CN102220400B - Method for synthesis of glutathione in vitro - Google Patents
Method for synthesis of glutathione in vitro Download PDFInfo
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- CN102220400B CN102220400B CN201110122890.XA CN201110122890A CN102220400B CN 102220400 B CN102220400 B CN 102220400B CN 201110122890 A CN201110122890 A CN 201110122890A CN 102220400 B CN102220400 B CN 102220400B
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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 synthesis of glutathione in vitro, be specifically related to a kind of method of enzyme process synthesizing glutathion.
Background technology
Gsh (Glutathione, GSH), as a kind of biological activity tripeptide compound, has multiple important physiological function, most important for maintaining redox environment suitable in organism.At present the industrial application value of gsh is, as biologically active additives, is applied to field of food, and, radioprotective anti-oxidant with it, the health care function of waiting for a long time is applied to medicines and health protection field, its business demand is had to 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 is prepared gsh technique and is fallen behind and yield poorly; Chemical synthesis cost is high, reactions steps is many, long reaction time, complicated operation, needs optical resolution and has the problems such as environmental pollution; Fermentation method prepare aftertreatment trouble of gsh and transformation efficiency low, also exist product from separated difficult problem in born of the same parents.Compare with front several method, enzyme process has that catalysis specificity is strong, reaction conditions is gentle, transformation efficiency advantages of higher.
Production by Enzymes gsh is to utilize the required key enzyme-gamma-glutamylcysteine synthetase of synthesizing glutathion and glutathione synthetase catalysis, add the substrates such as Pidolidone, Cys and glycine simultaneously, and add a small amount of Triphosaden (ATP) and carry out synthesizing glutathion.Building-up reactions is divided into two steps, and the first step catalyzes and synthesizes intermediate gamma-glutamyl cysteine by gamma-glutamylcysteine synthetase (γ-Glutamylcysteine Synthetase, γ-GCS); Second step is by glutathione synthetase (Glutathione Synthetase, GS) catalysis, between the carboxyl of halfcystine end of gamma-glutamyl cysteine and the amino of glycine, forms peptide bond, obtains thus gsh, as follows:
The enzyme process of reporting in document is at present mainly to using wet thallus or fixation of microbial cell reaction generates gsh as enzyme source catalytic substrate.
Patent No. CN100540650C provides a kind of " by the method for enzyme engineering technology biosynthesis of glutathione ", and this patent discloses a kind of method that immobilized cell enzyme engineering technology is produced for gsh; In the method, adopt the yeast saccharomyces cerevisiae mutagenesis bacterium (CGMCC No 1917) of the high-yield glutathione that contriver filters out, by sodium alginate to embed method immobilized cell, and using Pidolidone, Cys, glycine as substrate, be aided with glucose and magnesium ion is prepared gsh.Adopt the method, 500mL substrate solution (20mM), through immobilized cell catalysis, obtains 0.95g gsh powder, yield 31% after final separation and purification.The 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 relevant enzyme in born of the same parents to be fully utilized, and glutathione synthesis efficiency 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 enzyme process synthesizing glutathion.The method is cultivated recombination bacillus coli E.coli WSH-KE1 cell as enzyme source by structure, and in reaction system, directly add organic solvent or the tensio-active agent of lower concentration, reduce the permeability barrier of epicyte, under Triphosaden (ATP) exists, catalysis Pidolidone, Cys and glycine synthesizing glutathion, react 2 hours glutathione synthesis amounts and can reach 4.8g/L.Penetrating dose of cell add the utilization ratio that can significantly improve relevant enzyme in microbe, but can cause key enzyme with the loss of substrate, cause immobilized cell can be reused as this advantage of catalysts and weaken.In addition, need to add a large amount of ATP in reaction system, ATP price is more expensive, so the method is not high for industrial feasibility.
