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High-throughput screening method of microorganism strain producing cytidine at high yield

A screening method and high-yield cytidine technology, applied in the field of microbial breeding and fermentation engineering, can solve the problems of large workload, high cost and long cycle, and achieve the effect of overcoming expensive equipment

Active Publication Date: 2016-04-20
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to overcome the problems of large workload, long period and high cost in the conventional method of screening high-producing strains of cytidine, the present invention provides a high-throughput screening based on miniaturized culture of deep-well plate and micro-detection based on microplate reader method

Method used

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  • High-throughput screening method of microorganism strain producing cytidine at high yield
  • High-throughput screening method of microorganism strain producing cytidine at high yield
  • High-throughput screening method of microorganism strain producing cytidine at high yield

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Effect test

Embodiment 1

[0069] A high-throughput screening method for cytidine high-yielding microbial strains is characterized in that it is performed according to the following steps:

[0070] (1) Pick a single colony of cytidine-producing bacteria, inoculate it in deep-well plate I with liquid seed medium, cover the deep-well plate, and culture it on a shaker at 30-40°C and 300-800rpm for 8 hours;

[0071] (2) Inoculate the seed liquid in well I of the deep well plate into the deep well plate II containing the fermentation medium, and incubate on a shaking table at 40°C and 300 rpm for 20 hours;

[0072] (3) Place the deep-well plate II in a well-plate centrifuge, centrifuge at 8000×g for 30 minutes, and dilute the supernatant of the fermentation broth by micro-gradient dilution method;

[0073] (4) Add the diluted fermentation broth supernatant, buffer solution and appropriate amount of cytidine deaminase to the microwells of the microplate, take the experiment without cytidine deaminase as the b...

Embodiment 2

[0082] H 2 Standard curve for cytidine in O.

[0083] Prepare reaction solution A and reaction solution B:

[0084] Reaction solution A: sodium hydroxide 26g / L, salicylic acid 68g / L and sodium nitroferricyanide 2g / L;

[0085] Reaction solution B: sodium hypochlorite 45ml / L.

[0086] The cytidine standard is dissolved in deionized water, and the final concentration of the cytidine standard is 0.01mM, 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM.

[0087] Add 25 μL of cytidine standards of different concentrations, 100 μL of 0.01M PBS buffer and 3UCDA (thawed on ice after taking it out from -20°C) into the microwells of the microtiter plate, and react in a warm bath at 37°C for 30 minutes; immediately after the reaction Add 50 μL of reaction solution A and 50 μL of reaction solution B with a row gun, and react in a warm bath at 37°C for 30 minutes, and then use a microplate reader to measure the absorbance value of the reaction solutio...

Embodiment 3

[0089] Standard curve of cytidine in M9 medium.

[0090] 1000×Trace elements: MnCl 2 1g / L; ZnCl 2 1.7g / L; CuCl 2 2H 2 O0.43g / L; CoCl 2 ·6H 2 O0.6g / L; FeCl 3 8.125g / L; Na 2 MoO 4 2H 2 O0.6g / L.

[0091] M9 medium formula: glucose 10g / L; Na 2 HPO 4 12H 2 O8.96g / L; KH 2 PO 4 1.5g / L; NaCl2.5g / L; Urea 2g / L; L-tryptophan 0.05g / L; 1000×trace elements 1mL; MgSO 4 ·7H 2 O0.49g / L; CaCl 2 0.011g / L.

[0092] The preparation method of reaction solution A and B is the same as embodiment 1.

[0093] Dissolve the cytidine standard with M9, dilute the cytidine standard with M9 to a final concentration of 0.01mM, 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM , 9mM, 10mM. .

[0094] Add 25 μL of cytidine standards of different concentrations, 100 μL of 0.01M PBS buffer and 3UCDA (thawed on ice after taking it out from -20°C) into the microwells of the microtiter plate, and react in a warm bath at 37°C for 30 minutes; immediately after the reaction Ad...

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Abstract

The invention discloses a high-throughput screening method of a microorganism strain producing cytidine at high yield. The method includes the steps of (a) inoculating single colony to a deep pore plate I filled with a liquid seed culture medium, and performing shaking table culture; (b) correspondingly inoculating the seed liquid in the deep pore plate I to a deep pore plate II filled with a fermenting culture medium, and performing shaking table culture; (c) performing centrifugation to the deep pore plate to obtain a supernate to detect the content of the cytidine; (d) adding the supernate of a fermentation liquid, a buffer liquid and a proper amount of cytidine deaminase in pores of an ELISA plate, and performing a reaction in a bath at 30-40 DEG C for 10-40 min; (e) instantly adding a reaction solution A and a reaction solution B after the reaction is finished, and performing a reaction in a bath at 30-40 DEG C for 20-50 min; and (f) measuring the absorbance of the reaction solution through an ELISA instrument, and calculating the yield of the cytidine according to a standard curve and dilution ratio of the supernate of the fermentation liquid. The method achieves high-throughput culturing, high-throughput preparation of fermentation liquid, and high-throughput measurement of the cytidine.

Description

technical field [0001] The invention relates to a high-throughput screening method for microbial strains with high cytidine production, and belongs to the technical field of microbial breeding and fermentation engineering. Background technique [0002] Cytidine (cytidine) is also known as cytosine nucleoside, cytosine, 1-β-D-nucleofuranoside cytosine. As a pyrimidine nucleoside, cytidine is mainly used as an intermediate in the production of antitumor and antiviral drugs, and is used to manufacture cytarabine (Ara-CR), cyclocytidine (CycloC), cytidine triphosphate (CTP), and cytidine The main raw material of drugs such as choline (CDP-Choline). At present, the production methods of cytidine include RNA hydrolysis method, combination of fermentation method and synthesis method, fermentation method with uracil as precursor, direct fermentation method, etc. (Zhang Kexu, Du Lianxiang, translation. Nucleic acid fermentation [M]. Beijing: China Light Industry Publishing Society,...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12Q1/34C12R1/19
Inventor 张大伟董会娜刘永飞李宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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