Enzyme mutant, enzyme composition or immobilized enzyme thereof and application and method for preparing cordycepin

Abstract

The present invention relates to the field of enzyme engineering, and more particularly to enzyme mutants, enzyme compositions or their immobilized enzymes and their applications and methods for preparing cordycepin. The method utilizes adenosine as a starting material, generates 3'-adenosine phosphate (3'-AMP) under the action of adenosine 3'-kinase (Cor3K), then utilizes cordycepin dephosphatase (CorDep) to generate dehydrated adenosine, and finally generates cordycepin under cordycepin reductase (CorRed) conversion. The route is very simple, has a high conversion rate, and the raw materials used are also very cheap. This conversion can be completed step by step or in a one-pot process. At the same time, for the coenzymes ATP and NADH required for the first and third step conversions, pyruvate kinase (PcPyk) and formate dehydrogenase (MspFDH) can also be introduced for recycling, thereby further reducing its production price.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering, and in particular to an enzyme mutant, an enzyme composition or an immobilized enzyme thereof, and an application and method for preparing cordycepin. Background Art

[0002] Cordycepin is also known as cordycepin, cordycepin, cordycepin, alias 3'-deoxyadenosine, chemical formula is C 10 H 13 N₅O₃ is the first nucleoside antibiotic isolated from fungi. It has a long history of application and exhibits diverse pharmacological activities, including antibacterial, anti-inflammatory, antiviral, anti-tumor, and immunomodulatory activities. Phase III clinical trial data also demonstrates the potential of cordycepin in treating patients with acute pre-B and pre-T lymphocytic leukemia. Recent biological research has further demonstrated that cordycepin can enhance human immunity and eliminate the increase in free radicals in the body caused by aging.

[0003] Currently, cordycepin is primarily obtained through extraction and isolation from cultivated Cordyceps militaris, a process that is costly, expensive, highly polluting, and limited in scale. Chemical synthesis, using adenosine as a raw material, involves a multi-step process of group protection, free radical reduction, and subsequent deprotection. This complex synthesis process results in low yields and a high production cost.

[0004] Cordycepin is a natural compound whose biosynthesis offers advantages such as environmental compatibility, high product quality, and affordability. The biosynthesis of cordycepin has been previously reported. Adenosine, the raw material, is phosphorylated by CNS3 kinase to produce 3'-adenosine monophosphate (3'-AMP), which is then dephosphorylated by CNS2 enzyme to produce 2'-glycosyl-3'-deoxyadenosine. Finally, CNS1 ketoreductase generates 3'-deoxyadenosine (cordycepin). However, the enzymes involved in this biosynthesis pathway are only found in a few fungi, and heterologous expression of these enzymes in industrial bacteria presents solubility issues.

[0005] Therefore, providing a biological method for preparing cordycepin has important practical significance. Summary of the Invention

[0006] In view of this, in order to solve the above problems, through long-term laboratory testing and research, we finally obtained cns3 and cns1 replacement enzymes with industrial performance, and then combined them with adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NADH) regeneration systems commonly used in enzyme catalysis applications, thereby realizing the full enzyme synthesis of cordycepin.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a mutant of an enzyme comprising one or more of cordycepin kinase (Cor3K), cordycepin dephosphatase (CorDep), cordycepin reductase (CorRed), pyruvate kinase (PcPyK) and / or formate dehydrogenase (MspFDH);

[0009] The mutation sites of the cordycepin kinase (Cor3K) include one or more of R13A, M14L, W32T, G35F, D63S, G64M, D65Q, R68I, G70M, N72Y, N73K, I89V, Q157T or K180S;

[0010] The mutation site of the cordycepin dephosphatase (CorDep) includes one or more of K130N, E149D or A175V;

[0011] The mutation sites of the cordycepin reductase (CorRed) include one or more of S68M, E69G, E70F, R81V, K89S, P92W, F127N, G128I, T133V, R184A, H187T or N198D;

[0012] The mutation sites of the pyruvate kinase (PcPyK) include one or more of F31I, P99N, G105L, P169M, D248T or W312F;

[0013] The mutation sites of the formate dehydrogenase (MspFDH) include one or more of P12C, Q35T or Y188L.

[0014] In some specific embodiments of the present invention, in the mutant of the enzyme, the cordycepin kinase (Cor3K) is derived from Bacteroides thetaiotaomicron (Uniprot ID: Q8AAQ1, EC 2.7.1.25);

[0015] The cordycepin dephosphatase (CorDep) is derived from Cordyceps militaris (UniprotID: G3JF09);

[0016] The cordycepin reductase (CorRed) is derived from Thermococcus sibiricus (Uniprot ID: C6A1Z0);

[0017] The pyruvate kinase (PcPyK) is derived from Pyrobaculum calidifontis (Uniprot ID: A3MS52, EC 2.7.1.40);

[0018] The formate dehydrogenase (MspFDH) is derived from Microbacterium sp.

