100kg, Sodium Glutamate 20kg, five aqueous ferrous sulfate 0.25kg, rape oil 100kg, CaCO
310kg, pH 7.0
Loading amount: the dress substratum is 5 tons in 10 tons of fermentor tanks
The seed liquor of inoculum size: 400L
Cultivate: 120rpm, 30 ℃, 140h
5. feeding medium during fermentation
Begin to mend precursor when pH is raised to 7.0 left and right sides for the first time in the fermentation culture process, the feed supplement amount is 0.03ml/L
Fermented liquid .h promptly per hour mends the 150ml precursor; Proceed to about 35 hours in fermentation until finish, the amount of feed supplement is 0.55ml/L fermented liquid .h, promptly per hour mends the 2750ml precursor.
According to above-mentioned fermentation condition and technology, carry out the test of 3 batch fermentations at 10 tons of fermentor tanks, fermentation unit is respectively 3780 μ g/ml, 3250 μ g/ml and 3420 μ g/ml.
Embodiment 4: the production fermentation culture of daptomycin
1. shake-flask seed is cultivated
Substratum: glucose 100g, molasses 40g, glycerine 20g, vegetable protein peptone 14g, steeping water 36g, KNO
326g adds water 2000ml, pH7.0
Cultivate: 30 ℃, 24h, 250rpm
Grow mycelia, have obvious wall cling phenomenon and bacterium dense about about 13%,, insert in the following seeding tank 2L shake-flask seed nutrient solution.
2. seeding tank seed culture
Seed tank culture: glycerine 26kg, glucose 50kg yeast powder 14.2kg gives birth to bean powder 5.2kg, pancreatin Sunlover 10 4kg, Fe (NH
4)
2(SO
4)
2.6H
2O 0.6kg, skimmer 0.16kg, pH7.0
Loading amount: dress substratum 800L in the 2000L seeding tank, 121 ℃ of sterilization 30min.
Inoculum size: 2L shake-flask seed liquid
Cultivate: 30 ℃, 32h
3. fermentor cultivation
Fermention medium: glucose 44kg, cane molasses 31kg, analysis for soybean powder 182kg, peptone 165kg, sodium acetate 48kg, ferrous ammonium sulphate 3kg adds 5 tons in water, pH7.2
Loading amount: the dress substratum is 6 tons in 10 tons of fermentor tanks
The seed liquor of inoculum size: 800L
Cultivate: 120rpm, 30 ℃, 190h
4. feeding medium during fermentation
Begin to mend precursor when pH is raised to 7.5 left and right sides for the first time in the fermentation culture process; The amount of precursor is 0.08ml/L fermented liquid .h; Proceed to about 38 hours in fermentation until finish, the amount of precursor is 0.45ml/L fermented liquid .h.But about fermentation was to 70 hours, the benefit of suspending precursor was gone into and disposable benefit is gone into rape oil, and the benefit amount by volume of rape oil is that about 0.5% of fermentor tank volume is 30kg, and air flow quantity suitably reduces 10～20%; The rape oil feed supplement finishes continuation in back 4 hours and mends precursor, and the feed supplement amount is 0.10ml/L fermented liquid .h; After mending rape oil 24 hours, to suspend once more and mend precursor and change benefit rape oil, disposable benefit is gone into, and feed supplement amount by volume is that about 1.5% of fermentor tank volume is 90kg, and air flow quantity continues suitably to reduce 10～15%; Then by foregoing benefit precursor, 0.45ml/L fermented liquid .h is until fermentation ends then.
Putting that a jar gained fermented liquid tires is 3100 μ g/ml.
According to above-mentioned fermentation condition and technology; If after for the second time mending rape oil 24 hours, mend rape oil for the third time, benefit amount by volume is that about 1.0% of fermentor tank volume is 60kg; Then then by foregoing benefit precursor, promptly with 0.25ml/L fermented liquid .h until fermentation ends.
When putting jar, fermentation unit is 3200 μ g/ml.
Embodiment 5: the pilot scale fermentation of daptomycin is cultivated
1. shake-flask seed is cultivated
Substratum: maltodextrin 20g, vegetable protein peptone 40g, K
2HPO
410g, the pH nature adds water 2000ml and cultivates: 30 ℃, cultivate: 30 ℃, 25h, 250rpm
Grow mycelia, have that obvious wall cling phenomenon and bacterium are dense to be about about 9%,, insert in the following seeding tank 2L shake-flask seed nutrient solution.
2. seeding tank seed culture
Substratum: glucose 1.7kg, potato dextrin 4.5kg, cane molasses 730g, soybean cake powder 2.6kg, Fe (NH
4)
2(SO
4)
2.6H
2O 1.2kg, skimmer 100g, pH7.0
Loading amount: dress substratum 100L in the 300L seeding tank, 121 ℃ of sterilization 30min.
