CN105624068A - Streptomyces tsukubaensis and application thereof in preparation of tacrolimus - Google Patents
Streptomyces tsukubaensis and application thereof in preparation of tacrolimus Download PDFInfo
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Abstract
The invention discloses streptomyces tsukubaensis HDW68 and application thereof. The strain is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC M 2016086, and the preservation date is March 7, 2016. The invention further discloses a method for preparing a tacrolimus intermediate from the strain through fermentation. According to the fermentation technology, production practice of a 1-ton fermentation tank, a 4-ton fermentation tank and a 20-ton fermentation tank proves that the strain is stable in production capacity, high in fermentation unit and fewer in fermentation byproduct (FK520), therefore, the post-extraction difficulty is greatly reduced, the strain is suitable for industrialized mass production, and the obtained tacrolimus product is high in quality.
Description
Technical field
The present invention relates to a kind of novel microorganism and purposes thereof and application, particularly relate to a kind of streptomyces tsukubaensis and in the application preparing in tacrolimus.
Background technology
Tacrolimus (Tacrolimus) has another name called FK506, and its chemical structure belongs to 23 membered macrolide class microbiotic, is the neotype immunosuppressant of a kind of brute force. At molecular level, the effect of this product mediates by the albumen FKBP12 combined with it in tenuigenin. FKBP12 makes this product enter in cell, and form mixture, this mixture is combined specifically with calmodulin competitively and suppresses calmodulin, in the latter mediates T cell-and Ca-dependent suppresses property signal transduction system, thus stops a series of lymphokine genetic transcription. Experiment in vivo and vitro proves, tacrolimus is a potent immunosuppressor, and the formation of poison lymphocyte capable of inhibiting cell, graft-rejection is mainly caused by the latter. The B cell proliferation of the activation of this medicine suppressor T cell and TH cell dependent antibody, and the generation of lymphokine is such as interleukin II, interleukin �� and beta-interferon, and the expression of interleukin 2 receptor. Tacrolimus can suppress the rejection of skin, heart, kidney, liver transplantation, extends the survival time of homotransplant, is confirmed with it rodent, dog, primate and the mankind. Since listing extensively for liver, kidney and bone marrow transplantation acute and chronic repel treatment.
The current report about tacrolimus production method mainly contains US Patent No. 4894366, US4929611, US5116756, US5264355, US5496727 and US5624842. These patent reports produces tacrolimus with different streptomyces strains, output is not very high, the tacrolimus of the streptomycestsukubaensis mentioned in streptomycessp.MA6858 and US5624842 mentioned in US5116756 patent produces bacterial strain, the output producing tacrolimus is all very low, the highest only 37.8mg/L of the concentration of tacrolimus in its fermented liquid. It is 385 �� g/ml that a kind of streptomycete of Chinese patent " method of fermentative production tacrolimus " (CN201310644714.1) report produces tacrolimus, and needs Feeding medium among process, and technique is loaded down with trivial details. The highest fermentation unit disclosed in Chinese patent " a kind of streptomycete and application thereof " (CN200810019003.4) is 550 �� g/ml. The highest fermentation unit disclosed in Chinese patent " preparation method of a kind of high purity tacrolimus " (CN201210447477.5) is 491 �� g/ml, but does not mention zymotechnique. Chinese patent " genetic engineering bacterium streptomyces tsukubaensis L20 and application thereof " (CN201510664086.2) is mentioned genetic engineering bacterium and can be reached 716mg/L by shake flask fermentation unit.
Foreign periodical " JournalofIndustrialMicrobiology&Biotechnology " (2009,36 (12): 1467-1471) mentioning the concentration 600-1600 �� g/ml by improving tacrolimus in substratum gradually in a literary composition " StraindevelopmentofStreptomycessp.fortacrolimusproductio nusingsequentialadaptation " and increase bacterial strain to the tolerance of product, the final bacterial strain obtained reaches 972 �� g/ml in 5L fermentor tank. Interim " seed selection of resistant mutation tacrolimus superior strain " of domestic periodical " microbiology magazine " volume October the 34th in 2014 the 5th is mentioned and is obtained superior strain H-493 by resistant mutation breeding, 50L fermentor tank can reach 918 �� g/ml, but produce as a trial without industrialization. These two kinds of bacterial strains all relate to resistant mutation seed selection, and the stability in its bacterial classification later stage needs to be investigated.
In sum, both at home and abroad in the tacrolimus fermentation production technology of report, adopt the fermentation technique unit height of resistant mutation bacterial strain, genetic engineering bacterium, but this fermentation technique only confirms without suitability for industrialized production through lab scale test; Adopt the zymotechnique unit of conventional bacterial strain low, and fermentation byproduct is more, particularly the content of isomers FK520 wherein is higher, because being that isometry is difficult to be removed by the purifying process in later stage, cause rear extraction process comparatively complicated, substantially increase follow-up purification difficulty, it is difficult to obtain the finished product of high purity, thus be difficult to carry out better industrialization production.