Fourth Radiance-of-fire etc. are at " GSH produce by GSH synthesis enzymes with Antarctic diatom Berkeleya rutilans GJ 01 " (Chinese aquatic science, 14 5 phase of volume 829-835 pages, 2007 are open) in a literary composition, delivered a kind of synthetase series that utilizes the thick zyme extract GSH-PX activity of Antarctic diatom GJ01 cell, and the Antarctic diatom GJ01 cell that adds acetone treatment in reaction substrate liquid to be to supply a small amount of ATP, under 30 ℃, pH7.5 reaction conditions, carry out glutathione synthesis reaction.Because of the enzyme limitation alive of South Pole diatom synthetase series self, under the 5L system 8mM concentration of substrate of employing this method, gsh output is only 0.702g/L, 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 1 phase of volume 24-28 pages, 2002 are open) in a literary composition, delivered a kind of by recombination bacillus coli and brewing yeast cell co-immobilization system, in packed bed, react, GSH resultant quantity reaches 1.24g/L.In this system, the glycolytic pathway of yeast saccharomyces cerevisiae can provide energy matter for glutathione synthesis reaction, and can utilize the synthetic a small amount of GSH of GSH synthetic enzyme of self.The defect of this system is still 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 (for ATP regeneration) to produce gsh, under 20mM concentration of substrate condition, gsh accumulation volume can reach 4.5g/L, to the transformation efficiency of halfcystine, can reach 70%.To be that in its acetyl phosphate/acetate kinase ATP regeneration system using, the stable not and price of acetylphosphate character is more expensive be unfavorable for that industry amplifies to its shortcoming.
In sum, microbial enzyme method is produced problem that gsh faces 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, therefore will realize the efficient synthetic of gsh, the effective supply of ATP is its essential condition.Ahn K etc. (Polyphosphate kinase from Escherichia coli Purification and demonstration of a phosphoenzyme intermediate[J] .Biol.Chem., 1990,265:11734-11739.) in intestinal bacteria, separation and purification has gone out a kind of polyphosphoric acid poly (P) that makes
n-1interact and generate poly (P) with ATP
nwith the polyphosphate kinase Polyphosphate Kinase (PPK) of ADP (adenosine diphosphate (ADP)), the catalyzed reaction of this enzyme possesses reversibility.Noguchi T etc. (Use of Escherichia coli polyphosphate kinase for oligosaccharide synthesis[J] Biosci.Biotech.Binchem, 1998,62:1594-1596.) with stable in properties, cheap poly (P)
nfor phosphodonor, utilize the PPK enzyme in intestinal bacteria to build ATP regenerating system, and this system is successfully applied to the synthetic of oligose.
Summary of the invention
The invention provides a kind of method of synthesis of glutathione in vitro, be included under polyphosphoric acid kinases (PPK) catalysis and produce ATP by ADP and polyphosphoric acid compound, and under the katalysis of gamma-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS), by L-glutamic acid, halfcystine and glycine, utilize energy that described ATP provides synthesizing glutathion in vitro.
The present invention's two main chemical reactions steps of the prior art that have been coupled, the ATP providing is continually gsh syntheticly provides energy assurance, " ADP generates ATP and " forms organic whole with " ATP is for producing gsh energy supply " two reactions, whole reactive system only consumes the phosphate in three seed amino acids and polyphosphoric acid, makes to produce gsh cost and declines to a great extent.
Gamma-glutamylcysteine synthetase described in the present invention and glutathione synthetase can be resolvase, can be also immobilized enzyme, and preferably the two is resolvase.The composition that preferably participates in synthesis of glutathione in vitro 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, conventional 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 L-glutamic acid and glycine price are lower than halfcystine, thereby in the present invention's test, to choose halfcystine be key ingredient, and the concentration of substrate glutamic acid and glycine is the concentration that is not less than substrate halfcystine.
In the present invention, preferred described ADP concentration is every liter of 0.2-100 mmole; More preferably ADP concentration is every liter of 2-10 mmole.Preferably described polyphosphoric acid kinases concentration is 20-1600mg/L; More preferably described polyphosphoric acid kinases concentration is 80-320mg/L.In the present invention, preferably the reaction times of described synthesizing glutathion is 3-35 hour; More preferably the described reaction times is 5-15 hour.When the reaction times, surpass 9-15 hour, synthetic glutathione concentrations no longer rises, but the reaction times reach 35 hours, glutathione concentrations drop-out value is also less.
The present invention introduces the polyphosphoric acid kinases (Polyphosphate kinase, PPK) for ATP regeneration in synthetic system, for glutathione synthesis reaction 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:
the polyphosphoric acid compound chemistry stable in properties of using, cheap is the promising phosphodonor thing of a kind of very tool.Take that this reacts as theoretical foundation and builds ATP regenerating system and for the Production by Enzymes of 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 in system, consume only has phosphate and an amino acid substrate in polyphosphoric acid compound, but reaches the object 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, preferably use free glutathione synthetases to react with amino acid substrate, synthesizing glutathion in vitro, has improved the utilization ratio of transformation efficiency and the enzyme of substrate.The present invention compared with prior art, can make enzyme-to-substrate contact completely, improves the utilization ratio of mass-transfer efficiency and enzyme; Reactive system is simple, controllability is strong; Can solve the problem that needs to drop in a large number ATP in production process, reduce production costs.
