CN103555785A - Process for fermenting rapamycin with high yield - Google Patents
Process for fermenting rapamycin with high yield Download PDFInfo
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Abstract
The invention provides a process for fermenting rapamycin with high yield, and belongs to a method for preparing antibiotics by utilizing fermentation of microorganism in the technical field of biology. According to the method, enzymolytic cotton seed powder, dextrin, glycerinum, yeast extract, L-lysine, KH2PO4, K2HPO4, (NH4)2SO4 and trace elements as culture media, wherein the trace elements contain Co<2+>, glycerinum and phosphate buffer are fed in the early phase of the fermenting culture process, and L-pipecolic acid and shikimic acid are fed in the synthesizing phase. By adopting the method, the methyl derivative can be reduced in the growing phase, and the content of rapamycin is increased; intermediate products can be reduced, and rapamycin can generate key enzyme activity; the beneficial effect of high yield of rapamycin can be achieved.
Description
Technical field
The invention belongs in biological technical field and utilize the antibiotic method of microorganism fermentative production, is a kind of high yield rapamycin zymotechnique specifically.
Background technology
Rapamycin (Rapamycin, RAPA), has another name called sirolimus (Sirolimus), molecular structure as the molecular formula of Fig. 1 RAPA be C
51h
79nO
13, molecular weight 99lKD, is white crystalline solid, fusing point is 183～185 ℃, specific rotatory power [ɑ]
25 d=153
0(methyl alcohol), lipotropy, is dissolved in the organic solvents such as methyl alcohol, ethanol, acetone, chloroform, atomic water-soluble, is dissolved in hardly ether, and in blood plasma, pH is low neutrality, fast especially 37 ℃ of degradeds, and in blood plasma, the transformation period is 1～5h.The novel immunosuppressor of nitrogenous triolefin Macrolide that rapamycin is comprised of three sixteen-rings, it has antimycotic, antiproliferative and antitumor action, rapamycin is successfully applied to clinically in recent years, as treating organs transplant rejection medicine, is the immunosuppressor that renal toxicity is minimum.
It is generally acknowledged, rapamycin is combined with FKBP12, RAPA-FKBP12 mixture and mammals rapamycin target protein (mTOR) combination, but it is the generation of blockage of T cells growth factor receptor not, but block the hyperplasia signal that these factors provide, suppress T lymphocyte that multiple stimulation causes and the hyperplasia of bone-marrow-derived lymphocyte, make cell rest on cell cycle Gl late period phase, stop these cells to enter the S phase.
Rapamycin preparation is applied to clinical gradually: the rejection that transplant for preventing and treating liver kidney organ (1).(2) there is anti-mycotic activity, particularly anti-candida albicans.(3) there is antitumor properties.(4) for smooth muscle cell proliferation and the endo cell hyperplasia of preventing or systemic lupus erythematosus, insulin-dependent diabetes mellitus, blood vessel injury cause.(5) be coated on support for preventing the treatment of coronary restenosis.
Now, rapamycin derivative is also applied to clinical gradually, FDA approval temsirolimus (Temsirolimus) in 2007 listing, treatment for renal cell carcinoma in late period, everolimus (Everolimus) was applied to clinical in 2009, the sub-phosphono rapamycin of dimethyl has restraining effect to multiple human body tumour cells such as fibrosarcoma, glioma, prostate cancer, mammary cancer and adenomyomas.
Rapamycin has potential applicability in clinical practice widely, but mainly adopt at present fermentative Production rapamycin, general rapamycin produces bacterium and adopts streptomyces hygroscopicus (Streptomyces hygrocscopicus NRRL5491), and fermentation unit is 150 to 400ug/ml.CN102433364 discloses a kind of employing fermentative Production rapamycin technique, the bacterial strain adopting is CGMCC No.5145, to take NRRL5491 to be that starting strain obtains through the method screening of ultraviolet mutagenesis protoplastis, and substratum is optimized to the highest rapamycin that obtains 815mg/L in 100L fermentor tank.
