CN102532208B - Method for continuously separating sialic acid - Google Patents

Abstract

The invention discloses a method for continuously separating sialic acid. The sialic acid is prepared by microbial fermentation using N-acetylglucosamine and sodium pyruvate as substrates, and the fermented liquid is filtered and ultrafiltered to obtain saliva Acid clear liquid, pumped into the continuous ion exchange system equipped with OH ^- type anion exchange resin for adsorption, deionized water for washing impurities, sodium chloride solution for elution, sodium hydroxide solution for resin regeneration, and collected sialic acid containing The eluate was leached with ethanol aqueous solution, and then ethyl acetate was added to dissolve the crystals, and the crystals were dried under reduced pressure to obtain pure sialic acid. The separation and purification method of the present invention is simple and easy to implement, has good effect, not only has low operating cost, but also can realize separation from kilogram level to ton level according to the amount of separation. Excellent results and guaranteed product quality.

Description

A method for continuous separation of sialic acid

technical field

The invention relates to a method for continuously separating sialic acid, which belongs to the technical field of separation and purification of biological products.

Background technique

Sialic acid (Sialic Acid, SA) is a general term for nine-carbon sugar neuraminic acid acylate, which widely exists in a variety of biological tissues. The sialic acid found in most mammalian tissues is mainly N-acetylneuraminic acid, so N-acetylneuraminic acid is usually called sialic acid. Sialic acid is widely distributed in nature. It has been found that sialic acid exists in many organisms. It is usually located at the key position of the sugar part of the outermost layer of the cell membrane and the secreted sugar complex (glycolipid, glycoprotein and lipopolysaccharide). An important material basis for complex structure and function diversification.

There are two types of biological functions of sialic acid, that is, the receptor function that sialic acid itself can be recognized and the masking function that covers 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. , Inhibition of sialidase and anti-virus, inhibition of leukocyte adhesion and anti-inflammation, etc. In addition, sialic acid also plays a great role in controlling the concentration of cell mucus, anti-recognition, anti-tumor and other physiological functions.

The industrial preparation methods of sialic acid mainly include natural product extraction and enzymatic synthesis. Since the content of sialic acid in natural raw materials is relatively low, the separation and purification process of natural product extraction is relatively complicated, the yield is low, and some special equipment is required at the same time, causing large pollution, which will inevitably affect the production of sialic acid derivatives. development and utilization. Enzymatic production of sialic acid has the advantages of high conversion rate, simple extraction, and high product purity, but it requires high raw materials for synthesis and is relatively expensive. At the same time, sialic acid aldolase is not easy to obtain, which limits the expansion of production scale.

In the sialidase conversion solution, in addition to the main components such as the reaction substrate sodium pyruvate, N-acetylglucose (GlcNAc) amine and the reaction product sialic acid, N-acetyl mannosamine (ManNAc), there are microbial cells and Its fragments, miscellaneous proteins, inorganic salts and pigments and other components. Therefore, it is very important to choose an economical and reasonable sialic acid extraction process to improve the market competitiveness of sialic acid. The reported extraction methods and separation methods mainly include:

1 Han Xiaoqing (patent 02110607.x) invented a method for extracting sialic acid from egg yolk powder. Firstly, the egg yolk powder is hydrolyzed, and the hydrolyzate is heated and flocculated to remove protein, and then deionized by strong acid and weak base resin; then, sialic acid is adsorbed by strong base resin, and formic acid is used as eluent; finally, the eluate is concentrated and crystallized, and dried to obtain Sialic acid finished product.

2 Han Xiaoda (patent 03115456.5) selects whey powder as raw material, removes protein by ultrafiltration, and the subsequent extraction steps are similar to Han Xiaoqing's.

3. Xu Ping (patent 200410024222.3) and others invented a method of using cheap sodium lactate as a precursor, synthesizing sialic acid through multi-step coupling biotransformation and separating and purifying sialic acid by ion exchange. After the conversion solution is pretreated by acidification and centrifugation, sialic acid is directly separated by an ion exchange column, and the resins used are commercial strong base resins such as HZ201x8, 330 and 717. The resin needs to be treated with 1N-2N NaOH solution first; then washed with water; finally treated with 1N-3N formic acid solution to convert the resin into formic acid type. During elution, gradient elution is carried out with water first, and then 0.5-3N formic acid solution, and the elution effluent of 1N-3N is collected. The effluent is subjected to vacuum freezing and crystallization to obtain the finished sialic acid product.

