CN112852911A - Preparation method of ursodeoxycholic acid - Google Patents
Preparation method of ursodeoxycholic acid Download PDFInfo
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- CN112852911A CN112852911A CN202010715401.0A CN202010715401A CN112852911A CN 112852911 A CN112852911 A CN 112852911A CN 202010715401 A CN202010715401 A CN 202010715401A CN 112852911 A CN112852911 A CN 112852911A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/02—Dehydrogenating; Dehydroxylating
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
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Abstract
In order to solve the problem that the biological preparation of ursodeoxycholic acid requires excessive co-substrate to realize cofactor regeneration, the invention takes riboflavin as a substrate under the action of flavin reductase to realize the addition of a catalytic amount of the co-substrate, and the production cost of oxidizing chenodeoxycholic acid into 7-carbonyl lithocholic acid is greatly reduced by taking oxygen in the air as the substrate. The concentration of the substrate can be effectively improved by using 7-carbonyl lithocholic acid as the substrate and isopropanol as a co-substrate. The invention provides a cheap biological catalysis process for green production of ursodeoxycholic acid.
Description
Technical Field
The invention relates to a method for preparing ursodeoxycholic acid by using various oxidoreductases as catalysts and chenodeoxycholic acid as a substrate, belonging to the field of bioengineering.
Background
Cholic acid and its derivatives have wide biological activity and are endogenous natural products. Due to its high degree of optical purity and structural characteristics of amphiphilic molecules, biological activity studies have been one of the research hotspots in the fields of pharmacology and drug discovery. Cholic acid drugs have been used clinically, for example, ursodeoxycholic acid and adeoxycholic acid of cattle have been used for treating liver diseases (the effect of ursodeoxycholic acid combination on treating fatty liver complicated with hyperlipidemia, Weihong, 2017, 30, 133). In 2015, two cholic acid drugs, namely a Cholham capsule (the main component is cholic acid) for treating rare diseases of bile acid synthesis disorder and a fat-dissolving injection Kybella (the main component is deoxycholic acid) for submental fat, are approved by FDA to be on the market.
Literature on traditional ursodeoxycholic acid preparation (NAD)+-Dependent Enzymatic Route for the infection of Hydroxysteroids, Fabio Toni, Linda G.Otten, Isabel W.C.E.Arends, 2018,11, 1-13; flavin oxidated product-media Regeneration of Nicotinamide Adenie with Dioxygen and Catalytic amino of Flavin monoglucoside for One-Point Multi-enzyme Preparation of Ursoxyolic Acid, Xi Chen, Yunfeng Cui, Jinhui Feng, Yu Wang, Xiangtao Liu, Qiaqing Wu, Dunming Zhu, Yanhe Ma,2019, DOI: 10.1002/adsc.201900111) although it is a One-Pot process, it often requires the use of co-substrates to achieve coenzyme Regeneration. Especially in the first oxidation step, it is often necessary to add an excess of co-substrate to drive the reaction.
The formula is the preparation of ursodeoxycholic acid from chenodeoxycholic acid
Disclosure of Invention
In order to effectively reduce the production cost of preparing the ursodeoxycholic acid from the chenodeoxycholic acid, the catalytic amount of the cosubstrate is added, so that the production cost of the ursodeoxycholic acid is reduced.
The invention has the beneficial effects that: by changing a coenzyme regeneration method, the usage amount of the cosubstrate is effectively reduced, so that the generation cost is reduced, and a cheap preparation method is provided for the preparation of the ursodeoxycholic acid.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention.
The flavin reductase gene used in the present invention was derived from riboflavin reductase (MFR) of Mycobacterium goodii (GenBank: ABX11274.1), the 7. alpha. -hydroxysteroid dehydrogenase (7. alpha. -HSDH) gene was derived from Brevundimonas sp. (GenBank: WP-046653274.1), the 7. beta. -hydroxysteroid dehydrogenase (7. beta. -HSDH) gene was derived from Clostridium sp. Marseille (GenBank: WP-066892209.1), and the TB alcohol dehydrogenase (TBADH) gene was derived from Thermoanaerobacter ethanolica (GenBank: X64841.1), which were constructed on pET21a or SFpRpR-1 expression vectors. The recombinant plasmid is transferred into host bacterial cells (preferably Escherichia coli BL21 (DE3)) to obtain corresponding engineering strains. The engineering strain is inoculated into an L-B culture medium and cultured for 16 hours at 25 ℃. And (4) centrifugally collecting thalli, and breaking the thalli to obtain a supernatant.
Example 1: preparation of 7-Keto-lithocholic acid by flavin reductase and 7 alpha-cholic acid dehydrogenase
In a 1mL reaction system, chenodeoxycholic acid (10mM), 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH), cofactor NAD + (0.1mM), flavin reductase (MFR) and riboflavin-5-phosphate (0.1mM) are added for reaction at 30 ℃, and the chenodeoxycholic acid conversion rate is more than 95 percent after 2 hours of detection.
