CN105624068B - Streptomyces tsukubaensis and its preparing the application in tacrolimus - Google Patents
Streptomyces tsukubaensis and its preparing the application in tacrolimus Download PDFInfo
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Abstract
The invention discloses a kind of streptomyces tsukubaensis (streptomyces tsukubaensis) HDW68 and its applications, the strain is by China typical culture collection center preservation, deposit number is CCTCC M 2016086, preservation date are as follows: on 03 07th, 2016.The invention also discloses a kind of methods that thus strain fermentation prepares tacrolimus intermediate.Production practices of the zymotechnique provided by the invention through 1 ton, 4 tons, 20 tons fermentor, prove its stable production capacity, fermentation unit is high and fermentation byproduct (FK520) is relatively fewer, the difficulty extracted after greatly reducing is suitable for industrialized production and the tacrolimus product quality height obtained.
Description
Technical field
The present invention relates to a kind of novel microbial and application thereof and application more particularly to a kind of streptomyces tsukubaensis and its making
Application in standby tacrolimus.
Background technique
Tacrolimus (Tacrolimus) also known as FK506, chemical structure belong to 23 membered macrolide antibiotic, for one kind
The neotype immunosuppressant of strength.In molecular level, the effect of this product is situated between by albumen FKBP12 in combination in cytoplasm
It leads.It is intracellular that FKBP12 enters this product, and forms compound, which competitively specifically ties with calmodulin
Merge and inhibits calmodulin, in the latter's mediate T cell-Ca-dependent inhibition signal transduction system, to prevent a series of lymphs
Factor gene transcription.Inside and outside is it is demonstrated experimentally that tacrolimus is a potent immunosuppressor, poison lymphocyte capable of inhibiting cell
Formation, graft-rejection mainly caused by the latter.The medicine inhibits T cell activation and the B cell of TH cell dependent antibody to increase
It grows and the generation of lymphokine such as interleukin 2, interleukin Ⅲ and beta-interferon and interleukin 2 receptor
Expression.Tacrolimus can inhibit skin, heart, kidney, liver transfer operation rejection, extend the time-to-live of alloplast,
It is confirmed with rodent, dog, primate and the mankind.Liver, kidney and marrow have been widely used in since listing
The acute and chronic of transplanting repels treatment.
The current report in relation to tacrolimus production method mainly has United States Patent (USP) US4894366, US4929611,
US5116756, US5264355, US5496727 and US5624842.These patent reports with different streptomyces strains produce him
Ke Mosi, yield be not it is very high, mentioned in the streptomycessp.MA6858 and US5624842 mentioned in US5116756 patent
The tacrolimus of the streptomyces tsukubaensis arrived produces bacterial strain, and the yield for producing tacrolimus is all very low, hair
The concentration highest of tacrolimus only has 37.8mg/L in zymotic fluid.Chinese patent " method of fermenting and producing tacrolimus "
(CN201310644714.1) reporting that a kind of streptomycete produces tacrolimus is 385 μ g/ml, and needs Feeding medium among process, and technique is cumbersome.In
Highest fermentation unit disclosed in state's patent " a kind of streptomycete and its application " (CN200810019003.4) is 550 μ g/ml.China
Highest fermentation unit disclosed in patent " a kind of preparation method of high-purity tacrolimus " (CN 201210447477.5) is 491 μ
G/ml, but zymotechnique is not referred to.Chinese patent " genetic engineering bacterium streptomyces tsukubaensis L20 and its application "
(CN201510664086.2) it mentions genetic engineering bacterium and 716mg/L can reach by shake flask fermentation unit.
Foreign periodical " Journal of Industrial Microbiology&Biotechnology " (2009,36
(12): 1467-1471) text " Strain development of Streptomyces sp.for a tacrolimus
Production using sequential adaptation " in mention by the way that the dense of tacrolimus in culture medium is gradually increased
600-1600 μ g/ml is spent to increase bacterial strain to the tolerance of product, and the bacterial strain finally obtained reaches 972 μ g/ in 5L fermentor
ml.Domestic periodical " JOURNAL OF MICROBIOLOGY " in October, 2014 volume 34 the 5th it is interim " resistant mutation tacrolimus superior strain
Breeding " it mentions superior strain H-493 is obtained by resistant mutation breeding, it can reach 918 μ g/ml in 50L fermentor, but without
Cross industrialization trial production.Both bacterial strains are directed to resistant mutation breeding, and the stability in strain later period needs to be investigated.
In conclusion in the tacrolimus fermentation production technology reported both at home and abroad, using resistant mutant strains, genetic engineering
The fermentation technique unit of bacterium is high, but the fermentation technique only passes through small-scale experiment and confirms without industrialized production；Using conventional bacterium
The zymotechnique unit of strain is low, and fermentation byproduct is more, and the content of isomer FK520 especially therein is higher,
Because being that isomerism is difficult to remove by the purifying process in later period, causes rear extraction process complex, substantially increase
Subsequent purification difficulty, it is difficult to the final products of high-purity are obtained, to be difficult to preferably carry out industrialization production.