Patent CN102229968 discloses in 7L fermentation cylinder for fermentation and has produced rapamycin, adopts the substratum of optimizing: glycerine, glucose, soybean cake powder, yeast extract, peptone, Methionin, inorganic salt.Earlier fermentation adopts stream to add glucose, and the later stage is controlled stream and adds shikimic acid and Methionin precursor, and rapamycin production peak can reach 700mg/L.
Patent CN101486976 discloses employing streptomyces hygroscopicus (being numbered CCTCC NO:207200) and has fermented, and rapamycin production peak can reach 600mg/L.Fermentative production rapamycin technique has been optimized in lijin's (Master's thesis 2012) research, considers the factors such as amino acid, precursor and cofactor, when cultivating 204h, tires and can reach 860.6ug/ml.
From above patent documentation, can obtain more than rapamycin fermentation unit reaches 600mg/L, but have following several respects problem: substratum is unreasonable, and viscosity is high, cause in large scale fermentation process dissolved oxygen content low, be unfavorable for thalli growth; No-feed supplement in fermenting process, cell density is low; Fermentation middle and later periods feed supplement is unreasonable, is unfavorable for the biosynthesizing of rapamycin, causes rapamycin content low; Mesostate is not controlled during the fermentation, fermenting process intermediate by-products is too much, and foreign matter content is high, and rapamycin is tired low, causes extraction cost too high.
Summary of the invention
The present invention aims to provide a kind of at generation minimizing demethyl derivative, increases rapamycin content; Reduce intermediate product, keep rapamycin to generate key enzyme activity; Thereby improve a kind of high yield rapamycin zymotechnique of rapamycin production.
In order to achieve the above object, the present invention adopts following technical scheme:
A rapamycin zymotechnique, adopts streptomyces hygroscopicus fermentation method to produce rapamycin, and as technical characterictic of the present invention, method therefor is divided into following step:
1. by strain transfer kind on slant medium, in the biochemical cultivation case of 28 ℃, cultivate 10～15 days, treat that inclined-plane is covered by spore completely, spore color is by turning in vain after light gray, at once uses or is stored under the environment of 4 ℃;
2. with inoculation shovel, from oneself cultured rapamycin generation bacterium inclined-plane, get a ring (or by 0.5% spore suspension amount) and be seeded in the Erlenmeyer flask that 30m/250ml seed culture medium is housed, shaking speed is 200r/m, and temperature is 30 ℃, cultivates 52～60h.
3. by 1%～3% inoculum size, seed culture fluid is seeded in the first class seed pot that 200L/400L seed culture fluid is housed, rotating speed 100r/min, cultivates 48h, 28～30 ℃ of culture temperature, air quantity 2.0～2.2m
3/ h, tank pressure 0.02MPa.
4. by 2%～10% inoculum size, seed culture fluid is seeded in the secondary seed tank that 1500L/2200L seed culture fluid is housed, rotating speed 80～100r/m, 28～30 ℃ of culture temperature, cultivate 48h, air quantity 2.0～2.2m
3/ h tank pressure 0.02MPa.
5. by secondary seed tank seed culture fluid according in 5～10% inoculum size access 15000L fermentor tank, the volumetric coefficient of fermentor tank is 75%, 25～27 ℃ of culture temperature, maintain tank pressure 0.05～0.07MPa, air quantity 35～46m
3/ h, rotating speed 80r/m～200r/m, suitably adjusts air quantity and revolution and guarantees that dissolved oxygen is not less than 20%, at earlier fermentation, maintains pH value 6.0～7.5, in synthesis phase, maintains pH value 4.5～6.0, and incubation time is 192～216h.
As technical characterictic of the present invention, described inclined-plane seed culture medium comprises following component and content: analysis for soybean powder 0.5 ~ 1.5g/L, Zulkovsky starch 8 ~ 12g/L, K
2hPO
40.2 ~ 0.8g/L, MgSO
47H
2o 0.5 ~ 1.5g/L, agar 19 ~ 21g/L.
