CN102121035B - Method for increasing yield of pyruvic acid - Google Patents
Method for increasing yield of pyruvic acid Download PDFInfo
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- CN102121035B CN102121035B CN 201010581532 CN201010581532A CN102121035B CN 102121035 B CN102121035 B CN 102121035B CN 201010581532 CN201010581532 CN 201010581532 CN 201010581532 A CN201010581532 A CN 201010581532A CN 102121035 B CN102121035 B CN 102121035B
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- pyruvic acid
- acid
- torulopsis glabrata
- vitamin
- fermentation
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Abstract
The invention discloses a method for increasing yield of pyruvic acid, belonging to the fermentation engineering field. The method is as follows: when Torulopsis glabrata (CCTCC NO: M202019) is used to ferment and produce pyruvic acid, aspartic acid is added to increase the acid stress resistance of Torulopsis glabrata. When the pH values are 5.5 and 4.5, the yields of pyruvic acid are separately 55.8g/L and 30.4g/L which are separately increased by 12.4% (49.6g/L) and 48.4% (20.5g/L) than the control groups without aspartic acid; and the method has better technical effect and wide application prospect.
Description
Technical field
The present invention relates to a kind of method that improves output of pyruvic acid, especially in cultivation, add energy cosubstrate aspartic acid, improve the interior ATP level of born of the same parents, strengthen bacterial strain acid tolerance, the growth of protection cell, the method for raising output of pyruvic acid belongs to field of fermentation engineering.
Background technology
The fermentative Production pyruvic acid began one's study from the fifties, obtained many achievements.Adopt optimizing fermentation and metabolic engineering principle, with pyruvic acid high yield, high yield and high production intensity mutually unification be target, the fermenting process of polyauxotroph bacterial strain torulopsis glabrata (Torulopsis glabrata) being produced pyruvic acid has carried out research work such as a large amount of optimizations and transformation, impels output of pyruvic acid to increase substantially.Along with output of pyruvic acid improves, consequent problem is that yeast is for the susceptibility of low pH value and high density pyruvic acid.
As a rule, in the organic acid fermentation process, stream adds alkaline matters such as a certain amount of NaOH in order to keep fermentation system pH to be in the suitableeest scope, and along with streams such as NaOH add, causes fermented liquid osmotic pressure constantly to raise; And when osmotic pressure reached finite concentration in the fermented liquid, cell further accumulated the organic acid ability and speed will descend gradually, is difficult to reach maximum theoretical yield.Can improve bioprocesses performance and efficient although biological respinse separates combination technique with product, the scope of application of this method and the cost of equipment of great number have limited its application in large-scale commercial production.For further improving output of pyruvic acid and productive rate, need the inhibition or the anti-low pH of raising cell that reduce pyruvic acid in the fermenting process to coerce ability.
Summary of the invention
Technical problem to be solved by this invention is a kind of method that improves output of pyruvic acid, serves as to produce bacterial strain with torulopsis glabrata (Torulopsisglabrata), and adding aspartic acid in initial medium, to make final concentration be 0-1.5g/L.
Described torulopsis glabrata is multiple vitamin defective type, and deposit number is CCTCC NO:M202019.
The content of aspartic acid is 1.5g/L in the initial medium.
The torulopsis glabrata fermentation is with substratum (g/L) being: glucose 100, NH
4SO
47, MgSO
47H
2O 0.8, KH
2PO
45, CH
3COONa 6, aspartic acid 1.5, liquid microelement 10mL, VITAMIN liquid 10mL; PH is adjusted to 5.5 and 4.5 with pyruvic acid solution respectively; The jar top fermentation is kept suitable pH with 8mol/LNaOH or 2mol/L HCl.
Described liquid microelement is: CaCl
22H
2O 2g, FeSO
47H
2O 2g, ZnCl
20.5g, MnCl
24H
2O 12g, CuSO
45H
2O 0.05g, 2mol/L HCl dissolving back is settled to 1L with deionized water.
Described VITAMIN liquid is: nicotinic acid 80mg, and VitB1 0.15mg, pyridoxol 40mg, vitamin H 4mg, riboflavin 10mg, 2mol/L HCl dissolving back is settled to 1L with deionized water.