Summary of the invention
Regarding the issue above, the present invention provides the tacrolimus that a kind of fermentation unit is high and by-product is few generates
Bacterium.
Streptomyces tsukubaensis HDW68 provided by the present invention, classification naming are streptomyces tsukubansis HDW68,
By China typical culture collection center preservation (abbreviation CCTCC), deposit number are as follows: CCTCC M2016086, preservation date are as follows:
On 03 07th, 2016.The deposit number is that the bacterial strain of CCTCC M 2016086 is from the soil around Yunnan Xinping phosphorus pools
Screening obtains in earth.
The deposit number is the colony characteristics of the bacterial strain of CCTCC M 2016086: bacterium colony Chinese red or red, nearly circle
Shape, colony diameter 2-4mm, bacterium colony swell and divide valve, and center subsides into hole.
According to the description of microbial morphology and external related data, the bacterium for being CCTCC M 2016086 in conjunction with deposit number
The various cultural characteristics and morphological feature of strain, the deposit number are that the bacterial strain of CCTCC M 2016086 should belong to streptomyces, are named
For streptomyces tsukubansis HDW68.
It is a further object of the present invention to provide the bacterial strains to prepare the application in tacrolimus.
The deposit number can be applied to fermentation for the bacterial strain of CCTCC M 2016086 and prepare tacrolimus.The preparation side
Method includes the following steps:
A. fermentation strain uses deposit number for the bacterial strain of CCTCC M 2016086；
B. strain culturing: the 30% glycerol mycelia cryovial (work strain library) prepared according to a conventional method access is shaken
Bottle carries out seed culture and obtains shake-flask seed liquid；Shake-flask seed liquid is inoculated in Medium of shaking flask fermentation or is cultivated in seeding tank；
Seed tank culture liquid is inoculated in fermentation tank culture medium culture, collects fermentation liquid；It is preferred that without carrying out during fermented and cultured
Feed supplement, after seeding tank mycelia well-grown accesses fermentor, according to fermentation liquid actual conditions from pH, bacterium is dense, speed of agitator, molten
Oxygen and mycelia microscopy situation etc. control fermentation process.
C. tunning extracts: extracting, is concentrated, is pre-crystallized, chromatographing, crystallizing and is dry.
The step of wherein described b are as follows: the 30% glycerol mycelia cryovial prepared according to a conventional method (work strain library) is pressed
0.8~1.2% access shake-flask seed culture medium of shake-flask seed culture medium volume, 250rpm cultivate 22~30h, obtain shaking flask kind
Sub- liquid；Shake-flask seed liquid is inoculated in seeding tank, stirring speed by 0.02~0.05% inoculum concentration of seed tank culture matrix product
150~250rpm is spent, 22~30h is cultivated；Seed tank culture liquid is inoculated in hair by 5~15% amount of fermentation medium volume
Fermentation tank culture medium, 150~250rpm of mixing speed fermented and cultured 6~7 days, collect fermentation liquid；Wherein cultivation temperature be 26~
30℃。
The invention also discloses culture mediums: volume basis by weight, and the carbon source during shaking flask, seed tank culture is 0.5
~4.0%, preferably 0.5~3.0%, most preferably 1.2~2.5%；Nitrogen source is 0.2~4.0%, preferably 0.5~3.5%, most
Preferably 0.5~2.0%；Inorganic salts are 0.2~2.0%, preferably 0.2~1.5%, most preferably 0.2~1.0%；Remaining is water.
Wherein carbon source is selected from soluble starch, soybean oil, glucose, cornstarch, glycerol, maltose, one in sucrose
Kind is several；It is preferred that soluble starch, soybean oil, one or more of glucose, cornstarch, glycerol, maltose；It is optimal
Select one or more of glucose, cornstarch, glycerol.
Wherein nitrogen source selects yeast extract powder, corn flour, hot soybean cake powder, soyabean protein powder, bean cake powder, raw bean powder, beer
One or more of yeast powder, corn steep solids, ammonium sulfate；It is preferred that hot soybean cake powder, soyabean protein powder, raw bean powder, beer
One or more of yeast powder, corn steep solids, ammonium sulfate；More preferable hot soybean cake powder, beer yeast powder, in ammonium sulfate
It is one or more of.
Wherein inorganic salts CaCO3、K2HPO4、NaCl、KNO3, MgSO4、KCl、KH2PO4One or more of；It is preferred that
CaCO3、K2HPO4、NaCl、KNO3、KH2PO4One or more of；More preferable CaCO3、K2HPO4、KH2PO4One of or
It is several.