Summary of the invention
It is an object of the invention to for the problems referred to above, it is provided that a kind of fermentation unit height and the few tacrolimus producing strains of by product.
Streptomyces tsukubaensis HDW68 provided by the present invention, classification called after streptomycestsukubansisHDW68, by China typical culture collection center preservation (being called for short CCTCC), preserving number is: CCTCCM2016086, and preservation date is: on 03 07th, 2016. Described preserving number is the bacterial strain of CCTCCM2016086 is screen to obtain from the soil around the new flat phosphorus pools in Yunnan.
Described preserving number is the colony characteristics of the bacterial strain of CCTCCM2016086: bacterium colony orange or redness, subcircular, colony diameter 2-4mm, and bacterium colony protuberance and point lobe, center subsides into hole.
According to the description of microbial morphology and external relevant data, it is various cultural characteristic and the morphological specificity of the bacterial strain of CCTCCM2016086 in conjunction with preserving number, the bacterial strain that this preserving number is CCTCCM2016086 should belong to streptomyces, names as streptomycestsukubansisHDW68.
It is a further object of the present invention to provide this bacterial strain in the application preparing in tacrolimus.
The bacterial strain that described preserving number is CCTCCM2016086 can be applicable to fermentation for tacrolimus. This preparation method comprises the following steps:
A. fermentation strain adopts deposit number to be the bacterial strain of CCTCCM2016086;
B. strain culturing: the 30% glycerine mycelia freeze pipe (namely work strain library) prepared according to a conventional method is accessed shaking flask and carries out seed culture and obtain shake-flask seed liquid; Shake-flask seed liquid is inoculated in shake flask fermentation substratum or seeding tank is cultivated; Seed tank culture liquid is inoculated in fermentation tank culture medium cultivate, collects fermented liquid; Preferably without the need to carrying out feed supplement in fermentation culture process, after seeding tank mycelial growth well accesses fermentor tank, according to fermented liquid practical situation, from pH, bacterium, the aspect such as dense, mixing speed, molten oxygen and mycelia mirror inspection situation controls fermenting process.
C. tunning extracts: extract, concentrated, pre-crystallized, layer is analysed, crystallization and drying.
Wherein said step b is: by the 0.8��1.2% of shake-flask seed culture volume, the 30% glycerine mycelia freeze pipe (work strain library) prepared according to a conventional method is accessed shake-flask seed substratum, 250rpm, cultivate 22��30h, obtain shake-flask seed liquid; By shake-flask seed liquid by seed tank culture matrix long-pending 0.02��0.05% inoculum size be inoculated in seeding tank, stirring velocity 150��250rpm, cultivate 22��30h; Seed tank culture liquid is inoculated in fermentation tank culture medium by the amount of the 5��15% of fermention medium volume, stirring velocity 150��250rpm, fermentation culture 6��7 days, collects fermented liquid; Wherein culture temperature is 26��30 DEG C.
The invention also discloses substratum: volume basis by weight, the carbon source in shaking flask, seed tank culture process is 0.5��4.0%, it is preferable that 0.5��3.0%, it is most preferred that be 1.2��2.5%; Nitrogenous source is 0.2��4.0%, it is preferable that 0.5��3.5%, it is most preferred that be 0.5��2.0%; Inorganic salt are 0.2��2.0%, it is preferable that 0.2��1.5%, it is most preferred that 0.2��1.0%; All the other are water.
Wherein carbon source is selected from Zulkovsky starch, soybean oil, one or more in glucose, W-Gum, glycerine, maltose, sucrose; Preferred Zulkovsky starch, soybean oil, one or more in glucose, W-Gum, glycerine, maltose; Most preferably glucose, W-Gum, one or more in glycerine.
Wherein nitrogenous source selects yeast to leach one or more in powder, Semen Maydis powder, hot soybean cake powder, soybean protein powder, bean cake powder, raw bean powder, beer yeast powder, corn steep solids, ammonium sulfate; One or more in preferred hot soybean cake powder, soybean protein powder, raw bean powder, beer yeast powder, corn steep solids, ammonium sulfate; More preferably hot soybean cake powder, beer yeast powder, one or more in ammonium sulfate.
Wherein inorganic salt CaCO3��K2HPO4��NaCl��KNO3, MgSO4��KCl��KH2PO4In one or more; Preferred CaCO3��K2HPO4��NaCl��KNO3��KH2PO4In one or more; More preferably CaCO3��K2HPO4��KH2PO4In one or more.
Volume basis by weight, the carbon source in fermentation culture process is 3.0��12.0%, it is preferable that 3.0��10.0) %, it is most preferred that 7.0��9.0%; Nitrogenous source is 1.0��8.0%, it is preferable that 1.0��6.0%, it is most preferred that 2.9��4.5%; Inorganic salt are 0.2��2.0%, it is preferable that 0.2��1.5%, it is most preferred that 0.25��1.0%; Amino acid is 0.2��2.0%, it is preferable that 0.2��1.5%, it is most preferred that 0.5��1.0%; All the other are water.