However, from the economical analysis of the operation process and extraction, the above method has certain defects. First of all, in the existing sialic acid production process, the fixed bed ion exchange technology is adopted, which is a batch operation mode. There are many disadvantages in this technology. Because the process is discontinuous, the yield is low, and the utilization of the resin on the column is low. The disadvantages are low efficiency, large consumption of eluent, large amount of resin, and large amount of wastewater discharge. At the same time, formic acid-type resin is selected as the separation medium, and the steps of resin pretreatment and regeneration are cumbersome, and formic acid is corrosive and easy to contaminate sialic acid. Therefore, it is very important to develop high-efficiency and low-cost sialic acid extraction methods to promote the industrialization of sialic acid.

Contents of the invention

The main purpose of the present invention is to overcome the disadvantages of high production cost, cumbersome operation, poor separation and purification effect, difficult scale, only intermittent operation and unsuitable for industrial production in the process of using fixed bed ion exchange technology to separate sialic acid in the prior art . A new type of separation medium is proposed, and a method for continuous separation of sialic acid is developed based on this medium. The process can increase the utilization rate of the resin (close to 95%), reduce the consumption of eluent, avoid the activation and regeneration of the resin, save the consumption of acid and alkali, and reduce the discharge of waste liquid. Thereby realizing the purpose of low cost, high yield and continuous production of sialic acid.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A method for continuously separating sialic acid, wherein the sialic acid is prepared by microbial fermentation using N-acetylglucosamine and sodium pyruvate as substrates, and the fermented liquid is filtered and ultrafiltered to obtain a sialic acid serum, Pump into a continuous ion exchange system equipped with OH-type anion exchange resin for adsorption, deionized water for washing impurities, sodium chloride solution for elution, sodium hydroxide solution for resin regeneration, and collect the eluent containing sialic acid After leaching with ethanol aqueous solution, ethyl acetate was added to dissolve the crystals, and the crystals were dried under reduced pressure to obtain pure sialic acid.

Wherein, the microbial fermentation preparation method of sialic acid is to obtain two high-density cultures of recombinant Escherichia coli containing slr (expressing isomerase) and recombinant Escherichia coli containing NanA (expressing aldolase) respectively. A highly expressed Escherichia coli cell. At 37°C, with N-acetylglucosamine (GlcNac) and excess sodium pyruvate as substrates, the obtained two recombinant Escherichia coli were mixed and cultured for 36 hours to catalyze the synthesis of sialic acid, specific references (Yinan Zhang, Fei Tao, Miaofen Du et al (2010) An efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system ApplMicrobiol Biotechnol 86: 481-489).

Wherein, in the orotic acid hydrolyzate, the concentration of sialic acid is 1-20 g/L.

Wherein, the ultrafiltration treatment has a molecular weight cut-off of 10,000-80,000 Daltons.

Wherein, the anion exchange resin has a styrene-divinylbenzene copolymer as a skeleton and a quaternary ammonium group (-N ⁺ R ₃ ) as a functional group.

Wherein, the particle size of the anion exchange resin is 0.315-1.25mm, the wet apparent density is 0.66-0.75g/ml, and the water content is 42-48%.

Wherein, the continuous ion exchange system is a continuously operated ion exchange system with 6-60 ion exchange columns connected in series. After the resin is saturated, the ion exchange column in the adsorption section is immediately removed from the adsorption section and sent to the impurity washing section for impurity washing. After washing, immediately remove the impurity washing section and enter the elution section for elution. Immediately remove the elution section and send it to the regeneration section for regeneration. After the regeneration cleaning is completed, immediately remove the regeneration section and send it to the adsorption section for adsorption. This cycle of operation process, and the state switching of the first ion exchange column in each section is carried out synchronously , and ensure that at least one ion exchange column is in the adsorption phase.

The above continuous ion exchange system preferably has 5-40 ion exchange columns connected in series, more preferably 8-24 ion exchange columns connected in series.

