CN103540537A - Preparation method of uridine triphosphate - Google Patents
Preparation method of uridine triphosphate Download PDFInfo
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- CN103540537A CN103540537A CN201210247342.4A CN201210247342A CN103540537A CN 103540537 A CN103540537 A CN 103540537A CN 201210247342 A CN201210247342 A CN 201210247342A CN 103540537 A CN103540537 A CN 103540537A
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Abstract
The invention provides a preparation method of uridine triphosphate, belonging to the field of biosynthesis and bioconversion. According to the method provided by the invention, high permeability yeast cells which are obtained from wet yeasts at a temperature lower than -20 DEG C for over 72 hours can efficiently convert uridylic acid into uridine triphosphate. The method of preparing the permeable yeast can maintain the integrity of yeast cells without a complex treatment process, so that the method is very applicable to industrialized production on a large scale.
Description
Technical field
The invention belongs to biosynthesizing and bio-transformation field, be specifically related to a kind of preparation method of uridine triphosphate.
Background technology
Uridine triphosphate (Uridine triphosphate, UTP) is important biologically active substance in biomass cells, participates in multiple biochemical reaction in body.As synthesizing of polysaccharide (glycogen or starch), need UTP first to react with Cori ester and generate uridine diphosphoglucose (UDPG), and as substrate glycogen biosynthesis or starch chain; And for example the synthetic of Yeast Nucleic Acid (RNA) needs UTP to participate in directly.
In recent years, UTP also has some development clinically as medicine.As, UTP and derivative thereof can be used for treating lung's cystic fibrosis disease, promote the expectoration (USP 5292498, Richard C.Boucher, 1994) of consumptive's phlegm; Theodore Page use it for general autism children treatment (Theodore P., Pro.Neuro-Psycho.Biolo.Psyh., 2002,26:397-400).In addition, aspect the signal transduction of cell, also find UTP show important function (Christopher M.A., etc, TiPS, 1997,18:387-392).
UTP not only can be directly used in drug research, also can be used as intermediate in a large number for the synthesis of UDPG, and the effect of playing the part of in glycobiology research.For many years, investigator has great enthusiasm for UTP synthetic.
The synthetic main biological process that adopts of UTP, wherein common are and utilizes yeast cell carry out the synthetic of UTP or utilize genetic engineering bacterium to synthesize UTP.
Brewing yeast cell has the glycolysis-ability of high vigor, and its various bacterial classifications are widely used at aspects such as wine brewing, alcohol, food.By glycolysis-and the coupling of adenosine triphosphate (ATP) regeneration phase, can synthesize ATP or other ucleotides material, as synthetic ATP, cytidine diphosphate etc.Wherein, synthetic ATP (Qiu Weiran, Shanghai chemical industry, 1994,5) and the CTP (Qiu Weiran, meticulous and specialty chemicals, 2002,17) of having obtained of the applying immobilized yeast such as Qiu Weiran.Yeast is fixed, and through a series of processing, although can increase the reaction times of yeast and facilitate solid-liquid separation, but due in reaction process along with the increase of reaction times, the materials such as some micromolecular coenzyme, along with reaction solution runs off, need to add these expensive materials on the contrary in the reaction later stage; And solid-liquid separation development in recent years is very fast, for yeast cell, also can solve very easily; In addition, owing to using carrageenin as fixation support, immobilization temperature, in 50 degree left and right, causes a large amount of yeast inactivations, need to add more than 50% immobilization particle, and immobilization cost is high, has lost existing meaning in reaction process.
Utilizing genetic engineering bacterium synthesising biological class material is also the emphasis of development in recent years.Jozef N. utilized the intestinal bacteria of the polyphosphate kinase of expressing, and can synthesize very easily UTP, activity than use Brevibacterium ammoniagenes effective (Jozef N., Biotech.Lett., 2002,24:925-930).But except having compared activity, do not relate to concrete UTP synthetic in literary composition.
