CN111621534B - Method for preparing nucleotide by aqueous two-phase enzymolysis system - Google Patents
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Abstract
The invention discloses a method for preparing nucleotide by a double-aqueous phase enzymolysis system, wherein ribonucleic acid is used as a raw material in the double-aqueous phase system consisting of polyethylene glycol and ammonium sulfate, and enzymolysis is carried out by nuclease P1, and the obtained reaction liquid is the liquid containing the nucleotide. The invention reasonably integrates the process of preparing the nucleotide by the enzymolysis of the ribonucleic acid by the nuclease P1 and a two-aqueous-phase system, not only improves the recovery rate of the nuclease P1, but also improves the enzymolysis rate and the finished product yield of the nucleotide, reduces the production cost, and provides a new idea and a new process for the production and large-scale preparation of the nucleotide.
Description
Technical Field
The invention relates to a method for preparing nucleotide by a double-aqueous phase enzymolysis system, in particular to a method for preparing nucleotide by integrating the enzymolysis process of ribonucleic acid and the double-aqueous phase separation system.
Background
5' -nucleotides have been widely used in the food, biochemical and pharmaceutical industries. In the food industry, the original food freshness-improving agent is expanded into a functional food additive with the function of improving the immunity of organisms, and the food freshness-improving agent can be added into foods such as bread, biscuits and the like, has very obvious using effect particularly in infant foods, can effectively enhance the capability of infants for resisting bacillary dysentery and reduce the occurrence of diarrhea; in the field of medicine, 5' -nucleotide can be used as a medicine and also can be used as a production raw material of a plurality of antiviral and antitumor medicines; in addition, the nucleotide preparation can also be used as an animal and plant growth regulator, and has obvious effects of increasing yield and increasing weight.
Nucleotide production mainly takes three methods: chemical synthesis method, microbial fermentation method and enzymolysis method. The enzymolysis method can obtain the mixture of four kinds of nucleotides at one time due to degradation of RNA, and has high enzyme reaction yield, so that the method is adopted for the industrial production of nucleotides at home and abroad. The enzyme required in preparing nucleotide by an enzymatic hydrolysis method is nuclease Pl, which is phosphodiesterase capable of hydrolyzing RNA to obtain four 5' -nucleotides, the impurities of the enzymatic hydrolysis product are few, the subsequent separation process is simple, and the method is generally adopted for producing the nucleotide abroad.
The condition of the double aqueous phase extraction separation process is mild, the adjustable factor is more, the amplification and the operation are easy, the relevant theory and experience of the traditional solvent extraction can be used, the problem of organic solvent residue does not exist, and the method is particularly suitable for the separation and the purification of biological substances. At present, the aqueous two-phase extraction technology has been widely applied in the fields of biochemistry, cell biology, biochemical engineering and the like. In the biological catalysis process, the aqueous two-phase system can distribute reactants, products and enzyme into different aqueous phases, so that the enzyme activity is recovered to the maximum extent, the efficiency of the catalytic reaction is improved, the operation process is simplified, the energy consumption is low, the treatment capacity is large, the investment and the cost are reduced, more importantly, the process is easy to amplify, and the method is particularly suitable for the industrial biological catalysis process.
The existing nucleotide enzymolysis reaction has the problems of low recovery rate of nuclease P1, more process steps, difficult amplification of biological processing process and the like. Therefore, it is important to improve the recovery rate of nuclease P1, the enzymolysis yield and simplify the scale-up process.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a method for preparing nucleotide by a two-aqueous-phase enzymolysis system aiming at the defects of the prior art.
In order to solve the technical problem, the invention discloses a method for preparing nucleotide by a two-aqueous-phase enzymolysis system, ribonucleic acid is taken as a raw material in the two-aqueous-phase system consisting of polyethylene glycol and ammonium sulfate, and the enzymolysis is carried out by nuclease P1, and the obtained reaction liquid is the liquid containing the nucleotide.
Wherein, the number average molecular weight of the polyethylene glycol is 1000-; in the aqueous two-phase system, the mass percent of the polyethylene glycol is 1-50%, preferably 12-48%, and more preferably 24%.