Example 2: preparation of ursodeoxycholic acid from chenodeoxycholic acid
In a 10mL reaction system, chenodeoxycholic acid (30mM), 7 alpha-steroid dehydrogenase (7 alpha-HSDH), cofactor NAD + (0.1mM), flavin reductase (MFR) and riboflavin-5-phosphoric acid (0.1mM) are added for reaction at 30 ℃, and the conversion rate of the chenodeoxycholic acid is detected to be more than 95 percent after 4 hours. TBADH, 7 beta-HSDH and 45mM isopropanol are added, the reaction is continued at 30 ℃, and the generation rate of ursodeoxycholic acid is detected to be more than 95 percent after 24 hours.
Example 3: preparation of ursodeoxycholic acid from chenodeoxycholic acid
In a 10mL reaction system, chenodeoxycholic acid (100mM), 7 alpha-steroid dehydrogenase (7 alpha-HSDH), cofactor NAD + (0.1mM), flavin reductase (MFR) and riboflavin-5-phosphoric acid (0.1mM) are added for reaction at 30 ℃, and the conversion rate of the chenodeoxycholic acid is detected to be more than 95 percent after 8 hours. TBADH, 7 beta-HSDH and 150mM isopropanol are added, the reaction is continued at 30 ℃, and the generation rate of ursodeoxycholic acid is detected to be more than 95% after 24 hours.
Example 4: preparation of ursodeoxycholic acid from chenodeoxycholic acid
In a 1L reaction system, chenodeoxycholic acid (500mM), 7 alpha-steroid dehydrogenase (7 alpha-HSDH), cofactor NAD + (10mM), flavin reductase (MFR) and riboflavin-5-phosphoric acid (5mM) are added for reaction at 30 ℃, and the conversion rate of the chenodeoxycholic acid is more than 95 percent after 12 hours of reaction. TBADH, 7 beta-HSDH and 750mM isopropanol are added, the reaction is continued at 30 ℃, and the ursodeoxycholic acid generation rate is detected to be more than 95% after 48 hours.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> 12-hydroxycholate dehydrogenase and use thereof
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tttagcggcg gtctggaggc ggcgaaagag gaactggagc gtgatttcgg cagcgaagtt 180
tttaccattc tggcgaacgg tagcgtggag gaagaggttc gtgcggcggt tgaggcgggt 240
gcggaacact ttggcggtcg tatcgacgtg ctgattaaca acgcgcaggc gagcgcgagc 300
ggtctgaccc tggttcaaca cagcgaagag gactttgatc tggcggtgcg tagcggtctg 360
tacgcgacct tcttttatat gaagcacgcg tacccgtatc tgaaagaaac cgcgggcagc 420
gttattaact ttgcgagcgg tgcgggtatc ggcggtaacc cgggtcagag cagctacgcg 480
gcggcgaaag agggtatccg tggtatgagc cgtgttgcgg cgagcgaatg gggtccggat 540
aacatcaacg tgaacattgt ttgcccgatc gtgatgacca aggcgctgga agagtggcgt 600
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gacgcggaaa aagatgtggg tcgtgtgtgc gttttcctgg cgagcccgga cgcgagcttt 720
gttaccggcg ataccatcat ggtgcaaggc ggtagcggta tgaaaccgta actcgag 777
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Met Asp Met Gly Leu Lys Asp Lys Val Val Leu Ile Thr Gly Gly Gly
1 5 10 15
Gly Gly Ile Ala Arg Gly Ile Glu Arg Ala Phe Ala Thr Glu Gly Ala
20 25 30
Lys Phe Ile Leu Thr Asp Leu Phe Ser Gly Gly Leu Glu Ala Ala Lys
35 40 45
Glu Glu Leu Glu Arg Asp Phe Gly Ser Glu Val Phe Thr Ile Leu Ala
50 55 60
Asn Gly Ser Val Glu Glu Glu Val Arg Ala Ala Val Glu Ala Gly Ala
65 70 75 80
Glu His Phe Gly Gly Arg Ile Asp Val Leu Ile Asn Asn Ala Gln Ala
85 90 95
Ser Ala Ser Gly Leu Thr Leu Val Gln His Ser Glu Glu Asp Phe Asp
100 105 110
Leu Ala Val Arg Ser Gly Leu Tyr Ala Thr Phe Phe Tyr Met Lys His
115 120 125
Ala Tyr Pro Tyr Leu Lys Glu Thr Ala Gly Ser Val Ile Asn Phe Ala
130 135 140
Ser Gly Ala Gly Ile Gly Gly Asn Pro Gly Gln Ser Ser Tyr Ala Ala
145 150 155 160
Ala Lys Glu Gly Ile Arg Gly Met Ser Arg Val Ala Ala Ser Glu Trp
165 170 175
Gly Pro Asp Asn Ile Asn Val Asn Ile Val Cys Pro Ile Val Met Thr
180 185 190
Lys Ala Leu Glu Glu Trp Arg Glu Arg Glu Pro Glu Met Tyr Glu Lys
195 200 205
Asn Val Lys Ala Ile Pro Leu Gly Arg Phe Gly Asp Ala Glu Lys Asp
210 215 220
Val Gly Arg Val Cys Val Phe Leu Ala Ser Pro Asp Ala Ser Phe Val
225 230 235 240
Thr Gly Asp Thr Ile Met Val Gln Gly Gly Ser Gly Met Lys Pro
245 250 255
Claims (6)
1. A preparation method of ursodeoxycholic acid is characterized in that chenodeoxycholic acid is used as a raw material to synthesize the ursodeoxycholic acid through an intermediate 7-carbonyl lithocholic acid by a biological catalysis method.
