阅读说明：本技术 一种酶法制备尿苷酸的方法 (Method for preparing uridylic acid by enzyme method ) 是由 周浩 于 2019-10-29 设计创作，主要内容包括：本发明属于生物制药和生物化工技术领域,公开了一种尿苷酸生产用酶组合物及一种酶法制备尿苷酸的方法。本发明所述酶组合物由胞苷脱氨酶、多聚磷酸激酶和尿苷-胞苷激酶组成。三种酶合理组合可以高效催化制备尿苷酸。本发明所述酶组合物可循环利用,成本低,节能环保。本发明所述酶法制备尿苷酸的方法以胞苷为底物,通过添加尿苷酸生产用酶组合物,可以低成本、安全可靠制备尿苷酸,降低现有路线的成本,使其适应规模化生产,为尿苷酸在生物催化和药品领域的使用提供保证。(The invention belongs to the technical field of biological pharmacy and biochemical engineering, and discloses an enzyme composition for producing uridylic acid and a method for preparing the uridylic acid by an enzyme method. The enzyme composition provided by the invention comprises cytidine deaminase, polyphosphate kinase and uridine-cytidine kinase. The reasonable combination of the three enzymes can efficiently catalyze and prepare uridylic acid. The enzyme composition disclosed by the invention can be recycled, is low in cost, and is energy-saving and environment-friendly. The method for preparing uridylic acid by the enzyme method takes cytidine as a substrate, and the enzyme composition for producing uridylic acid is added, so that the uridylic acid can be safely and reliably prepared at low cost, the cost of the existing route is reduced, the method is suitable for large-scale production, and the use of the uridylic acid in the fields of biological catalysis and medicines is guaranteed.)

1. An enzyme composition comprises cytidine deaminase, polyphosphate kinase, and uridine-cytidine kinase.

2. The enzyme composition according to claim 1, wherein the activity of cytidine deaminase is 1000-2000U/L, the activity of polyphosphate kinase is 600-1000U/L, and the activity of uridine-cytidine kinase is 1100-1500U/L.

3. The enzyme composition according to claim 1 or 2, wherein the cytidine deaminase, polyphosphate kinase and uridine-cytidine kinase are free enzyme solutions, immobilized enzymes or immobilized recombinant cells.

4. A method for preparing uridylic acid by enzyme method comprises adding cytidine, sodium hexametaphosphate, ATP, MgCl into pH7.0 phosphate buffer solution₂·6H₂O, mixing, adding the enzyme composition of any one of claims 1 to 3, and catalyzingAnd (4) preparing uridylic acid.

5. The method of claim 4, wherein the cytidine is at a final concentration of 16-24g/L, the sodium hexametaphosphate is at a final concentration of 50-80g/L, the ATP is at a final concentration of 1-2g/L, and the MgCl is at₂·6H₂The final concentration of O is 8-12 g/L.

6. The method of claim 5, wherein the catalytic reaction is carried out at pH7.0 and at a temperature of 30-35 ℃ for 10-15 hours.

Technical Field

The invention belongs to the technical field of biological pharmacy and biochemical engineering, and particularly relates to a method for preparing uridylic acid by an enzyme method.

Background

Uridine Monophosphate (UMP), also known as uridylic acid, is an important intermediate in de novo synthesis of pyrimidine nucleotides in humans and one of the important nucleotides that make up RNA. De novo synthesis of pyrimidine nucleotides in humans is largely regulated by negative feedback from UMP; further UDP (uridine diphosphate) can be produced in vivo.

The sodium salt of uridylic acid, 5' -uridylic acid disodium, can be used as an important intermediate for producing nucleic acid medicines, health-care foods and biochemical reagents, is used for manufacturing uridine diphosphate glucose, uridine triphosphate, poly-adenine uridylic acid and other medicines, and plays an important role in treating various serious diseases.

At present, with the expansion of the application range of uridine triphosphate, uridine tetraphosphate, uridine phosphatase inhibitor and the like in the field of medicine and the application of uridine diphosphate glucose pyrophosphorylase gene, research and development efforts on disodium 5' -uridylate are also increased by domestic and foreign scientific research institutions and enterprises.

The existing production method of uridylic Acid mainly comprises a chemical synthesis method and a Ribonucleic Acid (RNA) degradation method. The chemical method is to dissolve uridine in an acid solution, protect 2 and 3-site hydroxyl groups and then use a toxic POCl3 reagent as a phosphorylation reagent to synthesize uridylic acid, so that the yield of the whole process is high, but the toxic reagent is used in the reaction process, and is high in temperature and pressure, explosive, poor in production safety and easy to cause pollution. RNA degradation is the use of 5' -phosphodiesterase degradation of RNA from yeast cells, the whole process is relatively environmental but product separation is difficult, and cell lysates are difficult to handle.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preparing uridylic acid by an enzymatic method, which solves the problems of the prior art.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

an enzyme composition comprises cytidine deaminase, polyphosphate kinase, and uridine-cytidine kinase.

