WO2021237950A1 - 一种人工熊胆粉的制作工艺 - Google Patents

一种人工熊胆粉的制作工艺 Download PDF

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WO2021237950A1
WO2021237950A1 PCT/CN2020/108657 CN2020108657W WO2021237950A1 WO 2021237950 A1 WO2021237950 A1 WO 2021237950A1 CN 2020108657 W CN2020108657 W CN 2020108657W WO 2021237950 A1 WO2021237950 A1 WO 2021237950A1
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hydroxysteroid dehydrogenase
enzyme
bile powder
bear bile
conversion
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PCT/CN2020/108657
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English (en)
French (fr)
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赵志斌
王丹丹
秦松柏
程雷
丁峰
罗德彬
张苏敏
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重庆极泽生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/413—Gall bladder; Bile
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles

Definitions

  • the invention relates to the field of biomedicine, and relates to a manufacturing process of bear bile powder, in particular to a manufacturing process of artificial bear bile powder.
  • Bear bile powder is obtained from the dry gall bladder bile of the bear family animal black bear or brown bear. It is a precious Chinese medicinal material. It has a history of more than 2,000 years as a medicine. A large number of prescriptions contain bear bile ingredients. Bear bile powder has antispasmodic, anticonvulsant, anti-inflammatory and gallstone dissolving effects. It is a good medicine for protecting liver and gallbladder. Various modern clinical practice applications also show that bear bile can treat a variety of liver and gallbladder diseases. Due to the limited resources of wild bears, the method of artificial breeding "drainage and bile extraction" is commonly used to produce bear bile powder (that is, natural bear bile powder). At present, the annual output of natural bear bile powder in my country is about 30 tons, which cannot meet people's growing health needs.
  • the composition of the substrate (poultry gallbladder powder) is complex, which has a greater impact on the enzyme activities of the two enzymes.
  • the enzyme used in the biotransformation needs to be immobilized on a chitosan carrier to make an immobilized enzyme column. The steps are cumbersome, the cost is high, and the reaction system is not easy to scale up. And before the enzyme is immobilized, the process of purifying and enriching the above two enzymes is also complicated and time-consuming.
  • the present invention intends to provide a manufacturing process of artificial bear bile powder.
  • a bacterial suspension or supernatant containing two enzymes as a transformation solution, the technology of complex purification steps and high cost of enzymes and difficult maintenance of enzyme activity are solved. problem.
  • the present invention adopts the following technical solutions:
  • a manufacturing process of artificial bear bile powder including a biotransformation step for converting taurochenodeoxycholic acid into tauroursodeoxycholic acid: including conversion containing poultry and livestock bile powder, a conversion buffer, and a conversion solution System; the conversion liquid is a bacterial suspension containing enzyme cells or a supernatant of protoplasts containing enzyme cells; the enzyme cells express 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase .
  • this scheme directly uses enzyme bacteria or the supernatant of enzyme bacteria to carry out the biotransformation of poultry gallbladder meal, and the reaction solution obtained after biotransformation can be artificial Bear bile powder.
  • the biotransformation process of this scheme converts a certain amount of taurochenodeoxycholic acid (TCDCA) in poultry and livestock bile powder into tauroursodeoxycholic acid (TUDCA).
  • TCDCA taurochenodeoxycholic acid
  • TUDCA tauroursodeoxycholic acid
  • the conversion process of taurochenodeoxycholic acid to tauroursodeoxycholic acid is specifically: under the catalysis of 7 ⁇ -hydroxysteroid dehydrogenase, taurochenodeoxycholic acid is converted to the intermediate tauroursodeoxycholic acid 7-ketone.
  • the general process of the construction of the engineered bacteria in this scheme is: construct the genes of the two enzymes of 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase on the expression vector, and then transfer the expression vector into the bacteria (such as E. coli ), thereby preparing and obtaining engineered bacteria.
  • Enzyme cells are formed by inducing engineering bacteria to multiply and expressing 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase.
  • the activities of 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase are greatly affected by the external environment (especially the environment in which the enzymatic reaction proceeds).
  • poultry gallbladder powder contains more impurities (especially some are toxic or inhibiting 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase)
  • the active substance which has a greater inhibitory effect on the activities of these two enzymes, and more seriously affects the biotransformation process of poultry and livestock bile meal.
  • impurities the part of poultry gall powder that is not taurochenodeoxycholic acid is considered as impurities
  • the combination of the target compound affects the catalytic efficiency.
  • the enzyme bacteria or protoplasts protect the enzymes and hinder the influence of impurities on the enzyme activity.
  • the enzyme bacteria or protoplasts did not hinder the enzymes.
  • the combination with taurochenodeoxycholic acid and other target components can still effectively catalyze the substrate.
  • This scheme uses two enzymes to simultaneously catalyze the biotransformation of poultry and livestock gall meal, which is different from the step-by-step transformation process in the prior art, and it is easy to produce more intermediate tauro 7-ketolithocholic acid.
  • the inventor found that in the presence of a large amount of substrate (poultry gallbladder powder), the activity of 7 ⁇ -hydroxysteroid dehydrogenase was greatly affected, resulting in the reaction intermediate taurine 7-ketone. Cholic acid cannot be converted into tauroursodeoxycholic acid by 7 ⁇ -hydroxysteroid dehydrogenase. The increase of intermediates affects the quality of the final product.
  • the active substances in the protoplasts of the engineered bacteria have a strong protective effect on 7 ⁇ -hydroxysteroid dehydrogenase (stronger than the protective effect on 7 ⁇ -hydroxysteroid dehydrogenase), which catalyzes the reaction
  • the intermediate tauro 7-ketolithocholic acid forms tauroursodeoxycholic acid more efficiently.
  • the transformation system of this scheme does not contain medium and other components, and the viscosity of the system is low.
  • a relatively large amount of substrate can be added to the system to realize the biotransformation of a large amount of substrates at one time.
  • the ratio of tauroursodeoxycholic acid and taurochenodeoxycholic acid is similar to that of natural bear bile powder.
  • the product of this process can be used as a substitute for natural bear bile powder for intensive processing.
  • the preparation method of the bacterial suspension is: dispersing the enzyme bacteria in the conversion buffer to obtain the bacterial suspension;
  • the preparation method of the supernatant is: performing the enzyme bacteria in the bacterial suspension After the crushing treatment, the supernatant was obtained by centrifugation.
  • the preparation method of the bacterial suspension and the supernatant is simple, and is suitable for expanded production.
  • the enzyme bacteria is a mixture of engineered bacteria A expressing 7 ⁇ -hydroxysteroid dehydrogenase and engineered bacteria B expressing 7 ⁇ -hydroxysteroid dehydrogenase; or the enzyme bacteria are both expressing 7 ⁇ -Engineered bacteria C of two enzymes, hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase.
  • engineered bacteria C co-expresses two enzymes, or engineered bacteria A and B express 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase respectively, both of which can achieve the catalytic conversion of substrates.
  • the above-mentioned amount of enzyme bacteria can provide sufficient 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase to catalyze the biotransformation process of poultry and livestock gallbladder meal.
