CN1259425C - Method for microbial fermentation synthesis of alpha- ketoglutaric acid - Google Patents
Method for microbial fermentation synthesis of alpha- ketoglutaric acid Download PDFInfo
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- CN1259425C CN1259425C CN 200310106298 CN200310106298A CN1259425C CN 1259425 C CN1259425 C CN 1259425C CN 200310106298 CN200310106298 CN 200310106298 CN 200310106298 A CN200310106298 A CN 200310106298A CN 1259425 C CN1259425 C CN 1259425C
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Abstract
The present invention relates to a method for synthesizing alpha-ketoglutarate by means of microorganism fermentation, which is realized by adding calcium carbonate to a culture medium and increasing biotin concentration so as to promote pyruvic acid to generate a large amount of alpha-ketoglutarate in a fermentation process. In the process of the fermentation of candida glabrata CCTCC M202019 for producing pyruvic acid, the generation of alpha-KG is inhibited, and the carbon mole ratio (C<PYR>/C<KG>) of the pyruvic acid and the alpha-KG is increased by delaying time for adding calcium carbonate; the accumulation of a large amount of the alpha-KG is promoted by increasing the concentration of the calcium carbonate in the culture medium, and the alpha-KG is preferably generated when the concentration of the calcium carbonate is 40 g/L. The concentration of the alpha-KG is continuously increased, but a C<PYR>/C<KG> value is continuously decreased by maintaining a constant concentration of the calcium carbonate and increasing the concentration of the biotin in the culture medium; when the concentration of the biotin is 60 mu g/L, the accumulation amount of the alpha-KG is 23.5 g/L. When ca<2+> exists, the activity of intracellular pyruvate carboxylase can be increased by 40%, and the activity of a pyruvic acid dehydrogenase system does not change obviously. The increase of the concentration of the ca<2+> and the biotin in the culture medium can obviously improve the activity of pyruvate carboxylase so that T. glabrate is transferred to synthesizing alpha-KG with a high concentration from producing pyruvic acid by means of fermentation.
Description
Technical field
The present invention relates to the method for the synthetic α-Tong Wuersuan of a kind of microbial fermentation.
Background technology
(α-ketoglutaric acid, α-KG), chemical name: 2-oxo-1,5 pentanedioic acid, structural formula is abbreviation α-Tong Wuersuan
Be the powdery substance of white or off-white color, soluble in water, a kind of organic intermediate in work side is widely used in the organic synthesis production; Be a kind of important biochemical reagents, can be used as matched reagent in addition and be used to measure liver function, be widely used in as nutrition-fortifying agent: the enhance immunity ability; Reduce postoperative patient and patients on long-term's body loss; At brain as aminobutyric acid and L-glutamic acid synthetic precursor; Relevant with the intravital energy product of body.As the composition of sports nutrition beverage, can promote the aerobic energy supply; Improve sportsmen's insulin level, promote the absorption of energy supply materials such as amino acid, blood sugar, improve muscle glycogen and protein synthesis, improve the endogenous testosterone levels, promote the motion back to recover.At present, the preparation method of α-Tong Wuersuan is a chemical synthesis.And the synthetic α-Tong Wuersuan of microbial fermentation is not seen bibliographical information as yet.
Summary of the invention
The method that the purpose of this invention is to provide the synthetic α-Tong Wuersuan of a kind of microbial fermentation.
Technical scheme of the present invention: utilize in multiple vitamin deficient strain torulopsis glabrata (Torulopsis glabrata) the CCTCC M202019 fermentation production of acetone acid process, the concentration of adding lime carbonate and increase vitamin H in the substratum can promote the generation of α-KG in the fermented liquid.
1 bacterial classification
Torulopsis glabrata (T.glabrata) CCTCC M202019, nicotinic acid, vitamin H, VitB1, four kinds of vitamin deficient strain of pyridoxine hydrochloride, and the active composing type of pyruvic carboxylase reduces, be this research department's seed selection bacterial strain, this bacterial classification has been applied for Chinese patent, application number is 02113142.2, publication number CN1392246A.
