CN105219822A - A kind of method of external Production by Enzymes gsh - Google Patents
A kind of method of external Production by Enzymes gsh Download PDFInfo
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Abstract
The present invention relates to a kind of method of synthesis of glutathione in vitro.By giving expression to glutathione synthesis bifunctional enzyme and polyphosphoric acid kinases and under its catalysis, realize regenerating ATP using four polyphosphoric acids, hexametaphosphate as phosphodonor, under glutathione synthesis bifunctional enzyme catalytic condition, generate gsh with L-glutamic acid, glycine, halfcystine for substrate.Compared with the method for existing production gsh, the product inhibition of the glutathione synthesis bifunctional enzyme applied in the present invention is low, and the glutathione content that reaction system produces is high; The sodium such as phosphodonor four polyphosphoric acid, hexa metaphosphoric acid that reaction process uses, sylvite low price and to be commonly easy to get, use it as polyphosphoric acid kinase whose substrate regeneration ATP and greatly reduce production cost; Amino acid ratio is low to moderate Gly:Gys:Gly=2:1:2, and in system, remaining amino acid amount is few, is easy to late-stage products and is separated.
Description
Technical field
The invention belongs to biological chemical field, particularly a kind of method of external Production by Enzymes gsh.
Background technology
Gsh is a kind of compound with various biological function, and by Cys, Pidolidone and glycine three seed amino acid are obtained by peptide bond condensation.It can not only remove interior free yl, also has protection liver, and improve the effect of immunity of organisms, therefore gsh is widely used in biological medicine and field of food, and the demand of domestic and international medical market to gsh also increases increasingly.
Before deadline, the method for industrial production gsh mainly contains fermentation method, Enzyme catalyzed synthesis method, chemical synthesis and biological extraction process four kinds.In the technological process of fermentation method product separation and aftertreatment cumbersome, chemical synthesis cost is high, complicated operation, reaction process are loaded down with trivial details, extraction process output is too low, and enzyme process compares the advantages such as other several methods have that reaction process controllability is strong, catalyzed reaction specificity is strong and post-processed is simple.
Production by Enzymes gsh is by adding a small amount of Triphosaden (ATP) in reaction system, at Cys, under the condition of Pidolidone and glycine three seed amino acid three seed amino acid substrate, the key enzyme catalysis of r-glutamyl cysteine synthetase and glutathione synthetase two kinds of synthesizing glutathions is utilized to carry out synthesizing glutathion.These two kinds of enzymes respectively catalysis generate the two-step reaction in gsh process.
Use now glutathione synthesis bifunctional enzyme to carry out catalysis in a lot of research and produce gsh, glutathione synthesis bifunctional enzyme is the enzyme having r-glutamyl cysteine synthetase and glutathione synthetase two kinds of catalytic site concurrently.And using polyphosphoric acid kinases under the condition of substrate polyphosphoric acid salt and ADP, catalysis ATP regenerates, and the reaction cost of generation gsh is reduced.But that has reported all exists obvious Product inhibiton effect to produce the glutathione synthesis bifunctional enzyme applied for the purpose of gsh, and then Product yields is caused to be difficult to improve.When being so both not easy to industrial production, the later stage of product is separated, can not well meets the need of market again.
This research method provides a kind of method that glutathione synthesis bifunctional enzyme by utilizing Pasteurellamultocida to originate produces gsh.The originate product inhibition of this glutathione synthesis bifunctional enzyme of Pasteurellamultocida is weak, K
mvalue is large, under being adapted at higher amino acid substrate concentration conditions, and the production gsh of high yield.
This research method provides a kind of polyphosphate kinase utilizing corynebacterium glutamicum to originate and utilizes hexametaphosphate for substrate, hexametaphosphate for the polyphosphate kinase utilization in catalytic regeneration ATP. corynebacterium glutamicum source is more common to be easy to get, with hexametaphosphate as phosphodonor, facilitate ATP regenerative process.
Compared with known technology, the present invention has the following advantages:
(1) the glutathione synthesis bifunctional enzyme Product inhibiton effect of the present invention's utilization is weak, is beneficial to the output improving gsh.
(2) use polyphosphate kinase with four common polyphosphoric acids, hexametaphosphate for phosphodonor for system provides ATP endlessly, reduce production cost.
(3) adopt inflated with nitrogen anaerobic oscillatory reaction mode, prevent the gsh oxidation generated, preserve the gsh in product, improve gsh output.
