A method for separating and purifying Bosein from an enzyme reaction solution

Abstract

This invention provides a method for separating and purifying Bosein from an enzyme reaction solution. The method involves first ultrafiltration of the Bosein enzyme-catalyzed reaction solution through a ceramic membrane to remove proteins, followed by decolorization with activated carbon, and then impurity removal via a simulated moving bed continuous chromatography system. Afterwards, high-purity concentration, high-temperature reconstitution, and crystallization are performed. This invention utilizes a continuous chromatography separation system for desalting and impurity removal to improve product purity, and a separation and purification process based on ethanol recovery and recycling for cooling crystallization, which helps reduce production costs. This separation and purification process offers low purification cost, high yield, and high extraction purity. Therefore, this invention provides a novel, continuously producible, low-pollution, low-cost, high-yield, and high-purity crystalline Bosein purification method, which is beneficial for promoting the industrial production of Bosein.

Description

Technical Field

[0001] This invention relates to the field of separation and purification, and particularly to a method for separating and purifying bosine from an enzyme reaction solution. Background Technology

[0002] Pro-Xylane is a xylose derivative with anti-aging active ingredients. It can promote collagen synthesis, making skin stronger and more elastic, improving fine lines on the neck, and preventing aging. Therefore, the production of Pro-Xylane is of great significance.

[0003] Currently, the production methods of Bosein are divided into chemical synthesis and enzymatic catalysis. Separation and purification after Bosein production are crucial steps in the process. Most commercially available purification methods are designed for Bosein prepared by chemical synthesis. However, because the product contains enantiomers and numerous byproducts, it yields an oily crude Bosein, leading to transportation difficulties, separation challenges, and the use of anion and cation exchange columns, generating large amounts of wastewater and requiring repeated column filtration, resulting in low yields. Currently disclosed methods using organic membranes to retain Bosein for desalination cannot completely remove borates, acetates, and the generated organic byproducts. Furthermore, the product can permeate, leading to low yields after washing with large amounts of water.

[0004] Compared to chemical synthesis, enzymatic catalysis is safer, more efficient, less expensive, and more environmentally friendly, thus attracting increasing attention. However, enzymatic catalysis inevitably introduces enzyme proteins and other substances during the reaction, and generates biocolloids, affecting the purity of Bosein. Therefore, purification of Bosein prepared by enzymatic catalysis is crucial. Existing methods for separating and purifying Bosein from the enzyme reaction solution use the enzyme catalytic reaction solution as the starting material and extract Bosein from water using lipid-soluble solvents. However, because Bosein is readily soluble in water, its partition coefficient in lipid-soluble solvents is small and unreliable. Therefore, currently, there is no effective separation and purification method for Bosein prepared by bio-enzymatic catalysis. Summary of the Invention

[0005] In view of this, the present invention provides a method for separating and purifying Bosein from an enzyme reaction solution. The method provided by the present invention can effectively improve the yield and purity of Bosein, reduce purification costs, and facilitate the industrial production of Bosein.

[0006] This invention provides a method for separating and purifying bosonic acid from an enzyme reaction solution, comprising the following steps:

[0007] A) Ultrafiltration:

[0008] Proteins were removed from the Boseinase-catalyzed reaction solution by ultrafiltration through a ceramic membrane to obtain reaction solution 1;

[0009] B) Decolorization with activated carbon:

[0010] The reaction solution 1 is passed through an activated carbon filter for activated carbon decolorization to obtain reaction solution 2;

[0011] C) Continuous chromatographic separation:

[0012] The reaction solution 2 was sent into a simulated moving bed continuous chromatography system to remove impurities, resulting in a Bosein separation solution.

[0013] D) High-purity concentration:

[0014] The Bosein separation solution is concentrated under vacuum to obtain a Bosein concentrate; then the Bosein concentrate is concentrated under vacuum membrane heating with steam to obtain a high-purity concentrate.

[0015] E) High-temperature reconstitution:

[0016] The high-purity concentrate was mixed with anhydrous ethanol and heated to redissolve, thus obtaining a redissolved solution.

[0017] F) Crystallization:

[0018] The complex solution is cooled, and Bosein seed crystals are added under stirring. The solution is cooled until crystals precipitate, and the solid and liquid are separated and dried to obtain crystalline Bosein.

