Method for purifying sialic acid from fermentation liquor

Abstract

The invention discloses a method for purifying sialic acid from fermentation liquor, and belongs to the field of biochemical engineering separation and purification. The method comprises the following steps: firstly, concentrating sialic acid biological fermentation liquor by adopting membrane separation, adjusting the pH value of the concentrated liquor to 7-11, loading a sample on a boric acid affinity chromatography column, respectively washing with water and acid water, and collecting a sialic acid eluate; adjusting the pH value of the saliva acid eluate to 5-10, loading the saliva acid eluate to anion exchange resin, washing with water and acid water to obtain a resin desorption solution, and adding the resin desorption solution into a spray dryer for drying to obtain a sialic acid finished product. The stable five-membered ring complex is generated by utilizing the action of a boric acid group and a dihydroxyl structure in sialic acid. According to the method, the sialic acid is subjected to complexing under an acidic condition, molecules are eluted from a medium, selective separation of the sialic acid is realized, the sialic acid is further refined and purified by adopting anion exchange resin, high-purity sialic acid is prepared, introduction of a crystallization process is avoided, and use of an organic solvent and an acid reagent is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the field of biochemical separation and purification, and specifically relates to a method for purifying sialic acid from fermentation broth. Background Art

[0002] Sialic acid is a derivative of neuraminic acid and is widely found at the end of the sugar chains of glycoproteins and glycolipids on the surface of animal cells. Microbial fermentation has become the main way of large-scale production at this stage because of its simple process and relatively low cost. The fermentation method generally obtains polysialic acid, which needs to be depolymerized and purified by strong acid hydrolysis, acetic acid crystallization and other processes in the later stage. A large amount of waste acid will be generated, which needs to be neutralized before it can be discharged, increasing the production cost. However, the crystallization process can effectively remove impurities, and sialic acid is stable and not easy to degrade after forming crystals. Therefore, the crystallization process is often a necessary means to purify sialic acid fermentation broth.

[0003] CN202410313729.8 discloses a method for directly synthesizing sialic acid using Escherichia coli, and purifying it by membrane technology, activated carbon decolorization, column chromatography and acetic acid crystallization. A large amount of waste acid will be generated, and the crystallization time takes 2 days, which is a long period. In order to solve the above problems, there are literature reports that ethanol / ethyl acetate mixture (DOL: 10.3321 / j.issn: 0253-990X.2006.05.015), acetone (CN202311832241.8) and other reagents are used to replace the crystallization process of acetic acid. Although this method avoids the generation of waste acid, it also requires the consumption of a large amount of organic reagents, and the yield is low, and the crystallization time is 8~10 h. The above technology still needs to use a crystallization process to achieve the separation and purification of sialic acid, and it is difficult to avoid the use of organic reagents and the long cycle of the purification process. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides a method for purifying sialic acid from fermentation broth. The method avoids the introduction of a crystallization process, greatly reduces the amount of acid and organic solvent used, and reduces production costs and cycles.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a method for purifying sialic acid from a fermentation broth, comprising the following steps:

[0007] S1: Concentrating the sialic acid biological fermentation broth by membrane separation to obtain a concentrated solution;

[0008] S2: Adjust the pH of the concentrate to 7-11, load it onto a boric acid affinity chromatography column, flow rate: 300-1800 cm / h; rinse with water and acid water, respectively, the rinse times are 2-6 BV, the acid dosage is 0.01%-0.1% (v / v), and collect the sialic acid eluate;

[0009] S3: Adjust the pH of the sialic acid eluate to 5-10, load it onto an anion exchange resin, and wash it with water and acid water, each washing multiple is 2-5 BV, and the acid dosage is 0.01%-0.1% (v / v);

[0010] S4: adding the resin solution to a spray dryer for drying to obtain a sialic acid product.

[0011] As a preferred embodiment of the present invention, in S1, the membrane separation adopts one or more of a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane, and the concentration of sialic acid in the concentrate is 30-100 g / L, preferably 80-100 g / L.

[0012] As a preferred embodiment of the present invention, in S2, one or more of ammonia water, sodium hydroxide, potassium hydroxide, sodium bicarbonate, ammonium bicarbonate and sodium chloride are used to adjust the pH of the concentrate. The acid is one of hydrochloric acid, formic acid, acetic acid and trifluoroacetic acid.