In relevant ribonucleoside triphosphote synthetic, utilize yeast cell still to there is extraordinary convenience as catalyzer because yeast cell can Cong Ru brew-house, bio-ethanol Chang Huo bread yeast factory obtains easily, and quantity is unrestricted.But directly use yeast cell to there will be a serious obstacle, ucleotides material can not directly pass in and out cell, although by immobilized method, can solve, a large amount of being fixed of yeast, technique is very complicated and impracticable; And the method for adding organic solvent also has a large amount of uses in actual production, but the organic solvent adding does not reclaim after synthetic, and this can increase environmental pollution, and the vigor of glycolytic ferment system is also had a great impact.
Summary of the invention
Design of the present invention is like this:
Prepare the yeast that permeability improves, it be take glucose and is obviously improved as the ability that the energy carries out glycolysis-regeneration ATP; The yeast cell that perviousness is improved, adds in the reaction solution that contains glucose, phosphoric acid salt, sal epsom, uridylic acid (or adenosine), and uridylic acid (or adenosine) is changed into corresponding uridine triphosphate (or adenosine triphosphate); Reclaim uridine triphosphate (or adenosine triphosphate).
One of technical problem to be solved by this invention is: the preparation method that a kind of high infiltration yeast is provided.
Two of technical problem to be solved by this invention is: provide a kind of and utilize high infiltration yeast to prepare the method for uridine triphosphate.
Therefore, the invention provides:
1. a preparation method for high infiltration yeast, is characterized in that, it is characterized in that, the yeast that will wet is processed at low temperatures, condition is: wet yeast water content 20%-80% weight ratio (w/w), approximately-200 ℃-Yue-15 ℃ for the treatment of temp, and treatment time 24-480h.
2. according to the preparation method of the height infiltration yeast described in 1, it is characterized in that wet yeast water content 30%-80% weight ratio (w/w).
3. according to the preparation method of the height infiltration yeast described in 2, it is characterized in that wet yeast water content 40%-70% weight ratio (w/w).
4. according to the preparation method of the height infiltration yeast described in 3, it is characterized in that wet yeast water content 50%-60% weight ratio (w/w).
5. according to the preparation method of the height infiltration yeast described in 4, it is characterized in that wet yeast water content 50% weight ratio (w/w).
6. according to the preparation method of the height infiltration yeast described in 1, it is characterized in that approximately-100 ℃-Yue 20 ℃ for the treatment of temps.
7. according to the preparation method of the height infiltration yeast described in 6, it is characterized in that approximately-40 ℃ for the treatment of temps.
8. according to the preparation method of the height infiltration yeast described in 1, it is characterized in that about 48-480h for the treatment of time.
9. according to the preparation method of the height infiltration yeast described in 8, it is characterized in that about 48-240h for the treatment of time.
10. according to the preparation method of the height infiltration yeast described in 9, it is characterized in that about 72-96h for the treatment of time.
11. according to the preparation method of the height infiltration yeast described in 10, it is characterized in that about 96h for the treatment of time.
12. according to the preparation method of the height infiltration yeast described in 1, it is characterized in that, it is characterized in that, the yeast that will wet is processed at low temperatures, and condition is: wet yeast water content 50% weight ratio (w/w), approximately-40 ℃ for the treatment of temps, and treatment time 96h.
The preparation method of 13. 1 kinds of uridine triphosphate, it is characterized in that, the height infiltration yeast of preparing according to the arbitrary described method of 1-12 is mixed with the solution that contains uridine, reaction, then reclaim uridine triphosphate, condition is: the consumption of yeast is 3%-60% (weight of wet yeast weight/reaction solution); The solution that contains uridine is glucose, the 10mmol/L sal epsom of 50mmol/L uridylic acid, 150mmol/L, the phosphate buffered saline buffer of 250mmol/L pH7.5; 30 ℃ of temperature of reaction; Reaction times 1.0-10h.
14. according to the preparation method of the uridine triphosphate described in 13, it is characterized in that, the consumption of yeast is 10%-30% (weight of wet yeast weight/reaction solution).