[0019] Preferably, in step A), the molecular weight cutoff of the ceramic membrane is 10,000 to 30,000 Da.

[0020] Preferably, in step B), the operating conditions for passing the reaction solution 1 through the activated carbon filter for activated carbon decolorization are: temperature 40-60℃, stirring speed 50-150rpm, and circulation 3-5 times.

[0021] Preferably, in step B), the activated carbon decolorization treatment conditions are: pH 3.0 to 5.0, activated carbon addition amount 0.1% to 1.0%, and decolorization time 15 to 60 min.

[0022] Preferably, in step B), the activated carbon in the activated carbon filter is powdered injection type activated carbon.

[0023] Preferably, in step C), the number of chromatographic columns in the simulated moving bed continuous chromatography system is 20.

[0024] The resin in the simulated moving bed continuous chromatography system is macroporous adsorption resin DM-301, with resin particle diameter of 50-100 mesh and resin particle uniformity of over 95%.

[0025] Preferably, in step D), the conditions for vacuum concentration are: temperature 40–75°C, vacuum degree 1–760 mmHg;

[0026] The goal of vacuum thin-film concentration using steam heating is to concentrate the volume to 20%–30% of its original volume.

[0027] Preferably, in step E), the ratio of the amount of high-purity concentrate to anhydrous ethanol is such that the concentration of Bosein in the anhydrous ethanol is 100-300 g / L.

[0028] Preferably, in step E), the temperature for heating and remelting is 60°C.

[0029] Preferably, in step F), the temperature of the complex solution is reduced to 40°C; the particle size of the bosine seed crystals is 60-80 mesh.

[0030] The method provided by this invention first removes proteins from the Boseinase-catalyzed reaction solution through ultrafiltration using a ceramic membrane, then decolorizes it with activated carbon, and finally sends it to a simulated moving bed continuous chromatography system for impurity removal. Following this, high-purity concentration, high-temperature reconstitution, and crystallization are performed. This invention utilizes a continuous chromatography separation system for desalting and impurity removal to improve product purity, and a separation and purification process based on ethanol recovery and recycling for cooling crystallization, which helps reduce production costs. This separation and purification process features low purification cost, high yield, and high extraction purity. In other words, this invention provides a novel, continuously producible, low-pollution, low-cost, high-yield, and high-purity crystalline Bosein purification method, which is beneficial for promoting the industrial production of Bosein.

[0031] Experimental results show that the method of the present invention enables the purity of Bosein to reach over 99.8% and the total product yield to reach over 68%. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0033] Figure 1 This is a schematic diagram of the partitions of the simulated moving bed continuous chromatography system used in step C) of the purification method of the present invention. Detailed Implementation

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0035] In this article, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions that include the listed features.

[0036] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0037] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0038] In this article, when referring to units of data ranges, if the unit is only followed by the right endpoint, it means that the units of the left and right endpoints are the same. For example, 10000~30000Da means that the units of the left endpoint "10000" and the right endpoint "30000" are both Da.

[0039] This document only specifically discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0040] This invention provides a method for separating and purifying bosonic acid from an enzyme reaction solution, comprising the following steps:

[0041] A) Ultrafiltration:

[0042] Proteins were removed from the Boseinase-catalyzed reaction solution by ultrafiltration through a ceramic membrane to obtain reaction solution 1;

[0043] B) Decolorization with activated carbon:

[0044] The reaction solution 1 is passed through an activated carbon filter for activated carbon decolorization to obtain reaction solution 2;

[0045] C) Continuous chromatographic separation:

[0046] The reaction solution 2 was sent into a simulated moving bed continuous chromatography system to remove impurities, resulting in a Bosein separation solution.

[0047] D) High-purity concentration:

[0048] The Bosein separation solution is concentrated under vacuum to obtain a Bosein concentrate; then the Bosein concentrate is concentrated under vacuum membrane heating with steam to obtain a high-purity concentrate.

[0049] E) High-temperature reconstitution:

[0050] The high-purity concentrate was mixed with anhydrous ethanol and heated to redissolve, thus obtaining a redissolved solution.

[0051] F) Crystallization:

[0052] The complex solution is cooled, and Bosein seed crystals are added under stirring. The solution is cooled until crystals precipitate, and the solid and liquid are separated and dried to obtain crystalline Bosein.