2 substratum and cultural method:
Substratum is formed (g/T): glucose 100, sodium acetate 6, ammonium chloride 7, KH
2PO
45, MgSO
47H
2O 0.8, KCl5, vitamin 20 μ g/L, vitamin H 40～60 μ g/L, nicotinic acid 4mg/L, pyridoxine hydrochloride 100 μ g/L, CaCO
320-40, liquid microelement 5mL, initial pH5.0, tap water preparation; Liquid microelement: CaCl
22H
2O 2g, FeSO
47H
2O 2g, ZnCl
20.5g, MnCl
24H
2O0.2g, CuSO
45H
2O 0.05g is settled to 1L after the 2mol/L HCl dissolving.
Cultural method is:
Shake flask fermentation, fermention medium is 50mL in the 500mL Erlenmeyer flask, and temperature is 30 ℃, and rotating speed 200r/min, fermentation time are 48h;
Ferment tank, 7L fermentation cylinder for fermentation culture volume is 4L, and temperature is 30 ℃, air flow 3L/ (Lmin), and mixing speed is 400r/min, and fermentation time is 60h.
3 analytical procedures
Determination of glucose: 3,5-dinitrosalicylic acid method.
The mensuration of pyruvic acid and α-Tong Wuersuan: adopt high pressure liquid chromatography (HPLC) to measure.Chromatographic condition is: StableBondC
18Reversed-phase column, column temperature: 28 ℃, detector: UV210nm, moving phase: 0.1%H
3PO
4, flow velocity: 1.0mL/min, sampling volume: 10uL.
The mensuration of pyruvate carboxylase and pyruvate dehydrogenase activity:
Cell sterilized water centrifuge washing 3 times of 24h will under 30 ℃, have been cultivated, be suspended in pH then and be in the potassium phosphate buffer of 7.5 0.1mol/L, with granulated glass sphere vibration 5min under 4 ℃, cell is mixed in suspension, in 4 ℃ of ultrasonic disruption 2min, working strength is 30%, and work 1s is 0.5s at interval; At 4 ℃, 10, centrifugal 3min removes cell debris under the 000r/min subsequently, gets supernatant liquor and measures.
The active mensuration of pyruvate carboxylase (PC): contain in the reaction mixture: the phosphoric acid salt of pH 7.8 (PBS) damping fluid 0.56mL, distilled water 1.7mL, 0.5mol/L NaHCO
3Solution 0.4mL, 0.1mol/LMgCl
2Acetyl-CoA solution 0.4mL, the 0.1mol/L5 of solution 0.2mL, 1.0mmol/L, Triphosaden (ATP) the solution 0.2mL of citrate synthase 0.02mL, the 0.1mol/L of ethanolic soln 0.1mL, the 1000 μ/mL of 5-dithio phenylbenzene M-nitro benzoic acid, cell extract 0.2mL is incubated 10min with above-mentioned mixed solution down in 30 ℃.The pyruvic acid that adds 0.2mL 0.1mol/L starts reaction, reacts the KOH termination reaction of adding the 1mol/L of 0.3mL behind the 60s, detects the growing amount of oxaloacetic acid then under 415nm with spectrophotometer.Calculate the activity of pyruvate carboxylase according to typical curve.1 enzyme unit representation alive is micromole's number that this enzyme catalysis product generates in the 1min.
The active mensuration of pyruvate dehydrogenase system (PDH): contain in the reaction mixture: pH 7.8 potassium phosphate buffer 1.0mL, 2.5mmol/L Reduced nicotinamide-adenine dinucleotide (NAD) 0.2mL, 0.2mmol/L diphosphothiamine 0.2mL, 0.1mmol/L acetyl-CoA 0.4mL, 0.3mmol/L dithiothreitol (DTT) 0.1mL, 1mmol/L magnesium chloride 0.2mL, 1mg/mL bovine serum albumin 0.1mL, 6mmol/L 2-iodophenyl-3-oil of mirbane tetrazolium chloride (INT) 0.4mL, 0.1mg/L lipoamide dehydrogenase 0.1mL, cell extract 0.5mL.Above-mentioned mixed solution is added 0.2mL 5mmol/L pyruvic acid start reaction behind 30 ℃ of insulation 10min, add the H of the 1mol/L of 0.5mL behind the reaction 60s
2SO
4The solution termination reaction is then in 30 ℃ of reductions that detect INT down with spectrophotometer under 500nm.1 enzyme unit representation alive is micromole's number that this substrate for enzymatic activity reduces in 1min.