Volume basis by weight, the carbon source during fermented and cultured are 3.0~12.0%, preferably 3.0~10.0) %, most
It is preferred that 7.0~9.0%；Nitrogen source is 1.0~8.0%, preferably 1.0~6.0%, most preferably 2.9~4.5%；Inorganic salts be 0.2~
2.0%, preferably 0.2~1.5%, most preferably 0.25~1.0%；Amino acid is 0.2~2.0%, preferably 0.2~1.5%, optimal
Select 0.5~1.0%；Remaining is water.
Wherein carbon source be selected from soluble starch, cornstarch, glycerol, sunflower oil, dextrin, glucose, potato starch,
One or more of maltose, sucrose, rape oil, mannitol；It is preferred that soluble starch, cornstarch, glycerol, sunflower oil,
One or more of dextrin, glucose, potato starch, maltose, rape oil, mannitol, most preferably cornstarch, glycerol, paste
One or more of essence, glucose.
Wherein nitrogen source is selected from bean cake powder, raw bean powder, meat peptone, beer yeast powder, Saccharomyces cerevisiae powder, hot soybean cake powder, solid
Corn pulp, ammonium sulfate, fish peptone, yeast leach cream, urea, cottonseed meal, yeast extract powder, dried silkworm chrysalis meal, in corn protein powder
It is one or more of；It is preferred that bean cake powder, raw bean powder, meat peptone, beer yeast powder, Saccharomyces cerevisiae powder, hot soybean cake powder, corn in solids
Slurry, ammonium sulfate, yeast leach cream, urea, one or more of in corn protein powder；More preferably beer yeast powder, Saccharomyces cerevisiae
It is powder, hot soybean cake powder, corn steep solids, ammonium sulfate, one or more of in meat peptone.
Wherein inorganic salts are selected from CaCO3、MgSO4、K2HPO4、NaCl、KNO3, ZnSO4、KCl、KH2PO4One of or it is several
Kind；It is preferred that CaCO3、MgSO4、K2HPO4、NaCl、KNO3、KH2PO4One or more of；Most preferably CaCO3、K2HPO4、NaCl
One or more of.
Wherein amino acid is selected from leucine, l-Isoleucine, Valine, glutamic acid, sodium glutamate, one in L-Histidine
Kind is several；It is l-Isoleucine, Valine, glutamic acid, one or more of in sodium glutamate preferably from leucine；It is optimal
It is selected as l-Isoleucine, one or more of in sodium glutamate.
After tank is put in fermented completion, tunning is extracted, is concentrated, is pre-crystallized, chromatography, crystallization and it is dry after can be with
Purity is obtained up to 99% or more tacrolimus.
Of the invention has the technical effect that:
1, streptomyces tsukubaensis disclosed in this invention is a kind of tacrolimus producing strains of high unit currently used for production,
Fermenting property is excellent, can be used for being mass produced.Zymotechnique provided by the invention by shaking flask, lab scale (1 ton of fermentor), in
Examination (4 tons of fermentors) and 20 tons of production fermentors are tested and are produced, stable production capacity.Through detecting, mentioned with the present invention
The tacrolimus potency of bacterial strain and the fermentation culture method preparation of confession reaches 1100 μ g/ml or more, to be subsequent industry
Metaplasia production provides good basis.
2, bacterial strain disclosed in this invention, which is screened from the soil around Yunnan Xinping phosphorus pools, obtains, in optimization technique
Afterwards, it is tested and is produced for many years, strain stability is good.The bacterial strain is proved through Physiology and biochemistry, cultural characteristic and gene sequencing
For streptomyces tsukubaensis.
3, the fermentation byproduct of streptomyces tsukubaensis disclosed in this invention is relatively fewer, especially the important pair of tacrolimus
Product is also that the content of isomer FK520 significantly reduces, the difficulty extracted after reducing, and is extracted opposite with the technique of purifying
Simply, yield and purity are substantially increased, advantage is had more in industrialized production.
Bacterial strain preservation situation: being preserved in China typical culture collection center (abbreviation CCTCC), and preservation address is China
Wuhan Wuhan University, deposit number are CCTCC NO:M 2016086, and classification naming is streptomyces tsukubaensis HDW68
(streptomyces tsukubansis HDW68).The deposit date is on 03 07th, 2016.
Detailed description of the invention
Fig. 1 is to be prepared by Chinese patent " a kind of streptomycete and its application " (CN200810019003.4) embodiment 6
Sample FK520 content HPLC figure；
Fig. 2,3,4 are the FK520 content HPLC figure for the different samples being prepared with the embodiment of the present application 7.
Specific embodiment
By specific examples presented below, it can be further apparent from the present invention, but they are not to this hair
Bright restriction.
Embodiment 1: deposit number is the cultural characteristic of the bacterial strain of CCTCC M 2016086
The strain inoculated that deposit number is CCTCC M 2016086 is used to see in Gause I, glucose asparagine etc.
