CN107574201A - Using the method for fermentation method production β thymidines - Google Patents
Using the method for fermentation method production β thymidines Download PDFInfo
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Abstract
The present invention discloses a kind of method that β thymidines are produced using fermentation method, produces β thymidines with Escherichia coli fermentation, includes following steps：（1）Slant medium bevel strain strain Escherichia coli being inoculated into test tube；（2）Slant strains are expanded into culture as seed liquor；（3）Seed liquor is accessed in fermentation tank and fermented；（4）Add reactant ammoniacal liquor and glucose；（5）β thymidines isolate and purify.The present invention is to pass through Escherichia coli（E.coli）Fermenting and producing β thymidines, reduce production cost, and without using organic solvent in production process, do not produce harmful substance in fermentation process, processing step is simple, improves production operation security.High using β thymidines yield produced by the invention, the fermentation unit of β thymidines reaches 15g/L, isolated and purified by macroporous absorbent resin, and purifying has substantial industrial meaning up to more than 99%.
Description
Technical field
The present invention relates to beta-thymidine preparation field technology, refers in particular to a kind of method that beta-thymidine is produced using fermentation method.
Background technology
At present, there is not been reported for the domestic document for bioanalysis synthesis beta-thymidine：The preparation method master of external beta-thymidine
If being beta-thymidine by using β glycosidases as catalyst, catalyzing and synthesizing ammoniacal liquor with glucose, because β glycosidases are expensive, institute
It is very high with the beta-thymidine cost of production, and the beta-thymidine yield prepared using the above method is not high, is brought to actual popularization and application
Very big difficulty.
The content of the invention
In view of this, in view of the existing deficiencies of the prior art, its main purpose is to provide one kind and uses fermentation method the present invention
The method for producing beta-thymidine, it to Escherichia coli by being screened, mutagenic obtained one plant of beta-thymidine yield is very high, anti-ammoniacal liquor energy
The strong strain Escherichia coli of power（E.coli）, cultivated certain time, added under optimal culture conditions from suitable culture medium
Reactant ammoniacal liquor and glucose, fermenting and producing beta-thymidine, zymotic fluid pass through large pore resin absorption column under optimum reaction condition
Separate, after purification, can obtain the beta-thymidine of high-purity.This method has that technological operation is simple, production cost is low, safe operation etc.
Advantage.
To achieve the above object, the present invention is using following technical scheme：
A kind of method that beta-thymidine is produced using fermentation method, beta-thymidine is produced with Escherichia coli fermentation, includes following steps：
（1）Slant medium bevel strain strain Escherichia coli being inoculated into test tube；
（2）Slant strains are expanded into culture as seed liquor；
（3）Seed liquor is accessed in fermentation tank and fermented；
（4）Add reactant ammoniacal liquor and glucose；
（5）Beta-thymidine isolates and purifies.
As a kind of preferred scheme, the step（1）In slant medium be made up of following parts by weight of component：Dusty yeast
15-30 parts, ammonium sulfate 3-6 parts, 1.8 parts of potassium dihydrogen phosphate, 1.8 parts of dipotassium hydrogen phosphate, 7.5 parts of sodium chloride, glycerine 50-10 parts and
1000 parts of water.
As a kind of preferred scheme, the process conditions for preparing slant strains are：PH value be 7.0-8.0,25-40 DEG C of temperature,
Incubation time is 24-48 hours.
As a kind of preferred scheme, the step（2）In seed liquor and step（3）In zymotic fluid by following parts by weight
Component forms：Glucose 400-800 parts, tryptone 10-20 parts, potassium dihydrogen phosphate 0.5-1.0 parts, 2 parts of dipotassium hydrogen phosphate, chlorine
Change 1000 parts of 7.5 parts of sodium, ammoniacal liquor 20-100 parts, 50 parts of glycerine and water.
As a kind of preferred scheme, the process conditions of fermentation are：Zymotic fluid inoculum concentration is 10-15%, pH value 7.0-7.5,
Cultivation temperature is 25-40 DEG C, and ventilation is 10-30 m3/ h, speed of agitator are 350-450 revs/min, incubation time 24-36
Hour.
As a kind of preferred scheme, the step（4）In reaction condition be：Reactant ammoniacal liquor amount of substance concentration is 30-
48mmol/l, glucose substance amount concentration are 100-200mmol/l, and reaction temperature is 30-55 DEG C, and the reaction time is that 36-72 is small
When, speed of agitator is 160-200 revs/min.