Dry cell weight (DCW) calculates according to the 1OD=0.23g dry mycelium with spectrophotometric determination OD value back; The mensuration of protein content is with improved Folin-phenol method.
The influence that 4 correlation technique conditions generate α-Tong Wuersuan
1. shake the contrast experiment of bottle and fermentor tank production pyruvic acid
In fermentor tank, substratum is sterilized, behind the inoculation mixing, aseptic being transferred in the sterilized 500mL triangular flask of the vaccinated substratum of 30mL carried out shake-flask culture, form the difference of identical situation bottom fermentation jar and shake-flask culture to investigate substratum.As shown in table 1, when not regulating pH in shaking bottle and fermentor tank, the cell growth is faint, and pyruvic acid accumulates also seldom.In shaking bottle, use CaCO
3During as buffer reagent, the cell normal growth, pyruvic acid accumulates in a large number, but has also produced the α-KG of higher concentration (6.8g/L) simultaneously.In fermentor cultivation, if regulate pH with NaOH, α in the fermented liquid-KG concentration is very low.If but use CaCO instead
3Regulate pH, the concentration of α-KG has then increased by 8 times when regulating pH with NaOH in the fermented liquid.In fermentor tank and shake-flask culture, use CaCO
3C when regulating pH
PYR/ C
KGBe worth closely, show CaCO
3Interpolation the accumulation of α-KG in the fermented liquid is had material impact.
Table 1 shakes the contrast of bottle and fermentor tank top fermentation
| DCW /(g/L) | Consumption sugar/(g/L) | Pyruvic acid/(g/L) | α-KG /(g/L) | C PYR/C KG? | |
| Shake flask fermentation (No CaCO 3) | 2.8 | 5.6 | 2.3 | 0 | ∞ |
| Shake flask fermentation (CaCO 3) | 10.9 | 84.8 | 37.8 | 6.8 | 6.96 |
| Ferment tank (pH does not control) | 4.9 | 14.3 | 5.1 | 0 | ∞ |
| Ferment tank (NaOH controls pH) | 14.8 | 90.7 | 69.4 | 1.3 | 67.2 |
| Ferment tank (CaCO 3Control pH) | 16.7 | 89.7 | 62.3 | 11.5 | 6.84 |
Illustrate: C
PYR/ C
KGRatio for the carbon mole number of the carbon mole number of pyruvic acid and α-KG.
2. CaCO
3The influence that interpolation time and concentration produce α-KG
For CaCO is described
3Interpolation whether be the principal element that causes α-KG to produce, the present invention has studied CaCO
3The influence that interpolation time and concentration produce α-KG.Under the constant prerequisite of vitamine concentration, postpone CaCO
3The interpolation time can obviously be suppressed the generation of α-KG and be improved C
PYR/ C
KGValue (Fig. 1), and the CaCO in the increase substratum
3Concentration can cause the accumulation of α-KG to increase (table 2).These results show CaCO
3(as having activated PDH or PC) in some way, making pyruvic acid enter TCA round-robin flux increases.