The enzyme composition provided by the invention can be used for enzymatic catalytic synthesis of UMP by reasonably combining three enzymes, namely cytidine deaminase (ccd), uridine-cytidine kinase (UCK) and polyphosphate kinase (ppk).

In some embodiments, the activity of the cytidine deaminase in the enzyme composition is 1000-2000U/L, the activity of the polyphosphate kinase is 600-1000U/L, and the activity of the uridine-cytidine kinase is 1100-1500U/L.

The cytidine deaminase, the polyphosphate kinase and the uridine-cytidine kinase in the enzyme composition can be high-quality purified free enzyme liquid which is obtained by amplifying target fragments through PCR (polymerase chain reaction) by using a genetic engineering technology, is connected with a vector and is transferred into host bacteria, and then induces protein expression and purification. Or immobilized enzyme or immobilized recombinant cells after being immobilized.

The invention also provides a method for preparing uridylic acid by using the enzyme composition.

A method for preparing uridylic acid by enzyme method comprises adding cytidine, sodium hexametaphosphate, ATP, MgCl into pH7.0 phosphate buffer solution₂·6H₂And O, mixing uniformly, adding the enzyme composition, and catalyzing to prepare uridylic acid.

The method of the invention uses cytidine as a substrate, and can prepare uridylic acid with low cost, safety and reliability by adding the enzyme composition for producing uridylic acid. The specific reaction formula is as follows:

in some embodiments, the enzymatic method of preparing uridylic acid according to the invention comprises an enzyme composition comprising a cytidine deaminase activity of 1000-2000U/L, a polyphosphate kinase activity of 600-1000U/L, and a uridine-cytidine kinase activity of 1100-1500U/L. In some embodiments, the cytidine deaminase activity is 1000U/L, the polyphosphate kinase activity is 600U/L, and the uridine-cytidine kinase activity is 100U/L. In some embodiments, the cytidine deaminase activity is 2000U/L, the polyphosphate kinase activity is 800U/L, and the uridine-cytidine kinase activity is 1500U/L. In other embodiments, the cytidine deaminase activity is 1500U/L, the polyphosphate kinase activity is 1000U/L, and the uridine-cytidine kinase activity is 1100U/L.

In some embodiments, in the enzymatic method for preparing uridylic acid according to the present invention, the cytidine is present at a final concentration of 16 to 24g/L and hexametaphosphateThe final concentration of sodium is 50-80g/L, the final concentration of ATP is 1-2g/L, and MgCl₂·6H₂The final concentration of O is 8-12 g/L. In some embodiments, the cytidine is at a final concentration of 20g/L, the sodium hexametaphosphate is at a final concentration of 80g/L, the ATP is at a final concentration of 1g/L, and the MgCl is present₂·6H₂The final concentration of O was 10 g/L. In some embodiments, the cytidine is at a final concentration of 16g/L, the sodium hexametaphosphate is at a final concentration of 60g/L, the ATP is at a final concentration of 1g/L, and the MgCl is present₂·6H₂The final concentration of O was 12 g/L. In other embodiments, the cytidine is at a final concentration of 24g/L, the sodium hexametaphosphate is at a final concentration of 50g/L, the ATP is at a final concentration of 2g/L, and the MgCl is at a final concentration of 2g/L₂·6H₂The final concentration of O was 8 g/L.

Preferably, in the method for enzymatically producing uridylic acid according to the present invention, the catalytic reaction is carried out at a reaction temperature of 30 to 35 ℃ at a pH of 7.0 for 10 to 15 hours.

According to the technical scheme, the invention provides an enzyme composition for producing uridylic acid and a method for preparing the uridylic acid by using an enzyme method. The enzyme composition provided by the invention comprises cytidine deaminase, polyphosphate kinase and uridine-cytidine kinase. The reasonable combination of the three enzymes can efficiently catalyze and prepare uridylic acid. The enzyme composition disclosed by the invention can be recycled, is low in cost, and is energy-saving and environment-friendly. The method for preparing uridylic acid by the enzyme method takes cytidine as a substrate, and the enzyme composition for producing uridylic acid is added, so that the uridylic acid can be safely and reliably prepared at low cost, the cost of the existing route is reduced, the method is suitable for large-scale production, and the use of the uridylic acid in the fields of biological catalysis and medicines is guaranteed.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the cycle activity decreasing curve of immobilized enzyme of example 5, the abscissa shows the number of cycles and the ordinate shows the enzyme activity.