In the continuous ion exchange system, the number of anion exchange columns in the adsorption section, impurity washing section, elution section and regeneration section is 1-20, preferably 1-10, more preferably 2-6.

The continuous ion-exchange chromatographic technology uses a turntable to drive the resin column to rotate continuously and pass through different separation zones. The resin column is continuously indexed and passes through each notch, and the material and flow through the notch are controlled separately by each notch. The dispensing valve rotates in tandem to distribute the liquid into the resin column and collect the final product. When the saturated resin column leaves the adsorption zone, the regenerated and washed fresh resin bed immediately enters the adsorption zone. In the test, the number of resin columns can be adjusted in series to ensure the continuous stability of the output quality and concentration.

Wherein, the concentration of the sodium chloride solution is 0.001-2M, preferably 0.1-0.8M; the concentration of the sodium hydroxide solution is 0.001-2M, preferably 0.1-2M.

Wherein, the concentration of the aqueous ethanol solution is 5-40 g/L, and the amount of the aqueous ethanol solution is 1-2 times the volume of the eluent.

Wherein, the amount of ethyl acetate added is 0.1-1 times of the mixed volume of ethanol aqueous solution and eluent.

Beneficial effects: the method for separating and purifying sialic acid by continuous ion exchange according to the present invention has the following advantages:

1) Compared with the traditional ion exchange system, its equipment is compact, the system is simplified, the pipeline is reduced, the floor area is reduced by 40%, it has better flexibility and less moving parts.

2) Resin consumption is saved by 65%. The resin is always in a state of performing a function. 20% of the resin is always in adsorption regeneration, 20% regeneration, 30% wash/elution.

3) Alkali regenerant is adopted, the utilization rate is improved and the dosage is reduced by more than 40%, and the product yield reaches more than 99%.

4) 30-40% regeneration solution, 40-50% eluent and 60% waste solution are reduced. The elution yield is increased by 2-3 times, and the concentration of the eluent is increased by 5-10 times.

Therefore the separation and purification method of the present invention is simple and easy, and effect is good, and not only its equipment investment, operating cost are low, and can realize the separation from kilogram level to ton level according to the amount of separation, by the obtained product of the present invention in yield and Excellent results have been obtained in terms of product quality, which guarantees product quality. At the same time, the amount of regeneration solution, washing solution and eluent is minimized. At the same time, the concentration of the isolated product is increased, and crystallization and purification can be directly carried out.

Description of drawings

FIG. 1 is a schematic diagram of the continuous ion exchange system of the present invention in state 1 .

FIG. 2 is a schematic diagram of the continuous ion exchange system of the present invention in state 2. FIG.

Fig. 3 is a schematic diagram of the effect of pH value on the adsorption amount of sialic acid.

Fig. 4 Elution profile of sialic acid.

Figure 5. Biosynthesis diagram of sialic acid. Among them, the two highly expressed E. coli cells were E.coli K12/pBVS and E.coli K12/pVN, the epimerase was GlcNAc 2-isomerase, and the aldolase was Neu5Ac aldolase.

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.

The purity of sialic acid was detected by high performance liquid chromatography. The best conditions for HPLC separation of sialic acid are chromatographic column: a Bio-Rad Aminex HPX-87H column (300×7.8mm), mobile phase: 10mM H ₂ SO ₄ , flow rate: 0.4ml/min, detection wavelength 210nm, column temperature: 55°C, injection volume: 20μL. Quantification by external standard method.

The anion-exchange resin used in the following examples is based on styrene-divinylbenzene copolymer as a skeleton, and a quaternary amino group is a functional group. The particle size is 0.315-1.25mm, the wet apparent density is 0.66-0.75g/ml, and the water content is 42-48%, synthesized by Nanjing Tongkai Zhaoye Biotechnology Co., Ltd.