As a kind of preferred scheme, the step（5）In beta-thymidine isolate and purify including following four step：
A, the zymotic fluid after the completion of fermentation is centrifuged 20 minutes under the conditions of 4000 revs/min；
B, the zymotic fluid after a steps is decolourized by low pole large pore resin absorption column；
C, the zymotic fluid Jing Guo b step is isolated and purified by polar macroporous adsorbent resin column；
D, the zymotic fluid Jing Guo step c is dried in vacuo, you can obtain the beta-thymidine that purity is more than 98%.
As a kind of preferred scheme, zymotic fluid centrifuges 20 minutes under the conditions of 4000 revs/min in the step a；Step b
In low pole large pore resin absorption column be HZ818 or AB-8 posts；Polar macroporous adsorbent resin column in step c is D301 or S-
8 posts；Vacuum drying vacuum in step d is 0.1Mpa, and temperature is 65 DEG C, and dried finished product purity is up to more than 99%.
The present invention has clear advantage and beneficial effect compared with prior art, specifically, by above-mentioned technical proposal
Understand：
The present invention is to pass through Escherichia coli（E.coli）Fermenting and producing beta-thymidine, production cost is reduced, and in production process not
Using organic solvent, harmful substance is not produced in fermentation process, processing step is simple, improves production operation security.Using
Beta-thymidine yield produced by the invention is high, and the fermentation unit of beta-thymidine reaches 15g/L, isolated and purified by macroporous absorbent resin, pure
Change up to more than 99%, there is substantial industrial meaning.
Embodiment
Present invention is disclosed a kind of method that beta-thymidine is produced using fermentation method, and beta-thymidine is produced with Escherichia coli fermentation,
Include following steps：
（1）Slant medium bevel strain strain Escherichia coli being inoculated into test tube；Slant medium is by following heavy
Measure part component composition：Dusty yeast 15-30 parts, ammonium sulfate 3-6 parts, 1.8 parts of potassium dihydrogen phosphate, 1.8 parts of dipotassium hydrogen phosphate, sodium chloride
1000 parts of 7.5 parts, glycerine 50-10 parts and water.Prepare slant strains process conditions be：PH value is 7.0-8.0, temperature 25-40
DEG C, incubation time be 24-48 hours.
（2）Slant strains are expanded into culture as seed liquor.
（3）Seed liquor is accessed in fermentation tank and fermented；The step（2）In seed liquor and step（3）In hair
Zymotic fluid is made up of following parts by weight of component：Glucose 400-800 parts, tryptone 10-20 parts, potassium dihydrogen phosphate 0.5-1.0 parts,
1000 parts of 2 parts of dipotassium hydrogen phosphate, 7.5 parts of sodium chloride, ammoniacal liquor 20-100 parts, 50 parts of glycerine and water.The process conditions of fermentation are：Hair
Zymotic fluid inoculum concentration is 10-15%, and pH value 7.0-7.5, cultivation temperature is 25-40 DEG C, and ventilation is 10-30 m3/ h, stirring turn
Speed is 350-450 revs/min, and incubation time is 24-36 hours.
（4）Add reactant ammoniacal liquor and glucose；Reaction condition is：Reactant ammoniacal liquor amount of substance concentration is 30-48mmol/
L, glucose substance amount concentration are 100-200mmol/l, and reaction temperature is 30-55 DEG C, and the reaction time is 36-72 hours, stirring
Rotating speed is 160-200 revs/min.
（5）Beta-thymidine isolates and purifies.Beta-thymidine is isolated and purified including following four step：
A, the zymotic fluid after the completion of fermentation is centrifuged 20 minutes under the conditions of 4000 revs/min；
B, the zymotic fluid after a steps is decolourized by low pole large pore resin absorption column；
C, the zymotic fluid Jing Guo b step is isolated and purified by polar macroporous adsorbent resin column；
D, the zymotic fluid Jing Guo step c is dried in vacuo, you can obtain the beta-thymidine that purity is more than 98%.
Zymotic fluid centrifuges 20 minutes under the conditions of 4000 revs/min in the step a；Low pole macropore in step b is inhaled
Attached resin column is HZ818 or AB-8 posts；Polar macroporous adsorbent resin column in step c is D301 or S-8 posts；It is true in step d
It is 0.1Mpa that sky, which dries vacuum, and temperature is 65 DEG C, and dried finished product purity is up to more than 99%.
With multiple embodiments, the present invention will be further described below.
