CN105603028B - The method that enzyme process prepares glutathione and adenylate simultaneously - Google Patents
The method that enzyme process prepares glutathione and adenylate simultaneously Download PDFInfo
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Abstract
The invention discloses a kind of methods that enzyme process prepares glutathione and adenylate simultaneously, and the method comprising the steps of: (1) glutathione and adenylate are generated in reactor tank using GshF enzyme and Adk enzyme；(2) GshF the and Adk enzyme of immobilization is separated in reactor tank, or free GshF enzyme and Adk enzyme are separated using filter plant；And (3) separation product GSH and AMP.The reaction condition of Optimization of preparation of the invention GSH production makes GSH generate concentration and reaches 30-50g/L, and rate of ultilization of amino acid is also higher；Meanwhile partial regeneration is carried out to the ATP consumed in reaction using Adk enzyme, reduce the consumption of ATP；And method of the invention can prepare GSH and AMP simultaneously.
Description
Technical field
The present invention relates to the preparation method of glutathione, in particular to a kind of enzyme process prepares glutathione and adenylate simultaneously
Method.
Background technique
Glutathione is widely present in animals and plants and microorganism, is most important non-protein sulfhydryl compound in organism
One of, there is reduced glutathione (GSH) and oxidized form of glutathione (GSSG), largely exist in organism and play main make
It is GSH, is widely used in treatment liver diseases, tumour, oxygen poisoning, aging and endocrine system disease, and add as bioactivity
Add agent and antioxidant for field of food.
Tripeptides made of GSH is condensed as glutamic acid (Glu), cysteine (Cys) and glycine (Gly) through peptide bond.Relatively
Molecular mass is 307.32, isoelectric point 5.93, is white crystal, soluble easily in water, low-concentration ethanol aqueous solution, liquefied ammonia under room temperature
And dimethylformamide.
The main preparation methods of glutathione have at present: solvent extraction, chemical synthesis, biological fermentation process and enzyme process.
GSH is extracted from grain germ, since GSH yield is low, at high cost, organic solvent pollution is serious, purity is not high, and is consumed big
Measure grain, less use.Chemical synthesis synthesizes GSH and needs chemical resolution, product since activated product is not readily separated
Purity is not high, it is difficult to promote.GSH production both at home and abroad uses fermentation method substantially at present, and principle is the base that will encode GSH synthetase series
Because being cloned into Escherichia coli or yeast, GSH is produced using microbial fermentation.Yeast fermentation method, technique is more mature, but produces week
Phase is long, and yield is relatively low, and excessive by-product keeps downstream process processing complicated.
Production by Enzymes GSH technology steps up in recent years, and large-scale production is made to become possible.Classical Production by Enzymes GSH
Dependent on two kinds of enzymes of gamma glutamyl cysteine synthetase (Gsh I) and glutathione synthetase (Gsh II), Gsh I catalysis
Pidolidone and L-cysteine synthesize gamma-glutamyl cysteine, and Gsh II is catalyzed gamma-glutamyl cysteine and glycine
Synthesize GSH.In GSH synthesis process, feedback inhibition due to Gsh I catalytic process by final product GSH makes to generate
The first step of gamma-glutamyl cysteine is reacted to the rate-limiting step of entire GSH synthesis.
With further research, people are in Listeria monocytogenes (Listeria monocytogenes) etc. ten
A kind of difunctional glutathione synthetase (GshF enzyme) has been had been found that in several bacteriums.The enzyme has Gsh I and Gsh II simultaneously
Activity, can step catalysis GSH synthesis, and the enzyme feedback inhibition is smaller, accommodates very much applied to enzymatic clarification gluathione
Peptide.
The greatest problem of enzymatic clarification GSH is a large amount of consumption of atriphos (ATP), and production 1kg GSH at least needs to make
With the ATP of 3-5kg, cost is excessively high.In order to solve the problems, such as this respect, the method for yeast glycolytic regeneration ATP can be used therewith
It is coupled, regenerates ATP, effect stability using this method in patent CN201210201691.2.But it can be in reaction system using yeast
The impurity such as middle introducing pigment, to increase difficulty is further purified, regenerating ATP using enzyme system is research direction in recent years.In addition,
ATP is regenerated using polyphosphate enzyme system in patent CN201510762184.X, achieves good effect.The enzyme system includes more
Polyphosphate kinase (Ppk), adenosine acid kinase (Adk) and polyphosphoric acids-adenylate phosphotransferase (Pap).Wherein, Ppk with
Pap enzyme need to add polyphosphate in reaction process, equal to GSH reaction conversion ratio, attainable maximum concentration and later-period purification
Have an impact.Therefore, the production for how being optimized GSH using Adk enzyme is reduced the usage amount of ATP, and is easy to pure in guarantee product GSH
On the basis of changing and separating, other products having an economic benefit can be prepared simultaneously, such as adenylate (AMP), become current
Research emphasis.
Summary of the invention
The present invention provides a kind of methods that enzyme process prepares glutathione (GSH) and adenylate (AMP) simultaneously, to overcome
The drawbacks described above of the prior art.
The present invention is achieved through the following technical solutions:
A kind of method that enzyme process prepares glutathione and adenylate simultaneously, method includes the following steps:
(1) GSH and AMP is generated in reactor tank using GshF enzyme and Adk enzyme:
ATP, GshF enzyme and Adk enzyme are added in the reaction system, and reaction generates GSH and AMP, wherein the reaction system is
Containing substrate A TP, glutamic acid (Glu) or its salt, cysteine (Cys) or its salt, glycine (Gly) or its salt and magnesium from
One or two kinds of aqueous solutions of son and manganese ion.In addition, also may include in reaction system potassium ion, sodium ion, ammonium ion and
The one or more of Tris and phosphate anion.Substrate, enzyme and all kinds of salt that the present invention adds can be added at one time reaction system,
It can also flow in batches plus fill into according to industrial manufacture process process.
(2) GshF enzyme and Adk enzyme are separated:
The GshF enzyme and Adk enzyme of immobilization are directly separated in reactor tank.Above-mentioned separation can be separated by filter bag, can also be
It is directly separated in reaction column.
