CN105861598A - Method for regenerating ATP (adenosine triphosphate) by enzyme process and application thereof - Google Patents
Method for regenerating ATP (adenosine triphosphate) by enzyme process and application thereof Download PDFInfo
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Abstract
The invention discloses a method for regenerating ATP (adenosine triphosphate) by an enzyme process. The method comprises the following steps: (1) preparing and obtaining ATP regeneration enzymes; (2) regenerating ATP in a reaction solution; and (3) separating the product from the ATP regeneration enzymes. By adopting the novel ATP regeneration enzymes Ppk, Adk and Pap, the ATP consumed in the enzymatic reaction can be cyclically regenerated so as to greatly lower the ATP consumption, and the regeneration process is simple and efficient and has the advantages of controllable reaction and high stability. The substrate polyphosphoric acid or salt thereof added in the reaction has the advantages of low price and low pollution. In the enzymatic reaction, the expensive ATP is not needed for initiating the reaction, and only the ADP (adenosine diphosphate) or cheap AMP (adenosine monophosphate) is added. The ATP regeneration enzyme recovery system is established, and thus, the method is suitable for industrialized large-scale production.
Description
Technical field
The present invention relates to biological technical field, particularly to method and the application thereof of a kind of enzyme process regeneration ATP.
Background technology
Adenosine triphosphate (ATP) is made up of an adenosine (A) and three phosphate groups (Pi), and molecular weight is 507, molecular formula C10H16N5O13P3.It is transducer and the reservoir of bio-energy, in the enzymatic reaction relating to energy, plays irreplaceable important function.ATP molecule is A-Pi～Pi～Pi structure, away from adenosine two phosphate bonds by～represent, for energy-rich phosphate bond.In course of reaction, the energy-rich phosphate bond of ATP molecule can rupture and discharge big energy: when an energy-rich phosphate bond fission, ATP molecule is converted into adenosine diphosphate (ADP) (ADP), and release energy 30.5kJ/mol；When second energy-rich phosphate bond fission, ADP molecule is converted into adenylic acid (AMP), and can release energy 27.6kJ/mol again.In major part enzymatic reaction, ATP is by being hydrolyzed to ADP energy supply, but in actual production, the ADP in reaction system may proceed to hydrolysis and produces AMP.
Due to current ATP high expensive, the most directly ATP is used to carry out enzymatic reaction income little.It addition, a large amount of interpolation of ATP causes course of reaction precipitate occur, add the purification difficulty of late-stage products simultaneously.Therefore, it is possible to a small amount of ATP put in reaction produces, by setting up stable, effective ATP regenerating system, ATP is made to recycle the research direction being to carry out industrialized production currently with ATP in course of reaction.
The method regeneration such as substrate level phosphorylation, oxidative phosphorylation and photophosphorylation that ATP mainly passes through, the enzyme system carrying out catalytic action is usually relatively complex, and needs to carry out in the competent cell containing organized enzyme system or competent cell device.The method that industrial ATP regeneration is commonly used is to utilize saccharomycetic glycolytic pathway, carries out substrate level phosphorylation mode and regenerates ATP.Although the method regeneration effect is good, but complicated through the course of reaction of yeast cells enzyme system catalytic regeneration ATP, and the enzyme participating in catalytic reaction is numerous, and course of reaction is wayward, and between product batches, quality differs greatly.Yeast enzyme system quality is often because the producer of supply is different, batch difference, even season are different and have the biggest difference simultaneously.Additionally course of reaction needs to add substantial amounts of yeast cells enzyme liquid, introduces the much impurity such as albumen, pigment and brings certain difficulty to later-period purification.In recent years, the research emphasis of ATP regeneration turns to use single enzyme or better simply enzyme system, it is thus achieved that the regeneration effect of efficient stable.Wherein, the enzyme such as Acetate kinase, ammonia kinase, pyruvate kinase all can effective regeneration ATP.But, the substrate that these enzymes are utilized is worth costliness, the phosphoenolpyruvic acid utilized such as pyruvate kinase, and the by-product generated has certain bio-toxicity and contaminative, if the product of Acetate kinase, ammonia kinase catalytic reaction is respectively acetic acid and ammonia, the most difficult use the most in a large number.
There are some researches show, exist in some bacterial body and utilize polyphosphoric acids or the enzyme system of its salt regeneration ATP.This enzyme system includes polyphosphoric acids kinases (EC 2.7.4.1, Ppk), adenylic acid kinases (EC 2.7.4.3, Adk) and polyphosphoric acids-adenylic acid phosphotransferase (EC 2.7.4.-, Pap), the present invention is referred to as three kinds of enzymes for " ATP regeneration enzyme ".Wherein, Ppk catalysis ADP generates ATP with polyphosphoric acids or its reactant salt, Adk is catalyzed 2 molecule ADP and generates 1 molecule ATP and 1 molecule AMP, and Pap is then catalyzed AMP and generates ADP with polyphosphoric acids or its reactant salt, and the reasonable combination of three kinds of enzymes is used equally to synthesize ATP (seeing Fig. 1).It addition, Ppk enzyme has two kinds of multi-forms of Ppk1 and Ppk2, Ppk1 has ATP regeneration at polyphosphoric acids or its salt degree of polymerization time higher, and Ppk2 may utilize polyphosphoric acids or its salt regeneration ATP of low polymerization degree, and two kinds of Ppk enzymes are used equally to ATP regeneration.Using the ATP in the mode regenerative response of Ppk, Adk and Pap enzyme combination, reaction substrate is cheap, and product pollution is relatively small, and the impact on reaction enzyme is less, therefore, it can be developed for industrialized production.
Summary of the invention
The invention provides method and the application thereof of a kind of enzyme process regeneration ATP, it, by using Ppk, Adk and Pap tri-kinds " ATP regeneration enzyme " regeneration ATP, overcomes the drawbacks described above of prior art.
The technical problem to be solved is achieved through the following technical solutions:
The method of a kind of enzyme process regeneration ATP, comprises the following steps:
(1) ATP regeneration enzyme is prepared:
Obtain ATP regeneration enzyme by genetic engineering modified, fermentation, purification, or obtain ATP regeneration enzyme with alternate manners such as natural extractions.ATP regeneration enzyme can make enzyme liquid or dry powder with the form of resolvase；Also can be further secured on fixation support, prepare immobilization ATP regeneration enzyme.
