CN112390851A - Method for purifying biochemical reaction liquid, purified biochemical reaction product and preparation method thereof - Google Patents

Method for purifying biochemical reaction liquid, purified biochemical reaction product and preparation method thereof Download PDF

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CN112390851A
CN112390851A CN201910744278.2A CN201910744278A CN112390851A CN 112390851 A CN112390851 A CN 112390851A CN 201910744278 A CN201910744278 A CN 201910744278A CN 112390851 A CN112390851 A CN 112390851A
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biochemical reaction
reaction solution
solution
pretreatment
purified
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孙梁
银龙
熊愉
叶凯熙
王振桁
王骏
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Yuyao Laifusben Health Technology Co.,Ltd.
Bioright Worldwide Co Ltd
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    • C07—ORGANIC CHEMISTRY
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
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    • 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
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

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Abstract

The invention provides a purification method of biochemical reaction liquid, a method for preparing biochemical reaction products and prepared biochemical products. The purification method includes (S1) providing a biochemical reaction solution containing ions and biochemical reaction products; (S2) performing a first pretreatment on the biochemical reaction solution to remove ions in the biochemical reaction solution, thereby obtaining a first pretreatment solution; and (S3) passing the pre-treatment solution through an ion exchange resin, thereby obtaining a purified biochemical reaction solution. The purification method can improve the productivity and reduce the consumption of pure water and/or acid and alkali.

Description

Method for purifying biochemical reaction liquid, purified biochemical reaction product and preparation method thereof
Technical Field
The invention belongs to the field of biochemistry, and particularly relates to a purification method of biochemical reaction liquid, a purified biochemical reaction product and a preparation method thereof.
Background
With the development of society and the popularization of green synthesis technology, various biochemical reactions (such as biochemical synthesis reactions) taking enzyme as a catalyst are increasing, and most of the subsequent purification processes take ion exchange resin purification as a main means of process purification. The ion exchange resin is a high molecular compound with a net structure of active groups, has the functions of exchange, selection, absorption, catalysis and the like, and is widely applied to the fields of preparation of industrial high-purity water, medical sanitation, bioengineering and the like.
According to the nature of the groups carried by the ion exchange resin, the ion exchange resin can be divided into strongly acidic cations, weakly acidic cations, strongly basic anions, weakly basic anions, chelation, amphiphilicity and redox resin. The ion exchange resins used for purification processes in the biomedical field as well as in the biochemical reaction field are mostly cationic and/or anionic resins. However, purification methods using ion exchange resins require the expenditure of large amounts of water and the use of large amounts of acids and/or bases.
Therefore, the purification method needs to be improved actively, the consumption of pure water is reduced, the consumption of acid and alkali is reduced, the pollution to the environment is reduced, and the idea of practicing green chemistry and environment-friendly production in the field of biological medicine is promoted.
Disclosure of Invention
In order to solve at least one of the above problems, the present invention provides a method for purifying a biochemical reaction solution, a purified biochemical reaction product, and a method for preparing the same.
Specifically, the present invention provides:
a purification method of biochemical reaction liquid comprises the following steps:
(S1) providing a biochemical reaction solution containing ions and biochemical reaction products;
(S2) performing a first pretreatment on the biochemical reaction solution to remove ions in the biochemical reaction solution, thereby obtaining a first pretreatment solution; and
(S3) passing the pre-treatment solution through an ion exchange resin to obtain a purified biochemical reaction solution.
Wherein the step (S2) may include performing the first pretreatment by at least one selected from the group consisting of an ion precipitation method, an acid-base precipitation method, an ion resin exchange method, a nanofiltration method, and an electrodialysis method.
Optionally, the ions may be selected from sulfate, potassium, sodium, chloride, calcium and/or phosphate ions.
Wherein the purification method may further comprise (S4) performing a second pretreatment on the first pretreatment solution to further remove monovalent ions in the biochemical reaction solution.
Optionally, the second pretreatment is selected from at least one of an ultrafiltration process and a nanofiltration process.
Optionally, the nanofiltration has a molecular weight cut-off of 150-300 Da.
Wherein the biochemical reaction solution is selected from at least one of an enzyme catalysis reaction solution and a fermentation reaction solution.
Wherein the method may further comprise a step of performing ultrafiltration to remove the protein before, simultaneously with or after the step (S3).