The measuring method of dry cell weight: get a certain amount of bacteria suspension and place the 10mL volumetric flask, add the calcium carbonate in the 2mL dissolving with hydrochloric acid bacteria suspension, be settled to 10mL with deionized water, shake up; With UV 7500 type visible spectrophotometers, survey the OD value in 660nm place colorimetric, with the dry cell weight typical curve calculate dry cell weight.
The mensuration of pyruvic acid concentration: adopt Agilent 1100 high performance liquid chromatographs (joining UV, visible light detector, parallax refraction detector and workstation), chromatographic column adopting C18 post, 5 μ m, 4.6mm * 250mm; Moving phase: 0.1%H
3PO
4Flow velocity: 1mL/min, column temperature: 28 ℃, sample size: 10 μ L, UV-detector wavelength: 215nm.
Sample treatment: the 5mL fermented liquid is centrifugal 10min under 10000rpm, gets supernatant liquor and moves in the test tube in order to surveying pyruvic acid usefulness.When surveying pyruvic acid, get the 1mL supernatant liquor and move in the 50mL volumetric flask, deionized water is settled to groove, and behind 0.45 μ m membrane filtration, filtrate feed flow analysis of hplc is used.
The present invention has increased the TCA flow by adding aspartic acid, has promoted strain growth, has improved the acidproof ability of bacterial strain.Adopt method of the present invention to be respectively at pH under 5.5 and 4.5 the condition, output of pyruvic acid is respectively 55.8g/L and 30.4g/L, compare with control group (not interpolation group) and to have improved 12.4% (49.6g/L) and 48.4% (20.5g/L) respectively, and have better growth performance.Method provided by the invention, simple, require low to operator's specified quality.
Embodiment
Embodiment 1 bacterial strain
One strain quadruple vitamin defective type, pyruvic carboxylase composing type reduce and a large amount of torulopsis glabrata CCTCC No.M202019 that accumulates pyruvic acid, taxonomy called after torulopsis glabrata (Torulopsis glabrata), be laboratory seed selection bacterial strain, this bacterial strain has been applied for Chinese patent, application number is 02113142.2, publication number CN1392246A.
Pyruvic acid is produced in embodiment 2 fermentations
With T.glabrata CCTCC M202019, the inclined-plane of transferring is earlier cultivated 16h and is carried out while still alive in 30 ℃ of incubators.Single colony inoculation after the picking activation 30 ℃, is cultivated 24h under the 200rpm to seed culture medium.Inoculum size with 10% changes seed over to fermention medium respectively, and adding aspartic acid, to make final concentration be 1.5g/L, and pH is set at 5.5,4.5 respectively, is the pH regulator agent with 8mol/LNaOH or 2mol/L HCl, cultivates 56h under 30 ℃, 200rpm condition.Measure cell concentration and output of pyruvic acid after the fermentation ends, the result is as shown in table 1.
Described seed culture medium (g/L) is: glucose 30, peptone 10, KH
2PO
41, MgSO
47H
2O 0.5; Slant medium adds agar 20g/L, and pH 5.5.
Table 1 cell concentration and pyruvic acid concentration determination
Be respectively at pH under 5.5,4.5 the condition, the final cell concentration of bacterial strain CCTCC M202019 has improved 7.4% and 28.7% than control group (adding) respectively; Output of pyruvic acid has improved 12.4% and 48.4% than control group (not adding) respectively.
Pyruvic acid is produced in embodiment 3 fermentations
It is 0.5g/L that the interpolation aspartic acid makes final concentration, and other are with embodiment 2.
When interpolation aspartic acid concentration is 1g/L,
Pyruvic acid is produced in embodiment 4 fermentations
It is 1.0g/L that the interpolation aspartic acid makes final concentration, and other are with embodiment 2.
