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Pretreatment method of RNA enzymatic hydrolysate

A technology of pretreatment and enzymatic hydrolysis, which is applied in the field of pretreatment of RNA enzymatic hydrolysis, can solve problems such as poor separation effect, achieve the effects of prolonging life, uniform distribution, and increasing exchange capacity

Pending Publication Date: 2021-10-22
NANTONG QIUZHIYOU BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And the separation effect is getting worse and worse

Method used

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  • Pretreatment method of RNA enzymatic hydrolysate
  • Pretreatment method of RNA enzymatic hydrolysate
  • Pretreatment method of RNA enzymatic hydrolysate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Take the reaction solution 001, see Table 1:

[0030] name A430 Concentration C Adsorption capacity Remark Reaction solution 001 1.083 32.75 1 Volume 3L

[0031] Table I

[0032] Reaction solution 001 is realized at (molecular weight 3000D, pump pressure 0.2Mpa), (molecular weight 6000D, pump pressure 0.2Mpa), (molecular weight 10000D, pump pressure 0.2Mpa), (molecular weight 20000D, pump pressure 0.2Mpa) , and get the following data, see Table 2:

[0033] molecular weight pump pressure A430 Concentration C time Adsorption capacity exchange adsorption increase Separation 3000D 0.2Mpa 0.390 25.29 50min 1.18 18％ improve 6000D 0.2Mpa 0.455 28.35 42min 1.13 13％ improve 10000D 0.2Mpa 0.622 29.58 30min 1.08 8％ improve 20000D 0.2Mpa 0.921 30.12 24min 1.05 5％ no change

[0034] Table II

[0035] In summary, through the ultrafiltration membranes with different mol...

Embodiment 2

[0038] The filtrate enters the ultrafiltration machine, and the membrane molecular weight of the ultrafiltration machine is set to 3000D, and the pump pressure is 0.2Mpa for ultrafiltration, and ultrafiltration is performed for 10, 20, 30, 40, and 50 minutes respectively, and samples are taken within the ultrafiltration time range Ultrafiltrate and effluent, detect the ultraviolet absorption wavelengths A250, A260, A280, A430 of the filtrate, ultrafiltrate and effluent, and calculate the ratio A250 / A260 (ratio 1) and A80 / A260 (ratio 2) and Concentration C;

[0039] RNA reaction liquid ultrafiltration (taking 3000 molecular weight as an example), see Table 3:

[0040] name A250 A260 A280 Ratio 1 Ratio 2 Concentration C A430 Reaction solution 002 0.405 0.483 0.307 0.84 0.64 32.75 1.083 Ultrafiltration 10min 0.670 0.805 0.502 0.83 0.62 27.29 0.481 Ultrafiltration 20min 0.335 0.401 0.252 0.84 0.63 27.19 0.517 Ultrafiltra...

Embodiment 3

[0044] Load the ultrafiltrate and the RNA reaction solution separately, if the leakage concentration C≥0.4g / L, and the ratio of the concentration of the sample solution to the concentration of the leakage solution is 2≥1, it is judged as a sample leakage, record the concentration of the sample and the concentration of the sample. amount, and sample loading time, etc., and calculate the amount of sample loading increase, see Table 4:

[0045]

[0046] Table four

[0047] To sum up, the increase in ultrafiltration loading of the RNA reaction solution in the small test was greater than 10%, and the maximum increase was 18%.

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Abstract

The invention discloses a pretreatment method of RNA (Ribonucleic Acid) enzymatic hydrolysate, which comprises the following steps: preparing an RNA dissolving solution with the total volume of 6L, the dosage of RNA being 2%, the dosage of solid enzyme being 5% and the dosage of zinc sulfate being 0.5%; RNA reaction: respectively taking two groups of 3L RNA dissolving solutions, then respectively carrying out RNA reaction at the reaction temperature of 70-72 DEG C for 3 hours, then sampling at the reaction time of 2.5 hours and 3 hours, and carrying out HPLC to detect the reaction conversion rate; RNA sample loading: loading the ultrafiltrate and the RNA reaction liquid respectively, determining that the sample is under leakage penetration when the leakage concentration C is greater than or equal to 0.4 g / L and the ratio 2 of the concentration of the sample loading liquid to the concentration of the leakage liquid is greater than or equal to 1, and recording the sample loading amount and the sample loading volume. According to the method, through a series of pretreatment combination of plate and frame filtration, microfiltration, ultrafiltration, cation resin and the like, macromolecular protein, particle impurities, pigments, soluble protein, calcium, magnesium, zinc ions and other impurities contained in RNA, enzyme and other raw materials can be removed, and the impurities adsorbed to anion exchange resin are reduced. According to the method, the anion exchange resin is effectively protected, so that the service life of the anion exchange resin is prolonged.

Description

technical field [0001] The invention relates to the technical field of nucleotide products, in particular to a pretreatment method for RNA enzymatic hydrolysis solution. Background technique [0002] Ribonucleic acid (RNA) is hydrolyzed by nuclease P1 or phosphodiesterase, separated by anion exchange resin chromatography, concentrated, crystallized, and dried to obtain adenosine (AMP) and guanosine (GMP) ), cytidylic acid (CMP) and uridylic acid (UMP) and other four single nucleotide products. [0003] When anion exchange resin is used for chromatographic separation, since RNA and enzymes contain impurities such as pigments and proteins, the color of the resin will become darker and the exchange performance will be reduced. Generally, after using 10 batches of resin, the original light yellow resin will turn brown, and the loading and exchange nucleotide capacity will decrease by 10%; after using 20 batches of resin, the resin will turn dark brown, and the loading and excha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12P19/32C07H21/02C07H1/06
CPCC12P19/305C12P19/32C07H21/02C07H1/06
Inventor 陈修足徐浩邱蔚然武琳陈红燕
Owner NANTONG QIUZHIYOU BIOSCI & BIOTECH
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