Examine the culture medium of morphological feature.28 DEG C the culture regular hour 5,7,10 days, observe and be described.
Deposit number is colony characteristics of the bacterial strain of CCTCC M 2016086 on various culture mediums
Embodiment 2: utilization of carbon source and physiological and biochemical property
Physiology and biochemistry property is using the standard method in streptomyces category.
Deposit number is the biochemical reactions of the bacterial strain of CCTCC M 2016086 are as follows: can be liquefied on gelatin culture medium bright
Glue, peptonized milk cannot solidify milk, and starch hydrolysis negative does not utilize cellulose, nitrate reduction negative.
Using Biolog (GEN III) automatic microbe identification systems to bacterial strain streptomyces tsukubaensis into
94 kinds of phenotypes of having gone are tested, including 71 kinds of utilization of carbon source situations detections and 23 kinds of chemosensitivities detections: by strain inoculated spy
Fixed plating medium, 33 DEG C constant temperature incubation 2 days, the thallus on plate is washed down with aseptic cotton carrier, it is mixed with inoculation liquid (IF-A)
It closes, bacteria suspension is made, is adjusted with nephelometer to 92%T/IF-A.Bacteria suspension is added in Biolog respectively with 8 hole electric plus liquid devices
In each hole of (GEN III) micropore, every 100 μ l of hole.Micropore identification plate is placed in 33 DEG C of incubators, respectively culture 12h, for 24 hours,
It is placed it in after 32h on Biolog readout instrument and reads result.
Metabolic Fingerprinting is analyzed through Biolog readout instrument, streptomyces tsukubaensis streptomyces tsukubaensis changes 3 kinds
Learn material sensitive；Bacterial strain can be weaker to other 54 kinds of utilization of carbon source abilities or cannot utilize more by force using wherein 17 kinds of carbon sources.Bacterium
Strain.Biolog system provides 36h qualification result.Biolog qualification result is shown in Tables 1 and 2.
Chemosensitivity of 1 streptomyces tsukubaensis of table to 23 kinds of chemical substances on III plate of Biolog GEN
Illustrate :+, it is positive；, negative；B is not known
Utilization ability of 2 streptomyces tsukubaensis of table to 71 kinds of carbon sources on Biolog (GEN III) plate
Illustrate :+, it is positive；, negative；B is not known
Example 3: the production fermented and cultured of tacrolimus
Using the bacterial strain of CCTCC M 2016086
1. shake-flask seed culture
Culture medium: soluble starch 0.3g, soybean oil 0.2g, yeast extract powder 0.1g, corn flour 0.1g, KH2PO4
0.1g, CaCO30.1g, pH are naturally, add water to 100ml.
Inoculum concentration: by 0.8ml/ bottles of inoculum concentration
Culture: 28 DEG C, for 24 hours, 250rpm
It is grown in mycelia, after having obvious wall built-up phenomenon, 100ml shake-flask seed culture solution is accessed in seeding tank and is cultivated.
2. seed tank culture
Culture medium: soluble starch 1.5kg, soybean oil 1kg, yeast extract powder 0.5kg, corn flour 0.5kg, KH2PO4
0.5kg, CaCO30.5kg, pH are naturally, add water to 0.5 ton.
Loading amount: 0.5 ton of the built-in culture medium of 1 ton of fermentor, 121 DEG C of sterilizing 30min.
Inoculum concentration: 100ml shake-flask seed.
Culture: 28 DEG C, 30h, 150rpm
3. fermentation tank culture
Culture medium: potato starch 10kg, glycerol 2.5kg, sunflower oil 2.5kg, bean cake powder 2kg, raw bean powder 2kg, meat peptone
1kg, leucine 1kg, CaCO3 0.5kg、MgSO40.5kg adds water to 0.5 ton, adjusts pH8.5.
Loading amount: 0.5 ton of the built-in culture medium of 1 ton of fermentor.
Inoculum concentration: the seed liquor of 75L
Cultivation temperature: 28 DEG C, starting adjusts tank and presses 0.05MPa, and air mass flow is in 1:(0.6 ± 0.2) vvm, speed of agitator
250rpm.In metabolic process when opposite oxygen dissolving value is lower than 30%, air mass flow is first tuned up to 1:(1 ± 0.2) vvm；When opposite
When oxygen dissolving value is again below 30%, tune up agitating paddle frequency conversion value (agitating paddle frequency conversion value is between (150~250) rpm)；If phase
To oxygen dissolving value again below 30%, then increasing tank pressure within the scope of (0.05~0.08) MPa, (tank pressure maintains in fermentation process
Between (0.02~0.08)).Fermentation period is 7 days.
