Method and process for synthesizing adenosine triphosphate through in-vitro multi-enzyme cascade catalysis and biological preparation of enzyme compound

Abstract

The invention relates to a method and a process for synthesizing adenosine triphosphate through in-vitro multi-enzyme cascade catalysis and biological preparation of an enzyme compound, which are characterized in that the novel enzyme cascade catalysis for synthesizing ATP (adenosine triphosphate) adopts single enzyme protein for full-process catalysis, the related enzymes comprise adenosine kinase (EC 2.7. 1.20), adenylate kinase (EC 2.7. 4.3) and ADP (adenosine diphosphate) kinase (EC 2.7. 4.1), each enzyme is connected by using a connecting protein Linker, and the whole catalytic reaction process is as follows: (1) converting adenosine under the catalysis of adenosine kinase to generate AMP (adenosine monophosphate); (2) converting the AMP into ADP under the catalysis of adenylate kinase; and (3) converting the ADP into ATP under the catalysis of ADP kinase. And the contact path between the enzyme and the substrate is shortened, so that the product catalyzed in the previous step can quickly enter the enzyme catalytic reaction in the next step. The enzyme protein is immobilized, so that the application frequency of the enzyme protein is increased, and the production cost is greatly reduced.

Description

Technical Field

[0001] The present invention relates to a method for the preparation of ATP by in vitro multi-enzyme cascade catalysis. The present patent protects a new in vitro synthesis method and process of ATP, including the preparation process of enzyme complexes and the subsequent complete enzyme-catalyzed process flow. Background Art

[0002] Adenosine triphosphate (ATP) is composed of adenine, ribose and three phosphate groups connected together. When hydrolyzed, it releases relatively more energy. It is the most direct energy source in living organisms and is also an unstable high-energy phosphate compound. ATP is the energy supply substance for almost all chemical reactions related to the intracellular metabolic network. Most enzyme catalyses require ATP to provide energy, which is the manifestation of energy storage in cells and is also known as the energy converter and storage device in living organisms. In addition, it plays an important role in human energy metabolism and participates in the metabolism of carbohydrates, proteins, nucleic acids, fats, etc. in living organisms as a metabolic intermediate and coenzyme. In clinical applications, ATP has a wide range of effects and has good therapeutic and adjuvant therapeutic effects on progressive muscular atrophy, sequelae of stroke, myocardial infarction, myocarditis, coronary arteriosclerosis, hepatitis and other diseases. Most of the medicinal adenosine triphosphate on the market exists in the form of sodium salt, that is, adenosine triphosphate disodium (C 10 H 14 N5Na2O 13 P3).

[0003] The synthesis of ATP mainly includes chemical synthesis method, biocatalytic method by enzymes, photophosphorylation and oxidative phosphorylation method, and microbial enzyme system fermentation and other methods. Considering factors such as the safety, environmental protection and production cost of drugs, the catalytic synthesis of adenosine triphosphate by microbial enzyme systems using adenosine as a raw material has become the main process for the industrial production of adenosine triphosphate. There are mainly two synthetic routes for microbial enzyme system catalysis. The first classic route is to convert ADP into ATP under the catalytic action of ADP kinase with ADP as the starting substrate. The other synthetic route is to use AMP as the starting substrate and first convert AMP into ADP through two-step enzyme catalysis, and then convert ADP into ATP.

[0004] Although the microbial enzyme system catalysis has good production effects and low costs, the reaction process for the catalytic synthesis of ATP by yeast cell enzyme systems is complex, there are many enzyme systems participating in the catalytic reaction, the reaction process is not easy to control, and there are large differences in the quality between product batches. At the same time, the quality of yeast often varies greatly due to different suppliers, different batches, or even different seasons. Moreover, the activity of yeast enzyme systems decreases rapidly and the service life is short. Generally, it is used once, and a large amount of waste yeast causes relatively large environmental pollution to the atmosphere, water source and soil. In addition, a large amount of yeast cell enzyme solution is added during the reaction process, introducing a large amount of impurities such as proteins and pigments, which brings many difficulties to the later purification of ATP.

[0005] The main defects and problems existing in the field of ATP synthesis at present include:

[0006] The mainstream ATP synthesis-related processes all focus on the application of ATP cycle, paying less attention to the direct synthesis of ATP. As ADP is relatively expensive as a raw material itself, its economic applicability is not high. Currently, the method for producing ATP generally uses fermentation to prepare ATP. Since cells themselves will consume a large amount of ATP, the overall yield in the actual production process is not high and cannot meet the needs of industrial production. Moreover, the existing routes still have too many steps and a long route for the synthesis of ATP, which limits the yield of ATP.