Free GshF enzyme and Adk enzyme passes through Ultra filtration membrane in filter.Wherein, filter has feed inlet, discharge port
And refluxing opening, inside set the ultrafiltration membrane that molecular cut off is less than 20kDa.Trapped fluid through filter is the enzyme solution of recycling, filter liquor
To isolate the reaction solution after enzyme；And
(3) separation product GSH and AMP:
By ion-exchange, the separation product GSH from the filter liquor of above-mentioned steps (2), being pierced by after ion exchange
Another product AMP is mainly contained in liquid.
Preferably, further comprising the steps of in above-mentioned technical proposal:
(4) the GshF enzyme that separates in step (2) and Adk enzyme recycle:
The GshF enzyme and Adk enzyme that will be separated are added in reactor tank the successive reaction for generating GSH and AMP and regeneration ATP.
(5) the continuous separation of GshF enzyme and Adk enzyme:
I.e. continuously the GshF enzyme and Adk enzyme of separation immobilization or continuously separated using filter plant free GshF enzyme and
Adk enzyme.
Preferably, further comprising the steps of in above-mentioned technical proposal:
(6) the continuous separation of product GSH.The GSH that reaction generates passes through filtering or ion-exchange process separation.
(7) the continuous separation of product AMP.The AMP that reaction generates passes through filtering or ion-exchange process separation.
Preferably, in above-mentioned technical proposal of the invention, step (1) to step (3) can be repeated at least once more.It is preferred that heavy
It is multiple repeatedly, such as it is repeatedly 2,3,4,5,6,7,8,9,10,15,20,25,30,40,50 inferior.
Preferably, in above-mentioned technical proposal, the reaction condition that step (1) generates GSH and AMP in reactor tank is as follows:
Reaction temperature is 25-55 DEG C, and preferable temperature is 30-50 DEG C；
Reaction pH is 5-10, and preferably pH is 6-9.
Preferably, in above-mentioned technical proposal, GshF enzyme, Adk enzyme are free or immobilization enzyme and/or regeneration enzyme, are pressed
It reacts optimal conditions to calculate, two kinds of enzyme addition mass ratios are preferably GshF:Adk=(0.2-30): 1, more preferably (0.5-
20):1.It is preferably 0.002-1g/L that GshF enzyme, which adds concentration,.Above-mentioned GshF enzyme, Adk enzyme can derive from any biology, or
It is by the artificial reconstructed enzyme with same catalysis.
Preferably, in above-mentioned technical proposal, amino acid or its salt are L-type amino acid or its salt.By its economic cost and
It reacts optimal conditions to calculate, three kinds of amino acid addition mass ratios are preferably Glu:Cys:Gly=(1-2.5): 1:(0.5-
1.5), more preferably Glu:Cys:Gly=(1.2-2): 1:(0.8-1.5).It is preferably 1-50g/L that cysteine, which adds concentration,.
Preferably, in above-mentioned technical proposal, the ATP concentration that the present invention adds is 1-150g/L；Magnesium ion concentration is 0.01-
0.1M；Manganese ion concentration is 0.01-0.1M；Potassium concentration is 0.01-0.3M；Na ion concentration is 0.01-0.3M；Ammonium ion
Concentration is 0.005-0.2M；Tris concentration is 1-12g/L, phosphate concn 1-15g/L.
Preferably, in above-mentioned technical proposal, magnesium ion is in magnesium chloride, magnesium sulfate, magnesium sulfite and magnesium nitrate
It is one or more；Manganese ion is selected from one of manganese chloride and manganese sulfate or a variety of；Potassium ion is selected from potassium chloride, sulfuric acid
Potassium, potassium nitrate, potassium hydroxide, potassium sulfite, potassium carbonate, saleratus, potassium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and lemon
One of lemon acid potassium is a variety of；Sodium ion is selected from sodium chloride, sodium sulphate, sodium nitrate, sodium hydroxide, sodium sulfite, carbonic acid
One of sodium, sodium bicarbonate, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium citrate are a variety of；Ammonium ion is selected from
One in ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium hydroxide, ammonium carbonate, ammonium hydrogen carbonate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate and ammonium acetate
Kind is a variety of.
Preferably, in above-mentioned technical proposal, immobilised enzymes is fixed on fixation support in the following manner in step (2):
Absorption, crosslinking, covalent bond, embedding or combinations thereof.Two kinds of enzymes of GshF and Adk are solid together after can fixing or mixing respectively in proportion
It is fixed.Fixation support is one or more selected from macromolecule carrier, inorganic carrier and magnetic macromolecular microsphere carrier.
Wherein, macromolecule carrier selected from cellulose, glucose gel, agarose, polyacrylamide, polyamino acid,
Polystyrene, polyacrylic acid, sodium alginate, chitosan, starch, polyvinyl alcohol, gelatin, carragheen, nylon and synthetic high polymer
One or more combinations；Inorganic carrier is selected from the one or more of cellular glass, silica, active carbon, silica gel and diatomite
Combination.When carrying out enzymatic reaction, immobilised enzymes can be dispersed in reaction solution and directly be reacted, passed through after reaction
Filter mode recycles enzyme；Or immobilised enzymes is packed into reaction column, reaction solution is pumped through cylinder by certain flow rate and is carried out
Enzymatic reaction.
Preferably, in above-mentioned technical proposal, the ultrafiltration membrane used in the method for the present invention step (2) is selected from cellulose acetate
Film, PS membrane, polyacrylonitrile film, polychloroethylene film, polyvinylidene fluoride film, PA membrane or ceramic membrane.