15. according to according to the preparation method of the uridine triphosphate described in 14, it is characterized in that, the consumption of yeast is 15% (weight of wet yeast weight/reaction solution).
16. according to the preparation method of the arbitrary described uridine triphosphate of 13-15, it is characterized in that, the reaction times is 2h.
17. according to the preparation method of the arbitrary described uridine triphosphate of 13-15, it is characterized in that, yeast is what according to 12 method, prepare.
The preparation method of 18. 1 kinds of adenosine triphosphates, it is characterized in that, the height infiltration yeast of preparing according to the arbitrary described method of 1-12 is mixed with the solution that contains adenosine, reaction, then reclaim adenosine triphosphate, condition is: the consumption of yeast is 3%-60% (weight of wet yeast weight/reaction solution); The solution that contains adenosine is glucose, the 10mmol/L sal epsom of 30mmol/L adenosine, 150mmol/L, the phosphate buffered saline buffer of 250mmol/L pH7.5; 30 ℃ of temperature of reaction; Reaction times 1.0-10h.
19. according to the preparation method of the adenosine triphosphate described in 18, it is characterized in that, the consumption of yeast is 10%-30% (weight of wet yeast weight/reaction solution).
20. according to according to the preparation method of the adenosine triphosphate described in 19, it is characterized in that, the consumption of yeast is 15% (weight of wet yeast weight/reaction solution).
21. according to the preparation method of the arbitrary described adenosine triphosphate of 18-20, it is characterized in that, the reaction times is 2h.
22. according to the preparation method of the arbitrary described adenosine triphosphate of 18-20, it is characterized in that, yeast is what according to 12 method, prepare.
What the yeast cell using in the present invention was selected from cereuisiae fermentum, bread yeast, distillery yeast, yeast saccharomyces cerevisiae or other source can be for the preparation of the biomass cells of ribonucleoside triphosphote.
The height infiltrationization yeast that uses method of the present invention to prepare, there is significant technical progress, at least solved with next technical problem: the problem that has improved yeast cell permeability, make nucleotide analog can freely pass in and out cell, the activity that has retained original cell, greatly reduce the treatment step of yeast cell, reduced complicacy and the production cost of operation, can be used for the preparation of the ribonucleoside triphosphotes such as uridine triphosphate.
Because infiltrationization yeast does not destroy cell walls and cytolemma completely, in cell, macromolecular substance is not lost in reaction solution, has alleviated greatly the pressure of subsequent disposal.And after finishing, reaction can use easily the centrifugation apparatus of some low speed, and as rapidly that yeast cell is separated with reaction solution in link-suspended basket centrifuge, solid bowl centrifuge etc., be particularly suitable for large-scale production application.
In the present invention, the transformation efficiency of Nucleotide adopts HPLC method to measure.Method is as follows:
Agilent liquid chromatograph, post YWG-C18 (250mm * 4.6mm); Moving phase: the phosphate buffered saline buffer of 5% acetonitrile+50mmol/L pH7.0; Flow velocity: 1.0ml/min; Wavelength: 254nm; Temperature: room temperature.
Below in conjunction with embodiment, describe the present invention in detail, but it must be understood that these all embodiments are all only illustratives, the scope not limiting the present invention in any way.Scope of the present invention is determined by the structure of the law of claim.
Embodiment:
Embodiment 1
Take water content and be about each 100g of yeast cell of 50%, be placed in table 1 after freezing at each temperature 4 days, take out as the synthetic ATP in enzyme source.Reaction solution proportioning is: the phosphate buffered saline buffer of the glucose of 30mmol/L adenosine, 150mmol/L, 10mmol/L sal epsom, 250mmol/LpH7.5, temperature yeast add-on is 15%, in 30 ℃ of oscillatory reactions 2 hours, get 1mL reaction solution, centrifugal removal yeast cell, 10000 times of deionized water dilutions for reaction solution, measure the ATP generating with HPLC.Result is as table 1.