(2) regeneration of ATP in reactant liquor:
In enzymatic reaction system, add the one in ATP, ADP or AMP, any two or three combination by demand, carry out enzymatic reaction.Meanwhile, add the one of three kinds of ATP regeneration enzyme, any two or three combination in proportion, and add polyphosphoric acids or its salt is phosphodonor, carry out ATP regenerative response.Reaction system also comprises magnesium ion and/or manganese ion, one or more in potassium ion, sodium ion, ammonium ion, Tris or phosphate anion.Substrate, enzyme and all kinds of salt that the present invention adds can disposably add reaction system, it is possible to add according to industrial manufacture process flow process stream in batches and fill into.
(3) product and ATP regeneration enzyme are separated:
Immobilized ATP regeneration enzyme is directly separated in retort.Above-mentioned separation can be separated by filter bag, it is possible to is directly separated in reaction column.Or
Free ATP regeneration enzyme is by Ultra filtration membrane in filter.Wherein, filter has charging aperture, discharging opening and refluxing opening, inside sets the ultrafilter membrane retained.Being the enzyme liquid reclaimed through the trapped fluid of filter, filter liquor is to isolate after enzyme the reactant liquor containing product.
The ATP regeneration enzyme of recovery is repeatable is used in step (2).
Preferably, in technique scheme, ATP regeneration enzyme described in step (1) is selected from polyphosphoric acids kinases (EC 2.7.4.1, Ppk, including Ppk1 enzyme and Ppk2 enzyme), adenylic acid kinases (EC 2.7.4.3, Adk), polyphosphoric acids-adenylic acid phosphotransferase (EC 2.7.4.-, Pap) one in, any two or three combination, i.e. it is used alone Ppk, Adk or Pap, or use Ppk and Adk combination, Adk and Pap combination or Ppk and Pap combination, or the combination of Ppk, Adk and Pap tri-kinds.It is furthermore preferred that any two or three combination in above-mentioned three kinds of ATP regeneration enzyme, i.e. use Ppk and Adk combination, Adk and Pap combination or Ppk and Pap combination and the combination of Ppk, Adk and Pap tri-kinds.
Wherein, when selecting two kinds of enzyme combinations of Ppk Yu Adk, Ppk Yu Adk activity ratio is (10-0.1): 1；
Wherein, when selecting two kinds of enzyme combinations of Ppk Yu Pap, the activity ratio of Ppk Yu Pap is (10-0.1): 1；
Wherein, when selecting two kinds of enzyme combinations of Pap Yu Adk, Pap Yu Adk activity ratio is (10-0.1): 1；
Wherein, when selecting tri-kinds of enzyme combinations of Ppk, Pap and Adk, Ppk, Pap and Adk activity ratio are (10-0.1): (10-0.1): 1.
Above-mentioned Ppk, Adk, Pap can derive from any biology or through the artificial reconstructed enzyme with same catalysis.Ppk can be Ppk1 enzyme and/or Ppk2 enzyme.
Preferably, in technique scheme, step (1) described fixation support is selected from one or more in macromolecule carrier, inorganic carrier, magnetic macromolecular microsphere carrier.Wherein, macromolecule carrier is selected from cellulose, glucose gel, agarose, polyacrylamide, polyamino acid, polystyrene, polyacrylic acid, sodium alginate, chitosan, starch, polyvinyl alcohol, gelatin, carrageenan, nylon or synthetic high polymer etc.；Inorganic carrier is selected from cellular glass, silicon oxide, silica gel, activated carbon or kieselguhr etc..
Preferably, in technique scheme, described immobilization ATP regeneration enzyme is fixed on fixation support in the following manner: adsorbs, embed, covalency, combine, cross-link or a combination thereof.Wherein, tri-kinds of enzymes of Pap, Adk, Ppk can be fixed after fixing or be mixed in proportion respectively together.
Preferably, in technique scheme, step (2) ATP regenerative response condition is as follows:
Reaction temperature is 25-60 DEG C, and preferable temperature is 30-50 DEG C；
Reaction pH be 5-10, preferably pH be 6-9.
Enzymatic reaction system includes: the one in ATP, ADP and AMP, any two or three combination, and three kinds of materials, according to enzymatic reaction demand, add according to arbitrary proportion；
Regeneration reaction system includes: polyphosphoric acids or its salt, including one or both in magnesium ion, manganese ion, ammonium ion, potassium ion, sodium ion one or more, Tris or phosphate anion.
Preferably, in technique scheme, substrate, enzyme and all kinds of salt that the present invention adds can disposably add reaction system, it is possible to add according to industrial manufacture process flow process stream in batches and fill into.
Preferably, in technique scheme, polyphosphoric acids or the molar concentration of its salt that the present invention adds are react ATP, ADP and AMP molar concentration summation added 0.01-40 times；Magnesium ion concentration is 0.01-0.2M；Manganese ion concentration is 0.01-0.15M；Potassium concentration is 0.01-0.5M；Na ion concentration is 0.01-0.5M；Ammonium concentration is 0.005-0.2M；Tris concentration is 0.01-0.1M, phosphate concn is 0.01-0.1M.
Preferably, in technique scheme, polyphosphoric acids or its salt are selected from one or more in sodium polyphosphate, potassium polyphosphate, ammonium polyphosphate, hexa metaphosphoric acid (sodium), four polyphosphoric acids (sodium) and tripolyphosphate (sodium)；Magnesium ion is selected from one or more in magnesium chloride, magnesium sulfate, magnesium sulfite and magnesium nitrate；Manganese ion is selected from one or more in manganese chloride and manganese sulfate；Potassium ion is selected from one or more in potassium chloride, potassium sulfate, potassium nitrate, potassium hydroxide, potassium sulfite, potassium carbonate, potassium bicarbonate, potassium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium citrate；Sodium ion is selected from one or more in sodium chloride, sodium sulfate, sodium nitrate, sodium hydroxide, sodium sulfite, sodium carbonate, sodium bicarbonate, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium citrate；Ammonium ion is selected from one or more in ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia, ammonium carbonate, ammonium hydrogen carbonate, diammonium phosphate, ammonium dihydrogen phosphate and ammonium acetate.