Wherein carbon source be selected from Zulkovsky starch, W-Gum, glycerine, sunflower seed oil, dextrin, glucose, potato starch, maltose, sucrose, rape oil, N.F,USP MANNITOL one or more; One or more in preferred Zulkovsky starch, W-Gum, glycerine, sunflower seed oil, dextrin, glucose, potato starch, maltose, rape oil, N.F,USP MANNITOL, it is most preferred that one or more in W-Gum, glycerine, dextrin, glucose.
Wherein nitrogenous source is selected from bean cake powder, raw bean powder, meat peptone, beer yeast powder, bread yeast powder, hot soybean cake powder, corn steep solids, ammonium sulfate, fish peptone, yeast leach cream, urea, cottonseed meal, yeast leach one or more in powder, dried silkworm chrysalis meal, Zein powder; Preferred bean cake powder, raw bean powder, meat peptone, beer yeast powder, bread yeast powder, hot soybean cake powder, corn steep solids, ammonium sulfate, yeast leach one or more in cream, urea, Zein powder; Be more preferably in beer yeast powder, bread yeast powder, hot soybean cake powder, corn steep solids, ammonium sulfate, meat peptone one or more.
Wherein inorganic salt are selected from CaCO3��MgSO4��K2HPO4��NaCl��KNO3, ZnSO4��KCl��KH2PO4In one or more; Preferred CaCO3��MgSO4��K2HPO4��NaCl��KNO3��KH2PO4In one or more; Most preferably CaCO3��K2HPO4, one or more in NaCl.
Wherein amino acid is selected from leucine, in ILE, Valine, L-glutamic acid, Sodium Glutamate, L-Histidine one or more; Be preferably from leucine, in ILE, Valine, L-glutamic acid, Sodium Glutamate one or more; Most preferably be in ILE, Sodium Glutamate one or more.
After having fermented and having put tank, tunning is extracted, concentrates, the analysis of pre-crystallized, layer, the tacrolimus that purity reaches more than 99% can be obtained after crystallization and drying.
The technique effect of the present invention is mainly reflected in:
1, streptomyces tsukubaensis disclosed in this invention be at present for the production of the tacrolimus producing strains of a kind of high unit, leavening property is excellent, can be used for scale operation. Zymotechnique provided by the invention carries out testing and producing through shaking flask, lab scale (1 ton of fermentor tank), pilot scale (4 tons of fermentor tanks) and 20 tons of production fermentor tanks, its stable production capacity. After testing, the tacrolimus prepared with bacterial strain provided by the invention and fermentation culture method is tired and is reached 1100 �� g/ml or more, thus provides good basis for follow-up suitability for industrialized production.
2, bacterial strain disclosed in this invention screens from the soil around the new flat phosphorus pools in Yunnan and to obtain, and after optimizing technique, through experiment for many years and produce, its strain stability is good. Prove that this bacterial strain is streptomyces tsukubaensis through Physiology and biochemistry, cultural characteristic and gene sequencing.
3, the fermentation byproduct of streptomyces tsukubaensis disclosed in this invention is relatively less, particularly the important by-products of tacrolimus is also that the content of isomers FK520 obviously reduces, the difficulty extracted after reducing, the technique of Isolation and purification is relatively simple, substantially increase receipts rate and purity, suitability for industrialized production has more advantage.
Bacterial strain preservation situation: be preserved in China typical culture collection center (being called for short CCTCC), preservation address is China. Wuhan. and Wuhan University, deposit number is CCTCCNO:M2016086, classification called after streptomyces tsukubaensis HDW68 (streptomycestsukubansisHDW68). Preservation date is on 03 07th, 2016.
Accompanying drawing explanation
Fig. 1 schemes for the FK520 content HPLC of the sample prepared by Chinese patent " one streptomycete and application " thereof (CN200810019003.4) embodiment 6;
The FK520 content HPLC that Fig. 2,3,4 are the different samples prepared with the embodiment of the present application 7 schemes.
Embodiment
By specific embodiment given below, it is possible to clearly understand the present invention further, but they are not limitation of the invention.
Embodiment 1: preserving number is the cultural characteristic of the bacterial strain of CCTCCM2016086
It it is the substratum that the inoculation of CCTCCM2016086 is used for observing morphological specificity in Gause I, glucose asparagine etc. by preserving number. Cultivate 5,7,10 days regular hours, observe and be described for 28 DEG C.
Preserving number is the bacterial strain of the CCTCCM2016086 colony characteristics on various substratum
Embodiment 2: utilization of carbon source and physiological and biochemical property
Physiology and biochemistry character adopts the standard method in streptomyces category.
The biochemical reactions that preserving number is the bacterial strain of CCTCCM2016086 is: energy liquefy gelatin on gelatine culture, and peptonized milk, can not solidify milk, and starch hydrolysis is negative, does not utilize Mierocrystalline cellulose, nitrate reduction negative.