As technical characterictic of the present invention, described seed culture medium comprises following component and content: peptone 10～15g/L, yeast extract paste 6～10g/L, glucose 20～25g/L, 1B 5～10g/L, NaH
2pO
40.5～1.0g/L, is adjusted to 6.5～7.0,121 ℃ of sterilizing 45min before pH sterilizing.
As technical characterictic of the present invention, described fermention medium comprises following component and content: enzymolysis cottonseed meal 14.8～17.8g/L, dextrin 5～10g/L, glycerine 60～80g/L, Semen Maydis powder 4.5～6.0g/L, 1B 2～4 g/L, KH
2pO
40.75～1.0 g/L, K
2hPO
40.75～1.0g/L, bubble enemy 1.6 ~ 2.1g/L, liquid microelement 1.2 ~ 1.8ml/L, Jia Shui supply 1L.
As technical characterictic of the present invention, in described preferred trace element solution, comprise CoCl
27H
2o 2.0～3.0g/L, preferably trace element is combined as MgSO
4120～130g/L, ZnSO
47H
2o 40～60g/L, FeSO
47H
2o 10～30g/L, CoCl
27H
2o 2.0～3.0g/L, CuSO
45H
2o 4.0～5.0g/L, concentrated hydrochloric acid 20～30ml/L, Jia Shui supply 1L.
As technical characterictic of the present invention, in the zymotechnique of described optimization, adopt L-pipecolic acid, shikimic acid, glycerine associating feed supplement, wherein after fermentation culture 24h, start to add the mixed liquor containing 48 ~ 52g/L glycerine and 0.7 ~ 0.8g/L phosphate buffered saline buffer, pH value is 6.5～7.0, each 200～500ml, adds once every 30min; At fermentation culture 60h, start to add the mixed aqueous solution of 50g/L glycerine, each 500～1000ml, adds once every 30min; In fermentation, 60h adds containing 0.3 ~ 1.7g/L L-pipecolic acid, 1.8 ~ 2.2g/L shikimic acid mixed aqueous solution 100ml, and every 24h adds once; At fermentation culture 72h, add again liquid microelement 0.45 ~ 0.55ml/L.
As technical characterictic of the present invention, described streptomyces hygroscopicus logarithmic phase keeps pH value 6.5～7.0, in rapamycin generation, adds ammoniacal liquor and makes pH value be controlled at 5.0～5.5.
As technical characterictic of the present invention, described seed culture temperature is 28～30 ℃, and fermentation culture temperature is 25～27 ℃.
As technical characterictic of the present invention, described streptomyces hygroscopicus (Streptomyces hygrocscopicus HD1001) is to be obtained through ultraviolet mutagenesis, chemomorphosis, Protoplast Mutation fusion, high flux screening by streptomyces hygroscopicus (Streptomyces hygrocscopicus NRRL5491).
The present invention, through above-mentioned technical characterictic, has reached following beneficial effect:
1. in fermenting process, in rapamycin generation, add Co
2+, Cu
2+, Mg
2+, Zn
2+, Fe
2+the trace element solution of ion population, thus the expression minimizing demethyl derivative that improves the active of the synthetic relevant enzyme of rapamycin and strengthen Rap M gene increases rapamycin content, and reduces metabolic intermediate, improves rapamycin production.
2. during the fermentation, add glycerine and phosphate buffered saline buffer early stage, in rapamycin generation, adopts L-pipecolic acid, shikimic acid, glycerine associating feed supplement, improves rapamycin growing amount.
3. in thalli growth phase pH value, be controlled at 6.0～7.5, guarantee thalline vigorous growth, in rapamycin generation, Feeding ammonia water, controls pH value 5.0～5.5, keeps the activity of the synthetic key enzyme of rapamycin, improves rapamycin content.
4. the zymotechnique control process that adopts streptomyces hygroscopicus (Streptomyces hygrocscopicus HD1001) and match, more than rapamycin content reaches 1100mg/L, is greater than at present the rapamycin content of report both at home and abroad.