[0019] In some specific embodiments of the present invention, the enzyme mutants include mutants of cordycepin kinase (Cor3K), mutants of cordycepin dephosphatase (CorDep), mutants of cordycepin reductase (CorRed), mutants of pyruvate kinase (PcPyK) and / or mutants of formate dehydrogenase (MspFDH);

[0020] Wherein, the mutant of cordycepin kinase (Cor3K) has:

[0021] (I), the amino acid sequence shown in SEQ ID No. 1;

[0022] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0023] (III) an amino acid sequence that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of (I) or (II); or

[0024] The mutant of cordycepin dephosphatase (CorDep) has:

[0025] (I), the amino acid sequence shown in SEQ ID No. 2;

[0026] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0027] (III) an amino acid sequence that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of (I) or (II); or

[0028] The mutant of cordycepin reductase (CorRed) has:

[0029] (I), the amino acid sequence shown in SEQ ID No. 3;

[0030] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0031] (III) an amino acid sequence that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of (I) or (II); or

[0032] The mutant of pyruvate kinase (PcPyK) has:

[0033] (I), the amino acid sequence shown in SEQ ID No. 4;

[0034] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0035] (III) an amino acid sequence that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more identical to the amino acid sequence of (I) or (II); or

[0036] The mutant of formate dehydrogenase (MspFDH) has:

[0037] (I), the amino acid sequence shown in SEQ ID No. 5;

[0038] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or

[0039] (III) an amino acid sequence that is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to the amino acid sequence described in (I) or (II).

[0040] In a second aspect, the present invention also provides an enzyme composition or an immobilized enzyme thereof, including a mutant of the enzyme.

[0041] In a third aspect, the present invention also provides a nucleic acid molecule encoding a mutant of the enzyme or the enzyme composition or the immobilized enzyme thereof.

[0042] In some specific embodiments of the present invention, the nucleic acid molecule includes a nucleic acid molecule encoding a mutant of the cordycepin kinase (Cor3K), a nucleic acid molecule encoding a mutant of the cordycepin dephosphatase (CorDep), a nucleic acid molecule encoding a mutant of the cordycepin reductase (CorRed), a nucleic acid molecule encoding a mutant of the pyruvate kinase (PcPyK) and / or a nucleic acid molecule encoding a mutant of the formate dehydrogenase (MspFDH);

[0043] The nucleic acid molecule encoding the mutant of cordycepin kinase (Cor3K) has:

[0044] (I), the nucleotide sequence shown in SEQ ID NO.6; or

[0045] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0046] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0047] (IV) a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of any one of (I) to (III); or

[0048] The nucleic acid molecule encoding the mutant of cordycepin dephosphatase (CorDep) has:

[0049] (I), the nucleotide sequence shown in SEQ ID NO.7; or

[0050] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0051] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0052] (IV) a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of any one of (I) to (III); or

[0053] The nucleic acid molecule encoding the mutant of cordycepin reductase (CorRed) has:

[0054] (I), the nucleotide sequence shown in SEQ ID NO.8; or

[0055] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0056] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0057] (IV) a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of any one of (I) to (III); or

[0058] The nucleic acid molecule encoding the mutant of pyruvate kinase (PcPyK) has:

[0059] (I), the nucleotide sequence shown in SEQ ID NO.9; or

[0060] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0061] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0062] (IV) a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of any one of (I) to (III); or

[0063] The nucleic acid molecule encoding the mutant of formate dehydrogenase (MspFDH) has:

[0064] (I), the nucleotide sequence shown in SEQ ID NO.10; or

[0065] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0066] (III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0067] (IV) A nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of any one of (I) to (III).

[0068] In a fourth aspect, the present invention further provides an expression vector or host, wherein the expression vector comprises the nucleic acid molecule;

[0069] The host is transfected or transformed with the expression vector.

[0070] In a fifth aspect, the present invention further provides the use of any of the following in the preparation of cordycepin:

[0071] (I), a mutant of the enzyme;

[0072] (II), the enzyme composition or the immobilized enzyme thereof;

[0073] (III), the nucleic acid molecule; or

[0074] (IV), the expression vector or host.

[0075] In a sixth aspect, the present invention further provides a method for preparing cordycepin, wherein adenosine is used as a raw material and cordycepin is obtained by any of the following conversions:

[0076] (I), a mutant of the enzyme;

[0077] (II), the enzyme composition or the immobilized enzyme thereof;

[0078] (III), the nucleic acid molecule; or

[0079] (IV), the expression vector or host.

[0080] In some specific embodiments of the present invention, the preparation method specifically comprises using adenosine as a starting material, generating 3'-adenosine phosphate (3'-AMP) by the action of a mutant of the cordycepin kinase (Cor3K), then generating dehydrated adenosine by the action of a mutant of the cordycepin dephosphatase (CorDep), and finally generating cordycepin by conversion with a mutant of the cordycepin reductase (CorRed);

[0081] The enzymatic activity of the mutant of cordycepin kinase (Cor3K) is 800-1500, 1200-2000 U or 1400-1600 U; or

[0082] The enzymatic activity of the mutant of cordycepin dephosphatase (CorDep) is 1200-1800 U, 1800-2400 U or 800-1200 U; or

[0083] The enzymatic activity of the mutant of cordycepin reductase (CorRed) is 800-1200 U, 1500-2200 U or 900-1100 U; or

[0084] The enzyme activity of the mutant of pyruvate kinase (PcPyK) is 1800-3000 U or 1800-2400 U; or

[0085] The enzyme activity of the formate dehydrogenase (MspFDH) mutant is 2800-3500 or 1800-2400 U.

[0086] In some specific embodiments of the present invention, the ratio of raw materials to enzyme mutants is:

[0087] Adenosine: adenosine triphosphate disodium salt: the mutant of cordycepin kinase (Cor3K) = 100 mM: (90-120 mM): (800-1500 U).

