WO2020248855A1 - 酶法反应组合物、增加酶法反应中三磷酸腺苷(atp)量的方法及其应用 - Google Patents

酶法反应组合物、增加酶法反应中三磷酸腺苷(atp)量的方法及其应用 Download PDF

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WO2020248855A1
WO2020248855A1 PCT/CN2020/093704 CN2020093704W WO2020248855A1 WO 2020248855 A1 WO2020248855 A1 WO 2020248855A1 CN 2020093704 W CN2020093704 W CN 2020093704W WO 2020248855 A1 WO2020248855 A1 WO 2020248855A1
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enzyme
adenosine
kinase
reaction
atp
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PCT/CN2020/093704
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French (fr)
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曾实现
潘永强
萧游龙
卢锦春
王骏
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百瑞全球有限公司
曾实现
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Priority to US17/618,091 priority Critical patent/US20220315905A1/en
Publication of WO2020248855A1 publication Critical patent/WO2020248855A1/zh

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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
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    • C12N15/09—Recombinant DNA-technology
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0102—Adenosine kinase (2.7.1.20)
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02008—Acetylglutamate kinase (2.7.2.8)
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C12Y207/03002—Creatine kinase (2.7.3.2)
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04001—Polyphosphate kinase (2.7.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04003—Adenylate kinase (2.7.4.3)

Definitions

  • the present invention relates to the field of biochemistry, in particular to a biochemical reaction using adenosine triphosphate (ATP), particularly an enzymatic reaction using adenosine triphosphate, an enzymatic reaction composition and a method for increasing the content of adenosine triphosphate (ATP) in the enzymatic reaction
  • ATP adenosine triphosphate
  • Adenosine triphosphate is a high-energy compound composed of three connected phosphate groups, ribose and adenine.
  • Adenosine triphosphate ATP is the most important coenzyme in the organism, providing energy for metabolism and providing phosphate groups and adenosine groups.
  • the important components of adenosine triphosphate (ATP) are three connected ⁇ , ⁇ and ⁇ -phosphate groups; the ⁇ -phosphate group is connected to adenosine, and the ⁇ and ⁇ phosphate groups are high-energy phosphate bonds.
  • ⁇ -phosphate group The only containing ⁇ -phosphate group is adenosine monophosphate (AMP); the compound containing ⁇ and ⁇ two phosphate groups is adenosine diphosphate (ADP); the compound containing ⁇ , ⁇ and ⁇ three phosphate groups is adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • synthetase can use one or two high-energy phosphate bonds in adenosine triphosphate (ATP) Or/and use phosphate groups as substrates.
  • ATP adenosine diphosphate
  • ADP adenosine diphosphate
  • AMP adenosine triphosphate
  • synthetase can use the adenosine part of adenosine triphosphate (ATP) as a substrate It is synthesized with other compounds, and the by-product is monophosphoric acid or pyrophosphoric acid.
  • the invention provides an enzymatic reaction composition, an enzymatic reaction, a method for increasing the content of adenosine triphosphate (ATP) in an enzymatic reaction, and applications thereof.
  • ATP adenosine triphosphate
  • the present invention provides:
  • a method for increasing the amount of adenosine triphosphate (ATP) in an enzymatic reaction wherein the first enzyme or enzyme group that produces adenosine monophosphate (AMP) and adenosine are added during the enzymatic reaction to add The amount of adenosine triphosphate (ATP), wherein the reaction substrate of the enzymatic reaction contains adenosine triphosphate (ATP) or a salt thereof.
  • ATP adenosine triphosphate
  • the method further includes adding a second enzyme or enzyme group responsible for regeneration of adenosine triphosphate (ATP) at the same time, before or after adding the first enzyme or enzyme group.
  • a second enzyme or enzyme group responsible for regeneration of adenosine triphosphate (ATP) at the same time, before or after adding the first enzyme or enzyme group.
  • the third enzyme or enzyme group is added at the same time, before or after adding the first enzyme or enzyme group and/or the second enzyme or enzyme group.
  • the first enzyme or group of enzymes comprises adenosine kinase (AK).
  • AK adenosine kinase
  • the reaction substrate further contains at least one of polyphosphoric acid or its salt and auxiliary ions,
  • the auxiliary ion is preferably at least one of magnesium ion, sodium ion, potassium ion, and chloride ion, and more preferably at least one of magnesium ion and potassium ion; the auxiliary ion may be in the state of an inorganic salt or an organic salt, preferably At least one of magnesium chloride hexahydrate, sodium chloride, manganese chloride, magnesium sulfate, and potassium carbonate, and more preferably at least one of magnesium chloride hexahydrate, sodium chloride, and potassium carbonate.
  • the second enzyme or enzyme group comprises at least one of polyphosphate: AMP phosphotransferase (PAP), polyphosphokinase (PPK) and adenylate kinase (ADK)
  • PAP AMP phosphotransferase
  • PPK polyphosphokinase
  • ADK adenylate kinase
  • the third enzyme or enzyme group comprises creatine kinase (CK), glutamate kinase (GK), coenzyme I kinase (NK) and/or adenosine triphosphate (ATP) as one of the substrates to perform Other enzymes or enzyme groups for phosphorylation, phosphorylation of amino acids, peptides or proteins, or polypeptide synthesis.
  • CK creatine kinase
  • GK glutamate kinase
  • NK coenzyme I kinase
  • ATP adenosine triphosphate
  • the enzymatic reaction includes at least one of the following:
  • the first enzyme or enzyme group includes adenosine kinase (AK), the reaction substrate includes adenosine, polyphosphate and adenosine triphosphate (ATP), and the reaction product includes adenosine monophosphate (AMP) and diphosphate Adenosine (ADP);
  • AK adenosine kinase
  • ATP adenosine triphosphate
  • AMP adenosine monophosphate
  • ADP diphosphate Adenosine
  • the first enzyme or enzyme group includes adenosine kinase (AK), and the second enzyme or enzyme group includes polyphosphate:AMP phosphotransferase (PAP), and the reaction substrate includes adenosine monophosphate (AMP ) And polyphosphoric acid, and the reaction product contains adenosine diphosphate (ADP) and polyphosphoric acid;
  • AK adenosine kinase
  • PAP polyphosphate:AMP phosphotransferase
  • AMP adenosine monophosphate
  • ADP adenosine diphosphate
  • the first enzyme or enzyme group includes adenosine kinase (AK), and the second enzyme or enzyme group includes adenylate kinase (ADK), and the reaction substrate includes adenosine diphosphate (ADP) and polymer Phosphoric acid, and the reaction product contains adenosine monophosphate (AMP) and adenosine triphosphate (ATP);
  • AK adenosine kinase
  • ADK adenylate kinase
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the first enzyme or enzyme group includes adenosine kinase (AK), and the second enzyme or enzyme group includes polyphosphokinase (PPK), and the reaction substrate includes adenosine diphosphate (ADP) And polyphosphoric acid, and the reaction product contains adenosine triphosphate (ATP) and polyphosphoric acid; and
  • the first enzyme or enzyme group includes adenosine kinase (AK), and the second enzyme or enzyme group includes polyphosphate: AMP phosphotransferase (PAP), polyphosphokinase (PPK) and adenylate Kinase (ADK) and the third enzyme or enzyme group includes creatine kinase (CK), the reaction substrate includes creatine and adenosine triphosphate (ATP), and the reaction product includes creatine phosphate, adenosine diphosphate (ADP) and Polyphosphate; the third enzyme or enzyme group also contains glutamate kinase (GK), the reaction substrate contains glutamate and adenosine triphosphate (ATP), and the reaction product contains glutamate 5-phosphate, adenosine diphosphate (ADP ) And polyphosphate; the third enzyme or enzyme group may also include Coenzyme I kinase (NK), the reaction substrate includes Coenzyme I and adenosine triphosphate (ATP), and
  • AMP phosphotransferase PAP
  • ADK adenylate kinase
  • ATP adenosine triphosphate
  • polyphosphate AMP phosphotransferase (PAP), adenosine kinase (AK) and polyphosphokinase (PPK) all or at least one and use adenosine triphosphate (ATP) as the reaction substrate in different
  • PAP AMP phosphotransferase
  • AK adenosine kinase
  • PPK polyphosphokinase
  • the second enzyme or enzyme group regenerates adenosine monophosphate (AMP) and adenosine diphosphate (ADP) to adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively; as well as
  • the first enzyme or set of enzymes synthesizes adenosine to adenosine monophosphate (AMP).
  • AMP adenosine monophosphate
  • the enzymatic reaction includes the synthesis of adenosine monophosphate (AMP) using adenosine and adenosine triphosphate (ATP) as substrates.
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the reaction substrate further includes creatine or its hydrate, sodium glutamate or its hydrate, and/or coenzyme I.
  • the first enzyme or enzyme group to be added is determined according to the level of adenosine triphosphate (ATP) degradation product produced in the enzymatic reaction.
  • the degradation product is preferably adenosine diphosphate (ADP), adenosine monophosphate (AMP) and/or adenosine.
  • the conditions of the enzymatic reaction are: the temperature is 28-40 degrees Celsius, preferably 30-38 degrees Celsius, more preferably 33-37 degrees Celsius; pH value is 5-9, preferably 6 -8.5, more preferably 7-7.75.
  • An enzymatic reaction composition comprising a substrate and a first enzyme or group of enzymes for producing adenosine monophosphate (AMP), wherein the substrate comprises adenosine triphosphate (ATP) or a salt thereof, and Adenosine.
  • AMP adenosine monophosphate
  • the substrate further comprises at least one of polyphosphoric acid or its salt, auxiliary ion and creatine or its hydrate,
  • the auxiliary ion is preferably at least one of magnesium ion, sodium ion, potassium ion, and chloride ion, and more preferably at least one of magnesium ion and potassium ion; the auxiliary ion may be in the state of an inorganic salt or an organic salt, preferably At least one of magnesium chloride hexahydrate, sodium chloride, manganese chloride, magnesium sulfate, and potassium carbonate, and more preferably at least one of magnesium chloride hexahydrate, sodium chloride, and potassium carbonate.
  • the enzymatic reaction composition further comprises a second enzyme or enzyme group and optionally a third enzyme or enzyme group.
  • the first enzyme or enzyme group comprises adenosine kinase AK
  • the third enzyme or enzyme group comprises creatine kinase (CK), glutamate kinase (GK), coenzyme I kinase (NK) or adenosine triphosphate (ATP) as One of the enzymatic reaction substrates to perform phosphorylation, phosphorylation of amino acids, peptides or proteins, or other enzymes or enzyme groups for polypeptide synthesis, and preferably, the second enzyme or enzyme group comprises polyphosphate: AMP At least one of phosphotransferase (PAP), polyphosphate kinase (PPK), and adenylate kinase (ADK).
  • PAP phosphotransferase
  • PPK polyphosphate kinase
  • ADK adenylate kinase
  • the first enzyme or enzyme group, the second enzyme or enzyme group and the third enzyme or enzyme group are purified or non-purified cell disruption, liquid enzyme, immobilized
  • the form of cells or immobilized enzymes is included in the enzymatic reaction composition.
  • the polyphosphate: AMP phosphotransferase (PAP), adenosine kinase (AK), polyphosphate kinase (PPK) and adenylate kinase (ADK) and Creatine kinases are each independently a recombinase, and are expressed in E. coli separately or in combination.
  • a method for phosphorylation or phosphorylation of amino acids, nucleic acids, peptides or proteins using adenosine triphosphate (ATP) as a substrate including any of the above methods.
  • the amino acid, nucleic acid, peptide or protein is creatine, glutamic acid, coenzyme I and the like.
  • the present invention can not only use polyphosphoric acid as a substrate, but also use inexpensive adenosine as a reaction substrate, so that the amount of adenosine triphosphate (ATP) of the reaction species can be increased and/or the by-products in the reaction can be reduced Adenosine phosphate (AMP) and adenosine diphosphate (ADP) regenerate adenosine triphosphate (ATP) to produce more adenosine triphosphate (ATP). Therefore, in the enzymatic reaction, the amount of adenosine triphosphate (ATP) increases as the reaction time increases Non-reduced.
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • concentration of adenosine triphosphate (ATP) in the reaction is too low, the reaction efficiency will not be ideal, which wastes production capacity, increases the pressure of purification and affects the recovery rate of the final product.
  • the amount of adenosine triphosphate (ATP) in the enzymatic reaction will continue to increase with the number of reaction cycles, the reaction efficiency will gradually increase with the reaction time, and the related reaction speed will increase accordingly.
  • the present invention uses cheap adenosine to synthesize adenosine monophosphate (AMP), which can further reduce the production cost of existing reactions that require the use of adenosine triphosphate (ATP).
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) degrading enzymes have high enzymatic activity in E. coli cells.
  • ATP adenosine triphosphate
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • the adenosine produced after hydrolysis can be re-synthesized into adenosine monophosphate (AMP), and then re-converted into adenosine triphosphate (ATP), so the invertase can be used without purification, which greatly saves the need for purification.
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the enzymatic process has many advantages compared with chemical synthesis methods.
  • the enzymatic process also has its shortcomings: the biological reaction sometimes needs to rely on the use price Expensive adenosine triphosphate (ATP) puts a significant pressure on production costs.
  • polyphosphoric acid is generally used as a substrate, and adenosine diphosphate (ADP) or adenosine monophosphate (AMP) after the reaction is regenerated into adenosine triphosphate (ATP) by phosphate conversion or synthetase, or glucose is used as adenosine triphosphate (ATP). )
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • adenosine triphosphate (ATP) regeneration technology can reduce the cost pressure of adenosine triphosphate (ATP), it faces another problem.
  • the amount of adenosine triphosphate (ATP) used should not be too high, which leads to low efficiency of the enzymatic synthesis reaction;
  • the recombinant enzyme expressed by Escherichia coli as a host contains a large amount of hydrolyzed adenosine triphosphate (ATP) and hydrolysis Adenosine diphosphate (ADP) and an enzyme that hydrolyzes adenosine monophosphate (AMP).
  • This type of hydrolase has a very high activity and can hydrolyze adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) to adenosine monophosphate (AMP) and adenosine in a short time.
  • Adenosine triphosphate (ATP) is degraded into adenosine and then disappears irreversibly. Therefore, if the industry wants to use adenosine triphosphate (ATP) regeneration technology to reduce costs, the recombinant enzyme needs to be purified first. Protein purification is expensive.
