CN105603028A

CN105603028A - Enzymic method for simultaneously preparing glutathione and adenylate

Info

Publication number: CN105603028A
Application number: CN201610167664.6A
Authority: CN
Inventors: 刘珊珊; 于铁妹; 黄庆军; 秦永发
Original assignee: Shenzhen Gute Xinsheng Biological Technology Co Ltd
Current assignee: Beijing Tiankai Yida Biological Science & Technology Co ltd
Priority date: 2016-03-22
Filing date: 2016-03-22
Publication date: 2016-05-25
Anticipated expiration: 2036-03-22
Also published as: CN105603028B

Abstract

The invention discloses an enzymic method for simultaneously preparing glutathione and adenylate. The enzymic method comprises the following steps: (1) generating glutathione and adenylate from GshF enzyme and Adk enzyme in a reaction tank; (2) separating the immobilized GshF enzyme and Adk enzyme in the reaction tank, or separating the free GshF enzyme and Adk enzyme by virtue of filtering equipment; and (3) separating products GSH and AMP. By virtue of the preparation method, the reaction condition in production of the GSH is optimized, the generation concentration of GSH reaches 30g/L-50g/L, and the utilization rate of amino acid is relatively high; meanwhile, by carrying out partial regeneration on ATP consumed in reaction by virtue of the Adk enzyme, the consumption of the ATP is reduced; and furthermore, the GSH and the AMP can be simultaneously prepared by virtue of the preparation method.

Description

Enzyme process is prepared the method for glutathione and adenylate simultaneously

Technical field

The present invention relates to the preparation method of glutathione, particularly a kind of enzyme process prepare simultaneously glutathione andThe method of adenylate.

Background technology

Glutathione is extensively present in animals and plants and microorganism, is most important non-albumen sulfydryl in organismOne of compound, has reduced glutathione (GSH) and oxidized form of glutathione (GSSG), biologyIn body, exist in a large number and that play a major role is GSH, be widely used in treatment liver diseases, tumour, oxygen poisoning,Aging and endocrine system disease, and be used for field of food as bioactive additive and antioxidant.

GSH is formed through peptide bond condensation by glutamic acid (Glu), cysteine (Cys) and glycine (Gly)Tripeptides. Relative molecular mass is 307.32, and isoelectric point is 5.93, under normal temperature, is white crystal, is soluble inWater, the low-concentration ethanol aqueous solution, liquefied ammonia and dimethyl formamide.

The main preparation methods of glutathione has at present: solvent extraction, chemical synthesis, biological fermentation processAnd enzyme process. From grain germ, extract GSH, because GSH yield is low, cost is high, organic solvent pollution is tightHeavy, purity is not high, and consumes a large amount of grain, now less use. Chemical synthesis is synthesized GSH, byNot easily separated in activated product, need chemistry to split, product purity is not high, is difficult to promote. At present both at home and abroadGSH produces the basic fermentation method that adopts, and principle is to Escherichia coli by the Gene cloning of coding GSH synthetase seriesOr in yeast, use microorganism fermenting and producing GSH. Yeast fermentation method, technique is more ripe, but the production cycleLong, output is on the low side, and too much accessory substance makes downstream process process complexity.

Production by Enzymes GSH technology progressively improves in recent years, makes large-scale production become possibility. Classical enzyme processProduce GSH and depend on gamma glutamyl cysteine synthetase (GshI) and glutathione synthetase (GshII)Two kinds of enzymes, GshI catalysis Pidolidone and Cys synthesize gamma-glutamyl cysteine, GshII catalysisGamma-glutamyl cysteine and glycine synthesize GSH. In GSH building-up process, due to GshI catalysisJourney is subject to the feedback inhibition of end-product GSH, therefore makes the first step that generates gamma-glutamyl cysteine be reacted intoFor the synthetic rate-limiting step of whole GSH.

Along with further research, people are at Listeria monocytogenes (Listeriamonocytogenes)Deng all having found a kind of difunctional glutathione synthetase (GshF enzyme) in tens kinds of bacteriums. This enzyme has simultaneouslyThe activity of GshI and GshII, can synthesize by a step catalysis GSH, and this enzyme feedback inhibition is less, non-Often accommodate and be applied to enzyme process synthesizing glutathion.

The greatest problem of the synthetic GSH of enzyme process is a large amount of consumption of atriphos (ATP), produces 1kgGSHAt least need to use the ATP of 3-5kg, high cost. In order to solve the problem of this respect, can use Yeast sugarThe method coupling with it of glycolysis regeneration ATP, is used the method regeneration ATP in patent CN201210201691.2,Effect stability. But use yeast can in reaction system, introduce the impurity such as pigment, giving to be further purified increases difficultyDegree, using the regeneration ATP of enzyme system is research direction in recent years. In addition patent CN201510762184.X,The middle use polyphosphate enzyme regeneration ATP of system, has obtained good effect. This enzyme system comprises polyphosphateKinases (Ppk), adenylate kinase (Adk) and polyphosphoric acids-adenylate phosphotransferase (Pap). Wherein,Ppk and Pap enzyme need add polyphosphate in course of reaction, to GSH reaction conversion ratio, can reachLarge concentration and later stage purifying all have impact. Therefore, how to use Adk enzyme to optimize the production of GSH, reduceThe use amount of ATP, and ensureing that product GSH is easy to, on the basis of purifying and separation, can prepare simultaneouslyThe product that other has an economic benefit, as adenylate (AMP), becomes current research emphasis.

