A method for biosynthesizing guanine nucleoside diphosphate by multi-enzyme method

Abstract

The invention discloses a method for biosynthesizing guanosine diphosphate by a multi-enzyme method, which comprises a step of catalytically synthesizing GDP, wherein the step of catalytically synthesizing GDP is performed according to a conversion reaction, wherein GMP and ATP are converted into ADP, GDP, and GTP under the catalytic action of a first enzyme, wherein before the conversion reaction, the molar concentration ratio of GMP to ATP is greater than or equal to 40, and the method further comprises the steps of ADP enzymatic conversion and GTP enzymatic conversion. The method for biosynthesizing guanosine diphosphate by a multi-enzyme method provided by the invention has the effect of reducing the difficulty of purification.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a method for biosynthesizing guanine nucleoside diphosphate using a multi-enzyme method. Background Art

[0002] Guanosine diphosphate (GDP) is a very important diphosphate nucleoside in the body, composed of a pyrophosphate group, a pentose, and the base guanine. GDP is not only involved in the fine regulation of cell energy metabolism and signal transduction, but also shows unique importance in molecular construction and disease treatment strategies. Its complex and exquisite mechanism of action constitutes an important cornerstone of life science research.

[0003] Guanosine diphosphate (GDP), as a highly versatile nucleotide molecule in organisms, has its core value deeply embedded in the most basic processes of life and cutting-edge biomedical applications. In the field of biosynthesis, GDP and its derivatives, like "chemical craftsmen" in organisms, participate in complex glycosylation modifications. For example, GDP-fucose and GDP-mannose, as donor molecules for sugar chain assembly, are essential for post-translational modifications of proteins and lipids, which affect cell recognition, immune response and even the invasion mechanism of pathogens. In addition, in emerging anti-tumor immunity research, GDP-mannose has been found to be able to regulate the stability of specific proteins, showing the potential to enhance the body's anti-tumor immune response.

[0004] Currently, the synthesis process of guanine nucleoside diphosphate is mainly divided into chemical method and biological method.

[0005] In the process of chemical synthesis of guanine nucleoside diphosphate, the basic structure guanosine is synthesized first, and guanosine is converted into guanylic acid (GMP) through chemical phosphorylation reaction, and then further converted into GDP. Commonly used catalysts include guanine nucleoside monophosphate kinase (GKase), etc. Finally, it needs to be purified by chromatography, extraction and other methods to remove by-products and unreacted raw materials, and finally high-purity GDP is obtained by crystallization.

[0006] The chemical synthesis of guanine nucleoside diphosphate has the following problems:

[0007] ① Environmental pollution: A large amount of organic solvents are usually required in the chemical synthesis process. These solvents are not only expensive, but also produce harmful wastes in the post-reaction treatment stage, causing environmental pollution. The treatment and disposal of these chemical wastes require additional costs and technologies, increasing the environmental burden of the entire synthesis process.

[0008] ② Raw material cost and source: The starting materials and reagents that chemical synthesis relies on, such as different phosphorylation reagents and protecting groups, may be expensive and unstable in supply, affecting the economy and sustainability of the synthesis.

[0009] ③ Step complexity: Compared with biosynthesis, the steps of chemical synthesis of GDP are more complicated, involving multiple chemical reactions and purification steps. Each step requires precise control of conditions to ensure high yield and product purity, which not only has high technical requirements, but may also result in a low overall yield.

[0010] ④By-product problem: A variety of by-products are easily produced during the chemical synthesis process. The separation and purification of these by-products are very difficult, which not only increases the production cost, but may also affect the quality and purity of the final GDP product.

[0011] ⑤Safety considerations: Some of the chemical reagents used, such as phosphorus oxychloride, are highly corrosive and toxic, and place extremely high demands on the safety standards of operators and the production environment.

[0012] ⑥ Regulatory compliance: Products from chemical synthesis routes may need to undergo strict regulatory approval to ensure that they meet safety standards for drugs or food additives, which may extend the timeline and add additional compliance costs.