  • the inventors of the present invention have conducted a lot of in-depth theoretical research and experimental exploration, using low-cost adenosine as a substrate for the production of adenosine monophosphate (AMP), using adenosine triphosphate (ATP)
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the regenerated and/or newly added reaction system produces a larger amount of adenosine triphosphate (ATP). Therefore, during the enzymatic reaction, the amount of adenosine triphosphate (ATP) increases with the reaction time, thereby improving reaction efficiency and reducing costs.
  • the present invention provides an enzymatic reaction composition
  • a substrate comprising a substrate and a first enzyme or enzyme group that produces adenosine monophosphate (AMP), wherein the substrate comprises adenosine and adenosine triphosphate (ATP) or its salt.
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the substrate further comprises polyphosphoric acid or a salt thereof, preferably the sodium salt of polyphosphoric acid.
  • the degree of polymerization of polyphosphoric acid may be 3-20,000; preferably, the degree of polymerization of polyphosphoric acid may be 3-7,000, more preferably 3-75.
  • the enzymatic reaction composition may further include auxiliary ions.
  • the auxiliary ion may be selected from at least one of magnesium ion, sodium ion, potassium ion, and chloride ion, and more preferably at least one of magnesium ion and potassium ion.
  • the auxiliary ion may be in the form of an inorganic salt or an organic salt, preferably at least one of magnesium chloride hexahydrate, sodium chloride, manganese chloride, magnesium sulfate and potassium carbonate, more preferably magnesium chloride hexahydrate, sodium chloride and carbonic acid At least one of potassium.
  • the reaction also includes the required substrate for the synthesis reaction; in this article, the enzymatic synthesis of creatine phosphate is used as an example, and the reaction substrate includes creatine, magnesium chloride hexahydrate and adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • the enzymatic reaction composition may also contain other additives, for example, pH adjusting agents, such as buffers/salts, preferably sodium phosphate buffer, potassium phosphate buffer, and tris buffer Liquid, more preferably sodium phosphate buffer and tris buffer.
  • pH adjusting agents such as buffers/salts, preferably sodium phosphate buffer, potassium phosphate buffer, and tris buffer Liquid, more preferably sodium phosphate buffer and tris buffer.
  • concentration of the pH adjusting agent may be 0.001M-1M, preferably 0.01M-0.5M, more preferably 0.05M-0.3M.
  • the enzymatic reaction composition further comprises a second enzyme or enzyme group and optionally a third enzyme or enzyme group.
  • the first enzyme or group of enzymes comprises adenosine kinase (AK).
  • the third enzyme or enzyme group includes at least one of creatine kinase (CK), glutamate kinase (GK) and coenzyme I kinase (NK).
  • the second enzyme or enzyme group comprises at least one of polyphosphate:AMP phosphotransferase (PAP), polyphosphate kinase (PPK) and adenylate kinase (ADK).
  • PAP polyphosphate:AMP phosphotransferase
  • PPK polyphosphate kinase
  • ADK adenylate kinase
  • the first enzyme or enzyme group, the second enzyme or enzyme group, and the third enzyme or enzyme group contained in the enzymatic reaction composition may be purified or non-purified cell disruption, liquid enzymes, immobilized cells Or the form of immobilized enzyme.
  • immobilized cells or immobilized enzymes the preparation of immobilized cells and immobilized enzymes can be carried out according to the method described in Chinese patent CN1982445B, and the carrier in this patent can be used.
  • the carrier is a porous organic foam material with open pores, and the shape of the carrier includes granular, block, column, sheet or strip shape.
  • the porous organic foam material is melamine sponge.
  • an enzymatic reaction composition refers to a reactable mixture capable of carrying out a biochemical reaction with the help of an enzyme or an enzyme group to produce a valuable product.
  • the reaction product may include one or more of adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), polyphosphate, creatine phosphate, glutamate 5-phosphate, and oxidized coenzyme II .
  • AMP adenosine monophosphate
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • polyphosphate creatine phosphate
  • glutamate 5-phosphate glutamate 5-phosphate
  • oxidized coenzyme II oxidized coenzyme II .
  • the first enzyme or enzyme group, the second enzyme or enzyme group, and the third enzyme or enzyme group may each independently be a recombinase, and are expressed in a vector separately or in combination.
  • the vector may include Escherichia coli and yeast. In this way, cells or fragments thereof containing recombinase can be used to perform the enzymatic reaction.
  • the cells may be E. coli cells or yeast cells.
  • the vector may include Escherichia coli, yeast, Bacillus and other methods commonly used in biological sciences to express the recombinase.
  • the enzyme or enzyme group can be used in the liquid state of cells, crushed liquid, supernatant or purified enzyme, or used in any way with its corresponding carrier to make immobilized cells or immobilized enzymes for enzyme or enzyme group reactions.
  • the present invention also provides a method for increasing the amount of adenosine triphosphate (ATP) in an enzymatic reaction, which comprises adding a first enzyme or enzyme that produces adenosine monophosphate (AMP) during the enzymatic reaction Group and adenosine, in the amount of newly added adenosine triphosphate (ATP), wherein the reaction substrate of the enzymatic reaction contains adenosine triphosphate (ATP) or its salt.
  • ATP adenosine triphosphate
  • the method may also include adding a second enzyme or enzyme group responsible for adenosine triphosphate (ATP) regeneration at the same time, before or after adding the first enzyme or enzyme group.
  • ATP adenosine triphosphate
  • a third enzyme or enzyme group may be added.
  • the first enzyme or group of enzymes may comprise adenosine kinase (AK).
  • the reaction substrate further comprises at least one of polyphosphoric acid or its salt and auxiliary ions.
  • the auxiliary ion is preferably at least one of magnesium ion, sodium ion, potassium ion, and chloride ion, and more preferably at least one of magnesium ion and potassium ion; the auxiliary ion may be in the state of an inorganic salt or an organic salt, preferably At least one of magnesium chloride hexahydrate, sodium chloride, manganese chloride, magnesium sulfate, and potassium carbonate, and more preferably at least one of magnesium chloride hexahydrate, sodium chloride, and potassium carbonate.
  • the second enzyme or group of enzymes may include at least one of polyphosphate:AMP phosphotransferase (PAP), polyphosphate kinase (PPK), and adenylate kinase (ADK) creatine kinase.
  • PAP polyphosphate:AMP phosphotransferase
  • PPK polyphosphate kinase
  • ADK adenylate kinase
  • the enzymatic reaction includes at least one of the following:
  • the first enzyme or enzyme group includes adenosine kinase (AK), the reaction substrate includes adenosine, polyphosphate and adenosine triphosphate (ATP), and the reaction product includes adenosine monophosphate (AMP) and diphosphate Adenosine (ADP);
  • AK adenosine kinase
  • ATP adenosine triphosphate
  • AMP adenosine monophosphate
  • ADP diphosphate Adenosine
  • the first enzyme or enzyme group includes adenosine kinase (AK), and the second enzyme or enzyme group includes polyphosphate:AMP phosphotransferase (PAP), and the reaction substrate includes adenosine monophosphate (AMP ) And polyphosphoric acid, and the reaction product contains adenosine diphosphate (ADP) and polyphosphoric acid;
  • AK adenosine kinase
  • PAP polyphosphate:AMP phosphotransferase
  • AMP adenosine monophosphate
  • ADP adenosine diphosphate
  • the first enzyme or enzyme group includes adenosine kinase (AK), and the second enzyme or enzyme group includes adenylate kinase (ADK), and the reaction substrate includes adenosine diphosphate (ADP) and polymer Phosphoric acid, and the reaction product contains adenosine monophosphate (AMP) and adenosine triphosphate (ATP);
  • AK adenosine kinase
  • ADK adenylate kinase
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the first enzyme or enzyme group includes adenosine kinase (AK), and the second enzyme or enzyme group includes polyphosphokinase (PPK), and the reaction substrate includes adenosine diphosphate (ADP) And polyphosphoric acid, and the reaction product contains adenosine triphosphate (ATP) and polyphosphoric acid; and
  • the first enzyme or enzyme group includes adenosine kinase (AK), and the second enzyme or enzyme group includes polyphosphate: AMP phosphotransferase (PAP), polyphosphokinase (PPK) and adenylate
  • the third enzyme or enzyme group includes creatine kinase (CK), the reaction substrate includes creatine and adenosine triphosphate (ATP), and the reaction product includes creatine phosphate, adenosine diphosphate (ADP) and poly Polyphosphate; the third enzyme or enzyme group also includes glutamate kinase (GK), the reaction substrate includes glutamate and adenosine triphosphate (ATP), and the reaction product includes glutamate 5-phosphate, adenosine diphosphate (ADP) And polyphosphate; the third enzyme or group of enzymes may also include Coenzyme I kinase (NK), the reaction substrate includes Coenzyme I and adenosine triphosphate (ATP), and the reaction product includes oxidized
  • polyphosphate AMP phosphotransferase (PAP), adenylate kinase (ADK) and phosphokinase (PPK) all or at least one, adenosine kinase (AK) and adenosine triphosphate (ATP) as the reaction
  • PAP AMP phosphotransferase
  • ADK adenylate kinase
  • PPK phosphokinase
  • AK adenosine kinase
  • ATP adenosine triphosphate
  • AK adenosine kinase
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the second enzyme or group of enzymes regenerates adenosine monophosphate (AMP) and adenosine diphosphate (ADP) to adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively; and
  • the first enzyme or set of enzymes synthesizes adenosine to adenosine monophosphate (AMP).
  • AMP adenosine monophosphate
  • the enzymatic reaction involves the synthesis of adenosine monophosphate (AMP) using adenosine and adenosine triphosphate (ATP) as substrates.
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the reaction substrate may also include creatine or a hydrate thereof.
  • the first enzyme or enzyme group added is determined according to the level of adenosine triphosphate (ATP) degradants produced in the enzymatic reaction, and the degradants are preferably adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / Or adenosine.
  • ATP adenosine triphosphate
  • the degradants are preferably adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / Or adenosine.
  • the first enzyme or enzyme group, or the second enzyme or enzyme group, or the third enzyme is added in the form of purified or non-purified cell disruption, liquid enzyme, immobilized cell or immobilized enzyme Or enzyme group.
  • the conditions of the enzymatic reaction can be: the temperature is 28-40 degrees Celsius, preferably 30-38 degrees Celsius, more preferably 33-37 degrees Celsius; pH value is 5-9, preferably 6-8.5, more preferably 7-7.75.
  • the method of the present invention can use the by-products in the reaction system (such as adenosine, adenosine diphosphate (ADP) or adenosine monophosphate (AMP)) or additional adenosine as a substrate to rapidly add adenosine triphosphate (ATP) or regenerate adenosine triphosphate (ATP) to produce a greater amount of adenosine triphosphate (ATP). Therefore, in the enzymatic reaction, the amount of adenosine triphosphate (ATP) increases with the reaction time instead of decreasing.
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • the enzymatic reaction composition may further include at least one of polyphosphoric acid or its salt, auxiliary ion, salt, and creatine or its hydrate.
  • the reaction product of the enzymatic reaction includes one or more of adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), polyphosphate and creatine phosphate.
  • AMP adenosine monophosphate
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • polyphosphate adenosine triphosphate
  • the polyphosphate AMP phosphotransferase (PAP), adenosine kinase (AK), polyphosphate kinase (PPK) and adenylate kinase (ADK), creatine kinase (CK), Glutamate kinase (GK) and Coenzyme I kinase (NK) are each independently a recombinase, and are expressed in E. coli separately or in combination.
  • PAP AMP phosphotransferase
  • AK adenosine kinase
  • PPK polyphosphate kinase
  • ADK adenylate kinase
  • CK creatine kinase
  • GK Glutamate kinase
  • NK Coenzyme I kinase
  • the present invention also provides a method for phosphorylation or phosphorylation of amino acids, nucleic acids, peptides or proteins using adenosine triphosphate (ATP) as a substrate, including any of the above methods. Therefore, the above methods can be used to synthesize amino acids or their derivatives, nucleic acids, peptides, proteins or their derivatives.
  • ATP adenosine triphosphate
  • the method involves a peptide synthesis or phosphorylation reaction using adenosine triphosphate (ATP) as a substrate, specifically including adding the substrate, auxiliary ion or salt required for the reaction, performing the synthesis reaction, and adding Rapidly add adenosine triphosphate (ATP) and rapidly regenerate adenosine triphosphate (ATP) synthetase and substrates, magnesium ions.
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • the amino acid, peptide, nucleic acid, protein or derivative thereof is creatine, glutamic acid, coenzyme I and the like.
  • the immobilized cells and the immobilized enzyme may be placed in an immobilization reaction device to perform an immobilization reaction.
  • the fixed flower reaction can be performed according to the steps described in Chinese Patent Application CN106032520A.
  • the immobilized reaction device may include a columnar reactor with an inlet and an outlet, a reaction regulating tank, a high-flow water pump, a pH regulating device and a pH detecting electrode, a stirring device, and a pH regulating tank.
  • Reaction control tank Gene Harbor (Hong Kong) Biotechnology Co., Ltd., BR-1L;
  • Adjustable flow type suction pump SURGEFLO company, FL-32;
  • PH control device Gene Harbor (Hong Kong) Biotechnology Co., Ltd., AR-1;
  • Creatine monohydrate Jiangsu Yuanyang Chemical Co., Ltd.;
  • Adenosine Zhejiang Chengyi Pharmaceutical
  • Adenosine triphosphate disodium salt Kaiping Morningniu Pharmaceutical Company
  • the creatine kinase PCR primer sequence design is based on the Chinese Patent Application Publication No. CN102808006, specifically:
  • the BamHI restriction site is underlined.
  • RNA of mouse skeletal muscle was extracted, and cDNA was prepared by reverse transcription.
  • the creatine kinase gene was amplified by PCR with the primers mentioned above, and connected to pGEX-2T (purchased from GE Healthcare, USA) to obtain pGEX-2T(+)-CK (SEQ NO. 15), which was transformed into E. coli BL21 (DE3) to obtain a recombinant expression strain of creatine kinase.
  • the 100L fermenter with 60L LB medium (containing 100ug/ml ampicillin) In the cultivation.
  • the initial fermentation conditions were 37°C, 200rpm, and pH7.0.
  • the fermentation was carried out for 9 hours and IPTG was added to a final concentration of 1 mM, and the fermentation was completed for 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 1.42 kg of E. coli cells containing creatine kinase.