2. The method of claim 1, wherein the coenzyme NAD is achieved+RegeneratedThe co-substrate riboflavin is a catalytic amount, and isopropanol not only serves as a cosolvent to increase the solubility of the substrate, but also serves as a co-substrate to realize the regeneration of coenzyme NADPH.
3. The method of claim 1, wherein the oxidation of chenodeoxycholic acid and the reduction of 7-carbonyl lithocholic acid are accomplished by a biological catalysis step by synthesizing ursodeoxycholic acid from chenodeoxycholic acid as a raw material through an intermediate 7-carbonyl lithocholic acid by an enzymatic method.
4. The method of claim 1, wherein the oxidation of chenodeoxycholic acid and the reduction of 7-carbonyl lithocholic acid are accomplished by a biocatalytic one-pot method by synthesizing ursodeoxycholic acid from chenodeoxycholic acid as a raw material through an intermediate 7-carbonyl lithocholic acid by an enzymatic method.
5. The method of claim 1, wherein the co-substrate riboflavin obtained by oxidation of chenodeoxycholic acid is 0.1-10% of the molar concentration of chenodeoxycholic acid.
6. The method of claim 1, wherein the chenodeoxycholic acid concentration as a substrate is 20-100 g/L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010715401.0A CN112852911A (en) | 2020-07-23 | 2020-07-23 | Preparation method of ursodeoxycholic acid |
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| CN202010715401.0A CN112852911A (en) | 2020-07-23 | 2020-07-23 | Preparation method of ursodeoxycholic acid |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736842A (en) * | 2021-09-02 | 2021-12-03 | 四川澄华生物科技有限公司 | Method for efficiently preparing tauroursodeoxycholic acid by multiple cells |
| CN114107237A (en) * | 2021-09-07 | 2022-03-01 | 伊犁川宁生物技术股份有限公司 | Method for producing chenodeoxycholic acid oxidase by fermentation |
| CN114107237B (en) * | 2021-09-07 | 2024-06-04 | 伊犁川宁生物技术股份有限公司 | Method for producing chenodeoxycholic acid oxidase by fermentation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011064404A1 (en) * | 2009-11-30 | 2011-06-03 | Pharmazell Gmbh | NOVEL 7β-HYDROXYSTEROID DEHYDROGENASES AND THEIR USE |
| CN110628849A (en) * | 2018-06-25 | 2019-12-31 | 中国科学院天津工业生物技术研究所 | Method for regenerating oxidation state nicotinamide cofactor |
| CN111378703A (en) * | 2020-03-27 | 2020-07-07 | 长兴制药股份有限公司 | Preparation method of (2S,3S) -2-hydroxy-4-phenylbutane derivative |
-
2020
- 2020-07-23 CN CN202010715401.0A patent/CN112852911A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011064404A1 (en) * | 2009-11-30 | 2011-06-03 | Pharmazell Gmbh | NOVEL 7β-HYDROXYSTEROID DEHYDROGENASES AND THEIR USE |
| CN110628849A (en) * | 2018-06-25 | 2019-12-31 | 中国科学院天津工业生物技术研究所 | Method for regenerating oxidation state nicotinamide cofactor |
| CN111378703A (en) * | 2020-03-27 | 2020-07-07 | 长兴制药股份有限公司 | Preparation method of (2S,3S) -2-hydroxy-4-phenylbutane derivative |
Non-Patent Citations (1)
| Title |
|---|
| MING-MINZHENG 等: "Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques", 《PROCESS BIOCHEMISTRY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736842A (en) * | 2021-09-02 | 2021-12-03 | 四川澄华生物科技有限公司 | Method for efficiently preparing tauroursodeoxycholic acid by multiple cells |
| CN113736842B (en) * | 2021-09-02 | 2024-04-19 | 四川澄华生物科技有限公司 | Method for efficiently preparing tauroursodeoxycholic acid by multiple cells |
| CN114107237A (en) * | 2021-09-07 | 2022-03-01 | 伊犁川宁生物技术股份有限公司 | Method for producing chenodeoxycholic acid oxidase by fermentation |
| CN114107237B (en) * | 2021-09-07 | 2024-06-04 | 伊犁川宁生物技术股份有限公司 | Method for producing chenodeoxycholic acid oxidase by fermentation |
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