Compared with prior art, the technology of the present invention has the advantages that
1) reaction condition for optimizing GSH production, can greatly improve concentration of substrate, so that GSH is generated concentration and reach 30-
50g/L, wherein when GSH production quantity reaches 30-40g/L, rate of ultilization of amino acid can reach 75-90%.And this method is due to initial
Concentration of substrate is high, and reaction rate is fast, saves the reaction time；
2) partial regeneration can be carried out to the ATP consumed in reaction using Adk enzyme.When GshF enzyme production GSH is used alone,
Production 1kg GSH at least needs the ATP using 3-5kg, and after adding Adk enzyme, the GSH for producing 1kg needs the ATP of about 1.5-2.5kg,
Greatly reduce the consumption of ATP；In addition, regenerative response is not required to add other substrates such as polyphosphate etc, do not influence
The enzymatic reaction of the generation GSH of GshF enzymatic, guarantees the production quantity and reaction rate of GSH；
3) after Adk enzymatic, only minute quantity ADP is remaining in reaction system, and 95% or more ADP a part is converted into
ATP is continued to use, and reduces the consumption of ATP, and another part generates a large amount of AMP.AMP and GSH is easy to point in purification process
From purification process is simple, can be used as another product during preparing GSH while preparing.By the market price and at one's duty
Analysis, the method are economical and practical；
4) establish the stabilization recovery system of suitable GshF enzyme and Adk enzyme, no matter immobilization or the GshF enzyme that dissociates and
The combination of Adk enzyme can be applied to large-scale continuous production；
5) operation of this preparation method is simple and feasible, and amplification effect is good, can be amplified to tonne.
Detailed description of the invention
Fig. 1 is the SDS-PAGE figure of GshF enzyme expressed by the present invention.
Fig. 2 is the SDS-PAGE figure of Adk enzyme expressed by the present invention.
Fig. 3 is the process flow chart that the method for the present invention prepares GSH and AMP using resolvase.
Fig. 4 is the process flow chart that the method for the present invention prepares GSH and AMP using immobilized enzyme.
Fig. 5 is the HPLC map that the present invention is reacted using GshF enzyme and Adk enzyme.
Fig. 6 is substrate and product relational graph when the method for the present invention reaches different GSH production quantities.
Fig. 7 is the HPLC map that the present invention is used only that GshF enzyme is reacted.
Specific embodiment
Specific embodiments of the present invention are described in detail with reference to the accompanying drawing, in order to further understand the present invention.
The preparation of 1 GshF enzyme of embodiment
GshF enzyme in the method for the present invention can be commercially available, or by artificial reconstructed with same catalysis
Enzyme.
The preparation process of GshF enzyme is as follows:
According to gshF gene order (GenBank:NC_008532), pair for amplification primer is designed, by the calm and peaceful biological skill of Sino-U.S.
The synthesis of art Co., Ltd, primer sequence are as follows:
GshF sense primer: 5 '-CCATATGACATTAAACCAACTTCTTCAAAAACTG-3 '；With
GshF antisense primer: 5 '-CGAATTCTTAAGTTTGACCAGCCACTATTTC-3 '；
Streptococcus thermophilus (Streptococcus thermophilus) bacterial strain (CGMCC 1.6472) DNA is extracted, with it
For template, gshF genetic fragment is gone out by PCR amplification, and is respectively connected to pET 22b carrier and (is purchased from Invitrogen public affairs
Department), after being sequenced correctly, it is transferred to E.coli BL21 (DE3) bacterial strain (being purchased from Tiangeng biochemical technology Co., Ltd) respectively.
E.coli BL21 (DE3) monoclonal after conversion is accessed into LB culture medium, after culture to logarithmic phase, it is different that 1mM is added
After propyl-β-D- Thiogalactopyranoside (IPTG) induces 5 hours, thallus, sodium dodecyl sulphate-polyacrylamide are collected
The high expression bacterial strain of gel electrophoresis (SDS-PAGE) screening.
The high expression bacterial strain filtered out is aseptically accessed into seed culture medium, is accessed after culture to logarithmic growth phase
In the fermentor of the fermentation medium containing 5L, accessed in the fermentor of the fermentation medium containing 50L after cultivating to logarithmic growth phase, culture
It is added after 5 hours after 1mM IPTG induces 5 hours, thalline were collected by centrifugation about 1000g.
Wherein LB medium component are as follows: 1% peptone, 0.5% yeast powder and 1%NaCl；Seed culture based component are as follows:
1% peptone, 0.5% yeast powder and 1% sodium chloride；Fermentation medium components are as follows: 1% peptone, 0.5% yeast powder, 1% chlorine
Change sodium, 5% disodium hydrogen phosphate, 1% sodium dihydrogen phosphate, 0.01% magnesium sulfate and 1% glycerol.
Supernatant is collected by centrifugation after ultrasound or high-pressure homogenization break bacterium in the thallus of harvest.Pass through ammonium sulfate precipitation, acid-base precipitation
And the GshF enzyme of preliminary purification can be obtained in bivalent metal ion intermediate processing.
Fig. 1 is that the SDS-PAGE of prepared enzyme schemes, as shown in the figure: swimming lane 1 is that protein marker 14.4-116kDa (is purchased from
Rui Tai Bioisystech Co., Ltd, BeiJing ZhongKe)；Swimming lane 2 is the thallus of the enzyme containing GshF, GshF enzyme about 85kDa；Swimming lane 3, swimming lane 4
For the GshF enzyme after preliminary purification；Swimming lane 5 is the GshF enzyme after purifying concentration.
Using the method for the well known measurement GshF enzymatic activity that the prior art is recorded, 1mg/ml GshF enzymatic activity is detected
About 3000U, wherein complete transformation definition is 1 active unit (U) in 1 minute by 1 μM of substrate.
The preparation of 2 Adk enzyme of embodiment
Adk enzyme in the method for the present invention can be commercially available, or by artificial reconstructed with same catalysis
Enzyme.
The preparation process of Adk enzyme is as follows:
According to adk gene order (GenBank:NC_000913), pair for amplification primer is designed, by the calm and peaceful biological skill of Sino-U.S.