  • the obtained E. coli cells containing creatine kinase are prepared into an enzyme solution, and each ml of the enzyme solution contains 0.2g cells. According to the determination method of creatine phosphate content of Chinese Patent Application Publication No. CN102808006, the enzyme activity of cells was detected, and the enzyme activity was about 2.1 U/g.
  • the BamHI restriction site is underlined.
  • Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1-VE13AGY71676 DNA was used as the substrate, and the polyphosphate:AMP transferase gene was amplified by PCR with the above primers.
  • the PCR product was treated with restriction enzymes BamH I and EcoRI and ligated to pGEX-2T (purchased from GE Healthcare, USA) to obtain pGEX-2T(+)-PAP (SEQ NO. 16).
  • This recombinant expression vector was transformed into Escherichia coli HB101 to obtain a polyphosphate:AMP phosphotransferase recombinant expression strain.
  • the 100L fermenter with 60L LB medium (containing 100ug/ml ampicillin) In the cultivation.
  • the initial fermentation conditions were 37°C, 200rpm, and pH7.0.
  • the fermentation was carried out for 9 hours and isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM, and the fermentation was completed for 20 hours.
  • IPTG isopropylthiogalactoside
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 1.18 kg of E. coli cells containing polyphosphate:AMP phosphotransferase.
  • the obtained E. coli cells containing polyphosphate: AMP phosphotransferase are prepared into an enzyme solution, and each ml of the enzyme solution contains 0.2g cells. According to its enzymatic reaction, the cell enzyme activity was tested, and the cell enzyme activity was about 1.1 U/g.
  • the BamHI restriction site is underlined.
  • PCR amplification was performed with the above primers to obtain the adenylate kinase gene, and the PCR product was treated with restriction enzymes BamHI and EcoRI and It is connected to pGEX-2T (purchased from GE Healthcare, USA) to obtain pGEX-2T(+)-ADK (SEQ NO. 17).
  • This recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant adenylate kinase expression strain.
  • the 100L fermenter with 60L LB medium (containing 100ug/ml ampicillin) In the cultivation.
  • the initial fermentation conditions were 37°C, 200rpm, and pH7.0.
  • the fermentation was carried out for 9 hours and IPTG was added to a final concentration of 1 mM, and the fermentation was completed for 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 1.18 kg of E. coli cells containing adenylate kinase.
  • the obtained E. coli cells containing adenylate kinase are prepared into an enzyme solution, each ml of the enzyme solution contains 0.2g cells, and the enzyme activity of the cells is detected according to the enzymatic reaction, and the enzyme activity is about 0.08 U/g.
  • the BamHI restriction site is underlined.
  • C. crescentus NA1000YP_002518902 DNA was used as the substrate, and the phosphokinase gene was amplified by PCR with the above primers.
  • the PCR product was treated with restriction enzymes BamHI and EcoRI and ligated to pGEX-2T (purchased from GE Healthcare) ,USA), pGEX-2T(+)-PPK (SEQ NO.18) is obtained.
  • This recombinant expression vector was transformed into Escherichia coli HB101 to obtain a polyphosphokinase recombinant expression strain.
  • the 100L fermenter with 60L LB medium (containing 100ug/ml ampicillin) In the cultivation.
  • the initial fermentation conditions were 37°C, 200rpm, and pH7.0.
  • the fermentation was carried out for 9 hours and IPTG was added to the final concentration of 1 mM, and the fermentation was completed for 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 1.73 kg of E. coli cells containing polyphosphokinase.
  • the obtained E. coli cells containing polyphosphokinase are prepared into an enzyme solution, each ml of the enzyme solution contains 0.2g cells, and the enzyme activity of the cells is detected according to the enzymatic reaction, and the enzyme activity is about 0.03 U/g.
  • the BamHI restriction site is underlined.
  • PCR amplification was performed with the above primers to obtain the adenosine kinase gene sequence, and the PCR product was treated with restriction enzymes BamHI and EcoRI, and the gene sequence obtained was ligated to pGEX-2T (Purchased from GE Healthcare, USA), pGEX-2T(+)-AK (SEQ NO.19) was obtained, and transformed into E. coli BL21(DE3) to obtain a recombinant adenosine kinase expression strain.
  • the 100L fermenter with 60L LB medium (containing 100ug/ml ampicillin) In the cultivation.
  • the initial fermentation conditions were 37°C, 200rpm, and pH7.0.
  • the fermentation was carried out for 9 hours and IPTG was added to a final concentration of 1 mM, and the fermentation was completed for 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 1.2 kg of E. coli cells containing adenosine kinase.
  • the obtained E. coli cells of adenosine kinase were suspended in 50 mM Tris-HCl hydrochloric acid buffer (pH 7.5) at a ratio of 1:5. Then lyse the bacterial cells with ultrasound. Centrifuge (10°C, 12,500 rpm, 15 minutes) and collect the supernatant as adenosine kinase solution. According to its enzymatic reaction, the cell's enzyme activity was detected, and the enzyme activity was about 0.08EU/g.
  • the BamHI restriction site is underlined.
  • PCR amplification was performed with the above primers to obtain the adenosine kinase gene sequence, and the PCR product was treated with restriction enzymes BamHI and EcoRI, and the gene sequence obtained was ligated to pGEX-2T (purchased from GE Healthcare, USA), obtained pGEX-2T(+)-GK (SEQ NO.20), transformed into E. coli BL21(DE3), and obtained adenosine kinase recombinant expression strain.
  • the 100L fermenter with 60L LB medium (containing 100ug/ml ampicillin) In the cultivation.
  • the initial fermentation conditions were 37°C, 200rpm, and pH7.0.
  • the fermentation was carried out for 9 hours and IPTG was added to a final concentration of 1 mM, and the fermentation was completed for 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 1.2 kg of E. coli cells containing glutamate kinase.
  • the obtained glutamate kinase E. coli cells were suspended in 50 mM Tris-HCl hydrochloric acid buffer (pH 7.5) at a ratio of 1:5. Then lyse the bacterial cells with ultrasound. Centrifuge (10°C, 12,500 rpm, 15 minutes) and collect the supernatant as glutamate kinase solution. According to its enzymatic reaction, the cell's enzyme activity was tested, and the enzyme activity was about 0.02EU/g.
  • the BamHI restriction site is underlined.
  • PCR amplification was performed with the above primers to obtain the Coenzyme I kinase gene sequence, and the PCR products were treated with restriction enzymes BamHI and EcoRI, and the resulting gene sequences were ligated In pGEX-2T (purchased from GE Healthcare, USA), pGEX-2T(+)-NK (SEQ NO.21) was obtained, and transformed into E. coli BL21(DE3) to obtain a recombinant expression strain of Coenzyme I kinase.
  • the 100L fermenter with 60L LB medium (containing 100ug/ml ampicillin) In the cultivation.
  • the initial fermentation conditions were 37°C, 200rpm, and pH7.0.
  • the fermentation was carried out for 9 hours and IPTG was added to a final concentration of 1 mM, and the fermentation was completed for 20 hours.
  • the fermentation broth was centrifuged at 12,500 rpm for 10 minutes at 4°C to obtain 1.2 kg of E. coli cells containing glutamate kinase.
  • the obtained glutamate kinase E. coli cells were suspended in 50 mM Tris-HCl hydrochloric acid buffer (pH 7.5) at a ratio of 1:5. Then lyse the bacterial cells with ultrasound. Centrifuge (10°C, 12,500 rpm, 15 minutes) and collect the supernatant as Coenzyme I kinase solution. According to its enzymatic reaction, the enzyme activity of the cells was detected, and the enzyme activity was about 0.045 EU/g.
  • Example 3 of Chinese patent CN1982445B a mixed immobilized Escherichia coli containing creatine kinase, adenosine kinase, polyphosphate:AMP phosphotransferase, adenylate kinase and polyphosphokinase was prepared on a solid-phase carrier Cells, the mixing weight ratio of each cell is based on the corresponding enzyme activity. Table 1 below shows the wet weight of each cell after centrifugation.
  • the shape of the carrier is a strip: length 24 cm, width 5 cm, thickness 5 mm, and actual weight 46.3 g.
  • the prepared carrier carrying mixed immobilized E. coli cells is installed in an immobilized enzyme or immobilized cell reactor.
  • the reactor is a cylinder made of organic glass with a height of 7 cm and a radius of 4.5 cm. Use a knife to trim off about 3 cm of the head and tail of the above-mentioned carrier at an inclination of 45°, tightly hold and roll into a homogeneous cylinder with a height of 5 cm and a radius of 4.5 cm, with a weight of 32.2 g. Insert the cylinder into the reactor so that its tightness meets the level 3 standard described in Table 1 of Chinese invention patent application CN106032520A, and there is no gap between the side wall and the inner wall of the reactor.
  • the capacity of the reaction control tank is 1L; the high-flow water pump is an adjustable flow suction pump with a flow rate of 3L/min; the pH control device adopts 0.3M hydroxide
  • the pH of the sodium solution is adjusted, and the flow rate of the dosing pump is 1 ml per minute.
  • the substrate contained in the reaction solution is 8.94 g/L of creatine monohydrate, 3 g/L of adenosine triphosphate disodium salt, 16.4 g/L of magnesium chloride hexahydrate, 6 g/L of adenosine, and 13.3 g/L of polyphosphoric acid.
  • polyphosphoric acid AMP phosphotransferase (PAP), adenylate kinase (ADK), polyphosphokinase (PPK), adenosine kinase (AK) and glutamate kinase (GK)
  • PAP AMP phosphotransferase
  • ADK adenylate kinase
  • PPK polyphosphokinase
  • AK adenosine kinase
  • GK glutamate kinase
  • Example 3 of Chinese Patent CN1982445B a mixed immobilized large intestine containing glutamate kinase, adenosine kinase, polyphosphate:AMP phosphotransferase, adenylate kinase and polyphosphokinase was prepared on a solid-phase carrier.
  • Bacillus cells the mixing weight ratio of each cell is based on the corresponding enzyme activity.
  • Table 2 shows the wet weight of each cell after centrifugation.
  • the shape of the carrier is a strip: 28 cm in length, 5 cm in width, and 5 mm in thickness, and its actual weight is 48.1 g.
  • the prepared carrier carrying mixed immobilized E is a strip: 28 cm in length, 5 cm in width, and 5 mm in thickness, and its actual weight is 48.1 g.
  • the reactor is a cylinder made of organic glass with a height of 7 cm and a radius of 4.5 cm. Use a knife to trim off about 3 cm of the head and tail of the above-mentioned carrier at an inclination of 45°, and tightly roll it into a homogeneous cylinder with a height of 5 cm and a radius of 4.5 cm, with a weight of 31.4 g. Insert the cylinder into the reactor so that its tightness meets the level 3 standard described in Table 1 of Chinese invention patent application CN106032520A, and there is no gap between the side wall and the inner wall of the reactor.
  • polyphosphate AMP phosphotransferase, adenylate kinase, polyphosphokinase, adenosine kinase, coenzyme I kinase, and recombinant expression E. coli cells expressing the gene and proceeding Ferment.
  • a solid-phase carrier containing coenzyme I kinase (NK), adenosine kinase (AK), polyphosphate: AMP phosphotransferase (PAP), adenylate kinase (ADK) was prepared on a solid-phase carrier.
  • NK coenzyme I kinase
  • AK adenosine kinase
  • PAP polyphosphate
  • ADK adenylate kinase
  • the mixing weight ratio of each cell is based on the corresponding enzyme activity. Table 3 below shows the wet weight of each cell after centrifugation.
  • the shape of the carrier is a bar: 21 cm long, 5 cm wide, 5 mm thick, and its actual weight is 40.8 g.
  • the prepared carrier carrying mixed immobilized E. coli cells is installed in an immobilized enzyme or immobilized cell reactor.
  • the reactor is a cylinder made of organic glass with a height of 7 cm and a radius of 4.5 cm. Use a knife to trim off about 3 cm of the head and tail of the above-mentioned carrier at an inclination of 45°, and tightly roll it into a homogeneous cylinder with a height of 5 cm and a radius of 4.5 cm, with a weight of 30.8 g. Insert the cylinder into the reactor so that its tightness meets the level 3 standard described in Table 1 of Chinese invention patent application CN106032520A, and there is no gap between the side wall and the inner wall of the reactor.
  • the capacity of the reaction control tank is 1L; the high-flow water pump is an adjustable flow suction pump with a flow rate of 3L/min; the pH control device adopts 0.3M hydroxide
  • the pH of the sodium solution is adjusted, and the flow rate of the dosing pump is 1 ml per minute.
  • the substrate contained in the reaction solution is oxidized nicotinamide adenine dinucleotide 20.3g/L, adenosine triphosphate disodium salt 3g/L, magnesium chloride hexahydrate 16.4g/L, adenosine 6g/L, and polyphosphoric acid 13.3g /L.
  • Example 11 Use adenosine as a substrate to quickly increase the content of adenosine triphosphate (ATP)
  • polyphosphoric acid AMP phosphotransferase (PAP), adenylate kinase (ADK), polyphosphokinase (PPK), adenosine kinase (AK) and adenosine kinase (AK)
  • PAP AMP phosphotransferase
  • ADK adenylate kinase
  • PPK polyphosphokinase
  • AK adenosine kinase
  • AK adenosine kinase
  • E. coli cells expressing the gene and fermented.
  • a solid phase carrier containing polyphosphoric acid: AMP phosphotransferase (PAP), adenylate kinase (ADK), polyphosphokinase (PPK), adenosine kinase (AK) mixed immobilized E. coli cells the mixing weight ratio of each cell is based on basically correspondingly matched enzyme activity.
  • Table 4 shows the wet weight of each cell after centrifugation.
  • the shape of the carrier is a strip: 18 cm long, 5 cm wide, 5 mm thick, and its actual weight is 37.5 g.
  • the prepared carrier carrying mixed immobilized E. coli cells is installed in an immobilized enzyme or immobilized cell reactor.
  • the reactor is a cylinder made of organic glass with a height of 7 cm and a radius of 4.5 cm. Use a knife to trim off about 3 cm of the head and tail of the above-mentioned carrier at an inclination of 45°, tightly hold and roll it into a homogeneous cylinder with a height of 5 cm and a radius of 4.5 cm, weighing 27.2 g. Insert the cylinder into the reactor so that its tightness meets the level 3 standard described in Table 1 of Chinese invention patent application CN106032520A, and there is no gap between the side wall and the inner wall of the reactor. After the installation is completed, perform the installation procedures of other equipment according to Figure 1 of CN106032520A.