[0013] Compared with chemical synthesis, the biosynthesis of GDP has a series of significant advantages, such as:

[0014] ① Environmentally friendly: The biosynthesis process is carried out under mild conditions, usually using water as a solvent, avoiding the use of large amounts of toxic organic solvents, reducing environmental pollution and processing costs.

[0015] ② Cost-effectiveness: Once an effective biological production system is established, large-scale production can be achieved at a relatively low cost by utilizing the self-replication and self-maintenance characteristics of microorganisms. Microorganisms use cheap carbon sources (such as sugars) for growth and convert them into high-value GDP, reducing the cost of raw materials.

[0016] ③ High selectivity and specificity: Biological enzymes have natural stereoselectivity and chemical specificity, and can accurately catalyze reactions in complex cellular environments to generate single, high-purity products, reducing subsequent purification steps.

[0017] ④Continuous production: Microorganisms can be cultured continuously or in a fixed form to achieve continuous production of GDP, thereby improving production efficiency and facility utilization.

[0018] ⑤Flexibility and adjustability: Through genetic engineering, the metabolic pathways of microorganisms can be flexibly adjusted to optimize production or introduce new functions, such as enhancing the activity of specific enzymes or introducing the release of feedback inhibition to further increase GDP production.

[0019] ⑥Safety and Renewability: The biosynthesis process is based on renewable resources, and genetically engineered strains are cultivated under a strictly controlled environment. Compared with chemical synthesis, it is generally regarded as a safer and more sustainable production method.

[0020] ⑦ Wide applicability: It is suitable for the production of various complex nucleotide derivatives that are difficult to obtain through chemical synthesis, especially some GDP derivatives containing special glycosylation or modification, such as GDP-mannose or GDP-fucose.

[0021] The biosynthesis of guanosine diphosphate usually converts GMP and adenosine triphosphate (ATP) into GDP under the action of nucleoside monophosphate kinase. In industry, due to the high purification cost of nucleoside monophosphate kinase, the enzyme solution of nucleoside monophosphate kinase is used for the conversion reaction after fermentation. However, the nucleoside diphosphate kinase enzyme solution obtained after fermentation inevitably contains miscellaneous enzymes such as nucleoside diphosphate kinase, which makes the biosynthesis of guanosine diphosphate produce by-products such as guanosine triphosphate (GTP). In addition, enzyme preparation catalysis is often in a dynamic equilibrium state, and it is difficult to achieve complete conversion of the substrate, which ultimately leads to the simultaneous presence of unreacted GMP substrate, GDP, guanosine triphosphate (GTP), adenosine triphosphate (ATP), adenosine diphosphate (ADP) and other substances in the GDP biosynthesis reaction solution. These substances are of many types and have similar physical and chemical properties. They require multiple chromatography columns to be separated, which seriously increases the difficulty of subsequent separation and extraction, resulting in low purification yield, large amount of wastewater, high production cost, and inability to achieve industrialization. Summary of the invention

[0022] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for biosynthesizing guanine nucleoside diphosphate by a multi-enzyme method.

[0023] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0024] A multi-enzyme biosynthesis method for guanine nucleoside diphosphate occurs through the following conversion reaction:

[0025]

[0026] Before the conversion reaction, the molar concentration ratio of the GMP to the ATP is ;

[0027] The steps include:

[0028] Catalytic synthesis of GDP: GMP and ATP are reacted by the first enzyme to obtain a first reaction solution;

[0029] ADP enzymatic conversion: adding a phosphate donor and a second enzyme to the first reaction solution to react to obtain a second reaction solution;

[0030] GTPase conversion: adding a third enzyme to the second reaction solution to catalyze a reaction to obtain a third reaction solution, and purifying the third reaction solution to obtain GDP;

[0031] The second enzyme can catalyze the conversion of ADP into ATP; the third enzyme is used to hydrolyze GTP, so that the GTP is converted into GDP.