The continuous ion exchange system used in the following examples is a continuously operated ion exchange system in which 6-60 ion exchange columns are connected in series. After the resin is saturated, the ion exchange column in the adsorption section is immediately removed from the adsorption section and sent to the impurity washing section for impurity washing. After washing, immediately remove the impurity washing section and enter the elution section for elution. Immediately remove the elution section and send it to the regeneration section for regeneration. After the regeneration cleaning is completed, immediately remove the regeneration section and send it to the adsorption section for adsorption. This cycle of operation process, and the state switching of the first ion exchange column in each section is carried out synchronously , and ensure that at least one ion exchange column is in the adsorption phase. For example, adopt the process form of 4 adsorption zones, 4 scrubbing zones, 6 elution zones, and 4 regeneration zones. Figures 1 and 2 show the operation of the continuous ion exchange system of this process, in which the adsorption area, impurity washing area, elution area, and regeneration area are marked respectively. In Figure 1, the ion exchange column in the adsorption area The fourth ion exchange column can protect the adsorption area of the entire ion exchange column from being penetrated by the upper column liquid and cause losses. When the first ion exchange column in the adsorption area, column 4, is saturated, it will be removed from the adsorption area. Enter the impurity washing area for impurity washing, becoming the last column in the impurity washing area (that is, the state in Figure 2), and the first ion exchange column 8 in the impurity washing area has just completed the impurity washing under the control of the impurity washing flow rate. , enter the elution zone, become the last column in the elution zone (i.e. the state of Figure 2) and make the first column 14 in the elution zone also eluted by controlling the elution flow rate, and move into the last column in the regeneration zone Carry out resin regeneration (that is, the state of FIG. 2), the first column in the regeneration section, that is, the column 18, is also regenerated, and the last one that moves into the adsorption zone is adsorbed (that is, the state of FIG. 2). In this way, by controlling the flow rate of each zone and the switching of valves, the four zones can maintain the same rhythm to work. The flow rate of each zone is affected by the conditions of each section. In principle, the synchronous operation of each section is guaranteed. Those skilled in the art can adjust the flow rate of the mobile phase according to the state of the resin. By controlling the flow rate of each zone and the number of ion exchange columns in each zone, the switching time of the inlet and outlet is determined, and finally the switching of each zone of the entire system is achieved synchronously.

The preparation method of following embodiment sialic acid fermented liquid is as follows:

The fermentation medium of cultivating escherichia coli is common LB medium (based on the weight percentage of medium, tryptone 1%, yeast extract 0.5%, sodium chloride 1%, pH value is 7.5, can add simultaneously if necessary 0.1g/L of penicillin). Cultivation method: E.coli K12 comes from the American Type Culture Collection (abbreviated as ATCC-25404, which is used as the initial strain to construct a recombinant plasmid, and these E. isomerase) and recombinant Escherichia coli containing NanA (expressing aldolase) were respectively carried out high-density culture to obtain two highly expressed Escherichia coli cells. Acetylglucosamine (GlcNac) and excess 2.2M sodium pyruvate were used as substrate and carbon source, and the obtained two recombinant Escherichia coli were mixed and cultured for 36 hours to catalyze the synthesis of sialic acid. The yield of sialic acid in the above reaction was 21.2g/L.

Example 1:

After the fermentation broth is filtered, most of the protein is removed through an ultrafiltration membrane with a molecular weight cut-off of 10,000 Daltons, and the supernatant is diluted to a concentration of sialic acid of about 5g/L, and then pumped into a continuous ion exchange machine filled with anion exchange resin. system. A continuous ion exchange system consisting of 12 anion exchange columns is adopted, with 3 columns in the adsorption area, 3 columns in the impurity washing area, 4 columns in the elution area, and 2 columns in the regeneration area. Each zone can be used in series to increase the concentration of the target product effluent. Each ion exchange column was loaded with 100 g of resin. The adsorption flow rate is 3ml/min, and the adsorption capacity is 0.341g/g wet resin; the washing solution is deionized water, and the flow rate is 3ml/min, and the washing is done until the effluent is free of impurities (use HPLC to determine the first place in the washing area). No impurity flows out from the root ion exchange column); The eluent is the sodium chloride solution of 0.1mol/L, and the elution flow rate is 4ml/min, and the elution is complete (the first ion exchange column in the elution area is determined to be eluted by HPLC detection) At the same time, a sodium hydroxide solution with a concentration of 1mol/L will be pumped into the ion exchange column located in the regeneration area for regeneration. After the regeneration is completed, it will be rinsed with deionized water until neutral. The eluent of sialic acid uses 10g/L ethanol aqueous solution, wherein the volume of ethanol aqueous solution is 1 times of eluent, after leaching, use 0.2 times volume of ethyl acetate to dissolve and crystallize, and obtain the sialic acid that purity is 96%. The rate is 97.2%. Among them, 216ml of 1.0mol/L sodium hydroxide is used for resin regeneration, which saves 40% of regeneration solution compared with single column, and 210ml of 0.1N sodium chloride is used for elution, which saves 30% of elution compared with single column liquid. The elution yield is 0.055g (sialic acid)/g (resin amount)*min, which is 1.375 times higher than the single column elution yield, and the concentration of eluted sialic acid is 4 times that of the original solution.