The synthesis of art Co., Ltd, primer sequence are as follows:
Adk sense primer: 5 '-CCATATGCGTATCATTCTGCTTGGCGCTCCGG-3 '；With
Adk antisense primer: 5 '-CGGATCCTTAGCCGAGGATTTTTTCCAGATC-3 '；
Escherichia coli (Escherichia coli) K12 bacterial strain (being purchased from Tiangeng biochemical technology Co., Ltd) DNA is extracted, with
It is template, goes out adk genetic fragment by PCR amplification, and is connected to pET 22b carrier (being purchased from Invitrogen company)
Sequence is transferred to E.coli BL21 (DE3) bacterial strain (being purchased from Tiangeng biochemical technology Co., Ltd) after being sequenced correctly.
E.coli BL21 (DE3) monoclonal after conversion is accessed into LB culture medium, after culture to logarithmic phase, it is different that 1mM is added
After propyl-β-D- Thiogalactopyranoside (IPTG) induces 5 hours, thallus, sodium dodecyl sulphate-polyacrylamide are collected
The high expression bacterial strain of gel electrophoresis (SDS-PAGE) screening.
The high expression bacterial strain filtered out is aseptically accessed into seed culture medium, is accessed after culture to logarithmic growth phase
In the fermentor of the fermentation medium containing 5L, accessed in the fermentor of the fermentation medium containing 50L after cultivating to logarithmic growth phase, culture
It is added after 5 hours after 1mM IPTG induces 5 hours, thalline were collected by centrifugation about 1000g.
Wherein LB medium component are as follows: 1% peptone, 0.5% yeast powder and 1%NaCl；Seed culture based component are as follows:
1% peptone, 0.5% yeast powder and 1% sodium chloride；Fermentation medium components are as follows: 1% peptone, 0.5% yeast powder, 1% chlorine
Change sodium, 5% disodium hydrogen phosphate, 1% sodium dihydrogen phosphate, 0.01% magnesium sulfate and 1% glycerol.
Supernatant is collected by centrifugation after ultrasound or high-pressure homogenization break bacterium in the thallus of harvest.Then plus 40-60% saturation sulfuric acid
Precipitating is collected by centrifugation in ammonium.After the dissolution of Tris pH of buffer 8.0, using G25 column, (being purchased from General Electric's medical treatment bioscience has
Limit company) desalination, then chromatograph and can be obtained through DEAE-Sepharose FF (being purchased from medical treatment Biology Science Co., Ltd, General Electric)
The Adk enzyme of preliminary purification.
Fig. 2 is that the SDS-PAGE of prepared enzyme schemes, as shown in the figure: swimming lane 1 is that protein marker 14.4-116kDa (is purchased from
Rui Tai Bioisystech Co., Ltd, BeiJing ZhongKe)；Swimming lane 3 is Adk enzyme, about 24kDa.
Using the method for the well known measurement enzymatic activity that the prior art is recorded, detect that 1mg/ml Adk enzymatic activity is about
1000U, wherein complete transformation definition is 1 active unit (U) in 1 minute by 1 μM of substrate.
Embodiment 3 prepares GSH and AMP using resolvase
Fig. 3 is the process flow chart that the method for the present invention prepares GSH using resolvase.It is produced according to the present invention referring to Fig. 3
The process flow chart of GSH prepares GSH and AMP using resolvase in accordance with the following steps:
(1) GSH and AMP is generated in reactor tank:
In reactor tank, the reaction system of 100L sterile water be containing substrate 2.8kg glutamic acid, 1.8kg cysteine and
2.0kg glycine and 5.1kgATP, 1.2kg disodium hydrogen phosphate, 0.7kg potassium chloride, 0.6kg sodium chloride, 0.1kg ammonium chloride
With the solution of 1.0kg magnesium chloride hexahydrate, uniform stirring prevents precipitating when preparation.PH value is adjusted to about 7.0, in reaction system
Middle addition 0.01kg GshF enzyme and 0.001kgAdk enzyme start to react.It is 7.0 that pH value is controlled during reaction, and temperature is 37 DEG C.
After reaction 6 hours, the production quantity of high performance liquid chromatography (HPLC) detection glutathione is about 30g/L, and 95% or more
ATP exhausts, and is converted into AMP, and AMP production quantity is about 30g/L.Please also refer to Fig. 5, for 6 hours 10 times of diluting reactions of reaction
The HPLC map of liquid, not marking peak in figure is amino acid.HPLC testing conditions are as follows: Kromasil C18 chromatographic column (is purchased from AKZO
NOBEL company) (150 × 4.6mm), Detection wavelength 210nm, 30 DEG C of temperature of detection.Mobile phase is to contain 6.8g/L biphosphate
The aqueous solution of potassium, 2.0g/L sodium heptanesulfonate and 3% methanol, pH=3.0.
(2) GshF enzyme and Adk enzyme are separated in the filter:
By hyperfiltration process, the reaction solution of the reaction system of step (1) is separated by filtration GshF enzyme and Adk by filter
Enzyme, the built-in film packet of filter (being purchased from Pall company, molecular cut off 8kDa), filter liquor is the reaction solution after isolating enzyme.
Using method is described in embodiment 1, the activity of detection 1mg/ml GshF enzyme is about 2600-2900U.
Using method is described in embodiment 2, the activity of detection 1mg/ml Adk enzyme respectively may be about 850-950U.
(3) separation product GSH and AMP:
The pH value for adjusting filter liquor using hydrochloric acid passes through D001 macropore strong acid styrene to 3.0 in ion exchange column
Cation exchanger resin, GSH, partial amino-acid and cation in solution are adsorbed, are pierced by liquid and mainly contain AMP, can
Direct condensing crystallizing after simple desalination, yield about 2.8kg.
Use the GSH on 0-0.8M NaCl gradient elution cation exchange resin, GSH yield about 2.6-2.8kg, yield
About 90%.
(4) reactor tank generates the successive reaction of GSH and AMP, the i.e. successive reaction of step (1):
The enzyme isolated in step (2) is added to reactor tank via the refluxing opening of filter, and adds protoenzyme amount 5-
15% new enzyme is reacted.Reaction liquid making method ibid states step (1).
