CN114014896B - Separation and purification method of high-purity 3' -sialyllactose - Google Patents

Abstract

The invention belongs to the field of separation and purification of sialyllactose, and specifically relates to a method for separation and purification of high-purity 3'-sialyllactose, which includes heating and enzymatic filtration of the transformation liquid, adjusting the pH of the filtrate to alkaline and then filtering, and adjusting the pH of the filtrate to Adjust to neutrality, and undergo steps such as concentration, ion adsorption, and drying to obtain separated and purified 3'-SL; the present invention uses two methods: heating to inactivate enzymes and adjusting pH to alkaline to comprehensively improve the concentration of proteins and other macromolecular substances in the conversion solution. The removal rate, while controlling the concentration of 3'-SL in the concentrated solution, plays an important role in further improving the purity of the product 3'-SL for anion and cation adsorption purification. The purity of the product 3'-SL obtained by combining the above methods is ≥96%.

Description

A method for separating and purifying high-purity 3`-sialyllactose

Technical field

The invention belongs to the field of separation and purification of sialyllactose, and specifically relates to a method for separation and purification of high-purity 3'-sialyllactose.

Background technique

Human milk oligosaccharides (HMOS) are the third most abundant component in human milk after lactose and fat and have important biological functions. Sialyl oligosaccharide (SL) is an acidic human milk oligosaccharide. It can be divided into 3'-sialyllactose (3'-SL) and 6'-sialyllactose (SL) according to the binding position of sialic acid and lactose moiety. 6'-SL), existing research shows that 3'-SL plays an important role in infants, including neutralizing toxins produced by intestinal bacteria and preventing bacteria or viruses from adhering to the intestinal epithelial surface. Although 3'-SL has important biological functions, there is still a lack of cost-effective industrial production methods. Therefore, the development of low-cost substrate production and purification methods for 3'-SL is important for the promotion and application of 3'-SL. significance.

The existing method for preparing sialyllactose is, for example, the application number is CN201910787973.7, and the patent name is a method for preparing sialyllactose. The invention uses a single-bacteria multi-enzyme method combined with a one-step purification and immobilization method to immobilize multiple enzymes to achieve The one-pot method of CTP regeneration and multi-enzyme recycling during the preparation of sialyllactose has the advantages of high yield, low cost, and short cycle time. However, the purity of 3'-SL produced by this method needs to be further improved. Therefore, Based on the multi-enzyme catalyzed conversion solution for preparing 3'-SL, this application developed a method for further separation and purification of 3'-SL in the conversion solution, in order to further improve the purity of the product 3'-SL.

Contents of the invention

In view of the problems existing in the prior art, the present invention aims to provide a method for separating and purifying high-purity 3'-sialyllactose, by further separating and purifying 3'-SL in the multi-enzyme catalyzed conversion solution, as described in the present invention. The purity of 3'-SL after separation and purification by the method is not less than 96%.

Based on the above objectives, the technical solutions adopted by the present invention are as follows:

A method for separating and purifying high-purity 3’-sialyllactose, including the following steps:

S1: Heat the transformation solution to inactivate the enzyme, filter it through a 30-100nm filter membrane, and collect the filtrate;

S2: Adjust the pH of the filtrate collected in step S1 to alkaline, filter it through a 3000Da~5000Da membrane, and collect the filtrate;

S3: Adjust the pH of the filtrate collected in step S2 to neutral, filter and concentrate through 800Da~1000Da membrane, and collect the concentrated liquid on the membrane;

S4: The concentrated solution on the membrane obtained in step S3 is adsorbed, concentrated and dried by anion and cation exchange resins in sequence to prepare 3’-SL.

The present invention performs heating and enzyme-killing treatment on the transformation solution through step S1, so that proteins such as enzymes, bacteria and other large particle impurities in the transformation solution are heated to precipitate out, and are filtered through a 30-100nm filter membrane to effectively remove bacteria in the transformation solution. , large particle impurities and precipitated enzymes and other proteins. When the pore size of the filter membrane exceeds this range, the removal of bacteria will be incomplete and the product purity will be low.

In step S2, the pH of the filtrate is adjusted to alkaline, so that the pH of the filtrate reaches the isoelectric point of the remaining protein in the transformation solution, further promoting protein precipitation, and filtering through a 3000Da to 5000Da membrane to further remove the protein in the transformation solution. . The reason why 3000Da to 5000Da membranes are selected for filtration and impurity removal is because the molecular weight of 3’-SL is less than 3000Da, and it can pass through the membrane and enter the filtrate during filtration, while impurities are trapped in the membrane. When the molecular weight of the membrane exceeds the range of 3000Da to 5000Da, it will cause macromolecular impurities to pass through the organic membrane together with the target 3’-SL, and the purpose of separating the target and impurities cannot be achieved.

