A bile conversion product and preparation method thereof

Abstract

The invention discloses a bile conversion product and a preparation method thereof, and belongs to the field of bioengineering technology. The preparation method comprises the following steps: mixing the culture fluid of 7α hydroxysteroid dehydrogenase and 7β hydroxysteroid dehydrogenase secreted and expressed by engineering bacteria with a buffer solution after adsorption, impurity removal, and ultrafiltration concentration, and adding the culture fluid to an ion exchange agar gel column to immobilize the enzyme after adjusting the pH, thereby obtaining an immobilized enzyme column; mixing the avian bile extract with NADP+, adjusting the pH, and then adding the mixture to the immobilized enzyme column for conversion, and obtaining the bile conversion product after the clear liquid is concentrated under reduced pressure and dried under vacuum. The invention uses an ion exchange agar gel column as a carrier for immobilizing hydroxysteroid dehydrogenase, which has good stability, high mechanical strength, and large loading capacity, and the enzyme molecules are not easy to fall off, and can react continuously and the reaction product can be separated from the enzyme in time, thereby reducing the occurrence of reverse reaction, thereby obtaining a bile conversion product with a higher ratio of TUDCA:TCDCA.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a bile conversion product and a preparation method thereof. Background Art

[0002] Tauroursodeoxycholic acid (TUDCA), chemically named 3α,7β dihydroxycholanoyl-N-taurine, is a conjugated bile acid formed by the condensation between the carboxyl group of ursodeoxycholic acid (UDCA) and the amino group of taurine. It has obvious therapeutic effects on gallstones and liver diseases. Therefore, bile conversion products containing TUDCA are of great significance and broad application prospects in protecting the liver and gallbladder. The traditional preparation method of bile conversion products is generally mainly chemical synthesis. In recent years, with the development of enzyme catalysis, some bile conversion products prepared by enzyme catalysis have emerged. However, enzyme protein molecules are sensitive to reaction conditions, have poor stability, and are easily inactivated, resulting in a reduction in their catalytic ability; and the production of enzymes by biotechnology methods leads to high costs and are difficult to recycle and reuse. These shortcomings have greatly limited the large-scale application of enzymes in industry. Chinese invention patent CN202010469657.8 uses enzyme bacterial suspension or supernatant after bacterial crushing for biotransformation. This method puts the bacteria in direct contact with the raw materials for a long time, and a subsequent impurity removal step is required. It is also difficult to ensure the effective removal of the bacteria and its fragments as well as the exogenous DNA, proteins and endotoxins inside them. In addition, the enzyme encapsulated inside the bacteria will affect the proximity between the enzyme and the substrate, thereby affecting the conversion efficiency. Chinese invention patent CN201410588581.5 uses livestock bile powder obtained by direct freeze-drying of livestock bile as raw material, and uses chitosan-immobilized 7α-hydroxysteroid dehydrogenase and immobilized 7β-hydroxysteroid dehydrogenase for biotransformation. This method requires the enzymes used for biotransformation to be fixed on a chitosan carrier to form an immobilized enzyme column. However, chitosan as a carrier has problems such as poor mechanical strength, small enzyme loading, low enzyme activity and non-specific adsorption. At the same time, it is easy to break during the continuous reaction operation, resulting in enzyme shedding, which affects the catalytic effect and is not conducive to repeated use. After breaking, it is difficult to separate from the product. In addition, before the enzyme is immobilized, the enzyme purification step is complicated and costly, and the enzyme activity is not easy to maintain. These problems make it difficult to scale up the process, seriously affecting the preparation efficiency and failing to meet market demand. There are also some patents that use the histidine tag of recombinant protein to bind to metal ion affinity chromatography resin or ion exchange resin to prepare immobilized enzymes. However, the metal ions on the metal ion affinity chromatography resin have poor binding force, low loading capacity, and are easy to fall off, which is not conducive to reuse, and the contaminated products are difficult to separate after falling off; and ion exchange resins are generally only suitable for amino acids, etc. Summary of the invention

[0003] In order to overcome the shortcomings of the prior art, one of the objects of the present invention is to provide a method for preparing a bile conversion product. The preparation method uses ion exchange agarose gel as a carrier for immobilizing hydroxysteroid dehydrogenase, has good stability, high mechanical strength, large loading capacity, and the enzyme molecules are not easy to fall off. The reaction can be continuous and the reaction product can be separated from the enzyme in time, reducing the occurrence of reverse reaction, thereby obtaining a bile conversion product with a higher TUDCA:TCDCA ratio.