Wherein, in the aqueous two-phase system, the mass percent of the ammonium sulfate is 1-50%, preferably 12-24%, and more preferably 18%.
Wherein, in the aqueous two-phase system, the concentration of ribonucleic acid is 1-100g/L, preferably 30-80g/L, and more preferably 50 g/L.
Wherein, the dosage of the nuclease P1 is 0.1-10L/kg ribonucleic acid, preferably 0.6-1.5L/kg ribonucleic acid, and more preferably 1L/kg ribonucleic acid; the enzyme activity of the nuclease P1 is 1000-6000U/mL.
Definition of the enzymatic activity of nuclease P1: 1.9mL of a substrate solution (containing 1% by mass of RNA; 0.2M of pH5.2 in acetic acid buffer and 0.0005M of ZnSO)4) After 10min in a constant-temperature water bath at 70 ℃, 0.1mL of nuclease P1 is added, after 15min of heat preservation at 70 ℃, 2.0mL of nucleic acid precipitator (0.25% ammonium molybdate-2.5% perchloric acid in mass ratio) is added, after 20min of ice water bath, centrifugation is carried out, supernatant is taken and diluted by certain times by distilled water, the determination value is between 0.2 and 0.6, and the light absorption value at 260nm is A260. The control was made by adding the precipitant first, and the other operations were the same as before. Under the above conditions, the difference in absorbance at 260nm of the amount of nucleotide generated per minute was1.0 is defined as 1 enzyme activity unit.
Wherein, the 1000-6000U/mL nuclease P1 is prepared by the following method.
The preparation method of the nuclease P1 comprises the following steps:
(1) the strain is as follows: penicillium citrinum (Penicillium citrinum) with preservation number of CGMCC No.2014 is described in Chinese patent CN 101067116A.
(2) Fermentation medium: 20g/L glucose, 2g/L peptone, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate and 0.2g/L calcium chloride.
(3) Scraping the strain in bottle into sterile water, inoculating 2 bottles into fermentation medium in 100L fermentation tank, fermenting at pH6 and 32 deg.C for 30 hr.
(4) After the fermentation is finished, obtaining clear liquid by suction filtration, wherein the nuclease P1 has the enzyme activity of 1000-
Preferably, the aqueous two-phase system further comprises zinc chloride; in the aqueous two-phase system, the concentration of zinc chloride is 0.1-10g/L, preferably 1-5g/L, and more preferably 3 g/L.
Wherein the enzymolysis is carried out at pH4.0-9.0 (adjusted by HCl or NaOH) at 40-90 deg.C for 2-12 h.
After the reaction is finished, standing and layering the reaction solution into a double-aqueous phase, wherein the upper phase is a hydrophobic phase (containing polyethylene glycol and nuclease), and the lower phase is a hydrophilic phase (containing micromolecular substances such as nucleotides, ammonium sulfate short-chain ribonucleic acid, oligonucleotides and the like); discharging the lower phase, separating the obtained liquid with ultrafiltration membrane, and separating the obtained permeate with nanofiltration membrane to obtain retentate; and concentrating and drying the obtained retentate to obtain the nucleotide.
Wherein the molecular weight cut-off of the ultrafiltration membrane is 1000-6000; the molecular weight cut-off of the nanofiltration membrane is 150-300.
Wherein, ammonium sulfate is added into the upper phase again to ensure that the mass percent of the ammonium sulfate is 1-50 percent, and a double water phase system is formed; simultaneously adding ribonucleic acid, nuclease P1 and zinc chloride again to make the concentrations of each substance respectively 1-100g/L, 0.1-10L/g ribonucleic acid and 0.1-10g/L, and performing enzymolysis at pH4.0-9.0 and 40-90 deg.C for 2-12 h. Among them, ammonium sulfate, ribonucleic acid and zinc chloride are all lost in the lower phase, and need to be added again, and nuclease P1 is added according to the recovery amount.
