Novel production process of high-purity 5' nucleotide

Abstract

The invention relates to a process for producing 5'nucleotide. A penicillium citrinum strain M-71 is cultured by submerged fermentation to generate extracellular nuclease P1, 2.5 to 5 percent ribonucleic acid solution is degraded, of which, the degradation rate is more than 80 percent; foreign protein and residual ribonucleic acid are removed by using a high frequency vibrating screen, clean 5' nucleotide solution after impurity removal is subjected to ultrafiltration by a ceramic membrane to remove the macromolecular residual matters; and four columns filled with strong alkaline anion exchange resin are connected in series to one-time adsorb and elute and collect in multiple steps, four mononucleotide solutions are collected, and a nano filter membrane with the molecular weight of 300 daltons is selected for cutting, desalting, concentration, decoloration by active carbon, crystallization and drying to obtain the product. The process has the advantages of better separating four nucleotides, removing inorganic phosphorus by elution and ensuring the preparation of high-purity 5'nucleotide product, and according to HPLC tests, the purity of the product can reach between 98 and 102 percent.

Description

1. Technical field:

[0001] The present invention relates to the production process of 5' nucleotide. 2. Background technology:

[0002] At present, only the specific nuclease P can be used to produce four kinds of 5' nucleotides at the same time. 1 Degradation of ribonucleic acid is obtained by resin adsorption, analysis, separation and purification. The preparation of specific, high-activity, and cheap enzymes is one of the key points of this process. At present, immobilized and solid-phase nuclease P is used at home and abroad. 1 method, but the technical requirements are very high, and the preparation cost is also high, so it is not suitable for large-scale production of 5' nucleotides in my country. It has been reported in China to use soaked malt to extract nuclease P 1 , but malt is a food resource, and it is difficult to preserve.

[0003] Nuclease P 1 After the ribonucleic acid is degraded into a 5' nucleotide solution, denatured enzymes, other miscellaneous prot...

Examples

Embodiment 1

[0027] Produce high-purity 5' nucleotides according to the following new production process of 5' nucleotides, including concentrating nuclease P1 to degrade ribonucleic acid (RNA) into a 5' nucleotide solution, which is subjected to vibrating sieve, ultrafiltration, resin adsorption, Stepwise elution, collection, nanofiltration, concentration, decolorization, crystallization, and drying to obtain four kinds of pure nucleotides 5'CMP, 5'AMP, 5'UMP, and 5'GMP with a content ≥ 98%.

[0028] nuclease P 1 : Penicillium citrinum strain M-71 was cultivated in liquid deep ventilation, and nuclease P was obtained after 30 hours of cultivation 1 , Enzyme activity 1200 units / ml. Concentrated Nuclease P by Nanofiltration 1 , the nuclease P 1 Concentrate through a 300 Dalton nanofiltration membrane into an enzyme solution greater than 7500 units / ml for use;

[0029] Concentrated Nuclease P 1 Carry out the degradation of ribonucleic acid: get pure 17.5kg RNA and make 2.5% solution, ad...

1. Technical field:

[0001] The present invention relates to the production process of 5' nucleotide. 2. Background technology:

[0002] At present, only the specific nuclease P can be used to produce four kinds of 5' nucleotides at the same time. 1 Degradation of ribonucleic acid is obtained by resin adsorption, analysis, separation and purification. The preparation of specific, high-activity, and cheap enzymes is one of the key points of this process. At present, immobilized and solid-phase nuclease P is used at home and abroad. 1 method, but the technical requirements are very high, and the preparation cost is also high, so it is not suitable for large-scale production of 5' nucleotides in my country. It has been reported in China to use soaked malt to extract nuclease P 1 , but malt is a food resource, and it is difficult to preserve.

[0003] Nuclease P 1 After the ribonucleic acid is degraded into a 5' nucleotide solution, denatured enzymes, other miscellaneous prot...

Examples

Embodiment 1

[0027] Produce high-purity 5' nucleotides according to the following new production process of 5' nucleotides, including concentrating nuclease P1 to degrade ribonucleic acid (RNA) into a 5' nucleotide solution, which is subjected to vibrating sieve, ultrafiltration, resin adsorption, Stepwise elution, collection, nanofiltration, concentration, decolorization, crystallization, and drying to obtain four kinds of pure nucleotides 5'CMP, 5'AMP, 5'UMP, and 5'GMP with a content ≥ 98%.

[0028] nuclease P 1 : Penicillium citrinum strain M-71 was cultivated in liquid deep ventilation, and nuclease P was obtained after 30 hours of cultivation 1 , Enzyme activity 1200 units / ml. Concentrated Nuclease P by Nanofiltration 1 , the nuclease P 1 Concentrate through a 300 Dalton nanofiltration membrane into an enzyme solution greater than 7500 units / ml for use;

[0029] Concentrated Nuclease P 1 Carry out the degradation of ribonucleic acid: get pure 17.5kg RNA and make 2.5% solution, ad...

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