CN114736257A

CN114736257A - Method for separating and extracting uridine from catalytic liquid containing uridine

Info

Publication number: CN114736257A
Application number: CN202210546132.9A
Authority: CN
Inventors: 应汉杰; 温庆仕; 陈勇; 魏荷芬; 王骏之
Original assignee: Jiangsu Jicui Industrial Biotechnology Research Institute Co ltd; Nanjing Tech University
Current assignee: Jiangsu Jicui Industrial Biotechnology Research Institute Co ltd; Nanjing Tech University
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2022-07-12
Anticipated expiration: 2042-05-18
Also published as: CN114736257B

Abstract

The invention discloses a method for separating and extracting uridine from catalytic liquid containing uridine, which comprises the following steps: (1) mixing the catalytic solution containing uridine with polyethylene glycol, and separating liquid to obtain a uridine catalytic solution at the lower layer; (2) carrying out nanofiltration on the obtained uridine catalytic liquid, and collecting nanofiltration permeate; (3) evaporating and concentrating the nanofiltration permeate to obtain a uridine concentrated solution; (4) adjusting pH of the obtained uridine concentrated solution, adding seed crystal, stirring and crystallizing. The crystallization system in the invention is a water system, and no reagent is needed to be added, so that the crystallization cost is reduced. The crystallization process of the invention is completed within 12h to obtain bright white crystals, the yield of one-time crystallization is more than 60%, the yield of crystals is over 90% after multiple crystallization or nested crystallization, meanwhile, the purity of the crystals is more than 98%, and the product quality, yield and cost are higher than those of the prior art.

Description

Method for separating and extracting uridine from catalytic solution containing uridine

Technical Field

The invention belongs to the technical field of biochemical separation, and particularly relates to a method for efficiently separating and extracting uridine from an enzyme catalysis solution at low cost.

Background

Uridine is a pyrimidine nucleoside, white or off-white crystalline powder, a substance naturally produced by the liver, is a uridine, and is a main constituent of ribonucleic acid involved in vivo protein synthesis. Uracil is produced when it adheres to ribofuranose. This compound can be further added to various phosphate groups to form a trinucleotide (a factor involved in regulating metabolism). In addition, it binds between uracil and deoxyribose to form deoxyuridine, but this compound rarely occurs naturally in living organisms.

Researchers have found uridine to have potential in the treatment of a variety of diseases. For example, many clinical trials involving cancer patients have demonstrated that nutritional supplements containing this compound can counteract the toxic effects of chemotherapy. In addition, there is some evidence that uridine has a preventive effect on cell damage and liver dysfunction. Uridine may also be treated for conditions including Parkinson's disease, Alzheimer's disease, bipolar disorder, and the like. Indeed, animal-based studies have focused primarily on the antidepressant effects of uridine in combination with omega-3 fatty acids, which have been shown to be comparable to conventional drugs. Other studies are also testing the efficacy of uridine in combination with omega-3 fatty acids, and cytidine and choline in the prevention of Alzheimer's disease.

Sources of uridine include tomato, beet, broccoli, meat, molasses, brewers yeast, and the like. It is also one of the components of human milk. Apart from human milk and yeast, the human body cannot absorb this substance from the diet. The invention provides a method for separating and extracting uridine from an enzyme catalysis solution with high efficiency and low cost.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for separating and extracting uridine from catalytic liquid containing uridine and synchronously recycling cytidine deaminase, aiming at the defects of the prior art.