Summary of the invention

The invention provides a kind of enzyme process and prepare the side of glutathione (GSH) and adenylate (AMP) simultaneouslyMethod, thus overcome the above-mentioned defect of prior art.

The present invention is achieved through the following technical solutions:

Enzyme process is prepared a method for glutathione and adenylate simultaneously, and the method comprises the following steps:

(1) utilize GshF enzyme and Adk enzyme in retort, to generate GSH and AMP:

In reaction system, add ATP, GshF enzyme and Adk enzyme, reaction generates GSH and AMP, whereinDescribed reaction system is for containing substrate A TP, glutamic acid (Glu) or its salt, cysteine (Cys) or itsThe aqueous solution of one or both of salt, glycine (Gly) or its salt and magnesium ion and manganese ion. In addition,In reaction system, also can comprise a kind of or several of potassium ion, sodium ion, ammonium ion and Tris and phosphate anionKind. Substrate, enzyme and all kinds of salt that the present invention adds can disposablely add reaction system, also can be raw according to industryProduction. art flow process in batches stream adds and fills into.

(2) separate GshF enzyme and Adk enzyme:

Immobilized GshF enzyme directly separates in retort with Adk enzyme. Above-mentioned separation can separate by filter bag,Also can in reaction column, directly separate.

Free GshF enzyme separates by milipore filter in filter with Adk enzyme. Wherein, filter have charging aperture,Discharging opening and refluxing opening, inside establish the milipore filter that molecular cut off is less than 20kDa. Trapped fluid through filter isThe enzyme liquid reclaiming, filter liquor is to isolate enzyme reactant liquor afterwards; And

(3) separated product GSH and AMP:

By ion-exchange, separated product GSH from the filter liquor of above-mentioned steps (2), through ion-exchangeAfter pass and in liquid, mainly contain another product A MP.

Preferably, in technique scheme, further comprising the steps of:

(4) recycling of the GshF enzyme separating in step (2) and Adk enzyme:

Being about to the GshF enzyme that separates and Adk enzyme is added into and in retort, generates GSH and AMP and regeneration ATPSuccessive reaction.

(5) continuous separate of GshF enzyme and Adk enzyme from:

Be continuous separate from immobilized GshF enzyme and Adk enzyme or use the continuous separated free of filter plantGshF enzyme and Adk enzyme.

Preferably, in technique scheme, further comprising the steps of:

(6) continuous separate of product GSH from. The GSH that reaction generates divides by filtration or ion-exchange processFrom.

(7) continuous separate of product A MP from. The AMP that reaction generates divides by filtration or ion-exchange processFrom.

Preferably, in technique scheme of the present invention, step (1) to step (3) can repeat at leastOnce. Preferably repeatedly, for example repeat 2,3,4,5,6,7,8,9,10,15,20,25,30,40,50 is inferior.

Preferably, in technique scheme, step (1) generates the reaction of GSH and AMP in retortCondition is as follows:

Reaction temperature is 25-55 DEG C, and preferably temperature is 30-50 DEG C;

Reaction pH is 5-10, and preferably pH is 6-9.

Preferably, in technique scheme, GshF enzyme, Adk enzyme are for free or immobilized enzyme and/or againRaw enzyme, calculates by reaction optimal conditions, and two kinds of enzymes add mass ratio and are preferably GshF:Adk=(0.2-30): 1,More preferably (0.5-20): 1. GshF enzyme adds concentration and is preferably 0.002-1g/L. Above-mentioned GshF enzyme, AdkEnzyme can derive from any biology, or through the artificial reconstructed enzyme with same catalysis.

Preferably, in technique scheme, amino acid or its salt are L-type amino acid or its salt. By its warpJi cost and reaction optimal conditions calculate, and three seed amino acids add mass ratio and are preferably Glu:Cys:Gly=(1-2.5): 1:(0.5-1.5), Glu:Cys:Gly=(1.2-2): 1:(0.8-1.5 more preferably). Cysteine adds denseDegree is preferably 1-50g/L.

Preferably, in technique scheme, the ATP concentration that the present invention adds is 1-150g/L; Magnesium ion is denseDegree is 0.01-0.1M; Manganese ion concentration is 0.01-0.1M; Potassium concentration is 0.01-0.3M; Sodium ionConcentration is 0.01-0.3M; Ammonium concentration is 0.005-0.2M; Tris concentration is that 1-12g/L, phosphate are denseDegree is 1-15g/L.

Preferably, in technique scheme, magnesium ion is selected from magnesium chloride, magnesium sulfate, magnesium sulfite and nitreOne or more in acid magnesium; Manganese ion is selected from one or more in manganese chloride and manganese sulfate; Potassium ionBe selected from potassium chloride, potassium sulfate, potassium nitrate, potassium hydroxide, potassium sulfite, potash, saleratus,One or more in potassium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium citrate; Sodium ion is selected fromSodium chloride, sodium sulphate, sodium nitrate, NaOH, sodium sulfite, sodium carbonate, sodium acid carbonate, sodium acetate,One or more in sodium hydrogen phosphate, sodium dihydrogen phosphate and natrium citricum; Ammonium ion be selected from ammonium chloride,Ammonium sulfate, ammonium nitrate, ammoniacal liquor, ammonium carbonate, carbonic hydroammonium, diammonium hydrogen phosphate, ammonium dihydrogen phosphate (ADP) and acetic acidOne or more in ammonium.

Preferably, in technique scheme, in step (2), immobilised enzymes is fixed on fixing by following mannerChange carrier: absorption, crosslinked, covalent bond, embedding or its combination. Two kinds of enzymes of GshF and Adk can be consolidated respectivelyDetermine or after mixing, fix together in proportion. Fixation support is selected from macromolecule carrier, inorganic carrier and magneticPolymer microsphere supported one or more.