Though the present invention with preferred embodiment openly as above; but it is not in order to limiting the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (1)
1. a method that improves output of pyruvic acid is characterized in that with torulopsis glabrata (Torulopsis glabrata) serving as to produce bacterial strain; Described torulopsis glabrata is multiple vitamin defective type, and deposit number is CCTCCNO:M202019; Described torulopsis glabrata fermentation is regulated pH to 4.5 with substratum with pyruvic acid solution; The jar top fermentation is kept pH with 8mol/L NaOH or 2mol/L HCl; Described fermentation consists of with substratum 1L's: glucose 100g, NH
4SO
47g, MgSO
47H
2O 0.8g, KH
2PO
45g, CH
3COONa 6g, aspartic acid 1.5g, liquid microelement 10mL, VITAMIN liquid 10mL; Liquid microelement is: CaCl
22H
2O 2g, FeSO
47H
2O 2g, ZnCl
20.5g, MnCl
24H
2O 12g, CuSO
45H
2O 0.05g, 2mol/L HCl dissolving back is settled to 1L with deionized water; VITAMIN liquid is: nicotinic acid 80mg, and VitB1 0.15mg, pyridoxol 40mg, vitamin H 4mg, riboflavin 10mg, 2mol/LHCl dissolving back is settled to 1L with deionized water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010581532 CN102121035B (en) | 2010-12-10 | 2010-12-10 | Method for increasing yield of pyruvic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010581532 CN102121035B (en) | 2010-12-10 | 2010-12-10 | Method for increasing yield of pyruvic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102121035A CN102121035A (en) | 2011-07-13 |
| CN102121035B true CN102121035B (en) | 2013-08-07 |
Family
ID=44249703
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010581532 Expired - Fee Related CN102121035B (en) | 2010-12-10 | 2010-12-10 | Method for increasing yield of pyruvic acid |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710274A (en) * | 2013-12-24 | 2014-04-09 | 江南大学 | Genetically engineered bacterium for increasing yield of extracellular pyruvic acid and application thereof |
| CN106635852A (en) * | 2016-12-07 | 2017-05-10 | 江南大学 | Recombinant torulopsis glabrata capable of co-producing pyruvic acid and alpha-ketoglutaric acid |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368884B (en) * | 2015-12-10 | 2019-01-04 | 江南大学 | The recombinant bacterium and its application that a kind of Pyruvate production intensity improves |
| CN106834364A (en) * | 2017-01-09 | 2017-06-13 | 通辽市黄河龙生物工程有限公司 | Pyruvic acid industrial production zymotechnique |
-
2010
- 2010-12-10 CN CN 201010581532 patent/CN102121035B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 周景文等.葡萄糖酸钠对光滑球拟酵母CCTCC M202019能量代谢和丙酮酸积累的影响.《华中农业大学学报》.2010,第29卷(第4期),第527-532页. |
| 氨基酸强化Torulopsis glabrata发酵生产丙酮酸;许庆龙等;《过程工程学报》;20081231;第8卷(第6期);第1201页 * |
| 葡萄糖酸钠对光滑球拟酵母CCTCC M202019能量代谢和丙酮酸积累的影响;周景文等;《华中农业大学学报》;20100831;第29卷(第4期);第527－532页 * |
| 许庆龙等.氨基酸强化Torulopsis glabrata发酵生产丙酮酸.《过程工程学报》.2008,第8卷(第6期),第1200-1203页. |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710274A (en) * | 2013-12-24 | 2014-04-09 | 江南大学 | Genetically engineered bacterium for increasing yield of extracellular pyruvic acid and application thereof |
| CN103710274B (en) * | 2013-12-24 | 2016-03-23 | 江南大学 | The genetic engineering bacterium that the outer output of pyruvic acid of a kind of born of the same parents improves and application thereof |
| CN106635852A (en) * | 2016-12-07 | 2017-05-10 | 江南大学 | Recombinant torulopsis glabrata capable of co-producing pyruvic acid and alpha-ketoglutaric acid |
| CN106635852B (en) * | 2016-12-07 | 2019-11-26 | 江南大学 | A kind of recombination torulopsis glabrata of cogeneration of propanone acid and α-ketoglutaric acid |
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| Publication number | Publication date |
|---|---|
| CN102121035A (en) | 2011-07-13 |
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