[0007] Patent CN201610165471.7 uses a relatively simple enzyme system, in an immobilized manner, and uses inexpensive polyphosphate as a phosphate donor to prepare ATP, obtaining relatively stable results. The enzymes used include polyphosphate kinase (EC 2.7.4.1, Ppk), adenylate kinase (EC 2.7.4.3, Adk), and polyphosphate-adenylate phosphotransferase (EC 2.7.4.-, Pap). The Ppk enzyme catalyzes the reaction of ADP with polyphosphate or its salts to generate ATP. The Adk enzyme catalyzes the reaction of 2 molecules of ADP to generate 1 molecule of ATP and 1 molecule of AMP. The Pap enzyme catalyzes the reaction of AMP with polyphosphate or its salts to generate ADP. The reasonable combination of these three enzymes can successfully synthesize ATP. However, the substrate used in this patent is AMP, and AMP itself is relatively expensive, so this route is not suitable for industrial application. Another patent CN106191170A uses adenosine kinase to produce AMP, which, together with the enzymes used in the patent, forms an ATP catalytic production system. This reaction system involves the co-catalysis of 4 enzymes, resulting in an overly cumbersome whole reaction and a low overall yield. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for in vitro multi-enzyme cascade catalyzed synthesis of adenosine triphosphate, a process, and a biological method for preparing an enzyme complex to solve the industrialization bottleneck proposed in the above background technology. The present invention uses a new enzyme cascade reaction system for continuous production of ATP. Three enzymes are directly connected with a small linker protein to form a complete single large protein, and then the combined protein is used for enzyme catalysis. The advantage of this scheme is that it shortens the contact path between the enzyme and the substrate, enabling the product of the previous step of catalysis to quickly enter the next step of enzyme-catalyzed reaction. After immobilizing it, the reuse times of the enzyme protein are increased, thereby greatly reducing the production cost.

[0009] To achieve the above purpose, the present invention provides the following technical solutions:

[0010] A method for in vitro multi-enzyme cascade catalyzed synthesis of adenosine triphosphate, its process and the biological preparation of enzyme complex, characterized in that: a new enzyme cascade catalysis for synthesizing ATP, with a single enzyme protein catalyzing the whole process, and the enzymes involved include three kinds: adenosine kinase (EC 2.7.1.20), adenylate kinase (EC 2.7.4.3) and ADP kinase (EC 2.7.4.1), and each enzyme is connected with a linker protein. The whole catalytic reaction process is as follows: see attachment Figure 1

[0011] (1) Adenosine is converted to AMP under the catalysis of adenosine kinase;

[0012] (2) AMP is converted to ADP under the catalysis of adenylate kinase;

[0013] (3) ADP is converted to ATP under the catalysis of ADP kinase.

[0014] As an optional item, the enzyme-catalyzed process adopts the following steps:

[0015] (1) At the gene level: splice the genes of the three enzymes adenosine kinase, adenylate kinase and ADP kinase together, and insert the gene sequence of the linker protein in the middle; (see attachment Figure 2 )

[0016] (2) Expression of enzyme protein: use model strains such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pichia pastoris to express the enzyme complex protein;

[0017] (3) Enzyme immobilization: adopt the sodium alginate embedding method for enzyme immobilization;

[0018] (4) Add the obtained single-protein immobilized enzyme into a 1L reaction system for reaction;

[0019] (5) Separate the product and the ATP-producing enzyme;

[0020] (6) Preparation of finished product, the reaction solution separated in step (5) is prepared into finished product ATP through ion exchange chromatography, concentration, crystallization, and drying steps.

[0021] As an optional item, the enzyme proteins are all obtained through microbial fermentation.

[0022] As an optional item, the reaction temperature is 20 - 40 °C.

[0023] As an optional item, the reaction system also includes Tris-HCl, which serves as a reaction buffer.

[0024] As an optional item, the reaction conditions for ATP synthesis: pH 6.7 - 7.6.

[0025] Biological preparation of an enzyme complex for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate, characterized in that: it includes a method and process for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate described in any one of claims 1-6. The process for obtaining the enzyme for synthesizing ATP is as follows:

[0026] (1) Add the gene sequence of the linker protein Linker to the C-terminus of the gene of adenosine kinase, add the gene sequence of the linker protein Linker to the N-terminus and C-terminus of the gene of adenylate kinase, add the gene sequence of the linker protein Linker to the N-terminus of the gene of ADP kinase, remove the stop codon TAA at the C-terminus of the adenosine kinase gene fragment, remove the stop codon TAA at the C-terminus of the adenylate kinase gene fragment, and retain the stop codon TAA at the C-terminus of the ADP kinase gene fragment;

[0027] (2) Synthesize the complete sequences of the gene fragments of adenosine kinase, adenylate kinase and ADP kinase containing the linker protein Linker sequence, and then insert the complete sequences into the plasmid pET28a and transfer them into Escherichia coli BL21;

[0028] (3) Transfer the positive clone of the transformed Escherichia coli BL21 to the liquid LB medium, culture it until the logarithmic growth phase (4-6 h), add 0.5 mM IPTG for induction for 6 h, then collect the bacteria, and perform SDS-PAGE protein electrophoresis to screen for highly expressing strains;

[0029] (4) Under sterile conditions, inoculate the screened highly expressing strains into 100 mL of the seed medium, culture them until the logarithmic growth phase and then inoculate them into a 5 L fermenter, continue to culture until the logarithmic growth phase and then inoculate them into a fermenter containing 50 L of the medium, culture for 6 hours and then add 0.5 mM IPTG for induction expression for 10 hours, centrifuge to collect the bacteria, and obtain about 1 kg of Escherichia coli bacteria;

[0030] (5) Ultrasonically or homogenize the collected bacteria under high pressure to break them, and centrifuge to collect the supernatant.