Accompanying drawing explanation
Fig. 1 is the concentration value of synthesizing glutathion in the embodiment of the present invention 1,2 and comparative example 1 to 3;
Fig. 2 is the concentration value of synthesizing glutathion in the embodiment of the present invention 4 to 9.
Embodiment
Below be only the preferred embodiment of the present invention, protection scope of the present invention is not limited to this, and any those skilled in the art is in technical scope disclosed by the invention, within can being easy to the change carried out or changing and be encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
One, the preparation of gamma-glutamylcysteine synthetase (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 to take the high intestinal bacteria of glutathione synthesis ability or S. cervisiae be Host Strains, by building gene engineering recombinant bacterium, express three kinds of enzymes for the preparation of glutathione synthesis reaction.The embodiment of the present invention is for 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 carry out after homology expression in E.coli BL21 (D3) by pET-28a carrier, ultrasonication is extracted crude enzyme liquid and is made.In building-up reactions, the addition of three kinds of enzymes adopts Xylene Brilliant Cyanine G survey protein method to demarcate.
Two, the detection method of glutathione concentrations:
Adopt tetraoxypyrimidine method to measure glutathion inside cell content, concrete operations are as follows: in control tube and mensuration pipe, adding respectively 3.5mL concentration is the phosphate buffered saline buffer that 0.24mM and pH are 72,0.5mL analytic sample, 0.5mL glycine; In control tube and mensuration pipe, respectively add 0.5mL deionization H
2the tetraoxypyrimidine solution that O and 0.5mL concentration are 1g/L, mixes, timing, and the control tube of take after reaction 20min is surveyed the light absorption value of each sample under 305nm as reference; By typical curve reading or calculate GSH concentration.
Three, enzyme process synthesis of glutathione in vitro:
Under the effect of gamma-glutamylcysteine synthetase and glutathione synthetase, coupling ADP and polyphosphoric acid compound are in the process of the kinase catalytic lower generation ATP of polyphosphoric acid, at Tris-Cl buffer salt system and cofactors Mg
2+under existence, L-glutamic acid, halfcystine and glycine be synthesizing glutathion in vitro.
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
2; Add gamma-glutamylcysteine synthetase 92.6mg/L and glutathione synthetase 94.8mg/L, polyphosphate kinase add-on is 93.2mg/L, 37 ℃ of reactions are respectively got 0.5mL sample and by tetraoxypyrimidine method, are carried out the content analysis of gsh when 3h, 5h, 7h and 9h, and measurement result is shown in Fig. 1.
All the other conditions of synthesizing glutathion are with embodiment 1; Only polyphosphate kinase add-on is 186.4mg/L.Measurement result is shown in Fig. 1.
Comparative example 1
This routine negative contrast, all the other conditions of synthesizing glutathion are with embodiment 1; Only polyphosphate kinase add-on is 0mg/L.Measurement result is shown in Fig. 1.
Comparative example 2
This routine positive contrast, all the other conditions of synthesizing glutathion are with embodiment 1; Only polyphosphate kinase add-on is 0mg/L, and take the ATPNa that concentration is 15mM
2aDPNa in alternate embodiment 1
2(NaPO
3) n.Measurement result is shown in Fig. 1.
Comparative example 3
This example is another positive control, and all the other conditions of synthesizing glutathion are with embodiment 1; Only polyphosphate kinase add-on is 0mg/L, and take the ATPNa that concentration is 2mM
2aDPNa in alternate embodiment 1
2(NaPO
3) n.Measurement result is shown in Fig. 1.