[0032] In the present invention, ADP undergoes the following changes:

[0033] ;

[0034] The following changes occur in GTP:

[0035] .

[0036] Preferably, the .

[0037] Preferably, the first enzyme comprises guanosine monophosphate kinase (GKase).

[0038] Preferably, the pH in the step of catalytic synthesis of GDP is 7.0-8.0.

[0039] Preferably, the first enzyme participates in the reaction in the state of enzyme liquid, the first enzyme liquid is obtained by fermentation, and when the OD600 of the obtained fermentation liquid of the first enzyme is 70-80, the first enzyme liquid is 5-20% of the GMP mass.

[0040] Preferably, the second enzyme is selected from one or more of acetate kinase (ACKase), pyruvate kinase (PKase), and polyphosphate kinase (PPKase).

[0041] In the present invention, the second enzyme is selected from polyphosphate kinase (PPKase), and the phosphate donor is selected from (NaPO3)6.

[0042] Preferably, the pH in the ADP enzymatic conversion step is 7.0-8.0.

[0043] Preferably, the second enzyme participates in the reaction in the state of enzyme liquid, the second enzyme liquid is obtained by fermentation, and when the OD600 of the fermentation liquid of the obtained second enzyme is 70-80, the second enzyme liquid is 5-20% of the GMP mass.

[0044] Preferably, the third enzyme comprises glucokinase (GLKase).

[0045] Preferably, the third enzyme participates in the reaction in the state of enzyme liquid, and the third enzyme liquid is obtained by fermentation. When the OD600 of the fermentation liquid of the obtained third enzyme liquid is 70-80, the third enzyme liquid is 2.5-10% of the GMP quality.

[0046] Preferably, the step of catalytic synthesis of GDP and the step of ADP enzymatic conversion are combined, that is:

[0047] Combined transformation: GMP, ATP, the first enzyme and the second enzyme are mixed and reacted to obtain a second reaction solution.

[0048] Preferably, the method further comprises the steps of:

[0049] Degradation of residual GMP: adding nucleoside enzyme to the third reaction solution for catalytic reaction and then purifying to obtain GDP;

[0050] The nucleosidase is used to break down GMP into guanine and ribose phosphate.

[0051] In the present invention, the residual GMP undergoes the following transformation:

[0052] .

[0053] In the present invention, the incompletely converted GMP is decomposed into guanine and ribose phosphate by nucleosidase; guanine has very poor water solubility and is very easy to separate; ribose phosphate and the target product GDP have great differences in physical and chemical properties and are easy to separate.

[0054] Preferably, the pH in the residual GMP degradation step is 6.0-8.0.

[0055] Preferably, the nucleosidase participates in the reaction in the form of an enzyme liquid, the nucleosidase liquid is obtained by fermentation, and when the OD600 of the obtained nucleosidase fermentation liquid is 70-80, the nucleosidase liquid is 2.5-10% of the GMP quality.

[0056] The main raw materials of the present invention are introduced as follows:

[0057] Guanylic acid (GMP): Guanylic acid, also known as guanosine monophosphate (GMP), is formed by the condensation of phosphoric acid and the hydroxyl group on the 5th carbon atom of the nucleotide ribose. It can be converted from guanosine through a series of biochemical reactions.

[0058] The GMP used in the present invention can also be obtained by converting guanosine.

[0059] Compared with the prior art, the advantages of the present invention include:

[0060] (1) The present invention provides a multi-enzyme method for biosynthesizing guanine nucleoside diphosphate, which uses a phosphate donor and a second enzyme to convert the generated ADP into ATP, so that a small amount of ATP added can maintain the continuous progress of the conversion reaction;

[0061] (2) The present invention provides a multi-enzyme method for biosynthesizing guanine nucleoside diphosphate. A smaller amount of ATP used is beneficial to reducing the residual ATP after the conversion reaction is completed, thereby reducing the difficulty of GDP purification and improving GDP purity;