Utilize Biolog (GEN III) automatic microbe identification systems that bacterial strain streptomycestsukubaensis has carried out 94 kinds of phenotype tests, comprise 71 kinds of utilization of carbon source situation detections and 23 kinds of chemosensitivity detections: by specific for inoculation plate culture medium, 33 DEG C of constant temperature culture 2 days, with aseptic cotton carrier, the thalline on flat board is washed down, mix with inoculation liquid (IF-A), make bacteria suspension, it is adjusted to 92%T/IF-A with turbidometer. With the 8 electronic liquid fillerss in hole, bacteria suspension is added in each hole of Biolog (GEN III) micropore respectively, every hole 100 �� l. Micropore identification plate is placed in 33 DEG C of incubators, it is placed on Biolog readout instrument to read result respectively after cultivating 12h, 24h, 32h.
Analyzing metabolism fingerprint through Biolog readout instrument, streptomyces tsukubaensis streptomycestsukubaensis is to 3 kinds of chemical substance sensitivities; Bacterial strain can utilize more by force wherein 17 kinds of carbon sources, and to other, 54 kinds of utilization of carbon source abilities are more weak maybe can not utilize. Bacterial strain. Biolog system provides 36h qualification result. Biolog qualification result is in table 1 and table 2.
Table 1 streptomyces tsukubaensis is to the chemosensitivity of 23 kinds of chemical substances on BiologGEN III plate
Illustrate :+, positive;-, negative; B, uncertain
Table 2 streptomyces tsukubaensis is to the Utilization ability of 71 kinds of carbon sources on Biolog (GEN III) plate
Illustrate :+, positive;-, negative; B, uncertain
Example 3: the production fermentation culture of tacrolimus
Adopt the bacterial strain of CCTCCM2016086
1. shake-flask seed is cultivated
Substratum: Zulkovsky starch 0.3g, soybean oil 0.2g, yeast leaches powder 0.1g, Semen Maydis powder 0.1g, KH2PO40.1g, CaCO30.1g, pH nature, adds water to 100ml.
Inoculum size: by the inoculum size of 0.8ml/ bottle
Cultivate: 28 DEG C, 24h, 250rpm
Rise in mycelia length, after having obvious wall cling phenomenon, 100ml shake-flask seed nutrient solution is accessed in seeding tank and cultivates.
2. seed tank culture
Substratum: Zulkovsky starch 1.5kg, soybean oil 1kg, yeast leaches powder 0.5kg, Semen Maydis powder 0.5kg, KH2PO40.5kg, CaCO30.5kg, pH nature, adds water to 0.5 ton.
Loading amount: 1 ton of in-built substratum of fermentor tank 0.5 ton, 121 DEG C of sterilizing 30min.
Inoculum size: 100ml shake-flask seed.
Cultivate: 28 DEG C, 30h, 150rpm
3. fermentor cultivation
Substratum: potato starch 10kg, glycerine 2.5kg, sunflower seed oil 2.5kg, bean cake powder 2kg, raw bean powder 2kg, meat peptone 1kg, leucine 1kg, CaCO30.5kg��MgSO40.5kg, adds water to 0.5 ton, adjusts pH8.5.
Loading amount: 1 ton of in-built substratum of fermentor tank 0.5 ton.
The seed liquor of inoculum size: 75L
Culture temperature: 28 DEG C, initial adjustment tank pressure 0.05MPa, air flow quantity is in 1:(0.6 �� 0.2) vvm, mixing speed 250rpm. In metabolic process when relative oxygen dissolving value is lower than 30%, first tune up air flow quantity to 1:(1 �� 0.2) vvm; When relative oxygen dissolving value is again lower than 30%, tune up stirring rake frequency conversion value (stirring rake frequency conversion value is between (150��250) rpm); If oxygen dissolving value is again lower than 30% relatively, then within the scope of (0.05��0.08) MPa, increase tank pressure (in fermenting process, tank pressure maintains between (0.02��0.08)). Fermentation period is 7 days.
Put tank unit: 1323 �� g/ml
Example 4: the production fermentation culture of tacrolimus
Adopt the bacterial strain of CCTCCM2016086
1. shake-flask seed is cultivated
Substratum: maltose 3g, glycerine 6g, raw bean powder 3g, soybean protein powder 1.5g, corn steep solids 6g, CaCO30.6g, NaCl2.4g, KNO31.5g, adds water and is settled to 300ml, pH nature
Inoculum size: by the inoculum size of 1ml/ bottle
Cultivate: 26 DEG C, 30h, 250rpm
Rise in mycelia length, after having obvious wall cling phenomenon, 300ml shake-flask seed nutrient solution is accessed in seeding tank and cultivates.
2. seed tank culture
Substratum: maltose 10kg, glycerine 20kg, raw bean powder 10kg, soybean protein powder 5kg, corn steep solids 20kg, CaCO32kg, NaCl8kg, KNO35kg, adds water and is settled to 1 ton, pH nature.
