Novel production process of high-purity 5' nucleotide
A production process and nucleotide technology, applied in the direction of microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as inability to desalt, lower yield, affect product purity, etc. Ease of use
Active Publication Date: 2012-09-05
大连珍奥生物技术股份有限公司
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Problems solved by technology
[0006] Finally, the concentration of the nucleotide resin separation solution is relatively dilute and contains salt, which cannot reach the crystallization concentration. Conventionally, vacuum membranes can be used to concentrate, but it cannot be desalted, which affects the purity of the product. Using activated carbon for adsorption and desalination can desalinate, but the process is complicated and the yield is reduced.
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Embodiment 1
[0027] High-purity 5' nucleotides are produced according to the following new 5' nucleotide production process, including concentrated nuclease P1 to degrade ribonucleic acid (RNA) into 5' nucleotide solution, vibrating sieve, ultrafiltration, resin adsorption, Stepwise elution, collection, nanofiltration, concentration, decolorization, crystallization and drying to obtain four pure nucleotides 5'CMP, 5'AMP, 5'UMP and 5'GMP with a content of ≥98%.
[0028] First prepare nuclease P 1 : liquid deep ventilation culture Penicillium citrinum strain M-71, after 30 hours of culture to obtain nuclease P 1 , the enzyme activity is 1200 units / ml. Nanofiltration concentrated nuclease P 1 , the nuclease P 1 Concentrated into an enzyme solution greater than 7500 units / ml through a 300-dalton nanofiltration membrane for later use;
[0029] Concentrated Nuclease P 1 Carry out the degradation of ribonucleic acid: take pure 17.5kg RNA to make a 2.5% solution, add concentrated enzyme 3.2% ...
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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 a production process for 5' nucleotides. 2. Background technology: [0002] At present, the simultaneous production of four 5' nucleotides can only use specific nuclease P 1 Degradation of ribonucleic acid is obtained by resin adsorption, analytical separation and purification. For this reason, the preparation of specific, high-activity, and inexpensive 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 However, the technical requirements are very high, and the preparation cost is also high, which is not suitable for the large-scale production of 5' nucleotides in my country. There are reports in China using soaked malt to extract nuclease P 1 , but malt is a food resource, and it is difficult to preserve it. [0003] Nuclease P 1 After degrading ribonucleic acid into 5' nucleotide solution, denatured enzymes, other miscellaneous pro...
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IPC IPC(8): C12P19/32C12P19/30C12R1/80
Inventor 乔宾福俞慧君赵嘉庆于喜庆刘万峰陈晓丽
Owner 大连珍奥生物技术股份有限公司