[0031] (6) Add 30-60% saturated (NH4)2SO4 to the supernatant, centrifuge, dissolve the collected precipitate with Tris-HCl buffer (pH = 7.8), and perform desalting treatment using a dialysis bag; finally, filter and sterilize with a 0.22 μm membrane to obtain the preliminarily purified enzyme, which is the ATP synthase after the connection of adenosine kinase, adenylate kinase and ADP kinase.

[0032] Preferably, add kanamycin to the liquid LB medium to a final concentration of 50 mg / L when transferring the positive clone of the transformed Escherichia coli BL21.

[0033] Preferably, the process for catalytic synthesis of ATP includes using free ATP synthase.

[0034] Preferably, the process for catalytic synthesis of ATP includes embedding ATP synthase with sodium alginate, and then using the obtained immobilized enzyme after embedding for the synthesis and preparation of ATP.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] (1) By adopting a novel enzyme cascade method and using linker proteins to connect various enzymes, the overall efficiency of enzyme catalysis is improved, making the production of ATP simpler and more efficient. Compared with the catalytic production of single free proteins, the final ATP yield is higher.

[0037] (2) Using inexpensive adenosine as a substrate to produce ATP further reduces the cost of enzyme-catalyzed ATP production.

[0038] (3) The added phosphate donor in the reaction is inexpensive, has little pollution, and has no impact on the enzyme itself.

[0039] (4) An enzyme recovery system is established through enzyme immobilization, which is suitable for industrial production.

[0040] (5) Since various enzymes are integrated into a single enzyme protein, there are almost no miscellaneous proteins in the reaction system, which is very suitable for the subsequent ATP purification work. Description of the Drawings

[0041] Figure 1 For the attached Figure 1 This is the ATP synthesis route involved in the design of the present invention, with a total of three steps of enzyme catalysis starting from adenosine as the substrate.

[0042] Figure 2 This is a schematic diagram of gene assembly and protein connection related to the ATP synthesis route designed by the present invention.

[0043] Figure 3 This is an agarose nucleic acid electrophoresis map of the key enzyme gene related to the ATP synthesis route designed by the present invention.

[0044] Figure 4 This is an SDS-PAGE protein electrophoresis map of the key enzyme related to the ATP synthesis route designed by the present invention.

[0045] Figure 5 This is the HPLC map in Example 2 of the present invention.

[0046] Figure 6 This is the HPLC map in Example 3 of the present invention.

[0047] Figure 7 This is the HPLC map in Example 4 of the present invention.

[0048] Figure 8 This is the HPLC chromatogram in Example 5 of the present invention.

[0049] Figure 9 This is the HPLC chromatogram in Example 6 of the present invention.

[0050] Figure 10 This is the HPLC chromatogram in Comparative Example 1 of the present invention.

[0051] Figure 11 This is the HPLC chromatogram in Comparative Example 2 of the present invention. Detailed implementation manners

[0052] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0053] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0054] Embodiment, please refer to Figure 1 - 11 , the present invention provides a technical solution:

[0055] Embodiment 1: Preparation of ATP synthase

[0056] This embodiment illustrates the complete process for obtaining ATP synthase (complex enzyme).

[0057] The relevant genes used in the present invention can be obtained by the PCR method well-known in the biological field. The agarose nucleic acid electrophoresis diagram of the relevant key enzyme genes is shown in the appendix Figure 3 .

[0058] All the enzyme proteins used in the present invention are obtained through microbial fermentation. The SDS-PAGE protein electrophoresis diagram of the relevant key enzymes is shown in the appendix Figure 4 .

[0059] In the present invention, all proteins are tagged with His tags for protein purification operations. The tag, the usage of the tag, and the protein purification method are general methods well-known in the art. The purification system includes eluting the purification column with imidazole salts. By changing the concentration of imidazole salts, the chelating ability of the His tag with nickel in the purification column can be controlled, thereby achieving the purpose of protein purification.

[0060] In the present invention, the formulated LB medium is used for culturing Escherichia coli and Bacillus subtilis. The formula of the LB liquid medium is: 10 g / L of tryptone, 5 g / L of yeast extract, and 5 g / L of NaCl; the formula of the LB solid medium: 10 g / L of tryptone, 5 g / L of yeast extract, 5 g / L of NaCl, and 15 g / L of agar.

[0061] In the present invention, the components of the seed medium are: 10 g / L of peptone, 5 g / L of yeast powder, and 10 g / L of NaCl.

[0062] In the present invention, the formulated fermentation medium is: 10 g / L of peptone, 15 g / L of yeast powder, 2 g / L of K2HPO4, 10 g / L of glycerol, and 1 g / L of MgSO4. This fermentation medium is used for the fermentative production of enzyme proteins by Escherichia coli and Bacillus subtilis.

[0063] In the present invention, the formulated YPD medium is used for the normal culture and fermentative culture of Saccharomyces cerevisiae. The formula of the YPD solid medium: 10 g / L of yeast extract, 20 g / L of peptone, 20 g / L of glucose, and 20 g / L of agar; the formula of the YPD liquid medium is: 10 g / L of yeast extract, 20 g / L of peptone, and 20 g / L of glucose.

