A method for purifying N-acetylneuraminic acid

Abstract

The application provides a purification method of N-acetylneuraminic acid, and belongs to the technical field of bioengineering, and comprises the following steps: mixing a conversion liquid containing N-acetylneuraminic acid with acid to perform acidification, to obtain an acidified liquid; sequentially performing ceramic membrane microfiltration and electrodialysis desalination on the acidified liquid, to obtain a desalted liquid; performing ion exchange resin adsorption, water top washing and elution on the desalted liquid, to obtain an eluate; sequentially performing nanofiltration concentration and re-concentration on the eluate, to obtain a concentrated liquid; and sequentially performing activated carbon decolorization and drying on the concentrated liquid, to obtain N-acetylneuraminic acid crystals. The method provided by the application can prepare N-acetylneuraminic acid crystals with a chromatographic purity of greater than or equal to 99%, a content of greater than or equal to 99%, a whiteness of greater than or equal to 90 and a water content of less than or equal to 2%. The method is simple to operate, environmentally friendly and convenient for industrialized production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a method for purifying N-acetylneuraminic acid. BACKGROUND

[0002] N-acetylneuraminic acid, also known as sialic acid, is widely distributed in nature. Sialic acid is widely present in various biological tissues and is an important component of glycoprotein, oligosaccharide and glycolipid. It usually exists in the form of glycoside at the end of glycoprotein and glycolipid, especially in the brain, nerve tissue, blood, submandibular gland, mucin and colostrum of mammals. However, free sialic acid can also be found in serum, body fluid and urine.

[0003] There are two major biological functions of sialic acid, i.e. receptor function of sialic acid itself and masking function of covering other molecules. In recent years, it has also been found that sialic acid and its derivatives play an important role in the regulation of various life activities and are closely related to many diseases. In the aspects of inhibiting sialyltransferase and anti-cancer metastasis, promoting nerve cell growth and anti-Alzheimer's disease, inhibiting sialidase and anti-virus, inhibiting leukocyte adhesion and anti-inflammation, in addition, sialic acid also has a great effect on physiological functions such as controlling cell mucus concentration, anti-recognition and anti-tumor.

[0004] The industrialized preparation methods of sialic acid mainly include natural product extraction method, chemical synthesis method and enzyme synthesis method. The natural product extraction method is the most original production method of sialic acid, which is obtained by special steps from natural products such as bird's nest, egg yolk and whey. Although sialic acid is widely distributed in nature, it is inevitably affected in development and utilization due to low content, complex structure, tedious extraction process, low recovery rate and serious environmental pollution. The chemical synthesis method is to synthesize sialic acid by using certain sugar or non-sugar substances as substrates and chemical catalysts. Although this method can realize large-scale production, the reaction conditions are harsh, the production process is complex, and most of the intermediate products are not conducive to the later separation and purification process, which is difficult to meet the demand of industrial production while ensuring the quality of the product. With the development of biotechnology, the enzyme synthesis method has become the most competitive production method of sialic acid in the market, which has obvious advantages such as mild conditions, strong specificity, high conversion rate and high purity of the obtained product. In the sialic acid enzyme conversion liquid, in addition to the main components such as sodium pyruvate, N-acetylglucosamine, N-acetylmannosamine and sialic acid, there are also microbial cells and their fragments, impurities, inorganic salts and pigments. Therefore, it is very important to develop a high-efficiency and low-cost sialic acid extraction method to promote the industrialization of sialic acid.

[0005] In the sialic acid enzymatic conversion solution, in addition to the main components such as the reaction substrate sodium pyruvate, N-acetylglucosamine, N-acetymannosamine, and the reaction product sialic acid, there are also various other components including microbial cells and their debris, miscellaneous proteins, inorganic salts, and pigments. Therefore, developing a highly efficient and low-cost sialic acid extraction method is crucial for promoting the industrialization of sialic acid. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a method for purifying N-acetylneuraminic acid. The method provided by this invention yields N-acetylneuraminic acid with high chromatographic purity, content, and whiteness, and the process is simple and environmentally friendly.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for purifying N-acetylneuraminic acid, comprising the following steps:

[0009] The conversion solution containing N-acetylneuraminic acid was mixed with acid and then acidified to obtain an acidified solution.

[0010] The acidified solution is sequentially passed through ceramic membrane microfiltration and electrodialysis to remove salt, resulting in a desalinated solution.

[0011] The desalination solution is adsorbed by ion exchange resin, washed with water, and eluted to obtain an eluent.

[0012] The eluent was subjected to nanofiltration concentration and further concentration in sequence to obtain a concentrated solution;

[0013] The concentrated solution was sequentially decolorized with activated carbon and dried to obtain N-acetylneuraminic acid crystals.