  • the capacity of the reaction control tank is 1L; the high-flow water pump is an adjustable flow suction pump with a flow rate of 1L/min; the pH control device adopts 0.3M hydroxide
  • the pH of the sodium solution is adjusted, and the flow rate of the dosing pump is 1 ml per minute.
  • the substrates contained in the reaction solution are adenosine 6g/L, magnesium chloride hexahydrate 16.4g/L, and polyphosphoric acid 13.3g/L. After adding the above substrate, stir and dissolve with 1 liter of deionized water at about 45°C until the reaction solution is clear, and adjust the pH to 7-7.5 with 5M sodium hydroxide solution.
  • adenosine triphosphate ATP
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • the adenosine triphosphate (ATP) content of the product obtained by the above-mentioned method is 20 mM. Therefore, the content of adenosine triphosphate (ATP) is rapidly increased during the reaction.
  • Example 12 Enzymatic production of creatine phosphate (without AK, PAP, ADK and PPK)
  • the creatine kinase gene and E. coli cells expressing the gene were obtained and fermented.
  • the obtained wet weight 35 g of creatine kinase was used to recombinantly express E. coli cells to prepare immobilized cells on a carrier.
  • the shape of the carrier is a bar shape: length 27 cm, width 5 cm, thickness 5 mm, and actual weight 58.4 g.
  • the substrates contained in the reaction solution are creatine monohydrate 8.94 g/L, adenosine triphosphate disodium salt 3 g/L, magnesium chloride hexahydrate 16.4 g/L, adenosine 6 g/L, and polyphosphoric acid 13.3 g/L.
  • After adding the above substrate stir and dissolve with 1 liter of deionized water at about 45°C until the reaction solution is clear, and adjust the pH to 8.5 with 2M sodium hydroxide solution.
  • Add 300ml of the above reaction solution to the reaction control tank the temperature is 37°C, the pH is adjusted to 8.35-8.60, the flow of the high-flow water pump is 3L/min, and the reaction lasts for 60 minutes.
  • adenosine triphosphate ATP
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • the product creatine phosphate content is 0.8mM, totaling 0.05g.
  • Example 13 Enzymatic production of creatine phosphate (without AK)
  • creatine kinase (CK) and polyphosphoric acid AMP phosphotransferase (PAP), adenylate kinase (ADK), polyphosphokinase (PPK) and separately The recombinant expressing the gene is expressed in E. coli cells and fermented.
  • polyphosphoric acid was prepared on the carrier: AMP phosphotransferase expressing E. coli cells, adenylate kinase expressing E. coli cells, polyphosphokinase expressing E. coli cells and creatine Kinase-expressing mixed immobilized cells of E. coli cells. Table 5 below shows the wet weight of each cell after centrifugation.
  • the mixing weight ratio of each cell is based on the corresponding corresponding enzyme activity.
  • the shape of the carrier is a bar: 34 cm long, 5 cm wide, 5 mm thick, and the actual weight is 46.6 g.
  • the substrates contained in the reaction solution were 8.94 g/L of creatine monohydrate, 3 g/L of adenosine triphosphate disodium salt, 16.4 g/L of magnesium chloride hexahydrate, 6 g/L of adenosine, and 13.3 g/L of polyphosphoric acid. After adding the above substrate, stir and dissolve with 1 liter of deionized water at about 45°C until the reaction solution is clear, and adjust the pH to 8.5 with 2M sodium hydroxide solution.
  • the reaction solution contains 300ml of the above reaction solution to the reaction control tank, the temperature is 37°C, the pH is adjusted to 8.35-8.60, the flow of the high-flow water pump is 3L/min, and the reaction lasts for 60 minutes. Since adenosine triphosphate (ATP) enzymatic regeneration and adenosine kinase (AK) in the newly added combination were not added, adenosine triphosphate (ATP) was slowly degraded to adenosine over time during the reaction. When the reaction was completed, the reaction solution contained the product creatine phosphate, A large amount of adenosine and a small amount of adenine, the product creatine phosphate content is 3.8 mM, a total of 0.24 g.
  • ATP adenosine triphosphate
  • AK adenosine kinase
  • creatine kinase (CK), adenosine kinase (AK), adenylate kinase (ADK), polyphosphokinase (PPK) were obtained, and the recombinant expression of the gene was expressed separately. Bacillus cells and fermentation.
  • creatine kinase recombinant expression E. coli cells, adenosine kinase recombinant expression E. coli cells, adenylate kinase recombinant expression E. coli cells and polyphosphokinase recombinant expression were prepared on the carrier.
  • the mixing weight ratio of each cell is based on the substantially correspondingly matched enzyme activity.
  • the shape of the carrier is a strip: 25 cm long, 5 cm wide, 5 mm thick, and its actual weight is 34.5 g.
  • the substrate contained in the reaction solution is 8.94 g/L of creatine monohydrate, 3 g/L of adenosine triphosphate disodium salt, 16.4 g/L of magnesium chloride hexahydrate, 6 g/L of adenosine, and 13.3 g/L of polyphosphoric acid. After adding the above substrate, stir and dissolve with 1 liter of deionized water at about 45°C until the reaction solution is clear, and adjust the pH to 8.5 with 2M sodium hydroxide solution.
  • adenosine triphosphate (ATP), adenosine monophosphate (AMP) and creatine phosphate continue to increase; during the reaction, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) will increase over time.
  • ATP adenosine triphosphate
  • AMP adenosine monophosphate
  • AMP adenosine triphosphate
  • ATP adenosine triphosphate
  • ADP adenosine diphosphate
  • AMP adenosine monophosphate
  • AMP phosphotransferase fails to regenerate adenosine monophosphate (AMP) to adenosine diphosphate (ADP) and cannot further add and regenerate adenosine triphosphate (ATP).
  • AMP adenosine monophosphate
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • the remaining part of adenosine monophosphate (AMP) a small amount of adenosine diphosphate (ADP), adenosine triphosphate (ATP), adenosine and a large amount of adenine at the end of the reaction
  • the content of the product creatine phosphate is 4.8 mM, a total of 0.3 g.
  • Enzyme Cell weight g
  • Creatine Kinase CK
  • AK Adenosine Kinase
  • ADK Adenosine Kinase
  • PPK2 Polyphosphokinase
  • CK creatine kinase
  • AK adenosine kinase
  • ADK adenylate kinase
  • PAP polyphosphate:AMP phosphotransferase
  • coli cells and polyphosphate: AMP were prepared on the carrier
  • the phosphotransferase is recombinantly expressed in mixed immobilized cells of Escherichia coli cells, and the mixing weight ratio of each cell is based on substantially correspondingly matched enzyme activities.