[0064] In the present invention, the formulated MD medium is used for the normal culture of Pichia pastoris. The formula of the MD plate (MD solid medium): 13.4 g / L of YNB (yeast nitrogen base, containing ammonium sulfate without amino acids), 4×10 -4 g / L of biotin, 20 g / L of glucose, and 15 g / L of agar.

[0065] In the present invention, the antibiotic used is kanamycin, and the final concentrations added are 50 mg / L and 20 mg / L respectively.

[0066] In the present invention, the reaction conditions are as follows: reaction temperature: 20 - 40 °C, enzyme addition amount: 10 - 500 mg / L, substrate addition of NAD + : 1 - 100 g / L. Any one of Tris-HCl and phosphate buffer can be used as the buffer. The reaction pH is 6.5 - 7.8. The reaction duration is 10 - 20 hours.

[0067] In the present invention, for the biosynthesis of ATP, relatively inexpensive adenosine is used as the starting material, and the target product ATP can be obtained through three-step enzyme catalysis. In the whole technological process, only a single enzyme protein needs to be added to complete the three-step enzyme catalysis.

[0068] Regarding the enzymatic catalysis process, a more detailed description is as follows: Adenosine is converted into AMP under the catalysis of adenosine kinase, then AMP is converted into ADP under the catalysis of adenylate kinase, and then continues to be converted into ATP under the catalysis of ADP kinase. It should be emphasized that the above enzymatic catalysis reaction can be completed with a complete enzyme protein, without adding three enzyme proteins, which reduces the operation difficulty of the entire production process and also greatly reduces the design difficulty of the reactor, making the entire reaction process more controllable.

[0069] In the present invention, preferably, sodium alginate is used for embedding and immobilizing the enzyme protein.

[0070] In the present invention, in the catalytic reaction system, an adenosine substrate is added as required for the enzymatic reaction. At the same time, an enzyme protein, that is, a single protein formed by linking adenosine kinase, adenylate kinase, and ADP kinase, is added in proportion. And a small amount of ATP, ADP, or AMP for initiating the reaction is added, and sodium pyrophosphate as a phosphate donor is added for the enzymatic synthesis of ATP. The reaction system also includes Tris-HCl as a reaction buffer.

[0071] The substrate, enzyme, and salts added in the present invention can be added to the reaction system at one time, or can be added in batches according to the actual situation and process of industrial production.

[0072] In the present invention, the immobilized enzyme can be directly separated in a reaction tank. The separation method can be filtration, or it can be directly separated in a reaction column, or separated by membrane ultrafiltration. The filtrate after filtration is an ATP reaction solution without enzyme.

[0073] In the present invention, the recovered immobilized enzyme can be reused.

[0074] In the present invention, regarding the preparation of the finished product, the separated filtrate is prepared into the finished product ATP through steps such as ion exchange chromatography, concentration, crystallization, and drying.

[0075] In the present invention, the reaction conditions for ATP synthesis are as follows: The reaction temperature is 20 - 40 °C. The preferred temperature is 30 - 40 °C, and the most preferred is 35 °C. The reaction pH is 6.5 - 7.8, the preferred pH is 6.7 - 7.6, and the most preferred is pH 7.2. 6. Specific Embodiments

[0077] The following describes the specific embodiments of the present invention in detail for further understanding of the present invention.

[0078] Example 1 Preparation of ATP Synthase

[0079] This example illustrates the complete process for obtaining ATP synthase (complex enzyme).

[0080] In the present invention, all enzyme proteins are obtained by microbial fermentation. The preparation process of the complete enzyme protein of ATP synthase is as follows:

[0081] The gene fragments of adenosine kinase, adenylate kinase and ADP kinase were synthesized externally (Sangon Biotech, Shanghai). The gene sequence of linker protein was added at the C-terminus of the adenosine kinase gene, at the N-terminus and C-terminus of the adenylate kinase gene, and at the N-terminus of the ADP kinase gene. The stop codon TAA at the C-terminus of the adenosine kinase gene fragment was removed, the stop codon TAA at the C-terminus of the adenylate kinase gene fragment was removed, and the stop codon TAA at the C-terminus of the ADP kinase gene fragment was retained. The complete sequences of the gene fragments of adenosine kinase, adenylate kinase and ADP kinase containing the linker protein sequence were synthesized externally (Sangon Biotech, Shanghai), and then the complete sequences were inserted into plasmid pET28a and transferred into Escherichia coli BL21(DE3) (the strain was purchased from Miaoling Biology).

[0082] The positive clones of the transformed Escherichia coli BL21(DE3) were transferred to LB liquid medium (added with kanamycin to a final concentration of 50 mg / L). After culturing to the logarithmic growth phase, 0.5 mM IPTG was added for induction for 6 h, and then the cells were collected and subjected to SDS-PAGE protein electrophoresis to screen for high-expression strains.

[0083] The selected high-expression strains were inoculated into 100 mL of seed medium under sterile conditions. After culturing to the logarithmic growth phase, they were inoculated into a 5 L fermenter (liquid volume: 2.5 L). After continuing to culture to the logarithmic growth phase, they were inoculated into a fermenter containing 50 L of medium. After culturing for 6 h, 0.5 mM IPTG was added for induction expression for 10 h, and the cells were collected by centrifugation to obtain about 1 kg of Escherichia coli cells.