  • the amount of enzyme used is too large, the catalytic reaction has reached saturation, and the increase in cost cannot increase the catalytic effect; the amount of enzyme used is too small, the conversion effect is poor, and the expected conversion ratio cannot be achieved.
  • A-type engineering bacteria and B-type engineering bacteria with a ratio of 1:10-1:2 can produce 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase in a reasonable ratio to catalyze the reaction to the tauron bear
  • Deoxycholic acid proceeds in the direction (this reaction is a reversible reaction).
  • the conversion buffer is 0-15 mM glycine buffer; the volume of the conversion liquid is 1/5-4/5 of the volume of the conversion system.
  • the conversion buffer is 0-15 mM glycine buffer, which reduces the concentration of glycine used in the conversion reaction and reduces the introduction of glycine impurities.
  • the inventor found that too high a concentration of glycine buffer will reduce enzyme activity and reduce the quality of the final product.
  • the volume of the conversion solution is 1/5-4/5 of the volume of the conversion system.
  • the conversion solution of the above amount contains sufficient amounts of 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase, which can catalyze poultry and livestock gallbladder powder Biotransformation process. If the amount is too small, the amount of the enzyme is too small, which is not enough to carry out a full enzymatic reaction; if the amount is too much, the catalytic effect of the enzyme has become saturated, resulting in waste of the enzyme.
  • the concentration of coenzyme I or coenzyme II in the transformation system is 0.1-5 mM, and the concentration of poultry and livestock gall powder in the transformation system is 50-250 g/L.
  • this scheme uses bacterial suspension or supernatant as the transformation solution and does not contain medium, the viscosity of the entire system is small, and a relatively large amount of substrate (poultry and animal gall powder) can also be realized in this transformation system. Disperse, and then enzymatically catalyze the substrate.
  • the pH value of the conversion system is 6.0-9.0; the pH value of the conversion system is 6.0-9.0; the temperature at which the conversion system performs the enzymatic reaction is 20-30° C., and the duration is 2-24 h.
  • the above parameter range meets the suitable reaction conditions of the enzymatic reaction, which can ensure the smooth progress of the catalytic reaction, so that the poultry and livestock gall powder is fully converted into the artificial bear gall powder.
  • sequence of the gene of 7 ⁇ -hydroxysteroid dehydrogenase is SEQ ID NO: 1; the sequence of the gene of 7 ⁇ -hydroxysteroid dehydrogenase is SEQ ID NO: 3; controls 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxyl
  • the operons for the expression of the steroid dehydrogenase gene are all lactose operons.
  • SEQ ID NO: 1 and SEQ ID NO: 3 are all codon-modified genes, so that 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase can be expressed in large quantities in E. coli to obtain enrichment. Enzyme bacteria that gather these two enzymes.
  • the lactose operon is an operon that conventionally controls gene expression and is easy to prepare and obtain.
  • the biotransformation step also includes a fermentation step: using IPTG to induce engineered bacteria A to express 7 ⁇ -hydroxysteroid dehydrogenase, and using IPTG to induce engineered bacteria B to express 7 ⁇ -hydroxysteroid dehydrogenase; or using IPTG to induce engineering Strain C expresses both 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase.
  • IPTG induces fermentation, so that 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase are expressed in large quantities for the subsequent biotransformation process.
  • the biotransformation step it also includes the preparation step of artificial bear bile powder: the conversion system obtains a reaction liquid through the enzymatic reaction of the biotransformation step, removes the precipitate in the reaction liquid, and then concentrates to obtain an extract; using ethanol The solution disperses the extract to obtain an extract dispersion; removes the precipitate in the extract dispersion, and obtains artificial bear bile powder after being concentrated and dried.
  • the 7 ⁇ hydroxysteroid dehydrogenase gene S1-a-1 and the 7 ⁇ hydroxysteroid dehydrogenase gene Y1-b-1 were optimized for expression in E. coli, and affinity tags were added, and the whole gene was synthesized.
  • the optimized 7 ⁇ -hydroxysteroid dehydrogenase gene S1-a-1 is referred to as 7 ⁇ -steroid dehydrogenase gene in this article, denoted as 7 ⁇ -HSDH (SEQ ID NO:1); optimized 7 ⁇ -hydroxysteroid dehydrogenase
  • the gene Y1-b-1 is referred to as the 7 ⁇ -steroid dehydrogenase gene in this article, and is denoted as 7 ⁇ -HSDH (SEQ ID NO: 3).
  • the 7 ⁇ -steroid dehydrogenase gene fragment and pET28a vector were ligated with ligase to obtain the ligation product.
  • the ligation product was used to transform DH5 ⁇ and spread on kanamycin-resistant LB plates for screening. After the colonies are formed, single clones are selected and inoculated into 5mL LB for overnight culture. Collect the bacterial cells, extract the plasmids with the Tiangen Plasmid Extraction Kit, and send them for sequencing. Save the sequenced plasmid to obtain plasmid pET28a-7 ⁇ -HSDH.
  • the ligation product was used to transform DH5 ⁇ and spread on kanamycin-resistant LB plates for screening. After the colonies are formed, single clones are selected and inoculated into 5mL LB for overnight culture. Collect the bacteria, extract the plasmid with the Tiangen Plasmid Extraction Kit, send it for sequencing, save the sequenced plasmid, and obtain the plasmid pET28a-7 ⁇ -HSDH.
  • 7 ⁇ -HSDH was amplified by PCR with primer pair SEQ ID NO: 5 and SEQ ID NO: 6, digested with NdeI and XhoI, and the template was digested with DpnI.
  • the pETDuet-1 vector that was sequenced correctly was cut with Nde I and Xho I, and the 7 ⁇ -steroid dehydrogenase gene fragment and the vector were ligated with ligase to obtain the ligated product.
  • the ligation product was used to transform DH5 ⁇ and spread on an ampicillin-resistant LB plate for screening. After the colony is formed, select a single clone and inoculate it into 5ml LB for overnight culture. Collect the bacterial cells, extract the plasmids with the Tiangen Plasmid Extraction Kit, and send them for sequencing. Save the sequenced plasmid to obtain the plasmid pETDuet-1-7 ⁇ -HSDH.
  • 7 ⁇ -HSDH was amplified by PCR with primer pair SEQ ID NO: 7 and SEQ ID NO: 8, digested with BamH I and EcoR I, and the template was digested with Dpn I enzyme.
  • the pETDuet-1-7 ⁇ -HSDH-vector was digested with BamH I and EcoR I.
  • Ligase is used to connect the 7 ⁇ -steroid dehydrogenase gene fragment and the vector to obtain the ligation product.
  • the ligation product was used to transform DH5 ⁇ and spread on an ampicillin-resistant LB plate for screening. Pick a single clone and inoculate it into 5mL LB for overnight culture.
  • the pET28a-7 ⁇ -HSDH and pET28a-7 ⁇ -HSDH, as well as pETDuet-1-7 ⁇ -HSDH/7 ⁇ -HSDH were transformed into competent cells of E. coli BL21 (DE3) to obtain three kinds of engineered bacteria, which were named as engineering Strain A (containing pET28a-7 ⁇ -HSDH), engineered strain B (pET28a-7 ⁇ -HSDH) and engineered strain C (containing pETDuet-1-7 ⁇ -HSDH/7 ⁇ -HSDH).