[0088] In some specific embodiments of the present invention, the ratio of raw materials to enzyme mutants is:

[0089] Adenosine: adenosine triphosphate disodium salt: phosphoenolpyruvate (PEP): the mutant of the cordycepin kinase (Cor3K): the mutant of the pyruvate kinase (PcPyK) = 200 mM: (5-20 mM): (180-250 mM): (1200-2000 U): (1800-3000 U).

[0090] In some specific embodiments of the present invention, the ratio of raw materials to enzyme mutants is:

[0091] 3'-Adenosine disodium phosphate: reduced nicotinamide adenine dinucleotide disodium salt (NADH): the mutant of the cordycepin dephosphatase (CorDep): the mutant of the cordycepin reductase (CorRed) = 100 mM: (80-120 mM): (1200-1800 U): (800-1200 U).

[0092] In some specific embodiments of the present invention, the ratio of raw materials to enzyme mutants is:

[0093] 3'-Adenosine monophosphate disodium salt: reduced nicotinamide adenine dinucleotide disodium salt (NADH): the mutant of the cordycepin dephosphatase (CorDep): the mutant of the cordycepin reductase (CorRed): the mutant of the formate dehydrogenase (MspFDH) = 150 mM: (2-6 mM):

[0094] (1800~2400U): (1500~2200U): (2800~3500U).

[0095] In some specific embodiments of the present invention, cordycepin is prepared in one pot using adenosine as raw material and liquid enzymes (Cor3K, CorDep, CorRed), ATP regenerating enzyme (PcPyK) and NADH regenerating enzyme (MspFDH).

[0096] The ratio of raw materials to enzyme mutants is:

[0097] Adenosine: adenosine triphosphate disodium salt: phosphoenolpyruvate: reduced nicotinamide adenine dinucleotide disodium salt (NADH): mutant of the cordycepin kinase (Cor3K): mutant of the cordycepin dephosphatase (CorDep): mutant of the cordycepin reductase (CorRed): mutant of the pyruvate kinase (PcPyK): mutant of the formate dehydrogenase (MspFDH) = 100 mM: (2.0-6.0 mM): (90-130 mM): (3.0-6.0 mM): (1400-1600 U): (1800-2400 U): (800-1200 U): (900-1100 U): (1800-2400 U).

[0098] The present invention provides a full bioconversion of cordycepin. The overall process uses adenosine as the starting material, generates 3'-adenosine phosphate (3'-AMP) under the action of adenosine 3'-kinase (Cor3K), then uses cordycepin dephosphatase (CorDep) to generate dehydrated adenosine, and finally generates cordycepin under the conversion of cordycepin reductase (CorRed). This route is very simple, has a high conversion rate, and the raw materials used are also very cheap. This conversion can be completed step by step or in one pot. At the same time, for the coenzymes ATP and NADH required for the first and third steps of the conversion, pyruvate kinase (PcPyk) and formate dehydrogenase (MspFDH) can also be introduced for recycling and regeneration, thereby further reducing its production price. Therefore, the patented synthetic cordycepin preparation process has many advantages such as being green, cheap, and easy to scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0100] Figure 1 2. The SDS-PAGE gel detection diagram of the enzyme prepared by the present invention is shown;

[0101] Figure 2 1H-NMR of cordycepin is shown, with D2O as solvent and Varian 600 MHz NMR. DETAILED DESCRIPTION

[0102] The present invention discloses enzyme mutants, enzyme compositions, or immobilized enzymes thereof, and their applications and methods for preparing cordycepin. Those skilled in the art may refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and it is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0103] The present invention provides a full bioconversion of cordycepin. The overall process uses adenosine as the starting material, generates 3'-adenosine phosphate (3'-AMP) under the action of adenosine 3'-kinase (Cor3K), then uses cordycepin dephosphatase (CorDep) to generate dehydrated adenosine, and finally generates cordycepin under the conversion of cordycepin reductase (CorRed). This route is very simple, has a high conversion rate, and the raw materials used are also very cheap. This conversion can be completed step by step or in one pot. At the same time, for the coenzymes ATP and NADH required for the first and third steps of the conversion, pyruvate kinase (PcPyk) and formate dehydrogenase (MspFDH) can also be introduced for recycling and regeneration, thereby further reducing its production price. Therefore, the patented synthetic cordycepin preparation process has many advantages such as being green, cheap, and easy to scale.

[0104]

[0105] Information about the enzymes used above:

[0106] Table 1

[0107] English abbreviations Full English name Chinese meaning Cor3K Cordycepin kinase Cordycepin kinase CorDep Cordycepin Dephosphatase Cordycepin dephosphatase CorRed Cordycepin Reductase Cordycepin reductase PcPy Pc Pyruvate kinase Crenarchaea pyruvate kinase MspFDH Ms Formate dehydrogenase Microbacterium formate oxidase

[0108] Cordycepin kinase (Cor3K): Derived from Bacteroides thetaiotaomicron (Uniprot ID: Q8AAQ1, EC 2.7.1.25), the native enzyme (WTCor3K) initially reacts with adenosine sulfate and exhibits weak cordycepin kinase activity. Systematic mutagenesis of Cor3K has resulted in a highly potent cordycepin kinase. The mutations are: R13A, M14L, W32T, G35F, D63S, G64M, D65Q, R68I, G70M, N72Y, N73K, I89V, Q157T, K180S.

[0109] Cordycepin dephosphatase (CorDep): Derived from Cordyceps militaris (Uniprot ID: G3JF09), the native enzyme (WTCorDep) possesses this activity. Mutational modification (CorDep) has enhanced its expression and stability. Mutations are located at: K130N, E149D, and A175V.

