CN102220400A - Method for synthesis of glutathione in vitro - Google Patents
Method for synthesis of glutathione in vitro Download PDFInfo
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- CN102220400A CN102220400A CN201110122890XA CN201110122890A CN102220400A CN 102220400 A CN102220400 A CN 102220400A CN 201110122890X A CN201110122890X A CN 201110122890XA CN 201110122890 A CN201110122890 A CN 201110122890A CN 102220400 A CN102220400 A CN 102220400A
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Abstract
The invention relates to a method for synthesis of glutathione in vitro, comprising the step of: generating ATP (adenosine triphosphate) from ADP (adenosine diphosphate) and polyphosphoric acid compound under the catalysis of polyphosphate kinase; under the catalysis of gamma-glutamylcysteine synthetase and glutathione synthetase, synthesizing glutathione in vitro by glutamic acid, cysteine and glycine with the energy provided by the ATP. Compared with the prior art, the method of the invention can bring enzymes into complete contact with a substrate so as to improve the mass transfer efficiency and the utilization rate of enzymes. And the reaction system of the method is simple and strong in controllability. In addition, the method can solve the problem of great investment of ATP during production and reduce the production cost.
Description
Technical field
The present invention relates to a kind of method of external synthesizing glutathion, be specifically related to a kind of method of enzyme process synthesizing glutathion.
Background technology
(Glutathione GSH) as a kind of biological activity tripeptide compound, has multiple important physical function to gsh, and redox environment suitable in the organism is most important for keeping.At present the industrial application value of gsh is, is applied to field of food as biologically active additives, and, radioprotective anti-oxidant with it, the health care function of waiting for a long time is applied to the medicines and health protection field, its business demand is had ever-increasing trend both at home and abroad.
At present, the production method of gsh mainly contains extraction process, chemical synthesis, fermentation method and enzyme process.Extraction process prepares gsh technology backwardness and yields poorly; Chemical synthesis cost height, reactions steps are many, long reaction time, complicated operation, and light requirement is learned and split and have a problem such as environmental pollution; Fermentation method prepare then aftertreatment trouble of gsh and transformation efficiency low, also exist product to separate difficult problem in the born of the same parents.Compare with preceding several method, enzyme process has that the catalysis specificity is strong, reaction conditions is gentle, the transformation efficiency advantages of higher.
The Production by Enzymes gsh is to utilize required key enzyme-gamma-glutamylcysteine synthetase of synthesizing glutathion and glutathione synthetase catalysis, add substrates such as L-L-glutamic acid, L-halfcystine and glycine simultaneously, and add a small amount of Triphosaden (ATP) and come synthesizing glutathion.Building-up reactions was divided into for two steps, and the first step is by gamma-glutamylcysteine synthetase (γ-Glutamylcysteine Synthetase, the catalysis synthetic intermediate gamma-glutamyl cysteine of γ-GCS); Second step by glutathione synthetase (Glutathione Synthetase, GS) catalysis form peptide bond between the amino of the carboxyl of the halfcystine end of gamma-glutamyl cysteine and glycine, obtain gsh thus, and is as follows:
The enzyme process of reporting in the document mainly is to react as enzyme source catalytic substrate with wet thallus or fixation of microbial cell to generate gsh at present.
That patent No. CN100540650C provides is a kind of " with the method for enzyme engineering technology biosynthesis of glutathione ", this patent disclosure a kind of immobilized cell enzyme engineering technology be used for the method that gsh is produced; The yeast saccharomyces cerevisiae mutagenesis bacterium (CGMCC No 1917) of the high-yield glutathione that the employing contriver filters out in this method, by sodium alginate to embed method immobilized cell, and with L-L-glutamic acid, L-halfcystine, glycine as substrate, be aided with glucose and magnesium ion prepares gsh.Adopt this method, 500mL substrate solution (20mM) obtains 0.95g gsh powder, yield 31% through immobilized cell catalysis after the final separation and purification.This method process stabilizing, product aftertreatment are simple; Shortcoming is that the mass transfer obstacle between enzyme-to-substrate that after birth and cell walls cause causes that relevant enzyme can't be fully utilized in the born of the same parents, and the gsh combined coefficient is lower.