The ultrafilter membrane used in the inventive method step (3) is selected from cellulose acetate membrane, polysulfone membrane, polyacrylonitrile film, polychloroethylene film, polyvinylidene fluoride film, polyamide membrane or ceramic membrane.
The application of the method for a kind of enzyme process regeneration ATP, described method, for the enzymatic reaction of multiple dependency ATP, is expressed for synthetic and acellular albumen.
Preferably, in technique scheme, the enzymatic reaction that the enzymatic reaction of described dependency ATP is participated in by the transferring enzyme of transfer phosphate groups, and the enzymatic reaction that part ligase is participated in.
Preferably, in technique scheme, the enzymatic reaction that the transferring enzyme of described transfer phosphate groups is participated in is enzymatic clarification phosphagen, L-Arginine phosphate., 6-hexosephosphate, fructose 1,6-diphosphate, glycerol 3-phosphate, oxidized form of nicotinamide-adenine dinucleotide I, the reaction of AMP, CT (D) P, GT (D) P, UT (D) P；The reaction that enzymatic reaction is enzymatic clarification S-acetyl-coenzyme-A, carnosine, enterochelin., glutamine and L-thiamine that described part ligase is participated in.
Technique scheme of the present invention, has the advantages that
(1) using tri-kinds of ATP regeneration enzyme of novel Ppk, Adk, Pap, the ATP consumed in enzymatic reaction by circular regeneration, thus can be substantially reduced the usage amount of ATP.And regenerative process is simply efficient, reaction is easily controlled, and stability is high.
(2) reaction need to be added substrate polyphosphoric acids or its salt are cheap, pollute little, and the impact on reaction enzyme is less.
(3) enzymatic reaction need not add the ATP initial action of high price, adds ADP or cheap AMP, and any two or three combination of three kinds of materials all can be normally carried out catalytic reaction.
(4) ATP regeneration enzyme recovery system is established, it is adaptable to industrialization large-scale production.
In addition, ATP regenerating system described in the present invention can be applicable to the enzymatic reaction of multiple dependency ATP, especially shift the enzymatic reaction that the transferring enzyme (EC 2.7) of phosphate groups is participated in, such as: phosphagen, L-Arginine phosphate., 6-hexosephosphate, 1, fructose-1, 6-diphosphate, glycerol 3-phosphate, oxidized form of nicotinamide-adenine dinucleotide I, AMP, CT (D) P, GT (D) P, the synthetic reaction of the materials such as UT (D) P, and the enzymatic reaction that part ligase (EC 6) is participated in, such as: S-acetyl-coenzyme-A, carnosine, enterobactin, the synthetic reaction of the material such as glutaminase and L-thiamine.Catalysis enzyme and substrate needed for the reaction of above-mentioned enzyme' s catalysis see table 1.Catalysis enzyme listed by table 1 can derive from any biology or through the artificial reconstructed enzyme with same catalysis, commercially available.
Finally, during ATP regenerating system described in the present invention applies also for the new technique that acellular albumen expression etc. consumes bio-energy.
Enzymatic reaction catalysis enzyme that table 1. is involved in the present invention and substrate
Accompanying drawing explanation
Fig. 1 is the schematic diagram of tri-kinds of enzyme regeneration ATP of Ppk, Adk, Pap of the present invention.
Fig. 2 is the SDS-PAGE figure of Ppk, Adk, Pap enzyme expressed by the present invention.
Fig. 3 is the reaction process flow chart that the present invention uses free ATP regeneration enzyme.
Fig. 4 is the reaction process flow chart that the present invention uses immobilization ATP regeneration enzyme.
Fig. 5 is creatine and the concentration change figure of phosphagen that the present invention is applied to that phosphagen produces.
Fig. 6 is the SDS-PAGE figure that the present invention is applied to acellular expression Adk enzyme.
Detailed description of the invention
Below the specific embodiment of the present invention is described in detail, in order to be further appreciated by the present invention.
Embodiment 1Ppk, the preparation of Adk and Pap enzyme
Ppk (including Ppk1 and Ppk2) in the inventive method, Adk and Pap enzyme can be commercially available, or through the artificial reconstructed enzyme with same catalysis.
The preparation process of Ppk1, Ppk2, Adk and Pap enzyme is as follows:
According to the sequence of four kinds of enzyme genes, design four, to amplimer, is synthesized by calm and peaceful Bioisystech Co., Ltd of Sino-U.S., and primer sequence is as follows:
Ppk1 sense primer: 5 '-CCATATGGGTCAGGAAAAGCTATACATCG-3 '；
Ppk1 antisense primer: 5 '-CGGATCCTTATTCAGGTTGTTCGAGTGATT-3 '；
Ppk2 sense primer: 5 '-TACATATGGCCAGCCCGGCGCAGAAG-3 '；
Ppk2 antisense primer: 5 '-TGAATTCAGGCCGGGATATCCAGGTTC-3 '；
Adk sense primer: 5 '-CCATATGCGTATCATTCTGCTTGGCGCTCCGG-3 '；
Adk antisense primer: 5 '-CGGATCCTTAGCCGAGGATTTTTTCCAGATC-3 '；
Pap sense primer: 5 '-GCCATGGATACAGAAACGATCGCCAGTGCAG-3 '；With
Pap antisense primer: 5 '-CGGATCCTTAATCCGTGTCGCGATCCGCTT-3 '；
Extract escherichia coli (Escherichia coli) K12 bacterial strain (be purchased from Tian Gen biochemical technology company limited) DNA, with it as template, amplify ppk1 Yu adk genetic fragment by PCR, and be respectively connecting to pET 22b carrier (being purchased from Novagene company)；Extract Pseudomonas aeruginosa (Pseudomonas aeruginosa) bacterial strain (CICC 10419) DNA, with it as template, amplify ppk2 genetic fragment by PCR, and be connected to pET22b carrier (being purchased from Novagene company)；Extract Acinetobacter johnsonii (Acinetobacter johnsonii) bacterial strain (CGMCC 1.8030) DNA, with it as template, amplify pap genetic fragment by PCR, and be connected to pET22b carrier (being purchased from Novagene company).4 sections of catenation sequences, after order-checking is correct, proceed to E.coli BL21 (DE3) bacterial strain (be purchased from Tian Gen biochemical technology company limited) respectively.