  • Table 6 shows the wet weight of each cell after centrifugation.
  • the shape of the carrier is a strip: 23 cm long, 5 cm wide, 5 mm thick, and its actual weight is 32.8 g.
  • the substrates contained in the reaction solution were 8.94 g/L of creatine monohydrate, 3 g/L of adenosine triphosphate disodium salt, 16.4 g/L of magnesium chloride hexahydrate, 6 g/L of adenosine, and 13.3 g/L of polyphosphoric acid. After adding the above substrate, stir and dissolve with 1 liter of deionized water at about 45°C until the reaction solution is clear, adjust the pH to 8.5 with 2M sodium hydroxide solution, and wait until the temperature of the reaction solution drops to room temperature for later use.
  • adenosine monophosphate (AMP), adenosine triphosphate (ATP) and creatine phosphate continue to increase; at the end of the reaction, adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine are left; the content of product creatine phosphate It is 4.4mM, a total of 0.28g.
  • Example 16 Enzymatic production of creatine phosphate (without PPK2 and ADK)
  • creatine kinase (CK), polyphosphate: AMP phosphotransferase (PAP), adenosine kinase (AK) and recombinant expression E. coli cells expressing the gene were obtained and performed Ferment.
  • CK creatine kinase
  • PAP polyphosphate: AMP phosphotransferase
  • AK adenosine kinase
  • E. coli cells expressing the gene were obtained and performed Ferment.
  • Example 3 of Chinese Patent CN1982445B a mixed immobilization of creatine kinase expressing E. coli cells, polyphosphate: AMP phosphotransferase recombinant expression E. coli cells and adenosine kinase recombinant expression E.
  • the coli cells were prepared on the carrier Cells, the mixing weight ratio of each cell is based on the basically correspondingly matched enzyme activity. Table 7 below shows the wet weight of each cell after centrifugation.
  • the shape of the carrier is a strip shape: length 24 cm, width 5 cm, thickness 5 mm, and actual weight 34.2 g.
  • the substrates contained in the reaction solution were 8.94 g/L of creatine monohydrate, 3 g/L of adenosine triphosphate disodium salt, 16.4 g/L of magnesium chloride hexahydrate, 6 g/L of adenosine, and 13.3 g/L of polyphosphoric acid.

Abstract

提供了酶法反应组合物、增加酶法反应中的三磷酸腺苷（ATP）的量的方法以及利用三磷酸腺苷（ATP）合成氨基酸或其衍生物、多肽、酶或蛋白的方法。所述方法在进行酶法反应时加入生产单磷酸腺苷（AMP）的第一酶或酶组，以新增三磷酸腺苷（ATP）的量。

Description

酶法反应组合物、增加酶法反应中三磷酸腺苷(ATP)量的方法及其应用 技术领域
本发明涉及生化领域，具体而言，涉及利用三磷酸腺苷(ATP)的生化反应，特别是利用三磷酸腺苷的酶法反应，酶法反应组合物以及增加酶法反应中三磷酸腺苷(ATP)含量的方法
背景技术
三磷酸腺苷(ATP)是由三个相连的磷酸基团、核糖和腺嘌呤组成的高能量化合物。三磷酸腺苷(ATP)是生物体内最重要的辅酶，为新陈代谢提供能量和提供磷酸基团和腺苷基团等。三磷酸腺苷(ATP)的重要组成部份是三个相连的α、β和γ-磷酸基团；α-磷酸基团和腺苷连接，β和γ磷酸基团均为高能磷酸键。仅含α-磷酸基团的为单磷酸腺苷(AMP)；化合物含有α和β两个磷酸基团为二磷酸腺苷(ADP)；含α、β和γ三个磷酸基团为三磷酸腺苷(ATP)。在利用三磷酸腺苷(ATP)(例如，以三磷酸腺苷(ATP)为能量或/和以磷酸基团为底物)的酶法反应中，合成酶可使用三磷酸腺苷(ATP)中的一个或两个高能磷酸键或/和以磷酸基团为底物。反应完成后(ATP)转化成二磷酸腺苷(ADP)或AMP、单磷酸等；生物反应中也会以三磷酸腺苷(ATP)作为底物；合成酶能利用三磷酸腺苷(ATP)的腺苷部分为底物，与其他化合物进行合成，副产物为单磷酸或焦磷酸。
通过酶法反应进行多肽合成和磷酸化生产为现代生物科技的重要手段。与化学合成方法相比，酶法生产拥有多项优势：在合成过程中，利用酶法生产时不使用对环境有害或有毒的有机化学物、进行反应时一般不产生副产品而因此有利纯化等。但是，目前使用三磷酸腺苷(ATP)的酶法工艺由于成本等原因尚无法广泛应用。
仍然需要进一步改善的使用三磷酸腺苷(ATP)的酶法工艺或者酶法反应。
发明内容
本发明提供了酶法反应组合物、酶法反应、增加酶法反应中的三磷酸腺苷(ATP)的含量的方法以及其应用。
具体而言，本发明提供了：
(1)一种增加酶法反应中的三磷酸腺苷(ATP)的量的方法，其中在进行酶法反应时加入生产单磷酸腺苷(AMP)的第一酶或酶组以及腺苷，以新增三磷酸腺苷(ATP)的量，其中所述酶法反应的反应底物包含三磷酸腺苷(ATP)或其盐。
(2)根据上述任意的方法，所述方法还包括在加入第一酶或酶组的同时、之前或者之后，加入负责三磷酸腺苷(ATP)再生的第二酶或酶组。
(3)根据上述任意的方法，在加入第一酶或酶组和/或加入第二酶或酶组的同时、之前或者之后，加入第三酶或酶组。
(4)根据上述任意的方法，所述的第一酶或酶组包含腺苷激酶(AK)。
(5)根据上述任意的方法，所述反应底物还包含多聚磷酸或其盐以及辅助离子中的至少一者，
所述辅助离子优选镁离子、钠离子、钾离子和氯离子中的至少一者，更优选为镁离子和钾离子中至少一者；辅助离子可为其无机盐或有机盐的状态，优选为六水氯化镁、氯化钠、氯化锰、硫酸镁和碳酸钾中的至少一者，更优选为六水氯化镁、氯化钠和碳酸钾中的至少一者。
(6)根据上述任意的方法，所述的第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)中的至少一者，
任选地，所述第三酶或酶组包含肌酸激酶(CK)、谷氨酸激酶(GK)、輔酶I激酶(NK)和/或以三磷酸腺苷(ATP)为其中之一底物以进行氨基酸、肽或蛋白的磷酸化、磷酸转移或多肽合成的其他酶或者酶组。
(7)根据上述任意的方法，所述酶法反应包括下列中的至少一者：
(i)所述的第一酶或酶组包含腺苷激酶(AK)，反应底物包含腺苷、多聚磷酸和三磷酸腺苷(ATP)，并且反应产物包含单磷酸腺苷(AMP)和二磷酸腺苷(ADP)；
(ii)所述的第一酶或酶组包含腺苷激酶(AK)，并且第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)，反应底物包含单磷酸腺苷(AMP)和多聚磷酸，并且反应产物包含二磷酸腺苷(ADP)和多聚磷酸；
(iii)所述的第一酶或酶组包含腺苷激酶(AK)，并且第二酶或酶组包含腺苷酸激酶(ADK)，反应底物包含二磷酸腺苷(ADP)和多聚磷酸，并且反应产物包含单磷酸腺苷(AMP)和三磷酸腺苷(ATP)；
(iv)所述的第一酶或酶组包含腺苷激酶(AK)，并且所述的第二酶或酶组包含多聚磷酸激酶(PPK)，反应底物包含二磷酸腺苷(ADP)和多聚磷酸，并且反应产物包含三磷酸腺苷(ATP)和多聚磷酸；以及
(v)所述的第一酶或酶组包含腺苷激酶(AK)，第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)并且所述的第三酶或酶组包含肌酸激酶(CK)，反应底物包含肌酸和三磷酸腺苷(ATP)，并且反应产物包含磷酸肌酸、二磷酸腺苷(ADP)和多聚磷酸；第三酶或酶组还包含谷氨酸激酶(GK)，反应底物包含谷氨酸和三磷酸腺苷(ATP)，并且反应产物包含谷氨酸5-磷酸、二磷酸腺苷(ADP)和多聚磷酸；第三酶或酶组还可包含辅酶I激酶(NK)，反应底物包含辅酶I和三磷酸腺苷(ATP)，并且反应产物包含氧化型烟酰胺腺嘌呤二核苷酸磷酸、二磷酸腺苷(ADP)和多聚磷酸；第三酶或酶组还可包含其他酶或者酶组以三磷酸腺苷(ATP)为其中之一的酶法反应底物以进行氨基酸、核酸、肽或蛋白的磷酸化、磷酸转移或多肽合成；
(vi)利用多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)和磷酸激酶的全部或至少一者、腺苷激酶和以三磷酸腺苷(ATP)为反应底物在同一反应体系中以混合、并联或串联的方式同时进行反应或在不同的反应体系中分别进行反应；
(vii)只利用多聚磷酸:AMP磷酸转移酶(PAP)、腺苷激酶(AK)和多聚磷酸激酶(PPK)的全部或至少一者和以三磷酸腺苷(ATP)为反应底 物在不同的反应体系中以混合、并联或串联的方式同时进行反应或在不同的反应体系中分别进行反应；和
(viii)只利用腺苷激酶以腺苷为底物生产单磷酸腺苷(AMP)，以新增三磷酸腺苷(ATP)。
(8)根据上述任意的方法，所述的第二酶或酶组分别将单磷酸腺苷(AMP)和二磷酸腺苷(ADP)再生至二磷酸腺苷(ADP)和三磷酸腺苷(ATP)；以及
任选地，所述第一酶或酶组将腺苷合成至单磷酸腺苷(AMP)。
(9)根据上述任意的方法，所述酶法反应包括利用腺苷和三磷酸腺苷(ATP)为底物合成单磷酸腺苷(AMP)。
(10)根据上述任意的方法，所述反应底物还包含肌酸或其水合物、谷氨酸鈉或其水合物和/或輔酶I等。
(11)根据上述任意的方法，按酶法反应中产生三磷酸腺苷(ATP)降解物的水平确定加入的第一酶或者酶组，降解物优选为二磷酸腺苷(ADP)、单磷酸腺苷(AMP)和/或腺苷。
(12)根据上述任意的方法，以纯化或非纯化的细胞破碎液、液酶、固定化细胞或固定化酶的形式加入所述第一酶或酶组、或者第二酶或酶组、或者第三酶或酶组。
(13)根据上述任意的方法，所述酶法反应的条件为：温度为28-40摄氏度，优选为30-38摄氏度，更优选为33-37摄氏度；pH值为5-9，优选为6-8.5，更优选为7-7.75。
(14)一种酶法反应组合物，包含底物以及生产单磷酸腺苷单磷酸腺苷(AMP)的第一酶或酶组，其中所述底物包含三磷酸腺苷三磷酸腺苷(ATP)或其盐以及腺苷。
(15)根据上述任意的酶法反应组合物，所述底物还包含多聚磷酸或其盐、辅助离子以及肌酸或其水合物中的至少一种，
所述辅助离子优选镁离子、钠离子、钾离子和氯离子中的至少一者，更优选为镁离子和钾离子中至少一者；辅助离子可为其无机盐或有机盐的状态，优选为六水氯化镁、氯化钠、氯化锰、硫酸镁和碳酸钾中的至少一者，更优选为六水氯化镁、氯化钠和碳酸钾中的至少一者。
(16)根据上述任意的酶法反应组合物，所述酶法反应组合物还包含第二酶或酶组和任选的第三酶或酶组。
(17)根据上述任意的酶法反应组合物，所述第一酶或酶组包含腺苷激酶AK，
优选地，当第三酶或酶组存在时，所述第三酶或者酶组包含肌酸激酶(CK)、谷氨酸激酶(GK)、輔酶I激酶(NK)或以三磷酸腺苷(ATP)为其中之一的酶法反应底物以進行氨基酸、肽或蛋白的磷酸化、磷酸转移或多肽合成的其他酶或者酶组，并且优选地，所述第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)中的至少一者。
(18)根据上述任意的酶法反应组合物，所述第一酶或酶组、第二酶或酶组和第三酶或酶组以纯化或非纯化的细胞破碎液、液酶、固定化细胞或固定化酶的形式包含在所述酶法反应组合物中。
(19)根据上述任意的酶法反应组合物，所述多聚磷酸:AMP磷酸转移酶(PAP)、腺苷激酶(AK)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)和肌酸激酶各自独立地为重组酶，并且分别或组合在大肠杆菌中表达。
(20)一种利用三磷酸腺苷(ATP)为底物以使氨基酸、核酸、肽或蛋白进行磷酸化或磷酸转移的方法，包括上述任意的方法。
(21)根据上述任意的方法，氨基酸、核酸、肽或蛋白为肌酸、谷氨酸和輔酶I等。
本发明具有以下优点和积极效果：
1.本发明不仅能利用多聚磷酸为底物，还可以利用价格低廉的腺苷作为反应底物，从而可以新增反应物种的三磷酸腺苷(ATP)的量并且和/或者将反应中的副产品单磷酸腺苷(AMP)、二磷酸腺苷(ADP)再生成三磷酸腺苷(ATP)，从而生产更多数量的三磷酸腺苷(ATP)，因此在酶法反应中，三磷酸腺苷(ATP)的量随反应时间增加而非降低。然而，现有的三磷酸腺苷(ATP)再生技术在最理想情况下只能维持稳定的三磷酸腺苷(ATP)量。三磷酸腺苷(ATP)价格昂贵，囿于成本，因此在工业生产上通常只可加入少量以控制生产成本。当反应中的三磷酸腺苷(ATP)浓度过低时，反 应效率也不会理想，徒耗费产能，还增加纯化的压力以至影响最终产品回收率。根据本发明，酶法反应中的三磷酸腺苷(ATP)量会随反应循环次数不断增加，反应效率也随反应时间逐步提升，有关的反应速度也随之加快。
2.本发明利用价格低廉的腺苷来合成单磷酸腺苷(AMP)，可将现有需使用三磷酸腺苷(ATP)的反应的生产成本进一步降低。