Detailed Description

The invention discloses a method for preparing uridylic acid by an enzyme method. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The method for measuring the activity of the cytidine deaminase comprises the following steps: cytidine deaminase pynM transformation: adding 10mL of E.coli BL21/pET-28a/pynM ultrasonication bacteria, 0.1g of cytidine or 0.1g of deoxycytidine into a 50mL conversion bottle, carrying out conversion reaction at 30 ℃ and 150r/min for 2-3h, and after the conversion is finished, centrifuging and taking a supernatant for subsequent detection. The detection method comprises the following steps: high performance liquid chromatography is used for quantitatively detecting (deoxy) uridine. Pretreatment: after the transformation liquid is centrifuged, the supernatant is used for high performance liquid chromatography detection through a 0.22 mu m microporous membrane. Conditions are as follows: a chromatographic column: agilent C18 column (4.6 mm. times.250 mm i.d.5 μm); column temperature: 35 ℃; sample introduction volume: 20 mu L of the solution; the flow rate is 1 mL/min; the detection wavelength is 260 nm; mobile phase: a: ultrapure water; b: methanol. Gradient elution: 0min, 15% B, keeping for 3 min; 3.0-3.5min, 15-24% B; 3.5min, keeping 24% for 5 min; 8.5-9.0min, 24-35% B; keeping 35% B for 6 min; 15.0-16.0min, 35-85% B; keeping 85% B for 6 min; 22.0-22.5min, 85% -15% B; 15% B for 5 min. And (3) preparing a standard curve: accurately weighing (deoxy) uridine standard substance with the volume of about 1.00mg-2.00mg and ultra-pure water to constant volume. 6 gradients (1. mu.g/mL, 30. mu.g/mL, 60. mu.g/mL, 90. mu.g/mL, 120. mu.g/mL, 150. mu.g/mL) were prepared for each standard. Samples were then sequentially loaded, 5 replicates for each concentration. A volume of the standard solution with the highest concentration is taken and mixed together, the volume is determined, and then HPLC analysis is carried out on the optimal conditions for (deoxy) uridine detection.

The method for measuring the activity of the polyphosphate kinase comprises the following steps: the enzyme reaction system and the reaction conditions are as follows: 100mM Tris-HCl (pH8.0), 20mM MgCl2, 1mM AMP, 1mM sodium hexametaphosphate, reacted at 30 ℃ for 15min, boiled in boiling water for 5min to inactivate the enzyme, centrifuged, and the supernatant was passed through a membrane. Detecting ATP content by HPLC, specifically C18HPLC column (250 mm × 4.6 mm); mobile phase: aqueous triethylamine phosphate solution (with a phosphoric acid content of 0.6% (v/v), pH adjusted to 6.6 with triethylamine): methanol 90: 10; an ultraviolet detector with the wavelength of 254nm, the column temperature of 30 ℃, the flow rate of 1mL/min and the sample injection amount of 20 mu L.

The method for measuring the enzyme activity of the uridine-cytidine kinase comprises the following steps: uridine-cytidine kinase pynF transformation system: e.coli BL21/pET-28a/pynF 10mL and 0.1g cytidine (or 0.1g uridine) are added into a 50mL transformation bottle, and then transformation reaction is carried out for 2-3h at 30 ℃ and 150r/min, and after the transformation is finished, the supernatant is centrifuged for later detection. The detection method comprises the following steps: and (3) quantitatively detecting the pyrimidine nucleotide accumulation condition by using a high performance liquid chromatography. Pretreatment: after the transformation liquid is centrifuged, the supernatant is used for high performance liquid chromatography detection through a 0.22 mu m microporous membrane. Conditions are as follows: a chromatographic column: agilent C18 column (4.6 mm. times.250 mm i.d.5 μm); column temperature: 35 ℃; sample introduction volume: 20 mu L of the solution; the flow rate is 1 mL/min; the detection wavelength is 260 nm; mobile phase: a: ultrapure water; b: methanol. Gradient elution: 0min, 15% B, keeping for 3 min; 3.0-3.5min, 15-24% B; 3.5min, keeping 24% for 5 min; 8.5-9.0min, 24-35% B; keeping 35% B for 6 min; 15.0-16.0min, 35-85% B; keeping 85% B for 6 min; 22.0-22.5min, 85% -15% B; 15% B for 5 min. And (3) preparing a standard curve: accurately weighing each nucleotide (uridylic acid, cytidylic acid) standard, about 1.00-2.00 mg, and diluting with ultrapure water to constant volume. 6 gradients (1. mu.g/mL, 30. mu.g/mL, 60. mu.g/mL, 90. mu.g/mL, 120. mu.g/mL, 150. mu.g/mL) were prepared for each standard. Samples were then sequentially loaded, 5 replicates for each concentration. And mixing a certain volume of standard solution with the highest concentration, fixing the volume, and analyzing the optimal conditions for pyrimidine nucleotide detection by HPLC.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. Wherein the formula of the seed culture medium is 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl10 g. The fermentation medium formula comprises 10g/L of peptone, 5g/L of yeast powder, 8g/L of NaCl, 10g/L of glycerol, 1.2g/L of monopotassium phosphate, 1.8g/L of monopotassium phosphate and 1g/L of magnesium sulfate.