[0013] Further preferably, in S2, the pH of the concentrate is adjusted to 7-8; the concentrate is loaded onto a boric acid affinity chromatography column at a flow rate of 500-800 cm / h; the column is rinsed with water and acid water, respectively, at a rinse multiple of 2-4 BV, and the acid is hydrochloric acid; the acid dosage is 0.08%-0.1% (v / v).

[0014] As a preferred embodiment of the present invention, in S3, one or more of ammonia water, sodium hydroxide, potassium hydroxide, sodium bicarbonate, and ammonium bicarbonate are used to adjust the pH of the sialic acid eluent. The acid is one of hydrochloric acid, formic acid, acetic acid, and trifluoroacetic acid.

[0015] Further preferably, in S3, the pH of the sialic acid eluent is adjusted to 7-8 using one of sodium hydroxide and potassium hydroxide, the sample is loaded onto an anion exchange resin, and the resin is washed with water and acid water, the washing multiples are both 2-3 BV, the acid type is hydrochloric acid, and the acid dosage is 0.08%-0.1% (v / v).

[0016] As a preferred embodiment of the present invention, the air inlet temperature of the spray dryer S4 is 150°C, and the air outlet temperature is 100°C.

[0017] It should be noted that the production of sialic acid by biological fermentation has been reported. In order to increase the yield of sialic acid, researchers have tried to construct some high-yield sialic acid engineering bacteria, which can obtain sialic acid-containing fermentation broth under certain fermentation conditions. The raw material of the method of the present invention can be any sialic acid fermentation broth obtained by biological fermentation, typically but not limited, for example, it can be a fermentation broth obtained by the strain disclosed in CN202410313729.8 "A biosynthesis method of N-acetylneuraminic acid" and its method, or it can be a fermentation broth obtained by other methods such as CN202211469338.2 "A kind of Escherichia coli engineering bacteria for producing N-acetylneuraminic acid and its application".

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention takes avoiding the use of organic solvents and crystallization processes as the starting point for purifying sialic acid fermentation broth. It is found that under alkaline conditions, the dihydroxy structure in sialic acid can react with the boric acid group to generate a stable five-membered ring complex, and the study of other components in the fermentation broth found that under the appropriate operating conditions of the present invention, other components are not prone to such reactions, so sialic acid molecules can be adsorbed by affinity media. Further, under suitable acidic conditions, this complex is opened, and the molecules are eluted from the medium, achieving efficient selective separation between sialic acid and other impurities, and the one-step HPLC purity can reach more than 95%. Further, anion exchange resin is used to purify sialic acid to prepare high-purity (HPLC purity ≥ 99%) sialic acid, avoiding the introduction of crystallization process, greatly reducing the use of organic solvents and acid reagents, and providing a feasible solution for the industrial production of high-purity sialic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a flow chart of the method of the present invention;

[0021] Figure 2 This is the HPLC analysis chart of sialic acid finished product. DETAILED DESCRIPTION

[0022] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0023] The sialic acid fermentation broth used in the subsequent embodiments of the present invention is obtained by fermentation using the strain disclosed in the invention patent "A method for biosynthesis of N-acetylneuraminic acid" with application number CN202410313729.8 and the disclosed method, wherein N-acetylneuraminic acid (Neu5Ac) is commonly known as sialic acid, which is named because its content accounts for more than 99% of the entire sialic acid species. The general fermentation method is to culture the recombinant Escherichia coli with high yield of N-acetylneuraminic acid constructed in the document at 32-34°C and 200-220rpm to obtain seed liquid, and the seed liquid is transferred to the fermentation medium at an inoculum of 4%-6%, and fermented and cultured at 32-34°C for 22-24 h. When OD 600 =32-36, increase the fermentation temperature to 38-42°C, and continue fermenting for 66-72 h to obtain a fermentation liquid containing a certain concentration of N-acetylneuraminic acid.