CaCO under the different liquid amounts of table 2
3Mass concentration is to the influence of pyruvate fermentation
| Culture volume | 30mL | 50mL | |||
| CaCO 3/(g/L) | 0 | 20 | 40 | 20 | 40 |
| Final pH | 2.1 | 3.9 | 5.1 | 3.9 | 5.0 |
| DCW/(g/L) | 3.8 | 12.1 | 17.3 | 13.4 | 15.3 |
| Pyruvic acid/(g/L) | 1.2 | 27.2 | 37.9 | 25.9 | 31.2 |
| α-KG/(g/L) | 0 | 3.6 | 15.8 | 3.2 | 9.9 |
| Residual sugar/(g/L) | 95.7 | 31.3 | 9.7 | 19.8 | 8.5 |
| C PYR/C KG | ∞ | 9.78 | 3.1 | 10.48 | 4.08 |
3. Ca
2+, CO
3 2-Influence to α-KG
CaCO in the fermented liquid
3Existence promoted the accumulation of α-KG, but specifically be Ca
2+Or CO
3 2-Have or both promoted the accumulation of α-KG jointly? in order to explain this problem, author designed 5 kinds of methods control the pH:(1 of 7L fermentor tank (liquid amount 4L) fermenting process automatically) stream adds the NaOH of 5mol/L; (2) stream adds the Na of 2.5mol/L
2CO
3(3) stream adds CaCO
3Suspension liquid (150g CaCO
3/ 100mL water); (4) adding concentration at fermentation beginning back stream is 40% CaCl
2(flow acceleration 25mL/h, stream adds 8h to concentrated solution altogether, makes CaCl in the fermented liquid
2Final concentration reaches 20g/L), stream adds the NaOH of 5mol/L simultaneously; (5) adding concentration at fermentation beginning back stream is 40% CaCl
2Concentrated solution (feeding method with (4)) is used the Na of 2.5mol/L simultaneously
2CO
3Regulate pH.Fermenting process curve under 5 kinds of situations as shown in Figure 2.
From Fig. 2 (A) as can be known, when the pH of fermenting process being regulated with NaOH, C
PYR/ C
KGValue can remain between 50～60; Use NaCO
3When regulating pH, the C in the fermenting process
PYR/ C
KGValue drops to 20～30.When using CaCO
3When regulating pH, perhaps in fermented liquid, a large amount of CaCl are arranged
2Exist, again with NaOH or NaCO
3When regulating pH, the C in the fermenting process
PYR/ C
KGValue all is lower than 10.Can think Ca in conjunction with Fig. 2 (B)
2+And CO
3 2-All promoted the accumulation of α-KG, wherein Ca
2+α-KG has been accumulated main promoter action.
In addition, find in Fig. 2 (C), compare that a large amount of Ca are arranged in substratum with the fermenting process of regulating pH with NaOH
2+When existing, the cell growth is very fast, and final cell concentration is also higher.This may be because Ca
2+As intracellular second messenger, can quicken the growth of cell; In addition, Ca is arranged
2+α when existing-KG concentration is higher, for synthetic more many cells material provides possibility, causes the final cell amount to increase.
It is also influential when research is also found with different substances adjusting pH to the accumulating rate and the output of pyruvic acid.Shown in Fig. 2 (D), as Ca
2+When existing, because the cell growth is very fast, the pyruvic acid accumulating rate is also very fast, but the output of pyruvic acid descends to some extent.Simultaneously because the generation of α-KG, pyruvic acid is not to glucose yield descend to some extent (data provide).
4. VitB1 and biotin concentration are to the influence of α-KG generation
In bacterial strain T.glabrata CCTCC M202019, PDH and PC are that control pyruvic acid degraded enters two important enzymes of TCA round-robin (being) (Fig. 3), and what of TCA cyclic carbon stream its active height determined to enter.Because this bacterial strain self can not synthesize VitB1 (B
1) and vitamin H (Bio), so PDH and PC activity in this bacterial strain born of the same parents can be respectively by the B in the substratum
1Control with Bio concentration.In order to investigate specifically is the generation which enzyme (being) influences α-KG, and the present invention is at CaCO
3Adding concentration is under the prerequisite of 40g/L, has studied different B
1With Bio concentration to α-KG effect of accumulation.Found that, change B in the substratum
1Concentration, to the accumulation of α-KG, particularly to C
PYR/ C
KGValue is influence (Fig. 4 A) not.And the concentration of Bio in the increase substratum then causes the concentration of α-KG constantly to rise and C
PYR/ C
KGValue constantly descends (Fig. 4 B), and the concentration of vitamin H is advisable with 40～60 μ g/L in the substratum.Because Bio is the cofactor of PC, can think that in view of the above the generation of α-KG is caused by the PC activity change.