E.coli BL21 (DE3) monoclonal after converting accesses LB culture medium, cultivate to logarithmic (log) phase, after addition 1mM isopropyl-beta D-thio galactopyranoside (IPTG) induces 5 hours, collect thalline, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) screening high expressed bacterial strain.
The high expressed bacterial strain filtered out aseptically is accessed seed culture medium, cultivate to exponential phase and access in the fermentation tank containing 5L fermentation medium, cultivate to exponential phase and access in the fermentation tank containing 50L fermentation medium, after adding 1mM IPTG induction after cultivating 5 hours 5 hours, centrifugal collection thalline about 1kg.
Wherein LB medium component is: 1% peptone, 0.5% yeast powder and 1%NaCl；Seed culture based component is: 1% peptone, 0.5% yeast powder and 1% sodium chloride；Fermentation medium components is: 1% peptone, 0.5% yeast powder, 1% sodium chloride, 5% disodium hydrogen phosphate, 1% sodium dihydrogen phosphate, 0.01% magnesium sulfate and 1% glycerol.
The thalline of results, respectively after ultrasonic or high-pressure homogenization break bacterium, is centrifuged and collects supernatant.Then the saturated ammonium sulfate of 40-60%, centrifugal collecting precipitation are added.After Tris pH of buffer 8.0 is dissolved, use G25 post (being purchased from medical treatment Biology Science Co., Ltd of General Electric) desalination, then the resolvase through the available preliminary purification of CM-or DEAE-Sepharose FF (being purchased from medical treatment Biology Science Co., Ltd of General Electric) chromatography.
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 2 is Ppk1 enzyme, about 60kDa；Swimming lane 3 is Ppk2 enzyme, about 45kDa；Swimming lane 4 is Adk enzyme, about 25kDa；Swimming lane 5 is Pap enzyme, about 55kDa.
The method using the known mensuration enzymatic activity of prior art record, detect that 1mg/ml Ppk1, Ppk2, Adk and Pap enzyme liquid activity respectively may be about 100U, 500U, 1000U and 800U, be wherein 1 active unit (U) by 1 μM of substrate complete transformation definition in 1 minute.
Embodiment 2 enzymatic method produces phosphagen coupling ATP regenerating system
Fig. 3 is that the inventive method uses resolvase to carry out enzymatic reaction the process chart of coupling ATP regenerating system.Seeing Fig. 3, the operating procedure that enzyme process prepares the free ATP regenerating system of phosphagen coupling simultaneously is as follows:
(1) in retort, synthesize phosphagen and regenerate the reaction of ATP:
In retort, the reaction system of 100L sterilized water is containing substrate 1.5kg creatine, and 1.0kg ATP, 0.5kg disodium hydrogen phosphate, 0.38kg potassium chloride, 0.3kg sodium chloride, 0.2kg ammonium sulfate, 1.0kg magnesium chloride hexahydrate and the solution of 2.0kg sodium tetrapolyphosphate, during preparation, uniform stirring prevents precipitation.Regulation pH value, to about 7.5, adds 500U/L creatine kinase and 500U/L Ppk2 enzyme, 500U/L Adk enzyme starts reaction in reaction system.During reaction, control ph is 7.5, and temperature is 35 DEG C.
Fig. 5 is the surplus every 1 hour high performance liquid chromatography (HPLC) detection creatine and the growing amount of phosphagen.After reacting 5 hours, phosphagen growing amount is about 22g/L, and about more than 50% ATP is converted into ADP, AMP.HPLC testing conditions is: Kromasil C18 chromatographic column (be purchased from AKZO NOBEL company) (150 × 4.6mm), detection wavelength 210nm, detection temperature 25 DEG C, detection flow velocity 1ml/min, flowing is containing 20mM potassium dihydrogen phosphate, 0.1%TFA and the aqueous solution of 5% acetonitrile mutually.
(2) creatine kinase and ATP regeneration enzyme are separated in the filter:
Pass through hyperfiltration process, the reactant liquor of the reaction system of step (1) is filtered through filter and separates creatine kinase and ATP regeneration enzyme, filter built-in film bag (is purchased from Pall company, molecular cut off 8kDa), filter liquor is to isolate the reactant liquor after enzyme, containing materials such as phosphagen, ATP, ADP, AMP and salt, can be further purified by modes such as ion-exchange chromatographies.
Detect that the activity of the creatine kinase of recovery, Ppk2 enzyme and Adk enzyme is relatively reacted front and reduced 5%-20%, after corresponding new enzyme can be added, be re-used for step (1) reaction.
Embodiment 3 enzymatic method produces 1,6-fructose diphosphate coupling ATP regenerating system (immobilized enzyme)
Fig. 4 is that the inventive method uses immobilized enzyme to carry out enzymatic reaction production the process chart of coupling ATP regenerating system.Seeing Fig. 4, the operating procedure that immobilized enzyme method prepares fructose 1,6-diphosphate coupling ATP regenerating system simultaneously is as follows:
(1) catalysis enzyme and ATP regeneration enzyme is fixing:
Catalysis enzyme fructokinase (FK) and phosphofructokinase (PFK), by commercially available, are together fixed with business-like epoxy radicals fixation support LX1000EP with ATP regeneration enzyme Ppk2 enzyme and the Pap enzyme of preliminary purification in embodiment 1.
According to the mixing of specific activity 1:1:1:1, FK, PFK, Ppk2 and Pap enzyme is made into mixed enzyme solution 10L, and in enzyme liquid, each enzymatic activity is each about 200U.Adding LX1000EP wet carrier 3kg in constant temperature agitator tank to mix with above-mentioned enzyme liquid, under the conditions of 20 DEG C, 150rpm stirs 12 hours.Carrier is collected by filtration, cleans 2 times with 0.02M pH 8.0 kaliumphosphate buffer, obtain immobilization mixed enzyme.After FK, PFK, Ppk2 and Pap enzyme immobilization, activity is reduced to the 30-40% of former activity.