[0024] The construction method of recombinant Escherichia coli with high production of N-acetylneuraminic acid in CN202410313729.8 is as follows: using plasmid pETDuet as a skeleton, deleting the lacI gene, replacing the original T7 promoter of plasmid pETDuet with constitutive promoter PJ23101, inserting glucosamine-6-phosphate acetyltransferase encoding gene gna1 and fructose-6-phosphate aminotransferase encoding gene glmS, and obtaining plasmid pETDuet-PJ23101-gna1-PJ23101-glmS; using plasmid pCDFDuet as a skeleton, deleting the lacI gene, replacing the original T7 promoter of plasmid pCDFDuet with thermosensitive promoter loop cI ts -pR-pL was replaced, and the N-acetylglucosamine-2 epimerase encoding gene age and the N-acetylneuraminic acid synthetase encoding gene neuB were inserted to obtain the plasmid pCDFDuet-cI ts -pR-pL-age- pR-neuB; the plasmid pETDuet-gna1-glmS and the plasmid pCDFDuet-age-neuB are introduced into the host Escherichia coli K-12 MG1655 to construct a recombinant Escherichia coli with high production of N-acetylneuraminic acid. The nucleotide sequence of the relevant gene is shown in CN202410313729.8. It should be noted that the fermentation broth obtained by the above method in the embodiment of the present invention and the separation and purification are only illustrative and do not constitute a limitation on the fermentation broth raw material used in the method of the present invention. The raw material of the method of the present invention can also be sialic acid fermentation broth obtained by other biological fermentation methods.

[0025] Example 1

[0026] like Figure 1As shown in the flow chart, 5 L of sialic acid fermentation broth (content 35 g / L, HPLC purity 49%) was sequentially passed through a 50nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100Da to remove macromolecular impurities such as mycelium and protein, and a concentrate with a concentration of 100 g / L was obtained. 200 mol / L sodium chloride and ammonia water were added to the concentrate to adjust the pH of the solution to 8, and the concentrate was loaded onto an NMPB-200L boric acid affinity chromatography medium, and 3BV was eluted with water and 0.1% hydrochloric acid aqueous solution (v / v) in sequence, with a flow rate of 800 cm / h, and the sialic acid fraction (HPLC purity 95.6%) was collected. The pH of the sialic acid fraction was adjusted to 7 with 1 mol / L sodium hydroxide aqueous solution, loaded onto a WS-260 anion exchange resin, and then 2BV was eluted with water and 0.08% hydrochloric acid aqueous solution (v / v) to obtain the sialic acid fraction. It was added to a spray dryer with an air inlet temperature of 150°C and an air outlet temperature of 100°C to obtain 122 g of white sialic acid solid (yield 69.7%). The purity was 99.52% as determined by HPLC. Figure 2 This is the HPLC purity analysis chart.

[0027] Example 2

[0028] 18 L of sialic acid fermentation broth with a content of 30 g / L and an HPLC purity of 54.7% was taken and passed through a 50 nm ceramic membrane and a nanofiltration membrane with a molecular weight cutoff of 100 Da in sequence to obtain an 80 g / L concentrate. 300 mol / L sodium chloride was added to the concentrate and the pH was adjusted to 7.5 with sodium bicarbonate. The concentrate was loaded onto NMPB-200L boric acid affinity chromatography medium and 4 BV was eluted with water and 0.08% hydrochloric acid aqueous solution (v / v) in sequence at a flow rate of 600 cm / h. The sialic acid fraction (HPLC purity 96.1%) was collected. The pH of the sialic acid solution was adjusted to 8 with 1 mol / L potassium hydroxide solution and loaded onto a WS-260 anion exchange resin. The concentrate was eluted and eluted with water and 0.1% hydrochloric acid aqueous solution (v / v) in sequence for 3 BV and the sialic acid fraction was collected. It was added to a spray dryer with an air inlet temperature of 150°C and an air outlet temperature of 100°C to obtain 381 g of white sialic acid solid with a yield of 70.55%. The purity was 99.49% as determined by HPLC.

[0029] Example 3

[0030] 50 L of sialic acid fermentation broth (content 38 g / L, HPLC purity 51%) was taken and passed through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and a nanofiltration membrane with a molecular weight cutoff of 100Da in sequence to obtain a 100 g / L concentrate. 500 mol / L sodium chloride was added to the concentrate and the pH of the solution was adjusted to 8 with ammonia water. The concentrate was loaded onto an NMPB-200L boric acid affinity chromatography medium, 4BV was washed with water to remove impurities, and then 4BV of 0.1% hydrochloric acid aqueous solution (v / v) was used to elute sialic acid at a flow rate of 500 cm / h to obtain a sialic acid fraction (HPLC purity 95.8%). 1 mol / L sodium hydroxide aqueous solution was added to adjust the pH of the sialic acid solution to 8, and the concentrate was loaded onto a WS-260 anion exchange resin. 3BV was washed and eluted with water and 0.1% hydrochloric acid aqueous solution (v / v) in sequence to collect the sialic acid fraction. It was added to a spray dryer, with an air inlet temperature of 150°C and an air outlet temperature of 100°C, to obtain 1292 g of white sialic acid solid with a yield of 68%. The purity was 99.35% as determined by HPLC. Table 1 shows the HPLC purity and yield of Examples 1 to 3 after purification.