Put tank unit: 1323 μ g/ml
Example 4: the production fermented and cultured of tacrolimus
Using the bacterial strain of CCTCC M 2016086
1. shake-flask seed culture
Culture medium: maltose 3g, glycerol 6g, raw bean powder 3g, soyabean protein powder 1.5g, corn steep solids 6g, CaCO3
0.6g, NaCl 2.4g, KNO31.5g, adds water to be settled to 300ml, and pH is natural
Inoculum concentration: by 1ml/ bottles of inoculum concentration
Culture: 26 DEG C, 30h, 250rpm
It is grown in mycelia, after having obvious wall built-up phenomenon, 300ml shake-flask seed culture solution is accessed in seeding tank and is cultivated.
2. seed tank culture
Culture medium: maltose 10kg, glycerol 20kg, raw bean powder 10kg, soyabean protein powder 5kg, corn steep solids 20kg,
CaCO32kg, NaCl 8kg, KNO35kg adds water to be settled to 1 ton, and pH is natural.
Loading amount: 1 ton of the built-in culture medium of 4 tons of fermentors, 121 DEG C of sterilizing 30min.
Inoculum concentration: 300ml shake-flask seed.
Culture: 26 DEG C, 27h, 200rpm
3. fermentation tank culture
Culture medium: soluble starch 150kg, rape oil 25kg, mannitol 75kg, corn protein powder 50kg, yeast leach cream
75kg, hot soybean powder 12.5kg, urea 12.5kg, l-Isoleucine 12.5kg, glutamic acid 12.5kg, Valine 12.5kg,
KH2PO412.5kg KNO312.5kg CaCO312.5kg adds water to be settled to 2.5 tons, adjusts pH8.8.
Loading amount: 4 tons of fermentors built-in culture medium 2.5t, 121 DEG C of sterilizing 30min.
Inoculum concentration: the seed liquor of 250L
Cultivation temperature: 27 DEG C, starting adjusts tank and presses 0.05MPa, and air mass flow is in 1:(0.6 ± 0.2) vvm, speed of agitator
200rpm.In metabolic process when opposite oxygen dissolving value is lower than 30%, air mass flow is first tuned up to 1:(1 ± 0.2) vvm；When opposite
When oxygen dissolving value is again below 30%, tune up agitating paddle frequency conversion value (agitating paddle frequency conversion value is between (150~250) rpm)；If phase
To oxygen dissolving value again below 30%, then increasing tank pressure within the scope of (0.05~0.08) MPa, (tank pressure maintains in fermentation process
Between (0.02~0.08)).Fermentation period is 7 days.
Fermentation liquid puts tank unit: 1288 μ g/ml.
Example 5: the production fermented and cultured of tacrolimus
Using the bacterial strain of CCTCC M 2016086
1. shake-flask seed culture
Culture medium: glucose 20g, sucrose 20g, beer yeast powder 10g, bean cake powder 30g, MgSO45g, KCl 10g,
CaCO35g, pH naturally, plus water be settled to 1000ml, dispense 10 bottles.
Inoculum concentration: by 1ml/ bottles of inoculum concentration
Culture: 30 DEG C, 22h, 250rpm
It is grown in mycelia, after having obvious wall built-up phenomenon, 1000ml shake-flask seed culture solution is accessed in seeding tank and is cultivated.
2. seed tank culture
Culture medium: glucose 40kg, sucrose 40kg, beer yeast powder 20kg, bean cake powder 60kg, MgSO410g, KCl
20g, CaCO310g, GPE 2kg, pH naturally, plus water be settled to 2 tons.
Loading amount: 2 tons of the built-in culture medium of 4 tons of fermentors, 121 DEG C of sterilizing 30min.
Inoculum concentration: 1000ml shake-flask seed.
Culture: 30 DEG C, 22h, 200rpm
3. fermentation tank culture
Culture medium: sucrose 125kg, glucose 25kg, maltose 150kg, fish peptone 25kg, cottonseed meal 75kg, yeast leaching
Powder 50kg out, dried silkworm chrysalis meal 50kg, glutamic acid 25kg, L-Histidine 25kg, ZnSO415kg, KCl 25kg, CaCO310kg adds
Water is settled to 2.5t, adjusts pH9.0
Loading amount: 2.5 tons of the built-in culture medium of 4 tons of fermentors
Inoculum concentration: the seed liquor of 375L
Cultivation temperature: 29 DEG C, starting adjusts tank and presses 0.05MPa, and air mass flow is in 1:(0.6 ± 0.2) vvm, speed of agitator
150rpm.In metabolic process when opposite oxygen dissolving value is lower than 30%, air mass flow is first tuned up to 1:(1 ± 0.2) vvm；When opposite
When oxygen dissolving value is again below 30%, tune up agitating paddle frequency conversion value (agitating paddle frequency conversion value is between (150~250) rpm)；If phase
To oxygen dissolving value again below 30%, then increasing tank pressure within the scope of (0.05~0.08) MPa, (tank pressure maintains in fermentation process
Between (0.02~0.08)).Fermentation period is 6 days.