(2) in reaction column, 1,6-fructose diphosphate is generated:
Preparation reactant liquor, every 50L contains substrate 0.9kg fructose, and 0.3kgATP, 0.2kgAMP, 0.15kg Tris, 0.1kg ammonium chloride, 0.6kg magnesium sulfate and the solution of 1.5kg sodium hexameta phosphate, and during preparation, uniform stirring prevents precipitation.Regulation pH value is to about 7.0, and temperature is upgraded to 35-40 DEG C.
Mixing immobilized enzyme about 3kg in above-mentioned steps (1) is loaded reaction column, after draining bubble, prepares enzyme reaction post.Using constant flow pump that with 15L/h flow velocity, reactant liquor is the most slowly pumped through enzyme reaction post, controlling temperature during reaction is 37 DEG C.After circular response about 6 hours, collecting reactant liquor, the growing amount of detection fructose 1,6-diphosphate is about 30g/L, and more than 70% ATP is converted into ADP, AMP.
Immobilized enzyme circular response more than 20 times or-4 DEG C stores January time more than, and enzymatic activity reduces about 10-15%, need to add in proportion or the new enzyme of removable parts.
Embodiment 4 enzymatic method produces carnosine coupling ATP regenerating system
Seeing Fig. 3, the operating procedure that enzyme process prepares the free ATP regeneration enzyme regeneration ATP of carnosine coupling simultaneously is as follows:
(1) in retort, synthesize carnosine and regenerate the reaction of ATP:
In retort, the reaction system of 100L sterilized water is containing substrate L-Histidine 1.8kg, Beta-alanine 1.0kg, and 2.0kgAMP, 0.6kg disodium hydrogen phosphate, 0.38kg potassium chloride, 0.3kg sodium chloride, 1.0kg magnesium chloride hexahydrate and the solution of 5.0kg sodium tetrapolyphosphate, during preparation, uniform stirring prevents precipitation.Regulation pH value, to about 7.0, adds 800U/L carnosine synthetase and 600U/L Adk enzyme, 600U/L Pap enzyme starts reaction in reaction system.During reaction, control ph is 7.0, and temperature is 37 DEG C.
After reacting 6 hours, carnosine growing amount is about 20g/L, and about more than 80% AMP is converted into ADP, ATP.HPLC testing conditions is: Kromasil C18 chromatographic column (be purchased from AKZO NOBEL company) (150 × 4.6mm), detection wavelength 210nm, detection temperature 25 DEG C, detects flow velocity 0.8ml/min, and flowing is containing 80mM phosphate buffer and the aqueous solution of 15% methanol mutually.
(2) carnosine synthetase and ATP regeneration enzyme are separated in the filter:
Pass through hyperfiltration process, the reactant liquor of the reaction system of step (1) is filtered through filter and separates carnosine synthetase and ATP regeneration enzyme, filter built-in film bag (is purchased from Pall company, molecular cut off 8kDa), filter liquor is to isolate the reactant liquor after enzyme, containing materials such as carnosine, ATP, ADP, AMP and salt, can be further purified by modes such as ion-exchange chromatographies.
Detect that the activity of the carnosine synthetase of recovery, Adk enzyme and Pap enzyme is relatively reacted front and reduced 10%-20%, after corresponding new enzyme can be added, be re-used for step (1) reaction.
Embodiment 5 enzymatic method produces theanine coupling ATP regenerating system
Seeing Fig. 3, the operating procedure that enzyme process prepares the free ATP regenerating system of theanine coupling simultaneously is as follows:
(1) in retort, synthesize theanine and regenerate the reaction of ATP:
In retort, the reaction system of 100L sterilized water is containing substrate 1.5kg sodium glutamate, 0.7kg ethylamine hydrochloride, and 1.0kg ATP, 0.5kg disodium hydrogen phosphate, 1.0kg magnesium chloride hexahydrate, 0.5kg mono-water manganese chloride and the solution of 2.0kg sodium tetrapolyphosphate, during preparation, uniform stirring prevents precipitation.Regulation pH value, to about 7.0, adds 500U/L theanine synthetase in reaction system and 600U/L Adk enzyme starts reaction.During reaction, control ph is 7.0, and temperature is 30 DEG C.
After reacting 5 hours, theanine growing amount is about 14g/L, and about more than 95% ATP is converted into AMP.HPLC testing conditions is: Kromasil C18 chromatographic column (be purchased from AKZO NOBEL company) (150 × 4.6mm), detection wavelength 203nm, detection temperature 30 DEG C, detects flow velocity 1ml/min, and flowing is containing 0.05%TFA and the aqueous solution of 5% acetonitrile mutually.
(2) theanine and ATP regeneration enzyme are separated in the filter:
Pass through hyperfiltration process, the reactant liquor of the reaction system of step (1) is filtered through filter and separates theanine synthetase and Adk enzyme, filter built-in film bag (is purchased from Pall company, molecular cut off 8kDa), filter liquor is to isolate the reactant liquor after enzyme, containing materials such as theanine, AMP and salt, can be further purified by modes such as ion-exchange chromatographies.
Detect that the theanine synthetase of recovery and the activity of Adk enzyme reduce 5%-10% before relatively reacting, after corresponding new enzyme can be added, be re-used for step (1) reaction.
Embodiment 6 enzymatic method produces phosphagen coupling ATP regenerating system
Seeing Fig. 3, the operating procedure that enzyme process prepares the free ATP regeneration enzyme regeneration ATP of phosphagen coupling simultaneously is as follows:
(1) in retort, synthesize phosphagen and regenerate the reaction of ATP:
In retort, the reaction system of 100L sterilized water is containing substrate 0.5kg creatine, and 0.26kgATP, 0.4kgADP, 0.7kgAMP, 0.14kg disodium hydrogen phosphate, 0.07kg potassium chloride, 0.03kg ammonium chloride, 0.15kg mono-water manganese chloride and the solution of 2.0kg ammonium polyphosphate (calculating by average degree of polymerization 500), during preparation, uniform stirring prevents precipitation.Regulation pH value, to about 10.0, adds 100U/L creatine kinase and 100U/L Ppk1 enzyme, 1000U/L Pap enzyme starts reaction in reaction system.During reaction, control ph is 10.0, and temperature is 60 DEG C.