Inoculum size: 2L shake-flask seed liquid
Cultivate: 30 ℃, 27h
3. fermentor cultivation
Fermention medium: glucose 45kg, maltodextrin 65kg, yeast powder 3.5kg, Fe (NH
4)
2(SO
4)
2X6H
2O 1.5kg, skimmer 2kg, pH 7.0
Loading amount: the dress substratum is 1 ton in 2 tons of fermentor tanks
The seed liquor of inoculum size: 100L
Cultivate: 120rpm, 30 ℃, 160h
4, feeding medium during fermentation
Begin to mend precursor when pH is raised to 8.5 left and right sides for the first time during the fermentation, the amount of feed supplement is 0.2ml/L fermented liquid .h; Proceed to about 45 hours in fermentation until finish, the amount of feed supplement is 0.10ml/L fermented liquid .h.
Monitor the concentration of reducing sugar simultaneously,, continue the concentration of monitoring reducing sugar, make it to maintain about 1% less than beginning to mend glucose 2L at 0.9% o'clock into 50%.
Putting that a jar gained fermented liquid tires is 3200 μ g/ml.
Embodiment 6: the production fermentation culture of daptomycin
1, shake-flask seed is cultivated
Shake-flask seed substratum: dextrin 180g, pancreatin Sunlover 10 8g, NaNO
31.0g, add 2000ml water, 240rpm, 30 ℃, 24～28h.
2, seed tank culture
Seed tank culture base: glucose 9kg, potato dextrin 36kg, cane molasses 9kg/L, soybean cake powder 9kg, Fe (NH
4) (SO)
2.6H
2O3kg, skimmer 300g, pH value 7.2
Culturing process pH value stream adds ammoniacal liquor to be controlled, and the pH value is controlled between 6.5～7.0.
Loading amount: dress substratum 600L in the 1000L seeding tank, 121 ℃ of sterilization 30min.
Inoculum size: 2L shake-flask seed liquid
Cultivate: 30 ℃, 30h
3, fermentor cultivation
Fermentation tank culture medium: glucose 20kg, potato dextrin 42kg, molasses 28kg/L, yeast powder 38kg, Fe (NH
4)
2SO
4.6H
2O 4kg,, add a small amount of alpha-amylase before the sterilization, general 3u/g, can liquefy gets final product.
Loading amount: the dress substratum is 6 tons in 10 tons of fermentor tanks
The seed liquor of inoculum size: 600L
Cultivate: 120rpm, 156～164h
Culture temperature: 30 ± 1 ℃, tank pressure: 0.03～0.05MPa, air flow quantity: 1: 1vvm, mixing speed 80～140rpm, fermenting process is regulated pH with strong aqua and is not less than 6.5, and dissolved oxygen is not less than 20% in the fermenting process,
4, feeding medium during fermentation
(fermented liquid is than thickness at this moment, and mycelial growth is vigorous, and bacterium is held more than 20%) beginning feed supplement stream adds capric acid ammonia (8% with the calculating of capric acid weight) flow velocity 1.5～2.0ml/L/h to fermentation ends about 28hr cultivating.Mycelia begins self-dissolving after the capric acid adding is a certain amount of, and dissolved oxygen rises, and can suitably reduce rotating speed, ventilation.
When putting jar, fermentation unit is 3100 μ g/ml.
The fermented liquid unit that is obtained in the above-described enforcement is high, and impurity is few, and fermented liquid through resin absorption and desorb, carries out chromatography and purifying through leaching filtrating again, can obtain purity at the daptomycin more than 98%.
Claims (9)
1. bacterial strain, classification called after streptomyces parvus SW0702 (Streptomyces parvus SW0702), by the typical culture collection center preservation of Wuhan China, deposit number is CCTCC NO:M2010136, preservation date is: on June 4th, 2010.
2. the application of bacterial strain in the fermentative prepn daptomycin of the CCTCC NO:M2010136 of streptomyces parvus according to claim 1.
3. application as claimed in claim 2 is characterized in that this preparation method comprises the following steps:
A. fermentation strain adopts the bacterial strain of streptomyces parvus CCTCC NO:M2010136;
B, strain culturing: by the inclined-plane of ordinary method preparation, take the inclined-plane lawn insert the shake-flask seed jar cultivate shake-flask seed liquid; Shake-flask seed liquid is inoculated in the seeding tank cultivates; Seed tank culture liquid is inoculated in fermentation tank culture medium cultivates, collect fermented liquid; It is characterized in that in the fermentation culture process, carrying out feed supplement,
C. tunning extracts: resin absorption and desorb, chromatography and purifying, crystallization.
4. application as claimed in claim 3 is characterized in that its described step b is: the inclined-plane by the ordinary method preparation takes 0.5 * 1.0cm
2The inclined-plane lawn insert the shake-flask seed substratum, 150～250rpm cultivated 16～32 hours, must shake-flask seed liquid; Shake-flask seed liquid is inoculated in seeding tank by the inoculum size of seed tank culture matrix long-pending 0.1～5%, and 80～120rpm cultivates 16～32h; Seed tank culture liquid is inoculated in fermentation tank culture medium by the amount of fermention medium volume 5～15%, 120～200rpm, fermentation culture 130～240 hours is collected fermented liquid; Wherein culture temperature is 28～32 ℃.