Patent application CN200510122930.5 provides " a kind of method that promotes microbial enzyme process to synthesizing glutathione ", wherein discloses a kind of technology of utilizing microorganism cells to carry out the enzyme process synthesizing glutathion.This method is cultivated recombination bacillus coli E.coli WSH-KE1 cell as the enzyme source by making up, and in reaction system, directly add the organic solvent or the tensio-active agent of lower concentration, reduce the permeability barrier of epicyte, catalysis L-L-glutamic acid, L-halfcystine and glycine synthesizing glutathion in the presence of Triphosaden (ATP) react 2 hours gsh resultant quantities and can reach 4.8g/L.The adding that cell is penetrating dose can significantly improve the utilization ratio of relevant enzyme in the microbe, but can cause the loss of key enzyme with substrate, causes immobilized cell can repeat to weaken as this advantage of catalysts.In addition, need to add a large amount of ATP in the reaction system, the ATP price is expensive, thus this method to be used for industrial feasibility not high.
Fourth Radiance-of-fire etc. are at " South Pole diatom GJ01 glutathione synthetase method is produced gsh " (Chinese aquatic science, 14 volumes, 5 phase 829-835 pages or leaves, 2007 are open) in the literary composition, delivered a kind of synthetase series that utilizes gsh in the thick zyme extract of South Pole diatom GJ01 cell, and the South Pole diatom GJ01 cell that adds acetone treatment in reaction substrate liquid to be supplying a small amount of ATP, carries out the gsh building-up reactions under 30 ℃, pH7.5 reaction conditions.Because of the enzyme of South Pole diatom synthetase series self limitation alive, gsh output only is 0.702g/L under the 5L system 8mM concentration of substrate of employing this method, and transformation efficiency only has 22.87%.
Shen Li is new to be waited at " immobilization E.coli BL21 (pTrc-gsh) cell catalysis synthesizing glutathion " (East China University of Science's journal, 28 volumes, 1 phase 24-28 page or leaf, 2002 are open) in the literary composition, delivered a kind of with recombination bacillus coli and brewing yeast cell co-immobilization system, react in packed bed, the GSH resultant quantity reaches 1.24g/L.The glycolytic pathway of yeast saccharomyces cerevisiae can provide energy matter for the gsh building-up reactions in this system, and can utilize the synthetic a small amount of GSH of GSH synthetic enzyme of self.The defective of this system still is the mass transfer obstacle between the enzyme-to-substrate that causes of cell multiplex film (and cell walls).
Kousaku Murata etc. (Overproduction of glutathione and its derivatives by genetically engineered microbial cells[J] .Biotechnology Advances, 1990,8 (1): 59-96) adopt the method for immobilization recombination bacillus coli and RC912 cell (being used for ATP regeneration) to produce gsh, the gsh accumulation volume can reach 4.5g/L under 20mM concentration of substrate condition, can reach 70% to the transformation efficiency of halfcystine.Its shortcoming be in its employed acetyl phosphate/acetate kinase ATP regeneration system the stable inadequately and price of acetylphosphate character costliness be unfavorable for that industry amplifies.
In sum, microbial enzyme method is produced problem that gsh faced and is mainly contained that mass transfer that cell wall or microbial film cause hinders and the ATP energy supply system of continuous and effective.In the building-up reactions of gsh, every production 1 molecule gsh needs 2 molecule ATP to participate in, so will realize the efficient synthetic of gsh, the effective supply of ATP is its essential condition.(Polyphosphate kinase from Escherichia coli Purification and demonstration of a phosphoenzyme interme such as Ahn K
Summary of the invention
The invention provides a kind of method of external synthesizing glutathion, be included under polyphosphoric acid kinases (PPK) catalysis and produce ATP by ADP and polyphosphoric acid compound, and at gamma-glutamylcysteine synthetase (γ-GCS) and under the katalysis of glutathione synthetase (GS) utilizes energy that described ATP provides at external synthesizing glutathion by L-glutamic acid, halfcystine and glycine.