3.三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和单磷酸腺苷(AMP)降解酶在大肠杆菌细胞中的酶活力甚高。当单磷酸腺苷(AMP)被降解成腺苷后，三磷酸腺苷(ATP)即在反应中逐渐消失，最终导致有关反应的停止。本发明可利用水解后产生的腺苷重新合成为单磷酸腺苷(AMP)，进而重新转化成三磷酸腺苷(ATP)，因此转化酶可在不纯化的情况下使用，这就大大节省了纯化的庞大投入成本和时间，使酶法生产工艺可以在成本与产能上得以工业上落实。
然而，目前利用三磷酸腺苷(ATP)再生的方法通常需要对有关的酶进行蛋白提纯，此手段费用极其高昂，将对生产成本构成重大压力，因此酶法生产技术未能普及。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
如上所述，与化学合成方法相比，酶法工艺具有很多优势。然而，酶法工艺也有其缺点：生物反应中有时需要依赖使用价格
Figure PCTCN2020093704-appb-000001
贵的三磷酸腺苷(ATP)，对生产成本构成重大压力。目前，普遍采用多聚磷酸为底物，以磷酸转换或合成酶将反应后的二磷酸腺苷(ADP)或单磷酸腺苷(AMP)再生成三磷酸腺苷(ATP)，或利用葡萄糖为三磷酸腺苷(ATP)的原材料，以降低三磷酸腺苷(ATP)的成本。这类三磷酸腺苷(ATP)再生技术虽可减轻三磷酸腺苷(ATP)的成本压力，但却面对另一难题。一是为降低成本，三磷酸腺苷(ATP)的使用量不可过高，因此导致酶法合成反应的效 率偏低；二是由大肠杆菌为宿体表达重组酶中含有大量的水解三磷酸腺苷(ATP)、水解二磷酸腺苷(ADP)和水解单磷酸腺苷(AMP)的酶。这类水解酶活力十分高，可在短时间将三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、单磷酸腺苷(AMP)水解至单磷酸腺苷(AMP)和腺苷。三磷酸腺苷(ATP)被降解成腺苷后便不可逆地消失。因此，工业上若希望使用三磷酸腺苷(ATP)再生技术降低成本，则重组酶需要先进行蛋白纯化。蛋白纯化成本不菲。
为了解决上述存在的至少一个问题，本发明的发明人进行了大量深入的理论研究和实验摸索，利用利用价格低廉的腺苷作为生产单磷酸腺苷(AMP)的底物，经三磷酸腺苷(ATP)再生和/或新增反应体系生产更多数量的三磷酸腺苷(ATP)，因此在酶法反应过程中，三磷酸腺苷(ATP)的量随反应时间增加，从而提高了反应效率并且降低了成本。
在一个方面中，本发明提供了一种酶法反应组合物，包含底物以及生产单磷酸腺苷(AMP)的第一酶或酶组，其中，所述底物包含腺苷和三磷酸腺苷三磷酸腺苷(ATP)或其盐。
任选地，所述底物还包含多聚磷酸或其盐，优选多聚磷酸的钠盐。多聚磷酸的聚合度可为3-20,000；优选地，多聚磷酸聚合度可为3-7,000，更优选为3-75。
任选地，酶法反应组合物还可以包含辅助离子。辅助离子可以选自镁离子、钠离子、钾离子和氯离子中的至少一者，更优选为镁离子和钾离子中至少一者。辅助离子可为其无机盐或有机盐的状态，优选为六水氯化镁、氯化钠、氯化锰、硫酸镁和碳酸钾中的至少一者，更优选为六水氯化镁、氯化钠和碳酸钾中的至少一者。
为了利用快速增新三磷酸腺苷(ATP)酶法反应组合物进行反应合成，其中还应包括利用三磷酸腺苷(ATP)为其底物以进行磷酸的交换、合成和以其为能量借以进行多肽合成或磷酸交换，因此，反应中还包含该合成反应的所需底物；本文中以利用酶法合成磷酸肌酸为例子，反应底物包含肌酸、六水氯化镁和三磷酸腺苷(ATP)。
此外，本领域公知的是，酶法反应组合物还可以包含其他添加剂，例如，pH调节剂，如缓冲液/盐，优选为磷酸钠缓冲液、磷酸钾缓冲液和 三羟甲基氨基甲烷缓冲液、更优选为磷酸钠缓冲液和三羟甲基氨基甲烷缓冲液。pH调节剂的浓度可以为0.001M-1M，优选为0.01M-0.5M，更优选为0.05M-0.3M。
任选地，所述酶法反应组合物还包含第二酶或酶组和任选的第三酶或酶组。优选地，所述第一酶或酶组包含腺苷激酶(AK)。优选地，当第三酶或酶组存在时，所述第三酶或者酶组包肌酸激酶(CK)、谷氨酸激酶(GK)和輔酶I激酶(NK)中的至少一者。
优选地，第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)中的至少一者。
任选地，包含在酶法反应组合物中的第一酶或酶组、第二酶或酶组和第三酶或酶组可以为纯化或非纯化的细胞破碎液、液酶、固定化细胞或固定化酶的形式。对于固定化细胞或固定化酶，可以按照中国专利CN1982445B中记载的方法进行固定化细胞和固定化酶的制备，并使用该专利中的载体。例如，所述的载体为开孔的多孔有机泡沫材料，载体的形状包括颗粒状、块状、柱状、片状或条带状。优选地，多孔有机泡沫材料为三聚氰胺海绵。
在文本中，酶法反应组合物是指能够借助于酶或酶组进行生化反应，从而生产有价值的产物的可反应混合物。反应产物可以包含单磷酸腺苷(AMP)、二磷酸腺苷(ADP)、三磷酸腺苷(ATP)、多聚磷酸、磷酸肌酸、谷氨酸5-磷酸、氧化型辅酶Ⅱ的一种或者多种。
在本文中，第一酶或酶组、第二酶或酶组和第三酶或酶组可以各自独立地为重组酶，并且分别或组合在载体中表达。载体可以包括大肠杆菌、酵母菌。这样，可以使用包含重组酶的细胞或其碎片来进行所述酶法反应。例如，细胞可以为大肠杆菌细胞、酵母菌细胞。载体可以包括大肠杆菌、酵母菌、芽胞杆菌等生物科学中常用的方式来进行重组酶的表达。酶或酶组可以在细胞、破碎液、上清液或纯化酶的液态中使用，或以任何方式和其对应载体制成固定化细胞或固定化酶进行行酶或酶组反应。
在另一个方面中，本发明还提供一种增加酶法反应中的三磷酸腺苷(ATP)的量的方法，其包括在进行酶法反应时加入生产单磷酸腺苷(AMP) 的第一酶或酶组以及腺苷，以新增三磷酸腺苷(ATP)的量，其中所述酶法反应的反应底物包含三磷酸腺苷(ATP)或其盐。
所述方法还可以包括在加入第一酶或酶组的同时、之前或者之后，加入负责三磷酸腺苷(ATP)再生的第二酶或酶组。
任选地，在加入第一酶或酶组和/或加入第二酶或酶组的同时、之前或者之后，可以加入第三酶或酶组。
如上所述，第一酶或酶组可以包含腺苷激酶(AK)。任选地，所述反应底物还包含多聚磷酸或其盐以及辅助离子中的至少一者。
所述辅助离子优选镁离子、钠离子、钾离子和氯离子中的至少一者，更优选为镁离子和钾离子中至少一者；辅助离子可为其无机盐或有机盐的状态，优选为六水氯化镁、氯化钠、氯化锰、硫酸镁和碳酸钾中的至少一者，更优选为六水氯化镁、氯化钠和碳酸钾中的至少一者。
如上所述，第二酶或酶组可以包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)肌酸激酶中的至少一者。
任选地，所述酶法反应包括下列中的至少一者：
(i)所述的第一酶或酶组包含腺苷激酶(AK)，反应底物包含腺苷、多聚磷酸和三磷酸腺苷(ATP)，并且反应产物包含单磷酸腺苷(AMP)和二磷酸腺苷(ADP)；
(ii)所述的第一酶或酶组包含腺苷激酶(AK)，并且第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)，反应底物包含单磷酸腺苷(AMP)和多聚磷酸，并且反应产物包含二磷酸腺苷(ADP)和多聚磷酸；
(iii)所述的第一酶或酶组包含腺苷激酶(AK)，并且第二酶或酶组包含腺苷酸激酶(ADK)，反应底物包含二磷酸腺苷(ADP)和多聚磷酸，并且反应产物包含单磷酸腺苷(AMP)和三磷酸腺苷(ATP)；
(iv)所述的第一酶或酶组包含腺苷激酶(AK)，并且所述的第二酶或酶组包含多聚磷酸激酶(PPK)，反应底物包含二磷酸腺苷(ADP)和多聚磷酸，并且反应产物包含三磷酸腺苷(ATP)和多聚磷酸；以及
(v)所述的第一酶或酶组包含腺苷激酶(AK)，第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶 (ADK)并且所述的第三酶或酶组包含肌酸激酶(CK)，反应底物包含肌酸和三磷酸腺苷(ATP),反应产物包含磷酸肌酸、二磷酸腺苷(ADP)和多聚磷酸；第三酶或酶组还包含谷氨酸激酶(GK)，反应底物包含谷氨酸和三磷酸腺苷(ATP)，并且反应产物包含谷氨酸5-磷酸、二磷酸腺苷(ADP)和多聚磷酸；第三酶或酶组还可包含辅酶I激酶(NK)，反应底物包含辅酶I和三磷酸腺苷(ATP)，并且反应产物包含氧化型烟酰胺腺嘌呤二核苷酸磷酸、二磷酸腺苷(ADP)和多聚磷酸；第三酶或酶组亦可包含其他酶或者酶组以三磷酸腺苷(ATP)为其中之一的酶法反应底物以进行氨基酸、核酸、肽或蛋白的磷酸化、磷酸转移或多肽合成。
(vi)利用多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)和磷酸激酶(PPK)的全部或至少一者、腺苷激酶(AK)和以三磷酸腺苷(ATP)为反应底物在同一反应体系中以混合、并联或串联的方式同时进行反应或在不同的反应体系中分别进行反应；
(vii)只利用多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)和磷酸激酶(PPK)的全部或至少一者和以三磷酸腺苷(ATP)为反应底物在不同的反应体系中以混合、并联或串联的方式同时进行反应或在不同的反应体系中分别进行反应；和
(viii)只利用腺苷激酶(AK)以腺苷为底物生产单磷酸腺苷(AMP)，以新增三磷酸腺苷(ATP)。
在一个实施方案中，第二酶或酶组分别将单磷酸腺苷(AMP)和二磷酸腺苷(ADP)再生至二磷酸腺苷(ADP)和三磷酸腺苷(ATP)；以及
任选地，所述第一酶或酶组将腺苷合成至单磷酸腺苷(AMP)。
在一个实施方案中，酶法反应包括利用腺苷和三磷酸腺苷(ATP)为底物合成单磷酸腺苷(AMP)。
在一个实施方案中，反应底物还可以包含肌酸或其水合物。
在一个实施方案中，按酶法反应中产生三磷酸腺苷(ATP)降解物的水平确定加入的第一酶或者酶组，降解物优选为二磷酸腺苷(ADP)、单磷酸腺苷(AMP)和/或腺苷。
在一个实施方案中，以纯化或非纯化的细胞破碎液、液酶、固定化细胞或固定化酶的形式加入所述第一酶或酶组、或者第二酶或酶组、或者第三酶或酶组。
任选地，酶法反应的条件可为：温度为28-40摄氏度，优选为30-38摄氏度，更优选为33-37摄氏度；pH值为5-9，优选为6-8.5，更优选为7-7.75。
这样，本发明的方法能够利用反应体系中的副产物(如腺苷、二磷酸腺苷(ADP)或单磷酸腺苷(AMP))或者额外加入的腺苷作为底物，快速新增三磷酸腺苷(ATP)或者再生成三磷酸腺苷(ATP)，从而产生更多数量的三磷酸腺苷(ATP)，因此在酶法反应中，三磷酸腺苷(ATP)的量随反应时间增加而非降低。
任选地，酶法反应组合物还可以包含多聚磷酸或其盐、辅助离子、盐以及肌酸或其水合物中的至少一种。
任选地，所述酶法反应的反应产物包含单磷酸腺苷(AMP)、二磷酸腺苷(ADP)、三磷酸腺苷(ATP)、多聚磷酸和磷酸肌酸中的一种或者多种。
在酶法反应中，所述多聚磷酸:AMP磷酸转移酶(PAP)、腺苷激酶(AK)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)、肌酸激酶(CK)、谷氨酸激酶(GK)和辅酶I激酶(NK)各自独立地为重组酶，并且分别或组合在大肠杆菌中表达。
在又一个方面中，本发明还提供一种利用三磷酸腺苷(ATP)为底物以使氨基酸、核酸、肽或蛋白进行磷酸化或磷酸转移的方法，包括上述的任意的方法。因此，可以利用上述方法来合成氨基酸或其衍生物、核酸、肽、蛋白或其衍生物。
在一个具体例子中，所述方法涉及到利用三磷酸腺苷(ATP)为底物的肽合成或加磷酸化反应，具体包括加入反应所需的底物、辅助离子或盐，进行其合成反应，同时加入快速新增三磷酸腺苷(ATP)及快速再生三磷酸腺苷(ATP)的合成酶和底物、镁离子，在多肽合成或加磷酸化适宜的反应条件下，同步进行三磷酸腺苷(ATP)快速新增及再生三磷酸腺苷(ATP)反应。
优选地，所述氨基酸、肽、核酸、蛋白或其衍生物为肌酸、谷氨酸、輔酶I等。
为了进行所述酶法反应，可以将所述的固定化细胞和固定化酶置于固定化反应装置中，以进行固定化反应。例如，可按照中国专利申请CN106032520A记载的步骤进行所述固定花反应。
固定化反应装置可以包括具有入口和出口的柱状反应器、反应调控罐、高流量水泵、pH调控装置与pH探测电极、搅拌装置和酸碱度调节罐。
以下通过例子的方式进一步解释或说明本发明内容，但这些例子不应被理解为对本发明保护范围的限制。
例子
以下例子中未注明具体条件的，均按常规条件或制造商建议的条件进行。除非特别说明，否则所述百分比为重量百分比。
下列例子中所用材料和设备的描述如下：
反应调控罐：基因港(香港)生物科技有限公司，BR-1L；
可调节流量式吸水泵：SURGEFLO公司，FL-32；
酸碱度调控装置：基因港(香港)生物科技有限公司，AR-1；
一水肌酸：江苏远洋化学股份有限公司；
腺苷：浙江诚意药业；
三磷酸腺苷二钠盐：开平牵牛医药公司；
多聚磷酸钠：西陇化工有限公司；
谷氨酸钠：味之素红碗牌味精；
輔酶I:Roche Inc.,USA；
例1：制备肌酸激酶(CK)
肌酸激酶PCR引物序列设计根据中国专利申请公开号CN102808006，具体为：
上游引物CPK1：
5’-ctgacc ggatccatgccgttcggtaacacccacaac-3’(SEQ NO.1)
其中，BamHI酶切点位以下划线表示。
下游引物CPK2：
5’-tatgcg gaattcttacttctgggcggggatcatgtc-3’(SEQ NO.2)
其中，EcoRI酶切点位以下划线表示。
根据中国专利申请公开号CN102808006所述的方法，提取小鼠骨骼肌总RNA，反转录制备cDNA，以小鼠骨骼肌cDNA为范本，以上述引物PCR扩增得到肌酸激酶基因，并连接至pGEX-2T(购自GE Healthcare,USA)，得到pGEX-2T(+)-CK(SEQ NO.15)，将其转化至大肠杆菌BL21(DE3)，获得肌酸激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含肌酸激酶的大肠杆菌细胞1.42kg。将所得含肌酸激酶的大肠杆菌细胞配制成酶液，每ml酶液中含0.2g细胞。根据中国专利申请公开号CN102808006的磷酸肌酸含量测定法对细胞进行酶活力检测，酶活性为约2.1U/g。
例2：制备多聚磷酸:AMP转移酶(PAP)
根据多聚磷酸:AMP磷酸转移酶序列设计PCR引物，具体为：
上游引物PAP1：
5’-ctgacc ggatccatgttcgaatccgcggaagttggc-3(SEQ NO.3)
其中，BamHI酶切点位以下划线表示。
下游引物PAP2：
5'-tatgcg aagcttttacttgtccttcttgtacgccgcctc-3'(SEQ NO.4)
其中，EcoRI酶切点位以下划线表示。
以绿脓杆菌(Pseudomonas aeruginosa)PAO1-VE13AGY71676DNA为底物，以上述引物进行PCR扩增得多聚磷酸:AMP转移酶基因， 利用限制性内切酶BamH I和EcoRI处理PCR产物并将其连接至pGEX-2T(购自GE Healthcare,USA)，得到pGEX-2T(+)-PAP(SEQ NO.16)。将此重组表达载体转化至大肠杆菌HB101中，得到多聚磷酸:AMP磷酸转移酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入异丙基硫代半乳糖苷(IPTG)至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含多聚磷酸:AMP磷酸转移酶的大肠杆菌细胞1.18kg。将所得含多聚磷酸:AMP磷酸转移酶的大肠杆菌细胞配制成酶液，每ml酶液中含0.2g细胞。根据其酶法反应对细胞进行酶活力检测，细胞酶活性为约1.1U/g。