(4) substrate ratios in the present invention is low to moderate Gly:Gys:Gly=(1-3): 1:(1-3), in system, remaining amino acid reduces, and is easy to product separation.
Summary of the invention
The object of the present invention is to provide a kind of glutathione synthesis bifunctional enzyme and polyphosphate kinase of heterogenous expression, with the L-glutamic acid of high density, halfcystine and glycine for substrate, there is provided phosphate group to generate ATP under the catalytic condition of polyphosphate kinase with hexametaphosphate and energy is provided, the final gsh producing high yield.
First the present invention builds genetic engineering bacterium, and heterogenous expression is from the glutathione synthesis bifunctional enzyme gene in Pasteurellamultocida.Be cloned in prokaryotic expression carrier pET22b by gene GS and obtain glutathione synthesis bifunctional enzyme expression vector, be i.e. recombinant plasmid pET22b-gs.Simultaneously we also by from polyphosphate kinase (PPK) gene clone in corynebacterium glutamicum in prokaryotic expression carrier pET28a, obtain recombinant plasmid pET28a-ppk.Then will be proceeded to by expression vector in e. coli bl21 (DE3) respectively, IPTG abduction delivering, can obtain glutathione synthesis bifunctional enzyme and polyphosphate kinase in born of the same parents.
After expressing, by ultrasonication, the glutathione synthesis bifunctional enzyme in intestinal bacteria is discharged, obtain crude enzyme liquid.Under the condition of phosphodonor is provided at hexametaphosphate, generate gsh with three kinds of substrate amino acid for substrate carries out reaction under crude enzyme liquid catalysis.Make substrate amino acid or gsh that oxidation occur in order to the oxygen in reaction system when preventing from reacting and then reduce the output of gsh, experiment adopts inflated with nitrogen anaerobic oscillatory reaction mode, preserves the gsh in product, improves gsh output.In order to improve the output of gsh further and be easy to late-stage products separation, this experiment brings up to 200mM concentration of substrate under the ratio condition of Gly:Gys:Gly=2:1:2.
Method provided by the invention, the polyphosphate kinase catalytic regeneration ATP that the corynebacterium glutamicum being substrate can utilize common hexametaphosphate is originated, the glutathione synthesis bifunctional enzyme that the product inhibition utilizing Pasteurellamultocida to originate is lower, produces gsh under the condition that substrate amino acid concentration is higher.Produce gsh at low cost for high yield and provide a kind of industrialized route.
Accompanying drawing explanation
Fig. 1: DNA agarose gel electrophoresis.Left side is the PCR primer of glutathione synthesis bifunctional enzyme gene gs, and right side is DNA size standards.
Fig. 2: the expression vector pET22b-gs built, by gs gene clone on pET22b carrier, there are Nde I and EcoR I restriction enzyme site in the two ends of gene, and it is numbered plasmid 1.
Fig. 3: DNA agarose gel electrophoresis.Left side is the PCR primer of polyphosphoric acid kinase gene ppk, and right side is DNA size standards.
Fig. 4: pET28a-ppk plasmid map, by ppk gene clone on pET28a carrier, there are Nco I and EcoR I restriction enzyme site in the two ends of gene, and it is numbered plasmid 2.
Fig. 5: abduction delivering glutathione synthesis bifunctional enzyme GS protein electrophoresis figure.Swimming lane 1 is born of the same parents' supernatant broken after the expression of pET22b empty plasmid, and swimming lane 2 is broken born of the same parents' supernatant after GS expression of enzymes.
Fig. 6: abduction delivering polyphosphoric acid kinases PPK protein electrophoresis figure.Swimming lane 1 is born of the same parents' supernatant broken after the expression of pET28a empty plasmid, and swimming lane 2 is broken born of the same parents' supernatant after PPK expression of enzymes.
Fig. 7: 20mM reaction system aerobic standing and reacting and inflated with nitrogen anaerobic reaction process contrast.
When adding 10mM, 20mM, 30mM, 40mM, 50mMATP in Fig. 8: 50mM reaction system respectively, the transformation efficiency contrast of day part." 10hGS ", for only adding GS enzyme in 50mM reaction system, the ATP that only outer source is added provides energy, and after reaction 10h, sampling detects the transformation efficiency obtained." 22hGS ", for only adding GS enzyme in 50mM reaction system, the ATP that only outer source is added provides energy, and after reaction 10h, sampling detects the transformation efficiency obtained." 10hGS+PPK ", for adding GS enzyme and PPK enzyme in 50mM reaction system, the ATP that outer source ATP and PPK enzyme are lived again provides energy, and samples the transformation efficiency of detection after reaction 10h." 22hGS+PPK ", for adding GS enzyme and PPK enzyme in 50mM reaction system, the ATP that outer source ATP and PPK enzyme are lived again provides energy, and samples the transformation efficiency of detection after reaction 22h.