  • the bacterial solution of engineered bacteria A and engineered bacteria B were respectively coated on a kana-resistant LB plate, and the bacterial fluid of engineered bacteria C was coated on an ampicillin-resistant LB plate.
  • 7 ⁇ -steroid dehydrogenase enzyme activity determination method using taurochenodeoxycholic acid as a substrate, add 2.97mL of 100mM phosphate buffer (pH8.0) to a 3mL reaction system, the final concentration of 0.5mM Taurochenodeoxycholic acid, 10 ⁇ L of gradient dilution crude enzyme solution, final concentration of 0.5mM NADP+, react at pH8.0 and 25°C for 1min, measure the change in absorbance at 340nm, and then calculate 7 ⁇ -steroid dehydrogenation Enzyme activity of enzymes.
  • phosphate buffer pH8.0
  • the preparation method of the crude enzyme solution is as follows: take 10 g of bacterial cells (enzyme bacterial cell A or B), resuspend in 100 mM phosphate buffer, ultrasonically break, filter and take the supernatant to obtain the crude enzyme solution.
  • 7 ⁇ -steroid dehydrogenase enzyme activity determination method Tauroursodeoxycholic acid is used as a substrate, and 2.97mL of 100mM phosphate buffer is added to a 3mL reaction system, and the final concentration of tauroursodeoxy is 0.5mM.
  • Cholic acid 10 ⁇ L of gradient dilution crude enzyme solution, final concentration of 0.5mM NADP+, react at pH8.0 and 25°C for 1min, measure the change in absorbance at 340nm, and then calculate the enzyme activity of 7 ⁇ -steroid dehydrogenase.
  • the enzyme activity meets the requirements of subsequent applications, indicating that the enzyme bacteria can be used for artificial production. Biotransformation of bear bile.
  • the preparation process of poultry gall powder is as follows: cut fresh poultry gall or thawed poultry gall with a meat grinder, pass a 100-mesh sieve to take the liquid part (mainly bile), and add 90-95 to the liquid part % Ethanol to a final ethanol concentration of 60-85%, centrifugation or filtration to remove the precipitate, the supernatant is concentrated under reduced pressure and vacuum dried to prepare poultry gall powder.
  • this embodiment it is specifically: take 20Kg of thawed chicken gallbladder, cut it with a meat grinder, filter with 100 mesh stainless steel filter screen and multi-layer 100 mesh nylon mesh, take the liquid phase, and depressurize the liquid phase to remove water to the paste (Density controlled at 1.10 ⁇ 0.05g/ml), add 25L 95% ethanol to the paste and mix overnight, centrifuge at 3800rpm for 5min to remove the precipitate, the supernatant is dewatered under reduced pressure to a paste, and dried in vacuum to prepare chicken gallbladder pink.
  • the process of converting poultry gall powder into bear gall powder is: dissolving poultry gall powder in 0-15mM glycine buffer, adding 0.1-5mM NADP+, adding the transformation solution, and then adding 20-100mM glycine buffer to the final Volume, adjust the pH to 6.0-9.0 with sodium hydroxide to obtain a transformation system, and then react at 20-30°C for 2-24 hours to complete the biotransformation and obtain the reaction solution that has completed the transformation.
  • the concentration of the substrate is 50-250g/L.
  • the conversion liquid is a bacterial suspension formed by dispersing enzyme cell A and enzyme cell B (mixed bacteria of two enzyme cells) in a glycine buffer.
  • Every 10g of mixed bacteria composed of enzyme cell A and enzyme cell B is dispersed with 20-100 ml of glycine buffer to obtain a bacterial suspension.
  • the mixed bacteria composed of enzyme cell A and enzyme cell B can be crushed, and the supernatant is taken after filtration, and the supernatant is the conversion liquid.
  • the mass ratio of enzyme cell A and enzyme cell B is 1:10-1:2.
  • the amount of conversion liquid added is 1/5-4/5 of the entire conversion system. It is also possible to use enzyme cell C instead of the mixture of enzyme cell A and enzyme cell B.
  • the preparation method of the transformation solution is to add enzyme bacteria to the glycine buffer, and use 50ml glycine buffer to disperse every 10g enzyme bacteria to obtain a bacterial suspension, which is the transformation solution.
  • the amount of conversion liquid added is 3/5 of the volume of the conversion system.
  • Enzyme cells include Enzyme Bacteria A (Type A engineered bacteria expressing 7 ⁇ -hydroxysteroid dehydrogenase) and Enzyme Bacteria (Type B engineering bacteria expressing 7 ⁇ -Hydroxysteroid dehydrogenase), both The mass ratio is 1:5.
  • the artificial bear bile powder in this example contains 32.0% TUDCA content, TCDCA content 26.5%, T-7 -KLCA content is 1.4%.
  • Example 5 to Example 11 and the preparation process of artificial bear bile powder are the same as that of Example 4. The difference lies in the selection of specific parameters. See Table 1 for details.
  • type I refers to dispersing the enzyme bacteria in the conversion buffer to obtain a bacterial suspension, which is the conversion solution
  • type II refers to dispersing the enzyme bacteria in the conversion buffer.
  • the bacterial suspension is obtained, and the enzyme cells are crushed, and then the supernatant is collected by centrifugation, and the supernatant is the conversion liquid.
  • type II transformation solution use a pressure of 800-1200bar to crush the enzyme cells, and then centrifuge at 12000-25000rpm for 0.5-2h to get the supernatant.
  • the above-mentioned conditions can effectively break the enzyme bacteria and separate the protoplasts and cell walls of the enzyme bacteria.
  • a specific parameter can be selected.
  • the above conditions can separate the protoplasts and cell walls of the enzyme bacteria to obtain a supernatant with catalytic activity.
  • the supernatant contains 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase.
  • the various active substances in the protoplasts form encapsulation and protection for these two enzymes, so that these two enzymes are protected from poultry and livestock gallbladder powder.
  • the interference of impurities in the product can smoothly catalyze the biotransformation process of poultry and livestock bile powder to artificial bear bile powder.
  • Example 10 the conversion buffer concentration is 0 mM, that is, deionized water is used.
  • the conversion system does not have the ability to self-adjust the pH value. It is necessary to monitor the pH value of the reaction system in real time and adjust the pH value to 7.0 (at least maintain Between 6.0-9.0).
  • enzyme cell C was used instead of a mixture of enzyme cell A and enzyme cell B.
  • Comparative Example 1 The biotransformation of Comparative Example 1 to Comparative Example 4 and the preparation process of artificial bear bile powder are the same as in Example 4. The difference lies in the selection of specific parameters. See Table 1 for details.
  • glycine buffer was not used, but a general medium was used to maintain the enzyme cell activity, and the medium was specifically LB medium.
  • Comparative Example 1 only 5 kg of poultry gall powder was added because the amount of enzyme bacteria was too large, and too much poultry gall powder could no longer be dissolved in the system.