Carry out generating the successive reaction of GSH and AMP under conditions of 37 DEG C, pH are 7.0；After 6 hours, HPLC detects GSH
Production quantity be about 30g/L, 95% or more ATP is converted into AMP, and AMP production quantity is about 30g/L.HPLC testing conditions are the same as above-mentioned
Step (1).In this step, enzyme is recycled.
The fixation of 4 enzyme of embodiment
GshF enzyme and Adk enzyme are fixed with commercialized epoxy group fixation support LX1000EP.
GshF enzyme described in above-described embodiment 1 and 2 and Adk enzyme are mixed according to mass ratio about 10:1 and are made into mixed enzyme solution.
It is 30:1 that the wet carrier of LX1000EP and above-mentioned enzyme solution are added in constant temperature stirred tank according to fixation support and enzyme mass ratio
Mixing, at 20 °C, 150rpm are stirred 12 hours.Carrier is collected by filtration, it is clear with 8.0 kaliumphosphate buffer of 0.02M pH
2 times are washed to get immobilization mixed enzyme.
After GshF enzyme and Adk enzyme immobilization, activity is reduced to original active about 30%.
Embodiment 5 prepares GSH and AMP using immobilized enzyme
Fig. 4 is the process flow chart that the method for the present invention prepares GSH using immobilized enzyme.Referring to fig. 4, produced according to the present invention
The process flow chart of GSH prepares GSH and AMP using immobilized enzyme in accordance with the following steps:
(1) glutathione GSH and AMP are generated in reaction column:
Reaction solution is prepared, every 100L contains substrate 2.8kg glutamic acid, 1.8kg cysteine and 2.0kg glycine, and
5.1kgATP, 1.2kg disodium hydrogen phosphate, 0.7kg potassium chloride, the six water chlorination of 0.6kg sodium chloride, 0.1kg ammonium chloride and 1.0kg
Magnesium, uniform stirring prevents precipitating when preparation, adjusts pH value to about 7.5, temperature is upgraded to 37-40 DEG C.
Mixing immobilized enzyme 50kg in above-described embodiment 4 is packed into reaction column equipment, enzyme reaction column is made after draining bubble.
Reaction solution is from bottom to top slowly pumped through enzyme reaction column with 16-20L/h flow velocity using constant flow pump, controls temperature during reaction
It is 37 DEG C.After reaction about 6 hours, reaction solution is collected, the production quantity of high performance liquid chromatography (HPLC) detection glutathione is about
30g/L, 95% or more ATP exhaust, and are converted into AMP, and AMP production quantity is about 30g/L.HPLC testing conditions are the same as 3 step of embodiment
(1)。
(2) separation product GSH and AMP:
It is strong to pass through D001 macropore to 3.0 in ion exchange column for the pH value for adjusting the reaction solution of above-mentioned collection using hydrochloric acid
Acid styrene type cation exchange resin, GSH, partial amino-acid and cation in solution are adsorbed, and are pierced by liquid main
Containing AMP, can direct condensing crystallizing after simple desalination, yield about 2.8kg.
Use the GSH on 0-0.8M NaCl gradient elution cation exchange resin, GSH yield about 2.6-2.8kg, yield
About 90%.
(3) reaction column generates the successive reaction of GSH and AMP, the i.e. successive reaction of step (1):
Same reaction liquid described in preparation steps (1), is continuously from bottom to top slowly pumped through enzyme with 16-20L/h flow velocity
Reaction column, controlled at 37 DEG C during reaction.
The production quantity that HPLC detects GSH is about 30g/L, and 95% or more ATP is converted into AMP, and AMP production quantity is about 30g/L.
HPLC testing conditions are the same as 3 step of embodiment (1).In this step, enzyme is recycled.
More than January, enzymatic activity reduces about 10% for immobilised enzymes circular response 20 times or more or -4 DEG C of storage times, needs
It adds in proportion or the new enzyme of removable parts.
Influence of 6 concentration of substrate of embodiment to GSH production quantity
In 100L reaction system, adding for disodium hydrogen phosphate, potassium chloride, sodium chloride, ammonium chloride and magnesium chloride hexahydrate is fixed
Dosage is respectively 1.2kg, 0.7kg, 0.6kg, 0.1kg and 1.0kg, the amount of fixed addition enzyme be 0.01kg GshF enzyme and
0.001kgAdk enzyme.The ATP and amino acid substrate for adding various concentration, wherein the amino acid masses ratio added is fixed are as follows:
Glu:Cys:Gly=1.6:1:1.1.PH value is adjusted to about 7.0, it is 6.5-7.0, temperature 35-38 that pH value is controlled during reaction
℃。
By analyzing multiple reaction result, the substrate when GSH production quantity can reach different value, added in reaction is summed up
ATP and amino acid (calculate) relationship of dosage and GSH conversion ratio with Cys.Fig. 6 is that the method for the present invention reaches different GSH lifes
At substrate when amount and product relational graph, when GSH production quantity successively reaches about 10,20,30,40 and 50g/L, required ATP
Amount is about 1.5-2.5 times of GSH production quantity.It is incrementally increased as required amino acid (calculates) dosage with Cys, reaction conversion
Rate successively reduces, and is 90.2%, 88.0%, 86.5%, 78.6% and 57.4%, it can be seen that when GSH production quantity reaches 30-
When 40g/L, rate of ultilization of amino acid is up to 75-90%, and when GSH production quantity reaches 50g/L, reaction can be normally carried out, and amino acid utilizes
Rate is still up to 50% or more.
Embodiment 7 prepares GSH and AMP
Referring to Fig. 3, the process flow chart of GSH produced according to the present invention prepared in accordance with the following steps using resolvase GSH and
AMP:
(1) glutathione GSH and AMP are generated in reactor tank:
In reactor tank, the reaction system of 100L sterile water be containing substrate 0.25kg glutamic acid, 0.1kg cysteine and
0.15kg glycine and 0.1kgATP, 0.1kg Tris, 0.075kg potassium chloride, 0.059kg sodium chloride, 0.03g ammonium chloride,
The solution of mono- water manganese chloride of 0.20kg magnesium chloride hexahydrate and 0.17kg, stirs evenly.PH value is adjusted to about 10.0, in reaction system
Middle addition 0.0002kg GshF enzyme and 0.001kgAdk enzyme start to react.It is 10.0 that pH value is controlled during reaction, temperature 55
℃。
After reaction 6 hours, the production quantity of high performance liquid chromatography (HPLC) detection glutathione is about 0.6g/L, and 90% or more
ATP exhausts, and is converted into AMP, and AMP production quantity is about 0.5g/L.HPLC testing conditions are the same as 3 step of embodiment (1).