It is preferable to perform ultrafiltration to remove proteins of the pretreatment liquid before the step (S3) and after the step (S2).
Preferably, the retentate protein of the ultrafiltration is 5,00-10,000 Da.
Wherein the biochemical reaction product is at least one selected from amino acids, peptides and proteins, preferably ademetionine, glutathione, adenosine triphosphate and/or S-adenosyl-L-homocysteine.
Wherein, in the biochemical reaction solution, the concentration of the biochemical reaction product is 4-800 mM, preferably 8-400 mM.
Wherein the biochemical reaction solution is provided in a circulating reaction mode and/or a continuous mode.
Wherein, the total yield of the purification method is 20 to 70 percent.
Wherein the purification method can generate 2000-4000 kg/kg of wastewater of biochemical reaction products, and/or 4-30 kg/kg of acid of biochemical reaction products is/are used.
Wherein the step (S2) further comprises adjusting the pH value of the biochemical reaction solution to 6-12.
Optionally, the purification method further comprises the step of adjusting the pH of the pretreatment liquid to 4-6 before the step (S3).
A method of preparing a biochemical reaction product, comprising the steps of:
(S11) providing a purified biochemical reaction solution by the method of any one of claims 1 to 11 and
(S12) drying the purified biochemical reaction solution, thereby obtaining a biochemical reaction product.
Wherein, the purity of the biochemical reaction product can reach more than 90 percent.
Wherein the method of preparing a biochemical reaction product further comprises a step of concentrating the purified biochemical reaction solution before the step (S12), preferably, the concentration step is performed by a nano-filtration method and/or a reverse osmosis method.
Wherein the drying is selected from at least one of crystallization drying, freeze drying and spray drying.
A purified biochemical reaction product obtained by any of the methods.
According to the invention, the purity of the biochemical reaction product can be more than 90%, even more than 95%.
The purification method is simple and feasible, and is universal and commercialized. In addition, the method of the invention also improves the productivity, increases and reduces the consumption of pure water and/or reduces the consumption of acid and alkali. For example, the applicant finds that, when 1kg of biochemical reaction product is produced, the method can improve the yield by 5-10%, reduce the wastewater by 10-40%, and save 60-80% of inorganic acid or inorganic base, such as concentrated hydrochloric acid or concentrated sulfuric acid.
In some cases, the methods of the invention also increase the purity of the biochemical reaction product, for example, to 80% -95%.
Brief description of the drawings
FIG. 1 shows a flow diagram of a purification process according to one embodiment of the present invention;
FIG. 2 shows a flow diagram of a purification process according to another embodiment of the present invention;
FIG. 3 shows a flow diagram of a method of preparing a biochemical reaction product, according to one embodiment of the present invention.
Detailed description of the preferred embodiments
The present invention is further illustrated by the following description of specific embodiments, which are not intended to limit the invention, and various modifications or improvements can be made by those skilled in the art based on the basic idea of the invention, but within the scope of the invention, without departing from the basic idea of the invention.
As mentioned above, many biochemical reaction products (particularly enzymatic reaction products) are purified using ion exchange resins. For example, Adenosine Triphosphate (ATP) can be purified using a weakly basic anion exchange resin. Separating and purifying S-adenosine-L-homocysteine with anion exchange resin and adsorption resin to obtain the product with purity up to 99.5%. However, before passing through the ion exchange resin, a large amount of anions and cations exist in the solution to be treated containing the products, so that the adsorption amount of the ion exchange resin to purified substances is reduced, and the effect of the ion exchange resin is reduced. And because acid or alkali is needed to regenerate the ion exchange resin after each use, the use amount of the acid and the alkali is increased. By statistical analysis of the data, a large amount of purified water is required for product purification using ion exchange resins, and about 3 to 6 tons of purified water are generally required for purification of 1kg of product. Therefore, the industrial mass production is carried out, the water consumption and the wastewater amount can cause great burden to water resources and ecological environment, and the method is contrary to the green and environment-friendly production concept at the present stage of China.
Applicants have found that increasing the loading of the ion exchange resin allows for a reduction in the amount of ion exchange resin used while producing the same amount of product. The method not only greatly reduces the dosage of acid and alkali and pure water, but also can improve the productivity. The invention removes the ions in the reaction solution before ion exchange, improves the adsorption capacity of the ion exchange resin to the purified substances, and enhances the effect of the ion exchange resin. Therefore, the purification method of the invention can improve the productivity, reduce the consumption of pure water and/or reduce the consumption of acid and alkali. For example, compared with the existing method without pretreatment of biochemical reaction liquid, 1kg of biochemical reaction product is produced, the method can improve the yield by 5-10%, reduce 10% -40% of waste water, and save 60% -80% of inorganic acid or inorganic base, such as concentrated hydrochloric acid or concentrated sulfuric acid.