Embodiment 1
（1）Strain inclined plane culture
By Escherichia coli（E.coli）Strain is inoculated into the slant medium bevel strain in test tube；
Wherein strain inclined plane culture medium is made up of following parts by weight of component：
It is dusty yeast 15-30 parts, ammonium sulfate 3-6 parts, 1.8 parts of potassium dihydrogen phosphate, 1.8 parts of dipotassium hydrogen phosphate, 7.5 parts of sodium chloride, sweet
1000 parts of oily 50-10 parts and water.
Prepare slant strains process conditions be pH value be 7.0,25-40 DEG C of temperature, incubation time be 36 hours.
（2）Inoculation fermentation
Inclined-plane bacterial strain is expanded into culture as seed liquor；
Seed liquor is accessed in fermentation tank by 6-12% inoculum concentration and fermented；
Wherein seed liquor and zymotic fluid are made up of following parts by weight of component：
Glucose 400-800 parts, tryptone 10-20 parts, potassium dihydrogen phosphate 0.5-1.0 parts, 2 parts of dipotassium hydrogen phosphate, sodium chloride
1000 parts of 7.5 parts, ammoniacal liquor 20-100 parts, 50 parts of glycerine and water.
The process conditions of fermentation are that zymotic fluid inoculum concentration is 10%, pH value 7.0, and cultivation temperature is 30 DEG C, and ventilation is
10-30m3/ h, speed of agitator are 130 revs/min, and incubation time is 36 hours.
（3）Produce beta-thymidine
Addition reactant ammoniacal liquor amount of substance concentration is 35mmol/l and glucose substance amount concentration is 150mmol/l；
Wherein reaction condition is：Reaction temperature is 35 DEG C, and the reaction time is 48 hours, and speed of agitator is 200 revs/min.Herein
Under reaction condition, the fermentation unit of beta-thymidine reaches 14.7g/L.
（4）Beta-thymidine isolates and purifies, including following four step：
A, the zymotic fluid after the completion of fermentation is centrifuged 20 minutes under the conditions of 4000 revs/min；
B, low pole large pore resin absorption column HZ818 or the AB-8 post in step, the zymotic fluid after step a pass through HZ818
Or after the absorption of AB-8 posts, zymotic fluid becomes colourless transparent liquid；
C, the polar macroporous adsorbent resin column in step is macroporous absorbent resin D301 or S-8 post；
D, the vacuum drying vacuum in step is 0.1Mpa, and temperature is 65 DEG C, and dried finished product is white powder, purity
Up to 99.0%.
Embodiment 2
（1）Strain inclined plane culture
By Escherichia coli（E.coli）Strain is inoculated into the slant medium bevel strain in test tube；
Wherein strain inclined plane culture medium is made up of following parts by weight of component：
It is dusty yeast 15-30 parts, ammonium sulfate 3-6 parts, 1.8 parts of potassium dihydrogen phosphate, 1.8 parts of dipotassium hydrogen phosphate, 7.5 parts of sodium chloride, sweet
1000 parts of oily 50-10 parts and water.
Prepare slant strains process conditions be pH value be 7.0,25-40 DEG C of temperature, incubation time be 36 hours.
（2）Inoculation fermentation
Inclined-plane bacterial strain is expanded into culture as seed liquor；
Seed liquor is accessed in fermentation tank by 6-12% inoculum concentration and fermented；
Wherein seed liquor and zymotic fluid are made up of following parts by weight of component：
Glucose 400-800 parts, tryptone 10-20 parts, potassium dihydrogen phosphate 0.5-1.0 parts, 2 parts of dipotassium hydrogen phosphate, sodium chloride
1000 parts of 7.5 parts, ammoniacal liquor 20-100 parts, 50 parts of glycerine and water.
The process conditions of fermentation are that zymotic fluid inoculum concentration is 10%, pH value 7.0, and cultivation temperature is 30 DEG C, and ventilation is
10-30m3/ h, speed of agitator are 130 revs/min, and incubation time is 36 hours.
（3）Produce beta-thymidine
Addition reactant ammoniacal liquor amount of substance concentration is 35mmol/l and glucose substance amount concentration is 150mmol/l；
Wherein reaction condition is：Reaction temperature is 35 DEG C, and the reaction time is 48 hours, and speed of agitator is 200 revs/min.Herein
Under reaction condition, the fermentation unit of beta-thymidine reaches 15g/L.