The present invention's two main chemical reactions steps of the prior art that have been coupled, the ATP that provides continually provides the energy assurance for gsh synthetic, " ADP generates ATP and " forms organic whole with " ATP is for producing the gsh energy supply " two reactions, the entire reaction system only consumes the phosphate in three seed amino acids and the polyphosphoric acid, makes that producing the gsh cost declines to a great extent.
Gamma-glutamylcysteine synthetase described in the present invention and glutathione synthetase can be resolvase, can be immobilized enzyme also, and preferably the two is resolvase.The preferred composition that participates in external synthesizing glutathion also comprises Tris-Cl buffer salt system and cofactors Mg
2+
Polyphosphoric acid compound general formula described in the present invention is (MPO
3) n, comprise two polymetaphosphate and four polymetaphosphate, commonly used is its sodium salt or sylvite.
The concentration of halfcystine described in the present invention is preferably every liter of 1-60 mmole; More preferably its concentration is every liter of 5-10 mmole.Because of L-glutamic acid and glycine price are lower than halfcystine, thereby to choose halfcystine in the present invention's test be key ingredient, and the concentration of substrate glutamic acid and glycine is the concentration that is not less than the substrate halfcystine.
Preferred described ADP concentration is every liter of 0.2-100 mmole among the present invention; More preferably ADP concentration is every liter of 2-10 mmole.Preferred described polyphosphoric acid kinases concentration is 20-1600mg/L; More preferably described polyphosphoric acid kinases concentration is 80-320mg/L.The reaction times of preferred described synthesizing glutathion is 3-35 hour among the present invention; The more preferably described reaction times is 5-15 hour.Surpass 9-15 hour when the reaction times, the synthetic glutathione concentrations no longer rises, but the reaction times reach 35 hours, the glutathione concentrations drop-out value is also less.
The present invention in synthetic system, introduce be used for ATP regenerated polyphosphoric acid kinases (Polyphosphate kinase, PPK), for the gsh building-up reactions provides lasting effectively energy source and power.Polyphosphoric acid kinases (PPK) can carry out the building-up reactions of ATP under the existence condition of polyphosphoric acid compound and ADP:
Employed polyphosphoric acid compound chemistry stable in properties, cheap is a kind of phosphodonor thing that has very much prospect.Reacting with this is the Production by Enzymes that theoretical foundation makes up ATP regenerating system and is used for gsh.At the initial interpolation polyphosphoric acid compound of reaction and a small amount of ADP, utilize the kinase whose effect of polyphosphoric acid that thereby ADP is converted into the building-up reactions that ATP starts gsh, along with reaction is carried out ATP and is constantly consumed and generate new ADP, so move in circles, what consume in the system only has phosphate and an amino acid substrate in the polyphosphoric acid compound, but reaches the purpose of gsh accumulation.In addition, the inventive method is eliminated the mass transfer that the existing cell wall of traditional microbial enzyme method and cytolemma cause and is hindered, preferred free glutathione synthetase system and the amino acid substrate used reacted, and at external synthesizing glutathion, improved the utilization ratio of the transformation efficiency and the enzyme of substrate.The present invention compared with prior art can make enzyme-to-substrate contact fully, improves the utilization ratio of mass-transfer efficiency and enzyme; Reactive system is simple, controllability is strong; Can solve the problem that needs a large amount of ATP of input in the production process, reduce production costs.
Description of drawings
Fig. 1 is the concentration value of synthesizing glutathion in the embodiment of the invention 1,2 and the Comparative Examples 1 to 3;
Fig. 2 is the concentration value of synthesizing glutathion in the embodiment of the invention 4 to 9.