Fig. 9: four polyphosphoric acids of different concns participate in coupling reaction as phosphodonor, with the GSH transformation efficiency that tetraoxypyrimidine method records after 24h.
Embodiment
1, ppk polyphosphoric acid kinase gene in Pasteurella (Pasteurellamultocida) bacterial strain GS glutathione synthesis bifunctional enzyme gene and corynebacterium glutamicum is cloned.
2, in Pasteurella (Pasteurellamultocida) bacterial strain GS glutathione synthesis bifunctional enzyme gene and corynebacterium glutamicum, ppk polyphosphate kinase gene recombined vector builds.
3, abduction delivering glutathione synthesis bifunctional enzyme gene GS and polyphosphoric acid kinases PPK.The recombinant expression vector pET22b-GS plasmid built is proceeded to competent escherichia coli cell E.coliBL21 (DE3), abduction delivering under IPTG effect.
4, thalline is expressed in ultrasonication, extracts crude enzyme liquid and reacts.Enzyme addition in building-up reactions adopts Xylene Brilliant Cyanine G to survey protein method and demarcates.
5, enzymatic clarification gsh.At Tris-HCl buffer salt system and cofactor Mg
2+under existence, with L-glutamic acid, glycine and halfcystine for substrate, hexametaphosphate or four polyphosphoric acids are phosphodonor, by glutathione synthesis bifunctional enzyme and polyphosphoric acid kinases synthesizing glutathion.
6, the detection of gsh.Adopt the content of GSH in tetraoxypyrimidine method assaying reaction liquid, under the buffer condition of pH7.2, reaction product is combined with tetraoxypyrimidine, measures the absorbance of binding substances under 305nm after 20min.The content of GSH in sample is calculated by GSH typical curve.
Below in conjunction with embodiment, concrete grammar of the present invention is further illustrated:
Embodiment 1
The clone of 1Pasteurellamultocida bacterial strain glutathione synthesis bifunctional enzyme gene gs and construction of recombinant vector
The clone of 1.1Pasteurellamultocida bacterial strain GS gene
Glutathione synthesis bifunctional enzyme GS gene DNA in the method synthesis Pasteurellamultocida bacterial strain utilizing full genome to synthesize.With this gene DNA for template, according to GenBank primers, and add corresponding restriction enzyme site (Nde I and EcoR I) and protection base.
Pcr amplification glutathione synthesis bifunctional enzyme GS gene the primer is as follows:
Upstream primer: 5 '-GCATATGATGGCCAAGAAGGAC-3 '
Downstream primer: 5 '-GCTCGAGTTACTTGGCGAG-3 '
PCR system reaction system contains 0.2 μ L genomic dna, each 0.5 μ L of upstream and downstream primer, Extaq mixed solution 10 μ L, adds distilled water and mends to 20 μ L.Reaction conditions is denaturation 94 DEG C of 5min, sex change 94 DEG C of 30s, and anneal 55 DEG C of 30s, extends 72 DEG C of 1min, circulates 30 times, 72 DEG C of 10min, 4 DEG C of preservations.Get final product clonal expansion like this to object glutathione synthesis bifunctional enzyme GS gene, nucleic acid electrophoresis figure is shown in Fig. 1.
The structure of 1.2 transfer vector plasmids 1
The GS gene of clone and pET22b carrier (being purchased from Novagen company) are carried out double digestion with restriction endonuclease Nde I and EcoR I respectively, 37 DEG C of enzymes cut 3h, the ratio of carrier and exogenous genetic fragment 1:3 in molar ratio, T4DNA ligase enzyme 16 DEG C of connections of NEB are utilized to spend the night, product conversion is connected to intestinal bacteria Top10 competent cell with this, coating is with amicillin resistance LB agar plate, after 37 DEG C of incubated overnight, picking list bacterium colony, incubated overnight in LB liquid nutrient medium, and carry out bacterium colony PCR checking.Test kit extracts plasmid, carries out the checking of plasmid double digestion, can obtain positive recombinant vector after sequencing result is correct.Measure sequence as shown in sequence 1, the aminoacid sequence of this sequence encoding as shown in sequence 2.The collection of illustrative plates of constructed transfer vector plasmid 1 as shown in Figure 2.