  • Table 1 Parameter selection list of Examples 4-11 and Comparative Examples 1-4
  • the artificial bear bile powder prepared in Example 1 to Example 11 and Comparative Example 1 to Comparative Example 4 were tested by HPLC, and the results are shown in Table 2. It can be seen from the experimental results that the content ratios of TUDCA and TCDCA in the artificial bear bile powder prepared in Examples 1-11 are in line with the content ratio range of natural bear bile powder, and the content of TUDCA is in line with the "Chinese Pharmacopoeia (2015)" for bear bile powder. Regulations and the low content of the intermediate product T-7-KLCA proves that the process has high bioconversion efficiency and the quality of the final product obtained is guaranteed. The amount of enzyme bacteria in Comparative Example 1 was larger, but it did not greatly improve the reaction efficiency.
  • Comparative Example 2 The amount of enzyme bacteria used in Comparative Example 2 was too small, resulting in insufficient catalysis, and the obtained artificial bear bile powder had a certain gap from those in Examples 1-11.
  • Comparative Example 3 a high-concentration glycine buffer was used, which inhibited the catalysis to a certain extent, resulting in that the obtained artificial bear bile powder did not meet the ideal requirements.
  • Comparative Example 4 uses culture medium instead of glycine buffer. The use of culture medium is used to maintain cell viability and avoid cells in a buffer-only environment from a stress state (which may affect enzyme activity). However, the addition of the culture medium did not improve the reaction efficiency, but made the quality of the final product worse.
  • TUDCA/TCDCA Whether it is the content of TUDCA/TCDCA or the intermediate, it is far from the ideal state. It shows that the culture medium is not conducive to the combination of enzyme and substrate, and the high activity of cells is not conducive to the catalysis of the 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase produced by it on the substrate.
  • the compounding process is: mix 7 ⁇ -hydroxysteroid dehydrogenase and 7 ⁇ -hydroxysteroid dehydrogenase according to the enzyme activity to ensure that the enzyme activity composition of the test substance in the first test group is 7 ⁇ -steroid dehydrogenase 2000U and 7 ⁇ -steroid dehydrogenase 100U.
  • the test objects of the second test group and the test objects of the third test group are compounded.
  • the catalytic reaction of this experimental example uses 2g poultry gall powder as a substrate, the conversion system is 10ml, pH6.5, reaction temperature 25°C, catalysis time 16h, buffer concentration 10mM, NADP+ content 2mM. The results are shown in Table 3.

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Abstract

本发明提供了一种人工熊胆粉的制作工艺，通过使用含细胞活性物质的转化液，解决了酶的纯化步骤复杂且成本高和酶活不易保持的技术问题。采用本方案进行禽畜胆粉的生物转化后，获得的人工熊胆粉的牛磺熊去氧胆酸和牛磺鹅去氧胆酸比例与天然熊胆粉近似。

Description

一种人工熊胆粉的制作工艺 技术领域
本发明涉及生物医药领域，涉及一种熊胆粉的制作工艺，具体涉及一种人工熊胆粉的制作工艺。
背景技术
熊胆粉，由熊科动物黑熊或棕熊干燥胆囊胆汁得到，是名贵中药材，有2000余年的入药历史，大量处方中都含有熊胆成分。熊胆粉具有解痉、抗惊厥、抗炎及溶胆石等作用，是保肝护胆的良药。各种现代临床实践应用也表明，熊胆能够治疗多种肝胆疾病。由于野生熊资源有限，现在普遍使用人工养殖“引流取胆”的方法生产熊胆粉(即天然熊胆粉)。目前，我国每年的天然熊胆粉产量约30吨，无法满足人们日益增长的健康需求。