[0110] Cordycepin reductase (CorRed): Derived from Thermococcus sibiricus (Uniprot ID: C6A1Z0), this native enzyme (WT CorRed) is weak at reducing cordycepin, but its expression and activity have been significantly improved through extensive mutational engineering (CorRed). The mutations are: S68M, E69G, E70F, R81V, K89S, P92W, F127N, G128I, T133V, R184A, H187T, N198D.

[0111] Pyruvate kinase (PcPyK): Derived from the archaeon Pyrobaculum calidifontis (Uniprot ID: A3MS52, EC 2.7.1.40), this native enzyme (WTPcPyK) can effectively regenerate adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP), but its expression and stability were poor. Through modification and screening (PcPyK), an enzyme with superior performance was obtained. The specific mutations are: F31I, P99N, G105L, P169M, D248T, and W312F.

[0112] Formate dehydrogenase (MspFDH): Derived from Microbacterium sp., this native enzyme (WTMspFDH) can effectively utilize formate to regenerate NADH (Uniprot ID: A0A0P0DRT5, EC 1.17.1.9). To further improve its expression and stability, it has been modified (MspFDH). Its mutation sites are: P12C, Q35T, Y188L.

[0113] Table 2

[0114]

[0115]

[0116] Table 3

[0117]

[0118]

[0119]

[0120]

[0121] The raw materials and reagents used in the enzyme mutant, enzyme composition or immobilized enzyme thereof and the application and method for preparing cordycepin provided by the present invention can be purchased from the market.

[0122] The present invention will be further described below in conjunction with the embodiments:

[0123] Preparation Example 1 Fermentation production of enzyme:

[0124] The enzymes used in this patent are all produced by in-house fermentation in the laboratory. The following is the basic process for preparing the enzyme. First, the gene sequence corresponding to the enzyme is synthesized by a genetic company (Anhui General Biotechnology). Then, it is subcloned into the pET28a plasmid using the NdeI / XhoI restriction sites. The plasmid is then transformed into E. coli (BL21) (Qingke Biotechnology) cells for plate culture. Finally, a single clone is selected for liquid amplification culture. The following is the basic process for cell amplification culture: First, a single colony on the plate is transferred to 5 ml of LB culture medium containing 50 μM kanamycin (37°C) for culture. When the cells reach the logarithmic phase, they are inoculated into 250 ml of LB culture medium containing the same antibiotic and finally transferred to a 5L culture fermenter for culture. When the cell OD reaches ~20, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added at 25°C to induce protein expression for 8 hours. Then, the wet cells are collected by centrifugation (4000 rpm, 15 minutes) to 30-50 g. To verify enzyme expression, a small amount of cells was first mixed with 50 mM Tris-HCl buffer (pH 8.0). Cells were then disrupted by freeze-thaw, centrifuged at high speed, and the supernatant was run on an SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel) to confirm soluble protein expression. The remaining cells, confirmed to be correct, were then mixed with buffer (10 g of wet cells in approximately 200 ml of commercially available buffer), followed by high-pressure cell disruption and high-speed centrifugation (16,000 rpm, 45 min) to remove cell walls. The resulting enzyme-containing supernatant was either used directly (the liquid enzyme activity ranged from 220 to 500 U / ml, where U is the amount of enzyme required to convert 1 μmol of substrate per minute at room temperature) or further purified and immobilized (for solid enzyme reactions). LB medium consisted of 1% tryptone, 0.5% yeast extract, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate, and 5% glycerol.

[0125] Table 4 Summary of enzyme properties covered by this patent

[0126]

[0127] Example 1: Preparation of 3'-adenosine phosphate using adenosine as raw material and liquid enzyme (Cor3K)

[0128]

[0129] To 1 L of 50 mM Tris-HCl (pH 8.0), add 26.7 g of adenosine (100 mM), 2.1 g of magnesium chloride hexahydrate (10 mM), 1.5 g of potassium chloride (20 mM), and 60.8 g of adenosine triphosphate disodium salt (110 mM). The pH of the reaction solution is then adjusted back to 8.0 using 0.1 N sodium hydroxide. Finally, 1000 U of Cor3K crude enzyme solution is added to initiate the reaction. During the reaction, sodium hydroxide is continuously added to maintain the pH between 7.0 and 8.5. The reaction is essentially complete after 3 hours. A dilute aqueous HCl solution was added to adjust the pH of the reaction solution to 2.0 to denature Cor3K and terminate the reaction. The pH was then adjusted back to 7.0, and D201 anion exchange resin (Tianjin Yunkai Resin Technology Co., Ltd.) was used to separate the product 3'-adenosine phosphate and the impurity adenosine diphosphate ADP. Finally, the product was desalted using a reverse osmosis membrane and concentrated to obtain 36 g of crude 3'-adenosine disodium phosphate (off-white solid, yield 92%, which can be directly used for the next step without purification).