[0062] (3) The present invention provides a multi-enzyme method for biosynthesizing guanine nucleoside diphosphate, which uses a third enzyme to convert the GTP produced by the reaction into the target product GDP and converts the by-products into the target product, thereby reducing the by-products while increasing the concentration and purity of the target product GDP, thereby reducing the difficulty of GDP purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0064] Figure 1 Schematic diagram of a multi-enzyme biosynthesis method of guanine nucleoside diphosphate in Example 1 of the present invention;

[0065] Figure 2 This is a graph showing the HPLC test results of the solution to be reacted obtained in step S1 in Example 1 of the present invention;

[0066] Figure 3 This is a graph showing the HPLC test results of the second reaction solution obtained in step S2 of Example 1 of the present invention;

[0067] Figure 4 This is a graph showing the HPLC test results of the third reaction solution obtained in step S3 of Example 1 of the present invention;

[0068] Figure 5 This is a graph showing the HPLC test results of the fourth reaction solution obtained in step S4 in Example 1 of the present invention;

[0069] Figure 6 This is a graph showing the HPLC detection results of GDP obtained in step S5 in Example 1 of the present invention. DETAILED DESCRIPTION

[0070] In order to enable those skilled in the art to understand the characteristics and effects of the present application, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the common meanings understood by those skilled in the art for the present application. In case of conflict, the definitions in this specification shall prevail.

[0071] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present application in any way, that is, the content of the present application can be implemented without being limited by any specific theory or mechanism.

[0072] As used herein, "this application" means "the present invention" or "the present disclosure".

[0073] The use of "one", "an", "a kind" or similar expressions to describe the components and technical features described in this application is merely for the convenience of expression and to provide a general meaning to the scope of this application. Therefore, such description should be understood to include one or at least one, and the singular also includes the plural, unless it is obvious that it refers to another meaning.

[0074] In this document, "or its combination" means "or any combination thereof", and "any one", "any one" means "any one", "any one" or "any one".

[0075] As used herein, the terms "comprise", "include", "have", "contain" or any other similar terms are open-ended transitional terms, which are intended to cover non-exclusive inclusions. For example, a composition or product containing multiple elements is not limited to the elements listed herein, but may also include other elements that are not explicitly listed but are generally inherent to the composition or product. In addition, unless otherwise expressly stated, the term "or" refers to an inclusive "or" rather than an exclusive "or". For example, any of the following situations satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and A and B are both true (or exist). In addition, in this article, the terms "comprising", "including", "having", and "containing" should be interpreted as being specifically disclosed and simultaneously covering closed conjunctions such as "consisting of", "consisting of", and "the remainder is", as well as conjunctions such as "consisting essentially of", "mainly consisting of", "mainly consisting of", "basically containing", "essentially consisting of", "essentially consisting of", and "essentially containing".

[0076] In this article, all features or conditions such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have covered and specifically disclosed all possible sub-ranges and individual values ​​within the range (including integers and fractions), especially integer values. For example, the range description of "1.0 to 8.0" or "between 1.0 and 8.0" or "between 1.0 and 8.0" should be deemed to have specifically disclosed all sub-ranges such as 1.0 to 8.0, 1.0 to 7.0, 2.0 to 8.0, 2.0 to 6.0, 3.0 to 6.0, 4.0 to 8.0, 3.0 to 8.0, etc., and should be deemed to cover endpoint values, especially sub-ranges defined by integer values, and should be deemed to have specifically disclosed individual values ​​such as 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, etc. Unless otherwise indicated, the foregoing method of interpretation applies to all contents of the entire application, regardless of whether the scope is broad or not.

[0077] If the quantity, concentration or other numerical value or parameter is expressed as a range, a preferred range (or a better range) or a series of upper and lower limits, it should be understood that all ranges consisting of any pair of the upper limit or preferred value (or a better value) of the range and the lower limit or preferred value (or a better value) of the range have been specifically disclosed herein, regardless of whether these ranges are disclosed separately. In addition, if a numerical range is mentioned herein, unless otherwise specified, the range should include its endpoints and all integers and fractions within the range.