为了解决天然熊胆粉短缺的问题，大量人工熊胆粉应运而生，例如：以其他动物胆为原料，将其中的牛磺鹅去氧胆酸(TCDCA)通过化学或生物方法转化为牛磺熊去氧胆酸(TUDCA)，获得人工熊胆粉。中国发明专利CN201410588581.5，以禽畜胆汁直接冷冻干燥制得的禽畜胆粉为原料，用固定化的7α-羟基类固醇脱氢酶和固定化的7β-羟基类固醇脱氢酶进行生物转化后，制得人工熊胆粉。此种方法，底物(禽畜胆粉)成分复杂，对两种酶的酶活造成了较大影响，需要将生物转化所用到的酶固定到壳聚糖载体上制成固定化酶柱，步骤繁琐，成本高昂，反应体系不易放大。并且在对酶进行固定化处理之前，纯化富集上述两种酶的过程也较为复杂和耗时。现有技术的人工熊胆的生物转化存在酶的纯化步骤复杂且成本高和酶活不易保持的问题，使得工艺不易放大，严重影响了人工熊胆粉的制备效率，无法满足市场对人工熊胆粉的需求。
发明内容
本发明意在提供一种人工熊胆粉的制作工艺，通过使用含有两种酶的菌悬液或者上清液作为转化液，解决了酶的纯化步骤复杂且成本高和酶活不易保持的技术问题。
为达到上述目的，本发明采用如下技术方案：
一种人工熊胆粉的制作工艺，包括用于将牛磺鹅去氧胆酸转换为牛磺熊去氧胆酸的生物转化步骤：包括含有禽畜胆粉、转化缓冲液和转化液的转化体系；所述转化液为包含酶菌体的菌悬液或者包含酶菌体的原生质体的上清液；所述酶菌体表达有7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶。
本方案的原理及优点是：本方案直接使用酶菌体或酶菌体裂解后的上清液进行禽畜胆粉的生物转化，经生物转化后获得的反应液经后续处理之后，可获得人工熊胆粉。本方案的生物转化过程使得禽畜胆粉中的一定量的牛磺鹅去氧胆酸(TCDCA)转化为牛磺熊去氧胆酸(TUDCA)。牛磺鹅去氧胆酸向牛磺熊去氧胆酸转化的过程具体是：在7α-羟基类固醇脱氢酶的催化作用下，牛磺鹅去氧胆酸转换为中间体牛磺7-酮石胆酸(T-7-KLCA)；牛磺7-酮石胆酸再在7β-羟基类固醇脱氢酶的催化下转换成牛磺熊去氧胆酸。经转化后的禽畜胆粉(即人工熊胆粉)的功效成分组成和天然熊胆粉的功效成分保持一致(在天然熊胆粉中， TUDCA/TCDCA＝1.1-1.5：1)。在本方案中，禽畜胆粉是指从禽类或者畜类动物的胆中提取胆汁等物质，再制备获得禽畜胆粉。本方案的工程菌的构建的大致过程为：将7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶两种酶的基因构建在表达载体上，再将表达载体转入菌(例如大肠杆菌)中，从而制备获得工程菌。酶菌体是诱导工程菌大量繁殖并表达7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶而形成。
7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶的活性受外界环境(特别是酶促反应进行的环境)影响较大。特别是在本方案的含有大量禽畜胆粉的转化体系中，由于禽畜胆粉含有较多杂质(特别是一些对7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶具有毒性或抑制作用的物质)，对这两种酶的活性带来了较大的抑制作用，较为严重地影响了禽畜胆粉的生物转化过程。现有技术中，为了减少禽畜胆粉对酶活的影响，研究人员采用了用壳聚糖固定酶的方法。但是，现有的方案步骤繁琐，成本高昂，反应体系不易放大。发明人尝试了多种方法来避免禽畜胆粉中的杂质对7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶的酶活的影响，发现酶菌体的原生质体(其中含有大量生物活性物质)对抵抗转化体系中的毒性环境有较好的作用。传统观念认为，将酶提取纯化和富集之后，足量的酶可以和底物充分结合。但是在含有禽畜胆粉的转化体系中，杂质(禽畜胆粉中非牛磺鹅去氧胆酸的部分被认为是杂质)对这两种酶形成一定的抑制，阻碍了两种酶与目的化合物(牛磺鹅去氧胆酸以及反应中间体牛磺7-酮石胆酸)的结合，影响了催化效率。当使用菌悬液或者上清液作为两种酶的载体的时候，酶菌体或者原生质体对酶形成保护，阻碍了杂质对酶活性的影响，但是，酶菌体或者原生质体并未阻碍酶和牛磺鹅去氧胆酸等目的成分的结合，仍然可以对底物进行有效催化。
本方案使用两种酶同时催化禽畜胆粉的生物转化，不同于现有技术中的分步转化的过程，容易产生较多的中间体牛磺7-酮石胆酸。发明人在生产实践的过程中发现，在大量底物(禽畜胆粉)存在的情况下，7β-羟基类固醇脱氢酶的活性受到较大的影响，导致反应中间体牛磺7-酮石胆酸不能被7β-羟基类固醇脱氢酶转化为牛磺熊去氧胆酸，中间体增多，影响最终产物的品质。但是由于本方案使用转化液，工程菌的原生质体内的活性物质对7β-羟基类固醇脱氢酶具有较强保护作用(比对7α-羟基类固醇脱氢酶的保护作用更强)，该酶催化反应中间体牛磺7-酮石胆酸形成牛磺熊去氧胆酸的效率更高。采用本方案的制备方法，中间体牛磺7-酮石胆酸在最终产物中的含量非常低，提升了产品质量。
综上所示，本方案的有益效果在于：
(1)由于转化液的使用，酶的活性得到保证，催化禽畜胆粉生物转化的效率提升，提高了人工熊胆粉中的牛磺熊去氧胆酸的含量。
(2)在禽畜胆粉溶液中，加入菌悬液或者上清液进行转化，不用再进行酶的纯化步骤，避免纯化过程中引入外源性杂质，节约了时间和经济成本。
(3)相对于壳聚糖固定化酶的现有技术中的方案，本方案的转化过程能够按比例进行放大，放大后转化效果可控，易于工业化放大生产。
(4)本方案的转化体系中不含有培养基等成分，体系黏度低，可以在体系中加入较为大量的底物(禽畜胆粉)，实现一次性对大量底物的生物转化。
(5)生物转化后，牛磺熊去氧胆酸和牛磺鹅去氧胆酸比例与天然熊胆粉近似，本工艺的产品可以作为天然熊胆粉的替代品进行精深加工。
进一步，所述菌悬液的制备方法为：将酶菌体分散于转化缓冲液中，获得菌悬液；所述上清液的制备方法为：对所述菌悬液中的酶菌体进行破碎处理后，离心获得上清液。
采用上述方案，菌悬液和上清液的制备方法简单，适合于扩大生产。
进一步，所述酶菌体为表达有7α-羟基类固醇脱氢酶的工程菌A和表达有7β-羟基类固醇脱氢酶的工程菌B组成的混合物；或者所述酶菌体为同时表达有7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶两种酶的工程菌C。
采用上述方案，工程菌C共表达两种酶，或者工程菌A和B分别表达7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶，两种方式均可实现对底物的催化转化。
进一步，每10g酶菌体使用20-100ml转化缓冲液分散，获得菌悬液；表达有7α-羟基类固醇脱氢酶的工程菌A和表达有7β-羟基类固醇脱氢酶的工程菌B的质量比为1:10-1:2。
采用上述方案，上述用量的酶菌体可提供足量的7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶，以催化禽畜胆粉的生物转化过程。使用的酶菌体量过大，催化反应已达到饱和，成本的增加并不能增加催化效果；用量过少转化效果差，达不到预期的转化比例。
采用配比为1:10-1:2的A类工程菌和B类工程菌，可产生合理配比的7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶，催化反应向牛磺熊去氧胆酸方向进行(该反应是一种可逆反应)。
进一步，所述转化缓冲液为0-15mM的甘氨酸缓冲液；所述转化液的体积为转化体系的体积的1/5-4/5。