In one aspect, as shown in FIG. 1, the present invention provides a method for purifying a biochemical reaction solution, comprising the steps (S1) to (S3) of the following steps.
The step (S1) includes providing a biochemical reaction solution containing ions and biochemical reaction products. The reaction liquid can be obtained by a method such as an immobilized enzyme catalytic reaction, a fermentation reaction involving an enzyme, or the like. The biochemical reaction can be a circulating reaction mode and a continuous reaction mode.
The ions may be selected from sulfate, potassium, sodium, chloride, calcium and/or phosphate ions.
For example, the biochemical reaction solution may be a reaction solution obtained by preparing adenosylmethionine using ATP and L-methionine as raw materials under the catalysis of an immobilized enzyme of adenosylmethionine synthetase; glutathione reaction liquid prepared by using glutamic acid, cysteine, glycine and the like as raw materials in the presence of immobilized enzyme, and the like.
In the biochemical reaction solution, the concentration of the biochemical reaction product can be 8-400 mM. The biochemical reaction solution may further comprise an anion, a cation, or a combination thereof.
Before the biochemical reaction solution containing anions and/or cations is subjected to the ion exchange treatment, the biochemical reaction solution may be subjected to a first pretreatment to remove ions (anions and/or cations) therein. Therefore, as shown in FIG. 1, the purification method of the present invention may include a step (S2) of subjecting the biochemical reaction solution to a first pretreatment to remove ions from the biochemical reaction solution, thereby obtaining a first pretreatment solution.
Preferably, the first pretreatment may be performed by at least one selected from the group consisting of a nanofiltration method, an ion precipitation method, an acid-base precipitation method, an ion resin exchange method, and an electrodialysis method. The ions may be anions and/or cations, and may be sulfate ions, calcium ions, phosphate ions, sodium ions, potassium ions, chloride ions, and the like.
In other words, the ions in the biochemical reaction solution can be removed by at least one selected from the group consisting of an ion precipitation method, an acid-base precipitation method, an ion resin exchange method, and an electrodialysis method.
As shown in fig. 2, the purification method may further include (S4) removing monovalent ions including cations and/or anions from the biochemical reaction solution by performing a second pretreatment on the biochemical reaction solution and/or the first pretreatment solution.
Preferably, the second pretreatment is selected from at least one of an ultrafiltration method and a nanofiltration method. The nanofiltration method is more preferable to remove monovalent anions and/or cations in the biochemical reaction solution, and the molecular weight cut-off of the nanofiltration can be 150-300 Da.
The first treatment and the second treatment may be performed only one of them, may be performed simultaneously, or may be performed first with the first pretreatment followed by the second pretreatment or with the second pretreatment followed by the first treatment.
Then, as shown in FIG. 1, the purification method of the present invention may further include a step (S3) of passing the pre-treated solution through an ion exchange resin to thereby obtain a purified biochemical reaction solution. The ion exchange resin may include various types of ion exchange resins such as anion exchange resins, cation exchange resins, strongly acidic cations, weakly acidic cations, strongly basic anions, weakly basic anions, chelating, amphoteric, and redox resins, preferably DK110 resin, HZ016 resin.
The dosage of the ion exchange resin is 1 to 5 percent of the product amount (biochemical reaction solution). The eluent can be pure water, sulfuric acid solution, hydrochloric acid solution, butanedisulfonic acid solution, etc.
It is also possible to perform ultrafiltration to remove proteins (step S5) before, simultaneously with or after the step (S3), and it is preferable to perform ultrafiltration to remove proteins of the pretreatment liquid before the step (S3) and after the step (S2). Preferably, the retentate protein of the ultrafiltration is 5,000-10,000 Da.
In one embodiment, the steps of first pretreatment to remove ions, ultrafiltration to remove proteins, nanofiltration to remove monovalent ions, and ion exchange resin purification may be sequentially performed.