Accompanying drawing explanation
Fig. 1 is rapamycin molecular structure;
Fig. 2 adds Co in fermenting process
2+front rapamycin is tired and impurity component HPLC figure;
Fig. 3 adds Co in fermenting process
2+rear rapamycin is tired and impurity component HPLC figure;
Fig. 4 is fermentation contrast broken line graph.
Embodiment
By being diluted to certain density rapamycin spore suspension, evenly coat in the plate of the slant medium that fills 30ml, substratum is analysis for soybean powder 1g/L, Zulkovsky starch 10g/L, K
2hPO
40.5g/L, MgSO
47H
2o 1g/L, agar 20g/L cultivate 11 days in the biochemical cultivation case of 28 ℃, and picking list bacterium colony is cultivated in test tube slant.
With inoculation shovel, from oneself cultured rapamycin generation bacterium inclined-plane, get a ring and be seeded in the 250ml Erlenmeyer flask that 30ml seed culture medium is housed, shaking speed is 200r/m, and temperature is 30 ℃, cultivates 52h.
1% inoculum size is seeded to seed culture fluid in the 400L first class seed pot that 200L seed culture fluid is housed by volume, air quantity 2.0m
3/ h, tank pressure 0.02MPa, suitably adjusts mixing speed, makes dissolved oxygen be not less than 10%, and 30 ℃ of culture temperature are cultivated 48h.Seed culture medium comprises following component and content: peptone 15g/L, yeast extract paste 10g/L, glucose 25g/L, 1B 10g/L, NaH
2pO
41.0g/L, is adjusted to 6.5,121 ℃ of sterilizing 45min before pH sterilizing.
2% inoculum size is seeded to seed culture fluid in the 2200L secondary seed tank that 1500L seed culture fluid is housed by volume, air quantity 2.2m
3/ h, tank pressure 0.02MPa, suitably adjusts mixing speed, makes dissolved oxygen be not less than 10%, and 30 ℃ of culture temperature are cultivated 48h.
Secondary seed tank seed culture fluid is equipped with in the 15000L fermentor tank of 11250L fermentation culture according to the inoculum size access of volume ratio 5%, and the volumetric coefficient of fermentor tank is 75%, and 26 ℃ of culture temperature, maintain tank pressure 0.07MPa, air quantity 46m
3/ h, suitably adjusts and guarantees that dissolved oxygen is not less than 20%, and fermention medium comprises following component and content: enzymolysis cottonseed meal 17.8g/L, dextrin 10g, glycerine 80g/L, Semen Maydis powder 6.0g/L, 1B 4 g/L, KH
2pO
41.0 g/L, K
2hPO
41.0g/L, bubble oppose 1.9g/L, liquid microelement 1.5ml/L, all the other are water.Trace element is combined as MgSO
4130g/L, ZnSO
47H
2o 60g/L, FeSO
47H
2o 30g/L, CoCl
27H
2o 3.0g/L, CuSO
45H
2o5.0g/L, concentrated hydrochloric acid 30ml/L, Jia Shui supply 1L.
After fermentation culture 24h, start to add the mixed liquor containing 50g/L glycerine and 0.75g/L phosphate buffered saline buffer, each 500ml, adds once every 30min; At fermentation culture 60h, start to add the mixed aqueous solution of 20～50g/L glycerine, each 500ml, adds once every 30min, according to the suitable Feeding ammonia water of pH value changing conditions, makes it to maintain 5.0.At 60h, add containing 0.5g/L L-pipecolic acid and 2g/L shikimic acid mixed aqueous solution 100ml, every 24h adds once.At fermentation culture 72h, add again liquid microelement 0.50ml/L.Culture cycle is 216 hours, and after fermentation ends, sampling detects rapamycin content in fermented liquid.
Embodiment 2
By the seed of preparation by volume 3% inoculum size seed culture fluid is seeded in the 400L first class seed pot that 250L seed culture fluid is housed, air quantity 2.0m
3/ h, tank pressure 0.02MPa, suitably adjusts mixing speed, makes dissolved oxygen be not less than 10%, and 30 ℃ of culture temperature are cultivated 48h.Seed culture medium comprises following component and content: peptone 10g/L, yeast extract paste 6g/L, glucose 20g/L, 1B 5g/L, NaH
2pO
40.5g/L, is adjusted to 6.8,121 ℃ of sterilizing 45min before pH sterilizing.