[0004] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0005] A method for preparing a bile conversion product comprises the following steps:

[0006] S1. Preparation of immobilized enzyme:

[0007] Adding activated carbon with a mass fraction of 0.5-2% to the culture solution of 7α-hydroxysteroid dehydrogenase secreted and expressed by the engineered bacteria and the culture solution of 7β-hydroxysteroid dehydrogenase, fully mixing for 0.5-1h, centrifuging or filtering to remove impurities, collecting the first enzyme-containing clear solution and ultrafiltration concentration, adding a buffer solution with a final concentration of 20-100mM to mix to obtain a second enzyme-containing clear solution, and adjusting the pH of the second enzyme-containing clear solution to 7.5-9.0; adding the second enzyme-containing clear solution to an ion exchange agarose gel column, discarding the flow-through liquid, and washing with a buffer solution to obtain a 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase co-immobilized enzyme column;

[0008] S2. Preparation of poultry bile refined solution:

[0009] The fowl gall is crushed to extract juice, filtered, concentrated, added with ethanol and mixed overnight, centrifuged or filtered to remove precipitation, and the clear liquid is concentrated until there is no alcohol taste to obtain the fowl gall refined liquid;

[0010] S3. Preparation of bile conversion products:

[0011] The avian bile extract is added with NADP+ and mixed, the pH is adjusted to 7.5-8.5, and added to a 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase co-immobilized enzyme column at 20-30°C, and the effluent reaction supernatant is concentrated under reduced pressure and dried in vacuum to obtain a bile conversion product.

[0012] As a preferred embodiment of the present invention, the engineered bacteria is one of Bacillus subtilis, yeast, and Escherichia coli.

[0013] As a preferred embodiment of the present invention, the amount of the target enzyme contained in the immobilized enzyme column is 0.05-0.2 g / ml, wherein the mass ratio between 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase is 1:1-5.

[0014] As a preferred embodiment of the present invention, the buffer is at least one of a phosphate buffer, a Tris-NaCl buffer, and a glycine buffer.

[0015] As a preferred embodiment of the present invention, the molecular weight cut-off of the ultrafiltration concentration in step S1 is 30KD, which can maintain a suitable filtration speed and avoid enzyme loss.

[0016] As a preferred embodiment of the present invention, the ion exchange agarose gel column is washed with pure water for 2 to 5 column volumes before use and then balanced with buffer for 2 to 5 column volumes.

[0017] As a preferred embodiment of the present invention, the final concentration of ethanol added in step S2 is 60-85%.

[0018] As a preferred embodiment of the present invention, the solid content of the poultry bile refined liquid in step S2 is 100-300 g / L.

[0019] As a preferred embodiment of the present invention, the final concentration of NADP+ in step S3 is 0.8-4 g / L.

[0020] As a preferred embodiment of the present invention, the mass ratio of TUDCA to TCDCA in the reaction supernatant is 1.3-2.0:1; the content of TUDCA in the bile conversion product is ≥28%, and the sum of the contents of TUDCA and TCDCA is ≥45%.

[0021] A second object of the present invention is to provide a bile conversion product, wherein the bile conversion product is prepared by the preparation method as described in any one of claims 1 to 9.

[0022] Compared with the prior art, the above technical solution has the following beneficial effects:

[0023] 1. The enzyme protein is secreted and expressed in the culture medium through engineered bacteria, which is conducive to the correct formation of disulfide bonds, thereby reducing the degradation of the enzyme protein by intracellular proteases. It does not require cell disruption and has a low content of impurities and no bacterial endotoxin residues. It has a relatively high purity and does not require enzyme purification. The enzyme-containing culture medium can be directly immobilized, and the process is simpler;

[0024] 2. The immobilized enzymes can realize complex reactions catalyzed by multiple enzyme systems, which are easy to separate from the products after the reaction, thus simplifying the separation and purification process;

[0025] 3. The ion exchange agarose gel column is used as the immobilization carrier of hydroxysteroid dehydrogenase. The ion exchanger of the ion exchange agarose gel is covalently bound to the agarose gel and is not easy to fall off. At the same time, it has strong antimicrobial ability, good stability, high mechanical strength, large loading capacity, low nonspecific adsorption, strong regeneration ability, and can be recycled. It binds to the hydroxysteroid dehydrogenase through electrostatic force and has good binding force, which reduces the conformational distortion of the enzyme and avoids the damage of the active center of the enzyme. The activity of the fixed enzyme is guaranteed to the greatest extent, and the bile conversion product can be prepared by continuous reaction, which improves the utilization rate of the enzyme and reduces the cost of industrial production. It is suitable for industrial automation and large-scale production;

[0026] 4. Both 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase have the ability to catalyze reactions in both directions. By continuously adding substrate solution, the products after the reaction are separated from the enzyme in time, which reduces the product inhibition and the occurrence of reverse reaction, thereby obtaining bile conversion products with a higher TUDCA:TCDCA ratio. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific implementation modes.