In one embodiment, the step (S2) may further include adjusting the pH of the biochemical reaction solution to 6 to 12, preferably 9 to 11. The pH of the biochemical reaction solution may be adjusted with an alkaline reagent or a buffer solution such as ammonia, an alkali metal hydroxide, an alkali metal carbonate, or the like.
In one embodiment, the purification method may further include the step of adjusting the pH of the pretreatment liquid to 4 to 6 before the step (S3).
The pH of the pretreatment solution can be adjusted with hydrochloric acid, sulfuric acid, a buffer solution, or the like. The buffer solution is one or more selected from phosphate, oxalate, acetate, citrate and glycine.
In another aspect, as shown in FIG. 3, the present invention also provides a method for preparing a biochemical reaction product, comprising the steps of: (S11) providing a purified biochemical reaction solution by the above-mentioned purification method and (S12) drying the purified biochemical reaction solution, thereby obtaining a biochemical reaction product. The drying is selected from at least one of crystallization drying, freeze drying and spray drying.
As shown in fig. 3, the method of preparing a biochemical reaction product may further include a step S13 of concentrating the purified biochemical reaction solution before the step S12. The concentration step may be performed by a nanofiltration method and/or a reverse osmosis method.
Specifically, the method for preparing a biochemical reaction product of the present invention may sequentially comprise the steps of:
(i) step S1 → step S2 → step S5 → step S4 → step S3 → step S13 → step S12;
(ii) step S1 → step S5 → step S4 → step S3 → step S13 → step S12;
(iii) step S1 → step S2 → step S3 → step S13 → step S12;
(iv) step S1 → step S4 → step S3 → step S12;
(v) step S1 → step S2 → step S5 → step S3 → step S13 → step S12;
(vi) step S1 → step S2 → step S4 → step S3 → step S13 → step S12;
(vii) step S1 → step S2 → step S3 → step S13 → step S12; or
(viii) Step S1 → step S2 → step S3 → step S12.
In one embodiment, the method for preparing a biochemical reaction product of the present invention may include steps S2 and/or S4 through S3.
According to the present invention, the purity of the biochemical reaction product may be 90% or more, preferably 95% or more.
The biochemical reaction product may be selected from at least one of amino acids, peptides and proteins, preferably ademetionine, glutathione, adenosine triphosphate and/or S-adenosyl-L-homocysteine.
By the method, the total yield of the purification method can reach 40-60%.
The purification method can produce 2000-4000 Kg/Kg of wastewater of biochemical reaction products, and/or uses 4-30 Kg/Kg of inorganic acid or organic acid of biochemical reaction products.
In yet another aspect, the invention also provides a purified biochemical reaction product obtained by any one of the methods described above.
The present disclosure is further explained or illustrated below by way of examples, which should not be construed as limiting the scope of the present disclosure.
Examples of the present invention
The following examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
The materials and equipment used in the examples are described below:
the instrument used in the purity analysis is a high performance liquid chromatograph (HPLC Shimadzu LC-20AH, chromatographic column Elite C184.6mm x 250mm), and the measurement is carried out according to the high performance liquid chromatography (four parts 0512 of Chinese pharmacopoeia 2015 edition);
the immobilized enzymes are all provided by the Biotech company of hong Kong (hong Kong);
ultrafiltration, nanofiltration, reverse osmosis devices and membrane cores thereof are all purchased from Hangzhou Tanna membrane technologies, Inc.;
ion exchange resins are all purchased from Shanghai Huazhen science and technology Limited;
the other reagents are conventional in the art and are commercially available.
Preparation example 1 preparation of an ademetionine reaction solution
Loading an immobilized enzyme of the adenosylmethionine synthetase into an enzyme column, connecting into a pipeline, and setting the reaction temperature to be 45-65 ℃. ATP, L-methionine, magnesium chloride and KCl are prepared into a solution according to a specified ratio (10:50:60:300), and the pH value is adjusted to 6.0-7.0 by ammonia water. The prepared reaction stock solution passes through an enzyme column at a certain speed (2BV/h), the reaction temperature is maintained at 45-65 ℃, and the reaction solution is received as the feed solution to be treated in the following examples when the concentration of the adenosylmethionine in the reaction solution reaches more than 4.0 mM.
