Enzyme composition, preparation method and application

Abstract

The invention discloses an enzyme composition, a preparation method and application, the enzyme composition comprises deoxynucleotide kinase and polyphosphate kinase, the enzyme composition is prepared by mixing after expression, extraction and purification of recombinant microorganisms, and the enzyme composition can be used for synthesizing deoxycytidine triphosphate. The enzyme composition is simple to prepare, a cyclic regeneration system is introduced when the enzyme composition is used for synthesizing deoxycytidine triphosphate, the adding amount of initial deoxycytidine triphosphate is small, and the cost is remarkably reduced; the reaction system generates few impurities, and the product is easy to purify; compared with the existing method, the yield is obviously improved and can reach 68.5%; reaction conditions are mild, and the process is simple and environment-friendly.

Description

Technical Field

[0001] The present invention relates to enzymes, and particularly to an enzyme composition, a preparation method and applications thereof. Background Art

[0002] Deoxycytidine triphosphate (dCTP) is one of the essential raw materials in the processes of DNA replication and repair. It acts synergistically with deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP) to synthesize new DNA strands by polymerase according to the base complementary pairing principle. In addition, dCTP can also bind to biotin to form an important tool molecule in synthetic biology, which is used for the modification and synthesis of DNA or RNA in vitro and in vivo, and serves as a cofactor of ligase under specific circumstances, showing broad application potential.

[0003] Traditional methods for preparing dCTP mainly include nucleic acid degradation, chemical synthesis and microbial fermentation. These methods have deficiencies in terms of efficiency, cost and environmental sustainability. In recent years, enzymatic synthesis methods have received attention, especially the technology based on kinase catalysis has been proven to achieve efficient synthesis of dCTP. For example, for the all-enzymatic synthesis technology of all deoxynucleoside triphosphates (dNTPs), deoxynucleoside triphosphates are synthesized starting from deoxynucleoside monophosphate through two consecutive enzymatic phosphorylation reactions, and the whole process is completed in a bioreactor. Specifically, first, nucleotide kinase is used to convert deoxynucleoside monophosphate into deoxynucleoside diphosphate, and then deoxynucleoside diphosphate is further converted into deoxynucleoside triphosphate by acetate kinase. The traditional way of kinase-catalyzed dNTP synthesis mimics the in vivo metabolic process in vitro, and its phosphorylation reaction requires a large amount of phosphate donors (such as nucleoside triphosphates). However, nucleoside triphosphates are expensive and unstable in nature, and are easily decomposed into nucleoside diphosphates, which not only increases the reaction cost but also interferes with product separation. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide an enzyme composition and a preparation method that can be used for synthesizing deoxycytidine triphosphate; the second object is to provide the application of the enzyme composition in the synthesis of deoxycytidine triphosphate.

[0005] Technical Solution: The enzyme composition described in the present invention includes deoxynucleotide kinase and polyphosphate kinase, and the molar ratio is 9 - 11:1.

[0006] Preferably, the deoxynucleotide kinase has the amino acid sequence shown in SEQ ID NO: 1, and the polyphosphate kinase has the amino acid sequence shown in SEQ ID NO: 2.

[0007] The preparation method of the enzyme composition described in the present invention includes:

[0008] (1) Construct a recombinant vector for expressing deoxynucleotide kinase or polyphosphate kinase;

[0009] (2) Prepare a recombinant microorganism containing the recombinant vector obtained in step (1);

[0010] (3) Culture the recombinant microorganism obtained in step (2) to express, extract and purify deoxynucleotide kinase and polyphosphate kinase;

[0011] (4) Mix the deoxynucleotide kinase and polyphosphate kinase obtained in step (3).

[0012] Use of the enzyme composition described in the present invention in the synthesis of deoxycytidine triphosphate.

[0013] Preferably, the use of the enzyme composition in the synthesis of deoxycytidine triphosphate includes:

[0014] (1) Mix the enzyme composition according to any one of claims 1-3 with deoxycytidine, deoxycytidine triphosphate, metal cofactor, phosphate donor and tris(hydroxymethyl)aminomethane hydrochloride buffer;

[0015] (2) Carry out an enzymatic reaction to synthesize deoxycytidine triphosphate.