[0078] In this document, numerical values ​​should be understood to have the accuracy of the number of significant digits of the numerical value, provided that the purpose of the invention can be achieved. For example, the number 40.0 should be understood to cover the range of 39.50 to 40.49.

[0079] In this article, for the use of Markush groups or optional terms to describe the features or examples of the present application, those skilled in the art should understand that all subgroups or any individual elements in the Markush group or option list can also be used to describe the present application. For example, if X is described as "selected from the group consisting of X1, X2 and X3", it also means that the claim that X is X1 and the claim that X is X1 and / or X2 and / or X3 have been fully described. Furthermore, for the use of Markush groups or optional terms to describe the features or examples of the present application, those skilled in the art should understand that any combination of subgroups or individual members of all elements in the Markush group or option list can also be used to describe the present application. Accordingly, for example, if X is described as "selected from the group consisting of X1, X2 and X3", and Y is described as "selected from the group consisting of Y1, Y2 and Y3", it means that the claim that X is X1 and / or X2 and / or X3 and Y is Y1 and / or Y2 and / or Y3 has been fully described.

[0080] It should be understood that the features disclosed in the various embodiments herein may be arbitrarily combined to form the technical solution of the present application, as long as there is no contradiction in the combination of these features.

[0081] The present application will be described below with specific implementations and examples. It should be understood that these specific implementations and examples are merely illustrative and are not intended to limit the scope of the present application and its use.

[0082] Unless otherwise stated, the methods, reagents and conditions used in the following preparation examples, comparative examples and examples are conventional methods, reagents and conditions in the art.

[0083] In the following descriptions:

[0084] Disodium 5'-guanylate: CAS No.: 5550-12-9, active ingredient content ≥ 99%,

[0085] Adenosine triphosphate disodium: CAS: 2964-48-9, active ingredient content ≥ 99%, density 2.63g / cm 3 ,

[0086] (NaPO3)6: CAS number: 10124-56-8 Active ingredient content ≥ 99%.

[0087] In the present invention, the recombinant engineering strains of guanosine monophosphate kinase (GKase), glucokinase (GLKase), nucleosidase (Nucleosidase) and polyphosphate kinase (PPKase) can use eukaryotic hosts such as Saccharomyces cerevisiae and Pichia pastoris, or prokaryotic expression systems such as Escherichia coli and Bacillus subtilis.

[0088] The present invention preferably uses Escherichia coli BL21 (DE3) as the recombinant protein expression host, and constructs guanosine monophosphate kinase (GKase), glucose kinase (GLKase), nucleosidase (Nucleosidase) polyphosphate kinase (PPKase) recombinant engineering strains respectively. The specific construction method refers to the "Molecular Cloning Experiment Guide".

[0089] The above recombinant enzyme protein fermentation process:

[0090] The fermentation medium used was TB liquid medium, which contained: 12 g / L peptone, 24 g / L yeast powder, 4 g / L glycerol, 2.31 g / L KH2PO4, and 16.43 g / L K2HPO4·3H2O;

[0091] The recombinant enzyme protein fermentation process uses isopropylthiogalactoside (IPTG) or lactose to induce protein expression; the culture temperature for protein induction expression is 20~40℃, and the culture is continued for 16~24 hours. The fermentation can be stopped when the OD600 of the fermentation liquid reaches 70, and the fermentation liquid bacteria can be collected.

[0092] The above-mentioned recombinant enzyme protein can be a fermented bacterial cell containing the recombinant enzyme protein, or a cell-disrupted enzyme solution (referred to as enzyme solution) containing the recombinant enzyme protein, or a lyophilized powder containing the recombinant enzyme protein. Of course, in other experiments, the enzyme protein can also be combined with a corresponding carrier material to become an immobilized enzyme of the recombinant enzyme protein. Example Example 1

[0093] The raw materials used in this embodiment include: disodium 5'-guanylate, disodium adenosine triphosphate, (NaPO3)6, Gkase enzyme solution, PPKase enzyme solution, GLKase enzyme solution, and nucleoside enzyme solution.