Specifically, the method for preparing the nucleotide by the aqueous two-phase enzymolysis system comprises the following steps:
(i) in an enzymolysis tank, a double water phase system of polyethylene glycol and ammonium sulfate is prepared, so that the mass percent of the polyethylene glycol is 1-50%, and the mass percent of the ammonium sulfate is 1-50%;
(ii) adding a nuclease P1 solution, ribonucleic acid and zinc chloride into the aqueous two-phase system in the step (i) to ensure that the concentration of the ribonucleic acid in the aqueous two-phase system reaches 1-100g/L, the concentration of the zinc chloride reaches 0.1-10g/L, the addition amount of the nuclease P1 is 0.1-10L/g of the ribonucleic acid, and carrying out enzymolysis reaction for 2-12h under stirring at the pH value of 4.0-9.0 and the temperature of 40-90 ℃;
(iii) standing the reaction solution to form a double-aqueous phase, discharging a lower phase, separating the lower phase by an ultrafiltration membrane with the MWCO of 1000-; further concentrating the nanofiltration retentate by 5 times, and spray drying to obtain a high-purity nucleotide product; wherein, the ultrafiltration retentate is spray dried to obtain a byproduct containing protein, short chain nucleic acid and oligonucleotide;
(iv) adding ammonium sulfate into the enzymolysis tank to form a two-water-phase system; returning to the step (ii), supplementing ribonucleic acid, nuclease P1 and zinc chloride again to make the concentrations of each substance respectively 1-100g/L, 0.1-10L/g ribonucleic acid and 0.1-10g/L, and performing enzymolysis again at pH4.0-9.0 and 40-90 deg.C for 2-12 h.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the invention reasonably integrates the process of preparing the nucleotide by the enzymolysis of the ribonucleic acid by the nuclease P1 and a two-aqueous-phase system, not only improves the recovery rate of the nuclease P1, but also improves the enzymolysis rate and the finished product yield of the nucleotide, reduces the production cost, and provides a new idea and a new process for the production and large-scale preparation of the nucleotide.
2. The aqueous two-phase system adopted by the invention has rapid phase formation and is easy to scale up.
3. The zinc chloride is added into the aqueous two-phase system, and the existence of zinc ions improves the enzymolysis rate of nucleotide; moreover, nuclease P1 can be recovered by the aqueous two-phase system, and the enzyme recovery rate can be improved by the existence of chloride ions.
Drawings
FIG. 1 is a high performance liquid phase diagram of the product obtained in example 2, with the first four peaks being CMP, UMP, GMP and AMP, respectively.
Detailed Description
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
In the following examples, the substance for adjusting the pH is HCl or NaOH.
In the following examples, methods for detecting nucleotides in liquid phase:
1, chromatographic column: tin-free and lexan Lichrospher C18(250mm×4.6mm i.d.，5μm)；
2, mobile phase: c: methanol; d, buffer solution: 2.3g (NH)4)H2PO4+35mL of methanol, made up to 1L with distilled water. Gradient elution procedure: 0-2 min, 100% of phase D; 2-8 min: 100% phase D decreased to 89% phase D and 11% phase C increased; 8-9 min: maintaining 89% phase D and 11% phase C; 9-10 min: 89% D phase added to 100% D phase and 11% C phase removed; 10-15 min: maintaining 100% phase D;
3, flow rate: 1.0 mL/min;
4, detection wavelength: 254 nm;
5 column temperature: room temperature;
6, sample injection volume: 20 μ L.
The molecular weights of the polyethylene glycols in the following examples are all number average molecular weights.