[0053] Regarding step A) :

[0054] A) Ultrafiltration: The protein in the Boseinase-catalyzed reaction solution is removed by ultrafiltration through a ceramic membrane to obtain reaction solution 1.

[0055] In this invention, the source of the Boseinase catalytic reaction solution is not particularly limited, and it is a reaction solution obtained by the enzyme catalytic method for preparing Bosein known in the art; in some embodiments of this invention, the Boseinase catalytic reaction solution is an enzyme catalytic reaction solution obtained by the enzyme catalytic method disclosed in CN1137179991A.

[0056] In this invention, the molecular weight cutoff of the ceramic membrane is preferably 10,000 to 30,000 Da, specifically 10,000 Da, 20,000 Da, 30,000 Da, or 40,000 Da. Protein is removed by ultrafiltration through the ceramic membrane to obtain reaction solution 1.

[0057] Regarding step B) :

[0058] B) Activated carbon decolorization: The reaction solution 1 is passed through an activated carbon filter for activated carbon decolorization to obtain reaction solution 2.

[0059] In this invention, the preferred operating conditions for passing the reaction solution 1 through an activated carbon filter for activated carbon decolorization are: temperature 40–60°C, stirring speed 50–150 rpm, and 3–5 cycles. Specifically, the temperature can be 40°C, 45°C, 50°C, 55°C, or 60°C. The stirring speed can be 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, or 150 rpm. The number of cycles can be 3, 4, or 5.

[0060] In this invention, the preferred treatment conditions for activated carbon decolorization are: pH 3.0–5.0, activated carbon addition amount 0.1%–1.0%, and decolorization time 15–60 min. Specifically, the pH value can be 3.0, 3.5, 4.0, 4.5, or 5.0. The activated carbon addition amount (i.e., the mass ratio of activated carbon to the introduced reaction solution 1) can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. The decolorization time can be 15 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

[0061] In this invention, the activated carbon used in the activated carbon filter is preferably powdered injection type activated carbon.

[0062] In this invention, the preferred indicator for decolorizing activated carbon in step B) is that the transmittance (440nm) of the resulting reaction solution 2 is greater than 98%.

[0063] Regarding step C) :

[0064] C) Continuous chromatographic separation: The reaction solution 2 is sent to a simulated moving bed continuous chromatography system to remove impurities, and the Bosein separation solution is obtained.

[0065] In this invention, the simulated moving bed continuous chromatography system preferably has 20 columns. The resin in the simulated moving bed continuous chromatography system is preferably macroporous adsorption resin DM-301, with a resin particle diameter preferably of 50-100 mesh and a resin particle uniformity of over 95%. The simulated moving bed continuous chromatography system is divided into an elution zone, a main separation zone, and a post-separation zone (e.g., ...). Figure 1 As shown in the figure, the system columns are connected in series. The system uses pure water for elution. Bosein travels slowly while impurities travel quickly. After impurity removal by a simulated moving bed continuous chromatography system, a high-purity Bosein separation solution is obtained.

[0066] After separation using the simulated moving bed method of this invention, Bosein and byproduct components are effectively separated. This step is also the key to this invention. If the separation of Bosein and byproducts is not thorough, high-purity crystalline Bosein products cannot be obtained.

[0067] Regarding step D) :

[0068] D) High-purity concentration: The Bosein separation solution is concentrated under vacuum to obtain a Bosein concentrate; the Bosein concentrate is then concentrated under vacuum membrane heating with steam to obtain a high-purity concentrate.

[0069] In this invention, the Bosein separation solution is first concentrated under vacuum. The preferred conditions for vacuum concentration are: temperature 40–75°C and vacuum degree 1–760 mmHg. Specifically, the temperature can be 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or 75°C. The vacuum degree can be 1 mmHg, 100 mmHg, 200 mmHg, 300 mmHg, 400 mmHg, 500 mmHg, 600 mmHg, 700 mmHg, or 760 mmHg. After the above vacuum concentration, the volume of the concentrated solution reaches 30%–50% of the original volume (i.e., the volume of the Bosein separation solution obtained in step C), specifically 30%, 35%, 40%, 45%, or 50%.

