CN114014896A - Separation and purification method of high-purity 3' -sialyllactose - Google Patents
Separation and purification method of high-purity 3' -sialyllactose Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Peptides Or Proteins (AREA)
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Abstract
The invention belongs to the field of separation and purification of sialyllactose, and particularly relates to a separation and purification method of high-purity 3 '-sialyllactose, which comprises the steps of heating a conversion solution to inactivate enzyme, filtering, adjusting the pH of a filtrate to be alkaline, filtering, adjusting the pH of the filtrate to be neutral, concentrating, adsorbing ions, drying and the like to obtain separated and purified 3' -SL; the method comprehensively improves the removal rate of macromolecular substances such as protein in the conversion solution by adopting two modes of heating to inactivate enzyme and regulating pH to alkalinity, simultaneously controls the concentration of 3 ' -SL in the concentrated solution, has an important effect on further improving the purity of the product 3 ' -SL by anion-cation adsorption purification, and comprehensively obtains the product 3 ' -SL with the purity more than or equal to 96 percent.
Description
Technical Field
The invention belongs to the field of separation and purification of sialyllactose, and particularly relates to a separation and purification method of high-purity 3' -sialyllactose.
Background
Human Milk Oligosaccharides (HMOS) are the third most abundant component in human milk, following lactose and fat, and have important biological functions. Sialyloligosaccharide (SL) is an acidic human milk oligosaccharide, which can be classified into 3 ' -sialyllactose (3 ' -SL) and 6 ' -sialyllactose (6 ' -SL) according to the position of the sialic acid bonded to the lactose moiety, and studies have shown that 3 ' -SL has an important role in infants, including neutralizing toxins produced by intestinal bacteria, preventing bacteria or viruses from adhering to the epithelial surface of the intestine, and the like. Although 3 ' -SL has important biological functions, at present, an economical and efficient industrial production method is still lacked, so that the development of a method for producing and purifying 3 ' -SL by using a low-cost substrate has important significance for popularization and application of 3 ' -SL.
The invention discloses a method for preparing sialyllactose, which is characterized in that a single-bacterium multi-enzyme method is combined with a further purification and immobilization method to immobilize multi-enzyme, so that CTP regeneration and multi-enzyme recycling in a one-pot sialyllactose preparation process are realized, the method has the advantages of high yield, low cost, short period and the like, but the purity of 3 '-SL prepared by the method needs to be further improved, so that a method for further separating and purifying 3' -SL in a conversion solution is developed based on the conversion solution for preparing 3 '-SL through multi-enzyme catalysis, and the purity of a product 3' -SL is further improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for separating and purifying high-purity 3 ' -sialyllactose, which further separates and purifies 3 ' -SL in a conversion solution catalyzed by multiple enzymes, and the purity of the 3 ' -SL separated and purified by the method is not lower than 96%.
Based on the above purpose, the technical scheme adopted by the invention is as follows:
a method for separating and purifying high-purity 3' -sialyllactose comprises the following steps:
s1: heating the conversion solution to inactivate enzyme, filtering through a 30-100 nm filter membrane, and collecting filtrate;
s2: adjusting the pH value of the filtrate collected in the step S1 to be alkaline, filtering the filtrate by a 3000 Da-5000 Da membrane, and collecting the filtrate;
s3: adjusting the pH value of the filtrate collected in the step S2 to be neutral, filtering and concentrating the filtrate by a membrane of 800 Da-1000 Da, and collecting the concentrated solution on the membrane;
s4: and (4) sequentially adsorbing the concentrated solution on the membrane obtained in the step S3 by anion and cation exchange resin, concentrating and drying to obtain the 3' -SL.
In the invention, the conversion solution is heated and enzyme-deactivated in the step S1, so that proteins such as enzyme and the like in the conversion solution, bacteria and other large-particle impurities are precipitated by heating, the proteins are filtered by a 30-100 nm filter membrane, the bacteria, the large-particle impurities and the precipitated proteins such as the enzyme and the like in the conversion solution are effectively removed, when the aperture of the filter membrane exceeds the range, the bacteria are incompletely removed, and the purity of the product is low.
In step S2, the pH of the filtrate is adjusted to be alkaline, so that the pH of the filtrate reaches the isoelectric point of the residual protein in the conversion solution, the protein is further precipitated and separated out, and the protein in the conversion solution is further removed by filtering through a 3000 Da-5000 Da membrane. The reason that the 3000 Da-5000 Da membrane is selected for filtration and impurity removal is that the molecular weight of the 3 '-SL is less than 3000Da, the 3' -SL can penetrate through the membrane to enter filtrate during filtration, and impurities are trapped in the membrane. When the molecular weight of the film exceeds the range of 3000 Da-5000 Da, macromolecular impurities and the target 3' -SL permeate the organic film together, and the aim of separating the target from the impurities cannot be achieved.