Wherein, macromolecule carrier is selected from cellulose, glucose gel, agarose, polyacrylamide, manyPolyaminoacid, polystyrene, polyacrylic acid, sodium alginate, shitosan, starch, polyvinyl alcohol, gelatin,One or more combinations of carragheen, nylon and synthetic high polymer; Inorganic carrier is selected from cellular glass, oxidationSilicon, active carbon, silica gel and diatomaceous one or more combinations. In the time carrying out enzymatic reaction, can be by fixingChange enzyme is dispersed in reactant liquor and directly reacts, and after reaction finishes, reclaims enzyme by filter type; Or willImmobilised enzymes is packed in reaction column, by certain flow rate, reactant liquor is pumped into by cylinder and carries out enzymatic reaction.

Preferably, in technique scheme, the milipore filter adopting in the inventive method step (2) is selected from vinegarAcid cellulose film, PS membrane, polyacrylonitrile film, polychloroethylene film, polyvinylidene fluoride film, PA membrane orCeramic membrane.

Compared with prior art, the technology of the present invention has following beneficial effect:

1) optimized the reaction condition that GSH produces, can greatly improve concentration of substrate, GSH has been generated denseDegree reaches 30-50g/L, and wherein, in the time that GSH growing amount reaches 30-40g/L, rate of ultilization of amino acid can reach75-90%. And the method is because initial substrate concentration is high, and reaction rate is fast, has saved the reaction time;

2) use Adk enzyme to carry out partial regeneration to the ATP consuming in reaction. Use separately GshF enzymeWhile producing GSH, produce 1kgGSH and at least need to use the ATP of 3-5kg, add after Adk enzyme, produceThe GSH of 1kg need to about 1.5-2.5kg ATP, greatly reduce the consumption of ATP; In addition, regenerationReaction does not need to add as other substrate of polyphosphate and so on, does not affect the enzymatic generation of GshF GSHEnzymatic reaction, ensure growing amount and the reaction rate of GSH;

3), after Adk enzymatic, in reaction system, only minute quantity ADP remains, more than 95% ADP mono-Partial Conversion is that ATP continues to use, and has reduced the consumption of ATP, and another part generates a large amount of AMP.AMP is easy in purge process with GSH separate, and purge process is simple, can be used as another product in preparationPreparation simultaneously in GSH process. By the market price and cost analysis, the method is economical and practical;

4) set up the stable recovery system of applicable GshF enzyme and Adk enzyme, no matter immobilization is still freeGshF enzyme and the combination of Adk enzyme can be applied to large-scale continuous production;

5) this preparation method operates simple and feasible, and amplification effect is good, can be amplified to tonne.

Brief description of the drawings

Fig. 1 is the SDS-PAGE figure of the expressed GshF enzyme of the present invention.

Fig. 2 is the SDS-PAGE figure of the expressed Adk enzyme of the present invention.

Fig. 3 is that the inventive method is used resolvase to prepare the process chart of GSH and AMP.

Fig. 4 is that the inventive method is used immobilized enzyme to prepare the process chart of GSH and AMP.

Fig. 5 is the HPLC collection of illustrative plates that the present invention uses GshF enzyme and Adk enzyme to react.

Fig. 6 is the inventive method substrate and product graph of a relation while reaching different GSH growing amount.

Fig. 7 is the HPLC collection of illustrative plates that the present invention only uses GshF enzyme to react.

Detailed description of the invention

Below in conjunction with accompanying drawing, specific embodiments of the invention are described in detail, so that further understand thisInvention.

The preparation of embodiment 1GshF enzyme

GshF enzyme in the inventive method can be commercially available, or pass through the artificial reconstructed same catalysis that hasThe enzyme of function.

The preparation process of GshF enzyme is as follows:

According to gshF gene order (GenBank:NC_008532), design pair for amplification primer, by Sino-U.S.Calm and peaceful Bioisystech Co., Ltd is synthetic, and primer sequence is as follows:

GshF sense primer: 5 '-CCATATGACATTAAACCAACTTCTTCAAAAACTG-3 ';With

GshF antisense primer: 5 '-CGAATTCTTAAGTTTGACCAGCCACTATTTC-3 ';

Extract streptococcus thermophilus (Streptococcusthermophilus) bacterial strain (CGMCC1.6472) DNA,Taking it as template, go out gshF genetic fragment by pcr amplification, and it is connected to respectively to pET22b carrier(being purchased from Invitrogen company), after checking order correctly, proceeds to respectively E.coliBL21 (DE3) bacterial strain and (purchasesIn Tian Gen biochemical technology Co., Ltd).

By E.coliBL21 (DE3) the monoclonal access LB culture medium after transforming, be cultured to after logarithmic phase, addEnter 1mM isopropyl-β-D-sulfo-galactopyranoside (IPTG) induction after 5 hours, collect thalline, tenDialkyl group sodium sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) screening high expressed bacterial strain.

The high expressed bacterial strain filtering out is accessed to seed culture medium under aseptic condition, be cultured to exponential phaseRear access, containing in the fermentation tank of 5L fermentation medium, is cultured to the access of logarithmic growth after date containing 50L fermentation trainingSupport in the fermentation tank of base, cultivate after 5 hours and add 1mMIPTG induction after 5 hours, centrifugal collection thallineAbout 1000g.