5. adopt different adjustment pH material to PC and the active influence of PDH
It is constant to keep in the substratum VITAMIN mass concentration, proceeds to cell log during vegetative period (20h) in fermentation, measures PC and the active variation of PDH in the cell when adopting different substances to regulate in the fermented liquid pH.Found that (Fig. 5), as Ca
2+When existing, the activity of PC is the highest in the born of the same parents improves 40%, and the activity of PDH does not have considerable change.Show Ca
2+Can improve the activity of PC, increase thereby make pyruvic acid enter TCA round-robin flux by pyruvic acid carboxylation approach.Because α-KG desaturase is also with B
1Be cofactor, at B
1Under the condition of restriction, α-KG dehydrogenase activity is also very low, causes thus that α-KG accumulates in a large number in the fermented liquid.The present invention finds that first zymic PC activity might be by Ca
2+Activate, thereby make T.glabrata turn to the α-KG of synthetic high density from fermentation production of acetone acid.
In sum, add CaCO in the substratum
3Mass concentration be 20～40g/L, biotin concentration is 40～60 μ g/L, the growing amount of α-Tong Wuersuan is 15～40g/L.
Beneficial effect of the present invention: find CaCO shaking in bottle and the fermentor tank experiment of multiple vitamin deficient strain torulopsis glabrata CCTCCM202019 fermentation production of acetone acid
3Interpolation (accumulation of α-KG) has material impact to α-Tong Wuersuan in the fermented liquid.Under the constant and sufficient prerequisite of oxygen supply, postpone CaCO at vitamine concentration
3The interpolation time can obviously be suppressed the generation of α-KG, and improves the carbon mol ratio (C of pyruvic acid and α-KG
PYR/ C
KG); And the CaCO in the increase substratum
3Concentration can cause the increase of α-KG accumulation.Regulate the experiment confirm of pH in the fermented liquid with different substances: in the pyruvate fermentation process, Ca
2+Accumulation to α-KG plays a major role, CO
3 2-Help out, both have synergistic effect to the accumulation of α-KG.Keep CaCO in the substratum
3Concentration is constant, changes the concentration of VitB1 in the substratum, to the accumulation of α-KG, particularly to C
PYR/ C
KGValue is influence not; And the concentration of vitamin H in the increase substratum then causes the concentration of α-KG constantly to rise and C
PYR/ C
KGValue constantly descends.As Ca
2+When existing, the activity of pyruvate carboxylase can improve 40% in the born of the same parents, and the activity of pyruvate dehydrogenase system does not have considerable change, Ca in the substratum
2+Can significantly improve the activity of pyruvate carboxylase with the increase of biotin concentration, thereby make T.glabrata turn to the α-KG of synthetic high density from fermentation production of acetone acid.
Description of drawings
Fig. 1 CaCO
3(40g/L) the interpolation time is to the influence of α-KG generation
The influence that when Fig. 2 different substances is regulated pH α-KG is produced
The metabolism of pyruvic acid among Fig. 3 Torulopsis glabrata
Fig. 4 VitB1, biotin concentration are to α-KG effect of accumulation
The material of Fig. 5 different adjustment pH is to PC and the active influence of PDH
The biological material specimens preservation
Torulopsis glabrata, preservation date on April 29th, 2002, depositary institution's title and abbreviation: Chinese typical culture collection center C CTCC, deposit number NO:M 202019.
Embodiment
Embodiment 1
In the 7L fermentor tank, substratum is sterilized, according to 10% (volume ratio) inoculating microbe seeds, behind the inoculation mixing, be transferred in the sterilized 500mL triangular flask the vaccinated substratum of 50mL is aseptic, add 40g/L lime carbonate simultaneously and carry out shake-flask culture, 30 ℃, 200rpm are cultivated 48h, and recording α-Tong Wuersuan concentration is 15.8g/L, C
PYR/ C
KGBe 3.1.