[0028] A method for preparing a bile conversion product comprises the following steps:

[0029] S1. Preparation of immobilized enzyme:

[0030] The 7α-hydroxysteroid dehydrogenase culture fluid and 7β-hydroxysteroid dehydrogenase culture fluid secreted and expressed by the engineered bacteria were mixed at a ratio of 7α-hydroxysteroid dehydrogenase culture fluid: 7β-hydroxysteroid dehydrogenase culture fluid = 1:1-5 and 0.5-2% activated carbon by mass, and fully mixed for 0.5-1h, centrifuged or filtered to remove impurities such as host bacteria, collected the first enzyme-containing clear liquid and concentrated it by ultrafiltration, collected the concentrate, added a buffer solution with a final concentration of 20-100mM and mixed it to obtain a second enzyme-containing clear liquid, and adjusted the pH of the second enzyme-containing clear liquid to 7.5-9.0; the ion exchange agarose gel column was rinsed with pure water for 2-5 column volumes before use, and then balanced with buffer for 2-5 column volumes, and then the second enzyme-containing clear liquid was added to the ion exchange agarose gel column to combine the enzyme with the ion exchange agarose gel column, the flow-through liquid was discarded, and the buffer solution was used to wash for 2-3 column volumes to remove the residual free enzyme, thereby obtaining a 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase co-immobilized enzyme column;

[0031] S2. Preparation of poultry bile refined solution:

[0032] The poultry gall is crushed to extract juice, filtered with a 100-mesh filter and a multi-layer nylon mesh, concentrated, ethanol is added to a final concentration of 60-85%, mixed overnight, centrifuged or filtered to remove precipitation, and the clear liquid is concentrated until there is no alcohol taste, to obtain a poultry gall refined liquid with a solid content of 100-300 g / L;

[0033] S3. Preparation of bile conversion products:

[0034] The avian bile extract is added with NADP+ with a final concentration of 0.8-4 g / L and mixed, the pH is adjusted to 7.5-8.5, and added to a 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase co-immobilized enzyme column at 20-30° C. The outflowing reaction clear liquid is concentrated under reduced pressure and vacuum dried to obtain a bile conversion product. The mass ratio of TUDCA to TCDCA in the reaction clear liquid is 1.3-2.0:1; the TUDCA content in the bile conversion product is ≥28%, and the sum of the contents of TUDCA and TCDCA is ≥45%.

[0035] In step S1, the engineered bacteria that secrete and express hydroxysteroid dehydrogenase is one of Bacillus subtilis, yeast, and Escherichia coli. 7α hydroxysteroid dehydrogenase and 7β hydroxysteroid dehydrogenase can be immobilized on an ion exchange agarose gel column at the same time, or one of the hydroxysteroid dehydrogenases can be fixed first and then the other hydroxysteroid dehydrogenase can be fixed. The pH value of the system can be adjusted according to the different isoelectric points of different hydroxysteroid dehydrogenases, so that different enzymes can be selectively fixed. Preferably, the amount of target enzyme contained in the immobilized enzyme column is 0.05-0.2 g / ml, wherein the mass ratio between 7α hydroxysteroid dehydrogenase and 7β hydroxysteroid dehydrogenase is 1:1-5. The buffer is at least one of phosphate buffer, Tris-NaCl buffer, and glycine buffer. The molecular weight cutoff of ultrafiltration concentration is 30KD.

[0036] Example 1

[0037] A method for preparing a bile conversion product comprises the following steps:

[0038] Preparation of co-immobilized enzyme column containing S1, 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase

[0039] 0.4 L of 7α-hydroxysteroid dehydrogenase culture medium and 1.6 L of 7β-hydroxysteroid dehydrogenase culture medium secreted and expressed by Escherichia coli were added with 1% activated carbon by mass, mixed thoroughly for 0.5-1 h, centrifuged at 3800 rpm for 5 min to remove host bacteria and other impurities, and the first enzyme-containing clear solution was collected. The first enzyme-containing supernatant was concentrated by ultrafiltration (molecular weight cutoff 30KD) to 100ml, 5ml of buffer (1M Tris+1M NaCl, pH 8.0) was added and mixed to obtain a second enzyme-containing supernatant, and the second enzyme-containing supernatant was loaded onto an ion exchange agarose gel column Q-FF-1ml at a flow rate of 1ml / min (Q-FF was rinsed with 3ml of pure water and then balanced with 3ml of buffer (50mM Tris+50mM NaCl, pH8.0)), the flow-through was discarded, and 3ml was washed with buffer (50mM Tris+50mM NaCl, pH8.0) to obtain 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase co-immobilized enzymes.