In the following examples, the calculation method of each parameter is as follows:
(1) the enzymolysis rate is 100 × CLower part*VLower part/M％
CLower partNucleotide concentration in the lower phase, g/L; vLower partLower phase volume, L; m, riboseWeight of nucleic acid, g
(2) The recovery rate of enzyme activity is 100UOn the upper part*VOn the upper part*/(UFirst stage*VFirst stage)％
UOn the upper partThe upper phase enzyme activity is U/mL; vOn the upper partUpper phase volume, L; u shapeFirst stageInitial enzyme activity, U/mL; vFirst stageInitial volume of enzyme solution, L
(3) Yield 100 × M1/M％
M1Nucleotide product quality, g; m, ribonucleic acid weight, g
Example 1:
adding 2kg of polyethylene glycol 2000, 1.2kg of ammonium sulfate, 0.4kg of ribonucleic acid and 0.02kg of zinc chloride into a double aqueous phase enzymolysis tank, supplementing deionized water until the total weight of the system is 10kg, adding 20% (w/w) of polyethylene glycol 2000 and 12% (w/w) of ammonium sulfate into a polyethylene glycol/ammonium sulfate system, adjusting the pH to 6.0, raising the temperature to 65 ℃, and stirring until the mixture is fully dissolved. Adding 0.6L of nuclease P1(3000U/mL), starting enzymolysis, finishing after 4h, and standing the reaction solution for 2h to form two phases. The upper phase contains nuclease P1 with the volume of 4.8L, the detected enzyme activity is 346U/mL, and the recovery rate of the enzyme activity is 92.27%. The volume of the lower phase is 5.6L, the nucleotide concentration is 48.86g/L, and the enzymolysis rate is 68.4%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide with ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain byproduct 68.3g (the byproduct can be used as feed additive). Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the retentate by 5 times volume, and spray drying to obtain 282.4g of nucleotide mixed product with yield of 70.6% and total content of 94.0% after high performance liquid detection of four nucleotides. The upper phase in the enzymolysis tank is supplemented with lost 7.73% nuclease P1, and then other raw materials with the same quantity as the previous round are added for a new round of enzymolysis preparation process of nucleotide.
Example 2:
2.4kg of polyethylene glycol 2000 and 1.8kg of ammonium sulfate are added into a double aqueous phase enzymolysis tank. Then 0.5kg of ribonucleic acid and 0.03kg of zinc chloride are added, deionized water is added until the total weight of the system is 10kg, the concentration of polyethylene glycol 2000 in a polyethylene glycol/ammonium sulfate system is 24 percent (w/w), the concentration of ammonium sulfate is 18 percent (w/w), the pH is adjusted to 5.5, the temperature is raised to 70 ℃, and the mixture is stirred until the mixture is fully dissolved. 0.5L of nuclease P1(2500U/mL) was added to start the enzymatic hydrolysis, and the reaction was terminated after 5 hours. The reaction solution is kept stand for 2 hours to form two phases. The upper phase contains nuclease P1 with the volume of 4.6L, the detected enzyme activity is 263U/mL, and the recovery rate of the enzyme activity is 96.78%. The volume of the lower phase is 5.9L, the nucleotide concentration is 64.07g/L, and the enzymolysis rate is 75.6%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain by-product 76.6 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the nanofiltration retentate by 5 times volume, and spray drying to obtain 372.8g of nucleotide mixed product with yield of 74.56%, wherein the total content of four nucleotides is 96.4% after high performance liquid detection (figure 1). The lost 3.22% nuclease P1 is supplemented from the upper phase in the enzymolysis tank, and other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation by enzymolysis.
Example 3:
1.6kg of polyethylene glycol 2000 and 2.4kg of ammonium sulfate are added into a double aqueous phase enzymolysis tank. Then 0.8kg of ribonucleic acid and 0.05kg of zinc chloride are added, and deionized water is added until the total weight of the system is 10 kg. In the polyethylene glycol/ammonium sulfate system, the concentration of polyethylene glycol 2000 is 16% (w/w), and the concentration of ammonium sulfate is 24% (w/w). Adjusting pH to 6.5, heating to 75 deg.C, stirring to dissolve completely, adding 0.5L nuclease P1(4000U/mL), performing enzymolysis, standing for 7 hr, and standing for 2 hr to obtain two phases. The upper phase contains nuclease P1 with the volume of 3.8L, the detected enzyme activity is 464U/mL, and the recovery rate of the enzyme activity is 88.2%. The volume of the lower phase is 6.7L, the nucleotide concentration is 79.28g/L, and the enzymolysis rate is 66.4%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain byproduct 202.4 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the retentate by 5 times volume, and spray drying to obtain 514.8g of nucleotide mixed product with yield of 64.25% and total content of 90.2% after high performance liquid detection of four nucleotides. The upper phase in the enzymolysis tank is supplemented with 11.8 percent of nuclease P1 which is lost, and then other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation by enzymolysis.