Loading amount: 4 tons of in-built substratum of fermentor tank 1 ton, 121 DEG C of sterilizing 30min.
Inoculum size: 300ml shake-flask seed.
Cultivate: 26 DEG C, 27h, 200rpm
3. fermentor cultivation
Substratum: Zulkovsky starch 150kg, rape oil 25kg, N.F,USP MANNITOL 75kg, Zein powder 50kg, yeast leaches cream 75kg, hot analysis for soybean powder 12.5kg, urea 12.5kg, ILE 12.5kg, L-glutamic acid 12.5kg, Valine 12.5kg, KH2PO412.5kg, KNO312.5kg, CaCO312.5kg, adds water and is settled to 2.5 tons, adjusts pH8.8.
Loading amount: 4 tons of in-built substratum 2.5t of fermentor tank, 121 DEG C of sterilizing 30min.
The seed liquor of inoculum size: 250L
Culture temperature: 27 DEG C, initial adjustment tank pressure 0.05MPa, air flow quantity is in 1:(0.6 �� 0.2) vvm, mixing speed 200rpm. In metabolic process when relative oxygen dissolving value is lower than 30%, first tune up air flow quantity to 1:(1 �� 0.2) vvm; When relative oxygen dissolving value is again lower than 30%, tune up stirring rake frequency conversion value (stirring rake frequency conversion value is between (150��250) rpm); If oxygen dissolving value is again lower than 30% relatively, then within the scope of (0.05��0.08) MPa, increase tank pressure (in fermenting process, tank pressure maintains between (0.02��0.08)). Fermentation period is 7 days.
Fermented liquid puts tank unit: 1288 �� g/ml.
Example 5: the production fermentation culture of tacrolimus
Adopt the bacterial strain of CCTCCM2016086
1. shake-flask seed is cultivated
Substratum: glucose 20g, sucrose 20g, beer yeast powder 10g, bean cake powder 30g, MgSO45g, KCl10g, CaCO35g, pH nature, adds water and is settled to 1000ml, packing 10 bottles.
Inoculum size: by the inoculum size of 1ml/ bottle
Cultivate: 30 DEG C, 22h, 250rpm
Rise in mycelia length, after having obvious wall cling phenomenon, 1000ml shake-flask seed nutrient solution is accessed in seeding tank and cultivates.
2. seed tank culture
Substratum: glucose 40kg, sucrose 40kg, beer yeast powder 20kg, bean cake powder 60kg, MgSO410g, KCl20g, CaCO310g, bubble enemy's 2kg, pH nature, adds water and is settled to 2 tons.
Loading amount: 4 tons of in-built substratum of fermentor tank 2 tons, 121 DEG C of sterilizing 30min.
Inoculum size: 1000ml shake-flask seed.
Cultivate: 30 DEG C, 22h, 200rpm
3. fermentor cultivation
Substratum: sucrose 125kg, glucose 25kg, maltose 150kg, fish peptone 25kg, cottonseed meal 75kg, yeast leaches powder 50kg, dried silkworm chrysalis meal 50kg, L-glutamic acid 25kg, L-Histidine 25kg, ZnSO415kg, KCl25kg, CaCO310kg, adds water and is settled to 2.5t, adjusts pH9.0
Loading amount: 4 tons of in-built substratum of fermentor tank 2.5 tons
The seed liquor of inoculum size: 375L
Culture temperature: 29 DEG C, initial adjustment tank pressure 0.05MPa, air flow quantity is in 1:(0.6 �� 0.2) vvm, mixing speed 150rpm. In metabolic process when relative oxygen dissolving value is lower than 30%, first tune up air flow quantity to 1:(1 �� 0.2) vvm; When relative oxygen dissolving value is again lower than 30%, tune up stirring rake frequency conversion value (stirring rake frequency conversion value is between (150��250) rpm); If oxygen dissolving value is again lower than 30% relatively, then within the scope of (0.05��0.08) MPa, increase tank pressure (in fermenting process, tank pressure maintains between (0.02��0.08)). Fermentation period is 6 days.
Fermented liquid puts tank unit: 1363 �� g/ml.
Example 6: the production fermentation culture of tacrolimus
Adopt the bacterial strain of CCTCCM2016086
1. shake-flask seed is cultivated
Substratum: glycerine 6g, W-Gum 9g, beer yeast powder 1.2g, ammonium sulfate 1.8g, K2HPO43g, CaCO33g, pH nature, adds water and is settled to 600ml, packing 6 bottles.
Inoculum size: by the inoculum size of 1.2ml/ bottle
Cultivate: 29 DEG C, 28h, 250rpm
Rise in mycelia length, after having obvious wall cling phenomenon, 600ml shake-flask seed nutrient solution is accessed in seeding tank and cultivates.
2. seed tank culture
Substratum: glycerine 15kg, W-Gum 22.5kg, beer yeast powder 3kg, ammonium sulfate 4.5kg, K2HPO47.5kg, CaCO37.5kg, bubble enemy's 1.5kg, pH nature, is settled to 1.5 tons.