[0084] The collected cells were disrupted by ultrasonic or high-pressure homogenization, and the supernatant was collected by centrifugation. 30-60% saturated (NH4)2SO4 was added to the supernatant and centrifuged. The collected precipitate was dissolved in Tris-HCl buffer (pH = 7.8), desalted by using a dialysis bag, and finally filtered and sterilized through a 0.22 μm membrane to obtain a preliminarily purified enzyme, namely the ATP synthase (complex enzyme) after the connection of adenosine kinase, adenylate kinase and ADP kinase.

[0085] Example 2 Catalytic synthesis of ATP using a free enzyme complex system

[0086] This example describes the preparation process of synthesizing ATP using free ATP synthase (complex enzyme).

[0087] (1) Synthesize ATP in a reaction tank

[0088] Add 100 L of sterile 10 mM phosphate buffer (pH = 7.5) to the reaction tank, and add the following materials: 2.5 kg of adenosine (as the substrate), 2.5 kg of MgSO4, 0.1 kg of ATP, 2 kg of sodium pyrophosphate, and 0.2 kg of NaCl. Add 500 g of ATP synthase (complex enzyme) to the reaction system, and control the pH to remain at 7.5 during the reaction, and control the temperature at 40 °C.

[0089] After reacting for 10 hours, the amount of ATP produced is 40 g / L, and the adenosine conversion rate is about 88%. See the HPLC chromatogram in Figure 5 . HPLC detection is carried out using a C18 column, the detection temperature is 35 °C, the flow rate is 1 mL / min, and the mobile phase is a phosphate buffer containing 5% methanol and 20 mM, pH = 3.2.

[0090] (2) Isolation of ATP synthase

[0091] Recover by ultrafiltration. Use an ultrafiltration membrane with a molecular weight cut-off of 20 kDa. The filtrate is the reaction solution without enzyme, which contains ATP, ADP, AMP, and a small amount of salts.

[0092] (3) Isolation operation of ATP

[0093] The filtrate obtained in step (2) is subjected to ion exchange chromatography, concentration, crystallization, and drying to obtain the finished product ATP, with a purity of over 98% and a total yield of 86%.

[0094] Example 3: Catalytic synthesis of ATP using free enzymes

[0095] This example describes the synthesis preparation process of ATP using adenosine kinase, adenylate kinase, and ADP kinase in a single free state.

[0096] (1) Preparation of single free enzymes

[0097] Referring to the process flow of Example 1, without using the linker protein Linker, adenosine kinase, adenylate kinase, and ADP kinase are respectively inserted into a single pET28a plasmid and then transferred into three strains of Escherichia coli.

[0098] Transfer the positive clones of the transformed Escherichia coli BL21(DE3) to the LB liquid medium (add kanamycin to a final concentration of 50 mg / L). After culturing to the logarithmic growth phase, add 0.5 mM IPTG for induction for 6 h, then collect the bacteria, and perform SDS-PAGE protein electrophoresis to screen for high-expression strains.

[0099] The selected highly-expressing strains were inoculated into 100 mL of seed medium under sterile conditions. After culturing until the logarithmic growth phase, they were transferred into a 5 L fermenter (liquid volume: 2.5 L). After continuous culturing until the logarithmic growth phase, they were transferred into a fermenter containing 50 L of medium. After culturing for 6 hours, 0.5 mM IPTG was added to induce expression for 10 hours. The cells were collected by centrifugation to obtain approximately 1 kg of Escherichia coli cells.

[0100] The collected cells were disrupted by ultrasonic treatment or high-pressure homogenization, and the supernatant was collected by centrifugation. 30 - 60% saturated (NH4)2SO4 was added to the supernatant, followed by centrifugation. The collected precipitate was dissolved in Tris-HCl buffer (pH = 7.8), desalted using a dialysis bag, and finally filtered through a 0.22 μm membrane to remove bacteria, obtaining a preliminarily purified enzyme, namely adenosine kinase, adenylate kinase, and ADP kinase.

[0101] (2) Synthesis of ATP in a reaction vessel

[0102] 100 L of sterile 10 mM phosphate buffer (pH = 7.0) was added to the reaction vessel, and the following materials were added: 2.0 kg of adenosine (as a substrate), 2.5 kg of MgSO4, 0.1 kg of ATP, 1.2 kg of sodium pyrophosphate, and 0.2 kg of NaCl. 200 g of adenosine kinase, 200 g of adenylate kinase, and 200 g of ADP kinase were added to the reaction system. During the reaction, the pH was maintained at 7.0, and the temperature was controlled at 35°C.

[0103] After reacting for 12 hours, the ATP production was 31 g / L, and the adenosine conversion rate was 89%. The HPLC chromatogram is shown in Figure 6 . HPLC detection was performed using a C18 column, the detection temperature was 35°C, the flow rate was 1 mL / min, and the mobile phase was a solution containing 5% methanol and 20 mM phosphate buffer, pH = 3.2.

[0104] (3) Isolation of free enzymes

[0105] Recovery was carried out by ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 20 kDa. The filtrate was the reaction solution without enzymes, which contained ATP, ADP, AMP, and a small amount of salts.