And then in step S3, adjusting the pH of the filtrate to be neutral, filtering and concentrating by using a 800 Da-1000 Da membrane, adsorbing the obtained concentrated solution by anion-cation exchange resin, concentrating and drying to obtain the 3' -SL. Similarly, when the molecular weight of the organic membrane exceeds the range of 800Da to 1000Da, small molecular impurities such as lactose, glycerol, sialic acid and the like cannot be separated from the target 3 '-SL, so that the purity of the 3' -SL is difficult to further improve.
In the purification process of the membrane-passing impurity removal of the S1-S3, the membrane-passing sequence is carried out according to the particle size of impurities in the conversion solution, firstly, macroscopic impurities with large particle size are filtered out, then, some residual protein is removed, and finally, lactose, glycerol, sialic acid and parts smaller than the target object are removed.
In addition, tests show that the effective removal of the protein in the conversion solution plays an important role in further improving the purity of the product 3 '-SL, the protein is usually removed by adopting a heating denaturation precipitation mode due to the complexity of components in the conversion solution, and the conversion solution further contains heat-insensitive protein through further tests, but the heat-insensitive protein can be further removed by adjusting the pH to the isoelectric point, and the step is a step which is very neglectable in the existing sialyllactose separation and purification process, so the invention adopts two modes of heat treatment and pH adjustment to effectively remove the protein in the conversion solution, and the purity of the product 3' -SL is obviously improved.
Further, in the step S2, the pH of the filtrate collected in the step S1 is adjusted to 10-12.
In the present invention, protein precipitation tests were performed by adjusting the pH of the filtrate collected in step S1 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, and as a result, it was found that when the pH of the filtrate was 8 or less, no precipitate was precipitated, when the pH was 9, a small amount of precipitate was precipitated, and when the pH was 10 to 12, a large amount of precipitate was precipitated. Therefore, the pH of the filtrate collected in the step S1 is selected to be adjusted to 10-12.
Further, in step S2, the pH of the filtrate collected in step S1 is adjusted to 10.
Since the previous experiment proves that when the pH of the filtrate collected in the step S1 is adjusted to 10, a large amount of precipitate is already precipitated, and since the alkali in the filtrate needs to be neutralized by acid in the later period, if the pH is adjusted to be too high in the previous period, the salt amount formed by the subsequent acid-alkali neutralization is too large, the pressure for subsequent desalination is increased, and even the purity of the target product is reduced, so that it is appropriate to control the pH of the filtrate collected in the step S1 to 10.
Further, the concentration of 3' -SL in the concentrated solution on the membrane in the step S3 is 10-30 g/L.
Experiments show that the concentration of the 3 ' -SL in the concentrated solution has important influence on the adsorption and purification process of the anion-cation exchange resin and the purity of the final product 3 ' -SL, when the concentration of the 3 ' -SL in the concentrated solution is higher than 30g/L, the resin loss is caused, the desalting effect is poor, a large amount of salt is remained to influence the yield and the purity of the product, the yield of the product is reduced to 85%, and the purity is not higher than 90%; when the concentration of 3' -SL in the concentrated solution is lower than 10g/L, the treatment system of the anion-cation exchange resin is enlarged, and the treatment efficiency is greatly reduced; and when the concentration of the 3 '-SL in the concentrated solution is 10-30 g/L, the yield of the product 3' -SL is not lower than 91%, and the purity of the 3 '-SL is not lower than 96%, so that the concentration of the 3' -SL in the concentrated solution on the membrane of the step S3 is adjusted to be 10-30 g/L, and simultaneously, higher yield and higher purity of the product can be obtained.
Further, the method of heating and inactivating the enzyme of the conversion solution in step S1 is as follows: heating the conversion solution at 80-100 ℃ for 8-15 min.
Furthermore, the purity of the 3' -SL prepared by purifying and drying in the steps S1-S4 is more than or equal to 96 percent.
Compared with the prior art, the invention has the following beneficial effects:
the method comprises the steps of heating, inactivating enzyme, filtering, adjusting the pH value of filtrate to be alkaline, filtering, adjusting the pH value of the filtrate to be neutral, concentrating, adsorbing ions, and drying to obtain the purified 3 '-SL, wherein the method comprehensively improves the removal rate of macromolecular substances such as protein in the conversion solution by adopting two modes of heating, inactivating enzyme and adjusting the pH value to be alkaline, controls the concentration of the 3' -SL in the concentrated solution, plays an important role in further improving the purity of the product 3 '-SL by anion and cation adsorption purification, and the purity of the product 3' -SL obtained by the comprehensive method is more than or equal to 96%.