Loading amount: 4 tons of in-built cultivation based 1.5 tons of fermentor tank, 121 DEG C of sterilizing 30min.
Inoculum size: 600ml shake-flask seed.
Cultivate: 28 DEG C, 28h, 250rpm
3. fermentor cultivation
Substratum: glycerine 140kg, glucose 280kg, W-Gum 210kg, meat peptone 70kg, beer yeast powder 105kg, hot analysis for soybean powder 35kg, corn steep solids 70Kg, ammonium sulfate 35kg, ILE 35Kg, Sodium Glutamate 35kg, K2HPO421kg, NaCl28kg, CaCO321kg, bubble enemy 14kg, adds water and is settled to 7 tons, adjust pH9.5.
Loading amount: 20 tons of in-built substratum of fermentor tank 7 tons
Inoculum size: the seed liquor of 0.35 ton
Culture temperature: 30 DEG C, initial adjustment tank pressure 0.05MPa, air flow quantity is in 1:(0.6 �� 0.2) vvm, mixing speed 150rpm. In metabolic process when relative oxygen dissolving value is lower than 30%, first tune up air flow quantity to 1:(1 �� 0.2) vvm; When relative oxygen dissolving value is again lower than 30%, tune up stirring rake frequency conversion value (stirring rake frequency conversion value is between (150��250) rpm); If oxygen dissolving value is again lower than 30% relatively, then within the scope of (0.05��0.08) MPa, increase tank pressure (in fermenting process, tank pressure maintains between (0.02��0.08)). Fermentation period is 7 days.
Fermented liquid puts tank unit: 1184 �� g/ml.
Example 7: the production fermentation culture of tacrolimus
Adopt the bacterial strain of CCTCCM2016086
1. shake-flask seed is cultivated
Substratum: glucose 1g, W-Gum 5g, hot soybean cake powder 10g, CaCO37.5g, pH nature, adds water and is settled to 500ml, packing 5 bottles.
Inoculum size: by the inoculum size of 1.0ml/ bottle
Cultivate: 28 DEG C, 26h, 250rpm
Rise in mycelia length, after having obvious wall cling phenomenon, 500ml shake-flask seed nutrient solution is accessed in seeding tank and cultivates.
2. seed tank culture
Substratum: glucose 3kg, W-Gum 15kg, hot soybean cake powder 30kg, CaCO33kg, bubble enemy's 1.5kg, pH nature, is settled to 1.5 tons.
Loading amount: 4 tons of in-built cultivation based 1.5 tons of fermentor tank, 121 DEG C of sterilizing 30min.
Inoculum size: 500ml shake-flask seed.
Cultivate: 28 DEG C, 25h, 200rpm
3. fermentor cultivation
Substratum: dextrin 910kg, bread yeast powder 78kg, beer yeast powder 221kg, corn steep solids 65Kg, ammonium sulfate 13kg, CaCO332.5kg, CaCO36.5kg, bubble enemy 42kg, adds water and is settled to 13 tons, adjust pH8.9.
Loading amount: 20 tons of in-built substratum of fermentor tank 13 tons
Inoculum size: the seed liquor of 1.5 tons
Culture temperature: 26 DEG C, initial adjustment tank pressure 0.05MPa, air flow quantity is in 1:(0.6 �� 0.2) vvm, mixing speed 150rpm. In metabolic process when relative oxygen dissolving value is lower than 30%, first tune up air flow quantity to 1:(1 �� 0.2) vvm; When relative oxygen dissolving value is again lower than 30%, tune up stirring rake frequency conversion value (stirring rake frequency conversion value is between (150��250) rpm); If oxygen dissolving value is again lower than 30% relatively, then within the scope of (0.05��0.08) MPa, increase tank pressure (in fermenting process, tank pressure maintains between (0.02��0.08)). Fermentation period is 6.5 days.
According to above-mentioned fermentation condition and technique, carrying out 3 batch fermentation tests at 20 tons of fermentor tanks, fermentation unit is respectively 1158 �� g/ml, 1161 �� g/ml and 1216 �� g/ml.
Embodiment 8:FK520 assay
Measuring method: HPLC method detects
Sample: the fermented liquid in embodiment 7
Control sample: by the sample of Chinese patent " a kind of streptomycete and application thereof " (CN200810019003.4) embodiment 6 preparation.
Chromatographic condition:
Chromatographic column: C8 post (4.6 �� 150mm, 5 ��m);
Flow velocity: 1.0mL/min;
Sampling volume: 20 �� l;
Determined wavelength: 210nm;
Post temperature: 60 DEG C;
Writing time: 65min (wherein the interval time of sample must not less than 1min);
Moving phase: the mixed solution of solution A and solution B, gradient timetable programsheet sees the following form.
Solution A: 0.02mol/L potassium dihydrogen phosphate, adjusts pH to 3.5 with phosphoric acid.
Solution B: acetonitrile.
Blank: acetone.