Example 2:

After the fermentation broth is filtered, most of the protein is removed through an ultrafiltration membrane with a molecular weight cut-off of 30,000 Daltons, the supernatant is diluted to a concentration of sialic acid of about 10 g/L, and pumped into a continuous ion exchange machine filled with anion exchange resin. system. A continuous ion exchange system consisting of 14 anion exchange columns is adopted, 4 in the adsorption area, 3 in the impurity washing area, 4 in the elution area, and 3 in the regeneration area. Each zone can be connected in series to increase the concentration of the target product effluent. Each ion exchange column was packed with 400g resin. The adsorption flow rate is 15ml/min, and the adsorption capacity is 0.244g/g wet resin; the washing solution is deionized water, and the washing flow rate is 25ml/min, and the washing is performed until the effluent is free of impurities (use HPLC to determine the area of the washing area). The first ion-exchange column has no impurity to flow out); The eluent is the sodium chloride solution of 0.5mol/L, and the elution flow rate is 15ml/min, and the elution is complete (the first ion-exchange column in the elution area is determined by HPLC detection). The column is completely eluted); at the same time, a sodium hydroxide solution with a concentration of 2mol/L will be pumped into the ion exchange column located in the regeneration area for regeneration, and after the regeneration is completed, it will be rinsed with deionized water until neutral. The eluent of sialic acid uses 15g/L ethanol aqueous solution, wherein the volume of ethanol aqueous solution is 1.2 times of eluent, with 0.5 times of volume ethyl acetate dissolution crystallization after leaching, obtains the sialic acid that purity is 97.5%. The rate is 98.1%. Among them, 208ml of 1.0mol/L sodium hydroxide is used for resin regeneration, which saves 42% of the regeneration solution compared with the single column, and 200ml of 0.1N sodium chloride is used for elution at the same time, which saves 34% of the elution compared with the single column liquid. The elution yield is 0.075g (sialic acid)/g (resin amount)*min, which is 1.875 times higher than the single column elution yield, and the concentration of eluted sialic acid is 5 times that of the original solution.

Example 3:

After the fermentation broth is filtered, most of the protein is removed through an ultrafiltration membrane with a molecular weight cut-off of 40,000 Daltons, and the supernatant is diluted to a concentration of sialic acid of about 15g/L, and then pumped into a continuous ion exchange machine filled with anion exchange resin. system. A continuous ion exchange system consisting of 14 anion exchange columns is adopted, 4 in the adsorption area, 3 in the impurity washing area, 4 in the elution area, and 3 in the regeneration area. Each zone can be connected in series to increase the concentration of the target product effluent. Each ion exchange column is filled with 1.2Kg resin. The adsorption flow rate is 60ml/min, and the adsorption capacity is 0.244g/g wet resin; the washing liquid is deionized water, and the washing flow rate is 50ml/min. An ion-exchange column has no impurity to flow out); The eluent is the sodium chloride solution of 0.8mol/L, and the elution flow rate is 60ml/min, and the elution is complete (the first ion-exchange column in the elution area is determined by HPLC detection Complete elution); at the same time, pump a sodium hydroxide solution with a concentration of 2 mol/L into the ion exchange column located in the regeneration area for regeneration, and rinse with deionized water until neutral after regeneration is completed. The eluent of sialic acid uses 20g/L ethanol aqueous solution, wherein the volume of ethanol aqueous solution is 1.5 times of eluent, after leaching, use 0.8 times volume of ethyl acetate to dissolve and crystallize, obtain the sialic acid that purity is 98%, receive The rate is 98.1%. Among them, 200ml of 1.0mol/L sodium hydroxide is used for resin regeneration, which saves 44% of regeneration solution compared with single column, and 192ml of 0.1N sodium chloride is used for elution, which saves 36% of elution compared with single column liquid. The elution yield is 0.085g (sialic acid)/g (resin amount)*min, which is 2.125 times higher than the single column elution yield, and the concentration of eluted sialic acid is 6 times that of the original solution.