[0070] After the above vacuum concentration, the vacuum membrane is further concentrated using steam heating until it reaches 20%–30% of the original volume (i.e., the volume of the Bosein separated solution obtained in step C), specifically 20%, 25%, or 30%, which is lower than the concentration achieved in the previous vacuum concentration step (i.e., if the concentration in the previous step was 30%, then this step should be lower than 30%). After the above concentration treatment, a high-purity concentrate is obtained. The HPLC purity of the high-purity concentrate is >99.0%.

[0071] Regarding step E) :

[0072] E) High-temperature redissolution: The high-purity concentrate is mixed with anhydrous ethanol and heated to redissolve, resulting in a redissolution solution.

[0073] In this invention, when mixing the high-purity concentrate with anhydrous ethanol, the preferred ratio of their amounts is such that the concentration of Bosein in the anhydrous ethanol is 100–300 g / L, specifically 100 g / L, 150 g / L, 200 g / L, 250 g / L, or 300 g / L. The preferred temperature for the heating and reconstitution is 60°C. After heating and reconstitution, a reconstituted solution is obtained.

[0074] Regarding step F) :

[0075] F) Crystallization: Cool the complex solution, add Bosein seed crystals under stirring, cool until crystals precipitate, separate the solid and liquid and dry to obtain crystalline Bosein.

[0076] In this invention, the temperature of the complex solution is preferably lowered to 40°C. After cooling, Bosein seed crystals are added under stirring. The particle size of the Bosein seed crystals is preferably 60-80 mesh, specifically 60 mesh, 65 mesh, 70 mesh, 75 mesh, or 80 mesh. There are no special restrictions on their source; they can be commercially available or prepared using conventional methods in the art. The amount of Bosein seed crystals added is preferably 0.1% to 1% of the mass of the complex solution, specifically 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. After adding the seed crystals, the temperature is slowly lowered until crystals precipitate, followed by solid-liquid separation. The solid-liquid separation is preferably performed by filtration. After the solid-liquid separation, the solution is dried to obtain pure crystalline Bosein.

[0077] The method provided by this invention first removes proteins from the Boseinase-catalyzed reaction solution through ultrafiltration using a ceramic membrane, then decolorizes it with activated carbon, and finally sends it to a simulated moving bed continuous chromatography system for impurity removal. Following this, high-purity concentration, high-temperature reconstitution, and crystallization are performed. This invention utilizes a continuous chromatography separation system for desalting and impurity removal to improve product purity, and a separation and purification process based on ethanol recovery and recycling for cooling crystallization, which helps reduce production costs. This separation and purification process features low purification cost, high yield, and high extraction purity. In other words, this invention provides a novel, continuously producible, low-pollution, low-cost, high-yield, and high-purity crystalline Bosein purification method, which is beneficial for promoting the industrial production of Bosein.

[0078] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses moving bed continuous chromatography separation and uses a specific type of separation resin to achieve complete separation between Bosein and by-products, and the resin utilization rate is high; (2) Anhydrous ethanol is easily volatilized and removed in subsequent stages and can be recycled multiple times; (3) The enzyme-catalyzed Bosein purification method provided by the present invention is simple to operate, low in cost, high in purity, and high in recovery rate, and is suitable for large-scale industrial enzyme-catalyzed production and purification of crystalline Bosein.

[0079] Experimental results show that the method of the present invention enables the purity of Bosein to reach over 99.8% and the total product yield to reach over 68%.

[0080] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. In the following examples and comparative examples, the starting material for the Boseinase catalytic reaction solution is an enzyme catalytic reaction solution obtained according to the enzyme catalytic method disclosed in CN1137179991A. The activated carbon used in the activated carbon decolorization is powdered injection type activated carbon.

[0081] Example 1

[0082] A) Ultrafiltration:

[0083] Proteins were removed by ultrafiltration of the Boseinase-catalyzed reaction solution through a ceramic membrane (with a molecular weight cutoff of 10,000 Da) to obtain reaction solution 1.

[0084] B) Decolorization with activated carbon:

[0085] Reaction solution 1 was passed through an activated carbon filter at 45°C and a stirring speed of 100 rpm, and circulated 3 times. The decolorization conditions were: pH 3.0, activated carbon addition of 0.5%, and decolorization time of 60 min. Reaction solution 2 was obtained, with a transmittance (440 nm) > 98%.

