Recombinant escherichia coli for high yield of cytidine monophosphate and application of recombinant escherichia coli

A technology for recombinant Escherichia coli and Escherichia coli, which is applied in the field of cytidylic acid preparation, can solve the problems of complicated separation operation, corrosion equipment, low yield, etc., and achieve the effects of simplified process, cost saving and mild reaction process

Pending Publication Date: 2020-06-12
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the first method can obtain four products at one time, except for cytidylic acid, the other three become by-products, and the source of RNA is more restricted, and the separation operation in the production process is complicated and the yield is low; At present, chemical methods are mainly used to synthesize 5'-cytidylic acid in industry. The main disadvantage of this method is that there are many wastes, which corrode equipment and are not conducive to the health of personnel and industrial production.

Method used

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  • Recombinant escherichia coli for high yield of cytidine monophosphate and application of recombinant escherichia coli
  • Recombinant escherichia coli for high yield of cytidine monophosphate and application of recombinant escherichia coli
  • Recombinant escherichia coli for high yield of cytidine monophosphate and application of recombinant escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0021] Example 1 Construction of BL21(DE3)-PET28a-UCK expression strain

[0022] Using the whole genome of Escherichia coli MG1655 as a template, the nucleotide coding sequence of cytidine kinase protein was amplified by conventional PCR.

[0023] The upstream primers used had Nco I restriction site, the sequence is: CATGCCATGGCAatgactgatcagtctcatcagt.

[0024] downstream primers with EcoR I Restriction site, the sequence is CCGGAATTCttaGTGGTGGTGGTGGTGGTGttcaaagaactgacttattttcgct.

[0025] The reaction conditions are: 95°C for 2min, 95°C for 20s, 50°C for 20s, 72°C for 10s, a total of 30 cycles; 72°C for 5min. The obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragments were recovered. The sequence and the expression vector pET28a were used by Takara company Nco I and EcoR I enzyme digestion, enzyme digestion reaction system: 10 × buffer 1 μL, Nco I 1 μL, EcoR I 1 μL, gene fragment or pET28a vector 7 μL. The enzyme digestio...

Embodiment 2

[0027] Example 2 Induced expression of BL21(DE3)-PET28a-UCK and cell disruption

[0028] 1. Induced expression of BL21(DE3)-PET28a-UCK

[0029] The positive strain BL21(DE3)-PET28a-UCK was inoculated into 100 mL LB / KanR liquid medium, and cultured with shaking at 37 °C and 200 rpm until OD 600 ≈1. Inoculate in 500 mL of fresh LB / KanR liquid medium at a ratio of 10:100, culture with shaking at 37°C and 200 rpm until OD600≈0.5~0.7, add IPTG to a final concentration of 0.5‰~1‰, at 20~30 ℃ (eg image 3 As shown, the soluble expression is more between 25~30℃, and 30℃ is the best), shake culture at 200rpm for 13~15h. Centrifuge at 6000rpm for 10min to collect the cells.

[0030] 2. Bacteria fragmentation

[0031] The bacterial cells were resuspended with 100 mM TrisHcl8.0 buffer, and broken up with a high-pressure homogenizer under the conditions of 12000 psi, 4°C, and four cycles. Centrifuge at 6000 rpm for 10 min to collect the supernatant, namely the crude enzyme solution, ...

Embodiment 3

[0032] Example 3 Catalytic synthesis of CAMP

[0033] Mix the following reaction system in a test tube: Add 30-35 mM cytidine, 30-35 mM adenosine triphosphate, 8-10 mM MgCl to the cytidine kinase crude enzyme solution with a protein concentration of 6-8 g / L 2 , Potassium phosphate buffer solution, stirred evenly, reacted at pH 7.5~8.0, 35~37°C for 7~9h, and completed the enzymatic reaction to synthesize cytidylic acid (cytidylic acid is detected by liquid phase as shown in Figure 1(d) It shows that the conversion rate of the substrate cytidine and ATP reaches more than 85%). Use Agilent HC-C18 chromatographic column (150 mm×4.6 mm, 5 μm), mobile phase is 0.05 mol / L potassium dihydrogen phosphate; flow rate 0.8 ml min -1 ; The detection wavelength is 260 nm.

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Abstract

The invention discloses recombinant escherichia coli for high yield of cytidine monophosphate and application of the recombinant escherichia coli. According to the invention, cytidine kinase gene is obtained by cloning from a cytidine monophosphate production strain; crude enzyme liquid obtained by crushing recombinant bacteria has good catalytic activity and stability; cytidine, adenosine triphosphate (ATP) and Mg < 2 + > are added, the reaction system is simple, the conditions are mild, the period is short, byproducts are fewew, the method is clean and free of pollution, the application is asimple, rapid and efficient production way, and the conversion rate of substrate cytidine and ATP reaches 85% or above.

Description

technical field [0001] The invention belongs to the field of cytidylic acid preparation, and in particular relates to a recombinant Escherichia coli with high cytidylic acid production and application thereof. Background technique [0002] 5'-cytidine (Cytidine 5'-Monophosphate) alias: cytarabine 5'-monophosphate; monophosphate cytarabine, etc., which are mostly white or off-white crystalline powder, soluble in water, insoluble in ethanol. 5'-cytidylic acid is an important biochemical substance in biological cells and participates in various physiological and biochemical reactions. In industry, 5'-cytidylic acid is mainly used in the manufacture of citicoline, cytidine triphosphate, cytarabine, polyinosin and other drugs. In recent years, it can also be added to milk powder in conjunction with other nucleotides to enhance the immunity of infants. [0003] There are two main production methods for 5'-cytidylic acid, 1) degraded from RNA (ribonucleic acid), and then separat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/70C12P19/30C12R1/19
CPCC12N9/1205C12N15/70C12P19/305C12Y207/01074
Inventor 王昕王静陈可泉马琛欧阳平凯
Owner NANJING UNIV OF TECH
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