CN103436455A - S. cerevisiae strain for producing citicoline through bioconversion and application of S. cerevisiae strain - Google Patents
S. cerevisiae strain for producing citicoline through bioconversion and application of S. cerevisiae strain Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine and relates to S. cerevisiae strain for producing citicoline through bioconversion and application of the S. cerevisiae strain, namely the S. cerevisiae strain of high activity CMP (cytidine monophosphate) kinase and high activity phosphoryl choline-cytidyltransferase and the application of the S. cerevisiae strain in the production of citicoline through bioconversion. The S. cerevisiae strain provided by the invention has a classification name of S. cerevisiae, and has the Preservation No. of CGMCC7978 in CGMCC (China General Microbiological Culture Collection Center).
Description
Technical field
The invention belongs to biomedicine field.The bacterial classification that relates to a kind of bio-transformation citicoline of seed selection, i.e. yeast saccharomyces cerevisiae (S.cerevisiae) bacterial strain and the application in the bio-transformation citicoline thereof of high vigor cytidylic acid (CMP) kinases and high vigor phosphorylcholine-cytidyl-transferase.
Background technology
The manufacture of citicoline adopts three kinds of methods such as chemosynthesis, double-bacterium ferment and yeast bio conversion at present.Because the chemical synthesis cost is high, environmental pollution is more serious, only has Italy to utilize this method to produce on a small quantity.After the nineties, Japanese refined agate mountain mainly adopts the yeast bio conversion method to be produced, but, because main raw material CMP price is more expensive, after 2000, Japan has developed the method for double-bacterium ferment.It utilizes the genetic engineering bacteriums such as intestinal bacteria and product ammonia rod bacillus.The vitamin B13 (or uridylic) of take is main raw material, manufactures citicoline, and its transformation efficiency is 56%.China is also that to utilize discarded cereuisiae fermentum be biological catalyst for the yeast bio method for transformation that adopts at present.Because the existing several different methods of CMP is produced in a large number, the prices amplitude is large, and at China's double-bacterium ferment, with yeast bio, transforming to compare does not have advantage.And also there is complex process in double-bacterium ferment, low conversion rate, and the high more formidable bottleneck of separating difficulty.The defects such as the discarded cereuisiae fermentum bio-transformation of utilization exists yeast to collect difficulty, and the catalytic activity fluctuation of waste yeast is larger, and transformation efficiency is unstable.Particularly during the citicoline large-scale production, its unfavorable factor can highlight more.
Summary of the invention
The present invention overcomes the bottleneck that the bio-transformation citicoline is produced, the phosphorylcholine-cytidyl-transferase Wine brewing yeast strain of seed selection high vigor CMP kinases and high vigor, and the transformation efficiency of original citicoline 60～70% is brought up to more than 85%.
The applying biological transformation technology can be by CMP and phosphorylcholine bio-transformation citicoline.CMP kinases and phosphorylcholine-cytidyl-transferase are the key enzymes of this reaction.Yeast is higher to glucose utilization efficiency, and in the Embden-Meyerhof-Parnaspathway warp, a part glucose can discharge two molecule ATP for the CTP bioconversion reaction.The height of this reaction conversion ratio, depend primarily on the activity that CMP kinases and phosphorylcholine cytidine acyl shift.The contriver has designed a kind of method of strain improvement for this reason.First by the method for chemomorphosis, filter out yeast saccharomyces cerevisiae Repone K (KCl) sensitive strain, then according to the height of two kinds of enzyme activities, screen the higher bacterial classification of CDP-C transformation efficiency.
The present invention be take yeast saccharomyces cerevisiae CGMCC2842 as starting strain, first by the chemomorphosis method, filters out the KCL sensitive strain.The bacterial classification that screening obtains is further measured the vigor of above two key enzymes.On this basis, obtained yeast saccharomyces cerevisiae bacterial classification of the present invention, Classification And Nomenclature: Saccharomyces Cerevisiae in S accharomyces cerevisiae, its preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC No.7978, is called for short QY131.Preservation day: 2013.8.2.
