TWI734095B - DNMP KINASE AND METHOD FOR MANUFACTURING NUCLEIC ACIDS USING THE dNMP KINASE - Google Patents

Abstract

The invention provides a dNMP Kinase obtained from Thermus P23 phage having the nucleic acids of SEQ ID NO: 1. The invention further provides a method for manufacturing NDP or dNDP using the dNMP kinase, comprising mixing (a) NMP or dNMP, (b) ATP or dATP, and (c) dNMP kinase under a high temperature and basic condition to product NDP or dNDP. In addition, a method for manufacturing dNTP is also provided.

Description

dNMP kinase and method for preparing nucleotide using this kinase

The present invention relates to the preparation of a ribonucleic acid, and particularly to a novel dNMP kinase (deoxynucleoside monphosphate), the use of this dNMP kinase to prepare (deoxy)ribonucleoside diphosphate (NDP or dNDP), (deoxy)ribose Nucleoside triphosphate (NTP or dNTP) method.

Nucleotides are compounds composed of a five-carbon sugar, a nitrogen-containing base, and one to three phosphates. They are very important molecules in organisms. At the genetic level, deoxyribonucleoside triphosphates (dNTPs) can be used to form DNA, and ribonucleotides can be used to form RNA. Commercially, nucleotides have been used in food flavor enhancers, animal feed, wound healing, cosmetics, etc., and their applications are quite wide.

With the development of PCR and the application of biotechnology, etc., the demand for dNTPs in research units and commercial applications is increasing year by year. However, the dNTPs sold on the market today are mainly synthesized by chemical methods, and only a few documents have recorded them. Biologically made.

The chemical synthesis method is to treat the dNMP salt with dimethylformamide, which is very soluble, and then use tri-n-butylamine for preliminary phosphate reduction, and then add the nitrogen-containing base to synthesize dNTP Finally, the organic phase and the water phase are separated with pyridine. The overall reaction time is about 2 hours. The yield varies from 20% to 20% depending on the type of dNTP. The difference is about 50%.

The chemical synthesis method has high complexity in the purification and separation steps, and it is necessary to separate unreacted salts, phosphoric acid solution, DMF and by-products produced in the reaction. In addition, the pyridine and DMF used in the reaction are organic poisons, which are harmful to the human body and the environment.

In view of this, in order to solve the above problems, the industry urgently needs a dNTP synthesis method and system that is free of environmental pollution, low reaction time, high yield, and simple. Therefore, the present invention provides a novel protein with dNMP kinase activity, a method for preparing (deoxy)ribonucleoside diphosphate using this protein, and a method for preparing (deoxy)ribonucleoside triphosphate using this protein method.

The present invention provides an isolated protein which has dNMP kinase (NMK) activity and has the amino acid sequence of SEQ ID NO:1.

The amino acid sequence of SEQ ID NO: 1 is shown below:

In an embodiment of the present invention, the protein is a protein derived from thermophilic bacteriophage.

In an embodiment of the present invention, the protein is a protein derived from Thermus P23 phage.

The present invention provides a use of the aforementioned protein as a kinase, which is used for conversion A ribonucleoside monophosphate (NMP) is used as a ribonucleoside diphosphate (NDP), or used to convert a deoxyribonucleoside monophosphate (dNMP) to a deoxyribonucleoside diphosphate (dNDP) use.

The present invention further provides a method for preparing ribonucleoside bisphosphate or deoxyribonucleoside bisphosphate using the aforementioned protein, comprising: (i) mixing (a) a ribonucleoside monophosphate (NMP) or a deoxyribonucleoside Monophosphate (dNMP), (b) adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) and (c) the aforementioned protein; and (ii) reacting under a high temperature and an alkaline environment , To produce ribonucleoside diphosphate (NDP) or deoxyribonucleoside diphosphate (dNDP).

The present invention further provides a method for preparing deoxyribonucleoside triphosphate (dNTP), which comprises: (i) mixing a deoxyribonucleoside monophosphate (dNMP), an adenosine triphosphate (ATP) and the aforementioned protein, Reacting under a high temperature and an alkaline environment to produce deoxyribonucleoside diphosphate (dNDP); and (ii) mixing the deoxyribonucleoside diphosphate (dNDP), monophosphate and monophosphotransferase , To produce deoxyribonucleoside triphosphate (dNTP).