10% inoculum size is seeded to seed culture fluid in the 2200L secondary seed tank that 1500L seed culture medium is housed by volume, air quantity 2.2m
3/ h, tank pressure 0.02MPa, suitably adjusts mixing speed, makes dissolved oxygen be not less than 10%, and 30 ℃ of culture temperature are cultivated 48h.
Secondary seed tank seed culture fluid is equipped with in the 15000L fermentor tank of 11250L fermentation culture according to the inoculum size access of volume ratio 10%, and the volumetric coefficient of fermentor tank is 75%, and 26 ℃ of culture temperature, maintain tank pressure 0.07MPa, air quantity 46m
3/ h, suitably adjusts and guarantees that dissolved oxygen is not less than 20%, at earlier fermentation, maintains pH value 6.8, in synthesis phase, maintains pH value 5.5, cultivates 196 hours.Fermention medium comprises following component and content: enzymolysis cottonseed meal 14.8g/L, dextrin 5g, glycerine 60g/L, Semen Maydis powder 4.5g/L, 1B 2 g/L, KH
2pO
40.75 g/L, K
2hPO
40.75g/L, bubble oppose 1.9g/L, liquid microelement 1.5ml/L, all the other are water.Trace element is combined as MgSO
4120g/L, ZnSO
47H
2o 40g/L, FeSO
47H
2o 10g/L, CoCl
27H
2o 2.0g/L, CuSO
45H
2o 4.0g/L, concentrated hydrochloric acid 30ml/L, Jia Shui supply 1L.After fermentation ends, sampling detects rapamycin content in fermented liquid.
Embodiment 3
By the seed of preparation by volume 3% inoculum size seed culture fluid is seeded in the 400L first class seed pot that 250L seed culture fluid is housed, air quantity 2.0m
3/ h, tank pressure 0.02MPa, suitably adjusts mixing speed, makes dissolved oxygen be not less than 10%, and 30 ℃ of culture temperature are cultivated 48h.Seed culture medium comprises following component and content: peptone 10g/L, yeast extract paste 6g/L, glucose 20g/L, 1B 5g/L, NaH
2pO
40.5g/L, is adjusted to 6.8,121 ℃ of sterilizing 45min before pH sterilizing.
10% inoculum size is seeded to seed culture fluid in the 2200L secondary seed tank that 1500L seed culture medium is housed by volume, air quantity 2.2m
3/ h, tank pressure 0.02MPa, suitably adjusts mixing speed, makes dissolved oxygen be not less than 10%, and 30 ℃ of culture temperature are cultivated 48h.
Secondary seed tank seed culture fluid is equipped with in the 15000L fermentor tank of 11250L fermentation culture according to the inoculum size access of volume ratio 8%, and the volumetric coefficient of fermentor tank is 75%, and 27 ℃ of culture temperature, maintain tank pressure 0.07MPa, air quantity 46m
3/ h, suitably adjusts and guarantees that dissolved oxygen is not less than 20%, at earlier fermentation, maintains pH value 7.0, in synthesis phase, maintains pH value 5.2, cultivates 196 hours.Fermention medium comprises following component and content: enzymolysis cottonseed meal 16.0g/L, dextrin 8.0g, glycerine 70g/L, Semen Maydis powder 5.5g/L, 1B 3 g/L, KH
2pO
40.85 g/L, K
2hPO
40.85g/L, bubble oppose 1.9g/L, liquid microelement 1.5ml/L, all the other are water.Trace element is combined as MgSO
4120g/L, ZnSO
47H
2o 40g/L, FeSO
47H
2o 10g/L, CoCl
27H
2o 2.0g/L, CuSO
45H
2o 4.0g/L, concentrated hydrochloric acid 30ml/L, Jia Shui supply 1L.After fermentation ends, sampling detects rapamycin content in fermented liquid.