(2) GshF enzyme and Adk enzyme are separated in the filter:
By hyperfiltration process, the reaction solution of the reaction system of step (1) is separated by filtration GshF enzyme and Adk by filter
Enzyme, the built-in film packet of filter (being purchased from Pall company, molecular cut off 8kDa), filter liquor is the reaction solution after isolating enzyme.
(3) separation product GSH and AMP:
The pH value for adjusting filter liquor using hydrochloric acid passes through D001 macropore strong acid styrene to 3.0 in ion exchange column
Cation exchanger resin, GSH, partial amino-acid and cation in solution are adsorbed, are pierced by liquid and mainly contain AMP, produce
Measure about 50g.
Use the GSH on 0-0.8M NaCl gradient elution cation exchange resin, GSH yield about 55g, yield about 90%.
(4) reactor tank generates the successive reaction of GSH and AMP, the i.e. successive reaction of step (1):
The enzyme isolated in step (2) is added to reactor tank via the refluxing opening of filter, and adds protoenzyme amount 5-
15% new enzyme is reacted.Reaction liquid making method ibid states step (1).
Carry out generating the successive reaction of GSH and AMP under conditions of 55 DEG C, pH are 10.0；After 6 hours, HPLC detects GSH
Production quantity be about 0.6g/L, 90% or more ATP is converted into AMP, and AMP production quantity is about 0.5g/L.HPLC testing conditions are the same as real
Apply 3 step of example (1).In this step, enzyme is recycled.
Embodiment 8 prepares GSH and AMP
Referring to Fig. 3, the process flow chart of GSH produced according to the present invention prepared in accordance with the following steps using resolvase GSH and
AMP:
(1) glutathione GSH and AMP are generated in reactor tank:
In reactor tank, the reaction system of 100L sterile water be containing substrate 5.0kg glutamic acid, 5.0kg cysteine and
2.5kg glycine and 15kgATP, 1.5kg disodium hydrogen phosphate, 2.24kg potassium chloride, 1.76kg sodium chloride, 1.07kg chlorination
The solution of mono- water manganese chloride of ammonium, 2.03kg magnesium chloride hexahydrate and 1.7kg, uniform stirring prevents precipitating when preparation.Adjust pH
Value adds 0.1kg GshF enzyme in the reaction system and 0.0033kgAdk enzyme starts to react to about 5.0.PH is controlled during reaction
Value is 5.0, and temperature is 25 DEG C.
After reaction 7 hours, the production quantity of high performance liquid chromatography (HPLC) detection glutathione is about 30g/L, and 70% or so
ATP exhausts, and is converted into AMP, and AMP production quantity is about 60g/L.HPLC testing conditions are the same as 3 step of embodiment (1).
(2) GshF enzyme and Adk enzyme are separated in the filter:
By hyperfiltration process, the reaction solution of the reaction system of step (1) is separated by filtration GshF enzyme and Adk by filter
Enzyme, the built-in film packet of filter (being purchased from Pall company, molecular cut off 8kDa), filter liquor is the reaction solution after isolating enzyme.
(3) separation product GSH and AMP:
The pH value for adjusting filter liquor using hydrochloric acid passes through D001 macropore strong acid styrene to 3.0 in ion exchange column
Cation exchanger resin, GSH, partial amino-acid and cation in solution are adsorbed, are pierced by liquid and mainly contain AMP, produce
Measure about 6kg.
Using the GSH on 0-0.8M NaCl gradient elution cation exchange resin, GSH yield about 2.8kg, yield is about
90%.
(4) reactor tank generates the successive reaction of GSH and AMP, the i.e. successive reaction of step (1):
The enzyme isolated in step (2) is added to reactor tank via the refluxing opening of filter, and adds protoenzyme amount 5-
15% new enzyme is reacted.
Carry out generating the successive reaction of GSH and AMP under conditions of 25 DEG C, pH are 5.0；After 7 hours, HPLC detects GSH
Production quantity be about 30g/L, 70% or so ATP is converted into AMP, and AMP production quantity is about 60g/L.HPLC testing conditions are the same as implementation
3 step of example (1).In this step, enzyme is recycled.
Embodiment 9 prepares GSH and AMP
Referring to Fig. 3, the process flow chart of GSH produced according to the present invention prepared in accordance with the following steps using resolvase GSH and
AMP:
(1) glutathione GSH and AMP are generated in reactor tank:
In reactor tank, the reaction system of 100L sterile water be containing substrate 2.8kg glutamic acid, 1.8kg cysteine and
The solution of mono- water manganese chloride of 2.0kg glycine and 5.1kgATP and 0.83kg, uniform stirring prevents precipitating when preparation.
PH value is adjusted to about 7.0,0.01kg GshF enzyme is added in the reaction system and 0.001kgAdk enzyme starts to react.During reaction
Controlling pH value is 7.0, and temperature is 37 DEG C.
After reaction 6 hours, the production quantity of high performance liquid chromatography (HPLC) detection glutathione is about 28g/L, and 95% or more
ATP exhausts, and is converted into AMP, and AMP production quantity is about 30g/L.HPLC testing conditions are the same as 3 step of embodiment (1).
(2) GshF enzyme and Adk enzyme are separated in the filter:
By hyperfiltration process, the reaction solution of the reaction system of step (1) is separated by filtration GshF enzyme and Adk by filter
Enzyme, the built-in film packet of filter (being purchased from Pall company, molecular cut off 8kDa), filter liquor is the reaction solution after isolating enzyme.