Wherein LB medium component is: 1% peptone, 0.5% dusty yeast and 1%NaCl; Seed cultureBased component is: 1% peptone, 0.5% dusty yeast and 1% sodium chloride; Fermented and cultured based component is: 1% albumenPeptone, 0.5% dusty yeast, 1% sodium chloride, 5% sodium hydrogen phosphate, 1% sodium dihydrogen phosphate, 0.01% magnesium sulfate and1% glycerine.

The thalline of results breaks after bacterium through ultrasonic or high-pressure homogenization, centrifugal collection supernatant. By ammonium sulfate precipitation,Acid-base precipitation and bivalent metal ion intermediate processing can obtain the GshF enzyme of preliminary purification.

Fig. 1 is the SDS-PAGE figure of prepared enzyme, as shown in the figure: swimming lane 1 is protein marker 14.4-116KDa (being purchased from Rui Tai Bioisystech Co., Ltd of BeiJing ZhongKe); Swimming lane 2 is the thalline containing GshF enzyme, GshFThe about 85kDa of enzyme; Swimming lane 3, swimming lane 4 are the GshF enzyme after preliminary purification; Swimming lane 5 is for purifying is after concentratedGshF enzyme.

The method that uses the known mensuration GshF enzymatic activity of prior art record, detects 1mg/mlGshFEnzymatic activity is about 3000U, is wherein 1 active unit by 1 μ M substrate complete transformation definition in 1 minute(U)。

The preparation of embodiment 2Adk enzyme

Adk enzyme in the inventive method can be commercially available, or pass through the artificial reconstructed same catalysis that hasThe enzyme of function.

The preparation process of Adk enzyme is as follows:

According to adk gene order (GenBank:NC_000913), design pair for amplification primer, by Sino-U.S.Calm and peaceful Bioisystech Co., Ltd is synthetic, and primer sequence is as follows:

Adk sense primer: 5 '-CCATATGCGTATCATTCTGCTTGGCGCTCCGG-3 '; With

Adk antisense primer: 5 '-CGGATCCTTAGCCGAGGATTTTTTCCAGATC-3 ';

Extract Escherichia coli (Escherichiacoli) K12 bacterial strains (being purchased from Tian Gen biochemical technology Co., Ltd)DNA, taking it as template, goes out adk genetic fragment by pcr amplification, and is connected to pET22b and carriesBody (being purchased from Invitrogen company) sequence, after checking order correctly, proceeds to E.coliBL21 (DE3) bacterial strain and (purchasesIn Tian Gen biochemical technology Co., Ltd).

The thalline of results breaks after bacterium through ultrasonic or high-pressure homogenization, centrifugal collection supernatant. Then add 40-60% saturatedAmmonium sulfate, centrifugal collecting precipitation. After Tris pH of buffer 8.0 is dissolved, use G25 post (to be purchased from logicalWith electric medical Biology Science Co., Ltd) desalination, then (be purchased from General Electric through DEAE-SepharoseFFMedical treatment Biology Science Co., Ltd) chromatography can obtain the Adk enzyme of preliminary purification.

Fig. 2 is the SDS-PAGE figure of prepared enzyme, as shown in the figure: swimming lane 1 is protein marker 14.4-116KDa (being purchased from Rui Tai Bioisystech Co., Ltd of BeiJing ZhongKe); Swimming lane 3 is Adk enzyme, about 24kDa.

The method that uses the known mensuration enzymatic activity of prior art record, detects 1mg/mlAdk enzymatic activityBeing about 1000U, is wherein 1 active unit (U) by 1 μ M substrate complete transformation definition in 1 minute.

Embodiment 3 uses resolvase to prepare GSH and AMP

Fig. 3 is that the inventive method is used resolvase to prepare the process chart of GSH. Referring to Fig. 3, according to thisThe process chart of invention preparation GSH uses resolvase to prepare in accordance with the following steps GSH and AMP:

(1) in retort, generate GSH and AMP:

In retort, the reaction system of 100L sterilized water is for containing substrate 2.8kg glutamic acid, 1.8kg halfCystine and 2.0kg glycine, and 5.1kgATP, 1.2kg sodium hydrogen phosphate, 0.7kg potassium chloride,The solution of 0.6kg sodium chloride, 0.1kg ammonium chloride and 1.0kg magnesium chloride hexahydrate, when preparation, uniform stirring preventsThere is precipitation. Regulate pH value to approximately 7.0, in reaction system, add 0.01kgGshF enzyme and 0.001kgAdkEnzyme starts reaction. Between the stage of reaction, controlling pH value is 7.0, and temperature is 37 DEG C.

React after 6 hours, the growing amount that high performance liquid chromatography (HPLC) detects glutathione is about 30g/L,95% above ATP exhausts, and is converted into AMP, and AMP growing amount is about 30g/L. Please also refer to Fig. 5,It is the HPLC collection of illustrative plates of 6 hours 10 times of dilute reaction solutions of reaction, and in figure, not marking peak is amino acid. HPLCTesting conditions is: KromasilC18 chromatographic column (being purchased from AKZONOBEL company) (150 × 4.6mm),Detect wavelength 210nm, 30 DEG C of detected temperatures. Mobile phase is for containing 6.8g/L potassium dihydrogen phosphate, 2.0g/LThe aqueous solution of sodium heptanesulfonate and 3% methyl alcohol, pH=3.0.

(2) in filter, separate GshF enzyme and Adk enzyme:

By hyperfiltration process, by the reactant liquor process filter isolated by filtration GshF of the reaction system of step (1)Enzyme and Adk enzyme, the in-built film bag of filter (being purchased from Pall company, molecular cut off 8kDa), filter liquor isIsolate enzyme reactant liquor afterwards.