[0040] S2. Preparation of chicken gallbladder refined solution

[0041] Take 1kg of chicken bile, crush it to extract the juice, filter it with a 100-mesh filter and a multi-layer nylon mesh, concentrate it to 250g, add 1.2L of ethanol with an initial concentration of 95% to the paste, mix it overnight, centrifuge it at 3800rpm for 5min to remove the precipitate, and concentrate the clear liquid until there is no alcohol taste, which is the chicken bile refined liquid; take 10ml of the chicken bile refined liquid and dry it into powder, weighing 2.1g.

[0042] S3. Preparation of bile conversion products

[0043] Add 0.01 g NADP+ to 10 ml of chicken bile extract and mix well. Adjust the pH to 7.5 with 5 M NaOH and add the mixture to a 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase co-immobilized enzyme column at 25°C. Concentrate the effluent supernatant under reduced pressure and dry it in vacuum to obtain 1.9 g of bile conversion product, in which TUDCA: TCDCA=1.68:1 (mass ratio), the TUDCA content is 32.5%, and the (TUDCA+TCDCA) content is 51.8%.

[0044] Examples 2-16 and Comparative Examples 1-5

[0045] The difference between Examples 2-16 and Comparative Examples 1 to 5 and Example 1 is that the pH value of the immobilized system, the volume of the immobilized enzyme column, the enzyme dosage ratio, the volume of the avian bile refined liquid, the solid content of the avian bile refined liquid, the pH and temperature of the conversion system are different. The specific process conditions are shown in Table 1.

[0046] Table 1 Process parameters of Examples 2 to 16 and Comparative Examples 1 to 5

[0047]

[0048] Comparative Example 6

[0049] A method for preparing bile conversion products, which uses an immobilized enzyme column with chitosan as a matrix to prepare bile conversion products, and the specific steps are as follows:

[0050] Add appropriate amount of acetic acid to a 1% chitosan (water-soluble) solution, stir magnetically for 30 minutes to obtain chitosan acetic acid solution. Inject the solution into a sodium tripolyphosphate solution and solidify for 2 hours to obtain chitosan spheres, filter, and wash with deionized water for multiple times. Put the prepared chitosan spheres into a beaker containing 0.125% glutaraldehyde aqueous solution, place in a shaking table at 37°C and 170rpm, activate for 4 hours, and obtain activated chitosan microspheres. Weigh 0.8g of activated chitosan microspheres and load into a 1ml chromatography column. The subsequent enzyme fixation and bile conversion product preparation conditions refer to Example 1.

[0051] Comparative Example 7

[0052] A method for preparing bile conversion products, which uses an immobilized enzyme column with chitosan as a matrix to prepare bile conversion products, and the specific steps are as follows:

[0053] Take 0.8g of the activated chitosan microspheres in Comparative Example 5, add 20mg 7α hydroxysteroid dehydrogenase solution and 80mg 7β hydroxysteroid dehydrogenase solution (equivalent to 0.4L 7α hydroxysteroid dehydrogenase culture solution and 1.6L 7β hydroxysteroid dehydrogenase culture solution), vertically mix and combine for 6h, release the enzyme solution after fixation, load the immobilized enzyme into a 1ml chromatography column, rinse the fixed enzyme column 5 times with PBS buffer, add 1ml chicken bile refined solution (containing 0.0095g NADP+) to the immobilized enzyme column, react at 25°C for 6 hours, and release the reaction solution. The subsequent preparation conditions of bile conversion products refer to Example 1.

[0054] The bile conversion products obtained in Examples 2 to 16 and Comparative Examples 1 to 7 were assayed, and the mass ratio of TUDCA to TCDCA, the TUDCA content, and the TUDCA+TCDCA content were calculated. The results are shown in Table 2.