Example 1
5.35L of the enzyme reaction solution of preparation example 1 containing 15.2g of ademetionine was taken, 14.0g of sodium dihydrogenphosphate was added, the pH was adjusted to 10 with ammonia water, and the pH of the filtered filtrate was adjusted to 4 to 6 with hydrochloric acid to obtain 5.4L of a solution. Performing ultrafiltration with pressure of 0.2-0.6Mpa, and adding water 10L to obtain permeate 14.7L and waste water 0.7L. Nanofiltration is carried out on the obtained permeate, the pressure is 0.6-1.0Mpa, concentration is carried out after 28L of water is supplemented, 2.5L of concentrated solution is obtained, and 40L of produced wastewater is obtained. 2.5L of the resulting concentrate was eluted through an ion exchange resin (DK110) having a resin content of 500g at a flow rate of 1.5-2.5 BV/h. After the sample is loaded, pure water is used for eluting to remove impurities, sulfuric acid solution is used for eluting, 1.32L of product eluent is collected, and 6.88L of waste water containing 109.5g of concentrated hydrochloric acid is generated.
4g of p-toluenesulfonic acid and 8ml of 1M sulfuric acid were added to 1.32L of the collected eluate. Stirring, and freeze drying to obtain lyophilized product 17.5 g. The total yield is 60%, the purity is 92.3%, and the total wastewater amount is 48L.
Example 2
1.75L of the enzyme reaction solution obtained in preparation example 1 containing 4.75g of ademetionine was taken, 2.75g of sodium dihydrogen phosphate was added, the pH was adjusted to 10 with ammonia water, and the pH of the filtered filtrate was adjusted to 4 to 6 with hydrochloric acid to obtain 1.8L of a solution. The solution was eluted through an ion exchange resin (DK110) with a resin content of 500g at a flow rate of 1.5-2.5 BV/h. After the sample is loaded, pure water is used for eluting to remove impurities, sulfuric acid solution is used for eluting, 0.67L of product eluent is collected, and 5.43L of waste water containing 100.4g of concentrated hydrochloric acid is generated. Nanofiltration is carried out on 0.67L of collected eluent under the pressure of 0.6-1.0Mpa, 10L of water is supplemented, and then concentration is carried out to obtain 0.8L of concentrated solution, and 9.87L of generated wastewater is obtained.
0.8L of the collected eluate was added to 1.1g of p-toluenesulfonic acid and 5.8ml of 1M sulfuric acid. Stirring, and freeze drying to obtain lyophilized product 4.51 g. The total yield is 49.5%, the purity is 89.8%, and the total wastewater amount is 16.2L.
Example 3
Taking 3.6L of the enzyme reaction liquid obtained in the preparation example 1, containing 10.2g of adenosylmethionine, performing electrodialysis on the reaction liquid, controlling the temperature at 10-15 ℃ and the pressure at 0.05-0.4 Mpa, and circulating for 1h to obtain 1.2L of concentrated liquid and 2.4L of generated wastewater. The concentrated solution is eluted by ion exchange resin (DK110) with the resin amount of 500g at the flow rate of 1.5-2.5 BV/h. After the sample is loaded, pure water is used for eluting and removing impurities, sulfuric acid solution is used for eluting, 0.65L of product eluent is collected, and 6.4L of waste water containing 100.6g of concentrated hydrochloric acid is generated. Nanofiltration is carried out on 0.65L of the collected eluent under the pressure of 0.6-1.0Mpa, 25L of water is supplemented, and then concentration is carried out to obtain 0.75L of concentrated solution, and 25.1L of generated wastewater is obtained.
To 0.8L of the collected eluate were added 2.3g of p-toluenesulfonic acid and 10.9ml of 1M sulfuric acid. Stirring, and freeze drying to obtain lyophilized product 10.1 g. The total yield is 51.6%, the purity is 90.5%, and the total wastewater amount is 33.5L.
Example 4
3L of the enzyme reaction solution of preparation example 1 containing 8.35g of ademetionine was taken, and the pH was adjusted to 4 to 6 with hydrochloric acid to obtain 4.2L of a solution. And (3) carrying out nanofiltration on the reaction liquid under the pressure of 0.6-1.0Mpa, supplementing water for 20L, and then concentrating to obtain 1.5L of concentrated solution and 22.6L of generated wastewater. 1.5L of the obtained concentrate was eluted through an ion exchange resin (DK110) having a resin content of 500g at a flow rate of 1.5-2.5 BV/h. After the sample is loaded, pure water is used for eluting and removing impurities, sulfuric acid solution is used for eluting, 0.66L of product eluent is collected, and 6.5L of waste water containing 99.6g of concentrated hydrochloric acid is generated.