In order to solve the technical problem, the invention discloses a method for separating and extracting uridine from a catalytic solution containing uridine, which comprises the following steps:

(1) fully and uniformly mixing a catalytic solution containing uridine with polyethylene glycol, standing for naturally layering, wherein the lower phase of the double water phase system is a uridine product without introducing new impurities, the upper phase is PEG 1000-1000, cytidine deaminase is obtained from the upper layer, and the lower phase is a uridine catalytic solution; taking the lower-layer filtrate through a separating funnel to obtain a uridine catalytic solution;

(2) filtering the obtained uridine catalytic liquid by nanofiltration, continuously washing the uridine catalytic liquid by pure water, and collecting nanofiltration permeate;

(3) concentrating the nanofiltration permeate through vacuum evaporation to obtain a uridine concentrate;

(4) after the pH of the obtained uridine concentrated solution is adjusted, a small amount of seed crystals are added to be used as an inducer, and the mixture is stirred until uridine crystals are separated out;

(5) filtering the crystals obtained in the step (4), leaching with ethanol, and collecting the crystals and the mother liquor; vacuum drying the obtained crystals to obtain pure uridine crystals; and (5) repeating the step (4) to crystallize the mother liquor, so as to obtain the uridine crystals.

In some examples, the mother liquor obtained is subjected to the crystallization of steps (3) to (5) to obtain pure uridine crystals.

The uridine in the invention is derived from an enzyme catalytic solution using cytidine as a precursor. The initial yield of uridine was 180-260 g/L. In the fermentation liquid, there are a large amount of cell fragments and pigments, and the production cost of the enzyme is high.

In the step (1), the polyethylene glycol is 1000-10000, preferably PEG 6000.

In the step (1), the concentration of the polyethylene glycol in the mixed solution of the uridine-containing catalytic solution and the polyethylene glycol is 2% -5% g/mL, preferably 3% g/mL.

In the step (1), the concentration of uridine in the uridine-containing catalyst solution is 150-260 g/L, preferably 200 g/L.

In the step (2), ultrafiltration membrane of 250-3000 Da is adopted for nanofiltration, and the ultrafiltration membrane is used for decoloring and removing PEG 1000-10000; the preferable range is 300-500 Da, and the small molecular pigment substances can be removed while the large molecular impurities are intercepted.

In the step (2), the pressure of the filter membrane is 1-3 MPa.

In the step (3), the vacuum evaporation concentration condition is that the vacuum degree is lower than 100mbar, preferably lower than 50 mbr; the water bath temperature is 45-60 ℃, and preferably 50 ℃.

In the step (3), the concentration of uridine in the uridine concentrated solution is 400-900 g/L, preferably 800 g/L.

In the step (4), the pH is adjusted to 5.0-9.0, preferably 7.0.

In the step (4), after the pH of the obtained uridine concentrated solution is adjusted, adding seed crystals, stirring for crystallization (after stirring for 1-2 hours, crystals are separated out), cooling, and continuing crystallization; preferably, the temperature is reduced to 0-10 ℃ within 10-24 h, preferably to 2-6 ℃ within 8-12 h, and preferably to 4 ℃ within 8-12 h.

In the step (5), the drying is forced air drying, double cone drying or vacuum drying.

In the step (5), the drying temperature is 40-100 ℃, preferably 50-80 ℃; the drying time is 2-6 h, preferably 4 h.

Wherein, the vacuum degree of the vacuum drying is 0-200 mbar, preferably 10-100 mbar.

In the step (5), in order to improve the purity of the crystals and reduce the influence of impurities in the crystallization mother liquor, the crystals are washed by ethanol solution with the concentration of 95-99 percent, and the crystals are collected by centrifugation or suction filtration.

According to the uridine separation and extraction method, the crystallization system is a water system, and the workshop modification and production cost is reduced.

The purity of the uridine crystal prepared and extracted by the method is more than or equal to 98 percent, and the yield is more than or equal to 90 percent. The invention innovatively adopts a double water-phase system to recover cytidine deaminase and further recycle a catalytic enzyme preparation, removes impurities by single-membrane system separation, adopts a water system crystallization process, has the whole separation yield of more than 90 percent and the purity of more than 99 percent, and has the cost and the purity superior to those of the prior art. The invention not only successfully realizes the problem of difficult separation of more impurities, but also has the advantages of high efficiency, low energy consumption and low cost; wherein, the catalytic enzyme is cytidine deaminase obtained by expression with escherichia coli as an expression vector.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the invention firstly adopts a double water phase system to reuse the cytidine deaminase, reduces the production cost, and simultaneously takes the uridine as the component of the double water phases, reduces the introduction of impurities and reduces the separation difficulty.