[0014] Preferably, the conversion solution containing N-acetylneuraminic acid is a conversion solution containing N-acetylneuraminic acid obtained by conversion using immobilized dual enzymes.

[0015] Preferably, the pH value of the acidification solution is 1.8 to 2.5.

[0016] Preferably, the diameter of the pores in the ceramic membrane is 50–100 nm.

[0017] Preferably, the conductivity of the desalination solution is 2–4 mS / cm. 2 .

[0018] Preferably, the ion exchange resin is a strong base anion exchange resin; the degree of water washing is such that the concentration of N-acetylglucosamine in the resulting wash solution is ≤50μg / ml; and the eluent is an aqueous formic acid solution.

[0019] Preferably, the nanofiltration membrane used for nanofiltration concentration has a molecular weight cutoff of 50 to 300 Daltons; and the mass concentration of N-acetylneuraminic acid in the concentrate obtained by nanofiltration concentration is 10 to 20%.

[0020] Preferably, the mass concentration of N-acetylneuraminic acid in the concentrate obtained after the second concentration is 30-40%.

[0021] Preferably, the amount of activated carbon used is 1-5% of the volume of the concentrated liquid; the temperature for decolorization of the activated carbon is 40-70°C, and the time is 15-50 min.

[0022] Preferably, the N-acetylneuraminic acid crystals have a chromatographic purity ≥99%, a content ≥99%, a whiteness ≥90, and a moisture content ≤2%.

[0023] Beneficial technical effects:

[0024] This invention provides a method for purifying N-acetylneuraminic acid, comprising the following steps: mixing a conversion solution containing N-acetylneuraminic acid with an acid and then acidifying it to obtain an acidified solution; sequentially passing the acidified solution through ceramic membrane microfiltration and electrodialysis for desalination to obtain a desalted solution; passing the desalted solution through ion exchange resin adsorption, water top washing, and elution to obtain an eluent; sequentially subjecting the eluent to nanofiltration concentration and further concentration to obtain a concentrated solution; and sequentially subjecting the concentrated solution to activated carbon decolorization and drying to obtain N-acetylneuraminic acid crystals. The method provided by this invention yields N-acetylneuraminic acid crystals with a chromatographic purity ≥99%, a content ≥99%, a whiteness ≥90, and a water content ≤2%. The method is simple to operate, environmentally friendly, and suitable for industrial production. Detailed Implementation

[0025] This invention provides a method for purifying N-acetylneuraminic acid, comprising the following steps:

[0026] The conversion solution containing N-acetylneuraminic acid was mixed with acid and then acidified to obtain an acidified solution.

[0027] The acidified solution is sequentially passed through ceramic membrane microfiltration and electrodialysis to remove salt, resulting in a desalinated solution.

[0028] The desalination solution is adsorbed by ion exchange resin, washed with water, and eluted to obtain an eluent.

[0029] The eluent was subjected to nanofiltration concentration and further concentration in sequence to obtain a concentrated solution;

[0030] The concentrated solution was sequentially decolorized with activated carbon and dried to obtain N-acetylneuraminic acid crystals.

[0031] In this invention, a conversion solution containing N-acetylneuraminic acid is mixed with acid and then acidified to obtain an acidified solution.

[0032] In this invention, the conversion solution containing N-acetylneuraminic acid is preferably a conversion solution containing N-acetylneuraminic acid obtained by conversion with immobilized dual enzymes, and more preferably an N-acetylneuraminic acid conversion solution prepared by mixing N-acetylglucosamine and sodium pyruvate with immobilized N-acetylglucosamine-2-epomerase and immobilized N-acetylneuraminic acid aldolase at pH 7 and temperature 30°C.

[0033] The N-acetylneuraminic acid conversion solution preferably comprises the following components in weight percentage: 25-30% sodium pyruvate, 40-45% N-acetylglucosamine, and the remainder being suspended matter and miscellaneous proteins.

[0034] In this invention, the acid is preferably concentrated hydrochloric acid or concentrated sulfuric acid. There is no particular limitation on the amount of acid used, as long as it is sufficient to achieve the required pH value of the acidification solution. The pH value of the acidification solution in this invention is preferably 1.8–2.5, more preferably 2.0–2.3. This invention uses acidification to bring the pH value to near the isoelectric point of electrodialysis, facilitating electrodialysis treatment.

[0035] After obtaining the acidified solution, the present invention sequentially passes the acidified solution through ceramic membrane microfiltration and electrodialysis to desalinate it, thereby obtaining a desalinated solution.

[0036] In this invention, the diameter of the pores in the ceramic membrane is preferably 50-100 nm, more preferably 75-85 nm.