Use describing method in embodiment 1, the activity that detects 1mg/mlGshF enzyme is about 2600-2900U.

Use describing method in embodiment 2, the activity that detects 1mg/mlAdk enzyme is about respectively 850-950U.

(3) separated product GSH and AMP:

Use hydrochloric acid to regulate the pH value to 3.0 of filter liquor, in ion exchange column, pass through D001 large porous strong acidProperty styrene type cation exchange resin, GSH, partial amino-acid and cation in solution are adsorbed, and wearIn fluid, mainly contain AMP, simply direct condensing crystallizing after desalination, the about 2.8kg of output.

Use the GSH on 0-0.8MNaCl gradient elution cationic ion-exchange resin, the about 2.6-2.8 of GSH outputKg, yield approximately 90%.

(4) retort generates the successive reaction of GSH and AMP, i.e. the successive reaction of step (1):

Isolated enzyme in step (2) is added into retort via the refluxing opening of filter, and adds protoenzymeThe new enzyme of amount 5-15% reacts. The same above-mentioned steps of reactant liquor compound method (1).

Under the condition that is 7.0 at 37 DEG C, pH, generate the successive reaction of GSH and AMP; After 6 hours,The growing amount that HPLC detects GSH is about 30g/L, and 95% above ATP is converted into AMP, and AMP generatesAmount is about 30g/L. The same above-mentioned steps of HPLC testing conditions (1). In this step, enzyme is recycled.

Fixing of embodiment 4 enzymes

GshF enzyme and Adk enzyme are fixed with business-like epoxy radicals fixation support LX1000EP.

Above-described embodiment 1 is mixed and is made into according to the about 10:1 of mass ratio with the enzyme of GshF described in 2 and Adk enzymeMixed enzyme solution. In constant temperature stirred tank, add the wet carrier of LX1000EP and above-mentioned enzyme liquid according to immobilizationCarrier is that 30:1 mixes with enzyme mass ratio, and under 20 DEG C of conditions, 150rpm stirs 12 hours. Filter and collectCarrier, with 0.02MpH8.0 kaliumphosphate buffer cleaning 2 times, obtains immobilization mixed enzyme.

After GshF enzyme and Adk enzyme immobilization, active approximately 30% of the former activity that is all reduced to.

Embodiment 5 uses immobilized enzyme to prepare GSH and AMP

Fig. 4 is that the inventive method is used immobilized enzyme to prepare the process chart of GSH. Referring to Fig. 4, according to thisThe process chart of invention preparation GSH uses immobilized enzyme to prepare in accordance with the following steps GSH and AMP:

(1) in reaction column, generate glutathione GSH and AMP:

Preparation reactant liquor, it is sweet that every 100L contains substrate 2.8kg glutamic acid, 1.8kg cysteine and 2.0kgPropylhomoserin, and 5.1kgATP, 1.2kg sodium hydrogen phosphate, 0.7kg potassium chloride, 0.6kg sodium chloride, 0.1Kg ammonium chloride and 1.0kg magnesium chloride hexahydrate, when preparation, uniform stirring prevents precipitation, regulates pH value extremelyApproximately 7.5, temperature is upgraded to 37-40 DEG C.

Pack the mixing immobilized enzyme 50kg in above-described embodiment 4 into reaction column equipment, make enzyme after draining bubbleReaction column. Use constant flow pump that reactant liquor is from bottom to top slowly pumped into by enzyme reaction post with 16-20L/h flow velocity,Between the stage of reaction, controlling temperature is 37 DEG C. React after approximately 6 hours, collect reactant liquor, high performance liquid chromatography (HPLC)The growing amount that detects glutathione is about 30g/L, and 95% above ATP exhausts, and is converted into AMP, AMPGrowing amount is about 30g/L. HPLC testing conditions is with embodiment 3 steps (1).

(2) separated product GSH and AMP:

Use hydrochloric acid to regulate the pH value to 3.0 of the reactant liquor of above-mentioned collection, in ion exchange column, pass through D001Large hole strong acid styrene system cation exchange resin, GSH, partial amino-acid and cation quilt in solutionAbsorption, passes and in liquid, mainly contains AMP, simply direct condensing crystallizing after desalination, the about 2.8kg of output.

(3) reaction column generates the successive reaction of GSH and AMP, i.e. the successive reaction of step (1):

Same reaction liquid described in preparation steps (1), continuously with from bottom to top slow pump of 16-20L/h flow velocityEnter by enzyme reaction post, between the stage of reaction, controlling temperature is 37 DEG C.

The growing amount that HPLC detects GSH is about 30g/L, and 95% above ATP is converted into AMP, AMPGrowing amount is about 30g/L. HPLC testing conditions is with embodiment 3 steps (1). In this step, enzyme is followedRing utilizes.

Immobilised enzymes circular response 20 times is above or-4 DEG C more than January in storage time, and enzymatic activity reduces approximately10%, need add in proportion or the new enzyme of removable parts.

The impact of embodiment 6 concentration of substrate on GSH growing amount

In 100L reaction system, fixing sodium hydrogen phosphate, potassium chloride, sodium chloride, ammonium chloride and six water chlorineThe addition of changing magnesium is respectively 1.2kg, 0.7kg, 0.6kg, 0.1kg and 1.0kg, fixing amount of adding enzymeFor 0.01kgGshF enzyme and 0.001kgAdk enzyme. Add ATP and the amino acid substrate of variable concentrations, itsThe amino acid masses ratio of middle interpolation is fixed as: Glu:Cys:Gly=1.6:1:1.1. Regulate pH value to approximately 7.0,Between the stage of reaction, control pH value for 6.5-7.0, temperature is 35-38 DEG C.