Drawings
FIG. 1 is a schematic view of the process of the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified. The conversion solution used in the present invention was derived from the conversion solution obtained by the method described in the patent application No. CN201910787973.7, entitled a method for preparing sialyllactose.
Example 1
This example provides a method for separating and purifying high-purity 3' -sialyllactose, which comprises the following steps, as shown in fig. 1:
s1: heating the conversion solution at 90 ℃ for 10min, inactivating the activity of enzyme in the conversion solution, promoting the protein denaturation and precipitation of the conversion solution, simultaneously promoting the thallus, large-particle impurities and non-enzyme protein in the conversion solution to be denatured and precipitated, then filtering and removing the precipitate by using a 30nm ceramic filter membrane, adding water with the minimum circulation volume 2 times that of the ceramic membrane for further filtering by using a circulation membrane after the minimum circulation volume of the ceramic membrane is reached, further removing the precipitated precipitate to obtain a clear solution containing 3 '-SL, wherein the yield of the 3' -SL in the conversion solution in the step is up to 95%.
S2: adding alkali into the clear liquid collected in the step S1, adjusting the pH value of the clear liquid to 10, promoting proteins in the clear liquid such as CMP kinase, polyphosphate kinase, CMP-sialic acid synthetase and other foreign proteins released by cell disruption to reach the isoelectric point of the proteins to aggregate and precipitate, then filtering the solution by a 3000Da organic membrane to remove protein precipitates and macromolecular impurities such as sodium hexametaphosphate, adding water with 4-6 times of the initial membrane volume into the filtering system to perform circulating membrane filtration, collecting filtrate, and effectively removing macromolecular substances such as sodium hexametaphosphate in the clear liquid.
In the process of adjusting the pH of the clear liquid collected in step S1, since the components in the clear liquid are complex, it is difficult to ascertain the type of protein therein and determine the isoelectric point thereof, the inventors adjusted the pH of the clear liquid collected in step S1 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, respectively, and found that the pH of the clear liquid is 8 or less and no precipitate is precipitated; when the pH is 9, a small amount of precipitate is precipitated, and when the pH is 10-12, a large amount of precipitate is precipitated, so that the pH of the clear liquid collected in the step S1 is selectively adjusted to 10-12, a large amount of protein impurities can be further removed, and in order to avoid the problem that the amount of subsequent acid-base neutralization and salt formation is too large due to too high pH, the pressure and desalting effect of subsequent desalting are poor, and the purity of the target product is reduced, so that the pH of the clear liquid collected in the step S1 is adjusted to 10.
S3: and (4) adding acid into the filtrate obtained in the step S2 to adjust the pH of the filtrate to be neutral, then filtering and removing impurities through a 1000Da organic membrane, concentrating, and collecting the concentrated solution on the membrane, wherein the filtering and removing impurities mainly refer to removal of small molecular substances such as salt, magnesium chloride heptahydrate, sialic acid and the like. In the step, water with 5-10 times of the initial membrane passing volume is added into a filtering system for circulating membrane filtration, concentrated solution on the membrane is collected, the concentration of 3 '-SL in the concentrated solution on the membrane is detected in real time by using high performance liquid chromatography until the concentration of 3' -SL in the concentrated solution on the membrane is 10-30 g/L, and the circulating membrane filtration and concentration are stopped.
S4: deeply desalting the concentrated solution obtained in the step S3 by using an anion-cation exchange method: firstly, the salt substances in the concentrated solution are further removed by utilizing cation resin adsorption and purification and then utilizing anion adsorption and purification, and the conductivity in the conversion solution can be reduced by about 90 percent through the anion and cation exchange desalination. And concentrating and drying (freeze-drying or spray-drying) the desalted liquid to obtain 3 '-SL, wherein the purity of the 3' -SL separated after the treatment of the steps S1-S4 is not lower than 96%.
Experiments show that the concentration of 3 ' -SL in the concentrated solution obtained in the step S3 has important influence on the adsorption and purification process of the anion-cation exchange resin and the purity of the final product 3 ' -SL, when the concentration of 3 ' -SL in the concentrated solution is higher than 30g/L, resin loss is caused, the desalting effect is poor, a large amount of salt remains to influence the yield and the purity of the product, the yield of the product is reduced to 85%, and the purity is not higher than 90%; when the concentration of 3' -SL in the concentrated solution is lower than 10g/L, the treatment system of the anion-cation exchange resin is enlarged, and the treatment efficiency is greatly reduced; and when the concentration of the 3 '-SL in the concentrated solution is 10-30 g/L, the yield of the product 3' -SL is not lower than 91%, and the purity of the 3 '-SL is not lower than 96%, so that the concentration of the 3' -SL in the concentrated solution on the membrane of the step S3 is controlled to be 10-30 g/L, and simultaneously, higher yield and higher purity of the product can be obtained.