Example 4:

After the fermentation broth is filtered, most of the protein is removed through an ultrafiltration membrane with a molecular weight cut-off of 60,000 Daltons, and the supernatant is diluted to a concentration of sialic acid of about 20g/L, and then pumped into a continuous ion exchange machine filled with anion exchange resin. system. A continuous ion exchange system consisting of 16 anion exchange columns is adopted, including 4 in the adsorption area, 4 in the impurity washing area, 4 in the elution area, and 4 in the regeneration area. Each zone can be connected in series to increase the concentration of the target product effluent. Each ion exchange column is packed with 4Kg resin. The adsorption flow rate is 200ml/min, and the adsorption capacity is 0.308g/g wet resin; the washing solution is deionized water, and the washing flow rate is 160ml/min, and the washing is done until there is no impurities (use HPLC to determine the first place in the washing area). No impurity flows out from the root ion exchange column); The eluent is the sodium chloride solution of 0.5mol/L, and the elution flow rate is 200ml/min, and the elution is complete (the first ion exchange column in the elution area is determined to be eluted by HPLC detection) deionized completely); at the same time, a sodium hydroxide solution with a concentration of 2mol/L will be pumped into the ion exchange column located in the regeneration area for regeneration, and after the regeneration is completed, it will be rinsed with deionized water until neutral. The eluent of sialic acid uses 30g/L ethanol aqueous solution, wherein the volume of ethanol aqueous solution is 1.8 times of eluent, after leaching, use 1 times volume of ethyl acetate to dissolve and crystallize, and obtain the sialic acid that purity is 99.4%. The rate is 98.8%. Among them, 194ml of 1.0mol/L sodium hydroxide is used for resin regeneration, which saves 46% of regeneration solution compared with single column, and 185ml of 0.1N sodium chloride is used for elution, which saves 38.5% of elution compared with single column liquid. The elution yield is 0.105g (sialic acid)/g (resin amount)*min, which is 2.265 times higher than the single column elution yield, and the concentration of eluted sialic acid is 8 times that of the original solution.

Example 5:

After the fermentation broth is filtered, most of the protein is removed through an ultrafiltration membrane with a molecular weight cut-off of 80,000 Daltons, and the supernatant is diluted to a concentration of sialic acid of about 10g/L, and then pumped into a continuous ion exchange machine filled with anion exchange resin. system. A continuous ion exchange system consisting of 18 anion exchange columns is adopted, 4 in the adsorption area, 4 in the impurity washing area, 6 in the elution area, and 4 in the regeneration area. Each zone can be connected in series to increase the concentration of the target product effluent. Each ion exchange column is filled with 8Kg resin. The adsorption flow rate is 400ml/min, the adsorption capacity is 0.328g/g wet resin, the cleaning fluid is deionized water, and the cleaning flow rate is 320ml/min; The first ion-exchange column has no impurity to flow out); The eluent is a solution of 0.8mol/L sodium chloride, and the elution flow rate is 400ml/min, and the elution is complete (detection with HPLC to determine the first ion-exchange column in the elution zone The column is completely eluted); at the same time, a sodium hydroxide solution with a concentration of 1.5mol/L will be pumped into the ion exchange column located in the regeneration area for regeneration, and after the regeneration is completed, it will be rinsed with deionized water until neutral. The eluent of sialic acid uses 40g/L ethanol aqueous solution, wherein the volume of ethanol aqueous solution is 2 times of eluent, after leaching, use 0.8 times volume of ethyl acetate to dissolve and crystallize, and obtain the sialic acid that purity is 99.5%. The rate is 99.1%. Among them, 180ml of 1.0mol/L sodium hydroxide is used for resin regeneration, which saves 50% of regeneration solution compared with single column, and 180ml of 0.1N sodium chloride is used for elution, which saves 40% of elution compared with single column liquid. The elution yield is 0.125g (sialic acid)/g (resin amount)*min, which is 3.125 times higher than the single column elution yield, and the concentration of eluted sialic acid is 10 times that of the original solution.