[0130] Example 2 Preparation of 3'-adenosine phosphate using adenosine as raw material and liquid enzyme (Cor3K)

[0131]

[0132] To 1 L of 50 mM Tris-HCl (pH 8.0), add 26.7 g of adenosine (100 mM), 2.1 g of magnesium chloride hexahydrate (10 mM), 1.5 g of potassium chloride (20 mM), and 49.7 g of adenosine triphosphate disodium salt (90 mM). The pH of the reaction solution is then adjusted back to 8.0 using 0.1 N sodium hydroxide. Finally, 800 U of Cor3K crude enzyme solution is added to initiate the reaction. During the reaction, sodium hydroxide is continuously added to maintain the pH between 7.0 and 8.5. The reaction is essentially complete after 3 hours. Dilute HCl aqueous solution was added to adjust the pH of the reaction solution to 2.0 to denature Cor3K and terminate the reaction. The pH was then adjusted back to 7.0 and D201 anion exchange resin (Tianjin Yunkai Resin Technology Co., Ltd.) was used to separate the product 3'-adenosine phosphate and the impurity adenosine diphosphate ADP. Finally, the product was desalted using a reverse osmosis membrane and concentrated to obtain 34.8 g of crude 3'-adenosine disodium phosphate (off-white solid, yield 89%, the solid could be directly used for the next reaction without purification).

[0133] Example 3 Preparation of 3'-adenosine phosphate using adenosine as raw material and liquid enzyme (Cor3K)

[0134]

[0135] To 1 L of 50 mM Tris-HCl (pH 8.0), add 26.7 g of adenosine (100 mM), 2.1 g of magnesium chloride hexahydrate (10 mM), 1.5 g of potassium chloride (20 mM), and 66.3 g of adenosine triphosphate disodium salt (120 mM). The pH of the reaction solution is then adjusted back to 8.0 using 0.1 N sodium hydroxide. Finally, 1500 U of Cor3K crude enzyme solution is added to initiate the reaction. During the reaction, sodium hydroxide is continuously added to maintain the pH between 7.0 and 8.5. The reaction is essentially complete after 3 hours. A dilute aqueous HCl solution was added to adjust the pH of the reaction solution to 2.0 to denature Cor3K and terminate the reaction. The pH was then adjusted back to 7.0, and D201 anion exchange resin (Tianjin Yunkai Resin Technology Co., Ltd.) was used to separate the product 3'-adenosine phosphate and the impurity adenosine diphosphate ADP. Finally, the product was desalted using a reverse osmosis membrane and concentrated to obtain 36.8 g of crude 3'-adenosine disodium phosphate (off-white solid, yield 94%, the solid could be directly used for the next step without purification).

[0136] Example 4: Preparation of 3'-adenosine phosphate using adenosine as raw material, liquid enzyme (Cor3K) and ATP regenerating enzyme (PcPyK)

[0137]

[0138] Similar to Example 1, 53.4 g adenosine (200 mM), 4.2 g magnesium chloride hexahydrate (20 mM), 3.8 g potassium chloride (50 mM), 5.5 g adenosine triphosphate disodium salt (10 mM), and 39.9 g phosphoenolpyruvate (PEP, 210 mM) were added to 1 L of 50 mM Tris-HCl solution (pH 8.0). The pH of the reaction solution was then adjusted back to 8.0 using 0.1 N sodium hydroxide solution. Finally, 1500 U of crude Cor3K enzyme solution and 2000 U of crude PcPyK enzyme solution were added to initiate the reaction. During the reaction, sodium hydroxide solution was continuously added to maintain the pH between 7.0 and 8.5. The reaction was substantially complete after 4 hours. The pH of the reaction solution was adjusted to 2.0 by adding dilute HCl solution to denature the Cor3K and PcPyK enzymes, thereby terminating the reaction. The pH was then adjusted back to 7.0. 54g of barium oxalate (240mM) was added to the reaction solution to precipitate the phosphate-containing compound, and the precipitated solid was then collected by centrifugation and dissolved in a pH 1.0 HCl aqueous solution. 35.5g of anhydrous sodium sulfate (250mM) was then added to precipitate the barium ions in the solution, and the solid BaSO4 was removed by centrifugation. Finally, the pH value of the solution was adjusted back to 7.0, and then the solution was desalted using a reverse osmosis membrane and concentrated to obtain 68g of 3'-adenosine disodium phosphate (white solid, yield 87%, the solid can be directly used for the next reaction without purification). Example 5 Preparation of 3'-adenosine phosphate using adenosine as a raw material, liquid enzyme (Cor3K) and ATP regeneration enzyme (PcPyK)

[0139]

[0140] Similar to Example 2, 53.4 g adenosine (200 mM), 4.2 g magnesium chloride hexahydrate (20 mM), 3.8 g potassium chloride (50 mM), 2.8 g adenosine triphosphate disodium salt (5 mM), and 34.2 g phosphoenolpyruvate (PEP, 180 mM) were added to 1 L of 50 mM Tris-HCl solution (pH 8.0). The pH of the reaction solution was then adjusted back to 8.0 using 0.1 N sodium hydroxide solution. Finally, 1200 U of crude Cor3K enzyme solution and 1800 U of crude PcPyK enzyme solution were added to initiate the reaction. During the reaction, sodium hydroxide solution was continuously added to maintain the pH between 7.0 and 8.5. The reaction was substantially complete after 4 hours. The pH of the reaction solution was adjusted to 2.0 by adding dilute HCl solution to denature the Cor3K and PcPyK enzymes, thereby terminating the reaction. The pH was then adjusted back to 7.0. 5.4g of barium oxalate (240mM) was added to the reaction solution to precipitate the phosphate-containing compound. The precipitated solid was then collected by centrifugation and dissolved in a pH 1.0 aqueous HCl solution. 28.4g of anhydrous sodium sulfate (200mM) was then added to precipitate the barium ions in the solution, and the solid BaSO4 was removed by centrifugation. Finally, the solution pH was adjusted back to 7.0, and the salt was removed using a reverse osmosis membrane and concentrated to yield 64g of 3'-phosphated adenosine disodium salt (a white solid, 82% yield). This solid was carried directly to the next reaction without purification.