In an embodiment of the present invention, the aforementioned phosphate includes phosphoenolpyruvate (PEP) or acetyl phosphate (AcP).

In an embodiment of the present invention, the phosphotransferase includes pyruvate kinase or acetate kinase.

In an embodiment of the present invention, the aforementioned high temperature environment is above 50°C.

In an embodiment of the present invention, the aforementioned high-temperature environment is 50-75°C.

In an embodiment of the present invention, the aforementioned high temperature environment is 80-100°C.

In an embodiment of the present invention, the aforementioned alkaline environment is above pH 6.5.

In an embodiment of the present invention, the aforementioned alkaline environment is pH 6.5-9.

In an embodiment of the present invention, the aforementioned alkaline environment is pH 5-7.

Figure 1 is a schematic diagram of an embodiment of the biological reaction synthesis method of the present invention.

Figure 2 shows the enzyme activity of the thermophilic bacterium P23 phage NMK of the present invention at different pH values.

Figure 3 shows the enzyme activity of the thermophilic bacterium P23 phage NMK of the present invention at different temperatures.

Figures 4A-4D are the ultraviolet (UV) spectra of the high performance liquid chromatograph. Analysis of deoxyadenosine diphosphate (dADP), deoxythymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP) and deoxygenated by the thermophilic bacterium P23 phage NMK by high performance liquid chromatography Guanosine Diphosphate (dGDP).

Figures 5A-5D are the ultraviolet (UV) spectra of the high performance liquid chromatograph. Analyze NDP kinase (NDK) by high performance liquid chromatography, and use dNDP produced by thermophilic bacterium P23 phage NMK as the raw material to generate deoxyadenosine diphosphate (dATP) and deoxythymidine Diphosphate (dTTP), Deoxycytidine Diphosphate (dCTP) and Deoxyguanosine Diphosphate (dGTP).

Figures 6A-6B are 1% agarose gel electrophoresis analysis images. The dNTP synthesized by the thermophilic bacterium P23 phage NMK of the present invention can be subjected to PCR reaction.

The present invention provides an isolated protein which has the activity of dNMP kinase (NMK). The isolated protein of the present invention is derived from Thermus P23 phage and has the amino acid sequence shown in SEQ ID NO:1.

The amino acid sequence of SEQ ID NO:1 is shown below:

The present invention provides an isolated protein comprising the amino acid sequence shown in SEQ ID NO: 1, and further comprising variants of the protein. The variant system of the protein refers to having one or more amino acid substitutions, deletions or additions. The derived protein, and the protein also has the activity of dNMP kinase, the variants of the protein and its use should be regarded as equivalent to the scope of the present invention.

The amino acid sequence of the dNMP kinase of the present invention has a high proportion of arginine (Arg), and has good thermal stability. The guanidinium group in the side chain branch of Arg uses resonance to form a stable hydrogen bond with the surrounding amino acid, and Arg also contains a methylene group, which can strengthen the amino acid The hydrophobic force, these bonds are helpful to the thermal stability of the structure. In addition, Arg has a dissociation constant (pKa) of up to 12, and can exist in a charged form at high temperatures, maintaining the bond and force with nearby amino acids to stabilize the structure and thermal stability of the protein.

In one embodiment, the Arg ratio of the dNMP kinase of the present invention is 8.5% or more, preferably 6-8%, more preferably 8-12%.

The dNMP kinase of the present invention has good activity and can convert dNMP or NMP into dNDP or NDP. In one embodiment, the dNMP kinase of the present invention has thermal stability and activity, especially good activity at 50°C to 65°C.

In another embodiment, the present invention has good activity at pH 6.5 to 7.0.

The present invention also provides a method for preparing ribonucleoside diphosphate (NDP) or deoxyribonucleoside diphosphate (dNDP) using the dNMP kinase of the present invention. The method of the present invention includes: mixing (a) ribonucleoside monophosphate (NMP) or deoxyribonucleoside monophosphate (dNMP), (b) monoadenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) ) And (c) The dNMP kinase of the present invention reacts in a high temperature and alkaline environment to produce ribonucleoside diphosphate (NDP) or deoxyribonucleoside diphosphate (dNDP).