Fermentation liquid puts tank unit: 1363 μ g/ml.
Example 6: the production fermented and cultured of tacrolimus
Using the bacterial strain of CCTCC M 2016086
1. shake-flask seed culture
Culture medium: glycerol 6g, cornstarch 9g, beer yeast powder 1.2g, ammonium sulfate 1.8g, K2HPO43g, CaCO33g,
PH naturally, plus water be settled to 600ml, dispense 6 bottles.
Inoculum concentration: by 1.2ml/ bottles of inoculum concentration
Culture: 29 DEG C, 28h, 250rpm
It is grown in mycelia, after having obvious wall built-up phenomenon, 600ml shake-flask seed culture solution is accessed in seeding tank and is cultivated.
2. seed tank culture
Culture medium: glycerol 15kg, cornstarch 22.5kg, beer yeast powder 3kg, ammonium sulfate 4.5kg, K2HPO47.5kg
CaCO37.5kg, GPE 1.5kg, pH are naturally, be settled to 1.5 tons.
Loading amount: 1.5 tons of the built-in culture medium of 4 tons of fermentors, 121 DEG C of sterilizing 30min.
Inoculum concentration: 600ml shake-flask seed.
Culture: 28 DEG C, 28h, 250rpm
3. fermentation tank culture
Culture medium: glycerol 140kg, glucose 280kg, cornstarch 210kg, meat peptone 70kg, beer yeast powder 105kg,
Hot soybean powder 35kg, corn steep solids 70Kg, ammonium sulfate 35kg, l-Isoleucine 35Kg, sodium glutamate 35kg, K2HPO4
21kg, NaCl 28kg, CaCO321kg, GPE 14kg add water to be settled to 7 tons, adjust pH9.5.
Loading amount: 7 tons of the built-in culture medium of 20 tons of fermentors
Inoculum concentration: 0.35 ton of seed liquor
Cultivation temperature: 30 DEG C, starting adjusts tank and presses 0.05MPa, and air mass flow is in 1:(0.6 ± 0.2) vvm, speed of agitator
150rpm.In metabolic process when opposite oxygen dissolving value is lower than 30%, air mass flow is first tuned up to 1:(1 ± 0.2) vvm；When opposite
When oxygen dissolving value is again below 30%, tune up agitating paddle frequency conversion value (agitating paddle frequency conversion value is between (150~250) rpm)；If phase
To oxygen dissolving value again below 30%, then increasing tank pressure within the scope of (0.05~0.08) MPa, (tank pressure maintains in fermentation process
Between (0.02~0.08)).Fermentation period is 7 days.
Fermentation liquid puts tank unit: 1184 μ g/ml.
Example 7: the production fermented and cultured of tacrolimus
Using the bacterial strain of CCTCC M 2016086
1. shake-flask seed culture
Culture medium: glucose 1g, cornstarch 5g, hot soybean cake powder 10g, CaCO37.5g, pH naturally, plus water be settled to
500ml dispenses 5 bottles.
Inoculum concentration: by 1.0ml/ bottles of inoculum concentration
Culture: 28 DEG C, 26h, 250rpm
It is grown in mycelia, after having obvious wall built-up phenomenon, 500ml shake-flask seed culture solution is accessed in seeding tank and is cultivated.
2. seed tank culture
Culture medium: glucose 3kg, cornstarch 15kg, hot soybean cake powder 30kg, CaCO33kg, GPE 1.5kg, pH are certainly
So, 1.5 tons are settled to.
Loading amount: 1.5 tons of the built-in culture medium of 4 tons of fermentors, 121 DEG C of sterilizing 30min.
Inoculum concentration: 500ml shake-flask seed.
Culture: 28 DEG C, 25h, 200rpm
3. fermentation tank culture
Culture medium: dextrin 910kg, Saccharomyces cerevisiae powder 78kg, beer yeast powder 221kg, corn steep solids 65Kg, ammonium sulfate
13kg, CaCO332.5kg CaCO36.5kg, GPE 42kg add water to be settled to 13 tons, adjust pH8.9.
Loading amount: 13 tons of the built-in culture medium of 20 tons of fermentors
Inoculum concentration: 1.5 tons of seed liquor
Cultivation temperature: 26 DEG C, starting adjusts tank and presses 0.05MPa, and air mass flow is in 1:(0.6 ± 0.2) vvm, speed of agitator
150rpm.In metabolic process when opposite oxygen dissolving value is lower than 30%, air mass flow is first tuned up to 1:(1 ± 0.2) vvm；When opposite
When oxygen dissolving value is again below 30%, tune up agitating paddle frequency conversion value (agitating paddle frequency conversion value is between (150~250) rpm)；If phase
To oxygen dissolving value again below 30%, then increasing tank pressure within the scope of (0.05~0.08) MPa, (tank pressure maintains in fermentation process
Between (0.02~0.08)).Fermentation period is 6.5 days.