(3) separation product GSH and AMP:
The pH value for adjusting filter liquor using hydrochloric acid passes through D001 macropore strong acid styrene to 3.0 in ion exchange column
Cation exchanger resin, GSH, partial amino-acid and cation in solution are adsorbed, are pierced by liquid and mainly contain AMP, can
Direct condensing crystallizing after simple desalination, yield about 2.8kg.
Using the GSH on 0-0.8M NaCl gradient elution cation exchange resin, GSH yield about 2.6kg, yield is about
90%.
(4) reactor tank generates the successive reaction of GSH and AMP, the i.e. successive reaction of step (1):
The enzyme isolated in step (2) is added to reactor tank via the refluxing opening of filter, and adds protoenzyme amount 5-
15% new enzyme is reacted.Reaction liquid making method ibid states step (1).
Carry out generating the successive reaction of GSH and AMP under conditions of 37 DEG C, pH are 7.0；After 6 hours, HPLC detects GSH
Production quantity be about 28g/L, 95% or more ATP is converted into AMP, and AMP production quantity is about 30g/L.HPLC testing conditions are the same as implementation
3 step of example (1).In this step, enzyme is recycled.
Embodiment 10 prepares GSH and AMP
Referring to fig. 4, the process flow chart of GSH produced according to the present invention prepared in accordance with the following steps using immobilized enzyme GSH and
AMP:
(1) glutathione GSH and AMP are generated in reaction column:
Reaction solution is prepared, every 100L contains substrate 2.8kg glutamic acid, 1.8kg cysteine and 2.0kg glycine, and
5.1kgATP, 1.2kg disodium hydrogen phosphate and 1.0kg magnesium chloride hexahydrate, uniform stirring prevents precipitating when preparation, adjusts pH value
To about 7.5, temperature is upgraded to 37-40 DEG C.
Mixing immobilized enzyme 50kg in above-described embodiment 4 is packed into reaction column equipment, enzyme reaction column is made after draining bubble.
Reaction solution is from bottom to top slowly pumped through enzyme reaction column with 16-20L/h flow velocity using constant flow pump, controls temperature during reaction
It is 37 DEG C.After reaction about 6 hours, reaction solution is collected, the production quantity of high performance liquid chromatography (HPLC) detection glutathione is about
27g/L, 95% or more ATP exhaust, and are converted into AMP, and AMP production quantity is about 30g/L.HPLC testing conditions are the same as 3 step of embodiment
(1)。
(2) separation product GSH and AMP:
It is strong to pass through D001 macropore to 3.0 in ion exchange column for the pH value for adjusting the reaction solution of above-mentioned collection using hydrochloric acid
Acid styrene type cation exchange resin, GSH, partial amino-acid and cation in solution are adsorbed, and are pierced by liquid main
Containing AMP, can direct condensing crystallizing after simple desalination, yield about 2.9kg.
Using the GSH on 0-0.8M NaCl gradient elution cation exchange resin, GSH yield about 2.5kg, yield is about
90%.
(3) reaction column generates the successive reaction of GSH and AMP, the i.e. successive reaction of step (1):
Same reaction liquid described in preparation steps (1), is continuously from bottom to top slowly pumped through enzyme with 16-20L/h flow velocity
Reaction column, controlled at 37 DEG C during reaction.
The production quantity that HPLC detects GSH is about 27g/L, and 95% or more ATP is converted into AMP, and AMP production quantity is about 30g/L.
HPLC testing conditions are the same as 3 step of embodiment (1).In this step, enzyme is recycled.
More than January, enzymatic activity reduces about 10% for immobilised enzymes circular response 20 times or more or -4 DEG C of storage times, needs
It adds in proportion or the new enzyme of removable parts.
Embodiment 11 prepares GSH and AMP using amino-acid salt
Referring to Fig. 3, the process flow chart of GSH produced according to the present invention prepared in accordance with the following steps using resolvase GSH and
AMP:
(1) glutathione GSH and AMP are generated in reactor tank:
In reactor tank, the reaction system of 100L sterile water is to contain substrate 3.2kg sodium glutamate, 2.3kg cysteine
Hydrochloride and 2.6kg Sodium Glycinate and 5.1kgATP, 1.2kg disodium hydrogen phosphate, 0.7kg potassium chloride, 0.6kg sodium chloride,
The solution of 0.1kg ammonium chloride and 1.0kg magnesium chloride hexahydrate, uniform stirring prevents precipitating when preparation.PH value is adjusted to about
7.0, it adds 0.01kg GshF enzyme in the reaction system and 0.001kgAdk enzyme starts to react.PH value is controlled during reaction is
7.0, temperature is 37 DEG C.
After reaction 6 hours, the production quantity of high performance liquid chromatography (HPLC) detection glutathione is about 30g/L, and 95% or more
ATP exhausts, and is converted into AMP, and AMP production quantity is about 30g/L.HPLC testing conditions are the same as 3 step of embodiment (1).
(2) GshF enzyme and Adk enzyme are separated in the filter:
By hyperfiltration process, the reaction solution of the reaction system of step (1) is separated by filtration GshF enzyme and Adk by filter
Enzyme, the built-in film packet of filter (being purchased from Pall company, molecular cut off 8kDa), filter liquor is the reaction solution after isolating enzyme.
(3) separation product GSH and AMP:
The pH value for adjusting filter liquor using hydrochloric acid passes through D001 macropore strong acid styrene to 3.0 in ion exchange column
Cation exchanger resin, GSH, partial amino-acid and cation in solution are adsorbed, are pierced by liquid and mainly contain AMP, can
Direct condensing crystallizing after simple desalination, yield about 2.8kg.
Use the GSH on 0-0.8M NaCl gradient elution cation exchange resin, GSH yield about 2.6-2.8kg, yield
About 90%.
(4) reactor tank generates the successive reaction of GSH and AMP, the i.e. successive reaction of step (1):
The enzyme isolated in step (2) is added to reactor tank via the refluxing opening of filter, and adds protoenzyme amount 5-
15% new enzyme is reacted.Reaction liquid making method ibid states step (1).