After reacting 6 hours, phosphagen growing amount is about 3g/L, and about more than 30% ATP is converted into ADP, AMP.HPLC testing conditions is with embodiment 2 step (1).
(2) creatine kinase and ATP regeneration enzyme are separated in the filter:
Pass through hyperfiltration process, the reactant liquor of the reaction system of step (1) is filtered through filter and separates creatine kinase and ATP regeneration enzyme, filter built-in film bag (is purchased from Pall company, molecular cut off 20kDa), filter liquor is to isolate the reactant liquor after enzyme, containing materials such as phosphagen, ATP, ADP, AMP and salt, can be further purified by modes such as ion-exchange chromatographies.
Detect that the activity of the creatine kinase of recovery, Ppk1 enzyme and Pap enzyme is relatively reacted front and reduced 30%-50%, after corresponding new enzyme can be added, be re-used for step (1) reaction.
Embodiment 7 enzymatic method produces phosphagen coupling ATP regenerating system
Seeing Fig. 3, the operating procedure that enzyme process prepares the free ATP regeneration enzyme regeneration ATP of phosphagen coupling simultaneously is as follows:
(1) in retort, synthesize phosphagen and regenerate the reaction of ATP:
In retort, the reaction system of 100L sterilized water is containing substrate 2.0kg creatine, and 0.30kg AMP, 1.2kg Tris, 2.92kg sodium chloride, 1.33kg ammonium sulfate, 4.1kg magnesium chloride hexahydrate and the solution of 21.0kg sodium hexameta phosphate, during preparation, uniform stirring prevents precipitation.Regulation pH value, to about 5.0, adds 800U/L creatine kinase and 1000U/L Ppk2 enzyme, 100U/L Adk enzyme, 800U/L Pap enzyme start reaction in reaction system.During reaction, control ph is 5.0, and temperature is 25 DEG C.
After reacting 10 hours, phosphagen growing amount is about 19g/L, and about more than 20% AMP is converted into ADP, ATP.HPLC testing conditions is with embodiment 2 step (1).
(2) creatine kinase and ATP regeneration enzyme are separated in the filter:
Pass through hyperfiltration process, the reactant liquor of the reaction system of step (1) is filtered through filter and separates creatine kinase and ATP regeneration enzyme, filter built-in film bag (is purchased from Pall company, molecular cut off 8kDa), filter liquor is to isolate the reactant liquor after enzyme, containing materials such as phosphagen, ATP, ADP, AMP and salt, can be further purified by modes such as ion-exchange chromatographies.
Detect that the activity of the creatine kinase of recovery, Ppk2 enzyme, Adk enzyme and Pap enzyme is relatively reacted front and reduced 15%-40%, after corresponding new enzyme can be added, be re-used for step (1) reaction.
Embodiment 8 enzymatic method produces phosphagen coupling ATP regenerating system
Seeing Fig. 3, the operating procedure that enzyme process prepares the free ATP regeneration enzyme regeneration ATP of phosphagen coupling simultaneously is as follows:
(1) in retort, synthesize phosphagen and regenerate the reaction of ATP:
In retort, the reaction system of 100L sterilized water is containing substrate 1.8kg creatine, and 4.0kg ATP, 0.6kg Tris, 1.05kg ammonium chloride, 2.0kg magnesium chloride hexahydrate and the solution of 0.4kg sodium polyphosphate (calculating by average degree of polymerization 50), during preparation, uniform stirring prevents precipitation.Regulation pH value, to about 6.5, adds 100U/L creatine kinase in reaction system and 300U/L Pap enzyme starts reaction.During reaction, control ph is 6.5, and temperature is 37 DEG C.
After reacting 7 hours, phosphagen growing amount is about 21g/L, and about more than 90% ATP is converted into ADP.HPLC testing conditions is with embodiment 2 step (1).
(2) creatine kinase and ATP regeneration enzyme are separated in the filter:
Pass through hyperfiltration process, the reactant liquor of the reaction system of step (1) is filtered through filter and separates creatine kinase and ATP regeneration enzyme, filter built-in film bag (is purchased from Pall company, molecular cut off 20kDa), filter liquor is to isolate the reactant liquor after enzyme, containing materials such as phosphagen, ATP, ADP and salt, can be further purified by modes such as ion-exchange chromatographies.
Detect that the creatine kinase of recovery and the activity of Pap enzyme reduce 10%-20% before relatively reacting, after corresponding new enzyme can be added, be re-used for step (1) reaction.
Embodiment 9 enzymatic method produces 1,6-fructose diphosphate coupling ATP regenerating system (immobilized enzyme)
Seeing Fig. 4, the operating procedure that immobilized enzyme method prepares fructose 1,6-diphosphate coupling ATP regenerating system simultaneously is as follows:
(1) catalysis enzyme and ATP regeneration enzyme is fixing:
Catalysis enzyme fructokinase (FK) and phosphofructokinase (PFK), by commercially available, are fixed with business-like epoxy radicals fixation support LX1000EP or the synthetic high polymer carrier LX1000HA Han amino respectively with ATP regeneration enzyme Adk enzyme and the Pap enzyme of preliminary purification in embodiment 1.
FK, PFK, Adk and Pap enzyme is made enzyme liquid, each 3L respectively, and 4 kinds of enzyme liquid activity are 200-400U.
Adding LX1000EP wet carrier 1kg and FK enzyme liquid 3L in constant temperature agitator tank to mix, under the conditions of 20 DEG C, 150rpm stirs 12 hours.Carrier is collected by filtration, cleans 2 times with 0.02M pH 8.0 kaliumphosphate buffer, obtain immobilization FK enzyme.Adk enzyme is fixed in the same way.
Adding LX1000HA wet carrier 1kg and PFK enzyme liquid 3L in constant temperature agitator tank to mix, under the conditions of 20 DEG C, 150rpm stirs 12 hours.Carrier is collected by filtration, cleans 2 times with 0.02M pH 8.0 kaliumphosphate buffer, obtain immobilization PFK enzyme.Pap enzyme is fixed in the same way.