Comparative example 1:

Compared with Example 5, single-column fixed-bed adsorption, impurity washing and elution experiments were carried out, and the filling amount of resin was 100g. The column concentration of sialic acid fermentation liquid is 5g/L, the adsorption flow rate is 3mL/min, and the adsorption capacity is 0.289g/g wet resin; Up to impurities (use HPLC to confirm that there is no flow out of impurities); the eluent is 0.1mol/L sodium chloride solution, the elution flow rate is 4ml/min, and the elution is complete (use HPLC to determine that elution does not flow out of sialic acid) ;Finally pump a sodium hydroxide solution with a concentration of 1.0mol/L to the ion exchange column to be regenerated for regeneration, and rinse with deionized water after the regeneration is completed. The eluate of sialic acid was leached with 10 g/L absolute ethanol and crystallized with 0.2 times the volume of ethyl acetate to obtain sialic acid with a purity of 98.6% and a yield of 97.9%. And the sodium hydroxide 200ml of 1.0mol/L is used for the resin regeneration of embodiment 5, and 360ml is used for the comparative example, and 180ml of 0.1N sodium chloride is used for elution in the embodiment 5, and 300ml is used for the comparative example. Now the elution yield rate of single column is 0.04g (sialic acid)/g (resin amount)*min.

Claims (2)

1. continuous separate is from a sialic method, and described sialic acid is to take N-Acetyl-D-glucosamine and Sodium.alpha.-ketopropionate as substrate is fermented and made by microorganism, it is characterized in that, by fermented liquid by filtering, after uf processing sialic acid clear liquid, pump into OH be housed ^-in the continuous ionic exchange system of type anionite-exchange resin, adsorb, deionized water is washed assorted, sodium chloride solution carries out wash-out, sodium hydroxide solution carries out resin regeneration, collection contains sialic elutriant with adding ethyl acetate dilution crystallization after aqueous ethanolic solution leaching again, obtains sialic acid sterling after crystal drying under reduced pressure;

In described sialic acid clear liquid, sialic concentration is 1～20g/L;

Described anionite-exchange resin be take styrene-divinylbenzene copolymer as skeleton, take quaternary amine base as functional group;

Described anion exchange beads degree is 0.315-1.25mm, and wet volume density is 0.66-0.75g/ml, and water content is 42-48%;

Described continuous ionic exchange system is the ion exchange system by the operate continuously of 8-18 radical ion exchange column series connection, by the absorption in Combined valve goalkeeper ion exchange process, wash assorted, between wash-out and regeneration Si Ge workshop section, switch in order, adsorption stage ion exchange column being shifted out at once after resin absorption is saturated to adsorption stage sends into and washes assorted section and wash assorted, washing to shift out at once after assorted end and washing assorted section and enter wash-out section and carry out wash-out, after wash-out completes, shifting out at once wash-out section sends into RS Regenerator Section and regenerates, after regeneration washing completes, shifting out at once RS Regenerator Section sends into adsorption stage and adsorbs, so operating process of circulation, and the state of the 1st radical ion exchange column of each workshop section switches and synchronously carries out, and guarantee to have a radical ion exchange column at least in absorption phase,

In described continuous ionic exchange system, adsorption stage, the anion-exchange column of washing assorted section, wash-out section and the RS Regenerator Section 2-6 root of respectively doing for oneself;

Described concentration of sodium chloride solution is 0.001-2M; Described concentration of sodium hydroxide solution is 0.001-2M;

Described aqueous ethanolic solution concentration is 5-40g/L, and the 1-2 that the add-on of aqueous ethanolic solution is effluent volume doubly;

Ethyl acetate add-on is aqueous ethanolic solution and elutriant mixed volume 0.1-1 times.

2. continuous separate according to claim 1, from sialic method, is characterized in that, described uf processing is removed most of protein, and adopting molecular weight cut-off is 10,000～80,000 dalton.