Embodiment 4
By the seed of preparation by volume 3% inoculum size seed culture fluid is seeded in the 400L first class seed pot that 250L seed culture fluid is housed, air quantity 2.0m
3/ h, tank pressure 0.02MPa, suitably adjusts mixing speed, makes dissolved oxygen be not less than 10%, and 30 ℃ of culture temperature are cultivated 48h.Seed culture medium comprises following component and content: peptone 10g/L, yeast extract paste 6g/L, glucose 20g/L, 1B 5g/L, NaH
2pO
40.5g/L, is adjusted to 6.8,121 ℃ of sterilizing 45min before pH sterilizing.
10% inoculum size is seeded to seed culture fluid in the 2200L secondary seed tank that 1500L seed culture medium is housed by volume, air quantity 2.2m
3/ h, tank pressure 0.02MPa, suitably adjusts mixing speed, makes dissolved oxygen be not less than 10%, and 30 ℃ of culture temperature are cultivated 48h.
Secondary seed tank seed culture fluid is equipped with in the 15000L fermentor tank of 11250L fermentation culture according to the inoculum size access of volume ratio 8%, and the volumetric coefficient of fermentor tank is 75%, and 25 ℃ of culture temperature, maintain tank pressure 0.05MPa, air quantity 46m
3/ h, suitably adjusts and guarantees that dissolved oxygen is not less than 20%, cultivates 210 hours.Fermention medium comprises following component and content: enzymolysis cottonseed meal 16.0g/L, dextrin 8.0g, glycerine 70g/L, Semen Maydis powder 5.5g/L, 1B 3 g/L, KH
2pO
40.85 g/L, K
2hPO
40.85g/L, bubble oppose 1.9g/L, liquid microelement 1.5ml/L, all the other are water.Trace element is combined as MgSO
4130g/L, ZnSO
47H
2o 60g/L, FeSO
47H
2o 30g/L, CoCl
27H
2o 3.0g/L, CuSO
45H
2o 5.0g/L, concentrated hydrochloric acid 30ml/L, Jia Shui supply 1L.
After fermentation culture 24h, start to add the mixed liquor containing 50g/L glycerine and 0.75g/L phosphate buffered saline buffer, each 300ml, adds once every 30min, at earlier fermentation, maintains pH value 7.0; At fermentation culture 60h, start to add the mixed aqueous solution of 50g/L glycerine, each 800ml, adds once every 30min, according to the suitable Feeding ammonia water of pH value changing conditions, makes it to maintain 5.2.At 60h, add containing 0.5g/L L-pipecolic acid and 2g/L shikimic acid mixed aqueous solution 100ml, every 24h adds once.At fermentation culture 72h, add again liquid microelement 0.50ml/L.Culture cycle is 216 hours, and after fermentation ends, sampling detects rapamycin content in fermented liquid.
Rapamycin detected result is as follows:
1. the content of rapamycin
Mensuration for rapamycin content in fermented liquid and other impurity adopts high performance liquid chromatography, with octadecylsilane chemically bonded silica, is weighting agent, and the methanol-water-acetonitrile (61:26:13) of take is moving phase, 40 ℃ of column temperatures, detection wavelength is 277nm, sample size 20ul, and flow velocity is 1ml/min.Reference substance is purchased from Sigma company.Detected result is in Table 1, more than showing in fermented liquid that rapamycin content reaches 1100mg/L, compares raising at least 27.9% with the 860mg/L of existing report, improves rapamycin production, reduces production costs.
Rapamycin detected result in table 1 fermented liquid
2, in fermenting process, add Co
2+before and after rapamycin tire and the variation of impurity component
In thalline in rapamycin biosynthetic process, after the polyketide skeleton closed loop of rapamycin, the rear modification that enters composite structure, this process relates to a plurality of non-PKS genes, wherein rapM, rapI and rapQ are the genes of coding methyltransgerase, are responsible for C in total
7, C
32and C
41methoxyl group modify.Co
2+the coenzyme of methyltransgerase, as can be known from Fig. 2, the appropriate Co that adds in substratum
2+can reduce the mesostates such as demethyl derivative, increase rapamycin content, shorten extraction process, improve product recovery rate and qualification rate.
