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Method for preparing nucleotide by reaction separation coupling technology

A reaction separation coupling and nucleotide technology, applied in the field of biocatalysis and bioseparation, can solve the problems of yield reduction, achieve the effects of reducing energy consumption, eliminating product inhibition, improving enzyme utilization and reaction yield

Inactive Publication Date: 2009-08-12
NANJING UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since nuclease P1 is both an endonuclease and an exonuclease, while mononucleotides are obtained by enzymatic hydrolysis, some small fragments of oligonucleotides are also obtained, which sometimes also This results in a decrease in yield as mononucleotides are separated out

Method used

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  • Method for preparing nucleotide by reaction separation coupling technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Put the nuclease P1 with a concentration of 3000μ / mL into a 5L enzymatic hydrolysis reactor, and use a peristaltic pump to continuously pump the RNA solution with a concentration of 20g / L and pH5.0 to carry out the enzymatic hydrolysis reaction, and control the enzymatic hydrolysis temperature to 70°C , the RNA solution was pumped in at a flow rate of 20 mL / min. After 1 hour of reaction, start to pump the reaction liquid into the polysulfone hollow fiber ultrafiltration membrane for ultrafiltration. 2 . The ultrafiltrate flows into the polysulfone hollow fiber nanofiltration membrane for nanofiltration separation. The molecular weight cut-off of the nanofiltration membrane is 1000 Daltons, and the filtrate is nucleotides, which flow into the storage tank for collection. The ultrafiltration and nanofiltration residues flow back into the reactor to continue enzymatic hydrolysis. The total reaction time is 4 hours, the final RNA hydrolysis rate reaches 96%, and the nucle...

Embodiment 2

[0028] Put nuclease P1 with a concentration of 3000μ / mL into a 50L reactor, and use a peristaltic pump to continuously pump an RNA solution with a concentration of 50g / L and pH 6.0 to carry out the enzymatic hydrolysis reaction. The solution was pumped in at a flow rate of 50 mL / min. After one hour of reaction, start to pump the reaction liquid into the polysulfone hollow fiber ultrafiltration membrane for ultrafiltration. The molecular weight cut-off of the ultrafiltration membrane is 7000 Daltons, the flow rate is 70mL / min, and the pressure does not exceed 0.5Kg / cm 2 . The ultrafiltrate then flows into the polysulfone hollow fiber nanofiltration membrane for nanofiltration separation. The molecular weight cut-off of the nanofiltration membrane is 300 Daltons, and the filtrate is nucleotides, which flow into the storage tank for collection. The ultrafiltration and nanofiltration residues flow back into the reactor to continue enzymatic hydrolysis. The total reaction time is...

Embodiment 3

[0030] Put nuclease P1 with a concentration of 3500μ / mL into a 100L reactor, and use a peristaltic pump to continuously pump an RNA solution with a concentration of 100g / L and a pH of 7.0 for enzymolysis reaction. The enzymolysis temperature is controlled at 75°C. The solution was pumped in at a flow rate of 70mL / min. After one hour of reaction, pump the reaction liquid into the polysulfone hollow fiber ultrafiltration membrane for ultrafiltration. The molecular weight cut-off of the ultrafiltration membrane is 7000 Daltons, the flow rate is 100mL / min, and the pressure does not exceed 0.5Kg / cm 2 . The ultrafiltrate flows into the polysulfone hollow fiber nanofiltration membrane for nanofiltration separation. The molecular weight cut-off of the nanofiltration membrane is 400 Daltons, and the filtrate is nucleotides, which flow into the storage tank for collection. The ultrafiltration and nanofiltration residues flow back into the reactor to continue enzymatic hydrolysis. The ...

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Abstract

The invention discloses a method for preparing nucleotide by utilizing a reaction separation coupling technique, namely the nucleotide is prepared by using nuclease P1 for enzymatic hydrolysis of ribonucleic acid and the reaction separation coupling technique combining an enzymatic hydrolysis reactor, an ultrafiltration device and a nanofiltration device. The method utilizes the ultrafiltration device to separate an enzymatic hydrolysis product from an RNA phase, overcomes the product inhibiting effect in the production of the nucleotide, improves the enzymatic hydrolysis rate of the RNA and the concentration of the nucleotide, simultaneously utilizes the nanofiltration technique to recover the oligonucleotide lost in the ultrafiltration process, and further improves the enzymatic hydrolysis rate of the RNA. The method has the advantages of realizing the continuous separation production of products, shortening the process flow, breaking through the limitation of chemical balance, greatly improving the utilization ratio and the reaction yield of the enzyme, lowering energy consumption, and reducing production cost.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis and bioseparation, and relates to a method for preparing nucleotides by using reaction separation coupling technology. Background technique [0002] Nucleotides and their derivatives are important biochemical raw materials and have been widely used in genetic engineering, medicine, food, agricultural production and scientific research. It can generally be obtained by extracting ribonucleic acid (RNA) from yeast and then enzymatically hydrolyzing it with nuclease P1. [0003] At present, the nuclease P1 and RNA are generally mixed in a batch reactor, and the RNA is enzymatically hydrolyzed under the condition of stirring and heating, and then the enzyme P1 and RNA are precipitated with a precipitant to separate the nucleotides (CN1049521A). Because this method does not immediately separate the nucleotides obtained by enzymatic hydrolysis, a certain product inhibition effect is produced, so th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30
Inventor 欧阳平凯应汉杰张磊陈晓春熊健柏建新黄小权杨蕴毅
Owner NANJING UNIV OF TECH
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