[0031] Comparative Example 1

[0032] Take 5 L of sialic acid fermentation broth (content 38 g / L, HPLC purity 52%), pass through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100Da, remove macromolecular impurities such as mycelium and protein, and obtain a concentrate with a concentration of 92 g / L. The pH of the sialic acid concentrate was adjusted to 7 with a 1 mol / L sodium hydroxide aqueous solution, loaded on a WS-260 anion exchange resin, and then rinsed and eluted 2BV with water and 0.08% hydrochloric acid aqueous solution (v / v) to obtain a sialic acid fraction. Add it to a spray dryer, the air inlet temperature is 150 ° C, the air outlet temperature is 100 ° C, and 171 grams of orange-yellow sialic acid viscous material is obtained, the HPLC purity is 85.21%, and the yield is 90%. The difference between this comparative example and Example 1 is that it is not selectively separated by a boric acid affinity chromatography column. As can be seen from the results, the purity of the sialic acid sample obtained by using only anion exchange chromatography is lower than that of the sialic acid sample obtained by one-step boric acid affinity chromatography (greater than 95%). In order to obtain high-purity sialic acid, for the sialic acid viscous substance obtained in Comparative Example 1, it is necessary to further cooperate with the crystallization process for separation and purification in the prior art. Obviously, the affinity chromatography in Examples 1-3 can quickly obtain samples of higher purity, and the separation and purification efficiency is significantly improved, which is the key point of the present invention. It should be noted that if the sialic acid fraction obtained by eluting the anion exchange resin in Comparative Example 1 is purified by NMPB-200L boric acid affinity chromatography medium, the single effective load and effluent purity of the anion exchange resin will be reduced due to the presence of a large number of impurities such as electronegative pigments in the fermentation broth, and then purified by affinity chromatography and spray dried, the final product is a light yellow solid, HPLC purity 94.5%, total yield 75%. Obviously, the product properties and purity are not as good as Examples 1~3.

[0033] Comparative Example 2

[0034] 5 L of sialic acid fermentation broth (content 32 g / L, HPLC purity 48%) was sequentially passed through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100Da to remove macromolecular impurities such as mycelium and protein, and a concentrate with a concentration of 95 g / L was obtained. 200 mol / L sodium chloride was added to the concentrate, and the pH of the solution was 6.5. The concentrate was loaded onto NMPB-200L boric acid affinity chromatography medium, and 3 BV was washed and eluted with water and 0.1% hydrochloric acid aqueous solution (v / v) in sequence, with a flow rate of 800 cm / h, and the sialic acid fraction (HPLC purity 75%) was collected. The pH of the sialic acid fraction was adjusted to 7 with 1 mol / L sodium hydroxide aqueous solution, loaded onto WS-260 anion exchange resin, and then 2 BV was washed and eluted with water and 0.08% hydrochloric acid aqueous solution (v / v) to obtain the sialic acid fraction. It was added to a spray dryer, the air inlet temperature was 150 ° C, the air outlet temperature was 100 ° C, and 65.6 grams of white sialic acid solid (yield 41.0%) were obtained. The purity was 97.5% as determined by HPLC. Compared with Example 1, in the boric acid affinity chromatography, the pH of the loading solution was lower than 7, resulting in insufficient binding force between sialic acid and the boric acid affinity material. During the water elution, some sialic acid and impurities co-elute, affecting the impurity removal effect of this step, reducing the purity of the eluent, and at the same time, bringing challenges to the subsequent anion exchange chromatography, resulting in low purity and yield of the finished product.