（4）Beta-thymidine isolates and purifies, including following four step：
A, the zymotic fluid after the completion of fermentation is centrifuged 20 minutes under the conditions of 4000 revs/min；
B, low pole large pore resin absorption column HZ818 or the AB-8 post in step, the zymotic fluid after step a pass through HZ818
Or after the absorption of AB-8 posts, zymotic fluid becomes colourless transparent liquid；
C, the polar macroporous adsorbent resin column in step is macroporous absorbent resin D301 or S-8 post；
D, the vacuum drying vacuum in step is 0.1Mpa, and temperature is 65 DEG C, and dried finished product is white powder, purity
Up to 99.2%.
Embodiment 3
（1）Strain inclined plane culture
By Escherichia coli（E.coli）Strain is inoculated into the slant medium bevel strain in test tube；
Wherein strain inclined plane culture medium is made up of following parts by weight of component：
It is dusty yeast 15-30 parts, ammonium sulfate 3-6 parts, 1.8 parts of potassium dihydrogen phosphate, 1.8 parts of dipotassium hydrogen phosphate, 7.5 parts of sodium chloride, sweet
1000 parts of oily 50-10 parts and water.
Prepare slant strains process conditions be pH value be 7.0,25-40 DEG C of temperature, incubation time be 36 hours.
（2）Inoculation fermentation
Inclined-plane bacterial strain is expanded into culture as seed liquor；
Seed liquor is accessed in fermentation tank by 6-12% inoculum concentration and fermented；
Wherein seed liquor and zymotic fluid are made up of following parts by weight of component：
Glucose 400-800 parts, tryptone 10-20 parts, potassium dihydrogen phosphate 0.5-1.0 parts, 2 parts of dipotassium hydrogen phosphate, sodium chloride
1000 parts of 7.5 parts, ammoniacal liquor 20-100 parts, 50 parts of glycerine and water.
The process conditions of fermentation are that zymotic fluid inoculum concentration is 10%, pH value 7.0, and cultivation temperature is 30 DEG C, and ventilation is
10-30m3/ h, speed of agitator are 130 revs/min, and incubation time is 36 hours.
（3）Produce beta-thymidine
Addition reactant ammoniacal liquor amount of substance concentration is 35mmol/l and glucose substance amount concentration is 150mmol/l；
Wherein reaction condition is：Reaction temperature is 35 DEG C, and the reaction time is 48 hours, and speed of agitator is 200 revs/min.Herein
Under reaction condition, the fermentation unit of beta-thymidine reaches 14.9g/L.
（4）Beta-thymidine isolates and purifies, including following four step：
A, the zymotic fluid after the completion of fermentation is centrifuged 20 minutes under the conditions of 4000 revs/min；
B, low pole large pore resin absorption column HZ818 or the AB-8 post in step, the zymotic fluid after step a pass through HZ818
Or after the absorption of AB-8 posts, zymotic fluid becomes colourless transparent liquid；
C, the polar macroporous adsorbent resin column in step is macroporous absorbent resin D301 or S-8 post；
D, the vacuum drying vacuum in step is 0.1Mpa, and temperature is 65 DEG C, and dried finished product is white powder, purity
Up to 99.1%.
The design focal point of the present invention is：The present invention is to pass through Escherichia coli（E.coli）Fermenting and producing beta-thymidine, reduce
Production cost, and without using organic solvent in production process, harmful substance is not produced in fermentation process, processing step is simple,
Improve production operation security.High using beta-thymidine yield produced by the invention, the fermentation unit of beta-thymidine reaches 15g/L, leads to
Cross macroporous absorbent resin to isolate and purify, purifying has substantial industrial meaning up to more than 99%.
The above described is only a preferred embodiment of the present invention, be not intended to limit the scope of the present invention,
Therefore any subtle modifications, equivalent variations and modifications that every technical spirit according to the present invention is made to above example, still
Belong in the range of technical solution of the present invention.
Claims (8)
- A kind of 1. method that beta-thymidine is produced using fermentation method, it is characterised in that：Beta-thymidine is produced with Escherichia coli fermentation, including There are following steps：（1）Slant medium bevel strain strain Escherichia coli being inoculated into test tube；（2）Slant strains are expanded into culture as seed liquor；（3）Seed liquor is accessed in fermentation tank and fermented；（4）Add reactant ammoniacal liquor and glucose；（5）Beta-thymidine isolates and purifies.