例3：制备腺苷酸激酶(AK)
根据腺苷酸激酶序列设计PCR引物，具体为：
上游引物AK1：
5’-ctgacc ggatccatggcagtcgattcctccaactcg-3(SEQ NO.5)
其中，BamHI酶切点位以下划线表示。
下游引物AK2：
5’-tatgcg gaattcttaacacggaagtgaagtgaagct-3'(SEQ NO.6)
其中，EcoRI酶切点位以下划线表示。
以肠炎沙门氏菌亚种Salmonella enterica subsp.enterica serovar ATCC700720(ATCC,USA)DNA为底物，以上述引物进行PCR扩增得腺苷酸激酶基因，利用限制性内切酶BamHI和EcoRI处理PCR产物并将其连接至pGEX-2T(购自GE Healthcare,USA)中，得到pGEX-2T(+)-ADK(SEQ NO.17)。将此重组表达载体转化至大肠杆菌HB101中，得到腺苷酸激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含腺苷酸激酶的大肠杆菌细胞1.18kg。将所得含腺苷酸激酶的大肠杆菌细胞配制成酶液，每ml酶液中含0.2g细胞，根据其酶法反应对细胞进行酶活力检测，酶活约为0.08U/g。
例4：制备多聚磷酸激酶(PPK)
根据多聚磷酸激酶序列设计PCR引物，具体为：
上游引物PPK-1：
5’-ctgacc ggatccatgagcaagtccgacgacgacgag-3(SEQ NO.7)
其中，BamHI酶切点位以下划线表示。
下游引物PPK-2：
5’-tatgcg gaattcttaccgcgccaaccgcccatcttc-3'(SEQ NO.8)
其中，EcoRI酶切点位以下划线表示。
以新月柄杆菌C.crescentus NA1000YP_002518902DNA为底物，以上述引物进行PCR扩增得磷酸激酶基因，利用限制性内切酶BamHI和EcoRI处理PCR产物并将其连接至pGEX-2T(购自GE Healthcare,USA)中，得到pGEX-2T(+)-PPK(SEQ NO.18)。将此重组表达载体转化至大肠杆菌HB101中，得到多聚磷酸激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最 终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含多聚磷酸激酶的大肠杆菌细胞1.73kg。将所得含多聚磷酸激酶的大肠杆菌细胞配制成酶液，每ml酶液中含0.2g细胞，根据其酶法反应对细胞进行酶活力检测，酶活约为0.03U/g。
例5:制备腺苷激酶(ADK)
根据腺苷激酶序列设计PCR引物，具体为：
上游引物ADK1：
5’-ctgacc ggatccatgaatatcattttgatgggttta-3(SEQ NO.9)
其中，BamHI酶切点位以下划线表示。
下游引物ADK2：
5’-tatgcg gaattcttacaaatgatctaaaatatcaat-3'(SEQ NO.10)
其中，EcoRI酶切点位以下划线表示。
以耻垢分枝杆菌Mycobacterium smegmatis MC2 155DNA为底物，以上述引物进行PCR扩增得到腺苷激酶基因序列，利用限制性内切酶BamHI和EcoRI处理PCR产物，把所得基因序列连接到pGEX-2T(购自GE Healthcare,USA)中，得到pGEX-2T(+)-AK(SEQ NO.19)，转化至大肠杆菌BL21(DE3)中，得到腺苷激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含腺苷激酶的大肠杆菌细胞1.2kg。将所得到的腺苷激酶的大肠杆菌细胞以1对5比例悬浮于50mM Tris-HCl盐酸缓冲液(pH 7.5)中。然后用超声波裂解细菌细胞。离心(10℃，12,500rpm，15分钟)并收集上清液为腺苷激酶液。根据其酶法反应对细胞进行酶活力检测，酶活约为0.08EU/g。
例6:制备谷氨酸激酶(GK)
根据谷氨酸激酶序列设计PCR引物，具体为：
上游引物GK1：
5’-ctgacc ggatccatgcgggacaaggtgactggcgcg-3'(SEQ NO.16)
其中，BamHI酶切点位以下划线表示。
下游引物GK2：
5’-tatgcg gaattcttagaccagaaccagattgtcgcg-3'(SEQ NO.17)
其中，EcoRI酶切点位以下划线表示。
以绿脓杆菌Pseudomonas aeruginosa为底物，以上述引物进行PCR扩增得到腺苷激酶基因序列，利用限制性内切酶BamHI和EcoRI处理PCR产物，把所得基因序列连接到pGEX-2T(购自GE Healthcare,USA)中，得到pGEX-2T(+)-GK(SEQ NO.20)，转化至大肠杆菌BL21(DE3)中，得到腺苷激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含谷氨酸激酶的大肠杆菌细胞1.2kg。将所得到的谷氨酸激酶的大肠杆菌细胞以1对5比例悬浮于50mM Tris-HCl盐酸缓冲液(pH 7.5)中。然后用超声波裂解细菌细胞。离心(10℃，12,500rpm，15分钟)并收集上清液为谷氨酸激酶液。根据其酶法反应对细胞进行酶活力检测，酶活约为0.02EU/g。
例7:制备輔酶I激酶(NK)
根据輔酶I激酶序列设计PCR引物，具体为：
上游引物NK1：
5’-ctgacc ggatccatgcgggacaaggtgactggcgcg-3'(SEQ NO.18)
其中，BamHI酶切点位以下划线表示。
下游引物NK2：
5’-tatgcg gaattcttagaccagaaccagattgtcgcg-3'(SEQ NO.19)
其中，EcoRI酶切点位以下划线表示。
以结核分枝杆菌Mycobacterium tuberculosis variant bovis BCG str.Tokyo 172为底物，以上述引物进行PCR扩增得到輔酶I激酶基因序列，利用限制性内切酶BamHI和EcoRI处理PCR产物，把所得基因序列连接到pGEX-2T(购自GE Healthcare,USA)中，得到pGEX-2T(+)-NK(SEQ NO.21)，转化至大肠杆菌BL21(DE3)中，得到輔酶I激酶重组表达菌株。
将上述菌株挑选单一种落接种到4mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养16小时作为初级种子，完成后按1％接种比例接到100mL LB培养基(含100ug/ml氨苄青霉素)，在37℃、200rpm摇床中培养10小时作为二级种子，完成后按1％接种比例接到60L LB培养基(含100ug/ml氨苄青霉素)的100L发酵罐中培养。发酵初始条件为37℃、200rpm、pH7.0。发酵进行至9小时加入IPTG至最终浓度为1mM，发酵20小时结束。发酵液在4℃下以12,500rpm离心10分钟，得含谷氨酸激酶的大肠杆菌细胞1.2kg。将所得到的谷氨酸激酶的大肠杆菌细胞以1对5比例悬浮于50mM Tris-HCl盐酸缓冲液(pH 7.5)中。然后用超声波裂解细菌细胞。离心(10℃，12,500rpm，15分钟)并收集上清液为輔酶I激酶液。根据其酶法反应对细胞进行酶活力检测，酶活约为0.045EU/g。
例8：制备磷酸肌酸
按例1-5的制备方法得肌酸激酶基因、多聚磷酸:AMP磷酸转移酶、腺苷酸激酶、多聚磷酸激酶和腺苷激酶并且分别表达该基因的重组表达大肠杆菌细胞并且进行发酵。
根据中国专利CN1982445B的实施例3的方法，在固相载体上制备含有肌酸激酶、腺苷激酶、多聚磷酸:AMP磷酸转移酶、腺苷酸激酶和多 聚磷酸激酶的混合固定化大肠杆菌细胞，各细胞的混合重量比例依据基本相应匹配的酶活性。下表1为各细胞在离心后的湿重量。载体的形状为条形：长24cm、宽5cm、厚5mm，实重46.3g。将上述制得的承载有混合固定化大肠杆菌细胞的载体安装于固定化酶或固定化细胞反应器中。该反应器为有机玻璃制成的圆柱体，高7cm、半径4.5cm。用刀将上述载体头尾端约3cm以斜度45°整齐削去，紧握卷成高5cm、半径4.5cm的均质圆柱体，重量为32.2g。将该圆柱体插入反应器中，使其松紧程度符合中国发明专利申请CN106032520A中表1所述的3级标准，并且使其侧壁与反应器的内壁之间不留空隙。完成安装后，按CN106032520A的图1进行其它配备装置的安装程序，其中反应调控罐容量为1L；高流量水泵是可调节流量式吸水泵，流速为3L/分钟；酸碱度调控装置采用0.3M氢氧化钠溶液进行pH调控，其加液泵的流量为每分钟1ml。反应溶液所含的底物为一水肌酸8.94g/L，三磷酸腺苷二钠盐3g/L、六水氯化镁16.4g/L，腺苷6g/L，多聚磷酸13.3g/L。加入以上底物后用1升约45℃的去离子水搅拌溶解，至反应溶液澄凊，用2M氢氧化钠溶液调节pH至8.5。加入300ml上述反应溶液至反应调控罐中，温度为37℃，pH调控为8.35-8.60，高流量水泵的流量为3L/分钟，反应60分钟，产物磷酸肌酸含量为6.5mM，反应90分钟，产物磷酸肌酸含量为11.3mM，反应120分钟结束时，产物磷酸肌酸含量为15.7mM。
表1
Figure PCTCN2020093704-appb-000002
例9：制备谷氨酸5-磷酸
按例2-6的制备方法得多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)、多聚磷酸激酶(PPK)、腺苷激酶(AK)和谷氨酸激酶(GK)并且分别表达该基因的重组表达大肠杆菌细胞并且进行发酵。
根据中国专利CN1982445B的实施例3的方法，在固相载体上制备含有谷氨酸激酶、腺苷激酶、多聚磷酸:AMP磷酸转移酶、腺苷酸激酶和多聚磷酸激酶的混合固定化大肠杆菌细胞，各细胞的混合重量比例依据基本相应匹配的酶活性。下表2为各细胞在离心后的湿重量。载体的形状为条形：长28cm、宽5cm、厚5mm，实重48.1g。将上述制得的承载有混合固定化大肠杆菌细胞的载体安装于固定化酶或固定化细胞反应器中。该反应器为有机玻璃制成的圆柱体，高7cm、半径4.5cm。用刀将上述载体头尾端约3cm以斜度45°整齐削去，紧握卷成高5cm、半径4.5cm的均质圆柱体，重量为31.4g。将该圆柱体插入反应器中，使其松紧程度符合中国发明专利申请CN106032520A中表1所述的3级标准，并且使其侧壁与反应器的内壁之间不留空隙。完成安装后，按CN106032520A的图1进行其它配备装置的安装程序，其中反应调控罐容量为1L；高流量水泵是可调节流量式吸水泵，流速为3L/分钟；酸碱度调控装置采用0.3M氢氧化钠溶液进行pH调控，其加液泵的流量为每分钟1ml。反应溶液所含的底物为谷氨酸鈉3.5g/L，三磷酸腺苷二钠盐3g/L、六水氯化镁16.4g/L，腺苷6g/L，多聚磷酸13.3g/L。加入以上底物后用1升约30℃的去离子水搅拌溶解，至反应溶液澄凊，用2M氢氧化钠溶液调节pH至7.5。加入300ml上述反应溶液至反应调控罐中，温度为32℃，pH调控为7.25-7.5，高流量水泵的流量为3L/分钟，反应60分钟，产物谷氨酸5-磷酸含量为6.8mM，反应90分钟，产物谷氨酸5-磷酸含量为8.4mM，反应120分钟结束时，产物谷氨酸5-磷酸含量为9.8mM。
表2
Figure PCTCN2020093704-appb-000003
例10：制备氧化型烟酰胺腺嘌呤二核苷酸磷酸
按例2-5和7的制备方法得多聚磷酸:AMP磷酸转移酶、腺苷酸激酶、多聚磷酸激酶、腺苷激酶、輔酶I激酶并且分别表达该基因的重组表达大肠杆菌细胞并且进行发酵。
根据中国专利CN1982445B的实施例3的方法，在固相载体上制备含有輔酶I激酶(NK)、腺苷激酶(AK)、多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)和多聚磷酸激酶(PPK)的混合固定化大肠杆菌细胞，各细胞的混合重量比例依据基本相应匹配的酶活性。下表3为各细胞在离心后的湿重量。载体的形状为条形：长21cm、宽5cm、厚5mm，实重40.8g。将上述制得的承载有混合固定化大肠杆菌细胞的载体安装于固定化酶或固定化细胞反应器中。该反应器为有机玻璃制成的圆柱体，高7cm、半径4.5cm。用刀将上述载体头尾端约3cm以斜度45°整齐削去，紧握卷成高5cm、半径4.5cm的均质圆柱体，重量为30.8g。将该圆柱体插入反应器中，使其松紧程度符合中国发明专利申请CN106032520A中表1所述的3级标准，并且使其侧壁与反应器的内壁之间不留空隙。完成安装后，按CN106032520A的图1进行其它配备装置的安装程序，其中反应调控罐容量为1L；高流量水泵是可调节流量式吸 水泵，流速为3L/分钟；酸碱度调控装置采用0.3M氢氧化钠溶液进行pH调控，其加液泵的流量为每分钟1ml。反应溶液所含的底物为氧化型烟酰胺腺嘌呤二核苷酸20.3g/L，三磷酸腺苷二钠盐3g/L、六水氯化镁16.4g/L，腺苷6g/L，多聚磷酸13.3g/L。加入以上底物后用1升约30℃的去离子水搅拌溶解，至反应溶液澄凊，用2M氢氧化钠溶液调节pH至7.5。加入300ml上述反应溶液至反应调控罐中，温度为35℃，pH调控为7.75-8，高流量水泵的流量为3L/分钟，反应60分钟，产物氧化型烟酰胺腺嘌呤二核苷酸磷酸含量为7.2mM，反应90分钟，产物氧化型烟酰胺腺嘌呤二核苷酸磷酸含量为9.4mM，反应120分钟结束时，产物氧化型烟酰胺腺嘌呤二核苷酸磷酸含量为13.1mM。
表3
Figure PCTCN2020093704-appb-000004
例11:以腺苷为底物，快速新增三磷酸腺苷(ATP)含量
按例2-5的制备方法得多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)、多聚磷酸激酶(PPK)、腺苷激酶(AK)和腺苷激酶(AK)和表达该基因的大肠杆菌细胞并且进行发酵。
根据中国专利CN1982445B的实施例3的方法，在固相载体上制备含有多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)、多聚磷酸激酶(PPK)、腺苷激酶(AK)的混合固定化大肠杆菌细胞，各细胞的混合重量比例依据基本相应匹配的酶活性。下表4为各细胞在离心后的湿重量。载体的形状为条形：长18cm、宽5cm、厚5mm，实重37.5g。将上述制得的承载有混合固定化大肠杆菌细胞的载体安装于固定化酶或固定化细胞反应器中。该反应器为有机玻璃制成的圆柱体，高7cm、半径4.5cm。用刀将上述载体头尾端约3cm以斜度45°整齐削去，紧握卷成高5cm、半径4.5cm的均质圆柱体，重量为27.2g。将该圆柱体插入反应器中，使其松紧程度符合中国发明专利申请CN106032520A中表1所述的3级标准，并且使其侧壁与反应器的内壁之间不留空隙。完成安装后，按CN106032520A的图1进行其它配备装置的安装程序，其中反应调控罐容量为1L；高流量水泵是可调节流量式吸水泵，流速为1L/分钟；酸碱度调控装置采用0.3M氢氧化钠溶液进行pH调控，其加液泵的流量为每分钟1ml。反应溶液所含的底物为腺苷6g/L、六水氯化镁16.4g/L和多聚磷酸13.3g/L。加入以上底物后用1升约45℃的去离子水搅拌溶解，至反应溶液澄凊，用5M氢氧化钠溶液调节pH至7-7.5。加入1L上述反应溶液至反应调控罐中，温度为37℃，pH调控为7-7.5,高流量水泵的流量为2L/分钟，反应持续120分钟，产物三磷酸腺苷(ATP)含量20mM。