[0106] (4) Isolation and purification of ATP product

[0107] The filtrate obtained in step (2) was subjected to ion exchange chromatography, concentration, crystallization, and drying to obtain a finished ATP product with a purity of over 97% and a total yield of 82%.

[0108] Example 4: Catalytic synthesis of ATP using immobilized enzymes

[0109] This example describes the process of entrapping ATP synthase (complex enzyme) with sodium alginate, and then using the obtained immobilized enzyme to synthesize ATP.

[0110] (1) Immobilization of enzymes

[0111] Take the complex enzyme obtained in Example 1. At 25 °C, mix an equal volume of the enzyme solution with 20 g / L sodium alginate and stir for 40 min. Then, drop it into a 1 wt% sterile CaCl2 solution and fix it at room temperature for 1 h under stirring conditions. Filter, wash, and dry to obtain spherical immobilized enzyme. Store the obtained immobilized enzyme in Tris-HCl buffer (pH = 7.1).

[0112] (2) Synthesis of ATP in a reaction column

[0113] Add 100 L of sterile 10 mM phosphate buffer (pH = 7.0) to the reaction column, and add the following materials: 3.0 kg of adenosine (as a substrate), 2.5 kg of MgSO4, 0.1 kg of ATP, 1.5 kg of sodium pyrophosphate, and 0.2 kg of NaCl. Add 1 kg of immobilized enzyme to the reaction system, evacuate the air bubbles, and prepare an enzyme reaction column. Use a constant flow pump to slowly pump the reaction solution into the enzyme reaction column from bottom to top at a flow rate of 10 L / h. During the reaction, control the pH to remain at 7.3 and the temperature to be controlled at 37 °C.

[0114] After 11 hours of cyclic reaction, the ATP production amount is 41 g / L, and the adenosine conversion rate is 90%. The HPLC chromatogram is shown in Figure 7 . HPLC detection uses a C18 column, the detection temperature is 35 °C, the flow rate is 1 mL / min, and the mobile phase uses a solution containing 5% methanol and 20 mM phosphate buffer, pH = 3.2.

[0115] The immobilized enzyme can be recycled for more than 25 times and stored at -4 °C for more than one month, and the enzyme activity decreases by 20%. It is necessary to regularly replace and supplement the immobilized enzyme particles.

[0116] (3) Isolation and purification of ATP products

[0117] The reaction solution in step (2) is prepared into a finished ATP product through ion exchange chromatography, concentration, crystallization, and drying. The purity is above 98%, and the total yield is 85%.

[0118] Example 5 uses an immobilized enzyme complex system to catalyze the synthesis of ATP

[0119] This example describes the process of immobilizing single adenosine kinase, adenylate kinase, and ADP kinase, and then using the obtained immobilized enzyme to synthesize ATP.

[0120] (1) Immobilization of enzymes

[0121] The adenosine kinase, adenylate kinase, and ADP kinase obtained in Example 3 were respectively mixed with an equal volume of enzyme solution and 20 g / L sodium alginate at 25°C and stirred for 40 min, and then dropped into a 1 wt% sterile CaCl2 solution and fixed at room temperature for 1 h under stirring conditions, filtered, washed, and dried to obtain three kinds of spherical immobilized enzymes. The obtained immobilized enzymes were stored in Tris-HCl buffer (pH = 7.1).

[0122] (2) Synthesis of ATP in a reaction column

[0123] 100 L of sterile 10 mM phosphate buffer (pH = 7.0) was added to the reaction column, and the following materials were added: 4.0 kg of adenosine (as a substrate), 2.5 kg of MgSO4, 0.1 kg of ATP, 2.0 kg of sodium pyrophosphate, and 0.2 kg of NaCl. 1 kg of immobilized adenosine kinase, adenylate kinase, and ADP kinase were respectively added to the reaction system. After exhausting the air bubbles, an enzyme reaction column was prepared. The reaction solution was slowly pumped into the enzyme reaction column from the bottom up at a flow rate of 10 L / h using a constant flow pump. During the reaction, the pH was controlled to be maintained at 7.3, and the temperature was controlled at 37°C.

[0124] After 10 hours of cyclic reaction, the ATP production amount was 52 g / L, and the adenosine conversion rate was 91%. The HPLC chromatogram is shown in Figure 8 . HPLC detection was carried out using a C18 column, the detection temperature was 35°C, the flow rate was 1 mL / min, and the mobile phase was a phosphate buffer containing 5% methanol and 20 mM, pH = 3.2.

[0125] The immobilized enzyme was cycled more than 25 times and stored at -4°C for more than one month, and the enzyme activity decreased by 20%. It is necessary to regularly replace and supplement the immobilized enzyme particles.

[0126] (3) Separation and purification of ATP products

[0127] The reaction solution in step (2) was prepared into a finished ATP product through ion exchange chromatography, concentration, crystallization, and drying. The purity was above 97%, and the total yield was 83%.

[0128] Example 6 uses immobilized enzymes to catalyze the synthesis of ATP

[0129] This example describes the technological process of embedding ATP synthase (complex enzyme) with sodium alginate and then synthesizing ATP using the obtained embedded immobilized enzyme.