[0086] C) Continuous chromatographic separation:

[0087] The reaction solution 2 was fed into a simulated moving bed continuous chromatography system with 20 columns. The resin used in the system was macroporous adsorption resin DM-301, with resin particles of 50-100 mesh in diameter and a particle uniformity of over 95%. Elution was performed using pure water. After purification using the above system, pigments and most byproducts were removed, yielding a high-purity Bosein separation solution.

[0088] D) High-purity concentration:

[0089] First, the Bosein separation solution is concentrated under vacuum (temperature 75℃, vacuum degree 1mmHg) until the volume of the concentrated solution reaches 30% of the original volume (i.e., the volume of the Bosein separation solution obtained in step C); then, the vacuum membrane is heated with steam to continue concentration until it reaches 20% of the original volume (i.e., the volume of the Bosein separation solution obtained in step C), resulting in a high-purity concentrated solution with HPLC purity > 99.0%.

[0090] E) High-temperature reconstitution:

[0091] The high-purity concentrate was mixed with anhydrous ethanol (the concentration of Bosein in the anhydrous ethanol was 150 g / L), and then heated at 60 °C to redissolve the solution, thus obtaining the redissolved solution.

[0092] F) Crystallization:

[0093] The complex solution was rapidly cooled to 40°C, and 60-mesh Bosein seed crystals (0.3% of the mass of the complex solution) were added while stirring. The solution was then slowly cooled until crystals precipitated. After filtration, the solution was vacuum dried to obtain pure crystalline Bosein.

[0094] The purity of the crystals was determined by HPLC, and the results showed that the purity was 99.8% and the total yield was 68.7%.

[0095] Example 2

[0096] A) Ultrafiltration:

[0097] Proteins were removed by ultrafiltration of the Boseinase-catalyzed reaction solution through a ceramic membrane (with a molecular weight cutoff of 15000 Da) to obtain reaction solution 1.

[0098] B) Decolorization with activated carbon:

[0099] Reaction solution 1 was passed through an activated carbon filter at 45°C and a stirring speed of 150 rpm, and circulated twice. The decolorization conditions were: pH 4.0, activated carbon addition of 1.0%, and decolorization time of 60 min. Reaction solution 2 was obtained, with a transmittance (440 nm) > 98%.

[0100] C) Continuous chromatographic separation:

[0101] The reaction solution 2 was fed into a simulated moving bed continuous chromatography system with 20 columns. The resin used in the system was macroporous adsorption resin DM-301, with resin particles of 50-100 mesh in diameter and a particle uniformity of over 95%. Elution was performed using pure water. After purification using the above system, pigments and most byproducts were removed, yielding a high-purity Bosein separation solution.

[0102] D) High-purity concentration:

[0103] First, the Bosein separation solution is concentrated under vacuum (temperature 40℃, vacuum degree 760mmHg) until the volume of the concentrated solution reaches 30% of the original volume (i.e., the volume of the Bosein separation solution obtained in step C); then, the vacuum membrane is heated with steam to continue concentration until it reaches 25% of the original volume (i.e., the volume of the Bosein separation solution obtained in step C), resulting in a high-purity concentrated solution with HPLC purity > 99.0%.

[0104] E) High-temperature reconstitution:

[0105] The high-purity concentrate was mixed with anhydrous ethanol (the concentration of Bosein in the anhydrous ethanol was 200 g / L), and then heated at 60 °C to redissolve the solution, thus obtaining the redissolved solution.

[0106] F) Crystallization:

[0107] The complex solution was rapidly cooled to 40°C, and 80-mesh Bosein seed crystals (0.5% of the mass of the complex solution) were added while stirring. The solution was then slowly cooled until crystals precipitated. After filtration, the solution was vacuum dried to obtain pure crystalline Bosein.

[0108] The purity of the crystals was determined by HPLC, and the results showed that the purity was 99.8% and the total yield was 72.5%.

[0109] Example 3

[0110] A) Ultrafiltration:

[0111] Proteins were removed by ultrafiltration of the Boseinase-catalyzed reaction solution through a ceramic membrane (with a molecular weight cutoff of 20,000 Da) to obtain reaction solution 1.

[0112] B) Decolorization with activated carbon:

[0113] Reaction solution 1 was passed through an activated carbon filter at 45°C and a stirring speed of 150 rpm, and circulated three times. The decolorization conditions were: pH 5.0, activated carbon addition of 0.1%, and decolorization time of 15 min. Reaction solution 2 was obtained, with a transmittance (440 nm) > 98%.