Embodiment
Be preferred implementation of the present invention only below, protection scope of the present invention is not limited thereto, and any those skilled in the art can be easy to the change of carrying out or change be encompassed within protection scope of the present invention in technical scope disclosed by the invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
One, gamma-glutamylcysteine synthetase (preparation of γ-GCS), glutathione synthetase (GS) and polyphosphoric acid kinases (PPK):
The preparation method of three kinds of enzymes adopts technology well-known to those skilled in the art; As being the host bacterium,, express three kinds of enzymes that preparation is used for the gsh building-up reactions by making up gene engineering recombinant bacterium with gsh synthesis capability high intestinal bacteria or S. cervisiae.The embodiment of the invention is to be the bacterium that sets out with E.coli BL21 (D3), difference clones coding gamma-glutamylcysteine synthetase, glutathione synthetase and the kinase whose gshA of polyphosphoric acid, gshB and ppk gene, and in E.coli BL21 (D3), carry out after homology expresses by the pET-28a carrier, ultrasonication is extracted crude enzyme liquid and is made.The addition of three kinds of enzymes adopts Xylene Brilliant Cyanine G survey protein method to demarcate in the building-up reactions.
Two, the detection method of glutathione concentrations:
Adopt the tetraoxypyrimidine method to measure glutathion inside cell content, concrete operations are as follows: adding 3.5mL concentration respectively in control tube and mensuration pipe is that 0.24mM and pH are 72 phosphate buffered saline buffer, 0.5mL analytic sample, 0.5mL glycine; Respectively add 0.5mL deionization H in control tube and the mensuration pipe
2O and 0.5mL concentration are the tetraoxypyrimidine solution of 1g/L, mix, and timing is that reference is surveyed the light absorption value of each sample under 305nm with the control tube behind the reaction 20min; By the typical curve reading or calculate GSH concentration.
Three, the external synthesizing glutathion of enzyme process:
Under the effect of gamma-glutamylcysteine synthetase and glutathione synthetase, coupling ADP and polyphosphoric acid compound are in the kinase catalytic process that produces ATP down of polyphosphoric acid, at Tris-Cl buffer salt system and cofactors Mg
2+Exist down, L-glutamic acid, halfcystine and glycine are at external synthesizing glutathion.
Embodiment 1
Preparation 5mM reaction substrate liquid, wherein: the Tris-Cl of 0.1M, 5mM halfcystine, 7.5mM glycine, 7.5mM L-glutamic acid, the ADPNa of 10mM
2, (the NaPO of 100mM
3) n, the MgCl of 20mM
2Add gamma-glutamylcysteine synthetase 92.6mg/L and glutathione synthetase 94.8mg/L, the polyphosphate kinase add-on is 93.2mg/L, 37 ℃ of reactions are respectively got the 0.5mL sample and are carried out the content analysis of gsh with the tetraoxypyrimidine method when 3h, 5h, 7h and 9h, measurement result is seen Fig. 1.
Embodiment 2
All the other conditions of synthesizing glutathion are with embodiment 1; Only the polyphosphate kinase add-on is 186.4mg/L.Measurement result is seen Fig. 1.
Comparative Examples 1
This routine negative contrast, all the other conditions of synthesizing glutathion are with embodiment 1; Only the polyphosphate kinase add-on is 0mg/L.Measurement result is seen Fig. 1.
Comparative Examples 2
This routine positive contrast, all the other conditions of synthesizing glutathion are with embodiment 1; Only the polyphosphate kinase add-on is 0mg/L, and is the ATPNa of 15mM with concentration
2ADPNa in the alternate embodiment 1
2(NaPO
3) n.Measurement result is seen Fig. 1.
Comparative Examples 3
This example is another positive control, and all the other conditions of synthesizing glutathion are with embodiment 1; Only the polyphosphate kinase add-on is 0mg/L, and is the ATPNa of 2mM with concentration
2ADPNa in the alternate embodiment 1
2(NaPO
3) n.Measurement result is seen Fig. 1.