By analyzing repeatedly reaction result, sum up in the time that GSH growing amount can reach different value, in reaction, addThe relation of the substrate A TP adding and amino acid (calculating with Cys) consumption and GSH conversion ratio. Fig. 6Substrate and product graph of a relation while reaching different GSH growing amount for the inventive method, when GSH growing amount is complied withInferior reaching is about 10,20,30,40 and when 50g/L, and required ATP amount is about the 1.5-2.5 of GSH growing amountDoubly. Along with the progressively increase of amino acid needed (calculating with Cys) consumption, reaction conversion ratio reduces successively,Be 90.2%, 88.0%, 86.5%, 78.6% and 57.4%, can find out, when GSH growing amount reaches 30-40When g/L, rate of ultilization of amino acid can reach 75-90%, and when GSH growing amount reaches 50g/L, reaction can normally be carried out,Rate of ultilization of amino acid still can reach more than 50%.

Embodiment 7 prepares GSH and AMP

Referring to Fig. 3, the process chart of GSH produced according to the present invention uses resolvase to make in accordance with the following stepsStandby GSH and AMP:

(1) in retort, generate glutathione GSH and AMP:

In retort, the reaction system of 100L sterilized water is for containing substrate 0.25kg glutamic acid, 0.1kg halfCystine and 0.15kg glycine, and 0.1kgATP, 0.1kgTris, 0.075kg potassium chloride, 0.059kgThe solution of sodium chloride, 0.03g ammonium chloride, 0.20kg magnesium chloride hexahydrate and 0.17kg mono-water manganese chloride, stirsEvenly. Regulate pH value to approximately 10.0, in reaction system, add 0.0002kgGshF enzyme and 0.001kgAdkEnzyme starts reaction. Between the stage of reaction, controlling pH value is 10.0, and temperature is 55 DEG C.

React after 6 hours, the growing amount that high performance liquid chromatography (HPLC) detects glutathione is about 0.6g/L,90% above ATP exhausts, and is converted into AMP, and AMP growing amount is about 0.5g/L. HPLC testing conditions is sameEmbodiment 3 steps (1).

(2) in filter, separate GshF enzyme and Adk enzyme:

(3) separated product GSH and AMP:

Use hydrochloric acid to regulate the pH value to 3.0 of filter liquor, in ion exchange column, pass through D001 large porous strong acidProperty styrene type cation exchange resin, GSH, partial amino-acid and cation in solution are adsorbed, and wearIn fluid, mainly contain AMP, the about 50g of output.

Use the GSH on 0-0.8MNaCl gradient elution cationic ion-exchange resin, the about 55g of GSH output,Yield approximately 90%.

Under the condition that is 10.0 at 55 DEG C, pH, generate the successive reaction of GSH and AMP; After 6 hours,The growing amount that HPLC detects GSH is about 0.6g/L, and 90% above ATP is converted into AMP, and AMP generatesAmount is about 0.5g/L. HPLC testing conditions is with embodiment 3 steps (1). In this step, enzyme is recycledUtilize.

Embodiment 8 prepares GSH and AMP

(1) in retort, generate glutathione GSH and AMP:

In retort, the reaction system of 100L sterilized water is for containing substrate 5.0kg glutamic acid, 5.0kg halfCystine and 2.5kg glycine, and 15kgATP, 1.5kg sodium hydrogen phosphate, 2.24kg potassium chloride,The solution of 1.76kg sodium chloride, 1.07kg ammonium chloride, 2.03kg magnesium chloride hexahydrate and 1.7kg mono-water manganese chloride,When preparation, uniform stirring prevents precipitation. Regulate pH value to approximately 5.0, in reaction system, add 0.1kgGshF enzyme and 0.0033kgAdk enzyme start reaction. Between the stage of reaction, controlling pH value is 5.0, and temperature is 25 DEG C.

React after 7 hours, the growing amount that high performance liquid chromatography (HPLC) detects glutathione is about 30g/L,70% left and right ATP exhausts, and is converted into AMP, and AMP growing amount is about 60g/L. HPLC testing conditions is sameEmbodiment 3 steps (1).

(2) in filter, separate GshF enzyme and Adk enzyme:

(3) separated product GSH and AMP:

Use hydrochloric acid to regulate the pH value to 3.0 of filter liquor, in ion exchange column, pass through D001 large porous strong acidProperty styrene type cation exchange resin, GSH, partial amino-acid and cation in solution are adsorbed, and wearIn fluid, mainly contain AMP, the about 6kg of output.

Use the GSH on 0-0.8MNaCl gradient elution cationic ion-exchange resin, the about 2.8kg of GSH output,Yield approximately 90%.

Isolated enzyme in step (2) is added into retort via the refluxing opening of filter, and adds protoenzymeThe new enzyme of amount 5-15% reacts.

Under the condition that is 5.0 at 25 DEG C, pH, generate the successive reaction of GSH and AMP; After 7 hours,The growing amount that HPLC detects GSH is about 30g/L, and 70% left and right ATP is converted into AMP, and AMP generatesAmount is about 60g/L. HPLC testing conditions is with embodiment 3 steps (1). In this step, enzyme is recycled profitWith.