Then in step S3, the pH of the filtrate is adjusted to neutral and filtered and concentrated using an 800Da to 1000Da membrane. The resulting concentrated liquid is adsorbed by anion and cation exchange resins, concentrated, and dried to prepare 3'-SL. Similarly, when the molecular weight of the organic membrane exceeds the range of 800Da to 1000Da, small molecule impurities such as lactose, glycerol, and sialic acid cannot be separated from the target 3’-SL, making it difficult to further improve the purity of 3’-SL.

In the membrane impurity removal and purification process of S1 to S3 of the present invention, the sequence of membrane passing is based on the particle size of the impurities in the transformation solution. First, filter to remove visible impurities with large particle sizes, then remove some residual proteins, and finally remove For lactose, glycerol, sialic acid and some parts that are smaller than the target substance, during the actual operation, I tried to change the order of membrane passing or omit part of the membrane passing process. It was found that after changing the order, it is easy to cause membrane clogging, which greatly reduces the membrane passing efficiency. Some parts were omitted. After the membrane process, the purity of the final product cannot reach the current level.

In addition, it was found through experiments that the effective removal of proteins in the transformation solution plays an important role in further improving the purity of the product 3'-SL. Due to the complexity of the components in the transformation solution, heating denaturation and precipitation are usually used to remove proteins. This application further tested It was found that the conversion solution also contained a heat-insensitive protein, but the heat-insensitive protein could be further removed by adjusting the pH to its isoelectric point. This step is easily ignored in the existing separation and purification process of sialyllactose. step, so the present invention adopts two methods: heat treatment and pH adjustment to effectively remove the protein in the transformation solution and significantly improve the purity of the product 3'-SL.

Further, in step S2, the pH of the filtrate collected in step S1 is adjusted to 10-12.

Since the components in the transformation solution are relatively complex, it is difficult to determine the type of protein and then determine its isoelectric point. In the present invention, the pH of the filtrate collected in step S1 is adjusted to 1, 2, 3, 4, 5, 6, 7, respectively. 8, 9, 10, 11, and 12 conducted protein precipitation tests. The results showed that when the pH of the filtrate was below 8, no precipitation precipitated. When pH = 9, a small amount of precipitation precipitated. When the pH was 10 to 12, there was a large amount of precipitation. Precipitate. Therefore, it is selected to adjust the pH of the filtrate collected in step S1 to 10-12.

Further, in step S2, the pH of the filtrate collected in step S1 is adjusted to 10.

Preliminary experiments have confirmed that when the pH of the filtrate collected in step S1 is adjusted to 10, a large amount of precipitates have already precipitated. Since it is necessary to use acid to neutralize the alkali in the filtrate in the later stage, if the pH is adjusted too high in the early stage, then If the amount of salt formed by subsequent acid-base neutralization is too large, it will increase the pressure of subsequent desalination and even cause a decrease in the purity of the target product. Therefore, it is more appropriate to control the pH of the filtrate collected in step S1 = 10.

Further, the concentration of 3’-SL in the concentrated solution on the membrane in step S3 is 10-30g/L.

It was found through experiments that the concentration of 3'-SL in the concentrated solution has an important impact on the anion and cation exchange resin adsorption and purification process and the purity of the final product 3'-SL. When the concentration of 3'-SL in the concentrated solution is higher than 30g/L , will not only cause the loss of resin, but also have poor desalination effect, resulting in a large amount of salt residue, which will affect the yield and purity of the product, reducing the yield of the product to 85% and the purity not higher than 90%; when 3'- in the concentrated solution When the concentration of SL is less than 10g/L, the treatment system of the anion and cation exchange resin becomes larger and the treatment efficiency is greatly reduced; when the concentration of 3'-SL in the concentrated solution is 10 to 30g/L, the product 3'- The yield of SL is not less than 91%, and the purity of 3'-SL is not less than 96%. Therefore, the concentration of 3'-SL in the concentrated solution on the membrane in step S3 is adjusted to 10-30g/L, and at the same time, a relatively high yield can be obtained. High yield and high purity of the product.

Further, the method of heating the transformation solution to inactivate the enzyme in step S1 is as follows: heating the transformation solution at 80-100°C for 8-15 minutes.

Further, the purity of 3’-SL obtained by purification and drying in steps S1 to S4 is ≥96%.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The present invention obtains purified 3'-SL by heating and enzyme-killing filtration of the conversion solution, adjusting the pH of the filtrate to alkaline and then filtering, and then adjusting the pH of the filtrate to neutral, concentrating, ion adsorbing, and drying. The two methods of heating to inactivate enzymes and adjusting the pH to alkaline are used to comprehensively improve the removal rate of proteins and other macromolecules in the conversion solution. At the same time, the concentration of 3'-SL in the concentrated solution is controlled to further improve the product 3'- for adsorption and purification of anions and cations. The purity of SL plays an important role. The purity of the product 3'-SL obtained by combining the above methods is ≥ 96%.