(2) in reaction column, 1,6-fructose diphosphate is generated:
Preparation reactant liquor, every 50L contains substrate 1.1kg fructose, and 0.75kgATP, 0.25kg potassium dihydrogen phosphate, 0.2kg potassium chloride, 0.6kg magnesium sulfate and the solution of 1.25kg sodium tetrapolyphosphate, and during preparation, uniform stirring prevents precipitation.Regulation pH value is to about 8.0, and temperature is upgraded to 40-45 DEG C.
Immobilized enzyme in above-mentioned steps (1) is mixed and loads reaction column, altogether about 4kg, after draining bubble, prepare enzyme reaction post.Using constant flow pump that with 10L/h flow velocity, reactant liquor is the most slowly pumped through enzyme reaction post, controlling temperature during reaction is 42 DEG C.After circular response about 6 hours, collecting reactant liquor, the growing amount of detection fructose 1,6-diphosphate is about 36g/L, and more than 50% ATP is converted into ADP, AMP.
Immobilized enzyme circular response more than 20 times or-4 DEG C stores January time more than, and enzymatic activity reduces about 10-15%, need to add in proportion or the new enzyme of removable parts.
Embodiment 10 acellular albumen expresses Adk enzyme coupling ATP regenerating system
The operating procedure that acellular albumen expresses Adk enzyme coupling ATP regenerating system simultaneously is as follows:
(1) structure of expression vector pIVEX2.4d-adk:
According to the sequence of adk gene, designing pair for amplification primer, calm and peaceful Bioisystech Co., Ltd of Sino-U.S. synthesize, primer sequence is as follows:
Adk sense primer: 5 '-TCCATGGGTATCATTCTGCTTGGCGCTCCGG-3 '；With
Adk antisense primer: 5 '-CGGATCCTTAGCCGAGGATTTTTTCCAGATC-3 '；
With reference to method described by embodiment 1, PCR expands adk genetic fragment, after using Nco I and BamH I double digestion, is connected to pIVEX2.4d carrier (being purchased from Roche company), is built into pIVEX2.4d-adk carrier, correct through order-checking.
(2) prepared by E. coli cell free extract:
E.coli A19 (disappearance nuclease I gene, non-degradable exogenous gene) monoclonal is accessed LB culture medium, cultivates to exponential phase and access in the fermentation tank containing 5L fermentation medium, cultivate to OD600Reach centrifugal collection thalline when 3.LB culture medium and fermentation medium components see embodiment 1.
With the resuspended thalline of S30 buffer, 4 DEG C of centrifuge washings 3 times, collect thalline, be stored in-80 DEG C.S30 buffer components is: 14mM magnesium acetate, 60mM potassium acetate, 1mM dithiothreitol, DTT (DTT) and 10mM Tris (pH value 8.1).
Weighing thalline weight, it is molten that every 10 grams of thalline add 100ml S30 buffer weight, is simultaneously introduced 1mM DTT.Under 75kPa pressure, high pressure homogenize crushes thalline, adds 1mM DTT, 4 DEG C of centrifugal collection supernatants.In constant-temperature table 100rpm, 37 DEG C hatch 30 minutes, prepare E. coli cell free extract.
(3) cell-free extract expressing protein coupling ATP regenerating system is used
In 100 μ l protein expression systems, add 20 μ l step (2) described E. coli cell free extracts, add 15 μ g/ml step (1) described expression vector pIVEX2.4d-adk, add 15mM magnesium acetate, 50mM ammonium acetate, 50mM HEPES-potassium hydroxide (pH value 7.5), 2%PEG8000,2mM DTT, 0.33mM NAD+, 0.27mM coenzyme A, 4mM oxalic acid, 1U/ μ l T7 RNA polymerase, 10mM ATP, 1mM GTP, 1mM CTP, 1mM UTP and 20 kinds of each 2mM of aminoacid, be simultaneously introduced 1U/ μ l Adk enzyme, 1U/ μ l Pap enzyme and 10mM tetra-Quadrafos and be used as ATP and regenerate.In constant-temperature table 200rpm, 30 DEG C react 6 hours.
Fig. 6 is the SDS-PAGE figure of expressed Adk 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 Adk enzyme of acellular expression, about 25kDa.
Comparative example 1 enzymatic method produces phosphagen
The operating procedure that enzyme process prepares phosphagen is as follows:
In retort, the reaction system of 100L sterilized water is containing substrate 1.5kg creatine, and 6.0kg ATP, 0.5kg disodium hydrogen phosphate, 0.38kg potassium chloride, 0.3kg sodium chloride, 0.2kg ammonium sulfate and the solution of 1.0kg magnesium chloride hexahydrate, during preparation, uniform stirring prevents precipitation.Regulation pH value, to about 7.5, adds 500U/L creatine kinase in reaction system and starts reaction.During reaction, control ph is 7.5, and temperature is 35 DEG C.
After reacting 5 hours, phosphagen growing amount is about 15g/L, and about more than 90% ATP is converted into ADP, AMP.HPLC testing conditions is with embodiment 2 step (1).
Comparative example 2 is not it can be seen that have coupling to be suitable for the ATP regenerating system of Production by Enzymes, and the ATP amount that reaction system needs is greatly increased, and the product amount that reaction generates significantly reduces, thus increases production cost.
Comparative example 2 enzymatic method produces phosphagen coupling single ATP regeneration enzyme system
The operating procedure that enzyme process prepares phosphagen coupling single ATP regeneration enzyme system is as follows:
In retort, the reaction system of 100L sterilized water is containing substrate 1.5kg creatine, and 1.0kg ATP, 0.5kg disodium hydrogen phosphate, 0.38kg potassium chloride, 0.3kg sodium chloride, 0.2kg ammonium sulfate, 1.0kg magnesium chloride hexahydrate and the solution of 2.0kg sodium tetrapolyphosphate, during preparation, uniform stirring prevents precipitation.Regulation pH value, to about 7.5, adds 500U/L creatine kinase in reaction system and 2500U/L Ppk2 enzyme starts reaction.During reaction, control ph is 7.5, and temperature is 35 DEG C.
After reacting 6 hours, phosphagen growing amount is about 20g/L, and about more than 90% ATP is converted into ADP, AMP.HPLC testing conditions is with embodiment 2 step (1).