[0141] Example 6: Preparation of 3'-adenosine phosphate using adenosine as raw material, liquid enzyme (Cor3K) and ATP regenerating enzyme (PcPyK)

[0142]

[0143] Similar to Example 3, 53.4 g adenosine (200 mM), 4.2 g magnesium chloride hexahydrate (20 mM), 3.8 g potassium chloride (50 mM), 11 g adenosine triphosphate disodium salt (20 mM), and 47.5 g phosphoenolpyruvate (PEP, 250 mM) were added to 1 L of 50 mM Tris-HCl solution (pH 8.0). The pH of the reaction solution was then adjusted back to 8.0 using 0.1 N sodium hydroxide solution. Finally, 2000 U of crude Cor3K enzyme solution and 3000 U of crude PcPyK enzyme solution were added to initiate the reaction. During the reaction, sodium hydroxide solution was continuously added to maintain the pH between 7.0 and 8.5. The reaction was substantially complete after 4 hours. The pH of the reaction solution was adjusted to 2.0 by adding dilute HCl solution to denature the Cor3K and PcPyK enzymes, thereby terminating the reaction. The pH was then adjusted back to 7.0. 54g of barium oxalate (240mM) was added to the reaction solution to precipitate the phosphate-containing compound. The precipitated solid was then collected by centrifugation and dissolved in a pH 1.0 aqueous HCl solution. 42.6g of anhydrous sodium sulfate (300mM) was then added to precipitate the barium ions in the solution, and the solid BaSO4 was removed by centrifugation. Finally, the solution pH was adjusted back to 7.0, and the salt was removed using a reverse osmosis membrane and concentrated to yield 70g of 3'-adenosine disodium phosphate (a white solid, 90% yield). This solid was carried directly to the next reaction without purification.

[0144] Example 7 Preparation of cordycepin using liquid enzymes (CorDep, CorRed) using 3'-adenosine phosphate as raw material

[0145]

[0146] To 1 L of 100 mM Tris-HCl (pH 7.0) solution, 39.1 g of 100 mM adenosine 3'-phosphate disodium salt (3'-phosphate) and 78.1 g of reduced nicotinamide adenine dinucleotide disodium salt (110 mM NADH) were added. The pH of the reaction solution was then adjusted back to 7.0. Finally, 1500 U of CorDep crude enzyme solution and 1000 U of CorRed crude enzyme solution were added to initiate the reaction. The pH was adjusted between 6.5 and 8.0 during the reaction. The reaction was complete after 3 hours. The pH of the reaction solution was adjusted to acidic to terminate the reaction. The pH was then returned to 7.0 and loaded onto D201 anion exchange resin to remove phosphate-containing impurities. The cordycepin solution was then desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to yield 20.8 g of cordycepin (white solid, 83% yield).

[0147] Example 8 Preparation of cordycepin using liquid enzymes (CorDep, CorRed) using 3'-adenosine phosphate as raw material

[0148]

[0149] To 1 L of 100 mM Tris-HCl (pH 7.0) solution, 39.1 g of 100 mM adenosine 3'-phosphate disodium salt (3'-phosphate) and 56.8 g of reduced nicotinamide adenine dinucleotide disodium salt (NADH, 80 mM) were added. The reaction mixture was then adjusted back to pH 7.0. Finally, 1200 U of CorDep crude enzyme solution and 800 U of CorRed crude enzyme solution were added to initiate the reaction. The pH was maintained between 6.5 and 8.0 during the reaction, and the reaction was complete after 3 hours. The pH of the reaction mixture was adjusted to acidic to terminate the reaction, and then the pH was returned to 7.0. D201 anion exchange resin was then applied to remove phosphate-containing impurities. Finally, the cordycepin-containing solution was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to yield 19.5 g of cordycepin (white solid, 75% yield).

[0150] Example 9 Preparation of cordycepin using liquid enzymes (CorDep, CorRed) using 3'-adenosine phosphate as raw material

[0151]

[0152] To 1 L of 100 mM Tris-HCl (pH 7.0) solution, 39.1 g of 100 mM adenosine 3'-phosphate disodium salt (3'-phosphate) and 85.2 g of reduced nicotinamide adenine dinucleotide disodium salt (NADH, 120 mM) were added. The reaction mixture was then adjusted back to pH 7.0. Finally, 1800 U of CorDep crude enzyme solution and 1200 U of CorRed crude enzyme solution were added to initiate the reaction. The pH was maintained between 6.5 and 8.0 during the reaction, and the reaction was complete after 3 hours. The pH of the reaction mixture was adjusted to acidic to terminate the reaction, and then the pH was returned to 7.0. D201 anion exchange resin was then applied to remove phosphate-containing impurities. The cordycepin solution was then desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to yield 21.3 g of cordycepin (white solid, 85% yield).