通常，在上述反应过程初段大量三磷酸腺苷(ATP)已被降解至二磷酸腺苷(ADP)、单磷酸腺苷(AMP)和腺苷，从而耗尽。然而，上述的方法得到产物三磷酸腺苷(ATP)含量20mM，因此，三磷酸腺苷(ATP)的含量在反应中快速得到增加。
表4
Figure PCTCN2020093704-appb-000005
例12：酶法生产磷酸肌酸(不含AK、PAP、ADK和PPK)
按例1的制备方法得肌酸激酶基因和表达该基因的大肠杆菌细胞并且进行发酵。根据中国专利CN1982445B的实施例3所述的方法，利用所得的湿重35g肌酸激酶重组表达大肠杆菌细胞在载体上制备固定化细胞。载体的形状为条形：长27cm、宽5cm、厚5mm，实重58.4g。反应溶液所含的底物为一水肌酸8.94g/L，三磷酸腺苷二钠盐3g/L和六水氯化镁16.4g/L，腺苷6g/L，多聚磷酸13.3g/L。加入以上底物后用1升约45℃的去离子水搅拌溶解，至反应溶液澄凊，用2M氢氧化钠溶液调节pH至8.5。加入300ml上述反应溶液至反应调控罐中，温度为37℃，pH调控为8.35-8.60，高流量水泵的流量为3L/分钟，反应持续60分钟。反应过程初段大量三磷酸腺苷(ATP)已被降解至二磷酸腺苷(ADP)、单磷酸腺苷(AMP)和腺苷并无法使用，产物磷酸肌酸含量为0.8mM，共0.05g。
例13：酶法生产磷酸肌酸(不含AK)
按例1-2和4-5的制备方法得肌酸激酶(CK)、多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)、多聚磷酸激酶(PPK)并分别地表达该基因的重组表达大肠杆菌细胞和进行发酵。根据中国专利CN1982445B的 实施例3所述的方法，在载体上制备多聚磷酸:AMP磷酸转移酶表达大肠杆菌细胞、腺苷酸激酶表达大肠杆菌细胞、多聚磷酸激酶表达大肠杆菌细胞和肌酸激酶表达大肠杆菌细胞的混合固定化细胞。下表5为各细胞在离心后的湿重量。
各细胞的混合重量比例依据基本相应匹配的酶活性，载体的形状为条形：长34cm、宽5cm、厚5mm，实重46.6g。反应溶液所含的底物为一水肌酸8.94g/L、三磷酸腺苷二钠盐3g/L、六水氯化镁16.4g/L、腺苷6g/L以及多聚磷酸13.3g/L。加入以上底物后用1升约45℃的去离子水搅拌溶解，至反应溶液澄凊，用2M氢氧化钠溶液调节pH至8.5。加入300ml上述反应溶液至反应调控罐中，温度为37℃，pH调控为8.35-8.60，高流量水泵的流量为3L/分钟，反应持续60分钟。由于没有加入三磷酸腺苷(ATP)酶法再生和新增组合中的腺苷激酶(AK)，反应过程中三磷酸腺苷(ATP)随时间缓慢降解至腺苷，反应完结时反应溶液中含有产物磷酸肌酸、大量腺苷和少量腺嘌呤，产物磷酸肌酸含量为产物磷酸肌酸含量为3.8mM，共0.24g。
表5
Figure PCTCN2020093704-appb-000006
例14：酶法生产磷酸肌酸(不含PAP)
按例1和3-5的制备方法得肌酸激酶(CK)、腺苷激酶(AK)、腺苷酸激酶(ADK)、多聚磷酸激酶(PPK)并分别地表达该基因的重组表达大肠杆菌细胞和进行发酵。根据中国专利CN1982445B的实施例3所述的方 法，在载体上制备肌酸激酶重组表达大肠杆菌细胞、腺苷激酶重组表达大肠杆菌细胞、腺苷酸激酶重组表达大肠杆菌细胞和多聚磷酸激酶重组表达大肠杆菌细胞的混合固定化细胞。下表6为各细胞在离心后的湿重量。
各细胞的混合重量比例是依据基本相应匹配的酶活性。载体的形状为条形：长25cm、宽5cm、厚5mm，实重34.5g。反应溶液所含的底物为一水肌酸8.94g/L，三磷酸腺苷二钠盐3g/L、六水氯化镁16.4g/L、腺苷6g/L以及多聚磷酸13.3g/L。加入以上底物后用1升约45℃的去离子水搅拌溶解，至反应溶液澄凊，用2M氢氧化钠溶液调节pH至8.5。加入300ml上述反应溶液至反应调控罐中，温度为37℃，pH调控为8.35-8.60，高流量水泵的流量为3L/分钟，反应持续60分钟。反应过程中三磷酸腺苷(ATP)、单磷酸腺苷(AMP)和磷酸肌酸不断增加；由于反应过程中三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、单磷酸腺苷(AMP)会随时间在杂酶影响下缓慢降解至腺苷，但多聚磷酸:AMP磷酸转移酶的缺乏下单磷酸腺苷(AMP)未能再生至二磷酸腺苷(ADP)而不可进一步新增和再生三磷酸腺苷(ATP)，反应结束时余下部分单磷酸腺苷(AMP)、少量二磷酸腺苷(ADP)、三磷酸腺苷(ATP)、腺苷和大量腺嘌呤；产物磷酸肌酸含量为4.8mM，共0.3g。
表6
酶 细胞重量(g)
肌酸激酶(CK) 9.1
腺苷激酶(AK) 8.3
腺苷酸激酶(ADK) 10
多聚磷酸激酶(PPK2) 7.5
例15：酶法生产磷酸肌酸(不含PPK)
按照例1-3和5的制备方法分别得肌酸激酶(CK)、腺苷激酶(AK)、腺苷酸激酶(ADK)、多聚磷酸:AMP磷酸转移酶(PAP)的基因和分别表达该基因的重组表达大肠杆菌细胞并且进行发酵。根据中国专利CN1982445B的实施例3所述的方法，在载体上制备肌酸激酶重组表达大肠杆菌细胞、腺苷激酶重组表达大肠杆菌细胞、腺苷酸激酶重组表达大肠杆菌细胞和多聚磷酸:AMP磷酸转移酶重组表达大肠杆菌细胞的混合固定化细胞，各细胞的混合重量比例是依据基本相应匹配的酶活性。下表6为各细胞在离心后的湿重量。载体的形状为条形：长23cm、宽5cm、厚5mm，实重32.8g。反应溶液所含的底物为一水肌酸8.94g/L、三磷酸腺苷二钠盐3g/L、六水氯化镁16.4g/L、腺苷6g/L和多聚磷酸13.3g/L。加入以上底物后用1升约45℃的去离子水搅拌溶解，至反应溶液澄凊，用2M氢氧化钠溶液调节pH至8.5，待反应液温度下降至室温备用。加入300ml上述反应溶液至反应调控罐中，温度为37℃，pH调控为8.35-8.60，高流量水泵的流量为3L/分钟，反应持续60分钟。反应过程中单磷酸腺苷(AMP)、三磷酸腺苷(ATP)和磷酸肌酸不断增加；反应结束时余下单磷酸腺苷(AMP)、二磷酸腺苷(ADP)和腺苷；产物磷酸肌酸含量为4.4mM，共0.28g。
表7
Figure PCTCN2020093704-appb-000007
例16：酶法生产磷酸肌酸(不含PPK2和ADK)
按例1-2和5的制备方法分别得肌酸激酶(CK)、多聚磷酸:AMP磷酸转移酶(PAP)、腺苷激酶(AK)和分别表达该基因的重组表达大肠杆菌细胞并且进行发酵。根据中国专利CN1982445B的实施例3所述的方法，在载体上制备肌酸激酶表达大肠杆菌细胞、多聚磷酸:AMP磷酸转移酶重组表达大肠杆菌细胞和腺苷激酶重组表达大肠杆菌细胞的混合固定化细胞，各细胞的混合重量比例是依据基本相应匹配的酶活性。下表7为各细胞在离心后的湿重量。载体的形状为条形：长24cm、宽5cm、厚5mm，实重34.2g。反应溶液所含的底物为一水肌酸8.94g/L、三磷酸腺苷二钠盐3g/L、六水氯化镁16.4g/L、腺苷6g/L以及多聚磷酸13.3g/L。加入以上底物后用1升约45℃的去离子水搅拌溶解，至反应溶液澄凊，用2M氢氧化钠溶液调节pH至8.5。加入300ml上述反应溶液至反应调控罐中，温度为37℃，pH调控为8.35-8.60，高流量水泵的流量为3L/分钟，反应持续60分钟。由于反应中缺少了三磷酸腺苷(ATP)酶法再生和增新组合中的PPK2和ADK，反应未能由二磷酸腺苷(ADP)再生至三磷酸腺苷(ATP)；反应过程中二磷酸腺苷(ADP)不断增加；反应结束时余下大量二磷酸腺苷(ADP)、少量单磷酸腺苷(AMP)和腺苷；产物磷酸肌酸含量为0.9mM，共0.06g。
表8
Figure PCTCN2020093704-appb-000008
本发明不受上述具体文字描述的限制，本发明可在权利要求书所概括的范围内做各种修改或改变。这些改变均在本发明要求保护的范围之内。

Claims (21)

  1. 一种增加酶法反应中的三磷酸腺苷(ATP)的量的方法，其特征在于在进行酶法反应时加入生产单磷酸腺苷(AMP)的第一酶或酶组以及腺苷，以新增三磷酸腺苷(ATP)的量，其中所述酶法反应的反应底物包含三磷酸腺苷(ATP)或其盐。
  2. 根据权利要求1所述的方法，其特征在于所述方法还包括在加入第一酶或酶组的同时、之前或者之后，加入负责三磷酸腺苷(ATP)再生的第二酶或酶组。
  3. 根据权利要求2所述的方法，其特征在于在加入第一酶或酶组和/或加入第二酶或酶组的同时、之前或者之后，加入第三酶或酶组。
  4. 根据权利要求1-3中任意一项所述的方法，其特征在于所述的第一酶或酶组包含腺苷激酶(AK)。
  5. 根据权利要求1-4中任意一项所述的方法，其特征在于所述反应底物还包含多聚磷酸或其盐以及辅助离子中的至少一者，
    所述辅助离子优选镁离子、钠离子、钾离子和氯离子中的至少一者，更优选为镁离子和钾离子中至少一者；辅助离子可为其无机盐或有机盐的状态，优选为六水氯化镁、氯化钠、氯化锰、硫酸镁和碳酸钾中的至少一者，更优选为六水氯化镁、氯化钠和碳酸钾中的至少一者。
  6. 根据权利要求2所述的方法，其特征在于所述的第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)中的至少一者；或者
    所述第三酶或酶组包含肌酸激酶(CK)、谷氨酸激酶(GK)、輔酶I激酶(NK)和/或以三磷酸腺苷(ATP)为其中之一底物以进行氨基酸、肽或蛋白的磷酸化、磷酸转移或多肽合成的其他酶或者酶组。
  7. 根据权利要求2所述的方法，其特征在于所述酶法反应包括下列中的至少一者：
    (i)所述的第一酶或酶组包含腺苷激酶(AK)，反应底物包含腺苷、多聚磷酸和三磷酸腺苷(ATP)，并且反应产物包含单磷酸腺苷(AMP)和二磷酸腺苷(ADP)；
    (ii)所述的第一酶或酶组包含腺苷激酶(AK)，并且第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)，反应底物包含单磷酸腺苷(AMP)和多聚磷酸，并且反应产物包含二磷酸腺苷(ADP)和多聚磷酸；
    (iii)所述的第一酶或酶组包含腺苷激酶(AK)，并且第二酶或酶组包含腺苷酸激酶(ADK)，反应底物包含二磷酸腺苷(ADP)和多聚磷酸，并且反应产物包含单磷酸腺苷(AMP)和三磷酸腺苷(ATP)；
    (iv)所述的第一酶或酶组包含腺苷激酶(AK)，并且所述的第二酶或酶组包含多聚磷酸激酶(PPK)，反应底物包含二磷酸腺苷(ADP)和多聚磷酸，并且反应产物包含三磷酸腺苷(ATP)和多聚磷酸；以及
    (v)所述的第一酶或酶组包含腺苷激酶(AK)，第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)并且所述的第三酶或酶组包含肌酸激酶，反应底物包含肌酸和三磷酸腺苷(ATP)，并且反应产物包含磷酸肌酸、二磷酸腺苷(ADP)和多聚磷酸；
    (vi)利用多聚磷酸:AMP磷酸转移酶(PAP)、腺苷酸激酶(ADK)和多聚磷酸激酶(PPK)的全部或至少一者、腺苷激酶(AK)和以三磷酸腺苷(ATP)为反应底物在同一反应体系中以混合、并联或串联的方式同时进行反应或在不同的反应体系中分别进行反应；
    (vii)只利用多聚磷酸:AMP磷酸转移酶(PAP)、腺苷激酶(AK)和多聚磷酸激酶(PPK)的全部或至少一者和以三磷酸腺苷(ATP)为反应底物在不同的反应体系中以混合、并联或串联的方式同时进行反应或在不同的反应体系中分别进行反应；和
    (viii)只利用腺苷激酶以腺苷为底物生产单磷酸腺苷(AMP)，以新增三磷酸腺苷(ATP)。
  8. 根据权利要求2所述的方法，其特征在于所述的第二酶或酶组分别将单磷酸腺苷(AMP)和二磷酸腺苷(ADP)再生至二磷酸腺苷(ADP)和三磷酸腺苷(ATP)；以及
    任选地，所述第一酶或酶组将腺苷合成至单磷酸腺苷(AMP)。
  9. 根据权利要求1到8中任意一项所述的方法，其特征在于所述酶法反应包括利用腺苷和三磷酸腺苷(ATP)为底物合成单磷酸腺苷(AMP)。
  10. 根据权利要求1到9中任意一项所述的方法，其特征在于所述反应底物还包含肌酸或其水合物、谷氨酸鈉或其水合物和輔酶I中的至少一者。
  11. 根据权利要求1到10中任意一项所述的方法，其特征在于按酶法反应中产生三磷酸腺苷(ATP)降解物的水平确定加入的第一酶或者酶组，降解物优选为二磷酸腺苷(ADP)、单磷酸腺苷(AMP)和/或腺苷。
  12. 根据权利要求1到11中任意一项所述的方法，其特征在于以纯化或非纯化的细胞破碎液、液酶、固定化细胞或固定化酶的形式加入所述第一酶或酶组、或者第二酶或酶组、或者第三酶或酶组。
  13. 根据权利要求1到12中任意一项所述的方法，其特征在于所述酶法反应的条件为：温度为28-40摄氏度，优选为30-38摄氏度，更优选为33-37摄氏度；pH值为5-9，优选为6-8.5，更优选为7-7.75。
  14. 一种酶法反应组合物，其特征在于包含底物以及生产单磷酸腺苷(AMP)的第一酶或酶组，其中所述底物包含三磷酸腺苷(ATP)或其盐以及腺苷。
  15. 根据权利要求14所述的酶法反应组合物，其特征在于所述底物还包含多聚磷酸或其盐、辅助离子以及肌酸或其水合物中的至少一种，
    所述辅助离子优选镁离子、钠离子、钾离子和氯离子中的至少一者，更优选为镁离子和钾离子中至少一者；辅助离子可为其无机盐或有机盐的状态，优选为六水氯化镁、氯化钠、氯化锰、硫酸镁和碳酸钾中的至少一者，更优选为六水氯化镁、氯化钠和碳酸钾中的至少一者。
  16. 根据权利要求14-15中任意一项所述的酶法反应组合物，其特征在于，所述酶法反应组合物还包含第二酶或酶组和任选的第三酶或酶组。
  17. 根据权利要求16所述的酶法反应组合物，其特征在于，所述第一酶或酶组包含腺苷激酶(AK)，
    优选地，所述酶法反应组合物还包含第三酶或酶组，所述第三酶或者酶组包含肌酸激酶(CK)、谷氨酸激酶(GK)、輔酶I激酶(NK)或其他酶或者酶组以三磷酸腺苷(ATP)为其中之一的酶法反應底物以進行氨基酸、肽或蛋白的磷酸化、磷酸转移或多肽合成，并且
    优选地，所述第二酶或酶组包含多聚磷酸:AMP磷酸转移酶(PAP)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)中的至少一者。
  18. 根据权利要求16所述的酶法反应组合物，其特征在于所述第一酶或酶组、第二酶或酶组和第三酶或酶组以纯化或非纯化的细胞破碎液、液酶、固定化细胞或固定化酶的形式包含在所述酶法反应组合物中。
  19. 根据权利要求17所述的酶法反应组合物，其特征在于所述多聚磷酸:AMP磷酸转移酶(PAP)、腺苷激酶(AK)、多聚磷酸激酶(PPK)和腺苷酸激酶(ADK)、肌酸激酶(CK)、谷氨酸激酶(GK)和輔酶I激酶(NK)各自独立地为重组酶，并且分别或组合在大肠杆菌中表达。
  20. 一种利用三磷酸腺苷(ATP)为底物以使氨基酸、核酸、肽或蛋白进行磷酸化或磷酸转移的方法，包括权利要求1-13中任意一项所述的方法。
  21. 根据权利要求20所述的方法，其特征在于氨基酸、核酸、肽或蛋白为肌酸、谷氨酸和辅酶I中的至少一者。
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