Method for preparing uridylic acid by enzyme method

A technology for uridylic acid and uridine, which is applied in the field of enzymatic preparation of uridylic acid, can solve the problems of difficult product separation, poor production safety, explosiveness and the like, and achieves the effects of reduced cost, low cost, and safe and reliable preparation.

Inactive Publication Date: 2020-03-17
杭州唯泰生物药业有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical method is to dissolve uridine in an acidic solution, protect the hydroxyl groups at the 2 and 3 positions, and then use toxic POCl3 reagents as phosphorylation reagents to synthesize uridine acid. The yield of the whole process is high, but toxic reagents are used in the reaction process , high

Method used

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  • Method for preparing uridylic acid by enzyme method
  • Method for preparing uridylic acid by enzyme method
  • Method for preparing uridylic acid by enzyme method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, preparation UMP production enzyme

[0030] According to the sequences of the three enzyme genes, three pairs of amplification primers were designed. Genomic DNA of Escherichia coli strains was extracted, using it as a template, PCR amplified cytidine deaminase (ccd) and polyphosphate kinase (PPK1) gene fragments, and respectively ligated them into the pET28a vector ( purchased from Novagene Company); extract Lactobacillus bulgaricus (Lactobacillus bulgaricus) genomic DNA, and use it as a template to amplify the uridine-cytidine kinase (UCK) gene fragment by PCR, and connect it to the pET 28a vector (purchased from Novagene Company ). After the three gene fragments were successfully connected and sequenced correctly, they were respectively transferred into E.coli BL21 (DE3) strains (Shanghai Weidi Biotechnology Co., Ltd.).

[0031] 其中,胞苷脱氨酶(cytidine deaminase,ccd)的序列为ATGCATCCACGTTTTCAAACCGCTTTTGCCCAACTTGCGGATAACTTGCAATCTGCACTGGAACCTATTCTGGCAGACAAGTACTTCCC...

Embodiment 2

[0038] Embodiment 2, use free enzyme to prepare UMP

[0039] Substrate cytidine 100g, sodium hexametaphosphate 400g, ATP5g, MgCl 2 ·6H 2 O 50g, added to 5L pH7.0 phosphate buffer solution, stirred evenly, and adjusted the pH to 7.0. Add cytidine deaminase: 1000U / L, polyphosphate kinase: 600U / L, uridine-cytidine kinase: 1200U / L to the reaction liquid, control the pH during the reaction and keep it at 7.0, and the reaction temperature is 30-35°C. After reacting for 10 hours, the amount of UMP produced in the reaction supernatant liquid detected by HPLC was 23.5 g / L, the purity was 70%, and the conversion rate of cytidine was 85%.

[0040] HPLC detection conditions: Octadecylsilane bonded silica gel is used as filler, mobile phase A is tetrabutylammonium hydrogen phosphate, B phase is acetonitrile, detection wavelength is 260nm, flow rate is 1ml / min, column temperature is 30°C, elution The program is shown in Table 1.

[0041] Table 1 Elution program

[0042] time ...

Embodiment 3

[0044] Embodiment 3, use immobilized cell to prepare UMP

[0045] Substrate cytidine 80g, sodium hexametaphosphate 300g, ATP5g, MgCl 2 ·6H 2 O60g, add 5L pH7.0 phosphate buffer solution, stir evenly, adjust pH to 7.0. Add the immobilized cells of each enzyme in the reaction solution, wherein the activities are respectively cytidine deaminase: 2000U / L, polyphosphate kinase: 800U / L, uridine-cytidine kinase: 1500U / L, and the pH is controlled during the reaction Keep it at 7.0, and the reaction temperature is 30-35°C. After reacting for 15 hours, the amount of UMP produced in the reaction supernatant was detected by HPLC to be 16.4 g / L, the conversion rate of cytidine was 75%, and the purity was 53%. HPLC detection condition is the same as embodiment 2.

[0046]The supernatant collected by filtration was subjected to ion-exchange chromatography on a macroporous strongly basic anion-exchange resin, concentrated, crystallized, and dried to obtain 82 g of finished product UMP wit...

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Abstract

The invention, which belongs to the technical field of biological pharmacy and biochemical engineering, discloses an enzyme composition for uridylic acid production and a method for preparing uridylicacid by an enzyme method. The enzyme composition is prepared by cytidine deaminase, polyphosphate kinase and uridine-cytidine kinase. The three enzymes are reasonably combined to efficiently catalyzeand prepare uridylic acid. The enzyme composition disclosed by the invention can be recycled and is low in cost; and energy-saving and environment-friendly effects are realized. According to the invention, the cytidine is used as a substrate and the enzyme composition for uridylic acid production is added, so that the uridylic acid is prepared with low cost and high safety and reliability; the cost of the existing route is reduced; and the large-scale production is realized. The application of uridylic acid in fields of biocatalysis and medicines is guaranteed.

Description

technical field [0001] The invention belongs to the technical fields of biopharmaceutical and biochemical industry, and in particular relates to a method for preparing uridine acid by enzymatic method. Background technique [0002] Uridine monophosphate (UMP), also known as uridine monophosphate, is an important intermediate product in the de novo synthesis of pyrimidine nucleotides in the human body and one of the important nucleotides that make up RNA. The de novo synthesis of pyrimidine nucleotides in the human body is mainly regulated by the negative feedback of UMP; UDP (uridine diphosphate) can be further generated in the body. [0003] [0004] The sodium salt of uridine acid, 5'-uridine acid disodium, can be used as an important intermediate in the production of nucleic acid drugs, health food and biochemical reagents, and used in the manufacture of uridine diphosphate glucose, uridine triphosphate, polyadenylic acid Drugs such as drugs play an important role in ...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N9/12C12P19/30
CPCC12N9/1229C12N9/78C12P19/305C12Y207/04001C12Y207/04014C12Y305/04005
Inventor 周浩
Owner 杭州唯泰生物药业有限公司
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