As can be seen from Fig. 1, under 5mM concentration of substrate, the positive control transformation efficiency when reaction proceeds to 7h left and right of ATP that directly adds 15mM is the highest, reaches 83.04%, and now GSH concentration is 1.30g/L.And reaction system speed of reaction before 3h of adding polyphosphate kinase (PPK enzyme) is slower, between 3-7 hour, GSH semi-invariant increases substantially, and reaches reaction end about 7 hours; Wherein embodiment 2 interpolation PPK enzyme amounts are 186.4mg/L, ADPNa
2in synthetic system for 10mM, the concentration of gsh reaches 119g/L, and transformation efficiency reaches 77.36%, and its synthetic effect is considerably close to directly adding the glutathione synthesis 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
2; Add gamma-glutamylcysteine synthetase 924mg/L and glutathione synthetase 939mg/L, polyphosphate kinase enzyme liquid add-on is 763mg/L, 37 ℃ of reactions; When 5h, 7h and 9h, gsh output is respectively 0.51g/L, 2.21g/L and 2.76g/L, and transformation efficiency is respectively 16.6%, 72.0% and 89.9%.
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
2; Add gamma-glutamylcysteine synthetase 68.2mg/L and glutathione synthetase 91.6mg/L, polyphosphate kinase add-on is 280mg/L, 37 ℃ of reactions are respectively got 0.5mL sample and by tetraoxypyrimidine method, are carried out the content analysis of gsh when 3h, 5h, 7h and 9h.Measurement result is shown in Fig. 2.
Embodiment 5
All the other conditions of synthesizing glutathion are with embodiment 4; Only the initial addition of ADP is 8mM.Measurement result is shown in Fig. 2.
All the other conditions of synthesizing glutathion are with embodiment 4; Only the initial addition of ADP is 6mM.Measurement result is shown in 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 shown in Fig. 2.
All the other conditions of synthesizing glutathion are with embodiment 4; Only the initial addition of ADP is 2mM.Measurement result is shown in Fig. 2; Wherein, when 3h, 5h, 7h and 9h, gsh output is respectively 0.45g/L, 1.22g/L, 1.42g/L and 1.47g/L, 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 shown in Fig. 2.
As can be seen from Fig. 2, even when ADP starting point concentration is low to moderate 2mM, present method still can realize the efficient synthetic of gsh.
Claims (7)
1. the method for a synthesis of glutathione in vitro, be included in polyphosphoric acid and by ADP and polyphosphoric acid compound, produce ATP under kinase catalytic, and under the katalysis of gamma-glutamylcysteine synthetase and glutathione synthetase, by L-glutamic acid, halfcystine and glycine, utilize energy that described ATP provides synthesizing glutathion in vitro; The concentration of wherein said halfcystine is every liter of 1-60 mmole, and described ADP concentration is every liter of 0.2-100 mmole, and described polyphosphoric acid kinases concentration is 20-1600mg/L.
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 5-10 mmole.
4. method according to claim 1, is characterized in that, described ADP concentration is every liter of 2-10 mmole.
5. method according to claim 1, is characterized in that, described polyphosphoric acid kinases concentration is 80-320mg/L.
6. according to the method described in any one in claim 1-5, it is characterized in that, the reaction times of described synthesizing glutathion is 3-35 hour.
7. method according to claim 6, is characterized in that, the described reaction times is 5-15 hour.
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| CN102978267B (en) * | 2012-06-15 | 2014-07-16 | 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 |
| KR101745346B1 (en) * | 2015-01-16 | 2017-06-09 | 서강대학교산학협력단 | A method for continuous production of glutathione by photosynthetic membrane vesicle |
| 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 |
| CN106086126B (en) * | 2016-08-29 | 2019-12-13 | 开平牵牛生化制药有限公司 | Method for synthesizing glutathione by enzyme catalysis |
| EP3620527A4 (en) * | 2017-05-01 | 2020-05-20 | Kaneka Corporation | Production method for substance using atp |
| CN109134594B (en) * | 2017-06-15 | 2022-06-17 | 安徽古特生物科技有限公司 | Method for preparing glutathione by enzyme method |
| CN111979206B (en) * | 2019-05-24 | 2021-08-17 | 深圳瑞德林生物技术有限公司 | Immobilized fusion enzyme and method for preparing glutathione by using same |
| CN115747089A (en) * | 2022-07-06 | 2023-03-07 | 河南省巴饲福微生物技术研究院 | Recombinant saccharomyces cerevisiae for producing glutathione and construction method thereof |
| 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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| Noguchi et al..Use of Escherichia coli Polyphosphate Kinase for Oligosaccharide Synthesis.《Biosci.Biotechnol.Biochem》.1998,第62卷(第8期),1594-1596. * |
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