Claims (9)
1. a high yield rapamycin zymotechnique, adopts streptomyces hygroscopicus fermentation method to produce rapamycin, it is characterized in that: method therefor is divided into following step:
1. by strain transfer kind on slant medium, in the biochemical cultivation case of 28 ℃, cultivate 10～15 days, treat that inclined-plane is covered by spore completely, spore color is by turning in vain after light gray, at once uses or is stored under the environment of 4 ℃;
2. with inoculation shovel, from oneself cultured rapamycin generation bacterium inclined-plane, get a ring (or by 0.5% spore suspension amount) and be seeded in the Erlenmeyer flask that 30m/250ml seed culture medium is housed, shaking speed is 200r/m, and temperature is 30 ℃, cultivates 52～60h;
3. by 1%～3% inoculum size, seed culture fluid is seeded in the first class seed pot that 200L/400L seed culture fluid is housed, rotating speed 100r/min, cultivates 48h, 28～30 ℃ of culture temperature, air quantity 2.0～2.2m
3/ h, tank pressure 0.02MPa;
4. by 2%～10% inoculum size, seed culture fluid is seeded in the secondary seed tank that 1500L/2200L seed culture fluid is housed, rotating speed 80～100r/m, 28～30 ℃ of culture temperature, cultivate 48h, air quantity 2.0～2.2m
3/ h tank pressure 0.02MPa;
5. by secondary seed tank seeding tank seed culture fluid according in 5～10% inoculum size access 15000L fermentor tank, the volumetric coefficient of fermentor tank is 75%, 25～27 ℃ of culture temperature, maintain tank pressure 0.05～0.07MPa, air quantity 35～46m
3/ h, rotating speed 80r/m～200r/m, suitably adjusts air quantity and revolution and guarantees that dissolved oxygen is not less than 20%, at earlier fermentation, maintains pH value 6.0～7.5, in synthesis phase, maintains pH value 4.5～6.0, and incubation time is 192～216h.
2. a kind of high yield rapamycin zymotechnique as claimed in claim 1, is characterized in that: inclined-plane seed culture medium comprises following component and content: analysis for soybean powder 0.5 ~ 1.5g/L, Zulkovsky starch 8 ~ 12g/L, K
2hPO
40.2 ~ 0.8g/L, MgSO
47H
2o 0.5 ~ 1.5g/L, agar 19 ~ 21g/L.
3. a kind of high yield rapamycin zymotechnique as claimed in claim 1, it is characterized in that: described seed culture medium comprises following component and content: peptone 10～15g/L, yeast extract paste 6～10g/L, glucose 20～25g/L, 1B 5～10g/L, NaH
2pO
40.5～1.0g/L, is adjusted to 6.5～7.0,121 ℃ of sterilizing 45min before pH sterilizing.
4. a kind of high yield rapamycin zymotechnique as claimed in claim 1, is characterized in that: fermention medium comprises following component and content: enzymolysis cottonseed meal 14.8～17.8g/L, dextrin 5～10g/L, glycerine 60～80g/L, Semen Maydis powder 4.5～6.0g/L, 1B 2～4 g/L, KH
2pO
40.75～1.0 g/L, K
2hPO
40.75～1.0g/L, bubble enemy 1.6 ~ 2.1g/L, liquid microelement 1.2 ~ 1.8ml/L, Jia Shui supply 1L.
5. a kind of high yield rapamycin zymotechnique as claimed in claim 1, is characterized in that: in described preferred trace element solution, comprise CoCl
27H
2o 2.0～3.0g/L, preferably trace element is combined as MgSO
4120～130g/L, ZnSO
47H
2o 40～60g/L, FeSO
47H
2o 10～30g/L, CoCl
27H
2o 2.0～3.0g/L, CuSO
45H
2o 4.0～5.0g/L, concentrated hydrochloric acid 20～30ml/L, Jia Shui supply 1L.