[0016] Preferably, the mass ratio of deoxycytidine to deoxycytidine triphosphate is 10-50:1; the molar ratio of deoxycytidine to deoxynucleotide kinase or polyphosphate kinase is 3-7:1; the metal cofactor is MgCl2 with a concentration of 20-100 mM; the phosphate donor is any one of sodium pyrophosphate, sodium trimetaphosphate, sodium hexametaphosphate, sodium polyphosphate with a concentration of 20-60 mM; the temperature of the enzymatic reaction is 30-45 °C, the pH is 7.0-9.5, and the time is 12-36 h.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. The preparation of the enzyme composition is simple; 2. By introducing a deoxycytidine triphosphate cycle regeneration system, the required starting amount of deoxycytidine triphosphate added is small, and the cost is significantly reduced; 3. Through the deoxycytidine triphosphate cycle regeneration, fewer impurities are generated in the reaction system, and the product is easy to purify; 4. The yield is significantly increased compared with the existing method, up to 68.5%; 5. The reaction conditions are mild, the process is simple, and it is environmentally friendly. Description of the Drawings

[0018] Figure 1 It is a diagram of the catalytic efficiency of the deoxynucleotide kinase and polyphosphate kinase composition at different temperatures;

[0019] Figure 2 It is a diagram of the catalytic efficiency of the deoxynucleotide kinase and polyphosphate kinase composition at different pH values;

[0020] Figure 3 Catalytic efficiency diagrams of the deoxynucleotide kinase and polyphosphate kinase compositions at different MgCl2 concentrations;

[0021] Figure 4 Catalytic efficiency diagrams of the deoxynucleotide kinase and polyphosphate kinase compositions at different polyP6 concentrations;

[0022] Figure 5 dCTP yield diagrams under different molar ratios of deoxycytidine to dCTP;

[0023] Figure 6 Diagrams showing the effects of different phosphate donors on the dCTP yield;

[0024] Figure 7 HPLC peak diagram of the dCTP standard;

[0025] Figure 8 HPLC peak diagram of the total product of the synthesis of dCTP by the deoxynucleotide kinase and polyphosphate kinase compositions. Detailed implementation manners

[0026] The technical solutions of the present invention will be further described below.

[0027] Example 1: Preparation of the deoxynucleotide kinase and polyphosphate kinase compositions

[0028] 1. Vector construction and cloning: The nucleotide sequences encoding deoxynucleotide kinase and polyphosphate kinase were respectively ligated to the vector pET28a by seamless cloning to obtain deoxynucleotide kinase-pET28a and polyphosphate kinase-pET28a. The following operations were performed on the aforementioned ligated vectors: Take 10 μl of the ligation product and add it to 100 μl of ice-bathed E. coli BL21(DE3) competent cells, then incubate on ice for 30 min, heat shock at 42 °C for 60 s, then incubate on ice for 5 min. Add 300 μl of antibiotic-free LB culture medium at 37 °C to the tube, resuscitate on a shaker at 37 °C and 200 rpm for 1 h, and then spread it on a solid LB plate supplemented with kanamycin. After culturing at 37 °C, pick single colonies;

[0029] 2. Verification: The picked single colonies were added to a 20 μl PCR Mix system containing T7 universal primers for PCR amplification. The PCR reaction conditions were: incubate at 95 °C for 15 min, denature at 94 °C for 15 s, anneal at 55 °C for 15 s, extend at 72 °C for 1 min, perform 30 cycles, and finally extend at 72 °C for 5 min. After PCR amplification, electrophoresis was performed to observe the results, and positive clones were confirmed to obtain E. coli strains containing the target enzyme sequences: deoxynucleotide kinase-E. coli and polyphosphate kinase-E. coli;

[0030] 3. Enzyme expression and extraction: The obtained Escherichia coli was respectively added to LB medium supplemented with kanamycin and cultured at 37°C until OD 600 = 0.7, then IPTG with a final concentration of 0.5 mM was added. Among them, the Escherichia coli containing the deoxynucleotide kinase sequence was induced to express at 16°C for 16 h, and the Escherichia coli containing the polyphosphate kinase sequence was induced to express at 20°C for 16 h. Then the bacterial solution was centrifuged at 5000 rpm for 10 min to collect the bacterial cells. After discarding the supernatant, the bacterial cells were resuspended according to 5 mL of pH 7.5, 50 mM Tris-HCl solution per gram of bacterial cells. The resuspended bacterial cells were disrupted by a high-pressure cell disruptor to obtain an enzyme-containing lysate. After centrifugation at 12500 rpm for 1 h, the supernatant was extracted to obtain a crude enzyme solution containing deoxynucleotide kinase and polyphosphate kinase, which was stored at 4°C for later use;

[0031] 4. The crude enzyme solutions of deoxynucleotide kinase and polyphosphate kinase obtained in step 3 were respectively subjected to affinity chromatography through Ni-chelate affinity agarose, and gradient elution was carried out using imidazole; The eluate of the pure enzyme was collected, transferred to a dialysis bag, and dialyzed in 50 mM Tris-HCl solution with pH 7.5, and the dialyzed protein sample was collected;

[0032] 5. Deoxynucleotide kinase and polyphosphate kinase were mixed at a molar ratio of 10:1 to obtain a deoxynucleotide kinase and polyphosphate kinase composition.