[0153] Example 10 Preparation of cordycepin using liquid enzymes (CorDep, CorRed) and NADH regenerating enzyme (MspFDH) using 3'-adenosine phosphate as raw material

[0154]

[0155] Similar to Example 7, 58.6 g of 150 mM adenosine 3'-phosphate disodium salt, 3.6 g of 5 mM nicotinamide adenine dinucleotide (NADH), and 11 g of 160 mM sodium formate were added to 1 L of a 100 mM Tris-HCl solution (pH 7.5). The pH of the reaction solution was then adjusted back to 7.5. Finally, 2000 U of crude CorDep enzyme solution, 2000 U of crude CorRed enzyme solution, and 3000 U of crude MspFDH enzyme solution were added to initiate the reaction. The pH was maintained between 7.0 and 8.5 during the reaction, and the reaction was complete after 4 hours. The pH of the reaction solution was adjusted to acidic to terminate the reaction, and then the pH of the solution was adjusted back to 7.0 and loaded onto D201 anion exchange resin to remove phosphoric acid-containing impurities. Finally, the cordycepin-containing solution was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to obtain 34.2 g of cordycepin (white solid, 91% yield).

[0156] Example 11 Preparation of cordycepin using 3'-adenosine phosphate as raw material using liquid enzymes (CorDep, CorRed) and NADH regenerating enzyme (MspFDH)

[0157]

[0158] Similar to Example 8, 58.6 g of 150 mM adenosine 3'-phosphate disodium salt, 1.4 g of reduced nicotinamide adenine dinucleotide disodium salt (NADH, 2 mM), and 11 g of 160 mM sodium formate were added to 1 L of a 100 mM Tris-HCl solution (pH 7.5). The pH of the reaction solution was then adjusted back to 7.5. Finally, 1800 U of CorDep crude enzyme solution, 1500 U of CorRed crude enzyme solution, and 2800 U of SpFDH crude enzyme solution were added to initiate the reaction. The pH was maintained between 7.0 and 8.5 during the reaction, and the reaction was complete after 4 hours. The pH of the reaction solution was adjusted to acidic to terminate the reaction, and then the pH of the solution was adjusted back to 7.0 and loaded onto D201 anion exchange resin to remove phosphoric acid impurities. Finally, the cordycepin-containing solution was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to obtain 32.7-35 g of cordycepin (white solid, 87% yield). Example 12: Cordycepin was prepared using liquid enzymes (CorDep, CorRed) and NADH regenerating enzyme (MspFDH) using 3'-adenosine monophosphate as the starting material.

[0159]

[0160] Similar to Example 9 above, 58.6 g of adenosine 3'-phosphate disodium salt (150 mM), 4.3 g of reduced nicotinamide adenine dinucleotide disodium salt (NADH, 6 mM), and 11 g of sodium formate (160 mM) were added to 1 L of a 100 mM Tris-HCl solution (pH 7.5). The pH of the reaction solution was then adjusted back to 7.5. Finally, 2400 U of CorDep crude enzyme solution, 2200 U of CorRed crude enzyme solution, and 3500 U of SpFDH crude enzyme solution were added to initiate the reaction. The pH was maintained between 7.0 and 8.5 during the reaction, and the reaction was complete after 4 hours. The pH of the reaction solution was adjusted to acidic to terminate the reaction, and then the pH of the solution was adjusted back to 7.0 and loaded onto D201 anion exchange resin to remove phosphoric acid-containing impurities. Finally, the cordycepin-containing solution was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to obtain 35 g of cordycepin (white solid, 93% yield).

[0161] Example 13: Preparation of cordycepin using adenosine as raw material using liquid enzymes (Cor3K, CorDep, CorRed) and ATP regenerating enzyme (PcPyK) and NADH regenerating enzyme (MspFDH) in a one-pot process

[0162]

[0163] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, add 26.7 g adenosine (100 mM), 2.1 g magnesium chloride hexahydrate (10 mM), 1.5 g potassium chloride (20 mM), 2.8 g adenosine triphosphate disodium salt (5 mM), 20.9 g phosphoenolpyruvate (PEP, 110 mM), 3.6 g nicotinamide adenine dinucleotide disodium salt (NADH, 5 mM), and 7.6 g sodium formate (110 mM). The pH of the solution was adjusted back to 8.0. Finally, 1500 U Cor3K, 2000 U PcPyK, 1000 U CorDep, 1000 U CorRed, and 2000 U MspFDH enzyme were added sequentially to initiate the reaction. The pH was maintained between 7.0 and 8.5 during the reaction, and the reaction was essentially complete after 4 hours. The reaction solution was then acidified to terminate the reaction, then adjusted back to pH 7.0 and separated using D201 anion exchange resin to remove phosphate-containing impurities. The cordycepin solution was then desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to yield 18.3 g of cordycepin (white solid, 73% yield).

[0164] Example 14: Preparation of cordycepin using adenosine as raw material using liquid enzymes (Cor3K, CorDep, CorRed) and ATP regenerating enzyme (PcPyK) and NADH regenerating enzyme (MspFDH) in a one-pot process

[0165]

[0166] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, add 26.7 g adenosine (100 mM), 2.1 g magnesium chloride hexahydrate (10 mM), 1.5 g potassium chloride (20 mM), 1.1 g adenosine triphosphate disodium salt (2.0 mM), 17.1 g phosphoenolpyruvate (PEP, 90 mM), 2.2 g nicotinamide adenine dinucleotide disodium salt (NADH, 3.0 mM), and 7.6 g sodium formate (110 mM). The pH of the solution was adjusted back to 8.0. Finally, 1400 U Cor3K, 1800 U PcPyK, 800 U CorDep, 900 U CorRed, and 1800 U MspFDH enzyme were added sequentially to initiate the reaction. The pH was maintained between 7.0 and 8.5 during the reaction, and the reaction was essentially complete after 4 hours. The reaction solution was then acidified to terminate the reaction, and then adjusted back to pH 7.0. Phosphoric acid-containing impurities were removed using D201 anion exchange resin. Finally, the cordycepin-containing solution was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to yield 17.5 g of cordycepin (white solid, 70% yield).