Example 2
This example provides a method for separating and purifying high-purity 3' -sialyllactose, comprising the following steps:
s1: heating the 3' -SL conversion solution at 90 ℃ for 10min to inactivate the activity of enzyme in the conversion system and to denature and precipitate thermosensitive protein; filtering the heated conversion solution by a 30nm ceramic membrane to remove thalli, large-particle impurities and precipitated impurity proteins to obtain clear liquid containing 3' -SL, and adding water for dialysis until the yield is not lower than 95%.
S2: adding alkali into the 3' -SL clear solution to adjust the pH value to 10, promoting the non-heat denatured protein to settle at the isoelectric point, then filtering by a 3000D organic membrane to further remove settled protein and some soluble macromolecular impurities (such as sodium hexametaphosphate in a conversion system, and the like), and adding water for dialysis until the yield is not lower than 90%.
S3: collecting clear liquid passing through a 3000D membrane, adding acid to adjust the pH value of the clear liquid to 7, then passing through a 1000D organic membrane, removing monovalent salt and small molecular impurities in the clear liquid, adding water for dialysis until the conductivity of the concentrated liquid is not changed, and collecting membrane supernatant, wherein the concentration of 3' -SL in the membrane supernatant is within the range of 10-30 g/L.
S4: then further desalting the membrane supernatant by using ion exchange resin; adding 0.1% active carbon into the desalted clear solution for decolorization, and then concentrating and spray drying to obtain the 3' -SL with the purity of 98%.
Comparative example 1
Referring to the method of example 2 for separating and purifying 3 '-SL, the comparative example only differs from example 2 in that the processes of "adjusting pH of 3' -SL clear solution to 10 by adding alkali" and "adjusting pH of clear solution collected through 3000D membrane to 7" are not performed in step S2 and step S3, and the rest steps are the same as example 2.
The purity of the 3' -SL isolated and purified by the method described in this comparative example was 95%.
Comparative example 2
Referring to the method of example 2, this comparative example is different from example 2 only in that step S1 is not treated by "heating 3' -SL conversion solution at 90 ℃ for 10 min", and the rest of the procedure is the same as example 2.
The purity of the 3' -SL isolated and purified by the method described in this comparative example was 80%.
Comparative example 3
Referring to the method described in example 2, this comparative example differs from example 2 only in that step S1 is not subjected to the "heating 3 '-SL conversion solution at 90 ℃ for 10 min" treatment, while steps S2 and S3 are not subjected to the "adjusting pH of the 3' -SL clear solution to 10 with alkali" and the "adjusting pH of the clear solution collected on a 3000D membrane to 7 with acid" treatment, and the rest of the steps are the same as example 2.
The purity of the 3' -SL isolated and purified by the method described in this comparative example was 60%.
As can be seen from the comparison of the results of example 2, comparative example 1, comparative example 2 and comparative example 3, the purity of 3' -SL finally isolated is comprehensively improved by adopting the way of heat treatment and pH adjustment of the conversion solution in the invention, although the components in the conversion solution are complex.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A method for separating and purifying high-purity 3' -sialyllactose is characterized by comprising the following steps:
s1: heating the conversion solution to inactivate enzyme, filtering through a 30-100 nm filter membrane, and collecting filtrate;
s2: adjusting the pH value of the filtrate collected in the step S1 to be alkaline, filtering the filtrate by a 3000 Da-5000 Da membrane, and collecting the filtrate;
s3: adjusting the pH value of the filtrate collected in the step S2 to be neutral, filtering and concentrating the filtrate by a membrane of 800 Da-1000 Da, and collecting the concentrated solution on the membrane;
s4: and (4) sequentially adsorbing the concentrated solution on the membrane obtained in the step S3 by anion and cation exchange resin, concentrating and drying to obtain the 3' -SL.
2. The method as claimed in claim 1, wherein the pH of the filtrate collected in step S1 is adjusted to 10-12 in step S2.
3. The method as claimed in claim 2, wherein the pH of the filtrate collected in step S1 is adjusted to 10 in step S2.
4. The method according to claim 1, wherein the concentration of 3' -SL in the concentrated solution on the membrane in the step S3 is 10-30 g/L.
5. The method of claim 1, wherein the step S1 comprises the following steps: heating the conversion solution at 80-100 ℃ for 8-15 min.
6. The method of any one of claims 1 to 5, wherein the purity of the 3' -SL obtained through steps S1 to S4 is not less than 96%.
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