Result:
After HPLC detects, as 3.58%, see accompanying drawing 1 press FK520 content in sample prepared by Chinese patent " a kind of streptomycete and application " thereof (CN200810019003.4) embodiment 6. The FK520 content of the sample prepared taking embodiment 7, as respectively 1.47%, 1.55%, 1.61%, is shown in accompanying drawing 2,3,4. Both compare, FK520 content decline 50%.
Claims (10)
1. streptomyces tsukubaensis (streptomycestsukubansis) HDW68, by China typical culture collection center preservation, preserving number is CCTCCM2016086, and preservation date is: on 03 07th, 2016.
2. preserving number as claimed in claim 1 be the bacterial strain of CCTCCM2016086 in fermentation for the application in tacrolimus.
3. application rights requires that preserving number described in 1 is the method that the strain fermentation of CCTCCM2016086 prepares tacrolimus, it is characterised in that comprise the following steps:
A. fermentation strain adopts deposit number to be the bacterial strain of CCTCCM2016086;
B. by the bacterial strain access shake-flask seed substratum of CCTCCM2016086,250rpm, cultivates 22��30h, obtains shake-flask seed liquid;
Shake-flask seed liquid is inoculated in seeding tank, stirring velocity 150��250rpm by the inoculum size of seed tank culture matrix long-pending 0.02��0.05%, cultivates 22��30h;
Seed tank culture liquid is inoculated in fermentation tank culture medium by the amount of fermention medium volume 5��15%, stirring velocity 150��250rpm, fermentation culture 6��7 days, collects fermented liquid;
Wherein culture temperature is 26��30 DEG C.
4. method as claimed in claim 3, it is characterised in that volume basis by weight in shaking flask, seed tank culture process in described step b, carbon source is 0.5��4.0%, it is preferable that 0.5��3.0%, it is most preferred that be 1.2��2.5%; Nitrogenous source is 0.2��4.0%, it is preferable that 0.5��3.5%, it is most preferred that be 0.5��2.0%; Inorganic salt are 0.2��2.0%, it is preferable that 0.2��1.5%, it is most preferred that 0.2��1.0%; All the other are water.
5. method as claimed in claim 3, it is characterised in that substratum volume basis by weight in fermentation culture process in described step b, carbon source is 3.0��12.0%, it is preferable that 3.0��10.0%, it is most preferred that 7.0��9.0%; Nitrogenous source is 1.0��8.0%, it is preferable that 1.0��6.0%, it is most preferred that 2.9��4.5%; Inorganic salt are 0.2��2.0%, it is preferable that 0.2��1.5%, it is most preferred that 0.25��1.0%; Amino acid is 0.2��2.0%, it is preferable that 0.2��1.5%, it is most preferred that 0.5��1.0%; All the other are water.
6. method as claimed in claim 4, it is characterised in that
Described carbon source is selected from Zulkovsky starch, soybean oil, one or more in glucose, W-Gum, glycerine, maltose, sucrose; Preferred Zulkovsky starch, soybean oil, one or more in glucose, W-Gum, glycerine, maltose; Most preferably glucose, W-Gum, one or more in glycerine;
Described nitrogenous source selects yeast to leach one or more in powder, Semen Maydis powder, hot soybean cake powder, soybean protein powder, bean cake powder, raw bean powder, beer yeast powder, corn steep solids, ammonium sulfate; One or more in preferred hot soybean cake powder, soybean protein powder, raw bean powder, beer yeast powder, corn steep solids, ammonium sulfate; More preferably hot soybean cake powder, beer yeast powder, one or more in ammonium sulfate;
Described inorganic salt CaCO3��K2HPO4��NaCl��KNO3, MgSO4��KCl��KH2PO4In one or more; Preferred CaCO3��K2HPO4��NaCl��KNO3��KH2PO4In one or more; More preferably CaCO3��K2HPO4��KH2PO4In one or more.
7. method as claimed in claim 5, it is characterised in that
Described carbon source be selected from Zulkovsky starch, W-Gum, glycerine, sunflower seed oil, dextrin, glucose, potato starch, maltose, sucrose, rape oil, N.F,USP MANNITOL one or more; One or more in preferred Zulkovsky starch, W-Gum, glycerine, sunflower seed oil, dextrin, glucose, potato starch, maltose, rape oil, N.F,USP MANNITOL, it is most preferred that one or more in W-Gum, glycerine, dextrin, glucose.
8. method as claimed in claim 5, it is characterised in that described nitrogenous source is selected from bean cake powder, raw bean powder, meat peptone, beer yeast powder, bread yeast powder, hot soybean cake powder, corn steep solids, ammonium sulfate, fish peptone, yeast leach cream, urea, cottonseed meal, yeast leach one or more in powder, dried silkworm chrysalis meal, Zein powder; Preferred bean cake powder, raw bean powder, meat peptone, beer yeast powder, bread yeast powder, hot soybean cake powder, corn steep solids, ammonium sulfate, yeast leach one or more in cream, urea, Zein powder; Be more preferably in beer yeast powder, bread yeast powder, hot soybean cake powder, corn steep solids, ammonium sulfate, meat peptone one or more.