Example 4:
3.2kg of polyethylene glycol 4000 and 2.4kg of ammonium sulfate are added into a double aqueous phase enzymolysis tank, then 0.3kg of ribonucleic acid and 0.01kg of zinc chloride are added, and deionized water is supplemented until the total weight of the system is 10 kg. In the polyethylene glycol/ammonium sulfate system, the concentration of polyethylene glycol 4000 is 32% (w/w), the concentration of ammonium sulfate is 24% (w/w), the pH is adjusted to 7.0, the temperature is raised to 68 ℃, and the mixture is stirred until the mixture is fully dissolved. Adding 0.2L nuclease P1(3500U/mL), starting enzymolysis, ending after 6h, and standing the reaction solution for 2h to form two phases. The upper phase contains nuclease P1 with the volume of 4.4L, the detected enzyme activity is 144U/mL, the recovery rate of the enzyme activity is 90.51%, the volume of the lower phase is 5.8L, the nucleotide concentration is 35.43g/L, and the enzymolysis rate is 68.5%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain by-product 82.3 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the nanofiltration retentate by 5 times volume, and spray drying to obtain 188.5g of nucleotide product with yield of 62.83%, wherein the total content of four nucleotides after high performance liquid detection is 91.2%. The lost 9.49% nuclease P1 is supplemented from the upper phase in the enzymolysis tank, and other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation by enzymolysis.
Example 5:
2.0kg of polyethylene glycol 5000 and 2.2kg of ammonium sulfate are added into a double aqueous phase enzymolysis tank, and then 0.6kg of ribonucleic acid and 0.04kg of zinc chloride are added, and deionized water is supplemented until the total weight of the system is 10 kg. In the polyethylene glycol/ammonium sulfate system, the concentration of polyethylene glycol 5000 is 20% (w/w), the concentration of ammonium sulfate is 22% (w/w), the pH is adjusted to 5.8, the temperature is raised to 72 ℃, and the mixture is stirred until the mixture is fully dissolved. Adding 0.5L of nuclease P1(3000U/mL), starting enzymolysis, ending after 5h, and standing the reaction solution for 2h to form two phases. The upper phase contains nuclease P1 with the volume of 3.4L, the detected enzyme activity is 415U/mL, and the recovery rate of the enzyme activity is 94.07%. The volume of the lower phase is 7.1L, the nucleotide concentration is 61.18g/L, and the enzymolysis rate is 72.4%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain by-product 116.5 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the retentate by 5 times volume, and spray drying to obtain nucleotide product 422.6g with yield of 70.44%, and total content of four nucleotides after high performance liquid detection of 93.2%. The lost 5.93 percent nuclease P1 is supplemented from the upper phase in the enzymolysis tank, and other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation process by enzymolysis.
Comparative example 1: non-aqueous two-phase system
Adding 0.5kg of ribonucleic acid and 0.03kg of zinc chloride into an enzymolysis tank, supplementing water by using deionized water until the total amount is 10kg, adjusting the pH to 5.5, heating to 70 ℃, and stirring until the ribonucleic acid and the zinc chloride are fully dissolved. Adding 0.5L nuclease P1(2500U/mL), starting enzymolysis, ending after 5h, the nucleotide concentration is 32.67g/L, and the enzymolysis rate is 68.6%. An ultrafiltration membrane with molecular weight cutoff of 2000 is adopted to cut off a solution containing protein, short chain nucleic acid and oligonucleotide, and the solution is sprayed and dried to obtain a byproduct of 94.2 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the nanofiltration retentate by 5 times volume, and spray drying to obtain 321.2g nucleotide product with yield of 64.24% and total content of 92.8% after four nucleotides are detected by high performance liquid chromatography. Nuclease P1 was not recycled.