Embodiment 9 prepares GSH and AMP

(1) in retort, generate glutathione GSH and AMP:

In retort, the reaction system of 100L sterilized water is for containing substrate 2.8kg glutamic acid, 1.8kg halfCystine and 2.0kg glycine, and the solution of 5.1kgATP and 0.83kg mono-water manganese chloride, when preparationUniform stirring prevents precipitation. Regulate pH value to approximately 7.0, in reaction system, add 0.01kgGshFEnzyme and 0.001kgAdk enzyme start reaction. Between the stage of reaction, controlling pH value is 7.0, and temperature is 37 DEG C.

React after 6 hours, the growing amount that high performance liquid chromatography (HPLC) detects glutathione is about 28g/L,95% above ATP exhausts, and is converted into AMP, and AMP growing amount is about 30g/L. HPLC testing conditions is sameEmbodiment 3 steps (1).

(2) in filter, separate GshF enzyme and Adk enzyme:

(3) separated product GSH and AMP:

Use the GSH on 0-0.8MNaCl gradient elution cationic ion-exchange resin, the about 2.6kg of GSH output,Yield approximately 90%.

Under the condition that is 7.0 at 37 DEG C, pH, generate the successive reaction of GSH and AMP; After 6 hours,The growing amount that HPLC detects GSH is about 28g/L, and 95% above ATP is converted into AMP, and AMP generatesAmount is about 30g/L. HPLC testing conditions is with embodiment 3 steps (1). In this step, enzyme is recycled profitWith.

Embodiment 10 prepares GSH and AMP

Referring to Fig. 4, the process chart of GSH produced according to the present invention uses immobilized enzyme to make in accordance with the following stepsStandby GSH and AMP:

(1) in reaction column, generate glutathione GSH and AMP:

Preparation reactant liquor, it is sweet that every 100L contains substrate 2.8kg glutamic acid, 1.8kg cysteine and 2.0kgPropylhomoserin, and 5.1kgATP, 1.2kg sodium hydrogen phosphate and 1.0kg magnesium chloride hexahydrate, evenly stir when preparationMix and prevent precipitation, regulate pH value to approximately 7.5, temperature is upgraded to 37-40 DEG C.

Pack the mixing immobilized enzyme 50kg in above-described embodiment 4 into reaction column equipment, make enzyme after draining bubbleReaction column. Use constant flow pump that reactant liquor is from bottom to top slowly pumped into by enzyme reaction post with 16-20L/h flow velocity,Between the stage of reaction, controlling temperature is 37 DEG C. React after approximately 6 hours, collect reactant liquor, high performance liquid chromatography (HPLC)The growing amount that detects glutathione is about 27g/L, and 95% above ATP exhausts, and is converted into AMP, AMPGrowing amount is about 30g/L. HPLC testing conditions is with embodiment 3 steps (1).

(2) separated product GSH and AMP:

Use hydrochloric acid to regulate the pH value to 3.0 of the reactant liquor of above-mentioned collection, in ion exchange column, pass through D001Large hole strong acid styrene system cation exchange resin, GSH, partial amino-acid and cation quilt in solutionAbsorption, passes and in liquid, mainly contains AMP, simply direct condensing crystallizing after desalination, the about 2.9kg of output.

Use the GSH on 0-0.8MNaCl gradient elution cationic ion-exchange resin, the about 2.5kg of GSH output,Yield approximately 90%.

The growing amount that HPLC detects GSH is about 27g/L, and 95% above ATP is converted into AMP, AMPGrowing amount is about 30g/L. HPLC testing conditions is with embodiment 3 steps (1). In this step, enzyme is followedRing utilizes.

Embodiment 11 uses amino-acid salt to prepare GSH and AMP

(1) in retort, generate glutathione GSH and AMP:

In retort, the reaction system of 100L sterilized water is for containing substrate 3.2kg sodium glutamate, 2.3kgCysteine hydrochloride and 2.6kg Sodium Glycinate, and 5.1kgATP, 1.2kg sodium hydrogen phosphate, 0.7kgThe solution of potassium chloride, 0.6kg sodium chloride, 0.1kg ammonium chloride and 1.0kg magnesium chloride hexahydrate, even when preparationStirring prevents precipitation. Regulate pH value extremely approximately 7.0, in reaction system, add 0.01kgGshF enzyme and0.001kgAdk enzyme starts reaction. Between the stage of reaction, controlling pH value is 7.0, and temperature is 37 DEG C.

React after 6 hours, the growing amount that high performance liquid chromatography (HPLC) detects glutathione is about 30g/L,95% above ATP exhausts, and is converted into AMP, and AMP growing amount is about 30g/L. HPLC testing conditions is sameEmbodiment 3 steps (1).

(2) in filter, separate GshF enzyme and Adk enzyme:

(3) separated product GSH and AMP:

Under the condition that is 7.0 at 37 DEG C, pH, generate the successive reaction of GSH and AMP; After 6 hours,The growing amount that HPLC detects GSH is about 30g/L, and 95% above ATP is converted into AMP, and AMP generatesAmount is about 30g/L. HPLC testing conditions is with embodiment 3 steps (1). In this step, enzyme is recycled profitWith.

As can be seen from the results: use glutamate, cysteine salt and glycinate substitute identical mole denseGlutamic acid, cysteine and the glycine of degree do not affect reaction result. In production, can use glutamate,Cysteine salt and glycinate substitute glutamic acid, cysteine and glycine.