Table 1
| Numbering | Disposal temperature (℃) | ATP(mmol/L) |
| 1 | 4 | 0 |
| 2 | -5 | 1.3 |
| 3 | -10 | 7.9 |
| 4 | -15 | 26.7 |
| 5 | -20 | 27.9 |
| 6 | -40 | 28.8 |
| 7 | ＜-100 | 28.3 |
Embodiment 2
Take the yeast 100g of each water content in table 2, be placed in-40 ℃ and place after 4 days, take out as the synthetic ATP in enzyme source.Reaction method is as embodiment 1, and result is as table 2.
Table 2
| Numbering | Yeast water content (%) | ATP(mmol/L) |
| 1 | 10 | 2.6 |
| 2 | 20 | 9.1 |
| 3 | 30 | 21.3 |
| 4 | 40 | 25.9 |
| 5 | 50 | 28.9 |
| 6 | 60 | 28.1 |
| 7 | 70 | 27.3 |
| 8 | 80 | 21.5 |
Embodiment 3
Take each 100g of yeast of water content approximately 50%, be placed in-40 ℃ of placements, the time is as shown in table 3, takes out as the synthetic ATP in enzyme source.
Reaction method is as embodiment 1, and result is as table 3.
Table 3
| Numbering | Freezing time (h) | ATP(mmol/L) |
| 1 | 12 | 10.6 |
| 2 | 24 | 19.3 |
| 3 | 48 | 22.7 |
| 4 | 72 | 26.1 |
| 5 | 96 | 28.4 |
| 6 | 120 | 27.5 |
| 7 | 240 | 27.4 |
| 8 | 480 | 27.1 |
Embodiment 4
Take 100 grams, the yeast of water content approximately 50%, be placed in-40 ℃ and place 96h, taking-up is as the synthetic uridine triphosphate in enzyme source, reaction method is as follows: the phosphate buffered saline buffer of the glucose of 50mmol/L uridylic acid, 150mmol/L, 10mmol/L sal epsom, 250mmol/L pH7.5, yeast add-on is 15%, in 30 ℃ of oscillatory reactions 2 hours, simultaneously with the yeast of freezing treatment not in contrast.Reaction solution, through the treatment process of embodiment 1, is measured the amount of the uridine triphosphate generating.Result is that the content of uridine triphosphate in reaction solution is 46.1mmol/L (transformation efficiency is 92.2%), and in contrast liquid, the content of uridine triphosphate is only 0.22mmol/L (transformation efficiency is 0.44%).
Claims (22)
1. a preparation method for high infiltration yeast, is characterized in that, it is characterized in that, the yeast that will wet is processed at low temperatures, and condition is: wet yeast water content weight ratio 20%-80%, and treatment temp-200 ℃--15 ℃, and treatment time 24-480h.
2. the preparation method of high infiltration yeast according to claim 1, is characterized in that, wet yeast water content 30%-80% weight ratio.
3. the preparation method of high infiltration yeast according to claim 2, is characterized in that, wet yeast water content weight ratio 40%-70%.
4. the preparation method of high infiltration yeast according to claim 3, is characterized in that, wet yeast water content weight ratio 50%-60%.
5. the preparation method of high infiltration yeast according to claim 4, is characterized in that, wet yeast water content weight ratio 50%.
6. the preparation method of high infiltration yeast according to claim 1, is characterized in that, ℃-20 ℃, treatment temp-100.
7. the preparation method of high infiltration yeast according to claim 6, is characterized in that treatment temp-40 ℃.
8. the preparation method of high infiltration yeast according to claim 1, is characterized in that treatment time 48-480h.
9. the preparation method of high infiltration yeast according to claim 8, is characterized in that treatment time 48-240h.
10. the preparation method of high infiltration yeast according to claim 9, is characterized in that treatment time 72-96h.
The preparation method of 11. high infiltration yeast according to claim 10, is characterized in that treatment time 96h.
The preparation method of 12. high infiltration yeast according to claim 1, is characterized in that, it is characterized in that, the yeast that will wet is processed at low temperatures, and condition is: wet yeast water content weight ratio 50%, treatment temp-40 ℃, and treatment time 96h.