1, the phosphorylcholine-cytidyl-transferase measuring method is as follows:
The reaction solution that adds 100ml in the 250ml shaking flask, wherein containing CTP25mmol/L, phosphoric acid buffer 200mmol/L, glucose 2g.Phosphorylcholine 5g, sal epsom 0.4g, barms 5g(dry weight).32 ℃, rotating speed 150rpm, oscillatory reaction 1h.Sampling, the content of mensuration citicoline.Adopt document (" medicine industry " the 2nd phase in 1985) agarose electrophoresis or HPLC method (Chinese Pharmacopoeia 2010 editions) to measure citicoline content, and be converted into its micromole's number.
2, the measuring method of cytidylate kinase is as follows:
The reaction solution that adds 100ml in the 250ml shaking flask, wherein containing CMP25mmol/L, phosphoric acid buffer 200mmol/L, glucose 2g, sal epsom 0.4g, barms 5g(dry weight).32 ℃, rotating speed 150rpm, oscillatory reaction 1h.Sampling, the content of mensuration CTP.CTP measures and can, with reference to aforesaid method, still should change original borate buffer into the pH3.6 citrate buffer solution, and do typical curve with the CTP standard specimen through electrophoresis.Sample records content by this method, and is converted into micromole's number and gets final product.
Screening through above-mentioned two kinds of enzyme activities height, finally obtain QY131 yeast saccharomyces cerevisiae bacterial classification.Its phosphorylcholine-cytidyl-transferase vigor can reach 17.7U, than the contrast strain, improves 106%.Its cytidylate kinase vigor improves 129%.This bacterial classification is compared as follows with the enzyme activity that contrasts strain, document [1] bacterial classification, the cereuisiae fermentum strain of industrial application at present.
| The enzyme title | The contrast strain | Document [2] bacterial classification * | The cereuisiae fermentum strain of industrial application at present | QY131 |
| CMP kinases (vigor) U | 28 | / | 30.6 | 64 |
| Phosphorylcholine-cytidyl-transferase | 8.6 | 0.25 | 12.6 | 17.7 |
* enzyme activity definition: micromole's number of per minute conversion of substrate under these conditions.
* document [2]: " Huadong Chemical College journal " the 2nd phase in 1986.
Above-mentioned QY131 yeast saccharomyces cerevisiae bacterial classification is collected to thalline by shaking flask and fermentor tank amplification culture and by inclined-plane, access shake-flask culture.Thalline be can be used for to CMP and phosphorylcholine bio-transformation citicoline after air-dry or quick-frozen broken wall.
The invention discloses a kind of method for preparing citicoline, comprising: the QY131 yeast saccharomyces cerevisiae be take to CMP and phosphorylcholine and carry out bio-transformation as substrate.
The citicoline bioconversion reaction is to react with the ATP energy coupling, therefore must guarantee the continual supply of ATP in the whole process of bio-transformation.And the ATP generation realizes by adding glucose.After bio-transformation is initial, add glucose and generally all be exhausted at 1-2 hour, subsequent reactions will lack ATP.If than glucosan, ATP is not in time for reaction in initial interpolation, it also can be degraded and consume, and excessive glucose has restraining effect to reaction.So, for guaranteeing that reaction process ATP constantly continues supply, glucose is added in the present invention's employing or stream adds the glucose method.
In the bioconversion reaction process, preferably add or stream adds glucose, the glucose concn of adding is 20～80%; Be 0.5～3h the interval time of adding; The concentration that stream adds glucose is 20～80%; The speed that stream adds is 20～80ml/h, all is weight percentage.
The preferred glucose concn of adding is 40～50%; Be 1～2h the interval time of adding; The concentration that stream adds glucose is 40～50%; The speed that stream adds is 40～50ml/h.
A kind of preferred method for preparing citicoline: substrate is 28～34 ℃ in temperature, and pH carries out bio-transformation under 6.5～8.5 conditions, in conversion reaction, adds or stream adds glucose, and described substrate is containing cytidylic acid 28～40mmol/L, MgSO
44H
2o7.5～15mmol/L and phosphorylcholine 75～90mmol/L form.
The glucose concn of preferably adding in above-mentioned bioconversion reaction is 20～80%; Be 0.5～3h the interval time of adding; The concentration that stream adds glucose is 20～80%; The speed that stream adds is 20～80ml/h, all is weight percentage.
The preferred glucose concn of adding is 40～50%; Be 1～2h the interval time of adding; The concentration that stream adds glucose is 40～50%; The speed that stream adds is 40～50ml/h.
The temperature of preferred bioconversion reaction is 32～34 ℃; PH is 6.5～7.2.