[0094] See also Figure 1 , the preparation method of this embodiment includes:

[0095] S1. Prepare reactants: at 37°C, prepare disodium 5'-guanylate, disodium adenosine triphosphate, and (NaPO3)6 into a solution with a concentration of 300 mM disodium 5'-guanylate, 7.5 mM disodium adenosine triphosphate, and 120 mM (NaPO3)6 to obtain a solution to be reacted;

[0096] S2, combined transformation: Gkase enzyme solution and PPKase enzyme solution are added to the reaction solution, and the amount of Gkase enzyme solution and PPKase enzyme solution added is 5% of the mass of 5'-disodium guanylate; the temperature is controlled to 37°C for reaction, and the pH of the solution is adjusted to 7.0-8.0 during the reaction and stirred continuously, and the GMP content is detected by HPLC every 20 minutes; when the GMP content no longer decreases, the pH of the solution is adjusted to 10 to obtain a second reaction solution;

[0097] S3, GTP enzyme conversion: the pH of the second reaction solution obtained in S2 is adjusted to 7.0, and then anhydrous glucose is added to an anhydrous glucose concentration of 50mM, and then GLKase enzyme solution is added, and the amount of GLKase enzyme solution added is 2.5% of the mass of 5'-disodium guanylate; the temperature is controlled to 37°C for reaction, and the solution pH is adjusted to 6.0-8.0 during the reaction and stirred continuously, and the GTP content is detected by HPLC every 20 minutes; when the GTP content no longer decreases, the solution pH is adjusted to 10 to obtain a third reaction solution;

[0098] S4, residual GMP degradation: the pH of the third reaction solution obtained in step S3 is adjusted to 7.0, and then nucleosidase solution is added, and the amount of nucleosidase solution added is 2.5% of the mass of 5'-disodium guanylate; the temperature is controlled to 37°C for reaction, and the solution pH is adjusted to 6.0-8.0 during the reaction and stirred continuously, and the GMP content is detected by HPLC every 20 minutes; when the GMP content no longer decreases, the solution pH is adjusted to 10 to obtain a fourth reaction solution;

[0099] S5. Purification: at 10° C., the supernatant obtained after centrifugation of the fourth reaction solution is chromatographed using an ion exchange chromatography column, and then eluted, crystallized, and vacuum dried to obtain GDP. Example 2

[0100] The difference between this embodiment and embodiment 1 is that the concentration of adenosine triphosphate disodium in step S1 is 5 mM, the amount of Gkase enzyme solution and PPKase enzyme solution added in step S2 is 12% of the mass of 5'-guanylate disodium, the amount of GLKase enzyme solution added in step S3 is 6% of the mass of 5'-guanylate disodium, and the amount of nucleoside enzyme solution added in step S4 is 6% of the mass of 5'-guanylate disodium. Example 3

[0101] The difference between this embodiment and embodiment 1 is that the concentration of adenosine triphosphate disodium in step S1 is 3 mM, the amount of Gkase enzyme solution and PPKase enzyme solution added in step S2 is 20% of the mass of 5'-guanylate disodium, the amount of GLKase enzyme solution added in step S3 is 10% of the mass of 5'-guanylate disodium, and the amount of nucleoside enzyme solution added in step S4 is 10% of the mass of 5'-guanylate disodium.