(2) The invention combines the two aqueous phase system with the membrane separation, greatly saves the time cost, simultaneously improves the recovery rate of biological products uridine and prevents the degradation of uridine.

(3) The crystallization system in the invention is a water system, and no reagent is needed to be added, so that the crystallization cost is reduced. The crystallization process of the invention is completed within 12h to obtain bright white crystals, the yield of one-time crystallization is more than 60%, the yield of crystals is over 90% after multiple crystallization or nested crystallization, meanwhile, the purity of the crystals is more than 98%, and the product quality, yield and cost are higher than those of the prior art.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is the uridine crystalline product of example 1 according to the present invention.

FIG. 2 is the crystal structure of uridine in example 1 of the present invention.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

In the invention, HPLC is adopted to measure uridine, and the specific conditions are as follows:

ZORBAX SD-C188 column (4.6X 150mm), mobile phase 0.6% triethylamine phosphate (pH adjusted to 6.6 with triethylamine): methanol 97: 3, the flow rate is 0.6mL/min, the detection wavelength is 270nm, and the column temperature is 25 ℃.

The uridine-containing catalyst solution described in the following examples was prepared as follows: 1g of cytidine deaminase corresponds to 100g of cytidine substrate, 0.1g of magnesium chloride, 0.1mol/L of sodium hydrogen phosphate and sodium dihydrogen phosphate buffer solution are added, and stirring is carried out at constant temperature of 37 ℃ for more than 4 hours. The enzyme activity of cytidine deaminase is defined as the amount of 1U of enzyme required to convert 1 micromole of cytidine in 1 minute, and the enzyme activity is 40000U/g

The mother liquor is concentrated and then crystallized again in the following examples, namely the mother liquor is subjected to the steps (5) to (7) repeatedly, namely the obtained mother liquor is concentrated to the uridine concentration, then the pH is adjusted, seed crystals are added, and the mother liquor is cooled, crystallized, filtered, washed and dried to carry out crystallization.

Example 1:

1) 3000mL of catalytic solution (uridine content 200g/L) containing uridine, wherein the components of the catalytic solution except uridine mainly comprise cytidine deaminase, escherichia coli broken protein, pigment, organelles and the like.

Wherein the catalytic solution containing uridine is cytidine deaminase used for converting cytidine to form uridine.

2) Adding 90g of PEG6000 into the catalytic liquid obtained in the step 1), fully stirring and uniformly mixing, and standing at normal temperature for 30 min.

3) Collecting the phase-separated solution in the step 2) by a separating funnel to obtain a lower phase solution, returning the upper phase solution to the upstream for enzyme supplementation, and then carrying out the next round of enzyme catalysis reaction, wherein the lower phase solution contains 540g of uridine, and the following yield is calculated.

4) Passing the lower phase solution obtained in the step 3) through a 500Da ultrafiltration membrane, removing macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain uridine, and collecting the filtrate of the filter membrane.

5) Concentrating the permeate obtained in the step 4) to obtain uridine with the concentration of 800g/L, adjusting the pH to 7.0 by using hydrochloric acid, adding 0.1% of seed crystal at the room temperature of 20 ℃, continuously stirring at 200rpm for 1h, precipitating crystals, and carrying out gradient cooling to 4 ℃ for 10 h;

6) and (3) carrying out suction filtration on the crystals obtained in the step 5), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.

7) Putting the crystals obtained in the step 6) into a vacuum drying oven at 40 ℃, and drying for 4h under 10mbar to obtain 378g of uridine crystals. The primary yield of uridine was 70%, the mother liquor was concentrated and crystallized twice to obtain 105.3g, the secondary mother liquor recovery was 65%, the mother liquor was concentrated and crystallized continuously to obtain 29.5g uridine crystals, the tertiary mother liquor crystallization yield was 52%, and the tertiary total recovery was 95%.