Comparative example 2: non-zinc chloride
2.4kg of polyethylene glycol 2000 and 1.8kg of ammonium sulfate are added into a double aqueous phase enzymolysis tank, 0.5kg of ribonucleic acid is added, and deionized water is supplemented until the total weight of the system is 10kg, wherein in a polyethylene glycol/ammonium sulfate system, the concentration of the polyethylene glycol 2000 is 24% (w/w) and the concentration of the ammonium sulfate is 18% (w/w). The pH was adjusted to 5.5, the temperature was raised to 70 ℃ and stirred until fully dissolved. Adding 0.5L nuclease P1(2500U/mL), starting enzymolysis, ending after 5h, and standing the reaction solution for 2h to form two phases. The upper phase contains nuclease P1 with the volume of 4.6L, the detected enzyme activity is 213U/mL, and the recovery rate of the enzyme activity is 78.38%. The volume of the lower phase is 5.9L, the nucleotide concentration is 58.95g/L, and the enzymolysis rate is 69.56%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain byproduct 92.4 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the nanofiltration retentate by 5 times volume, and spray drying to obtain nucleotide product 342.5g with yield of 68.5%, and total content of 91.3% after high performance liquid detection of four nucleotides. The lost 21.62 percent nuclease P1 is supplemented from the upper phase in the enzymolysis tank, and other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation process by enzymolysis.
Comparative example 3 polyethylene glycol-magnesium sulfate aqueous two-phase System
2.4kg of polyethylene glycol 2000 and 1.8kg of magnesium sulfate are added into a double aqueous phase enzymolysis tank, and then 0.5kg of ribonucleic acid and 0.03kg of zinc chloride are added, and deionized water is supplemented until the total weight of the system is 10 kg. In the polyethylene glycol/magnesium sulfate system, the concentration of polyethylene glycol 2000 is 24% (w/w), the concentration of magnesium sulfate is 18% (w/w), the pH is adjusted to 5.5, the temperature is raised to 70 ℃, and the mixture is stirred until the mixture is fully dissolved. Adding 0.5L nuclease P1(2500U/mL), starting enzymolysis, ending after 5h, and standing the reaction solution for 2h to form two phases. The upper phase contains nuclease P1 with the volume of 5.8L, the detected enzyme activity is 172U/mL, and the enzyme activity recovery rate is 79.81%. The volume of the lower phase is 4.7L, the nucleotide concentration is 74.83g/L, and the enzymolysis rate is 70.34%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain byproduct 79.3 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the nanofiltration retentate by 5 times volume, and spray drying to obtain 328.3g of nucleotide mixed product with yield of 65.66% and total content of 91.3% of four nucleotides after high performance liquid detection. The upper phase in the enzymolysis tank is supplemented with the lost 20.2 percent nuclease P1, and then other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation by enzymolysis.
Comparative example 4 No polyethylene glycol addition
Adding 1.8kg of ammonium sulfate, then adding 0.5kg of ribonucleic acid and 0.03kg of zinc chloride into a double aqueous phase enzymolysis tank, supplementing deionized water until the total weight of the system is 10kg, enabling the concentration of the ammonium sulfate to be 18% (w/w), adjusting the pH to 5.5, raising the temperature to 70 ℃, and stirring until the ammonium sulfate is fully dissolved. Adding 0.5L nuclease P1(2500U/mL), starting enzymolysis, finishing after 5h, the nucleotide concentration is 32.47g/L, and the enzymolysis rate is 68.18%. The reaction solution was allowed to stand for 2 hours to form two phases. An ultrafiltration membrane with molecular weight cutoff of 2000 is adopted to cut off a solution containing protein, short chain nucleic acid and oligonucleotide, and the solution is sprayed and dried to obtain a byproduct of 94.3 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the nanofiltration retentate by 5 times volume, and spray drying to obtain nucleotide product 320.3g with yield of 64.06%, and total content of 92.8% after high performance liquid detection of four nucleotides. Nuclease P1 could not be recovered.