[0102] Table 1 GDP content in each step of Examples 1 to 3

[0103] step Example 1 Example 2 Example 3 s1 0 0 0 s2 53.82% 54.11% 54.64% s3 46.35% 45.93% 45.81% s4 90.50% 90.72% 90.99% s5 95.59% 95.87% 95.92%

[0104] See also Figures 2 to 6 As shown in Table 1, for Example 1, the GDP content in the reaction solution obtained in step S1 is 0, and the GMP content is 97.81%;

[0105] The GDP content in the second reaction solution obtained in step S2 is 53.82%, and the GMP content is 28.20%, indicating that the conversion reaction has occurred;

[0106] The GDP content in the third reaction solution obtained in step S3 is 45.93%, and the GMP content is 46.84%, indicating that GTP is converted into GDP and GMP;

[0107] The GDP content in the fourth reaction solution obtained in step S4 is 90.50%, and the GMP content is significantly reduced, indicating that GMP undergoes a hydrolysis reaction.

[0108] It should be understood that the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, some simple deductions or substitutions can be made without departing from the concept of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for biosynthesizing guanine nucleoside diphosphate by a multi-enzyme method, characterized in that: The following conversion reaction occurs: ； Before the conversion reaction, the molar concentration ratio of the GMP to the ATP is ; The steps include: Catalytic synthesis of GDP: GMP and ATP are reacted by the first enzyme to obtain a first reaction solution; ADP enzymatic conversion: adding a phosphate donor and a second enzyme to the first reaction solution to react to obtain a second reaction solution; GTPase conversion: adding a third enzyme to the second reaction solution to catalyze a reaction to obtain a third reaction solution, and purifying the third reaction solution to obtain GDP; The second enzyme is capable of catalyzing the conversion of ADP into ATP; the third enzyme is used to hydrolyze the GTP, so that the GTP is converted into GDP; Also includes the steps: Degradation of residual GMP: adding nucleoside enzyme to the third reaction solution for catalytic reaction and then purifying to obtain GDP; The nucleosidase is used to decompose GMP into guanine and ribose phosphate; The third enzyme includes glucokinase; The step of catalytic synthesis of GDP and the step of ADP enzymatic conversion are combined, that is, combined conversion: GMP, ATP, the first enzyme and the second enzyme are mixed and reacted to obtain a second reaction solution.

2. The method for biosynthesizing guanine nucleoside diphosphate by a multi-enzyme method according to claim 1, characterized in that: Said .

3. The method for biosynthesizing guanine nucleoside diphosphate by a multi-enzyme method according to claim 1, characterized in that: The first enzyme includes guanosine monophosphate kinase; And / or, the pH in the step of catalyzing and synthesizing GDP is 7.0 to 8.0; And / or, the first enzyme participates in the reaction in the state of enzyme liquid, the first enzyme liquid is obtained by fermentation, and when the OD600 of the obtained fermentation liquid of the first enzyme is 70-80, the first enzyme liquid is 5-20% of the GMP mass.

4. The method for biosynthesizing guanine nucleoside diphosphate by a multi-enzyme method according to claim 1, characterized in that: The second enzyme is selected from one or more of acetate kinase, pyruvate kinase, and polyphosphate kinase; and / or, the pH in the ADP enzymatic conversion step is 7.0 to 8.0; And / or, the second enzyme participates in the reaction in the state of enzyme liquid, the second enzyme liquid is obtained by fermentation, and when the OD600 of the fermentation liquid of the obtained second enzyme is 70-80, the second enzyme liquid is 5-20% of the GMP mass.

5. The method for biosynthesizing guanine nucleoside diphosphate by a multi-enzyme method according to claim 1, characterized in that: The third enzyme participates in the reaction in the form of enzyme liquid, and the third enzyme liquid is obtained by fermentation. When the OD600 of the fermentation liquid of the third enzyme is 70-80, the third enzyme liquid is 2.5-10% of the GMP quality.

6. The method for biosynthesizing guanine nucleoside diphosphate by a multi-enzyme method according to claim 1, characterized in that: The pH in the residual GMP degradation step is 6.0 to 8.0; And / or, the nucleosidase participates in the reaction in the state of enzyme liquid, the nucleosidase liquid is obtained by fermentation, and when the OD600 of the obtained nucleosidase fermentation liquid is 70-80, the nucleosidase liquid is 2.5-10% of the GMP quality.

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