[0167] Example 15: Cordycepin was prepared in one pot using adenosine as raw material using liquid enzymes (Cor3K, CorDep, CorRed), ATP regenerating enzyme (PcPyK), and NADH regenerating enzyme (MspFDH).

[0168]

[0169] To 1 L of 100 mM Tris-HCl (pH 8.0) solution, add 26.7 g adenosine (100 mM), 2.1 g magnesium chloride hexahydrate (10 mM), 1.5 g potassium chloride (20 mM), 3.4 g adenosine triphosphate disodium salt (6.0 mM), 24.7 g phosphoenolpyruvate (PEP, 130 mM), 4.3 g nicotinamide adenine dinucleotide disodium salt (NADH, 6.0 mM), and 7.6 g sodium formate (110 mM). The pH of the solution was adjusted back to 8.0. Finally, 1600 U Cor3K, 2400 U PcPyK, 1200 U CorDep, 1100 U CorRed, and 2400 U MspFDH enzyme were added sequentially to initiate the reaction. The pH was maintained between 7.0 and 8.5 during the reaction, and the reaction was essentially complete after 4 hours. The reaction solution was then acidified to terminate the reaction, then adjusted back to pH 7.0 and separated using D201 anion exchange resin to remove phosphate-containing impurities. The cordycepin solution was then desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol:water, 2:1, v:v) to yield 18.8 g of cordycepin (white solid, 75% yield).

[0170] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A combination of enzyme mutants, characterized in that The enzyme mutants include a mutant of cordycepin kinase (Cor3K), a mutant of cordycepin dephosphatase (CorDep), and a mutant of cordycepin reductase (CorRed); Wherein, the amino acid sequence of the mutant of cordycepin kinase (Cor3K) is shown in SEQ ID No. 1; The amino acid sequence of the mutant of cordycepin dephosphatase (CorDep) is shown in SEQ ID No. 2; The amino acid sequence of the mutant of cordycepin reductase (CorRed) is shown in SEQ ID No.

3.

2. The combination of mutants according to claim 1, characterized in that Also included are mutants of pyruvate kinase (PcPyK) and formate dehydrogenase (MspFDH); The amino acid sequence of the mutant of pyruvate kinase (PcPyK) is shown in SEQ ID No. 4; The amino acid sequence of the mutant of formate dehydrogenase (MspFDH) is shown in SEQ ID No.

5.

3. An enzyme composition or an immobilized enzyme thereof, characterized in that A combination comprising mutants of the enzyme according to any one of claims 1 and 2.

4. A nucleic acid molecule encoding a combination of mutants of the enzyme according to claim 1 or 2 or an enzyme composition according to claim 3 or an immobilized enzyme thereof.

5. The nucleic acid molecule according to claim 4, wherein The nucleotide sequence of the nucleic acid molecule encoding the mutant of cordycepin kinase (Cor3K) is shown in SEQ ID NO.6; The nucleotide sequence of the nucleic acid molecule encoding the mutant of cordycepin dephosphatase (CorDep) is shown in SEQ ID NO.7; The nucleotide sequence of the nucleic acid molecule encoding the mutant of cordycepin reductase (CorRed) is shown in SEQ ID NO.8; The nucleotide sequence of the nucleic acid molecule encoding the mutant of pyruvate kinase (PcPyK) is shown in SEQ ID NO.9; The nucleotide sequence of the nucleic acid molecule encoding the mutant of formate dehydrogenase (MspFDH) is shown in SEQ ID NO.

10.

6. An expression vector or host, characterized in that The expression vector comprises the nucleic acid molecule according to claim 4 or 5; The host is transfected or transformed with the expression vector.

7. Use of any of the following in the preparation of cordycepin; (I) A combination of mutants of the enzyme according to claim 1 or 2; (II) The enzyme composition or immobilized enzyme thereof according to claim 3; (III), the nucleic acid molecule according to claim 4 or 5; or (IV) The expression vector or host according to claim 6.

8. A method for preparing cordycepin, characterized in that: Cordycepin can be obtained by using adenosine as raw material through any of the following transformations: (I) A combination of mutants of the enzyme according to claim 1 or 2; (II) The enzyme composition or immobilized enzyme thereof according to claim 3; (III), the nucleic acid molecule according to claim 4 or 5; or (IV) The expression vector or host according to claim 6.

9. The preparation method according to claim 8, wherein Adenosine is used as a starting material, 3'-adenosine phosphate (3'-AMP) is generated under the action of the mutant of the cordycepin kinase (Cor3K), dehydrated adenosine is generated using the mutant of the cordycepin dephosphatase (CorDep), and finally cordycepin is generated under the conversion of the mutant of the cordycepin reductase (CorRed); The enzymatic activity of the mutant of cordycepin kinase (Cor3K) is 800-1500 U, 1200-2000 U, or 1400-1600 U; The enzymatic activity of the mutant of cordycepin dephosphatase (CorDep) is 1200-1800 U, 1800-2400 U, or 800-1200 U; The enzymatic activity of the mutant of cordycepin reductase (CorRed) is 800-1200 U, 1500-2200 U, or 900-1100 U; The enzyme activity of the pyruvate kinase (PcPyK) mutant is 1800-3000 U or 1800-2400 U; The enzyme activity of the formate dehydrogenase (MspFDH) mutant is 2800-3500 U or 1800-2400 U.

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