According to above-mentioned fermentation condition and technique, 3 batches of fermentation tests are carried out in 20 tons of fermentors, fermentation unit is respectively 1158
μ g/ml, 1161 μ g/ml and 1216 μ g/ml.
Embodiment 8:FK520 assay
Measuring method: HPLC method detection
Sample: the fermentation liquid in embodiment 7
Control sample: it is prepared by Chinese patent " a kind of streptomycete and its application " (CN200810019003.4) embodiment 6
Sample.
Chromatographic condition:
Chromatographic column: C8 column (4.6 × 150mm, 5 μm)；
Flow velocity: 1.0mL/min；
Sampling volume: 20 μ l；
Detection wavelength: 210nm；
Column temperature: 60 DEG C；
Record the time: 65min (wherein the interval time of sample must not be less than 1min)；
Mobile phase: the mixed liquor of solution A and solution B, gradient timetable program list see the table below.
Solution A: 0.02mol/L potassium dihydrogen phosphate, with phosphoric acid tune pH to 3.5.
Solution B: acetonitrile.
Blank: acetone.
As a result:
After HPLC is detected, by Chinese patent " a kind of streptomycete and its application " (CN200810019003.4) embodiment
FK520 content is 3.58% in the sample of 6 preparations, sees attached drawing 1.The FK520 content for the sample being prepared with embodiment 7 is point
Other 1.47%, 1.55%, 1.61%, see attached drawing 2,3,4.The two is compared, FK520 content decline 50%.
Claims (35)
1. a kind of streptomyces tsukubaensis (streptomyces tsukubansis) HDW68, by China typical culture collection center
Preservation, deposit number are CCTCC M 2016086, preservation date are as follows: on 03 07th, 2016.
2. streptomyces tsukubaensis (streptomyces tsukubansis) HDW68 as described in claim 1 prepares him in fermentation
Application in Ke Mosi.
3. preparing him using streptomyces tsukubaensis described in claim 1 (streptomyces tsukubansis) HDW68 fermentation
The method of Ke Mosi, it is characterised in that include the following steps:
A. fermentation strain uses deposit number for the bacterial strain of CCTCC M 2016086；
B. the bacterial strain of CCTCC M 2016086 is accessed into shake-flask seed culture medium, 250rpm cultivates 22~30h, obtains shake-flask seed
Liquid；
Shake-flask seed liquid is inoculated in seeding tank, mixing speed by the inoculum concentration of seed tank culture matrix product 0.02~0.05%
150~250rpm cultivates 22~30h；
Seed tank culture liquid is inoculated in fermentation tank culture medium by the amount of fermentation medium volume 5~15%, mixing speed 150~
250rpm fermented and cultured 6~7 days, collects fermentation liquid；
Wherein cultivation temperature is 26~30 DEG C.
4. method as claimed in claim 3, it is characterised in that the shake-flask seed culture medium, seed tank culture base, by weight
Volume basis is measured, carbon source is 0.5~4.0%, and nitrogen source is 0.2~4.0%, and inorganic salts are 0.2~2.0%, remaining is water.
5. method as claimed in claim 4, it is characterised in that the shake-flask seed culture medium, seed tank culture base, by weight
Volume basis is measured, carbon source is 0.5~3.0%.
6. method as claimed in claim 4, it is characterised in that the shake-flask seed culture medium, seed tank culture base, by weight
Volume basis is measured, carbon source is 1.2~2.5%.
7. method as claimed in claim 4, it is characterised in that the shake-flask seed culture medium, seed tank culture base, by weight
Volume basis is measured, nitrogen source is 0.5~3.5%.
8. method as claimed in claim 4, it is characterised in that the shake-flask seed culture medium, seed tank culture, by weight
Volume basis, nitrogen source are 0.5~2.0%.
9. method as claimed in claim 4, it is characterised in that the shake-flask seed culture medium, seed tank culture base, by weight
Volume basis is measured, inorganic salts are 0.2~1.5%.
10. method as claimed in claim 4, it is characterised in that the shake-flask seed culture medium, seed tank culture base, by weight
Volume basis is measured, inorganic salts are 0.2~1.0%.
11. method as claimed in claim 3, it is characterised in that fermentation medium volume basis by weight, carbon source are
3.0~12.0%, nitrogen source is 1.0~8.0%, and inorganic salts are 0.2~2.0%, and amino acid is 0.2~2.0%, remaining is water.
12. method as claimed in claim 11, it is characterised in that fermentation medium volume basis by weight, carbon source are
3.0~10.0%.
13. method as claimed in claim 11, it is characterised in that fermentation medium volume basis by weight, carbon source are
7.0~9.0%.