6. a kind of high yield rapamycin zymotechnique as claimed in claim 1, it is characterized in that: in the zymotechnique of described optimization, adopt L-pipecolic acid, shikimic acid, glycerine associating feed supplement, wherein after fermentation culture 24h, start to add the mixed liquor containing 48 ~ 52g/L glycerine and 0.7 ~ 0.8g/L phosphate buffered saline buffer, pH value is 6.5～7.0, each 200～500ml, adds once every 30min; At fermentation culture 60h, start to add the mixed aqueous solution of 50g/L glycerine, each 500～1000ml, adds once every 30min; In fermentation, 60h adds containing 0.3 ~ 1.7g/L L-pipecolic acid, 1.8 ~ 2.2g/L shikimic acid mixed aqueous solution 100ml, and every 24h adds once; At fermentation culture 72h, add again liquid microelement 0.45 ~ 0.55ml/L.
7. a kind of high yield rapamycin zymotechnique as claimed in claim 1, is characterized in that: described streptomyces hygroscopicus logarithmic phase keeps pH value 6.5～7.0, in rapamycin generation, adds ammoniacal liquor and makes pH value be controlled at 5.0～5.5.
8. a kind of high yield rapamycin zymotechnique as claimed in claim 1, is characterized in that: described seed culture temperature is 28～30 ℃, and fermentation culture temperature is 25～27 ℃.
9. a kind of high yield rapamycin zymotechnique as described in claim 1 or 7, is characterized in that: described streptomyces hygroscopicus (Streptomyces hygroscopicus HD1001) is to be obtained through ultraviolet mutagenesis, chemomorphosis, Protoplast Mutation fusion, high flux screening by streptomyces hygroscopicus (Streptomyces hygroscopicus NRRL5491).
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Cited By (6)
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| CN104388492A (en) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | Method for producing rapamycin by using streptomyces hygroscopicus |
| CN107365811A (en) * | 2017-09-15 | 2017-11-21 | 常州兰陵制药有限公司 | Utilize the technique of actinoplanes fermenting and producing rapamycin |
| CN107557403A (en) * | 2017-10-31 | 2018-01-09 | 无锡福祈制药有限公司 | A kind of method for improving sirolimus fermentation yield |
| CN109485559A (en) * | 2018-11-22 | 2019-03-19 | 桂林莱茵生物科技股份有限公司 | A method of extracting shikimic acid from illiciumverum |
| CN109486877A (en) * | 2018-11-23 | 2019-03-19 | 北大方正集团有限公司 | The method of fed-batch fermentation technique production rapamycin |
| CN110343639A (en) * | 2019-07-23 | 2019-10-18 | 中国医药集团总公司四川抗菌素工业研究所 | The streptomycete of one plant of 15 (S)-O- ethyl rapamycin of production |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388492A (en) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | Method for producing rapamycin by using streptomyces hygroscopicus |
| CN107365811A (en) * | 2017-09-15 | 2017-11-21 | 常州兰陵制药有限公司 | Utilize the technique of actinoplanes fermenting and producing rapamycin |
| CN107557403A (en) * | 2017-10-31 | 2018-01-09 | 无锡福祈制药有限公司 | A kind of method for improving sirolimus fermentation yield |
| CN109485559A (en) * | 2018-11-22 | 2019-03-19 | 桂林莱茵生物科技股份有限公司 | A method of extracting shikimic acid from illiciumverum |
| CN109485559B (en) * | 2018-11-22 | 2022-02-11 | 桂林莱茵生物科技股份有限公司 | Method for extracting shikimic acid from star anise |
| CN109486877A (en) * | 2018-11-23 | 2019-03-19 | 北大方正集团有限公司 | The method of fed-batch fermentation technique production rapamycin |
| CN110343639A (en) * | 2019-07-23 | 2019-10-18 | 中国医药集团总公司四川抗菌素工业研究所 | The streptomycete of one plant of 15 (S)-O- ethyl rapamycin of production |
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