[0114] C) Continuous chromatographic separation:

[0115] The reaction solution 2 was fed into a simulated moving bed continuous chromatography system with 20 columns. The resin used in the system was macroporous adsorption resin DM-301, with resin particles of 50-100 mesh in diameter and a particle uniformity of over 95%. Elution was performed using pure water. After purification using the above system, pigments and most byproducts were removed, yielding a high-purity Bosein separation solution.

[0116] D) High-purity concentration:

[0117] First, the Bosein separation solution is concentrated under vacuum (temperature 50℃, vacuum degree 500mmHg) until the volume of the concentrated solution reaches 50% of the original volume (i.e., the volume of the Bosein separation solution obtained in step C); then, the vacuum membrane is heated with steam to continue concentration until the volume of the concentrated solution reaches 30% of the original volume (i.e., the volume of the Bosein separation solution obtained in step C), resulting in a high-purity concentrated solution with HPLC purity > 99.0%.

[0118] E) High-temperature reconstitution:

[0119] The high-purity concentrate was mixed with anhydrous ethanol (the concentration of Bosein in the anhydrous ethanol was 170 g / L), and then heated at 60 °C to redissolve the solution, thus obtaining the redissolved solution.

[0120] F) Crystallization:

[0121] The complex solution was rapidly cooled to 40°C, and 70-mesh Bosein seed crystals (0.1% of the mass of the complex solution) were added while stirring. The solution was then slowly cooled until crystals precipitated. After filtration, the solution was vacuum dried to obtain pure crystalline Bosein.

[0122] The purity of the crystals was determined by HPLC, and the results showed that the purity was 99.9% and the total yield was 70.5%.

[0123] Example 4

[0124] A) Ultrafiltration:

[0125] Proteins were removed by ultrafiltration of the Boseinase-catalyzed reaction solution through a ceramic membrane (with a molecular weight cutoff of 30,000 Da) to obtain reaction solution 1.

[0126] B) Decolorization with activated carbon:

[0127] Reaction solution 1 was passed through an activated carbon filter at 45°C and a stirring speed of 150 rpm, and circulated three times. The decolorization conditions were: pH 3.0, activated carbon addition of 0.5%, and decolorization time of 45 min. Reaction solution 2 was obtained, with a transmittance (440 nm) > 98%.

[0128] C) Continuous chromatographic separation:

[0129] The reaction solution 2 was fed into a simulated moving bed continuous chromatography system with 20 columns. The resin used in the system was macroporous adsorption resin DM-301, with resin particles of 50-100 mesh in diameter and a particle uniformity of over 95%. Elution was performed using pure water. After purification using the above system, pigments and most byproducts were removed, yielding a high-purity Bosein separation solution.

[0130] D) High-purity concentration:

[0131] First, the Boseine separation solution is concentrated under vacuum (temperature 60℃, vacuum degree 600mmHg) until the volume of the concentrated solution reaches 35% of the original volume (i.e., the volume of the Boseine separation solution obtained in step C); then, the vacuum membrane is heated with steam to continue concentration until it reaches 30% of the original volume (i.e., the volume of the Boseine separation solution obtained in step C), resulting in a high-purity concentrated solution with HPLC purity > 99.0%.

[0132] E) High-temperature reconstitution:

[0133] The high-purity concentrate was mixed with anhydrous ethanol (the concentration of Bosein in the anhydrous ethanol was 300 g / L), and then heated at 60 °C to redissolve the solution, thus obtaining the redissolved solution.

[0134] F) Crystallization:

[0135] The complex solution was rapidly cooled to 40°C, and 70-mesh Bosein seed crystals (1% of the mass of the complex solution) were added while stirring. The solution was then slowly cooled until crystals precipitated. After filtration, the solution was vacuum dried to obtain pure crystalline Bosein.

[0136] The purity of the crystals was determined by HPLC, and the results showed that the purity was 99.8% and the total yield was 88.5%.

[0137] Comparative Example 1

[0138] The method employing the key step of ethyl acetate extraction as described in patent CN111704595A is as follows:

[0139] 1) Ultrafiltration: Same as in Example 1.