14. method as claimed in claim 11, it is characterised in that fermentation medium volume basis by weight, nitrogen source are
1.0~6.0%.
15. method as claimed in claim 11, it is characterised in that fermentation medium volume basis by weight, nitrogen source are
2.9~4.5%.
16. method as claimed in claim 11, it is characterised in that fermentation medium volume basis by weight, inorganic salts
It is 0.2~1.5%.
17. method as claimed in claim 11, it is characterised in that fermentation medium volume basis by weight, inorganic salts
It is 0.25~1.0%.
18. method as claimed in claim 11, it is characterised in that fermentation medium volume basis by weight, amino acid
It is 0.2~1.5%.
19. method as claimed in claim 11, it is characterised in that fermentation medium volume basis by weight, amino acid
It is 0.5~1.0%.
20. method as claimed in claim 4, it is characterised in that
The carbon source be selected from soluble starch, soybean oil, one of glucose, cornstarch, glycerol, maltose, sucrose or
It is several；
The nitrogen source is selected from yeast extract powder, corn flour, hot soybean cake powder, soyabean protein powder, bean cake powder, raw bean powder, beer ferment
One or more of female powder, corn steep solids, ammonium sulfate；
The inorganic salts are selected from CaCO3、K2HPO4、NaCl、KNO3, MgSO4、KCl、KH2PO4One or more of.
21. method as claimed in claim 20, it is characterised in that the carbon source be selected from soluble starch, soybean oil, glucose,
One or more of cornstarch, glycerol, maltose.
22. method as claimed in claim 20, it is characterised in that the carbon source is in glucose, cornstarch, glycerol
It is one or more of.
23. method as claimed in claim 20, it is characterised in that the nitrogen source is selected from hot soybean cake powder, soyabean protein powder, life
One or more of bean powder, beer yeast powder, corn steep solids, ammonium sulfate.
24. method as claimed in claim 20, it is characterised in that the nitrogen source is selected from hot soybean cake powder, beer yeast powder, sulphur
One or more of sour ammonium.
25. method as claimed in claim 20, it is characterised in that the inorganic salts are selected from CaCO3、K2HPO4、NaCl、KNO3、
KH2PO4One or more of.
26. method as claimed in claim 20, it is characterised in that the inorganic salts are selected from CaCO3、K2HPO4、KH2PO4In one
Kind is several.
27. method as claimed in claim 11, it is characterised in that
The carbon source is selected from soluble starch, cornstarch, glycerol, sunflower oil, dextrin, glucose, potato starch, malt
One or more of sugar, sucrose, rape oil, mannitol；
The nitrogen source is selected from bean cake powder, raw bean powder, meat peptone, beer yeast powder, Saccharomyces cerevisiae powder, hot soybean cake powder, corn in solids
Slurry, ammonium sulfate, fish peptone, yeast leach one of cream, urea, cottonseed meal, yeast extract powder, dried silkworm chrysalis meal, corn protein powder
Or it is several；
The inorganic salts are selected from CaCO3、MgSO4、K2HPO4、NaCl、KNO3, ZnSO4、KCl、KH2PO4One or more of；
The amino acid is selected from leucine, one of l-Isoleucine, Valine, glutamic acid, sodium glutamate, L-Histidine
Or it is several.
28. method as claimed in claim 27, it is characterised in that the carbon source be selected from soluble starch, cornstarch, glycerol,
One or more of sunflower oil, dextrin, glucose, potato starch, maltose, rape oil, mannitol.
29. method as claimed in claim 27, it is characterised in that the carbon source is selected from cornstarch, glycerol, dextrin, glucose
One or more of.
30. method as claimed in claim 27, it is characterised in that the nitrogen source is selected from bean cake powder, raw bean powder, meat peptone, beer ferment
Female powder, Saccharomyces cerevisiae powder, hot soybean cake powder, corn steep solids, ammonium sulfate, yeast leach cream, urea, one in corn protein powder
Kind is several.
31. method as claimed in claim 27, it is characterised in that the nitrogen source is selected from beer yeast powder, Saccharomyces cerevisiae powder, heat
One or more of soybean cake powder, corn steep solids, ammonium sulfate, meat peptone.
32. method as claimed in claim 27, it is characterised in that the inorganic salts are selected from CaCO3、MgSO4、K2HPO4、NaCl、
KNO3、KH2PO4One or more of.
33. method as claimed in claim 27, it is characterised in that the inorganic salts are selected from CaCO3、K2HPO4, in NaCl one
Kind is several.
34. method as claimed in claim 27, it is characterised in that the amino acid is selected from leucine, l-Isoleucine, L- figured silk fabrics
One or more of propylhomoserin, glutamic acid, sodium glutamate.
35. method as claimed in claim 27, it is characterised in that the amino acid is in l-Isoleucine, sodium glutamate
It is one or more of.
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| 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 |
| 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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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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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 |