The preparation method of 13. 1 kinds of uridine triphosphate, it is characterized in that, the height infiltration yeast of preparing according to the arbitrary described method of claim 1-12 is mixed with the solution that contains uridine, reaction, then reclaim uridine triphosphate, condition is: the weight of the wet yeast weight/reaction solution of consumption of yeast is 3%-60%; The solution that contains uridine is glucose, the 10mmol/L sal epsom of 50mmol/L uridylic acid, 150mmol/L, the phosphate buffered saline buffer of 250mmol/L pH7.5; 30 ℃ of temperature of reaction; Reaction times 1.0-10h.
The preparation method of 14. uridine triphosphate according to claim 13, is characterized in that, the weight of the wet yeast weight/reaction solution of consumption of yeast is 10%-30%.
15. according to the preparation method of claim uridine triphosphate according to claim 14, it is characterized in that, the weight of the wet yeast weight/reaction solution of consumption of yeast is 15%.
16. according to the preparation method of the arbitrary described uridine triphosphate of claim 13-15, it is characterized in that, the reaction times is 2h.
17. according to the preparation method of the arbitrary described uridine triphosphate of claim 13-15, it is characterized in that, yeast is what according to the method for claim 12, prepare.
The preparation method of 18. 1 kinds of adenosine triphosphates, it is characterized in that, the height infiltration yeast of preparing according to the arbitrary described method of claim 1-12 is mixed with the solution that contains adenosine, reaction, then reclaim adenosine triphosphate, condition is: the weight of the wet yeast weight/reaction solution of consumption of yeast is 3%-60%; The solution that contains adenosine is glucose, the 10mmol/L sal epsom of 30mmol/L adenosine, 150mmol/L, the phosphate buffered saline buffer of 250mmol/L pH7.5; 30 ℃ of temperature of reaction; Reaction times 1.0-10h.
The preparation method of 19. adenosine triphosphates according to claim 18, is characterized in that, the weight of the wet yeast weight/reaction solution of consumption of yeast is 10%-30%.
20. according to the preparation method of claim adenosine triphosphate according to claim 19, it is characterized in that, the consumption of yeast is the weight 15% of wet yeast weight/reaction solution.
21. according to the preparation method of the arbitrary described adenosine triphosphate of claim 18-20, it is characterized in that, the reaction times is 2h.
22. according to the preparation method of the arbitrary described adenosine triphosphate of claim 18-20, it is characterized in that, yeast is what according to the method for claim 12, prepare.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109701A (en) * | 2014-05-05 | 2014-10-22 | 吉林英联生物制药股份有限公司 | Adenosine triphosphate preparation method |
| CN108486195A (en) * | 2018-02-26 | 2018-09-04 | 安徽翠鸟生物技术有限公司 | A method of preparing UDP with enzyme process |
| CN112129938A (en) * | 2019-06-25 | 2020-12-25 | 中国科学院分子细胞科学卓越创新中心 | Application of UDP-Glc in lung cancer metastasis assessment |
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2012
- 2012-07-17 CN CN201210247342.4A patent/CN103540537A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104109701A (en) * | 2014-05-05 | 2014-10-22 | 吉林英联生物制药股份有限公司 | Adenosine triphosphate preparation method |
| CN104109701B (en) * | 2014-05-05 | 2017-04-26 | 吉林英联生物制药股份有限公司 | Adenosine triphosphate preparation method |
| CN108486195A (en) * | 2018-02-26 | 2018-09-04 | 安徽翠鸟生物技术有限公司 | A method of preparing UDP with enzyme process |
| CN112129938A (en) * | 2019-06-25 | 2020-12-25 | 中国科学院分子细胞科学卓越创新中心 | Application of UDP-Glc in lung cancer metastasis assessment |
| CN112129938B (en) * | 2019-06-25 | 2023-08-18 | 中国科学院分子细胞科学卓越创新中心 | Use of UDP-Glc in lung cancer metastasis assessment |
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Application publication date: 20140129 |