Preferred substrate is containing cytidylic acid 28～32mmol/L, MgSO
44H
2o7.5～10mmol/L and phosphorylcholine 75～80mmol/L form.
Most preferred preparation condition is: wherein substrate is containing 28mmol/L cytidylic acid, 15mmol/L MgSO
44H
2o, 75mmol phosphorylcholine, temperature is 32 ℃, pH=7.2.
Embodiment
Embodiment 1
The screening of high vigor CMP kinases and high vigor phosphorylcholine-cytidyl-transferase Wine brewing yeast strain
1, bacterial classification
Yeast saccharomyces cerevisiae (S.cerevisiae QY131, culture presevation number is CGMCC7978) is obtained after mutagenesis by CGMCC2842.
2, substratum
(1), original culture medium A: glucose 3%, extractum carnis 0.3%, yeast extract paste 0.3%, urea 0.5%, FeSO
47H
2o0.01%, agar 2.5%.
(2), KCl sensitive strain screening culture medium B: add 1.5mol/LKCl in culture medium A.
3, induction mutation of bacterium screening method
Ethyl sulfate (DES) stoste is added to a small amount of alcohol dissolves, be mixed with 1%DES solution with the pH7.0 phosphoric acid buffer, getting fresh bacterium liquid 2ml and 1%DES2ml fully mixes, 30 ℃, 200rpm dark place vibration, process respectively 10,20,30,40,60,80min, add 1% Sulfothiorine 2ml termination reaction, 10-1 serial dilution four times, then get 200ul coating culture dish, overnight incubation.Utilize replica-plating method screening KCL susceptibility bacterial strain, picking list bacterium colony.Will be by the thalline of inclined-plane, shake-flask culture after air-dry or quick-frozen, detect the vigor of CMP kinases and citicoline cytidylyltransferase.Final acquisition QY131 yeast strain.The kinase whose vigor of CMP is 64U, than the contrast strain, has improved 129%, than the cereuisiae fermentum strain of current industrial application, has improved 109%.The vigor of citicoline cytidylyltransferase is 17.7U, than the contrast strain, has improved 106%, than the cereuisiae fermentum strain of current industrial application, has improved 40%.
The QY131 yeast strain on August 02nd, 2013 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment 2
The cultivation of QY131 yeast
Slant medium is the wort agar substratum, the shake-flask culture based formulas is molasses 30g/L, glucose 10g/L, sal epsom 1.2g/L, ammonium sulfate 1.2g/L, urea 0.2g/L, potassium primary phosphate 1.2g/L, zinc sulfate 0.2g/L, be dissolved in water, adjust pH5.4(± 0.1) finally be settled to 1L.Use inoculating needle, picking one ring slant strains access is equipped with in the 500ml shaking flask of 100ml substratum, 28 ℃, rotating speed 250rpm, shaking culture 16 hours, then access in the 5L Fermentation that 2000ml substratum (formula is with the shake-flask culture base) is housed and cultivate, 28 ℃ of temperature, mixing speed 300rpm, air quantity 0.5vvm, cultivate 20 hours.Then it is centrifugal, collect thalline, thalline be can be used for to CMP and phosphorylcholine bio-transformation citicoline after air-dry or quick-frozen broken wall.
Embodiment 3
The bio-transformation of citicoline in shaking flask
Add the 100mL reaction solution in the 250ml shaking flask, wherein containing CMP25mmol/L, phosphoric acid buffer 200mmol/L, glucose 4g, phosphorylcholine 5g, sal epsom 0.4g, QY131 yeast 5g (dry weight).32 ℃, rotating speed 150rpm, oscillatory reaction 6-8h.Every 2h feed supplement 2g glucose, can consider to mend sugar 2-3 time according to response situation, and every 2h measures its transformation efficiency.React under these conditions 6 hours, the citicoline transformation efficiency can reach 83%.
Embodiment 4
The bio-transformation of citicoline in bio-reactor
In the 5L bio-reactor, the 2L reaction solution of packing into, wherein containing 28mmol/L CMP, 200mmol/L phosphoric acid buffer, 80g glucose, 100g phosphorylcholine, 8g sal epsom, 100g (dry weight) QY131 yeast, start stream and add 50% glucose after 32 ℃, rotating speed 100rpm reaction 1h, flow acceleration 50ml/h, reaction 5h, citicoline transformation efficiency 85%.