Description of drawings

Figure 1 is a schematic diagram of the process flow of the present invention.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

Unless otherwise specified, the test methods used in the examples are conventional methods; unless otherwise specified, the materials and reagents used can be obtained from commercial sources. The transformation liquid used in the present invention is derived from the transformation liquid prepared by the method described in the application number CN201910787973.7 and the patent name is a method for preparing sialyllactose.

Example 1

This embodiment provides a method for separating and purifying high-purity 3’-sialyllactose, as shown in Figure 1, including the following steps:

S1: Heat the transformation solution at 90°C for 10 minutes to inactivate the activity of the enzyme in the transformation solution and promote the denaturation and precipitation of the protein. At the same time, the bacterial cells, large particle impurities and non-enzyme proteins in the transformation solution are denatured and precipitated, and then Use a 30nm ceramic filter membrane to filter to remove the above-mentioned precipitates. When the minimum circulation volume of the ceramic membrane is reached, add water twice the minimum circulation volume of the ceramic membrane to further circulate the membrane for filtration to further remove the above-mentioned precipitated precipitates to obtain 3'-SL. The yield of 3'-SL in the conversion solution in this step is as high as 95%.

S2: Add alkali to the clear liquid collected in step S1, adjust the pH of the clear liquid = 10, and promote proteins in the clear liquid such as CMP kinase, polyphosphate kinase, CMP-sialic acid synthase and other impurities released by cell disruption. Proteins, etc. reach their isoelectric point, aggregate and precipitate, and are then filtered through a 3000Da organic membrane to remove protein precipitates and macromolecular impurities, such as sodium hexametaphosphate. In this step, an initial membrane passage volume of 4 is added to the filtration system. ~6 times the water is used for circulating membrane filtration, and the filtrate is collected to effectively remove macromolecular substances such as sodium hexametaphosphate in the clear liquid.

During the pH adjustment process of the clear liquid collected in step S1, since the components in the clear liquid are relatively complex, it is difficult to determine the type of protein and then determine its isoelectric point. The inventor of this case adjusted the pH of the clear liquid collected in step S1. Adjusted to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively. It was found that the pH of the clear liquid was below 8 and no precipitation occurred; when pH = 9, a small amount of precipitation occurred. , when the pH is 10 to 12, there is a large amount of precipitation, so we choose to adjust the pH of the clear liquid collected in step S1 to 10 to 12, which can further remove a large amount of protein impurities. In order to avoid the subsequent acid-base neutralization caused by excessive pH Excessive amount of salt formation will further lead to poor subsequent desalination pressure and desalination effect, resulting in a decrease in the purity of the target product. Therefore, it is more appropriate to adjust the pH of the clear liquid collected in step S1 to 10.

S3: Add acid to the filtrate in step S2 to adjust the pH of the filtrate to neutral, and then filter and concentrate it through a 1000Da organic membrane to collect the concentrated liquid on the membrane. Filtration and impurity removal mainly refers to the removal of small molecular substances such as salt, Magnesium chloride heptahydrate, sialic acid and other substances. In this step, cyclic membrane filtration is performed by adding 5 to 10 times the initial membrane volume of water to the filtration system, collecting the concentrated liquid on the membrane, and using high performance liquid chromatography to measure the concentration of 3'-SL in the concentrated liquid on the membrane. Carry out real-time detection until the concentration of 3'-SL in the concentrated liquid on the membrane is 10-30g/L, and stop the circulating membrane filtration and concentration.

S4: Deeply desalinate the concentrated liquid obtained in step S3 using anion and cation exchange methods: first use cation resin adsorption and purification, and then use anion adsorption and purification to further remove salt substances in the concentrated liquid. Through this anion and cation exchange desalination, the salt can be The conductivity in the conversion solution is reduced by about 90%. The desalted liquid is then concentrated and dried (lyophilized or spray dried) to prepare 3’-SL. The purity of the 3’-SL isolated after steps S1 to S4 is not less than 96%.

It was found through experiments that the concentration of 3'-SL in the concentrated solution obtained in step S3 has an important influence on the anion and cation exchange resin adsorption and purification process and the purity of the final product 3'-SL. When the concentration of 3'-SL in the concentrated solution is higher than 30g /L, not only will it cause the loss of resin, but the desalination effect is poor, resulting in a large amount of salt residue, which will affect the yield and purity of the product, reducing the yield of the product to 85% and the purity not higher than 90%; when the concentrated solution When the concentration of 3'-SL is lower than 10g/L, the treatment system of the anion and cation exchange resin becomes larger and the treatment efficiency is greatly reduced; when the concentration of 3'-SL in the concentrated solution is 10-30g/L, the treatment system of the anion and cation exchange resin becomes larger and the treatment efficiency is greatly reduced. The yield of the product 3'-SL is not less than 91%, and the purity of 3'-SL is not less than 96%. Therefore, the concentration of 3'-SL in the concentrated solution on the membrane in step S3 is controlled to be 10 to 30g/L. At the same time, higher yield and higher purity of the product can be obtained.