Embodiment 2
Inoculation, culture condition are with embodiment 1, and other adds the vitamin H of 20～40 μ g/L, find that α-Tong Wuersuan concentration increases C along with the increase of biotin concentration
PYR/ C
KGReduce gradually.When the vitamin H total concn reached 60 μ g/L in substratum, recording α-Tong Wuersuan concentration was 23.5g/L, C
PYR/ C
KGBe 1.47.
In the 7L fermentor tank, sterilize behind the dress 4L fermention medium, insert torulopsis glabrata, be added with 40g/L lime carbonate in the substratum, contain 60 μ g/L vitamin Hs simultaneously according to 10% (volume ratio).30 ℃, air flow 3L/ (Lmin), recording α-Tong Wuersuan concentration behind the rotating speed 400rpm cultivation 60h is 38.4g/L, C
PYR/ C
KGBe 0.72.
Claims (1)
1. the method for the synthetic α-Tong Wuersuan of a microbial fermentation, it is characterized in that utilizing in multiple vitamin deficient strain torulopsis glabrata (Torulopsis glabrata) the CCTCC M202019 fermentation production of acetone acid process, the concentration of adding lime carbonate and increase vitamin H in the substratum can promote the generation of α-KG in the fermented liquid;
The substratum of fermenting process is formed (g/L): glucose 100, sodium acetate 6, ammonium chloride 7, KH
2PO
45, MgSO
47H
2O 0.8, KCl5, vitamin 20 μ g/L, vitamin H 40～60 μ g/L, nicotinic acid 4mg/L, pyridoxine hydrochloride 100 μ g/L, CaCO
340, liquid microelement 5mL, initial pH5.0, tap water preparation;
Liquid microelement: CaCl
22H
2O 2g, FeSO
47H
2O 2g, ZnCl
20.5g, MnCl
24H
2O 0.2g, CuSO
45H
2O 0.05g is settled to 1L after the 2mol/L HCl dissolving;
The cultural method of fermenting process is: shake flask fermentation, and fermention medium is 50mL in the 500mL Erlenmeyer flask, and temperature is 30 ℃, and rotating speed 200r/min, fermentation time are 48h;
Ferment tank, 7L fermentation cylinder for fermentation culture volume is 4L, and temperature is 30 ℃, air flow 3L/ (Lmin), and mixing speed is 400r/min, and fermentation time is 60h;
Add CaCO in the fermenting process substratum
3Mass concentration be 20～40g/L, biotin concentration is 40～60 μ g/L, the growing amount of α-Tong Wuersuan is 15～40g/L.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101157941B (en) * | 2007-09-19 | 2010-06-09 | 江南大学 | Method for controlling dextrose and vitamine concentration for improving fermentation and acetonic acid yield |
| CN101250563B (en) * | 2008-03-20 | 2010-11-03 | 江南大学 | Method for realizing excessive accumulation of alpha-ketoglutarate acid by adding alpha-ketoglutarate acid dehydrogenase inhibitor |
| CN102269737B (en) * | 2010-06-07 | 2013-01-30 | 北京嘉事联博医药科技有限公司 | HPLC (high performance liquid chromatography) detection method of arginine ketoglutarate |
| GB2485862B (en) * | 2010-11-29 | 2013-09-04 | Guangdong Huanxi Biolog Technology Co Ltd | Preparation process of L-arginine-alpha-ketoglutarate by direct crystallization from fermentation broth |
| CN102586128B (en) * | 2012-03-14 | 2014-04-09 | 江南大学 | High-yield alpha-ketoglutarate engineered saccharomyces cerevisiae and application thereof |
| CN103911400B (en) * | 2014-04-02 | 2016-04-27 | 江南大学 | A kind of method adopting resting cell to produce α-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 |
| TWI655184B (en) * | 2017-09-22 | 2019-04-01 | 中山醫學大學 | Method for producing 2-hydroxy-2 methyl-4 ketoglutarate |
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