[0055] Table 2 Content determination results of Examples 2 to 16 and Comparative Examples 1 to 7

[0056]

[0057]

[0058] As shown in Table 2, the greater the pH value of the immobilized system, the greater the amount of immobilized enzyme, which in turn promotes the conversion effect. The best effect is achieved when pH = 9.0, TUDCA: TCDCA = 2.02. Too large a pH value can easily cause enzyme inactivation. When the pH is close to the isoelectric point of the enzyme, the amount of immobilized enzyme decreases and the conversion effect becomes worse. TUDCA: TCDCA = 0.42; when the enzyme dosage ratio is 7α: 7β = 1:1-1:5, the conversion effect is better, and it increases with the increase of 7β enzyme dosage. The best effect is achieved at 1:5, TUDCA: TCDCA = 1.71, and when 7α: 7β = 1:0.5 The conversion effect did not meet the standard when the volume of the avian bile extract was increased by 10 times, TUDCA: TCDCA = 0.73; the type of engineered bacteria and buffer had no obvious effect on the reaction; the volume of the avian bile extract and the volume of the immobilized enzyme column had little effect on the reaction, the conversion effect still met the standard when the volume of the avian bile extract was increased by 10 times, and the conversion effect remained unchanged when the volume of the immobilized enzyme column was increased by 5 times; when the solid content of the avian bile extract was between 100g / L and 300g / L, the higher the content, the more stable the conversion effect, TUDCA: TCDCA>1.6, but when the solid content is 350g / L, TUDCA:TCDCA=1.04, the viscosity of the system increases and the conversion effect decreases; when the pH of the conversion system is 7.5-8.5, the conversion effect TUDCA:TCDCA>1.3, as the pH increases, the enzyme activity is affected and the conversion effect decreases, at pH 9.0, TUDCA:TCDCA=0.98; when the conversion temperature is 20℃-30℃, TUDCA:TCDCA>1.5, and increases with increasing temperature, at 30℃, TUDCA:TCDCA=1.71, but when the temperature reaches 35℃, the enzyme activity decreases, TUDCA: TCDCA=0.85.

[0059] It can be seen that the preparation method of the present invention uses ion exchange agarose gel as a carrier for immobilizing hydroxysteroid dehydrogenase, which has good stability, high mechanical strength, large loading capacity, and the enzyme molecules are not easy to fall off. The reaction can be continuous and the reaction product can be separated from the enzyme in time, reducing the occurrence of reverse reaction, thereby obtaining a bile conversion product with a higher TUDCA: TCDCA ratio.

[0060] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a bile conversion product, characterized in that: The following steps are involved: S1. Preparation of immobilized enzyme: Add activated carbon with a mass fraction of 0.5-2% to the culture solution of 7α-hydroxysteroid dehydrogenase secreted and expressed by the engineered bacteria and the culture solution of 7β-hydroxysteroid dehydrogenase, mix thoroughly for 0.5-1h, centrifuge or filter to remove impurities, collect the first enzyme-containing clear solution and ultrafiltration concentration, add a buffer solution with a final concentration of 20-100mM and mix to obtain a second enzyme-containing clear solution, and adjust the pH of the second enzyme-containing clear solution to 8.0-9.0; add the second enzyme-containing clear solution to an ion exchange agarose gel column, discard the flow-through liquid, and wash with a buffer solution to obtain a 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase co-immobilized enzyme column; the target enzyme amount contained in the immobilized enzyme column is 0.05-0.2g / ml, wherein the mass ratio between 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase is 1:4-5; S2. Preparation of poultry bile refined solution: The poultry gall is crushed to extract juice, filtered, concentrated, added with ethanol and mixed overnight, centrifuged or filtered to remove precipitation, and the clear liquid is concentrated until there is no alcohol taste to obtain a poultry gallbladder refined liquid; the poultry gallbladder refined liquid has a solid content of 100 to 300 g / L; S3. Preparation of bile conversion products: Add NADP+ to the avian bile extract and mix well, adjust the pH to 7.5-8.5, add the solution to the 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase co-immobilized enzyme column at 25-30°C, take the outflowing reaction clear solution, concentrate it under reduced pressure, and dry it in vacuum to obtain the bile conversion product; The buffer is at least one of a Tris-NaCl buffer and a glycine buffer; The engineering bacteria is one of Bacillus subtilis and Escherichia coli.

2. The method for preparing bile conversion products according to claim 1, characterized in that: The ion exchange agarose gel column is washed with pure water for 2 to 5 column volumes before use and then balanced with buffer for 2 to 5 column volumes.

3. The method for preparing bile conversion products according to claim 1, characterized in that: The final concentration of ethanol added in step S2 is 60-85%.

4. The method for preparing bile conversion products according to claim 1, characterized in that: The final concentration of NADP+ in step S3 is 0.8-4 g / L.

5. The method for preparing bile conversion products according to claim 1, characterized in that: The mass ratio of TUDCA to TCDCA in the reaction clear solution is 1.3-2.0:1; the content of TUDCA in the bile conversion product is ≥28%, and the sum of the contents of TUDCA and TCDCA is ≥45%.

6. A bile conversion product, characterized in that: The bile conversion product is obtained by the preparation method according to any one of claims 1 to 5.

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