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Technology for separating nucleotide by adopting continuous ion exchange chromatography technique

A technology of exchange chromatography and continuous ions, applied in the field of biological separation, can solve the problems of difficult separation of nucleotides and low yield, and achieve the effects of simple and easy process, low waste water discharge and good effect.

Active Publication Date: 2017-05-10
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a method for separating four kinds of 5'-nucleotides in the enzymolysis solution by using continuous ion exchange chromatography technology, so as to solve the difficult problems of nucleotide separation and low yield

Method used

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  • Technology for separating nucleotide by adopting continuous ion exchange chromatography technique
  • Technology for separating nucleotide by adopting continuous ion exchange chromatography technique
  • Technology for separating nucleotide by adopting continuous ion exchange chromatography technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1: Obtaining of enzymatic hydrolyzate.

[0086] Weigh 30-70g of RNA powder, dissolve it in ultrapure water, then raise the temperature to 70°C, add nuclease P1 accounting for 5-8wt% of the RNA powder to carry out enzymatic hydrolysis reaction, after enzymatic hydrolysis for 4 hours, add 0.2wt% activated carbon, continue enzymatic hydrolysis for 1 hour, and then add chitosan flocculant accounting for 10wt% of RNA powder to the solution, and the enzymatic hydrolysis solution is prepared. After centrifuging the hydrolyzate obtained by enzymolysis for 10 minutes, it was decolorized by decolorizing resin column SX-1, and after 10 hours of decolorization, the hydrolyzate we needed was obtained.

Embodiment 2

[0087] Example 2: Separation of four nucleotides in the enzymatic hydrolyzate by continuous ion exchange chromatography.

[0088] The number of resin columns in the continuous separation device is 7, and the method for separating four kinds of 5'-nucleotides by three-zone two-step continuous ion-exchange chromatography includes two steps of cyclic operation:

[0089] step 1( figure 1 ):

[0090] The 7 resin columns are divided into three areas: adsorption area, desorption area and regeneration area. Among them, the adsorption area is 1 resin column, the desorption area is 2 resin columns, and the regeneration area is 4 resin columns; each resin column is filled with 100g Resin (NH-1), resin column diameter 3.0cm, height 23cm.

[0091] Adsorption area: Adsorb the pretreated enzymolysis solution according to the loading amount of 0.036g 5'-nucleotide / g wet resin, the concentration of the enzymolysis solution: uracil nucleotide 4g / L, cytosine nucleotide 6g / L L, guanine nucleot...

Embodiment 3

[0102] Example 3: Separation of four nucleotides in the enzymatic hydrolyzate by continuous ion exchange chromatography.

[0103] The number of resin columns in the continuous separation device is 9, and the method for separating four kinds of 5'-nucleotides by three-zone two-step continuous ion-exchange chromatography includes two steps of cyclic operation:

[0104] step 1( image 3 ):

[0105] The resin column is divided into three areas: adsorption area, desorption area and regeneration area. Among them, the adsorption area is 1 resin column, the desorption area is 3 resin columns, and the regeneration area is 5 resin columns;

[0106] Adsorption area: Adsorb the pretreated enzymolysis solution according to the loading amount of 0.025g 5'-nucleotide / g wet resin, the concentration of the enzymolysis solution: uracil nucleotide 4g / L, cytosine nucleotide 6g / L L, guanine nucleotide 10g / L, adenine nucleotide 11g / L, sample loading time is 2h, sample loading flow rate is 0.26BV / ...

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Abstract

The invention discloses a technology for separating nucleotide by adopting a continuous ion exchange chromatography technique. After RNA enzymatic hydrolysate is subjected to de-coloring pretreatment, a three-zone two-step continuous on exchange chromatography is adopted for separating four 5'-nucleotides UMP, GMP, CMP and AMP. According to the invention, the ion exchange competitive effect between the four nucleotides and the resin is utilized to achieve the purpose of separating and purifying the four nucleotides on the basis of a continuous device; except for UMP, the other nucleotides in the product all have a purity above 98% and the purity of the UMP is 92%-95%; and besides, the average yield of the four nucleotides in the product is above 97%.

Description

technical field [0001] The invention belongs to the technical field of biological separation, and in particular relates to a technology for separating nucleotides by using continuous ion-exchange chromatographic technology. Background technique [0002] 5'-nucleotide is the basic unit involved in the composition of biological macromolecular nucleic acid, and has a wide range of uses in agriculture, food industry and pharmaceutical industry. Especially in the application of baby food and medicine, it has an irreplaceable function. In baby food, as an additive in baby food, it can significantly improve the baby's immunity, promote the maturity of the intestinal tract, promote the synthesis of lipoproteins and polyunsaturated fatty acids, reduce the occurrence of colds and diarrhea in babies, and benefit the normal health of babies. growth and development. As pharmaceutical intermediates, nucleotides can be used to synthesize many antiviral and antitumor drugs, and their corr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H1/06C07H19/10C07H19/20
CPCC07H1/06C07H19/10C07H19/20
Inventor 应汉杰王莹莹吴菁岚焦朋飞周精卫
Owner NANJING UNIV OF TECH