Embodiment 2
The clone of ppk polyphosphate kinase gene and construction of recombinant vector in 1 corynebacterium glutamicum bacterial strain
Ppk polyphosphate kinase gene in the method synthesis coli strain utilizing full genome to synthesize.With this bacterial strain full genome for template, according to GenBank primers, and add corresponding restriction enzyme site (Nco I and EcoR I) and protection base.
Pcr amplification polyphosphoric acid kinase gene ppk the primer is as follows:
Upstream primer: 5 '-GCATATGATGACCGCCACCGATTC-3 '
Downstream primer: 5 '-GAAGCTTCCGGTTGGTAGCTGTGAC-3 '
PCR system reaction system contains 0.2 μ L genomic dna, each 0.5 μ L of upstream and downstream primer, Extaq mixed solution 10 μ L, adds distilled water and mends to 20 μ L.Reaction conditions is denaturation 94 DEG C of 5min, sex change 94 DEG C of 30s, and anneal 55 DEG C of 30s, extends 72 DEG C of 1min, circulates 30 times, 72 DEG C of 10min, 4 DEG C of preservations.Get final product clonal expansion like this to object polyphosphoric acid kinase gene ppk, nucleic acid electrophoresis figure is shown in Fig. 3.
The structure of 1.2 transfer vector plasmids 2
Restriction endonuclease (Nco I and EcoR I) is used to carry out double digestion respectively the PPK gene of clone and pET28a carrier (being purchased from Novagen company), 37 DEG C of enzymes cut 3h, the ratio of carrier and exogenous genetic fragment 1:3 in molar ratio, T4DNA ligase enzyme 16 DEG C of connections of NEB are utilized to spend the night, product conversion intestinal bacteria Top10 competent cell is connected with this, coat with on kalamycin resistance LB agar plate, after 37 DEG C of incubated overnight, picking list bacterium colony, incubated overnight in LB liquid nutrient medium, and carry out bacterium colony PCR checking.Test kit extracts plasmid, carries out plasmid enzyme restriction checking, can obtain positive recombinant vector after sequencing result is correct.Measure sequence as shown in sequence 3, the aminoacid sequence of this sequence encoding as shown in sequence 4.The collection of illustrative plates of constructed transfer vector plasmid 2 as shown in Figure 4.
Embodiment 3
Expression vector will be built and pET22b-gs plasmid turns in BL21 (DE3) intestinal bacteria; use LB substratum; after 37 DEG C of incubation growth are about 0.4-1.0 to OD600, induce with the IPTG of 0.1mM – 1mM, under cultivating at 30 DEG C, carry out intracellular expression.To contrast empty carrier pET22bization to turn in BL21 (DE3) intestinal bacteria, condition same as described above carries out abduction delivering, and the recombinant protein after expressing is carried out protein electrophoresis checking expression of results, and its protein electrophoresis as shown in Figure 5.
Embodiment 4
Expression vector will be built and pET28a-ppk plasmid turns in BL21 (DE3) intestinal bacteria, under the induction of IPTG, carry out intracellular expression.Carrier pET28aization turned in BL21 (DE3) intestinal bacteria, condition same as described above carries out abduction delivering, and the crude enzyme liquid after expressing is carried out protein electrophoresis checking expression of results, and its protein electrophoresis as shown in Figure 6.
Embodiment 5
Adopt the content of GSH in tetraoxypyrimidine method assaying reaction liquid, concrete operations are as follows: in 96 orifice plates, add 175ul concentration is 0.24mM, PH is the phosphate buffered saline buffer of 7.2,25ul concentration is the glycine solution of 7.5g/L, the tetraoxypyrimidine 25ul of 1g/L, sample 25ul after dilution, blank well adds 25ul deionized water.Vibration mixing the accurate absorbance of test sample product under 305nm after timing 20min.Calculated the content of GSH in sample by GSH typical curve, and calculate transformation efficiency.