[0037] Specifically, the present invention involves microfiltration of the acidified solution using a ceramic membrane microfilter to obtain a concentrated solution and a first clarified solution; further washing of the concentrated solution to obtain a second clarified solution; and combining the first and second clarified solutions for electrodialysis desalination.

[0038] In this invention, the volume of the first clarified liquid is preferably 85-95% of the volume of the acidified liquid, more preferably 90%; the volume of the second clarified liquid is 25-35% of the volume of the acidified liquid, more preferably 30%.

[0039] In this invention, the conductivity of the desalination solution is preferably 2-4 mS / cm. 2 More preferably 3mS / cm 2 This invention uses a ceramic membrane to filter out suspended solids in the conversion solution, thus clarifying the material.

[0040] After obtaining the desalination solution, the present invention adsorbs the desalination solution through ion exchange resin, washes it with water, and elutes it to obtain an eluent.

[0041] In this invention, the ion exchange resin is preferably a strong base anion exchange resin, more preferably an anion exchange resin containing quaternary ammonium functional groups; the degree of water washing is such that the concentration of N-acetylglucosamine in the resulting washing solution is ≤50 μg / ml; the eluent is an aqueous formic acid solution; the mass concentration of the aqueous formic acid solution is preferably 5-20%, more preferably 10-15%.

[0042] After obtaining the eluent, the present invention performs nanofiltration concentration and further concentration on the eluent in sequence to obtain a concentrated solution.

[0043] In this invention, the nanofiltration membrane used for nanofiltration concentration has a molecular weight cutoff of 50-300 Daltons, more preferably 100-250 Daltons, and most preferably 150-200 Daltons; the mass concentration of N-acetylneuraminic acid in the concentrate obtained by nanofiltration concentration is preferably 10-20%, more preferably 15%; and the mass concentration of N-acetylneuraminic acid in the concentrate obtained by re-concentration is 30-40%, more preferably 35%.

[0044] After obtaining the concentrate, the present invention sequentially decolorizes and dries the concentrate with activated carbon to obtain N-acetylneuraminic acid crystals.

[0045] In this invention, the amount of activated carbon used is preferably 1-5% of the volume of the concentrated liquid, more preferably 2-3%; the decolorization temperature of the activated carbon is preferably 40-70℃, more preferably 50-60℃; and the decolorization time of the activated carbon is preferably 15-50 min, more preferably 20-40 min, and most preferably 25-30 min.

[0046] In this invention, the drying is preferably spray drying; the N-acetylneuraminic acid crystals have a chromatographic purity ≥99%, a content ≥99%, a whiteness ≥90, and a moisture content ≤2%.

[0047] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0048] Example 1

[0049] Take 2m 3 The N-acetylneuraminic acid conversion solution was adjusted to pH 2.0 with concentrated sulfuric acid and then microfiltered using a ceramic membrane microfiltration system (50 nm) to obtain a 1.8 m... 3 After clarifying the liquid, the concentrated solution is then washed with drinking water, followed by microfiltration and a 0.6m wash. 3 After water collection, a total of 2.4m was collected. 3 Filtrate. The microfiltrate was desalted by electrodialysis until the conductivity of the feed solution was 2 mS / cm. 2 2.3m of desalinated solution was obtained. 3The desalination solution was passed through an ion exchange column (packing volume 1m). 3 Anion exchange resin was loaded onto the column at a flow rate of 1 BV / h. After loading, 4 m... 3 The column was washed with water from the top, followed by elution with 5% formic acid. 3.3 ml of the eluent was collected. 3 The eluent was concentrated and purified by nanofiltration to obtain a 0.8 mL nanofiltration concentrate. 3 The nanofiltration concentrate was further concentrated in a single tank until the N-acetylneuraminic acid concentration reached 30%. After being transferred to a decolorization tank, 8 kg of activated carbon was added, and the solution was decolorized at 70°C for 30 min. The solution was then filtered to obtain a decolorized solution, which was spray-dried to obtain 98 kg of N-acetylneuraminic acid with a moisture content of 1.5%, a chromatographic purity of 99.5%, and a mass percentage of 99.2% as determined by HPLC external standard method, and a whiteness of 92.