9. method as claimed in claim 5, it is characterised in that described inorganic salt are selected from CaCO3��MgSO4��K2HPO4��NaCl��KNO3, ZnSO4��KCl��KH2PO4In one or more; Preferred CaCO3��MgSO4��K2HPO4��NaCl��KNO3��KH2PO4In one or more; Most preferably CaCO3��K2HPO4, one or more in NaCl.
10. method as claimed in claim 5, it is characterised in that described amino acid is selected from leucine, in ILE, Valine, L-glutamic acid, Sodium Glutamate, L-Histidine one or more; Be preferably from leucine, in ILE, Valine, L-glutamic acid, Sodium Glutamate one or more; Most preferably be in ILE, Sodium Glutamate one or more.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106947713A (en) * | 2017-03-16 | 2017-07-14 | 新疆肥力沃生物工程有限公司 | A kind of culture medium for being used to cultivate streptomyces microflavus and its method for cultivating yellow streptomycete |
| CN108192908A (en) * | 2018-01-29 | 2018-06-22 | 天津大学 | The method that tacrolimus fermentation yield is improved using streptomyces tsukubaensis |
| CN108384819A (en) * | 2017-02-03 | 2018-08-10 | 上海医药工业研究院 | A kind of culture medium and fermentation process for the tacrolimus that ferments |
| CN109735467A (en) * | 2019-01-30 | 2019-05-10 | 福建省微生物研究所 | A kind of streptomycete mutagenic strain of high yield tacrolimus and its application |
| CN112111482A (en) * | 2020-08-31 | 2020-12-22 | 浙江工业大学 | Method and strain for screening high-yield tacrolimus Streptomyces tsukubaensis by high-throughput mutagenesis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481662A (en) * | 2008-01-08 | 2009-07-15 | 杭州华东医药集团生物工程研究所有限公司 | Streptomycete and use thereof |
| CN103060248A (en) * | 2011-10-19 | 2013-04-24 | 中国科学院上海有机化学研究所 | Method for constructing gene engineering FK506 high-producing strain and streptomyces tsukubaensis high-producing strain |
| CN105154382A (en) * | 2015-10-15 | 2015-12-16 | 浙江大学 | Gene engineering strain streptomyces tsukubaensis L20 and application thereof |
-
2016
- 2016-03-10 CN CN201610135737.3A patent/CN105624068B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481662A (en) * | 2008-01-08 | 2009-07-15 | 杭州华东医药集团生物工程研究所有限公司 | Streptomycete and use thereof |
| CN103060248A (en) * | 2011-10-19 | 2013-04-24 | 中国科学院上海有机化学研究所 | Method for constructing gene engineering FK506 high-producing strain and streptomyces tsukubaensis high-producing strain |
| CN105154382A (en) * | 2015-10-15 | 2015-12-16 | 浙江大学 | Gene engineering strain streptomyces tsukubaensis L20 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| T, GOTO ET AL: "Discovery of FK-506, a novel immunosuppressant isolated from Streptomyces tsukubaensis.", 《TRANSPLANTATION PROCEEDINGS》 * |
Cited By (7)
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|---|---|---|---|---|
| CN108384819A (en) * | 2017-02-03 | 2018-08-10 | 上海医药工业研究院 | A kind of culture medium and fermentation process for the tacrolimus that ferments |
| CN108384819B (en) * | 2017-02-03 | 2021-06-25 | 上海医药工业研究院 | Culture medium for fermenting tacrolimus and fermentation method |
| CN106947713A (en) * | 2017-03-16 | 2017-07-14 | 新疆肥力沃生物工程有限公司 | A kind of culture medium for being used to cultivate streptomyces microflavus and its method for cultivating yellow streptomycete |
| CN108192908A (en) * | 2018-01-29 | 2018-06-22 | 天津大学 | The method that tacrolimus fermentation yield is improved using streptomyces tsukubaensis |
| CN109735467A (en) * | 2019-01-30 | 2019-05-10 | 福建省微生物研究所 | A kind of streptomycete mutagenic strain of high yield tacrolimus and its application |
| CN109735467B (en) * | 2019-01-30 | 2021-07-23 | 福建省微生物研究所 | Streptomyces mutant strain for high-yield tacrolimus and application thereof |
| CN112111482A (en) * | 2020-08-31 | 2020-12-22 | 浙江工业大学 | Method and strain for screening high-yield tacrolimus Streptomyces tsukubaensis by high-throughput mutagenesis |
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Application publication date: 20160601 Assignee: HUADONG MEDICINE Co.,Ltd. Assignor: HANGZHOU ZHONGMEIHUADONG PHARMACEUTICAL Co.,Ltd. Contract record no.: X2021330000109 Denomination of invention: Streptomyces Tsukuba and its application in the preparation of tacrolimus Granted publication date: 20190917 License type: Common License Record date: 20210820 |