Embodiment 6
20mM reaction system is prepared in 50ml screw socket bottle and 50ml centrifuge tube, called after experimental group 1 and experimental group 2, the L-glutamic acid of 80mM is included in two individual system, the halfcystine of 20mM, the glycine of 120mM, 2mlGS enzyme crude enzyme liquid (containing albumen 1.36mg), 1.5mlPPK enzyme crude enzyme liquid (containing albumen 0.49mg), the hexametaphosphate of 100mM, the MgCl of 20mM
2, 0.5MTris-HCl (pH8.0), 5.6mMATP, cumulative volume is 10ml.After experimental group 1 inflated with nitrogen rear enclosed, laying temperature is the shaking table concussion reaction of 30 degree, and experimental group 2 is still in 30 DEG C of thermostat container reactions.Two group reactions all sample at 1h, 2h, 3h, 6h, 9h, 23h place, diluted sample 20 times of absorbances detected afterwards at 305nm wavelength place, transformation efficiency contrast under two kinds of reaction conditionss as shown in Figure 7, the peak rate of conversion of anaerobic concussion reaction is 63.75%, and the peak rate of conversion of aerobic standing and reacting is 52.20%.
Embodiment 7
In screw socket bottle, prepare 50mM reaction system 5, system includes the L-glutamic acid of 200mM, the halfcystine of 50mM, the glycine of 300mM, 3mlGS enzyme crude enzyme liquid (containing albumen 2.76mg), 4ml deionized water, the hexametaphosphate of 200mM, the MgCl of 30mM
2.The difference of each system is, the ATP concentration added is respectively 10mM, 20mM, 30mM, 40mM, 50mM, and the corresponding Tris-HCl concentration adding pH8.0 is that 0.3M, 0.6M, 0.6M, 0.9M, 0.9M regulate PH to be 8.0.Reaction system cumulative volume is 10ml.After inflated with nitrogen rear enclosed, be positioned over the shaking table oscillatory reaction that temperature is 30 DEG C.Sample when reacting 10h, 22h, diluted sample 60 times detects the absorbance of GSH at 305nm wavelength place in sample afterwards.Transformation efficiency after conversion is shown in Fig. 8, and when ATP concentration is 20mM, GSH transformation efficiency is 23.35%.
Embodiment 8
50mM reaction system 5 is prepared in screw socket bottle, system includes the L-glutamic acid of 200mM, the halfcystine of 50mM, the glycine of 300mM, 3mlGS enzyme crude enzyme liquid (containing albumen 2.76mg), 4mlPPK enzyme crude enzyme liquid (containing albumen 2.24mg), the hexametaphosphate of 200mM, the MgCl of 30mM
2.The difference of each system is, the ATP concentration added is respectively 10mM, 20mM, 30mM, 40mM, 50mM, and the corresponding Tris-HCl concentration adding pH8.0 is that 0.3M, 0.6M, 0.6M, 0.9M, 0.9M regulate PH to be 8.0.Reaction system cumulative volume is 10ml.After inflated with nitrogen rear enclosed, be positioned over the shaking table oscillatory reaction that temperature is 30 DEG C.Sample when reacting 10h, 22h, diluted sample 60 times detects the absorbance of GSH at 305nm wavelength place in sample afterwards.As shown in Figure 8, when ATP concentration is 20mM, GSH transformation efficiency is 80.91% to transformation efficiency after conversion.
Embodiment 9
In screw socket bottle, prepare 4 reaction systems, in system, amino acid whose ratio is Gly:Gys:Gly=2:1:2, and wherein the amount of Gys is respectively 20mM, 50mM, 100mM, 200mM.4mlGS enzyme crude enzyme liquid (containing albumen 3.68mg) is added, the MgCl of 30mM in system
2.The difference of each system is, the ATP concentration added is respectively 30mM, 75mM, 150mM, 300mM, and the corresponding Tris-HCl concentration adding pH8.0 is that 0.3M, 0.6M, 0.9M, 1.2M regulate PH to be 8.0.Reaction system cumulative volume is 10ml.After inflated with nitrogen rear enclosed, be positioned over the shaking table oscillatory reaction that temperature is 30 DEG C.Sample when reacting 10h, diluted sample 60 times detects the absorbance of GSH at 305nm wavelength place in sample afterwards.Transformation efficiency after conversion is respectively 86.23%, and 87.98%, 75.41%, 63.30%.