[0033] Example 2: Use of the deoxynucleotide kinase and polyphosphate kinase composition for the preparation of deoxycytidine triphosphate

[0034] 1. Optimal reaction temperature of the deoxynucleotide kinase and polyphosphate kinase composition

[0035] The deoxynucleotide kinase and polyphosphate kinase composition solution obtained in Example 1 was mixed with 10 mM deoxycytidine, 1 mM deoxycytidine triphosphate, 20 mM MgCl2 solution, 20 mM sodium hexametaphosphate (polyP6) solution and Tris-HCl buffer, and reacted at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, pH 7.5 for 36 h. The results are as Figure 1 shown. The yield of dCTP gradually increased with the increase of temperature. At 35°C, the yield reached 4.34 mM. Subsequently, when the temperature was further increased, the yield began to decrease.

[0036] 2. Optimal reaction pH of the deoxynucleotide kinase and polyphosphate kinase composition

[0037] The deoxynucleotide kinase and polyphosphate kinase composition solution obtained in Example 1 was mixed with 10 mM deoxycytidine, 1 mM deoxycytidine triphosphate, 20 mM MgCl2 solution, 20 mM polyP6 solution, and Tris-HCl buffer. At 35 °C, the pH values were 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 respectively, and the reaction was carried out for 36 h. The results are as Figure 2 shown. In the range of pH 6.0 - 7.0, the generation of dCTP was relatively low. As the pH increased to 9.0, the catalytic effect was the best, and the production amount of dCTP was 4.95 mM. Subsequently, as the pH value continued to increase, the yield gradually decreased.

[0038] 3. Optimal MgCl2 concentration of the deoxynucleotide kinase and polyphosphate kinase composition

[0039] The deoxynucleotide kinase and polyphosphate kinase composition solution obtained in Example 1 was mixed with 10 mM deoxycytidine, 1 mM deoxycytidine triphosphate, 20 mM polyP6 solution, and Tris-HCl buffer, and MgCl2 solutions with concentrations of 5 mM, 10 mM, 25 mM, 40 mM, 70 mM, 100 mM, and 200 mM were added respectively. The reaction was carried out at 35 °C and pH 9.0 for 36 h. The results are as Figure 3 shown. The yield of dCTP increased with the increase of Mg 2+ concentration. When the Mg 2+ concentration was 70 mM, the yield reached the maximum, which was 38.7%.

[0040] 4. Optimal PolyP6 concentration of the deoxynucleotide kinase and polyphosphate kinase composition

[0041] The deoxynucleotide kinase and polyphosphate kinase composition solution obtained in Example 1 was mixed with 10 mM deoxycytidine, 1 mM deoxycytidine triphosphate, 70 mM MgCl2 solution, and Tris-HCl buffer, and polyP6 solutions with concentrations of 0 mM, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, and 150 mM were added respectively. The reaction was carried out at 35 °C and pH 9.0 for 36 h. The results are as Figure 4 shown. When there was no PolyP6 present, almost no dCTP was produced. As the concentration of PolyP6 increased, the yield also increased. When the concentration of PolyP6 was 40 mM, the yield reached the maximum, which was 42.8%.

[0042] 5. Optimal deoxycytidine - deoxycytidine triphosphate ratio of the deoxynucleotide kinase and polyphosphate kinase composition

[0043] The solution of the deoxynucleotide kinase and polyphosphate kinase composition obtained in Example 1 was mixed with 1 mM deoxycytidine triphosphate, 70 mM MgCl2 solution, 40 mM polyP6 solution and Tris-HCl buffer, and deoxycytidine with concentrations of 10, 20, 30, 40, 50 mM was added respectively. The reaction was carried out at 35 °C and pH 9.0 for 36 h. The results are as Figure 5 shown. When the molar ratio of deoxycytidine (dC) to deoxycytidine triphosphate (dCTP) was 10:1, the yield of dCTP reached 58%. Subsequently, as the molar ratio increased, the yield of dCTP decreased.