Comparative example 5 No ammonium sulfate addition
Adding 2.4kg of polyethylene glycol 2000, then adding 0.5kg of ribonucleic acid and 0.03kg of zinc chloride into a double aqueous phase enzymolysis tank, supplementing deionized water until the total weight of the system is 10kg, the concentration of the polyethylene glycol 2000 is 24% (w/w), adjusting the pH to 5.5, heating to 70 ℃, and stirring until the mixture is fully dissolved. Adding 0.5L nuclease P1(2500U/mL), starting enzymolysis, finishing after 5h, the nucleotide concentration is 31.14g/L, and the enzymolysis rate is 65.39%. The reaction solution was allowed to stand for 2 hours to form two phases. And (3) intercepting a solution containing protein, short-chain nucleic acid, polyethylene glycol and oligonucleotide by adopting an ultrafiltration membrane with the molecular weight cutoff of 2000, and spray-drying the solution to obtain a by-product of 82.3 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the nanofiltration retentate by 5 times volume, and spray drying to obtain 285.6g nucleotide product with yield of 57.12%, and total content of 89.24% after high performance liquid detection of four nucleotides. Nuclease P1 could not be recovered.
Comparative example 6: concentration of polyethylene glycol
1.2kg of polyethylene glycol 2000 and 1.8kg of ammonium sulfate are added into a double aqueous phase enzymolysis tank, and then 0.5kg of ribonucleic acid and 0.03kg of zinc chloride are added, and deionized water is supplemented until the total weight of the system is 10 kg. In the polyethylene glycol/ammonium sulfate system, the concentration of polyethylene glycol 2000 is 12% (w/w), the concentration of ammonium sulfate is 18% (w/w), the pH is adjusted to 5.5, the temperature is raised to 70 ℃, and the mixture is stirred until the mixture is fully dissolved. Adding 0.5L nuclease P1(2500U/mL), starting enzymolysis, ending after 5h, and standing the reaction solution for 2h to form two phases. The upper phase contains nuclease P1 with the volume of 3.5L, the detected enzyme activity is 263U/mL, and the recovery rate of the enzyme activity is 73.64%. The volume of the lower phase is 7L, the nucleotide concentration is 50.29g/L, and the enzymolysis rate is 70.41%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain byproduct 79.5 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the retentate by 5 times volume, and spray drying to obtain 336.4g of nucleotide mixed product with yield of 67.28% and total content of 92.4% after four nucleotides are detected by high performance liquid chromatography. The upper phase in the enzymolysis tank is supplemented with lost 26.4 percent nuclease P1, and other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation by enzymolysis.
Comparative example 7: concentration of polyethylene glycol
In a double aqueous phase enzymolysis tank, 3.6kg of polyethylene glycol 2000 and 1.8kg of ammonium sulfate are added, and then 0.5kg of ribonucleic acid and 0.03kg of zinc chloride are added, and deionized water is supplemented until the total weight of the system is 10 kg. In the polyethylene glycol/ammonium sulfate system, the concentration of polyethylene glycol 2000 is 36% (w/w), the concentration of ammonium sulfate is 18% (w/w), the pH is adjusted to 5.5, the temperature is raised to 70 ℃, and the mixture is stirred until the mixture is fully dissolved. Adding 0.5L nuclease P1(2500U/mL), starting enzymolysis, ending after 5h, and standing the reaction solution for 2h to form two phases. The upper phase contains nuclease P1 with the volume of 5.4L, the detected enzyme activity is 206U/mL, and the enzyme activity recovery rate is 88.99%. The volume of the lower phase is 5.1L, the nucleotide concentration is 70g/L, and the enzymolysis rate is 71.4%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain byproduct 71.1 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the retentate by 5 times volume, and spray drying to obtain nucleotide mixture product 342.2g with yield of 68.44%, and total content of four nucleotides after high performance liquid detection of 93.2%. The lost 10.94 percent nuclease P1 is supplemented from the upper phase in the enzymolysis tank, and other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation process by enzymolysis.