Embodiment 10
3 reaction systems are prepared in screw socket bottle, system includes the L-glutamic acid of 100mM, the halfcystine of 50mM, the glycine of 100mM, 3mlGS enzyme crude enzyme liquid (containing albumen 2.76mg), 4mlPPK enzyme crude enzyme liquid (containing albumen 2.24mg), the hexametaphosphate of 200mM, the MgCl of 30mM
2, the ATP of 20mM.The PH that the difference of each system is to add respectively different concns be 9.0 Tris-HCl regulation system PH be 7.0,8.0,9.0, reaction system cumulative volume is 10ml.After inflated with nitrogen rear enclosed, be positioned over the shaking table oscillatory reaction that temperature is 30 DEG C.Sample when reacting 10h, diluted sample 60 times detects the absorbance of GSH at 305nm wavelength place in sample afterwards.Transformation efficiency after conversion is respectively 75%, and 81%, 79%.
Embodiment 11
In screw socket bottle, prepare 5 reaction systems, in system, amino acid whose ratio is Gly:Gys:Gly=2:1:2, and wherein the amount of Gys is respectively 50mM.3mlGS enzyme crude enzyme liquid (containing albumen 2.76mg) is added, 4mlPPK enzyme crude enzyme liquid (containing albumen 2.24mg), the MgCl of 30mM in system
2, the ATP of 20mM.The difference of each system is, the concentration of four polyphosphoric acids added is respectively 10mM, 30mM, 50mM, 100mM, 200mM.Each reaction system inflated with nitrogen is positioned over the shaking table oscillatory reaction that temperature is 30 DEG C after closing.Sample when reacting 24h, diluted sample 50 times detects the absorbance of GSH at 305nm wavelength place in sample afterwards.Calculate transformation efficiency to obtain as shown in Figure 9.Obtain by map analysis four polyphosphoric acids can participate in glutathione synthesis as the kinase whose substrate of polyphosphoric acid coupling reaction when concentration is 10 ~ 50mM, peak rate of conversion is 78.3%, and corresponding GSH output is 12.02g/L.
Claims (14)
1., by producing a method for gsh with the polyphosphoric acid kinases of the glutathione synthesis bifunctional enzyme in Pasteurellamultocida source and corynebacterium glutamicum source, it is characterized in that, comprising:
Step 1, clone's glutathione synthesis bifunctional enzyme gene, to expression vector, obtain recombinant plasmid, through IPTG abduction delivering.
Step 2, clone's polyphosphoric acid kinase gene, to expression vector, obtain recombinant plasmid, through IPTG abduction delivering.
Step 3, by the restructuring glutathione synthesis bifunctional enzyme that obtains after cytoclasis and polyphosphoric acid kinases, at Tris-HCl buffer salt system and cofactor Mg
2+under existence, with L-glutamic acid, glycine and halfcystine for substrate, sodium polyphosphate is phosphodonor, by glutathione synthesis bifunctional enzyme and the kinase catalytic generation gsh of polyphosphoric acid.
2. method according to claim 1, is characterized in that the nucleotide sequence as shown in sequence 1 of glutathione synthesis bifunctional enzyme GS.
3. method according to claim 1, is characterized in that the aminoacid sequence as shown in sequence 2 of glutathione synthesis bifunctional enzyme GS.
4. method according to claim 1, is characterized in that the nucleotide sequence as shown in sequence 3 of polyphosphoric acid kinases PPK.
5. method according to claim 1, is characterized in that the aminoacid sequence as shown in sequence 4 of polyphosphoric acid kinases PPK.
6. method according to claim 1, is characterized in that the glutathione synthesis bifunctional enzyme GS catalytic production gsh that reaction system is originated with Pasteurellamultocida.
7. method according to claim 1, it is characterized in that the polyphosphoric acid kinases PPK catalytic regeneration ATP that reaction system is originated with corynebacterium glutamicum, ATP regeneration times reaches more than 5 times.
8. method according to claim 1, is characterized in that reaction system by common hexametaphosphate or its sodium salt, sylvite as the kinase whose substrate of polyphosphoric acid.
9. method according to claim 1, is characterized in that reaction system by four common polyphosphoric acids or its sodium salt, sylvite as the kinase whose substrate of polyphosphoric acid.
10. method according to claim 1, is characterized in that reaction process adopts the oscillatory reaction of inflated with nitrogen anaerobic.
11. methods according to claim 1, is characterized in that reaction system concentration of substrate can reach 200mM.
12. methods according to claim 1, is characterized in that three seed amino acid ratios can be reduced to Gly:Gys:Gly=(1-3): 1:(1-3).
13. methods according to claim 1, is characterized in that range of reaction temperature is 30 ~ 37 degree.
14. methods according to claim 1, it is characterized in that the PH scope of reacting is relatively more extensive, is 7 ~ 9.
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