Carry out generating the successive reaction of GSH and AMP under conditions of 37 DEG C, pH are 7.0；After 6 hours, HPLC detects GSH
Production quantity be about 30g/L, 95% or more ATP is converted into AMP, and AMP production quantity is about 30g/L.HPLC testing conditions are the same as implementation
3 step of example (1).In this step, enzyme is recycled.
As can be seen from the results: using the paddy of glutamate, cysteine salt and glycinate substitution same molar ratio
Propylhomoserin, cysteine and glycine do not influence reaction result.Glutamate, cysteine salt and sweet ammonia can be used in production
Hydrochlorate substitutes glutamic acid, cysteine and glycine.
Comparative example 1
In reactor tank, the reaction system of 100L sterile water be containing substrate 2.8kg glutamic acid, 1.8kg cysteine and
2.0kg glycine and 10.0kgATP, 1.2kg disodium hydrogen phosphate, 0.7kg potassium chloride, 0.6kg sodium chloride, 0.1kg ammonium chloride
With the solution of 1.0kg magnesium chloride hexahydrate, uniform stirring prevents precipitating when preparation.PH value is adjusted to about 7.0, in reaction system
Middle addition 0.01kg GshF enzyme starts to react.It is 7.0 that pH value is controlled during reaction, and temperature is 37 DEG C.
After reaction 6 hours, the production quantity of high performance liquid chromatography (HPLC) detection glutathione is about 28g/L, and 70% or so
ATP exhausts, and is converted into ADP and AMP.Please also refer to Fig. 7, for the HPLC map of 6 hours 10 times of dilute reaction solutions of reaction, figure
In do not mark peak be amino acid.HPLC testing conditions are the same as 3 step of embodiment (1).
As can be seen from the results: without coupling Adk enzyme in comparative example 1, therefore the ATP amount that reaction system needs increases.And
GSH production quantity reduces in same time.In addition, reaction generates a large amount of ADP, this undoubtedly increases the difficulty of purifying.
For comparative example 1, Optimization of preparation of the invention reaction condition can make the production quantity of product GSH
It improves from 10-20g/L to 30-50g/L, and keeps higher conversion ratio.The Adk enzyme added in reaction process can make to generate GSH
When a part of ADP for generating be converted into ATP and continue to use, ATP dosage can reduce 30-50%, the consumption of ATP is greatly reduced,
Meanwhile another part ADP can be catalyzed and generate a large amount of AMP, it is easy to the purifying in later period, is prepared simultaneously in reaction process a large amount of
AMP.AMP has significant peripheral vasodilation and antihypertensive effect in terms of medicine；In terms of food addition, it can be used as bitter taste and cover
Lid agent uses, and has certain market value.
Although the present invention is disclosed as above with embodiment, so it is not intended to limit the present invention, any those skilled in the art
Member, without departing from the spirit and scope of the present invention, can make a variety of different selections and modification, therefore protection model of the invention
It encloses and is limited by claims and its equivalents.
Claims (2)
1. a kind of method that enzyme process prepares glutathione and adenylate simultaneously, which is characterized in that the described method comprises the following steps:
(1) glutathione GSH and AMP are generated in reactor tank:
In reactor tank, the reaction system of 100L sterile water is to contain substrate 2.8kg glutamic acid, 1.8kg cysteine and 2.0kg
The solution of mono- water manganese chloride of glycine and 5.1kg ATP and 0.83kg, uniform stirring；PH value is adjusted to 7.0, in reactant
0.01kg GshF enzyme is added in system and 0.001kg Adk enzyme starts to react, and it is 7.0 that pH value is controlled during reaction, temperature 37
DEG C, the reaction time 6 hours；
(2) GshF enzyme and Adk enzyme are separated in the filter:
By hyperfiltration process, the reaction solution of the reaction system of step (1) is separated by filtration GshF enzyme and Adk enzyme by filter,
Filtering, filter liquor is the reaction solution after isolating enzyme；
(3) separation product GSH and AMP:
The pH value for adjusting filter liquor using hydrochloric acid passes through D001 macropore strong acid polystyrene sun to 3.0 in ion exchange column
Ion exchange resin, GSH, partial amino-acid and cation in solution are adsorbed, are pierced by liquid and mainly contain AMP, can be simple
Direct condensing crystallizing after desalination；
Use the GSH on 0-0.8M NaCl gradient elution cation exchange resin；
(4) reactor tank generates the successive reaction of GSH and AMP, the i.e. successive reaction of step (1):
The enzyme isolated in step (2) is added to reactor tank via the refluxing opening of filter, and adds protoenzyme amount 5-15%'s
New enzyme is reacted；
37 DEG C, pH be 7.0 under conditions of carry out generate GSH and AMP successive reaction, the reaction time 6 hours.
2. the method that enzyme process according to claim 1 prepares glutathione and adenylate simultaneously, which is characterized in that the step
Suddenly reaction liquid making method is identical as step (1) in (4) reaction.
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| US11939615B2 (en) | 2017-06-15 | 2024-03-26 | Anhui Gsh Bio-Tech Co., Ltd. | Production method of enzymatic reaction using adenosine instead of ATP |
| CN107190035B (en) * | 2017-07-20 | 2020-04-21 | 湖南福来格生物技术有限公司 | Method for preparing reduced glutathione by multienzyme system |
| CN109280680B (en) * | 2017-07-21 | 2023-01-10 | 北京天开易达生物科技有限公司 | Enzymatic co-production method |
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Effective date of registration: 20220929 Address after: Room 102, Building 1, No. 9 Hangfeng Road, Science City, Fengtai District, Beijing 100000 Patentee after: BEIJING TIANKAI YIDA BIOLOGICAL SCIENCE & TECHNOLOGY Co.,Ltd. Address before: 518103 Xinguobang Logistics Park, 87 Yongfu Road, Fuyong Town, Baoan District, Shenzhen City, Guangdong Province Patentee before: SHENZHEN GSH BIO-TECH CO.,LTD. |