[0130] (1) Immobilization treatment of enzymes

[0131] Take the composite enzyme obtained in Example 1. At 25 °C, mix an equal volume of the enzyme solution with 20 g / L sodium alginate and stir for 40 min. Then, drop it into a 1 wt% sterile CaCl2 solution and fix it at room temperature for 1 h under stirring conditions. Filter, wash, and dry to obtain spherical immobilized enzyme. Place the obtained immobilized enzyme in Tris-HCl buffer (pH = 7.1) for storage.

[0132] (2) Synthesize ATP in the reaction column

[0133] Add 100 L of sterile 10 mM phosphate buffer (pH = 7.0) to the reaction column, and add the following materials: 1 kg of adenosine (as the substrate), 2.5 kg of MgSO4, 0.1 kg of ATP, 1 kg of sodium pyrophosphate, and 0.2 kg of NaCl. Add 1 kg of immobilized enzyme to the reaction system. After exhausting the air bubbles, prepare an enzyme reaction column. Use a constant flow pump to slowly pump the reaction solution into the enzyme reaction column from the bottom up at a flow rate of 10 L / h. During the reaction, control the pH to remain at 7.3 and the temperature to be controlled at 37 °C.

[0134] After 11 hours of cyclic reaction, the ATP production amount is 15 g / L and the adenosine conversion rate is 93%. The HPLC chromatogram is shown in Figure 9 . HPLC detection uses a C18 column, the detection temperature is 35 °C, the flow rate is 1 mL / min, and the mobile phase uses a solution containing 5% methanol and 20 mM phosphate buffer, pH = 3.2.

[0135] The immobilized enzyme undergoes more than 25 cycles of reaction and is stored at -4 °C for more than one month, and the enzyme activity decreases by 20%. It is necessary to regularly replace and supplement the immobilized enzyme particles.

[0136] (3) Separation and purification of ATP product

[0137] The reaction solution in step (2) is prepared into a finished ATP product through ion exchange chromatography, concentration, crystallization, and drying. The purity is above 98% and the total yield is 84%.

[0138] In Comparative Example 1, three separate free enzymes were used for the catalytic production of ATP

[0139] This comparative example describes the process flow for catalytic production of ATP using single free adenosine kinase, adenylate kinase, and ADP kinase.

[0140] (1) Preparation of free enzyme

[0141] The obtaining of single free adenosine kinase, adenylate kinase, and ADP kinase is shown in Example 3.

[0142] (2) Synthesize ATP in the reaction tank

[0143] Add 100 L of sterile 10 mM phosphate buffer (pH = 7.0) to the reaction tank, and add the following materials: 1.5 kg of adenosine (as the substrate), 2.5 kg of MgSO4, 0.1 kg of ATP, 1.5 kg of sodium pyrophosphate, and 0.2 kg of NaCl. Add 200 g of adenosine kinase, 200 g of adenylate kinase, and 200 g of ADP kinase to the reaction system. During the reaction, control the pH to remain at 7.0 and the temperature to be controlled at 30 °C.

[0144] After reacting for 12 hours, the amount of ATP produced is 18 g / L, and the adenosine conversion rate is 61%. Compared with the complete single complex protein catalysis, the overall yield decreased by about 20%. The HPLC chromatogram is shown in Figure 10 . HPLC detection is carried out using a C18 column, the detection temperature is 35 °C, the flow rate is 1 mL / min, and the mobile phase is a phosphate buffer containing 5% methanol and 20 mM, pH = 3.2.

[0145] (3) Isolation of free enzymes

[0146] Recover by ultrafiltration. Use an ultrafiltration membrane with a molecular weight cut-off of 20 kDa. The filtrate is the reaction solution without enzymes, which contains ATP, ADP, AMP, and a small amount of salts.

[0147] Comparative Example 2 Catalytic production of ATP in the absence of adenosine kinase

[0148] This comparative example describes the process flow of using the complex enzyme formed by simply linking adenylate kinase and ADP kinase with a linker to catalyze the production of ATP.

[0149] (1) Preparation of immobilized enzymes

[0150] The method for obtaining immobilized adenylate kinase and ADP kinase can be found in Example 5.

[0151] (2) Synthesis of ATP in the reaction column

[0152] Add 100 L of sterile 10 mM phosphate buffer (pH = 7.0) to the reaction column, and add the following materials: 3.0 kg of adenosine (as the substrate), 2.5 kg of MgSO4, 0.1 kg of ATP, 3.0 kg of sodium pyrophosphate, and 0.2 kg of NaCl. Add 1 kg of immobilized adenylate kinase and ADP kinase to the reaction system respectively. After exhausting the air bubbles, an enzyme reaction column is prepared. Use a constant flow pump to slowly pump the reaction solution from the bottom up through the enzyme reaction column at a flow rate of 10 L / h. During the reaction, control the pH to remain at 7.3 and the temperature to be controlled at 37 °C.

[0153] After circulating the reaction for 10 hours, the amount of ATP produced is 0 g / L, and the adenosine conversion rate is 0%. The HPLC chromatogram is shown in Figure 11。HPLC detection was performed using a C18 column, with a detection temperature of 35 °C, a flow rate of 1 mL / min, and a mobile phase consisting of 5% methanol and 20 mM phosphate buffer, pH = 3.2.