The purity of the dried crystal was 99.38%, and the total yield was 95%. The resulting crystals are shown in FIGS. 1 and 2.

Example 2:

2) Adding 60g of PEG1000 into the catalytic liquid obtained in the step 1), fully stirring and uniformly mixing, and standing at normal temperature for 30 min.

3) Collecting the phase-separated solution in the step 2) by a separating funnel to obtain a lower phase solution, returning the upper phase solution to the upstream for enzyme supplementation, and then carrying out the next round of enzyme catalysis reaction, wherein the lower phase solution contains 420g of uridine, and the following yield is calculated.

4) Passing the lower phase solution obtained in the step 3) through an ultrafiltration membrane of 1000Da to remove macromolecular impurities such as pigments, continuously washing the trapped fluid until the trapped fluid does not contain uridine, and collecting the filtrate of the filter membrane.

5) Concentrating the permeate obtained in the step 4) to obtain uridine with the concentration of 700g/L, adjusting the pH to 8.0 by using hydrochloric acid, adding 0.1% of seed crystal at the room temperature of 20 ℃, continuously stirring at 200rpm for 1h, precipitating crystals, and carrying out gradient cooling to 4 ℃ for 10 h;

7) Putting the crystals obtained in the step 6) into a vacuum drying oven at 50 ℃, and drying for 3.5 hours at 50mbar to obtain 281.82g of uridine crystals. The primary yield of uridine was 67.1%, the mother liquor was concentrated and crystallized twice to obtain 86.09g, the secondary mother liquor recovery rate was 62.3%, the mother liquor was concentrated and crystallized continuously to obtain 24.3g uridine crystals, the tertiary mother liquor crystallization yield was 46.6%, and the tertiary total recovery rate was 93.4%.

The purity of the dried crystal was 99.18%, and the total yield was 93.4%.

Example 3:

Wherein the uridine-containing catalytic solution is formed by converting cytidine by cytidine deaminase to form uridine.

2) Adding 150g of PEG10000 into the catalytic liquid obtained in the step 1), fully stirring and uniformly mixing, and standing at normal temperature for 30 min.

3) Collecting the phase-separated solution in the step 2) by a separating funnel to obtain a lower phase solution, returning the upper phase solution to the upstream for enzyme supplementation, and then carrying out the next round of enzyme catalysis reaction, wherein the lower phase contains 554g of uridine, and the following yield is calculated.

4) Passing the lower phase solution obtained in the step 3) through a 3000Da ultrafiltration membrane, removing macromolecular impurities such as pigments, continuously washing the trapped fluid until the trapped fluid does not contain uridine, and collecting the filtrate of the filter membrane.

5) Concentrating the permeate obtained in the step 4) to obtain uridine with the concentration of 900g/L, adjusting the pH to 6.0 by using hydrochloric acid, adding 0.1% at the room temperature of 20 ℃, continuously stirring at 200rpm for 1h, precipitating crystals, and carrying out gradient cooling to 4 ℃ for 10 h;

6) and (4) carrying out suction filtration on the crystals obtained in the step 5), removing a mother solution, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.

7) Putting the crystals obtained in the step 6) into a vacuum drying oven at 40 ℃ and drying for 4h at 20mbar to obtain 411.4g of uridine crystals. The primary yield of uridine was 74.3%, the mother liquor was concentrated and crystallized twice to obtain 96.33g, the secondary mother liquor recovery rate was 67.6%, the mother liquor was concentrated and crystallized continuously to obtain 23.25g uridine crystals, the tertiary mother liquor crystallization yield was 50.2%, and the tertiary total recovery rate was 95.8%.

The purity of the dried crystal was 98.12%, and the total yield was 95.8%.