As can be seen from Fig. 1, under the 5mM concentration of substrate, the positive control transformation efficiency when reaction proceeds to the 7h left and right sides of ATP that directly adds 15mM is the highest, reaches 83.04%, and this moment, GSH concentration was 1.30g/L.And reaction system speed of reaction before 3h of adding polyphosphate kinase (PPK enzyme) is slower, and the GSH semi-invariant increases substantially between 3-7 hour, reaches reaction end about 7 hours; Wherein embodiment 2 interpolation PPK enzyme amounts are 186.4mg/L, ADPNa
2In the synthetic system for 10mM, the concentration of gsh reaches 119g/L, and transformation efficiency reaches 77.36%, and its synthetic effect considerably approaches directly to add the gsh synthetic system of ATP.
Preparation 10mM reaction substrate liquid, wherein: the Tris-Cl of 0.1M, 10mM halfcystine, 15mM glycine, 15mM L-glutamic acid, the ADPNa of 20mM
2, (the NaPO of 100mM
3) n, the MgCl of 20mM
2Add gamma-glutamylcysteine synthetase 924mg/L and glutathione synthetase 939mg/L, polyphosphate kinase enzyme liquid add-on is 763mg/L, 37 ℃ of reactions; Gsh output is respectively 0.51g/L, 2.21g/L and 2.76g/L when 5h, 7h and 9h, and transformation efficiency is respectively 16.6%, 72.0% and 89.9%.
Embodiment 4
Preparation 5mM reaction substrate liquid, wherein: the Tris-Cl of 0.1M, 5mM halfcystine, 7.5mM glycine, 7.5mM L-glutamic acid, the ADPNa of 10mM
2, (the NaPO of 100mM
3) n, the MgCl of 20mM
2Add gamma-glutamylcysteine synthetase 68.2mg/L and glutathione synthetase 91.6mg/L, the polyphosphate kinase add-on is 280mg/L, the 0.5mL sample carries out gsh with the tetraoxypyrimidine method content analysis is respectively got in 37 ℃ of reactions when 3h, 5h, 7h and 9h.Measurement result is seen Fig. 2.
All the other conditions of synthesizing glutathion are with embodiment 4; Only the initial addition of ADP is 8mM.Measurement result is seen Fig. 2.
Embodiment 6
All the other conditions of synthesizing glutathion are with embodiment 4; Only the initial addition of ADP is 6mM.Measurement result is seen Fig. 2.
Embodiment 7
All the other conditions of synthesizing glutathion are with embodiment 4; Only the initial addition of ADP is 4mM.Measurement result is seen Fig. 2.
All the other conditions of synthesizing glutathion are with embodiment 4; Only the initial addition of ADP is 2mM.Measurement result is seen Fig. 2; Wherein, gsh output is respectively 0.45g/L, 1.22g/L, 1.42g/L and 1.47g/L when 3h, 5h, 7h and 9h, and transformation efficiency is respectively 29.2%, 79.3%, 92.7% and 96.0%.
All the other conditions of synthesizing glutathion are with embodiment 4; Only the initial addition of ADP is 0.2mM.Measurement result is seen Fig. 2.
As can be seen from Fig. 2, even when the ADP starting point concentration is low to moderate 2mM, present method still can realize the efficient synthetic of gsh.
Claims (10)
1. the method for an external synthesizing glutathion, it is kinase catalytic down by ADP and polyphosphoric acid compound generation ATP to be included in polyphosphoric acid, and under the katalysis of gamma-glutamylcysteine synthetase and glutathione synthetase, utilize energy that described ATP provides at external synthesizing glutathion by L-glutamic acid, halfcystine and glycine.
2. method according to claim 1 is characterized in that described gamma-glutamylcysteine synthetase and glutathione synthetase are resolvase.
3. method according to claim 1 is characterized in that, the concentration of described halfcystine is every liter of 1-60 mmole.
4. method according to claim 3 is characterized in that, the concentration of described halfcystine is every liter of 5-10 mmole.
5. method according to claim 1 is characterized in that, described ADP concentration is every liter of 0.2-100 mmole.