Example 2

This embodiment provides a method for separating and purifying high-purity 3’-sialyllactose, which includes the following steps:

S1: Heat the 3'-SL transformation solution at 90°C for 10 minutes to inactivate the enzyme activity in the transformation system and denature and precipitate heat-sensitive proteins; filter the heated transformation solution through a 30nm ceramic membrane to remove bacteria and macrophages. Particulate impurities and precipitated impurity proteins are obtained to obtain a clear liquid containing 3'-SL, which is dialyzed with water until the yield is not less than 95%.

S2: Add alkali to the 3'-SL clear solution to adjust its pH to 10 to promote the precipitation of the unheated denatured protein at the isoelectric point, and then filter it through a 3000D organic membrane to further remove the precipitated protein and some soluble macromolecular impurities (such as in the transformation system sodium hexametaphosphate, etc.), add water and dialyze until the yield is not less than 90%.

S3: Collect the clear liquid that has passed through the 3000D membrane and add acid to adjust its pH to 7, then pass through the 1000D organic membrane to remove monovalent salts and small molecule impurities, add water and dialyze until the conductivity of the concentrate no longer changes, collect the membrane supernatant liquid, the concentration of 3'-SL in the membrane supernatant is in the range of 10 to 30g/L.

S4: Then use ion exchange resin to further desalt the membrane supernatant; add 0.1% activated carbon to the desalted supernatant to decolorize, and then concentrate and spray dry to obtain 3’-SL, with a purity of 98%.

Comparative example 1

3'-SL was separated and purified with reference to the method described in Example 2. The only difference between this comparative example and Example 2 is that in steps S2 and S3, "adding alkali to the 3'-SL clear liquid to adjust its pH to 10" and "Collect the clear liquid that has passed through the 3000D membrane and add acid to adjust its pH to 7", and the remaining steps are the same as in Example 2.

The purity of 3'-SL separated and purified by the method described in this comparative example is 95%.

Comparative example 2

Referring to the method described in Example 2, the only difference between this comparative example and Example 2 is that step S1 does not carry out the process of "heating the 3'-SL conversion solution at 90°C for 10 minutes", and the remaining steps are the same as in Example 2.

The purity of 3'-SL separated and purified by the method described in this comparative example is 80%.

Comparative example 3

Referring to the method described in Example 2, the only difference between this comparative example and Example 2 is that the process of "heating the 3'-SL transformation solution at 90°C for 10 minutes" was not performed in step S1, and the " Add alkali to the 3'-SL clear liquid to adjust its pH to 10" and "Collect the clear liquid that has passed the 3000D membrane and add acid to adjust its pH to 7". The remaining steps are the same as in Example 2.

The purity of 3'-SL separated and purified by the method described in this comparative example is 60%.

From the comparison of the results of Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3, it can be seen that although the components in the conversion liquid are complex, the present invention adopts the method of heat treatment and pH adjustment of the conversion liquid to comprehensively improve the final separation of 3 '-SL purity.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention may be modified or equivalently substituted without departing from the essence and scope of the technical solution of the present invention.

Claims (3)

1. A method for separating and purifying high-purity 3'-sialyllactose, which is characterized in that it includes the following steps: S1: Heat the transformation solution to inactivate the enzyme, filter it through a 30-100nm filter membrane, and collect the filtrate; the method for heating the transformation solution to inactivate the enzyme in step S1 is as follows: Heat the transformation solution at 80-100°C for 8-15 minutes ; S2: Adjust the pH of the filtrate collected in step S1 to alkaline, filter it through a 3000Da-5000Da membrane, and collect the filtrate; in step S2, adjust the pH of the filtrate collected in step S1 to 10-12; S3: Adjust the pH of the filtrate collected in step S2 to neutral, filter and concentrate through an 800Da-1000Da membrane, and collect the concentrated liquid on the membrane; the concentration of 3’-SL in the concentrated liquid on the membrane in step S3 is 10-30g/L; S4: The concentrated solution on the membrane obtained in step S3 is adsorbed, concentrated and dried by anion and cation exchange resins in sequence to prepare 3’-SL. 2. The method according to claim 1, characterized in that in step S2, the pH of the filtrate collected in step S1 is adjusted to 10. 3. The method according to claim 1 or 2, characterized in that the purity of 3’-SL prepared through steps S1 to S4 is ≥96%.