Comparative example 1

In retort, the reaction system of 100L sterilized water is for containing substrate 2.8kg glutamic acid, 1.8kg halfCystine and 2.0kg glycine, and 10.0kgATP, 1.2kg sodium hydrogen phosphate, 0.7kg potassium chloride,The solution of 0.6kg sodium chloride, 0.1kg ammonium chloride and 1.0kg magnesium chloride hexahydrate, when preparation, uniform stirring preventsThere is precipitation. Regulate pH value to approximately 7.0, in reaction system, add 0.01kgGshF enzyme and start reaction.Between the stage of reaction, controlling pH value is 7.0, and temperature is 37 DEG C.

React after 6 hours, the growing amount that high performance liquid chromatography (HPLC) detects glutathione is about 28g/L,70% left and right ATP exhausts, and is converted into ADP and AMP. Please also refer to Fig. 7, it is reaction 6 hoursThe HPLC collection of illustrative plates of 10 times of dilute reaction solutions, in figure, not marking peak is amino acid. HPLC testing conditions is with realExecute example 3 steps (1).

As can be seen from the results: in comparative example 1, there is no coupling Adk enzyme, the ATP that therefore reaction system needsAmount increases. And in same time, GSH growing amount reduces. In addition, reaction generates a large amount of ADP, and this increases undoubtedlyAdd the difficulty of purifying.

With respect to comparative example 1, Optimization of preparation of the present invention reaction condition, can make product GSHGrowing amount be increased to 30-50g/L from 10-20g/L, and keep higher conversion ratio. In course of reaction, addA part of ADP of producing can make to generate GSH time of Adk enzyme be converted into ATP and continue to use, ATP consumptionCan reduce 30-50%, significantly reduce the consumption of ATP, meanwhile, can generate large by catalysis another part ADPMeasure AMP, be easy to the purifying in later stage, in course of reaction, prepare a large amount of AMP simultaneously. AMP is doctorPrescription face, has significant peripheral vasodilation and hypotensive effect; Aspect food interpolation, can be used as bitter taste and coverLid agent is used, and has certain market value.

Although with embodiment openly as above, so it is not intended to limit the present invention in the present invention, any this areaTechnical staff, without departing from the spirit and scope of the present invention, all can do various selection and amendment,Therefore protection scope of the present invention is limited by claims and equivalents thereof.

Claims

1. enzyme process is prepared a method for glutathione and adenylate simultaneously, it is characterized in that, described method bagDraw together following steps:

(1) utilize GshF enzyme and Adk enzyme, in retort, generate glutathione and adenylate;

(2) in retort, separate immobilized GshF enzyme and Adk enzyme, or use filter plant to separate tripFrom GshF enzyme and Adk enzyme; And

(3) separated product glutathione and adenylate.

2. enzyme process according to claim 1 is prepared the method for glutathione and adenylate simultaneously, its featureBe, described method is further comprising the steps:

(4) recycling of the GshF enzyme separating in described step (2) and Adk enzyme, is about to separateGshF enzyme and Adk enzyme are added into the successive reaction that generates glutathione and adenylate and regeneration ATP in retort;And

(5) continuous separate of GshF enzyme and Adk enzyme from, continuous separate is from immobilized GshF enzyme and AdkGshF enzyme and the Adk enzyme of enzyme or the continuous separated free of use filter plant.

3. enzyme process according to claim 2 is prepared the method for glutathione and adenylate simultaneously, its featureBe, described method is further comprising the steps:

(6) continuous separate of product glutathione from.

(7) continuous separate of product adenylate from.

4. enzyme process according to claim 3 is prepared the method for glutathione and adenylate simultaneously, its featureBe, recycle GshF enzyme and Adk enzyme, repeating said steps (4) to described step (7) at leastOnce or repeatedly.

5. prepare the method for glutathione and adenylate according to the enzyme process described in claim 3 or 4 simultaneously, itsBe characterised in that, the glutathione that the reaction of step (6) and step (7) generates and adenylate by filter or fromSub-switching method separates.

6. enzyme process according to claim 1 is prepared the method for glutathione and adenylate simultaneously, its featureBe, the reaction condition that generates glutathione and adenylate in described step (1) in retort is as follows:

Reaction temperature is 25-55 DEG C;

Under the condition that reaction pH is 5-10;

Reaction system comprises following substrate: ATP, glutamic acid or its salt, cysteine or its salt and glycineOr its salt;

Reaction system comprises following ion: one or both combinations of magnesium ion and manganese ion;

In above-mentioned reaction system, add GshF enzyme and Adk enzyme, in retort, generate glutathione and adenosineAcid.

7. enzyme process according to claim 6 is prepared the method for glutathione and adenylate simultaneously, its featureBe, described reaction system also comprises following ion: potassium ion, sodium ion, ammonium ion, Tris and phosphate radicalOne or more of ion.

8. enzyme process according to claim 6 is prepared the method for glutathione and adenylate simultaneously, its featureBe, the mass ratio of described GshF enzyme and Adk is (0.2-30): 1, and described GshF enzyme concentration is 0.002-1g/L.

9. enzyme process according to claim 6 is prepared the method for glutathione and adenylate simultaneously, its featureBe, the mass ratio that described glutamic acid, cysteine, glycine add is (1-2.5): 1:(0.5-1.5), described inSemicystinol concentration is 1-50g/L.

10. enzyme process according to claim 1 is prepared the method for glutathione and adenylate simultaneously, its featureBe, in described step (2), immobilized GshF enzyme and Adk enzyme are by absorption, crosslinked, covalent bondBe fixed on fixation support with one or more modes of embedding; Described fixation support is selected from macromolecule and carriesOne or more combinations of body, inorganic carrier and magnetic macromolecular microsphere carrier.