Comparative example 8: concentration of polyethylene glycol
4.8kg of polyethylene glycol 2000 and 1.8kg of ammonium sulfate are added into a double aqueous phase enzymolysis tank, and then 0.5kg of ribonucleic acid and 0.03kg of zinc chloride are added, and deionized water is supplemented until the total weight of the system is 10 kg. In the polyethylene glycol/ammonium sulfate system, the concentration of polyethylene glycol 2000 is 48% (w/w), the concentration of ammonium sulfate is 18% (w/w), the pH is adjusted to 5.5, the temperature is raised to 70 ℃, and the mixture is stirred until the mixture is fully dissolved. Adding 0.5L nuclease P1(2500U/mL), starting enzymolysis, ending after 5h, and standing the reaction solution for 2h to form two phases. The upper phase contains nuclease P1 with the volume of 6.3L, the detected enzyme activity is 129U/mL, and the recovery rate of the enzyme activity is 65.02%. The volume of the lower phase is 4.2L, the nucleotide concentration is 67.18g/L, and the enzymolysis rate is 56.43%. Discharging the lower phase water solution, intercepting solution containing protein, short chain nucleic acid and oligonucleotide by ultrafiltration membrane with molecular weight cutoff of 2000, and spray drying the solution to obtain byproduct 85.24 g. Ultrafiltering the permeate, removing inorganic salt with nanofiltration membrane with molecular weight cutoff of 200, concentrating the nanofiltration retentate by 5 times volume, and spray drying to obtain 294.32g of nucleotide mixed product with yield of 58.86% and total content of 88.4% of four nucleotides after high performance liquid detection. The upper phase in the enzymolysis tank is supplemented with the lost 34.87 percent nuclease P1, and other raw materials with the same quantity as the previous round are added for a new round of nucleotide preparation process by enzymolysis.
The invention provides a method for preparing nucleotide by a two-aqueous phase enzymolysis system, and a method and a way for realizing the technical scheme are many, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and decorations can be made without departing from the principle of the invention, and the improvements and decorations should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (8)
1. A method for preparing nucleotide by a two-aqueous phase enzymolysis system is characterized in that ribonucleic acid is used as a raw material in the two-aqueous phase system containing zinc chloride and consisting of polyethylene glycol and ammonium sulfate, enzymolysis is carried out by nuclease P1, after the reaction is finished, the obtained reaction liquid is kept stand to be divided into two aqueous phases, and the lower phase is liquid containing nucleotide;
wherein, in the aqueous two-phase system, the mass percent of the polyethylene glycol is 24 percent;
wherein in the aqueous two-phase system, the mass percent of ammonium sulfate is 18 percent;
wherein the number average molecular weight of the polyethylene glycol is 1000-6000.
2. The method of claim 1, wherein the concentration of ribonucleic acid in the aqueous two-phase system is 1-100 g/L.
3. The method of claim 1, wherein the nuclease P1 is used in an amount of 0.1 to 10L/kg ribonucleic acid; the enzyme activity of the nuclease P1 is 1000-6000U/mL.
4. The method according to claim 1, wherein the concentration of zinc chloride in the aqueous two-phase system is 0.1-10 g/L.
5. The method of claim 1, wherein the conditions of enzymatic hydrolysis are at ph4.0-9.0, 40-90 ℃ for 2-12 h.
6. The method according to claim 1, wherein after the reaction is completed, the reaction solution is allowed to stand and stratify into a two-phase water phase; discharging the lower phase, separating the liquid discharged from the lower phase with ultrafiltration membrane, and separating the obtained ultrafiltration permeate with nanofiltration membrane to obtain retentate; and concentrating and drying the obtained retentate to obtain the nucleotide.
7. The method as claimed in claim 6, wherein the ultrafiltration membrane has a molecular weight cut-off of 1000-6000 Da; the molecular weight cut-off of the nanofiltration membrane is 150-300 Da.
8. The method as claimed in claim 6, wherein ammonium sulfate is added again to the upper phase to make the mass percent of ammonium sulfate be 1-50%, and a two-aqueous phase system is formed again; simultaneously adding ribonucleic acid, nuclease P1 and zinc chloride again to make the concentrations of each substance respectively 1-100g/L, 0.1-10L/g ribonucleic acid and 0.1-10g/L, and performing enzymolysis at pH4.0-9.0 and 40-90 deg.C for 2-12 h.
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