[0154] The immobilized enzyme was subjected to more than 25 cycles of reaction and stored at -4 °C for more than one month, resulting in a 20% decrease in enzyme activity. It is necessary to regularly replace and supplement the immobilized enzyme particles. Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method and process for synthesizing adenosine triphosphate by multi-enzyme cascade catalysis in vitro, characterized in that: Including the enzyme cascade catalysis of ATP synthesis, a single enzyme protein performs the whole process catalysis. The enzymes involved include three types: adenosine kinase (EC2.7.1.20), adenylate kinase (EC 2.7.4.3) and ADP kinase (EC 2.7.4.1). Each enzyme is connected by a linker protein. The whole catalytic reaction process is as follows: (1) Adenosine is converted into AMP under the catalysis of adenosine kinase; (2) AMP is converted into ADP under the catalysis of adenylate kinase; (3) ADP is converted into ATP under the catalysis of ADP kinase.

2. The method and process for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate according to claim 1, characterized in that: The enzymatic process uses the following steps: (1) At the genetic level: the genes of three enzymes, adenosine kinase, adenylate kinase, and ADP kinase, are spliced ​​together, and the gene sequence of the linker protein is inserted in the middle; (2) Expression of enzyme proteins: Use model strains such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pichia pastoris to express enzyme complex proteins; (3) Enzyme immobilization: Enzyme immobilization is performed using sodium alginate embedding method; (4) adding the obtained single protein immobilized enzyme into 1 L reaction system for reaction; (5) separation of products and ATP-producing enzyme; (6) Preparation of finished product: the reaction solution separated in step (5) is subjected to ion exchange chromatography, concentration, crystallization, and drying steps to prepare finished ATP.

3. The method and process for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate according to claim 1, characterized in that: Enzyme proteins are obtained through microbial fermentation.

4. The method and process for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate according to claim 1, characterized in that: The reaction conditions for ATP synthesis are as follows: reaction temperature 20-40°C.

5. The method and process for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate according to claim 1, characterized in that: The reaction system also includes Tris-HCl, which serves as a reaction buffer.

6. The method and process for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate according to claim 1, characterized in that: Reaction conditions for ATP synthesis: PH 6.7 ~ 7.

6.

7. A biological method for preparing an enzyme complex for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate, characterized in that: A method and process for synthesizing adenosine triphosphate by using an in vitro multi-enzyme cascade catalyzed synthesis of adenosine triphosphate according to any one of claims 1 to 6, wherein the process for obtaining the enzyme for synthesizing ATP is as follows: Add the gene sequence of the linker protein to the C-terminus of the adenosine kinase gene, add the gene sequence of the linker protein to the N-terminus and C-terminus of the adenylate kinase, add the gene sequence of the linker protein to the N-terminus of the ADP kinase, remove the stop codon TAA at the C-terminus of the adenosine kinase gene fragment, remove the stop codon TAA at the C-terminus of the adenylate kinase gene fragment, and retain the stop codon TAA at the C-terminus of the ADP kinase gene fragment; The complete sequences of adenosine kinase, adenylate kinase and ADP kinase gene fragments including the linker sequence were synthesized, and then the complete sequences were inserted into plasmid pET28a and transformed into Escherichia coli BL21; The positive clones of the transformed E. coli BL21 were transferred to LB medium liquid culture medium, cultured to the logarithmic growth phase, and then 0.5 mM IPTG was added for induction for 6 h, and then the bacteria were collected and SDS-PAGE protein electrophoresis was performed to screen the high-expression strains; The selected high-expression strain was inoculated into 100 mL seed culture medium under sterile conditions, cultured to the logarithmic growth phase, and then inoculated into a 5 L fermentation tank. After continuing to culture to the logarithmic growth phase, it was inoculated into a fermentation tank containing 50 L culture medium. After culturing for 6 hours, 0.5 mM IPTG was added to induce expression for 10 hours, and the cells were collected by centrifugation to obtain about 1 kg of Escherichia coli cells; The collected bacteria are disrupted by ultrasound or high-pressure homogenization, and the supernatant is collected by centrifugation. Add 30-60% saturated (NH4)2SO4 to the supernatant, centrifuge, dissolve the collected precipitate with Tris-HCl buffer (pH=7.8), and use a dialysis bag for desalting; finally, filter and sterilize with a 0.22μm membrane to obtain a preliminarily purified enzyme, which is the ATP synthase after the connection of adenosine kinase, adenylate kinase and ADP kinase.

8. The biological method for preparing an enzyme complex for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate according to claim 7, characterized in that: The positive clones of the transformed Escherichia coli BL21 were transferred to LB medium, and kanamycin was added to the liquid medium to a final concentration of 50 mg / L.

9. The biological method for preparing an enzyme complex for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate according to claim 7 or 8, characterized in that: The process of catalyzing the synthesis of ATP includes the use of free ATP synthase.

10. The biological method for preparing an enzyme complex for in vitro multi-enzyme cascade catalytic synthesis of adenosine triphosphate according to claim 7 or 8, characterized in that: The catalytic synthesis of ATP preparation process includes the process of embedding ATP synthase in sodium alginate, and then using the embedded immobilized enzyme to synthesize ATP.

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