0.66L of the collected eluate was added to 1.93g of p-toluenesulfonic acid and 3.8ml of 1M sulfuric acid. Stirring, and freeze drying to obtain 8.6g lyophilized product. The total yield is 53.6%, the purity is 91.2%, and the total wastewater amount is 29.7L.
Example 5 (comparative example)
1.75L of the enzyme reaction feed liquid of preparation example 1 containing 4.8g of ademetionine was taken, and the pH was adjusted to 4 to 6 with hydrochloric acid to obtain 2.4L of a solution. 2.4L of the obtained feed liquid is eluted by ion exchange resin (DK110) with the resin amount of 500g at the flow rate of 1.5-2.5 BV/h. After the sample is loaded, pure water is used for eluting and removing impurities, sulfuric acid solution is used for eluting, 0.7L of product eluent is collected, and 5.25L of waste water containing 102g of concentrated hydrochloric acid is generated. Nanofiltration is carried out on 0.7L of the collected eluent under the pressure of 0.6-1.0Mpa, and the concentrated solution is obtained after 7.8L of water is supplemented, so that 0.65L of concentrated solution is obtained, and 7.55L of waste water is produced.
0.75g of p-toluenesulfonic acid and 4.5ml of 1M sulfuric acid were added to 0.65L of the collected eluate. Stirring, and freeze drying to obtain lyophilized product 3.3 g. The total yield is 36%, the purity is 90%, and the total wastewater amount is 13.2L.
Example 6
2.2L of the enzyme reaction feed liquid of preparation example 1 containing 6.0g of ademetionine was taken, and pH was adjusted to 4 to 6 with hydrochloric acid to obtain 3L of a solution. Performing ultrafiltration with pressure of 0.2-0.6Mpa, and adding water of 4L to obtain 6.5L of permeate and 0.8L of waste water. Nanofiltration is carried out on the obtained permeate, the pressure is 0.6-1.0Mpa, water is supplemented for 11L, concentration is carried out, 1L of concentrated solution is obtained, and 16.2L of waste water is generated. 1L of the obtained concentrated solution was eluted through an ion exchange resin (DK110) having a resin content of 500g at a flow rate of 1.5-2.5 BV/h. After the sample is loaded, pure water is used for eluting and removing impurities, sulfuric acid solution is used for eluting, 0.68L of product eluent is collected, and 5.7L of waste water containing 99g of concentrated hydrochloric acid is generated. Nanofiltration is carried out on 0.68L of collected eluent under the pressure of 0.6-1.0Mpa, 4L of water is supplemented, and then concentration is carried out to obtain 2L of feed liquid and 3.5L of produced wastewater.
2L of the collected eluate was added to 1.37g of p-toluenesulfonic acid and 9ml of 1M sulfuric acid. Stirring, and freeze drying to obtain 6.06g lyophilized product. The total yield is 52.7%, the purity is 90.8%, and the total wastewater amount is 28.2L.
As can be seen, compared with the comparative example, 1kg of p-toluenesulfonic acid adenosine dithiosulfate methionine product is produced, the method of the invention can reduce 4% -23.6% of waste water and save 5-7 of concentrated hydrochloric acid.
Preparation example 2 preparation of glutathione reaction solution
According to the method for preparing the conversion solution described in example 3 of Chinese patent CN200710020902.1, glutamic acid, cysteine, glycine and the like are prepared into a solution, the solution is added into an immobilized enzyme reaction tank, reaction is carried out by keeping a specified pH (8.0) and temperature (37 ℃), and when the concentration of glutathione reaches more than 35mM, the reaction solution is discharged; then, continuously preparing a raw material solution, and adding the raw material solution into the reaction tank to continue the circulating reaction. The reaction solution was collected as a feed solution to be treated in the following examples.