[0044] 6. Optimal phosphorus donor of deoxynucleotide kinase and polyphosphate kinase composition

[0045] The pure enzyme solution of the deoxynucleotide kinase and polyphosphate kinase composition obtained in Example 1 was mixed with 10 mM deoxycytidine, 1 mM deoxycytidine triphosphate, 70 mM MgCl2 solution and Tris-HCl buffer, and 40 mM sodium pyrophosphate (NPP), or sodium trimetaphosphate (polyP3), or sodium hexametaphosphate (polyP6), or sodium polyphosphate (polyPn) was added respectively. The reaction was carried out at 35 °C and pH 9.0 for 36 h. The results are as Figure 6 shown. When the phosphorus donor was polyPn, the production amount of dCTP could reach 6.85 mM, and the yield was 68.5%. This indicates that the deoxynucleotide kinase and polyphosphate kinase composition prefers to utilize long-chain polyphosphates, and has a lower utilization rate for short-chain polyphosphates.

[0046] The total products after the reaction were analyzed by HPLC. Combining with the HPLC peak pattern of the dCTP standard product as Figure 7 shown, the results are as Figure 8 shown. The main component in the total products was dCTP, and the resolution was relatively high, indicating that there were few impurities generated in the reaction system and the product was easy to purify.

[0047] Comparative Example 1 Synthesis of deoxycytidine triphosphate by nucleotide kinase and acetate kinase composition

[0048] 1. Referring to the method provided in Example 1, cytidylate kinase was prepared according to the NCBI reference sequence: NP_415430.1, and acetate kinase was prepared according to GenBank: CAI36403.1, and they were mixed into an enzyme composition;

[0049] 2. Mix the enzyme composition obtained in step 1 with 5 mM deoxycytidine monophosphate, 0.25 mM adenosine triphosphate, 20 mM acetyl phosphate, 5 mM MgCl2 solution, and 50 mM Tris-HCl buffer solution at pH 7.5, and react at 37 °C for 1 h. The yield of deoxycytidine triphosphate obtained is 40.67%, and when the reaction time is extended to 4 h, the yield does not increase.

Claims

1. An enzyme composition, characterized in that, The enzyme composition comprises deoxynucleotide kinase and polyphosphate kinase, with a molar ratio of 9-11:

1.

2. The enzyme composition according to claim 1, characterized in that, The deoxynucleotide kinase has the amino acid sequence shown in SEQ ID NO: 1, and the polyphosphate kinase has the amino acid sequence shown in SEQ ID NO:

2.

3. A method for preparing the enzyme composition according to claim 1 or 2, characterized in that, Comprising: (1) Constructing a recombinant vector for expressing deoxynucleotide kinase or polyphosphate kinase; (2) Preparing a recombinant microorganism containing the recombinant vector obtained in step 1; (3) Culturing the recombinant microorganism obtained in step 2 to express, extract and purify deoxynucleotide kinase and polyphosphate kinase; (4) Mixing the deoxynucleotide kinase and polyphosphate kinase obtained in step 3.

4. Use of the enzyme composition according to claim 1 or 2 in the synthesis of deoxycytidine triphosphate.

5. The application according to claim 4, characterized in that, Comprising: (1) Mixing the enzyme composition according to claim 1 or 2 with deoxycytidine, deoxycytidine triphosphate, metal cofactor, phosphate donor and tris(hydroxymethyl)aminomethane hydrochloride buffer; (2) Performing an enzymatic reaction to synthesize deoxycytidine triphosphate.

6. The application according to claim 5, wherein The mass ratio of the deoxycytidine to the deoxycytidine triphosphate is 10-50:

1.

7. The application according to claim 5, characterized in that, The molar ratio of the deoxycytidine to the deoxynucleotide kinase or polyphosphate kinase is 3-7:

1.

8. The application according to claim 5, wherein The metal cofactor is MgCl2, with a concentration of 20-100 mM.

9. The application according to claim 5, wherein The phosphate donor is any one of sodium pyrophosphate, sodium metaphosphate, sodium hexametaphosphate, sodium polyphosphate, with a concentration of 20-60 mM.

10. The application according to claim 5, wherein The temperature of the enzymatic reaction is 30-45 °C, the pH is 7.0-9.5, and the time is 12-36 h.

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