[0050] Example 2

[0051] Take 2m 3 The N-acetylneuraminic acid conversion solution was adjusted to pH 2.0 with concentrated sulfuric acid and then microfiltered using a ceramic membrane microfiltration system (50 nm) to obtain a 1.8 m... 3 After clarifying the liquid, the concentrated solution is then washed with drinking water, followed by microfiltration and a 0.6m wash. 3 After water collection, a total of 2.4m was collected. 3 Filtrate. The microfiltrate was desalted by electrodialysis until the conductivity of the feed solution was 3 mS / cm. 2 2.3m of desalinated solution was obtained. 3 The desalination solution was passed through an ion exchange column (packing volume 1m). 3 Anion exchange resin was loaded onto the column at a flow rate of 1 BV / h. After loading, 4 m... 3 The column was washed with water from the top, followed by elution with 10% formic acid. 3.0 ml of the eluent was collected. 3 The eluent was concentrated and purified by nanofiltration to obtain a 0.8 mL nanofiltration concentrate. 3 The nanofiltration concentrate was further concentrated in a single tank until the N-acetylneuraminic acid concentration reached 30%. After being transferred to a decolorization tank, 5 kg of activated carbon was added, and the solution was decolorized at 70°C for 30 min. The solution was then filtered to obtain a decolorized solution. The decolorized solution was spray-dried to obtain 102 kg of N-acetylneuraminic acid with a moisture content of 1.6%, a chromatographic purity of 99.2%, and a mass percentage of 99.0% as determined by HPLC external standard method, and a whiteness of 90.

[0052] Example 3

[0053] Take 2m 3 The N-acetylneuraminic acid conversion solution was adjusted to pH 2.0 with concentrated sulfuric acid and then microfiltered using a ceramic membrane microfiltration system (50 nm) to obtain a 1.8 m... 3 After clarifying the liquid, the concentrated solution is then washed with drinking water, followed by microfiltration and a 0.6m wash. 3After water collection, a total of 2.4m was collected. 3 Filtrate. The microfiltrate was desalted by electrodialysis until the conductivity of the feed solution was 4 mS / cm. 2 2.3m of desalinated solution was obtained. 3 The desalination solution was passed through an ion exchange column (packing volume 1m). 3 Anion exchange resin was loaded onto the column at a flow rate of 1 BV / h. After loading, 4 m... 3 The column was washed with water from the top, followed by elution with 10% formic acid. 3.0 ml of the eluent was collected. 3 The eluent was concentrated and purified by nanofiltration to obtain a 0.8 mL nanofiltration concentrate. 3 The nanofiltration concentrate was further concentrated in a single tank until the N-acetylneuraminic acid concentration reached 40%. After being transferred to a decolorization tank, 10 kg of activated carbon was added, and the solution was decolorized at 70°C for 30 min. The solution was then filtered to obtain a decolorized solution. The decolorized solution was spray-dried to obtain 108 kg of N-acetylneuraminic acid with a moisture content of 1.4%, a chromatographic purity of 99.3%, and a mass percentage content of 99.2% as determined by HPLC external standard method, and a whiteness of 91.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for purifying N-acetylneuraminic acid, characterized in that, Includes the following steps: The conversion solution containing N-acetylneuraminic acid was mixed with acid and then acidified to obtain an acidified solution. The acidified solution is sequentially passed through ceramic membrane microfiltration and electrodialysis to remove salt, resulting in a desalinated solution. The desalination solution is adsorbed by ion exchange resin, washed with water, and eluted to obtain an eluent. The eluent was subjected to nanofiltration concentration and further concentration in sequence to obtain a concentrated solution; The concentrated solution was sequentially decolorized with activated carbon and dried to obtain N-acetylneuraminic acid crystals. The conversion solution containing N-acetylneuraminic acid is a conversion solution containing N-acetylneuraminic acid obtained by conversion with immobilized dual enzymes; the immobilized dual enzymes are immobilized N-acetylglucosamine-2-epimerase and immobilized N-acetylneuraminic acid aldolase. The pH value of the acidification solution is 1.8~2.5; The diameter of the filter pores in the ceramic membrane is 50~100nm; The conductivity of the desalination solution is 2~4 mS / cm 2 ; The ion exchange resin is a strong base anion exchange resin; the degree of water washing is such that the concentration of N-acetylglucosamine in the resulting wash solution is ≤50μg / ml; the eluent is an aqueous formic acid solution. The nanofiltration membrane used for nanofiltration concentration has a molecular weight cutoff of 50-300 Daltons; the mass concentration of N-acetylneuraminic acid in the concentrate obtained by nanofiltration concentration is 10-20%.

2. The purification method according to claim 1, characterized in that, The mass concentration of N-acetylneuraminic acid in the concentrate obtained after the second concentration is 30-40%.

3. The purification method according to claim 1, characterized in that, The amount of activated carbon used is 1-5% of the volume of the concentrated liquid; the decolorization temperature of the activated carbon is 40-70℃, and the time is 15-50 min.

4. The purification method according to claim 1, characterized in that, The N-acetylneuraminic acid crystals have a chromatographic purity ≥99%, a content ≥99%, a whiteness ≥90, and a water content ≤2%.