Comparative example 2 and comparative example 1 be not it can be seen that the regeneration effect of system of coupling a kind of ATP regeneration enzyme has the effective of two kinds or three kinds ATP regeneration enzyme combined systems of coupling: the overall enzyme amount needed of reaction needs to increase, and generation product amount has reduced, the response time increases.But the advantage of the method is to reduce the usage amount of ATP, makes the part of the ATP in reaction be regenerated, thus reduce production cost.The present invention uses tri-kinds of ATP regeneration enzyme of novel Ppk, Adk, Pap, and the ATP consumed in enzymatic reaction by circular regeneration, thus can be substantially reduced ATP consumption, and regenerative process is simply efficient, and reaction is easily controlled, and stability is high；Secondly, substrate polyphosphoric acids or its salt that reaction need to be added are cheap, pollute little, and the impact on reaction enzyme is less；Again, enzymatic reaction need not add the ATP initial action of high price, adds ADP or cheap AMP, and any two or three combination of three kinds of materials all can be normally carried out catalytic reaction；Finally, ATP regeneration enzyme recovery system is established, it is adaptable to industrialization large-scale production.
ATP regenerating system described in the present invention applies also for the enzymatic reaction of multiple dependency ATP.Also apply also in the new technique that acellular albumen synthesis etc. consumes bio-energy.
Although the present invention is open as above with embodiment; so it is not intended to limit the present invention, any those skilled in the art, without departing from the spirit and scope of the present invention; all can make various different selection and amendment, therefore protection scope of the present invention is limited by claims and equivalents thereof.
Claims (10)
1. the method for an enzyme process regeneration ATP, it is characterised in that said method comprising the steps of:
(1) prepare and obtain ATP regeneration enzyme:
By genetic engineering modified, fermentation, purification or naturally extraction obtain ATP regeneration enzyme；This ATP
Regeneration enzyme is to make enzyme liquid or dry powder with the form of resolvase, or is fixed on fixing with immobilized enzyme form
Change on carrier；
(2) regeneration of ATP in reactant liquor:
Add one or more combinations of ATP, ADP and AMP, carry out enzymatic reaction；
Add ATP regeneration enzyme；
Add polyphosphoric acids or its salt is phosphodonor, carry out ATP regenerative response；
(3) product and ATP regeneration enzyme are separated:
Immobilized ATP regeneration enzyme is directly separated in retort；Or
Free ATP regeneration enzyme is by Ultra filtration membrane in filter；It is reclaim through the trapped fluid of filter
ATP regeneration enzyme, filter liquor is to isolate after enzyme the reactant liquor containing product.
The method of enzyme process the most according to claim 1 regeneration ATP, it is characterised in that described method
Further comprising the steps of:
(4) the ATP regeneration enzyme reclaimed is re-used in step (2).
The method of enzyme process the most according to claim 1 regeneration ATP, it is characterised in that described ATP
Regeneration enzyme is selected from polyphosphoric acids kinases (Ppk), adenylic acid kinases (Adk) and polyphosphoric acids-adenylic acid
One or more combinations of phosphotransferase (Pap).
The method of enzyme process the most according to claim 3 regeneration ATP, it is characterised in that when selecting Ppk
During with two kinds of enzyme combinations of Adk, Ppk Yu Adk activity ratio is (10-0.1): 1；When select Ppk and
During two kinds of enzyme combinations of Pap, the activity ratio of Ppk Yu Pap is (10-0.1): 1；When selecting Pap and Adk
During two kinds of enzyme combinations, Pap Yu Adk activity ratio is (10-0.1): 1；When selecting Ppk, Pap and Adk
During three kinds of enzyme combinations, Ppk, Pap and Adk activity ratio are (10-0.1): (10-0.1): 1.
The method of enzyme process the most according to claim 3 regeneration ATP, it is characterised in that described fixing
Change ATP regeneration enzyme to be fixed on by absorption, embedding, covalency, one or more modes of combining and cross-linking
Fixation support；Three kinds of ATP regeneration enzyme can be fixed after fixing or be mixed in proportion respectively together.
The method of enzyme process the most according to claim 1 regeneration ATP, it is characterised in that described step
(2) in, ATP regenerative response condition is as follows:
Reaction temperature is 25-60 DEG C；
Reaction pH is 5-10；
Enzymatic reaction system includes: one or more combinations in ATP, ADP and AMP, three kinds of materials
Add the most in any proportion；
Regeneration reaction system includes: polyphosphoric acids or its salt, magnesium ion and/or manganese ion, ammonium ion, potassium
One or more of ion and sodium ion, Tris or phosphate anion.
The method of enzyme process the most according to claim 6 regeneration ATP, it is characterised in that polyphosphoric acids
Or one or more that the molar concentration of its salt is ATP, ADP and AMP of adding of reaction is mole dense
0.01-40 times of degree summation；Magnesium ion concentration is 0.01-0.2M；Manganese ion concentration is 0.01-0.15M；
Potassium concentration is 0.01-0.5M；Na ion concentration is 0.01-0.5M；Ammonium concentration is 0.005-0.2
M；Tris concentration is 0.01-0.1M, phosphate concn is 0.01-0.1M.
8. the application of the method for ATP is regenerated according to the enzyme process described in claim 1-7 any claim,
It is characterized in that, described method is for the enzymatic reaction of multiple dependency ATP, for synthetic and nothing
Cell expressing protein.
The application of the method for enzyme process the most according to claim 8 regeneration ATP, it is characterised in that institute
State the enzymatic reaction that the enzymatic reaction of dependency ATP is participated in by the transferring enzyme of transfer phosphate groups, and
The enzymatic reaction that part ligase is participated in.
The application of the method for enzyme process the most according to claim 9 regeneration ATP, it is characterised in that
The enzymatic reaction that the transferring enzyme of described transfer phosphate groups is participated in is enzymatic clarification phosphagen, phosphoric acid
Arginine, 6-hexosephosphate, 1,6-fructose diphosphate, glycerol 3-phosphate, oxidized form of nicotinamide-adenine dinucleotide I, AMP,
The reaction of CT (D) P, GT (D) P and UT (D) P；The enzymatic reaction that described part ligase is participated in is enzyme
Method synthesis of acetyl coenzyme A, carnosine, enterochelin., glutamine and the reaction of L-thiamine.
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