采用上述方案，转化缓冲液为0-15mM的甘氨酸缓冲液，降低了转化反应中甘氨酸的使用浓度，减少了甘氨酸杂质的引入。发明人发现甘氨酸缓冲液的浓度过高会降低酶活，降低最终产品质量。转化液的体积为转化体系的体积的1/5-4/5，上述用量的转化液中含有足够量的7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶，可催化禽畜胆粉的生物转化过程。用量过少，酶的量过少，不足以进行充分酶促反应；用量过多，酶的催化作用已趋于饱和，导致酶的浪费。
进一步，辅酶Ⅰ或者辅酶Ⅱ在转化体系中的浓度为0.1-5mM，禽畜胆粉在转化体系中的浓度为50-250g/L。
采用上述方案，由于本方案采用了菌悬液或者上清液作为转化液，并且不含培养基，整个体系黏度小，较为大量的底物(禽畜胆粉)也能在本转化体系中实现分散，进而对底物进行酶促催化。
进一步，所述转化体系的pH值为6.0-9.0；所述转化体系的pH值为6.0-9.0；所述转化体系进行酶促反应的温度为20-30℃，时长为2-24h。
采用上述方案，上述参数范围符合酶促反应适合的反应条件，可保证催化反应的顺利进行，使得禽畜胆粉充分转化为人工熊胆粉。
进一步，7α-羟基类固醇脱氢酶的基因的序列为SEQ ID NO:1；7β-羟基类固醇脱氢酶的基因的序列为SEQ ID NO:3；控制7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶的基因的表达的操纵子均为乳糖操纵子。
采用上述方案，SEQ ID NO:1和SEQ ID NO:3均为经密码子改造的基因，使得7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶可在大肠杆菌中大量表达，获得富集这两种酶的酶菌体。乳糖操纵子为常规控制基因表达的操纵子，易于制备和获取。
进一步，在所述生物转化步骤之前还包括发酵步骤：使用IPTG诱导工程菌A表达7α-羟基类固醇脱氢酶，并使用IPTG诱导工程菌B表达7β-羟基类固醇脱氢酶；或者使用IPTG诱导工程菌C同时表达7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶。
采用上述方案，经过IPTG诱导发酵，使得7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶大量表达，以供后续的生物转化过程使用。
进一步，在所述生物转化步骤之后还包括人工熊胆粉制备步骤：转化体系经生物转化步骤的酶促反应获得反应液，除去所述反应液中的沉淀，再经浓缩获得浸膏；使用乙醇溶液分散浸膏，获得浸膏分散液；除去浸膏分散液中的沉淀，经浓缩和干燥处理之后获得人工熊胆粉。
采用上述方案，通过乙醇处理，除去物料中的酶菌体和蛋白等成分，提高人工熊胆粉中的目的成分的浓度。
具体实施方式
实施例1：工程菌的制备
对7α羟基类固醇脱氢酶基因S1-a-1和7β羟基类固醇脱氢酶基因Y1-b-1进行大肠杆菌表达密码子优化，加入亲和标签，并进行全基因合成。优化后的7α羟基类固醇脱氢酶基因S1-a-1在本文中简称为7α-类固醇脱氢酶基因，记为7α-HSDH(SEQ ID NO:1)；优化后的7β羟基类固醇脱氢酶基因Y1-b-1在本文中简称7β-类固醇脱氢酶基因，记为7β-HSDH(SEQ ID NO:3)。
1.表达载体的构建
a)含有7α-类固醇脱氢酶基因的重组质粒pET28a-7α-HSDH的制备
将7α-HSDH(DNA序列：SEQ ID NO:1，编码的蛋白质序列：SEQ ID NO:2)，用引物对5′-G G A A T T C C A T A T G G G C A G C A G C C A T C A T C A-3′(SEQ ID NO:5)和5′-TCCCTCGAGTTAACGGCTGCGCTCCATCAT-3′(SEQ ID NO:6)通过PCR进行扩增，用Nde I和Xho I酶切，用Dpn I酶(甲基化模板消化酶)消化模板。用Nde I和Xho I酶切pET28a载体。用连接酶连接7α-类固醇脱氢酶基因片段和pET28a载体，获得连接产物。使用连接产物转化DH5α，涂布在卡那霉素抗性的LB平板进行筛选。待菌落形成后，挑选单克隆，接种到5mL LB中进行过夜培养。收集菌体，用天根质粒提取试剂盒提取质粒，送测序。保存测序正确的质粒，获得质粒pET28a-7α-HSDH。
b)含有7β-类固醇脱氢酶基因的重组质粒pET28a-7β-HSDH的制备
将7β-HSDH(DNA序列：SEQ ID NO:3，编码的蛋白质序列：SEQ ID NO:4)用引物对5′-C G G G A T C C A T G G G C A G C A G C C A T C A T C A-3′(SEQ ID NO:7)和5′-CGGAATTCTTATTTCTCGTAAAAGGAACC-3′(SEQ ID NO:8)通过PCR进行扩增，用BamH I和EcoR I酶切，用Dpn I酶消化模板。用BamH I和EcoR I酶切pET28a载体。用连接酶连接7β-类固醇脱氢酶基因片段和载体，获得连接产物。使用连接产物转化DH5α，涂布在卡那霉素抗性的LB平板进行筛选。待菌落形成后，挑选单克隆，接种到5mL LB中进行过夜培养。收集菌体，用天根质粒提取试剂盒提取质粒，送测序，保存测序正确的质粒，获得质粒pET28a-7β-HSDH。
c)含有7α-类固醇脱氢酶基因和7β-类固醇脱氢酶基因的重组质粒pETDuet-1-7α-HSDH/7β-HSDH的制备
将7α-HSDH用引物对SEQ ID NO:5和SEQ ID NO:6通过PCR进行扩增，用Nde I和XhoI酶切，用Dpn I酶消化模板。将上述测序正确的pETDuet-1载体用Nde I和Xho I酶切，用连接酶连接7α-类固醇脱氢酶基因片段和载体，获得连接产物。使用连接产物转化DH5α，涂布在氨苄抗性的LB平板进行筛选。待菌落形成后，挑选单克隆，接种到5ml LB中进行过夜培养。收集菌体，用天根质粒提取试剂盒提取质粒，送测序。保存测序正确的质粒，获得质粒pETDuet-1-7α-HSDH。
将7β-HSDH用引物对SEQ ID NO:7和SEQ ID NO:8通过PCR进行扩增，用BamH I和EcoR I酶切，用Dpn I酶消化模板。用BamH I和EcoR I酶切pETDuet-1-7α-HSDH-载体。用连接酶连接7β-类固醇脱氢酶基因片段和载体，获得连接产物。使用连接产物转化DH5α，涂布在氨苄抗性的LB平板进行筛选。挑选单克隆，接种到5mL LB中进行过夜培养。收集菌体，用天根质粒提取试剂盒提取质粒，送测序。保存测序正确的质粒，获得质粒pETDuet-1-7α-HSDH/7β-HSDH。
2、工程菌构建
将pET28a-7α-HSDH和pET28a-7β-HSDH，以及pETDuet-1-7α-HSDH/7β-HSDH分别转化入大肠杆菌BL21(DE3)的感受态细胞中，得到三种工程菌，分别命名为工程菌A(含pET28a-7α-HSDH)、工程菌B(pET28a-7β-HSDH)和工程菌C(含有pETDuet-1-7α-HSDH/7β-HSDH)。
实施例2：工程菌的发酵表达
(1)菌种制备
工程菌A和工程菌B的菌液分别涂布卡那抗性的LB平板，工程菌C的菌液涂布氨苄抗性的LB平板，待形成菌落后，挑选单克隆，分别接种至5mL含有卡那或氨苄的LB培养基中，37℃，220rpm，进行培养，OD值为0.8-1.2时，加入1mM IPTG诱导2h，SDS-PAGE检测表达量，选取表达量高的克隆，作为中间菌种(中间菌种A、中间菌种B和中间菌种C)进行保藏。