[0035] Comparative Example 3

[0036] 5 L of sialic acid fermentation broth (content 30 g / L, HPLC purity 51%) was sequentially passed through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100Da to remove macromolecular impurities such as mycelium and protein, and a concentrate with a concentration of 101 g / L was obtained. 200 mol / L sodium chloride was added to the concentrate, and the pH of the solution was adjusted to 9 with ammonia water. The concentrate was loaded onto NMPB-200L boric acid affinity chromatography medium, and 3 BV was eluted with water and 4 BV with 0.1% hydrochloric acid aqueous solution (v / v) at a flow rate of 800 cm / h. The sialic acid fraction (HPLC purity 90%) was collected. The pH of the sialic acid fraction was adjusted to 7 with 1 mol / L sodium hydroxide aqueous solution, loaded onto WS-260 anion exchange resin, and then 2 BV was eluted with water and 0.08% hydrochloric acid aqueous solution (v / v) to obtain the sialic acid fraction. It was added to a spray dryer with an air inlet temperature of 150°C and an air outlet temperature of 100°C to obtain 88.5 grams of white sialic acid solid (yield 59.0%). The purity was 99.1% as determined by HPLC. The difference between this comparative example and Example 1 is that the pH value of the loading solution is higher in the boric acid affinity chromatography step. Under this condition, the binding force between sialic acid and the boric acid affinity chromatography medium is stronger, and more volume of acid water is required for elution to ensure the yield of this step. The resulting problem is that some strongly retained impurities are also eluted with sialic acid, so that the purity of the eluent is only 90%. Although high-purity sialic acid can be obtained by anion exchange chromatography, the yield is reduced.

[0037] It can be seen from Comparative Examples 2 and 3 that the optimization of boric acid affinity chromatography conditions is also crucial to the purification effect and yield, and is also the key point of the present invention.

[0038] Table 1 - Purity and yield after purification of Examples 1 to 3

[0039]

[0040] It can be seen from Table 1 that the method of the present invention can obtain sialic acid with a purity of more than 99%, and combined with the operating process of the embodiment, it can be seen that there is no need to introduce a crystallization process in the embodiment, which greatly reduces the use of organic solvents and acid reagents.

[0041] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for purifying sialic acid from a fermentation broth, characterized in that: The steps include: S1: Concentrating the sialic acid biological fermentation broth by membrane separation to obtain a concentrated solution; S2: The pH of the concentrate was adjusted to 7-11, and the concentrate was loaded onto a boric acid affinity chromatography column at a flow rate of 300-1800 cm / h; the column was washed with water and acid water at a multiple of 2-6 BV, and the volume percentage of the acid in the acid water was 0.01%-0.1%, and the sialic acid eluate was collected; S3: adjusting the pH of the sialic acid eluate to 5-10, loading the sample onto an anion exchange resin, washing with water and acid water at a washing multiple of 2-5 BV, and the volume percentage of the acid in the acid water being 0.01%-0.1%, to obtain a resin analytical solution; S4: adding the resin solution to a spray dryer for drying to obtain a sialic acid product.

2. The method for purifying sialic acid from a fermentation broth according to claim 1, characterized in that: In S1, membrane separation uses one or more of a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane, and the concentration of sialic acid in the concentrate is 30-100 g / L.

3. The method for purifying sialic acid from a fermentation broth according to claim 1, characterized in that: In S2, one or more of ammonia water, sodium hydroxide, potassium hydroxide, sodium bicarbonate, ammonium bicarbonate and sodium chloride are used to adjust the pH of the concentrate.

4. The method for purifying sialic acid from a fermentation broth according to claim 1, characterized in that: In S2, the acid is one of hydrochloric acid, formic acid, acetic acid and trifluoroacetic acid.

5. The method for purifying sialic acid from a fermentation broth according to claim 1, characterized in that: In S2, the pH of the concentrate is adjusted to 7-8; the sample is loaded onto a boric acid affinity chromatography column at a flow rate of 500-800 cm / h; it is washed with water and acid water, respectively, with a washing multiple of 2-4 BV, and the acid is hydrochloric acid; the volume percentage of the acid in the acid water is 0.08%-0.1%.

6. The method for purifying sialic acid from a fermentation broth according to claim 1, characterized in that: In S3, the pH of the sialic acid eluent is adjusted by using one or more of ammonia water, sodium hydroxide, potassium hydroxide, sodium bicarbonate, and ammonium bicarbonate.

7. The method for purifying sialic acid from a fermentation broth according to claim 1, characterized in that: In S3, the acid is one of hydrochloric acid, formic acid, acetic acid and trifluoroacetic acid.

8. The method for purifying sialic acid from a fermentation broth according to claim 1, characterized in that: In S3, the pH of the sialic acid eluent is adjusted to 7-8 using one of sodium hydroxide and potassium hydroxide, and the sample is loaded onto an anion exchange resin, and washed with water and acid water, with the washing multiples being 2-3 BV. The type of acid is hydrochloric acid; the volume percentage of the acid in the acid water is 0.08%-0.1%.

9. The method for purifying sialic acid from a fermentation broth according to claim 1, characterized in that: The air inlet temperature of the spray dryer S4 is 150 °C and the air outlet temperature is 100 °C.

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