Example 7
69L of the feed liquid obtained in the preparation example 2 is taken, wherein the feed liquid contains 746g of glutathione, hydrochloric acid is added to adjust the pH value to 3.5-4.5, and the filtrate 73L is obtained by filtration. Nanofiltration is carried out on the filtrate, the pressure is 0.6-1.0Mpa, 240L of water is supplemented, and then concentration is carried out, so that 36L of concentrated solution is obtained, and 277L of waste water is produced. Passing 36.5L of the obtained concentrated solution through ion exchange resin (HZ016), wherein the resin amount is 18.5Kg, and the flow rate is 1.0-1.5 BV/h. After loading on the column, pure water was eluted to remove impurities, 0.2M hydrochloric acid was eluted, and 238L of the eluate was collected to produce 220L of wastewater containing 12.6kg of concentrated hydrochloric acid. And (3) carrying out nanofiltration on the collected eluent 238L, wherein the pressure is 0.6-1.0Mpa, supplementing water for 700L, and then concentrating to obtain 12L of nanofiltration concentrated solution, and generating wastewater 925L. Reverse osmosis is carried out on the nanofiltration concentrated solution to obtain 3.1L of concentrated solution and 9L of waste water. Adding 4 times volume of ethanol into the concentrated solution under stirring for crystallization, centrifuging, filtering and drying to obtain 470g of product. The total yield is 63%, the purity is 96.0%, and the total wastewater amount is 1430L.
Example 8
Taking 33.5L of the feed liquid obtained in the preparation example 2, wherein the feed liquid contains 370g of glutathione, adding hydrochloric acid to adjust the pH value to 3.5-4.5, and filtering to obtain 35.1L of filtrate. The filtrate is passed through ion exchange resin (HZ016) with a resin amount of 18.5Kg and a flow rate of 1.0-1.5 BV/h. After loading on the column, pure water was eluted to remove impurities, 0.2M hydrochloric acid was eluted, and 235L of the eluate was collected to give 219L of wastewater containing 12.45kg of concentrated hydrochloric acid. And (3) carrying out nanofiltration on the collected eluent 235L, wherein the pressure is 0.6-1.0Mpa, supplementing water for 700L, and then concentrating to obtain 14L of nanofiltration concentrated solution, and generating 920L of wastewater. The nanofiltration concentrate was subjected to reverse osmosis to obtain 1.35L of concentrate, yielding 12.5L of wastewater. Adding 4 times volume of ethanol into the concentrated solution under stirring for crystallization, centrifuging, filtering and drying to obtain 206g of product. The total yield is 55.6%, the purity is 94.8%, and the total wastewater amount is 1150L.
Comparative example 7 and example 8, it can be seen that when 1kg of glutathione product is produced in the same way, the method of the invention reduces the waste water by 4-5 and saves the concentrated hydrochloric acid by 5.
In conclusion, the purification method of the invention is simple and feasible, and is a general and commercially available purification. In addition, the method of the invention also improves the productivity, reduces the consumption of pure water and/or reduces the consumption of acid and alkali. For example, the applicant finds that, when 1kg of biochemical reaction product is produced, the method can improve the yield by 5-10%, reduce the wastewater by 10-40%, and save 60-80% of inorganic acid or inorganic base, such as concentrated hydrochloric acid or concentrated sulfuric acid.
The invention is not limited by the foregoing detailed description, and various modifications or changes may be made within the scope of the invention as outlined in the claims. Such variations are within the scope of the invention as claimed.

Claims (17)

1. A purification method of biochemical reaction liquid is characterized by comprising the following steps:
(S1) providing a biochemical reaction solution containing ions and biochemical reaction products;
(S2) performing a first pretreatment on the biochemical reaction solution to remove ions in the biochemical reaction solution, thereby obtaining a first pretreatment solution; and
(S3) passing the pre-treatment solution through an ion exchange resin to obtain a purified biochemical reaction solution.
2. The method according to claim 1, characterized in that the first pretreatment is performed by at least one selected from the group consisting of an ion precipitation method, an acid-base precipitation method, an ion resin exchange method, a nanofiltration method, and an electrodialysis method.
3. The method according to claim 1 or 2, further comprising (S4) performing a second pretreatment on the first pretreatment solution to remove monovalent ions from the biochemical reaction solution,
optionally, the second pretreatment is selected from at least one of ultrafiltration and nanofiltration, and
optionally, the nanofiltration has a molecular weight cut-off of 150-300 Da.
4. The method according to any one of claims 1 to 3, wherein the biochemical reaction solution is selected from at least one of an enzyme-catalyzed reaction solution and a fermentation reaction solution.
5. The method according to any one of claims 1 to 4, characterized in that the method further comprises a step of performing ultrafiltration to remove proteins before, simultaneously with or after step (S3), preferably before step (S3) and after step (S2), to remove proteins of the pretreatment solution, preferably ultrafiltration with a retentate of 5,00-10,000 Da.