5. application as claimed in claim 3 is characterized in that the feed supplement in the fermentation culture process among the described step b is:
1), treat that mycelial growth begins to mend capric acid and Witconol 2301 after good with 1: 1 mixture of volume ratio, the amount of being mended is 0.1ml/L～0.6ml/L fermented liquid h;
Or
2), treat to begin after mycelial growth well stream and add benefit 8% capric acid ammonia, flow velocity 1.0～3.0ml/L/h;
Or
3), pH is raised at 7.0～8.5 o'clock and begins to mend capric acid and Witconol 2301 for the first time with 1: 1 mixture of volume ratio during the fermentation, the amount of being mended is 0.03～0.2ml/L fermented liquid h; Proceed to 35～45 hours until finish in fermentation, the amount of being mended is 0.10～0.55ml/L fermented liquid h;
Wherein fermenting to 70～78 hours, suspending capric acid and Witconol 2301 with 1: 1 mixture feed supplement of volume ratio, disposable benefit is gone into rape oil, and the rape oil by volume of mending is 0.5～1.7% of a fermentor tank volume, and air flow quantity reduces 10～20%; The rape oil feed supplement finishes the back and continued in 4～8 hours to mend capric acid and Witconol 2301 with 1: 1 mixture of volume ratio according to preceding method; After mending rape oil 24～40 hours; Suspend benefit capric acid and Witconol 2301 with 1: 1 mixture of volume ratio; Disposable once more benefit is gone into rape oil; The rape oil amount by volume of mending is 0.5～1.5% of a fermentor tank volume, and air flow quantity continue to reduce 10～20%, and the rape oil feed supplement finishes the back and continued in 4～8 hours to mend capric acid and Witconol 2301 with 1: 1 mixture of volume ratio according to preceding method;
Or
4), treat that mycelial growth begins to mend capric acid and Witconol 2301 after good with 1: 1 mixture of volume ratio; The amount of being mended is 0.1ml/L～0.4ml/L fermented liquid h; When the reducing sugar in the substratum is reduced to below 1%, begin to mend glucose, with the concentration that keeps reducing sugar 1%.
6. the application of stating like claim 5; It is characterized in that described 3) in fermentation culture process feed supplement after for the second time mending rape oil 24 hours; Suspend capric acid and Witconol 2301 and go into the benefit of 1: 1 mixture of volume ratio, mend rape oil again one time, disposable benefit is gone into; Benefit amount by volume is 0.5～1.2% of a fermentor tank volume, and the rape oil feed supplement finishes the back and continued in 4～8 hours to mend capric acid and Witconol 2301 with 1: 1 mixture of volume ratio according to preceding method.
7. method as claimed in claim 6 is characterized in that total benefit amount by volume of described rape oil is 1.5～4.4% of a fermentor tank volume.
8. application as claimed in claim 3 is characterized in that the substratum among the described step b is: volume ratio meter by weight, and the carbon source of shaking in the Bottle & Can culturing process is 1.0～9.5%; Nitrogenous source is 0.4～3.8%; Inorganic salt are 0.2～2.5%, and the carbon source in the seed tank culture process is 1.0～9.5%, and nitrogenous source is 0.8～3.8%; Inorganic salt are 0.2～2.5%, and all the other are water; Wherein carbon source is selected from one or more in maltodextrin, starch, amylum hydrolysate of the sugar, molasses, glycerine, glucose, SANMALT-S, wort, potato leaching powder, potato dextrin, the rolled oats; Described nitrogenous source is selected from soybean cake powder, steeping water, zein, peptone, pancreatin Sunlover 10, urea, ferrous ammonium sulphate, yeast powder, YE, gives birth to several kinds in bean powder, nitrate salt, the monosodium glutamate; Inorganic salt are selected from K
2HPO
4, NaCl, Fe (NH
4)
2(SO
4)
2.6H
2O, NaNO
3, KNO
3, MgSO
4.7H
2O, FeSO
4.7H
2O, KH
2PO
4In one or more;
In the fermentation culture process, carbon source is 1.5～15%, and nitrogenous source is 0.5～7.0%, and inorganic salt are 0.1～2.0% amino acid 0～1.5%, and all the other are water; Wherein carbon source is selected from one or more in maltodextrin, Zulkovsky starch, amylum hydrolysate of the sugar, cane molasses, beet sirup, glycerine, rape oil, glucose, SANMALT-S, wort, potato leaching powder, potato dextrin, the rolled oats; Described nitrogenous source is selected from soybean cake powder, steeping water, zein, peptone, pancreatin Sunlover 10, urea, ferrous ammonium sulphate, yeast powder, YE, gives birth to several kinds in bean powder, nitrate salt, the monosodium glutamate; Inorganic salt are selected from K
2HPO
4, NaCl, Fe (NH
4)
2(SO
4)
2.6H
2O, NaNO
3, KNO
3, MgSO
4.7H
2O, FeSO
4.7H
2O, CH
3COONa, CaCO
3In one or more.