取20μL含有重组质粒的大肠杆菌BL21(DE3)菌种(三种中间菌种：中间菌种A、中间菌种B和中间菌种C)，分别接种至200mL卡那或氨苄抗性LB培养基中，37℃，220rpm，过夜培养，OD 600值为2.5-4.0。取20mL培养液接种至1L卡那或氨苄抗性培养基中，37℃，140rpm，培养3小时，OD 600值为1时，加入0.5mM IPTG诱导过夜表达，然后离心收集菌体，分别获得菌种A(含有pET28a-7α-HSDH)、菌种B(含有pET28a-7β-HSDH)和菌种C(pETDuet-1-7α-HSDH/7β-HSDH)。
(2)大量发酵表达
取20μL菌种A、菌种B或者菌种C，接种至200mL卡那或氨苄抗性LB培养基中，37℃，220rpm，过夜培养，OD 600值为2.5-4.0。取20mL培养液接种至1L卡那或氨苄抗性培养基中，37℃，140rpm，过夜培养。将10L种子液无菌接种至装有200L大肠杆菌高密度发酵培养基的发酵罐中，37℃，通气搅拌培养8小时后，向发酵罐中加入终浓度为0.5mM的IPTG溶液进行诱导，诱导10-12h后发酵结束，放液，离心收集菌体并4℃保存，分别获得酶菌体A(由菌种A经上述过程获得)、酶菌体B(由菌种B经上述过程获得)和酶菌体C(由菌种C经上述过程获得)。
7α-类固醇脱氢酶的酶活测定方法：以牛磺鹅去氧胆酸为底物，在一个3mL的反应体系中加入2.97mL的100mM磷酸缓冲液(pH8.0)，终浓度0.5mM的牛磺鹅去氧胆酸，10μL的梯度稀释的粗酶液，终浓度0.5mM的NADP+，在pH8.0和25℃反应1min，在340nm处测定吸光值变 化情况，然后计算7α-类固醇脱氢酶的酶活。粗酶液的制备方法为：取10g菌体(酶菌体A或B)重悬于100mM磷酸盐缓冲液中，超声破碎，过滤取上清得到粗酶液。7β-类固醇脱氢酶的酶活测定方法：以牛磺熊去氧胆酸为底物，在一个3mL的反应体系中加入2.97mL的100mM磷酸缓冲液，终浓度0.5mM的牛磺熊去氧胆酸，10μL的梯度稀释的粗酶液，终浓度0.5mM的NADP+，在pH8.0和25℃反应1min，在340nm处测定吸光值变化情况，然后计算7β-类固醇脱氢酶的酶活。经计算，酶菌体A中7α-类固醇脱氢酶的酶活与酶菌体B中7β-类固醇脱氢酶的酶活，酶活符合后续应用需求，说明酶菌体均能用于进行人工熊胆的生物转化。
实施例3：禽胆粉的制备
禽胆粉的制备的工艺路线为：将新鲜禽畜胆或解冻禽畜胆用绞肉机切开，过100目筛取液相部分(主要是胆汁)，在液相部分中加入90-95％乙醇至乙醇终浓度为60-85％，离心或过滤去除沉淀，上清液经减压浓缩和真空干燥制得禽胆粉。
在本实施例中具体为：取20Kg解冻鸡胆，用绞肉机切开，用100目不锈钢滤网及多层100目尼龙网过滤，取液相，将液相部分减压除水至膏状(密度控制在1.10±0.05g/ml)，膏体中加入25L 95％乙醇混匀后过夜，3800rpm离心5min去除沉淀，上清液经减压除水至膏状、真空干燥制得鸡胆粉。使用HPLC-ELSD，依照《中国药典(2015)》(GBT16631-2008高效液相色谱法通则)进行检测，鸡胆粉中TCDCA含量为61.3％。
实施例4：生物转化以及人工熊胆粉的制备(100L反应体系)
将禽胆粉转化成熊胆粉的工艺为：将禽畜胆粉溶解于0-15mM甘氨酸缓冲液中，加入0.1-5mM的NADP+，加入转化液，然后补加20-100mM甘氨酸缓冲液至最终体积，用氢氧化钠调节pH至6.0-9.0，获得转化体系，然后20-30℃，反应2-24h，即可完成生物转化，获得转化完成的反应液。以上转化反应过程中，底物(禽畜胆粉)的浓度为50-250g/L。其中，转化液为酶菌体A和酶菌体B(两种酶菌体的混合菌)分散在甘氨酸缓冲液后形成的菌悬液。每10g酶菌体A和酶菌体B组成的混合菌使用20-100ml甘氨酸缓冲液分散，获得菌悬液。也可以为对酶菌体A和酶菌体B组成的混合菌进行破碎处理，经过滤后取上清，上清即为转化液。酶菌体A和酶菌体B的质量比为1:10-1:2。转化液的加入量为整个转化体系的1/5-4/5。也可以使用酶菌体C代替酶菌体A和酶菌体B的混合物。
离心或过滤转化完成的反应液，去除沉淀，上清液减压浓缩至膏状(膏状反应液)，加入膏状反应液体积的4-10倍的90-95％乙醇至乙醇终浓度为60-85％，离心或过滤去除沉淀，上清液经减压浓缩、真空干燥得人工熊胆粉。
在本实施例中具体为：将20kg鸡胆粉溶解于40L的15mM甘氨酸缓冲液中，加入5mM NADP+，加入转化液，补加15mM甘氨酸缓冲液至100L，用5M NaOH调节pH至6.5，获得转化体系。25℃，反应16h。转化液经0.22um滤膜过滤澄清，上清液浓缩成膏状(50L，密度控制在1.10±0.05g/ml)，再加入250L 95％乙醇混匀后过夜，醇沉液经0.22um滤膜过滤澄清，上清液经减压浓缩、真空干燥制得人工熊胆粉。转化液的制备方法为在甘氨酸缓冲液中加入酶菌体，每10g酶菌体使用50ml甘氨酸缓冲液分散，获得菌悬液，菌悬液即为转化液。转化液的加入量为转化体系的体积的3/5。酶菌体包括酶菌体A(表达有7α-羟基类固醇脱氢酶的A类工程菌)和酶菌体B(表达有7β-羟基类固醇脱氢酶的B类工程菌)两种，两者的质量比为1:5。
使用HPLC-ELSD，依照《中国药典(2015)》(GBT16631-2008高效液相色谱法通则)进行检测，本实施例中的人工熊胆粉中TUDCA含量32.0％，TCDCA含量26.5％，T-7-KLCA含量 1.4％。
实施例5-实施例11的生物转化以及人工熊胆粉的制备过程同实施例4，不同点在于具体参数的选择，详见表1。在转化液类型中，Ⅰ型是指将酶菌体分散于转化缓冲液中，获得菌悬液，所述菌悬液为转化液，Ⅱ型是指将酶菌体分散于转化缓冲液中，获得菌悬液，然后对酶菌体进行破碎处理后，离心取上清液，所述上清液为转化液。在Ⅱ型转化液中，使用800-1200bar的压力对酶菌体进行破碎处理，然后12000-25000rpm离心0.5-2h取上清液。上述条件(参数范围)均能有效地将酶菌体破碎，并将酶菌体的原生质体和细胞壁分离，具体实施时，选择一具体参数即可。上述条件可以将酶菌体的原生质体和细胞壁分离，获得具有催化活性的上清液。上清液中含有7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶，原生质体中的各种活性物质对这两种酶形成包裹和保护，使得这两种酶免受禽畜胆粉中的杂质的干扰，顺利催化禽畜胆粉向人工熊胆粉的生物转化过程。在实施例10中，转化缓冲液浓度为0mM，即使用的是去离子水，转化体系不具有自调节pH值的能力，需要实时监测反应体系的pH值，并调整pH值至7.0(至少维持在6.0-9.0之间)。在实施例11中，使用的是酶菌体C，而不是酶菌体A和酶菌体B的混合物。
对比例1-对比例4的生物转化以及人工熊胆粉的制备过程同实施例4，不同点在于具体参数的选择，详见表1。在对比例4中，没有使用甘氨酸缓冲液，而是使用一般培养基以维持酶菌体活性，该培养基具体为LB培养基。在对比例1中，禽畜胆粉只加入了5kg，是因为酶菌体的用量过大，体系中无法再溶解过多的禽畜胆粉。
表1：实施例4-11，以及对比例1-4的参数选择列表
Figure PCTCN2020108657-appb-000001