6. The method according to any one of claims 1 to 5, characterized in that the biochemical reaction product is selected from at least one of amino acids, peptides and proteins, preferably ademetionine, glutathione, adenosine triphosphate and/or S-adenosyl-L-homocysteine.
7. The method according to any one of claims 1 to 6, wherein the concentration of the biochemical reaction product in the biochemical reaction solution is 4 to 800mM, preferably 8 to 400 mM.
8. The method according to any one of claims 1 to 7, wherein the biochemical reaction solution is supplied by a cyclic reaction mode and/or a continuous mode.
9. The method according to any one of claims 1 to 8, characterized in that the overall yield of the purification process is between 20% and 70%.
10. The method according to any one of claims 1 to 9, wherein the purification method produces 2000 to 4000kg/kg of wastewater of biochemical reaction products and/or uses 4 to 30kg/kg of acids of biochemical reaction products.
11. The purification method according to any one of claims 1 to 10, wherein the step (S2) further comprises a step of adjusting the pH of the biochemical reaction solution to 6 to 12,
optionally, the method further comprises the step of adjusting the pH of the pretreatment liquid to 4-6 before the step (S3).
12. A method of producing a biochemical reaction product, comprising the steps of:
(S11) providing a purified biochemical reaction solution by the method of any one of claims 1 to 11 and
(S12) drying the purified biochemical reaction solution, thereby obtaining a biochemical reaction product.
13. The method of claim 12, wherein the biochemical reaction product has a purity of 90% or greater.
14. The method according to any one of claims 12 to 13, characterized in that a step of concentrating the purified biochemical reaction solution is performed before step (S12), preferably by nanofiltration and/or reverse osmosis.
15. The method according to any one of claims 12 to 14, characterized in that the drying is selected from at least one of crystallization drying, freeze drying and spray drying.
16. A purified biochemical reaction product obtained by the method of any one of claims 12-15.
17. The biochemical reaction product according to claim 16, having a purity of 90% or more.
CN201910744278.2A 2019-08-13 2019-08-13 Method for purifying biochemical reaction liquid, purified biochemical reaction product and preparation method thereof Pending CN112390851A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113444757A (en) * 2021-06-23 2021-09-28 贵州卡本嘉泰生物科技产业发展有限公司 Preparation method of 1, 4-butanedisulfonic acid adenosine methionine
CN113499608A (en) * 2021-06-29 2021-10-15 贵州卡本嘉泰生物科技产业发展有限公司 Chromatographic system for enzymatic reaction and control method thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101429229A (en) * 2008-12-14 2009-05-13 甘肃正生生物科技有限公司 Method for producing high-purity glutathione
CN104130310A (en) * 2014-05-23 2014-11-05 烟台大学 Separation and purification method for glutathione
CN104418928A (en) * 2013-08-27 2015-03-18 上海医药工业研究院 Preparation method of 1, 4-succinic acid adenosine methionine
WO2018228246A1 (en) * 2017-06-15 2018-12-20 安徽古特生物科技有限公司 Method for enzymatic preparation of glutathione
CN109504718A (en) * 2019-01-23 2019-03-22 内蒙古拜克生物有限公司 A kind of production method of l-Isoleucine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101429229A (en) * 2008-12-14 2009-05-13 甘肃正生生物科技有限公司 Method for producing high-purity glutathione
CN104418928A (en) * 2013-08-27 2015-03-18 上海医药工业研究院 Preparation method of 1, 4-succinic acid adenosine methionine
CN104130310A (en) * 2014-05-23 2014-11-05 烟台大学 Separation and purification method for glutathione
WO2018228246A1 (en) * 2017-06-15 2018-12-20 安徽古特生物科技有限公司 Method for enzymatic preparation of glutathione
CN109504718A (en) * 2019-01-23 2019-03-22 内蒙古拜克生物有限公司 A kind of production method of l-Isoleucine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
李从军等: "生物产品分离纯化技术", vol. 1, 武汉：华中师范大学出版社, pages: 262 - 263 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113444757A (en) * 2021-06-23 2021-09-28 贵州卡本嘉泰生物科技产业发展有限公司 Preparation method of 1, 4-butanedisulfonic acid adenosine methionine
CN113499608A (en) * 2021-06-29 2021-10-15 贵州卡本嘉泰生物科技产业发展有限公司 Chromatographic system for enzymatic reaction and control method thereof

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