The ribonucleoside diphosphate (NDP) of the present invention includes adenosine diphosphate (ADP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and/or guanosine diphosphate (GDP); deoxy Ribonucleoside diphosphate (dNDP) includes deoxyadenosine diphosphate (dADP), deoxythymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP) and/or deoxyguanosine diphosphate (dGDP) .

In the present invention, adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) can be used as a source of phosphate. Under a high temperature and alkaline environment, it reacts with the dNMP kinase of the present invention to convert ribonucleoside monophosphate (NMP) or deoxyribonucleoside monophosphate (dNMP) into ribonucleoside diphosphate (NDP) or to remove Oxyribonucleoside diphosphate (dNDP).

The "high temperature" mentioned in the method of the present invention refers to above 50°C, 50~60°C, 60~70°C, 70~80°C, 80~90°C, and 90~100°C.

The "alkaline" mentioned in the method of the present invention refers to pH 5 or higher, pH 5 to 6.5, pH 6.5 to 7, pH 7 to 8, pH 8 to 9.

The present invention further provides a method for preparing deoxyribonucleoside triphosphate (dNTP). The method includes two steps. The first step is to produce deoxyribonucleoside diphosphate (dNDP), and the second step is to produce deoxyribonucleotide nuclei. Glycoside triphosphate (dNTP), as shown in Figure 1.

The first step is the same as described above using the dNMP kinase of the present invention to prepare deoxyribonucleoside double The method of phosphoric acid (dNDP) is the same. Mixing deoxyribonucleoside monophosphate (dNMP) with the dNMP kinase of the present invention, using deoxyadenosine triphosphate (dATP) as the source of phosphate, reacts under a high temperature and alkaline environment to produce deoxygenation Ribonucleoside Diphosphate (dNDP).

The second step is to mix and react the deoxyribonucleoside diphosphate (dNDP) obtained in the first step with monophosphate and monophosphotransferase in a 65°C water bath for 10 minutes to produce deoxyribonucleoside triphosphate (dNTP). The phosphate substance used in the second step may include, for example, phosphoenolpyruvate (PEP) or acetyl phosphate (AcP), and the phosphotransferase may include, for example, pyruvate kinase or acetate kinase. After the obtained deoxyribonucleoside triphosphate (dNTP) is purified, high-purity deoxyribonucleoside triphosphate (dNTP) can be obtained.

The method of the present invention is a biosynthetic method. Compared with the previous chemical synthesis, only two enzymes (dNMP kinase, pyruvate kinase or acetate kinase) are required to carry out dNTP biosynthesis, and the overall reaction time is also greatly reduced ( It only takes about 40 minutes). In addition, because the reaction is performed in a high temperature environment, it can inhibit the activity of enzymes in many environments (such as phosphohydrolase) and reduce other factors that may affect dNTP synthesis. In terms of yield, the yield of dNTPs can reach more than 50%, preferably 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80% or more. It is much higher than the previous enzyme synthesis method.

Example

Example 1. Preparation of thermophilic bacteria P23 phage NMK

After the NMK gene fragment of the thermophilic bacteriophage P23 phage was amplified by PCR, it was digested with restriction enzymes NdeI and XhoI, ligated to the protein expression vector pET-30a (Novagen), and transformed into E. coli BL21 For performance.

It is understandable that protein preparation is a common technique in the technical field to which the present invention belongs, and other methods for preparing proteins should be regarded as equivalent to the scope of the present invention.

Example 2. Determination of the best activity of thermophilic bacteria P23 phage NMK

Take 1μg of purified thermophilic bacteriophage P23 phage NMK and add reaction solutions (50mM Tris-HCl; 80mM KCL; 8mM MgCl ₂ ; 5mM) of different pH values (pH 6.5, pH 7, pH 7.5, pH 8 and pH 8.5) dNMP; 2mM ATP), react in a water bath at different temperatures (55°C, 60°C, 65°C, 70°C and 75°C) for 30 minutes. The product was analyzed by a high-performance chromatograph.

Referring to Figure 2, at a temperature of 65°C, it has the best activity at a pH of 8.5. Compared with the activity at pH 8.5 (100%), the activity at pH 8.0 was 86%, the activity at pH 7.5 was 85%, the activity at pH 7.0 was 74%, and the activity at pH 6.5 was 73%. The results showed that the thermophilic bacteriophage P23 phage NMK can retain about 70% of the activity between pH 7 and 8.5.