对实施例1-实施例11以及对比例1-对比例4中制备的人工熊胆粉进行HPLC检测， 结果如表2所示。由实验结果可知，实施例1-11制备的人工熊胆粉的TUDCA和TCDCA的含量比例均符合天然熊胆粉的含量比例范围，TUDCA的含量符合《中国药典(2015)》对于熊胆粉的规定，并且中间产物T-7-KLCA的含量低，证明了工艺生物转化效率高，获得的最终产品的品质有保证。对比例1的酶菌体用量较大，但是对反应效率并没有很大的提升作用。对比例2的酶菌体使用量过小，导致催化作用不能充分进行，获得的人工熊胆粉各项指标离实施例1-11有一定差距。对比例3中使用了高浓度的甘氨酸缓冲液，对催化作用产生了一定抑制，导致获得的人工熊胆粉各项指标不符合理想。对比例4利用培养基代替甘氨酸缓冲液，培养基的使用是用来维持细胞活性，避免细胞在仅有缓冲液的环境中出现应激状态(可能会影响酶的活性)。但是，培养基的加入并没有提升反应效率，反而使得最终产物的质量变差，不管是TUDCA/TCDCA还是中间体的含量，均离理想状态相差甚远。说明培养基并不利于酶与底物的结合，细胞的活性高并不利于其产生的7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶对底物的催化作用。
表2：HPLC检测以及计算结果
Figure PCTCN2020108657-appb-000002
实验例1
为验证使用本方案的转化液的催化效果与纯化的酶的催化效果的区别，在本实验例中，同样酶活的条件下，测试了两种转化液和纯化的酶的催化能力。
待测物的制备：
在第一测试组中，将酶菌体A或B分别加入15mM甘氨酸缓冲液中(每10g酶菌体A或B 中分别加入50ml甘氨酸缓冲液)，1000bar高压破碎，20000rpm离心1h取上清液，使用Ni柱亲和纯化(质粒构建的时候已经加入亲和标签)，即可获得纯化的酶(分别获得7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶)。在第二测试组中，将酶菌体A或B分别加入15mM甘氨酸缓冲液中(每10g酶菌体A或B中分别加入50ml甘氨酸缓冲液)，1000bar高压破碎，20000rpm离心1h取上清液即得(两种：含7α-羟基类固醇脱氢酶的上清液和含7β-羟基类固醇脱氢酶的上清液)。在第三测试组中，将酶菌体A或B加入15mM甘氨酸缓冲液中(每10g酶菌体A或B中分别加入50ml甘氨酸缓冲液)即得(两种：含7α-羟基类固醇脱氢酶的菌悬液和含7β-羟基类固醇脱氢酶的菌悬液)。参照实施例2的方法测定上述2×3种待测物的酶活，并按照酶活来两两复配三组中的两种溶液，获得三组待测物。复配的过程为：将7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶依照酶活混合，确保第一测试组的待测物中的酶活组成为7α-类固醇脱氢酶2000U和7β-类固醇脱氢酶100U。同理复配出第二测试组的待测物和第三测试组的待测物。然后使用三种待测物催化禽胆粉的生物转化。本实验例的催化反应使用2g禽胆粉作为底物，转化体系为10ml，pH6.5，反应温度25℃，催化时间16h，缓冲液浓度10mM，NADP+含量2mM。结果如表3所示。
表3：催化能力检测结果
Figure PCTCN2020108657-appb-000003
如上表所示，相同的反应体系中分别加入总酶活相等的三种待测物转化反应，第三测试组和第二测试组TUDCA/TCDCA和TUDCA的含量均大于第一测试组，可见在高浓度禽胆粉的环境中，菌悬液或上清液更能保障酶的活性和稳定性，对转化效果更有利。
以上所述的仅是本发明的实施例，方案中公知的具体技术方案和/或特性等常识在此未作过多描述。应当指出，对于本领域的技术人员来说，在不脱离本发明技术方案的前提下，还可以作出若干变形和改进，这些也应该视为本发明的保护范围，这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准，说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Figure PCTCN2020108657-appb-000004
Figure PCTCN2020108657-appb-000005
Figure PCTCN2020108657-appb-000006
Figure PCTCN2020108657-appb-000007
Figure PCTCN2020108657-appb-000008
Figure PCTCN2020108657-appb-000009

Claims (10)

  1. 一种人工熊胆粉的制作工艺，其特征在于，包括用于将牛磺鹅去氧胆酸转换为牛磺熊去氧胆酸的生物转化步骤：包括含有禽畜胆粉、转化缓冲液和转化液的转化体系；所述转化液为包含酶菌体的菌悬液或者包含酶菌体的原生质体的上清液；所述酶菌体表达有7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶。
  2. 根据权利要求1所述的一种人工熊胆粉的制作工艺，其特征在于，所述菌悬液的制备方法为：将酶菌体分散于转化缓冲液中，获得菌悬液；所述上清液的制备方法为：对所述菌悬液中的酶菌体进行破碎处理后，离心获得上清液。
  3. 根据权利要求2所述的一种人工熊胆粉的制作工艺，其特征在于，所述酶菌体为表达有7α-羟基类固醇脱氢酶的工程菌A和表达有7β-羟基类固醇脱氢酶的工程菌B组成的混合物；或者所述酶菌体为同时表达有7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶两种酶的工程菌C。
  4. 根据权利要求3所述的一种人工熊胆粉的制作工艺，其特征在于，每10g酶菌体使用20-100ml转化缓冲液分散，获得菌悬液；表达有7α-羟基类固醇脱氢酶的工程菌A和表达有7β-羟基类固醇脱氢酶的工程菌B的质量比为1:10-1:2。
  5. 根据权利要求4所述的一种人工熊胆粉的制作工艺，其特征在于：所述转化缓冲液为0-15mM的甘氨酸缓冲液；所述转化液的体积为转化体系的体积的1/5-4/5。
  6. 根据权利要求5所述的一种人工熊胆粉的制作工艺，其特征在于：辅酶Ⅰ或者辅酶Ⅱ在转化体系中的浓度为0.1-5mM，禽畜胆粉在转化体系中的浓度为50-250g/L。
  7. 根据权利要求6所述的一种人工熊胆粉的制作工艺，其特征在于：所述转化体系的pH值为6.0-9.0；所述转化体系进行酶促反应的温度为20-30℃，时长为2-24h。
  8. 根据权利要求1-7中任一项所述的一种人工熊胆粉的制作工艺，其特征在于：7α-羟基类固醇脱氢酶的基因的序列为SEQ ID NO:1；7β-羟基类固醇脱氢酶的基因的序列为SEQ ID NO:3；控制7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶的基因的表达的操纵子均为乳糖操纵子。
  9. 根据权利要求8所述的一种人工熊胆粉的制作工艺，其特征在于：在所述生物转化步骤之前还包括发酵步骤：使用IPTG诱导工程菌A表达7α-羟基类固醇脱氢酶，并使用IPTG诱导工程菌B表达7β-羟基类固醇脱氢酶；或者使用IPTG诱导工程菌C同时表达7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶。
  10. 根据权利要求9所述的一种人工熊胆粉的制作工艺，其特征在于：在所述生物转化步骤之后还包括人工熊胆粉制备步骤：转化体系经生物转化步骤的酶促反应获得反应液，除去所述反应液中的沉淀，再经浓缩获得浸膏；使用乙醇溶液分散浸膏，获得浸膏分散液；除去浸膏分散液中的沉淀，经浓缩和干燥处理之后获得人工熊胆粉。
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