Embodiment 5
The optimization of citicoline biotransformation condition
In above-mentioned bioconversion reaction system, by embodiment 3 methods, add under the phosphoric acid buffer condition of glucose and initial interpolation 200mmol/L, investigate the impact of the factor pair citicoline transformation efficiencys such as CMP, sal epsom, phosphorylcholine, temperature and pH.Designed L for this reason
16(4
5) orthogonal test.
L
16(4
5) the CDP-C orthogonal experiment
As seen from table, under above-mentioned price modification, the influence factor maximum be temperature, temperature is high, speed of response is fast, but can not be higher than 34 ℃.The second influence factor is concentration of substrate, and the concentration of substrate low-conversion is high, but considers the productive rate of primary first-order equation, need find a trim point.Can determine following top condition by orthogonal table: 28mmol/L CMP, 15mmol/L MgSO
44H
2o, 75mmol phosphorylcholine, 32 ℃, pH7.2, now the citicoline transformation efficiency can reach 90.2%.
Claims (10)
1. a Wine brewing yeast strain, its preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC7978.
2. a method for preparing citicoline comprises: the yeast saccharomyces cerevisiae of claim 1 be take CMP and phosphorylcholine and is carried out bio-transformation as substrate.
3. the method for claim 2 is wherein added in the bioconversion reaction process or stream adds glucose, and the glucose concn of adding is 20～80%; Be 0.5～3h the interval time of adding; The concentration that stream adds glucose is 20～80%; The speed that stream adds is 20～80ml/h, all is weight percentage.
4. the method for claim 3, the glucose concn of wherein adding is 40～50%; Be 1～2h the interval time of adding; The concentration that stream adds glucose is 40～50%; The speed that stream adds is 40～50ml/h.
5. the method for claim 2, comprising: substrate is 28～34 ℃ in temperature, and pH carries out bio-transformation under 6.5～8.5 conditions, in conversion reaction, add or stream adds glucose, and described substrate is containing cytidylic acid 28～40mmol/L, MgSO
44H
2o7.5～15mmol/L and phosphorylcholine 75～90mmol/L form.
6. the method for claim 5, wherein, the glucose concn of adding is 20～80%; Be 0.5～3h the interval time of adding; The concentration that stream adds glucose is 20～80%; The speed that stream adds is 20～80ml/h, all is weight percentage.
7. the method for claim 6, the glucose concn of wherein adding is 40～50%; Be 1～2h the interval time of adding; The concentration that stream adds glucose is 40～50%; The speed that stream adds is 40～50ml/h.
8. the method for claim 5, wherein the temperature of bioconversion reaction is 32～34 ℃; PH is 6.5～7.2.
9. the method for claim 5, wherein substrate is containing cytidylic acid 28～32mmol/L, MgSO
44H
2o7.5～10mmol/L and phosphorylcholine 75～80mmol/L form.
10. the method for claim 5, wherein substrate is containing 28mmol/L cytidylic acid, 15mmol/L MgSO
44H
2o, 75mmol phosphorylcholine, temperature is 32 ℃, pH=7.2.
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| CN107488603A (en) * | 2017-10-19 | 2017-12-19 | 南通秋之友生物科技有限公司 | One kind is overexpressed choline phosphate cytidylyltransferase Saccharomyces cerevisiae gene engineering bacteria and construction method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792786A (en) * | 2010-03-04 | 2010-08-04 | 南京工业大学 | Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis |
| CN102286386A (en) * | 2011-08-15 | 2011-12-21 | 江南大学 | Issatchenkia orientalis and method for producing citicoline by whole cell conversion of Issatchenkia orientalis |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792786A (en) * | 2010-03-04 | 2010-08-04 | 南京工业大学 | Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis |
| CN102286386A (en) * | 2011-08-15 | 2011-12-21 | 江南大学 | Issatchenkia orientalis and method for producing citicoline by whole cell conversion of Issatchenkia orientalis |
Non-Patent Citations (1)
| Title |
|---|
| 田三德等: "超声波对酿酒酵母诱变作用的初探", 《北京工商大学学报》, vol. 22, no. 1, 31 January 2004 (2004-01-31), pages 12 - 14 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488603A (en) * | 2017-10-19 | 2017-12-19 | 南通秋之友生物科技有限公司 | One kind is overexpressed choline phosphate cytidylyltransferase Saccharomyces cerevisiae gene engineering bacteria and construction method and application |
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