Example 4:

1) 3000mL of catalytic liquid containing uridine (uridine content 200g/L) contains, besides uridine, cytidine deaminase, broken proteins of Escherichia coli, pigments, organelles and the like.

2) Adding 120g of PEG3000 into the catalytic solution obtained in the step 1), fully stirring and uniformly mixing, and standing at normal temperature for 30 min.

3) Collecting the phase-separated solution in the step 2) by a separating funnel to obtain a lower phase solution, returning the upper phase solution to the upstream for enzyme supplementation, and then carrying out the next round of enzyme catalysis reaction, wherein the lower phase solution contains 480g of uridine, and the following yield is calculated.

4) Passing the lower phase solution obtained in the step 3) through a 2000Da ultrafiltration membrane, removing macromolecular impurities such as pigments, continuously washing the trapped fluid until the trapped fluid does not contain uridine, and collecting the filtrate of the filter membrane.

5) Concentrating the permeate obtained in the step 4) to obtain uridine with the concentration of 600g/L, adjusting the pH to 5.0 by using hydrochloric acid, adding 0.05% of seed crystal at the room temperature of 20 ℃, continuously stirring at 200rpm for 1h, precipitating crystals, and carrying out gradient cooling to 4 ℃ for 10 h;

7) The crystals from step 6) were dried in a vacuum oven at 40 ℃ and 60mbar for 4h, i.e. 329.76g of uridine crystals. The primary yield of uridine was 68.7%, the mother liquor was concentrated and crystallized twice to obtain 96.9g, the secondary mother liquor recovery rate was 64.5%, the mother liquor was concentrated and crystallized continuously to obtain 27.3g uridine crystals, the tertiary mother liquor crystallization yield was 51.2%, and the tertiary total recovery rate was 94.6%.

The purity of the dried crystal was 99.43%, and the total yield was 94.6%.

The present invention provides a method and a concept for separating and extracting uridine from catalytic solution containing uridine, and a method and a way for implementing the technical scheme are many, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. A method for separating and extracting uridine from a catalytic solution containing uridine, which is characterized by comprising the following steps of:

(1) mixing the catalytic solution containing uridine with polyethylene glycol, and separating liquid to obtain a uridine catalytic solution at the lower layer;

(2) carrying out nanofiltration on the obtained uridine catalytic liquid, and collecting nanofiltration permeate;

(3) evaporating and concentrating the nanofiltration permeate to obtain a uridine concentrated solution;

(4) adjusting pH of the obtained uridine concentrated solution, adding seed crystal, stirring and crystallizing.

2. The method according to claim 1, wherein in the step (1), the polyethylene glycol is 1000-10000.

3. The method according to claim 1, wherein in the step (1), the concentration of the polyethylene glycol in the mixed solution of the uridine-containing catalytic solution and the polyethylene glycol is 2% to 5% g/mL, preferably 3% g/mL.

4. The method according to claim 1, wherein in the step (1), the concentration of uridine in the catalytic solution containing uridine is 150-260 g/L, preferably 200 g/L.

5. The method of claim 1, wherein in the step (2), the nanofiltration is performed by using an ultrafiltration membrane of 250-3000 Da.

6. The method according to claim 1, wherein in step (3), the concentration of uridine in the uridine concentrate is 400-900 g/L, preferably 800 g/L.

7. The method according to claim 1, wherein in the step (4), the pH is adjusted to 5.0 to 9.0, preferably 7.0.

8. The method according to claim 1, wherein in the step (4), after the pH of the obtained uridine concentrate is adjusted, seed crystals are added, stirred for crystallization, cooled and crystallized continuously; preferably, the temperature is reduced to 2-6 ℃ for 8-12 h.

9. The method of claim 1, further comprising the steps of;

(5) filtering the crystals obtained in the step (4), leaching with ethanol, and collecting the crystals and the mother liquor; drying the obtained crystals to obtain pure uridine crystals.

10. The process according to claim 9, wherein the mother liquor obtained is crystallized by repeating step (4) again to obtain uridine crystals.