9. application as claimed in claim 8 is characterized in that describedly it is characterized in that the substratum among the described step b is: volume ratio meter by weight,
Carbon source in the shake-flask culture process is 8.0～9.0%, and nitrogenous source is 2.0～2.8%, and inorganic salt are 0.5～1.3%, and the carbon source in the seed tank culture process is 7.0～9.0%, and nitrogenous source is 2.0～3.0%, and inorganic salt are 0.5～1.3%, and all the other are water; Wherein carbon source is selected from one or more in amylum hydrolysate of the sugar, molasses, maltodextrin, glycerine, glucose, the potato dextrin; Described nitrogenous source make a living two kinds of bean powder, pancreatin Sunlover 10, three kinds of yeast powders or vegetable protein peptone, pancreatin Sunlover 10s; Inorganic salt are selected from Fe (NH
4)
2(SO
4)
2.6H
2O, KNO
3In one or more;
In the fermentation culture process, carbon source is 5～11%, and nitrogenous source is 0.7～3%, and inorganic salt are 0.5～1.3%, amino acid 0.3～0.9%, and all the other are water; Wherein carbon source is selected from one or more in cane molasses, Zulkovsky starch, maltodextrin, glucose, rape oil, the potato dextrin; Described nitrogenous source is four kinds of analysis for soybean powder, KNO3, three kinds of Sodium Glutamates or peptone, urea, ferrous ammonium sulphate, yeast powders; Inorganic salt are selected from Fe (NH
4)
2(SO
4)
2.6H
2O, NaNO
3, KNO
3, CH
3COONa, CaCO
3In one or more.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102181390B (en) * | 2011-03-21 | 2012-11-14 | 北京市农林科学院 | Streptomyces parvus strain and application thereof |
| CN103159830B (en) * | 2011-12-15 | 2017-06-20 | 上海来益生物药物研究开发中心有限责任公司 | Lipopeptide compound |
| CN103160556A (en) * | 2011-12-15 | 2013-06-19 | 上海来益生物药物研究开发中心有限责任公司 | Application of Streptomyces parvus |
| CN102675426B (en) * | 2012-04-26 | 2013-12-11 | 杭州华东医药集团生物工程研究所有限公司 | Extraction and purification method of daptomycin |
| CN103859401A (en) * | 2014-03-11 | 2014-06-18 | 苏州科大微龙信息技术有限公司 | Fruit scented tea enzyme capable of nourishing yin and beautifying and preparation method of fruit scented tea enzyme |
| CN108728505B (en) * | 2014-11-26 | 2021-10-26 | 丽珠集团福州福兴医药有限公司 | Method for producing vancomycin capable of improving yield |
| CN104450823B (en) * | 2014-12-18 | 2017-11-17 | 河北圣雪大成制药有限责任公司 | A kind of method for improving nimoctin fermentation yield |
| CN105112298A (en) * | 2015-08-26 | 2015-12-02 | 中国科学院微生物研究所 | Trichoderma voglmayrii producing mycelium cultivation medium, method for preparing same and application |
| CN108342435A (en) * | 2017-01-22 | 2018-07-31 | 江苏恒瑞医药股份有限公司 | A kind of method that fermentation prepares Daptomycin |
| CN108342436A (en) * | 2017-01-22 | 2018-07-31 | 江苏恒瑞医药股份有限公司 | A kind of method that fermentation prepares Daptomycin |
| CN109593808B (en) * | 2017-09-30 | 2022-10-04 | 上海医药工业研究院 | Daptomycin fermentation medium and preparation method thereof |
| CN110343638B (en) | 2019-07-01 | 2020-08-11 | 浙江大学 | Streptomyces strain capable of producing daptomycin and application thereof |
| CN111690700B (en) * | 2020-07-01 | 2022-03-18 | 上海农乐生物制品股份有限公司 | Shenqinmycin fermentation process |
| CN112111413B (en) * | 2020-09-27 | 2023-08-08 | 贵州省生物技术研究所(贵州省生物技术重点实验室、贵州省马铃薯研究所、贵州省食品加工研究所) | Trichoderma fermentation medium for antagonizing tea anthracnose pathogen and fermentation method |
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