It can be seen from Figure 3 that the thermophilic bacteriophage P23 phage NMK has the best activity at 70°C. Compared with the activity at 70°C (100%), the activity at 75°C is 45%, the activity at 65°C is 95%, the activity at 60°C is 72%, and the activity at 55°C is 55%. The results show that it has the best activity at a temperature between 70°C and 65°C, and the thermophilic bacteriophage P23 phage NMK is found to be very sensitive to temperature. At temperatures other than the optimal activity, the activity is reduced to about 50%.

Example 3. The activity of thermophilic bacteria P23 phage NMK

Take the purified NMK at a final concentration of 1μg/μL and add the reaction solution (50mM Tris-HCl; _{80mM KCL; 8mM MgCl 2} ; 5mM dNMP; 2mM ATP or dATP; pH 8.0), the total reaction volume is 100μl, the reaction temperature is 65 ℃, the product was obtained after 30 minutes of reaction in a water bath. Using Water X-BridgeTM C18 column (4.6 x 20mm, particle size 3μm), the product was analyzed with a high-performance chromatograph at a constant temperature of 37°C.

It can be seen from Figures 4A-4D that whether ATP or dATP is used as the source of phosphate and dAMP, dTMP, dCMP or dGMP is used as the substrate, the thermophilic bacteriophage P23 NMK can produce dADP, dTDP, dCDP or dGDP .

Example 4. Synthesis of dNTPs using thermophilic bacteriophage P23 phage NMK

Take the purified thermophilic bacteriophage P23 NMK at a final concentration of 4μg/μL and add it to the _{microcentrifuge tube containing the reaction solution (50mM Tris-HCl; 80mM KCL; 8mM MgCl 2} ; 5mM dNMP; 2mM ATP; pH 8.0). The total reaction volume is 400μl, react at 65°C for 30 minutes. Then add a final concentration of 4μg/μL purified Me.thermophile , Th.maritima acetate kinase (AK) or T.thermophilus hb8 pyruvate kinase (pyruvate kinase, PK), and provide a final concentration of 2mM phosphate ( AcP or PEP), and then react in a 65°C water bath for 10 minutes.

From Figures 5A-5D, it can be seen that when ATP provides phosphate through NDP kinase (NDK), and dADP, dTDP, dCDP or dGDP produced by the thermophilic bacteriophage P23 phage NMK as the substrate, it can be To produce dADP, dTDP, dCDP or dGDP, the reaction and results are the same as the prior art.

Take 20μl of the reaction product as the source of dNTP, and use PCR amplification for detection. The PCR products were analyzed by electrophoresis on 1% agar gel. Referring to Figures 6A-6B, the dNTP synthesized by the thermophilic bacterium P23 phage NMK of the present invention can indeed be used for PCR.

<110> National Tsing Hua University

<120> dNMP kinase and method for preparing nucleotides using this kinase

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 177

<212> PRT

<213> Thermus virus P23

<400> 1

Claims (1)

A method for preparing ribonucleoside triphosphate or deoxyribonucleoside triphosphate from separated protein, comprising: (i) mixing (a) monoribonucleoside monophosphate (NMP) or monodeoxyribonucleoside monophosphate (dNMP) ), (b) an adenosine triphosphate (ATP) or a deoxyadenosine triphosphate (dATP) and (c) an isolated protein; and (ii) a reaction under a high temperature environment to produce a ribonucleoside Diphosphate (NDP) or deoxyribonucleoside diphosphate (dNDP); wherein the isolated protein has the amino acid sequence shown in SEQ ID NO:1; the high temperature environment is 65~70°C; the high temperature environment PH 7~8.5; wherein the method includes the steps of preparing deoxyribonucleoside triphosphate (dNTP), including: (iii) mixing the deoxyribonucleoside diphosphate (dNDP), monophosphate and monophosphotransferase , To produce deoxyribonucleoside triphosphate (dNTP), the phosphate includes phosphoenolpyruvate (PEP) or acetyl phosphate (AcP); the phosphotransferase includes pyruvate kinase or acetate kinase.

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