[0140] 2) Activated carbon decolorization: The lower layer solution of the reaction solution obtained in step A) after settling is passed through an activated carbon filter at a temperature of 50°C and a stirring speed of 100 rpm, and circulated 3 times. The decolorization conditions are the same as in Example 1. Reaction solution 2 is obtained.

[0141] 3) Concentration: Dehydrate reaction solution 2 under vacuum at a temperature of 80°C and a vacuum degree of 10 mmHg to remove 90% of the volume of water.

[0142] 4) Centrifugal extraction: Add the concentrated solution obtained in step C) to ethyl acetate (the volume ratio of ethyl acetate to concentrated solution = 2:1), and introduce it into a centrifugal extractor for centrifugal extraction. The centrifugation speed is 100 rpm, the extraction temperature is 30℃, and the extraction is repeated 3 times to obtain the extract.

[0143] 5) Distillation: The extract was heated to 80°C and subjected to a vacuum of 5 mmHg to evaporate ethyl acetate, yielding pure Bosein. The purity was determined to be 95.6%, and the overall yield after purification was 11.4%.

[0144] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of these embodiments are merely to aid in understanding the method and core ideas of the present invention, including the best mode, and to enable any person skilled in the art to practice the present invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims. The scope of protection of this patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements similar to those expressed in the claims, or if they include equivalent structural elements that are not substantially different from those expressed in the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A method for separating and purifying a bovine somatotropin from an enzyme reaction solution, characterized by, It comprises the following steps: A) Ultrafiltration: The catalytic reaction solution of the Bose enzyme is subjected to ultrafiltration through a ceramic membrane to remove proteins, to obtain reaction solution 1; B) Activated carbon decolorization: The reaction solution 1 is introduced into an activated carbon filter for activated carbon decolorization, to obtain reaction solution 2; C) Continuous chromatographic separation: The reaction solution 2 is introduced into a simulated moving bed continuous chromatographic system for impurity removal, to obtain a Bose enzyme separation solution; The number of chromatographic columns of the simulated moving bed continuous chromatographic system is 20; the resin in the simulated moving bed continuous chromatographic system is macroporous adsorption resin DM-301, the particle diameter of the resin is 50-100 mesh, and the uniformity of the resin particles is more than 95%; D) High-purity concentration: The Bose enzyme separation solution is subjected to vacuum concentration, to obtain a Bose enzyme concentrated solution; the Bose enzyme concentrated solution is subjected to steam heating vacuum thin film concentration, to obtain a high-purity concentrated solution; E) High-temperature redissolution: The high-purity concentrated solution is mixed with anhydrous ethanol and heated for redissolution, to obtain a redissolved solution; F) Crystallization: The redissolved solution is cooled, and Bose enzyme crystal seeds are added under stirring, and the temperature is lowered to crystallization, followed by solid-liquid separation and drying, to obtain crystalline Bose enzyme.

2. The method of claim 1, wherein, In step A), the molecular weight cut-off of the ceramic membrane is 10,000-30,000 Da.

3. The method of claim 1, wherein, In step B), the operation conditions for introducing the reaction solution 1 into the activated carbon filter for activated carbon decolorization are as follows: temperature 40-60°C, stirring speed 50-150 rpm, and circulation 3-5 times.

4. The method of claim 1, wherein, In step B), the treatment conditions for activated carbon decolorization are as follows: pH 3.0-5.0, activated carbon addition amount 0.1%-1.0%, and decolorization time 15-60 min.

5. The method of claim 1, wherein, In step B), the activated carbon in the activated carbon filter is in the form of powder injection.

6. The method of claim 1, wherein, In step D), the conditions for vacuum concentration are as follows: temperature 40-75°C and vacuum degree 1-760 mmHg; The target for steam heating vacuum thin film concentration is to concentrate to 20%-30% of the original volume.

7. The method of claim 1, wherein, In step E), the amount of anhydrous ethanol used in relation to the high-purity concentrated solution is such that the concentration of Bose enzyme in the anhydrous ethanol is 100-300 g / L.

8. The method of claim 1, wherein, In step E), the temperature for heating redissolution is 60°C.

9. The method of claim 1, wherein, In step F), the cooling of the redissolved solution is to 40°C; The particle size of the Bose enzyme crystal seeds is 60-80 mesh.

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