- 2. the method according to claim 1 that beta-thymidine is produced using fermentation method, it is characterised in that：The step（1）In Slant medium be made up of following parts by weight of component：Dusty yeast 15-30 parts, ammonium sulfate 3-6 parts, 1.8 parts of potassium dihydrogen phosphate, phosphorus 1000 parts of sour 1.8 parts of hydrogen dipotassium, 7.5 parts of sodium chloride, glycerine 50-10 parts and water.
- 3. the method according to claim 1 or 2 that beta-thymidine is produced using fermentation method, it is characterised in that：Prepare inclined-plane bacterium Kind process conditions be：PH value is 7.0-8.0,25-40 DEG C of temperature, incubation time are 24-48 hours.
- 4. the method according to claim 1 that beta-thymidine is produced using fermentation method, it is characterised in that：The step（2）In Seed liquor and step（3）In zymotic fluid be made up of following parts by weight of component：Glucose 400-800 parts, tryptone 10-20 Part, potassium dihydrogen phosphate 0.5-1.0 parts, 2 parts of dipotassium hydrogen phosphate, 7.5 parts of sodium chloride, ammoniacal liquor 20-100 parts, 50 parts of glycerine and water 1000 parts.
- 5. the method that beta-thymidine is produced using fermentation method according to claim 1 or 4, it is characterised in that：The technique of fermentation Condition is：Zymotic fluid inoculum concentration is 10-15%, and pH value 7.0-7.5, cultivation temperature is 25-40 DEG C, and ventilation is 10-30 m3/ H, speed of agitator are 350-450 revs/min, and incubation time is 24-36 hours.
- 6. the method according to claim 1 that beta-thymidine is produced using fermentation method, it is characterised in that：The step（4）In Reaction condition be：Reactant ammoniacal liquor amount of substance concentration is 30-48mmol/l, and glucose substance amount concentration is 100-200mmol/ L, reaction temperature are 30-55 DEG C, and the reaction time is 36-72 hours, and speed of agitator is 160-200 revs/min.
- 7. the method according to claim 1 that beta-thymidine is produced using fermentation method, it is characterised in that：The step（5）In Beta-thymidine isolate and purify including following four step：A, the zymotic fluid after the completion of fermentation is centrifuged 20 minutes under the conditions of 4000 revs/min；B, the zymotic fluid after a steps is decolourized by low pole large pore resin absorption column；C, the zymotic fluid Jing Guo b step is isolated and purified by polar macroporous adsorbent resin column；D, the zymotic fluid Jing Guo step c is dried in vacuo, you can obtain the beta-thymidine that purity is more than 98%.
- 8. the method according to claim 7 that beta-thymidine is produced using fermentation method, it is characterised in that：Sent out in the step a Zymotic fluid centrifuges 20 minutes under the conditions of 4000 revs/min；Low pole large pore resin absorption column in step b is HZ818 or AB-8 Post；Polar macroporous adsorbent resin column in step c is D301 or S-8 posts；Vacuum drying vacuum in step d is 0.1Mpa, Temperature is 65 DEG C, and dried finished product purity is up to more than 99%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107629096A (en) * | 2017-09-27 | 2018-01-26 | 江西诚志生物工程有限公司 | Using the method for β thymidines in ultrafiltration and ion exchange technique refining fermenting liquid |
| CN110408667A (en) * | 2019-06-29 | 2019-11-05 | 赤峰蒙广生物科技有限公司 | A kind of zymotechnique improving beta-thymidine yield |
| CN110698536A (en) * | 2019-10-30 | 2020-01-17 | 江西诚志生物工程有限公司 | Novel method for producing glutathione by adopting fermentation method |
| CN112210577A (en) * | 2020-11-04 | 2021-01-12 | 赤峰蒙广生物科技有限公司 | Method for producing beta-thymidine by fermentation method |
| CN114032265A (en) * | 2021-12-03 | 2022-02-11 | 江西诚志生物工程有限公司 | Process for producing thymidine by fermentation method |
| CN114853823A (en) * | 2022-05-26 | 2022-08-05 | 宁夏华吉生物有限公司 | Method for extracting thymidine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107629096A (en) * | 2017-09-27 | 2018-01-26 | 江西诚志生物工程有限公司 | Using the method for β thymidines in ultrafiltration and ion exchange technique refining fermenting liquid |
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| CN114853823A (en) * | 2022-05-26 | 2022-08-05 | 宁夏华吉生物有限公司 | Method for extracting thymidine |
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Application publication date: 20180112 |