6. method according to claim 5 is characterized in that, described ADP concentration is every liter of 2-10 mmole.
7. method according to claim 1 is characterized in that, described polyphosphoric acid kinases concentration is 20-1600mg/L.
8. method according to claim 7 is characterized in that, described polyphosphoric acid kinases concentration is 80-320mg/L.
9. according to any described method among the claim 1-8, it is characterized in that the reaction times of described synthesizing glutathion is 3-35 hour.
10. method according to claim 9 is characterized in that, the described reaction times is 5-15 hour.
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| CN102978267A (en) * | 2012-06-15 | 2013-03-20 | 北京天开易达生物科技有限公司 | Method for preparing glutathione through enzyme method |
| CN104611396A (en) * | 2013-11-04 | 2015-05-13 | 中国科学院上海生命科学研究院 | Glutathione production method |
| CN105624238A (en) * | 2016-01-12 | 2016-06-01 | 北京化工大学 | Method for regenerating ATP using rationally designed enzyme |
| WO2016114618A1 (en) * | 2015-01-16 | 2016-07-21 | 서강대학교산학협력단 | Method for continuously producing glutathione using photosynthetic cell membrane vesicle |
| CN105861598A (en) * | 2016-04-27 | 2016-08-17 | 深圳市古特新生生物科技有限公司 | Method for regenerating ATP (adenosine triphosphate) by enzyme process and application thereof |
| CN106086126A (en) * | 2016-08-29 | 2016-11-09 | 开平牵牛生化制药有限公司 | A kind of method of Enzyme catalyzed synthesis glutathion |
| WO2018228246A1 (en) * | 2017-06-15 | 2018-12-20 | 安徽古特生物科技有限公司 | Method for enzymatic preparation of glutathione |
| JPWO2018203482A1 (en) * | 2017-05-01 | 2020-03-12 | 株式会社カネカ | Method for producing substance using ATP |
| CN111979206A (en) * | 2019-05-24 | 2020-11-24 | 深圳瑞德林生物技术有限公司 | Immobilized fusion enzyme and method for preparing glutathione by using same |
| CN117292748A (en) * | 2023-09-25 | 2023-12-26 | 河南大学 | Enzyme activity optimization method for producing glutathione by enzyme method |
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| CN102978267A (en) * | 2012-06-15 | 2013-03-20 | 北京天开易达生物科技有限公司 | Method for preparing glutathione through enzyme method |
| CN104611396B (en) * | 2013-11-04 | 2018-09-04 | 中国科学院上海生命科学研究院 | A method of producing glutathione |
| CN104611396A (en) * | 2013-11-04 | 2015-05-13 | 中国科学院上海生命科学研究院 | Glutathione production method |
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| CN105624238A (en) * | 2016-01-12 | 2016-06-01 | 北京化工大学 | Method for regenerating ATP using rationally designed enzyme |
| CN105624238B (en) * | 2016-01-12 | 2021-05-04 | 北京化工大学 | Method for regenerating ATP by rationally designed enzyme |
| CN105861598A (en) * | 2016-04-27 | 2016-08-17 | 深圳市古特新生生物科技有限公司 | Method for regenerating ATP (adenosine triphosphate) by enzyme process and application thereof |
| CN106086126A (en) * | 2016-08-29 | 2016-11-09 | 开平牵牛生化制药有限公司 | A kind of method of Enzyme catalyzed synthesis glutathion |
| JPWO2018203482A1 (en) * | 2017-05-01 | 2020-03-12 | 株式会社カネカ | Method for producing substance using ATP |
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| CN117292748A (en) * | 2023-09-25 | 2023-12-26 | 河南大学 | Enzyme activity optimization method for producing glutathione by enzyme method |
| CN117292748B (en) * | 2023-09-25 | 2024-06-07 | 程静为 | Enzyme activity optimization method for producing glutathione by enzyme method |
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| C06 | Publication | ||
| PB01 | Publication | ||
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