CN109897835A - A kind of novel 7-beta- hydroxysteroid dehydrogenase and its it is used for cholic acid synthetic method - Google Patents
A kind of novel 7-beta- hydroxysteroid dehydrogenase and its it is used for cholic acid synthetic method Download PDFInfo
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Abstract
The invention discloses a kind of novel 7-beta- hydroxysteroid dehydrogenase and its it is used for cholic acid synthetic method, field is synthesized more particularly to cholic acid, 12nd amino acids residue of the novel 7-beta- hydroxysteroid dehydrogenase and the 86th amino acids residue mutate, and the 12nd amino acids residue lysine sported by tryptophan, the 86th amino acids residue sports isoleucine by leucine.Compared to the prior art, in actual implementation, under same cost, available more destination proteins of the present invention, and then the promotion of the production cost of cholic acid is effectively reduced are more applicable for industrialized production to the present invention.
Description
Technical field
The present invention relates to cholic acid synthesis technical fields, it is more particularly related to a kind of novel 7-beta- hydroxyl
Steroid dehydrogenase and its be used for cholic acid synthetic method.
Background technique
Ursodesoxycholic acid (I, UDCA) is principle active component contained by rare Chinese medicine bear gall, it is its corresponding diastereomeric
Isomers chenodeoxycholic acid (II, CDCA) is used clinically for treating various cholelith diseases, and various acute, chronic liver diseases have
Good effect, it is low to extract UDCA yield from the bear gall of the bear of artificial breeding, limited source, and against in animal protection,
Thus artificial synthesized UDCA is of great significance.The synthetic method of UDCA mainly has the synthesis of full chemistry method and chemical-enzymatic to combine
Method, starting material be animal origin cholic acid (CA) or deoxycholic aicd (such as CDCA).
It is prominent that the patent of invention of 105274070 A of patent application publication CN discloses a kind of 7beta-Hydroxysteroid dehydrogenase
Varitron and its application and synthetic method；A kind of muton of 7 β-steroid dehydrogenase, it is characterised in that the amino of the muton
Acid sequence Seq ID NO:4, coding nucleotide sequence are Seq ID NO:3；Or the amino acid sequence Seq ID of the muton
NO:6, coding nucleotide sequence are Seq ID NO:5.Use efficient 7 β-steroid dehydrogenase and its muton enzyme, Yi Jifu
Enzyme regenerative system catalyzes and synthesizes cholic acid compound especially ursodesoxycholic acid, and concentration of substrate is up to 100g/L, conversion ratio is
99.2-99.5%, weight yield are up to 94-96%.Production cost and product quality are better than chemical synthesis process, are suitable for industry
Metaplasia produces.
But it is in actual use, still there are some disadvantages, if it is when carrying out industrialization cholic acid synthesis, needs to make
A large amount of 7 β-steroid dehydrogenase is used, when directly buying on the market, since quantity required is larger, will also result in
Larger economic expenditure, and then will lead to the promotion of the production cost of cholic acid.
Summary of the invention
The present invention overcomes the shortcomings of the prior art, technical problem to be solved are as follows: provides a kind of novel 7-
Beta- hydroxysteroid dehydrogenase and its be used for cholic acid synthetic method, by using Escherichia coli to novel 7-beta- hydroxyl
Base steroid dehydrogenase is recombinated, and then generates gst fusion protein using E. coli recombinant stain, then merge by GST
Destination protein is made in albumen, ursodesoxycholic acid finally is made using destination protein catalysis substrate, compared to the prior art, in reality
When implementation, under same cost, the present invention available more destination proteins, and then the production cost that cholic acid is effectively reduced mentions
It rises, is more applicable for industrialized production, to solve the problems mentioned in the above background technology.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows: a kind of novel 7-beta- hydroxy kind is solid
Alcohol dehydrogenase, the 12nd amino acids residue of the novel 7-beta- hydroxysteroid dehydrogenase and the 86th amino acids are residual
Base mutates, and the 12nd amino acids residue sports lysine by tryptophan, and the 86th amino acids residue is dashed forward by leucine
Become isoleucine.
The novel specific preparation method of 7-beta- hydroxysteroid dehydrogenase the following steps are included:
S1: cultivating e. coli host cell in the medium, to give expression to the novel 7-beta- hydroxy kind
Sterol dehydrogenase；
S2: by the novel 7-beta- hydroxysteroid dehydrogenase by being separated in host cell.
The e. coli host cell contains carrier, and the carrier contains polynucleotide molecule, the polynucleotides point
Son encodes novel 7-beta- hydroxysteroid dehydrogenase described in claim 1.
The present invention also provides a kind of novel 7-beta- hydroxysteroid dehydrogenases to be used for cholic acid synthetic method, tool
Body the following steps are included:
S1, recombination: using coli strain as recombinant expression carrier to the novel 7-beta- hydroxy steroid
Dehydrogenase is recombinated；
S2, culture: successful E. coli recombinant stain will be transferred to according to the additive amount of 3-5%, LB liquid medium is added
In cultivated, culture revolving speed be set as 180-260RPM, cultivation temperature is set as 25-42 DEG C；
S3, induction: detection E. coli recombinant stain 600 nanometer wave strong points light absorption value, when light absorption value be 0.5 when,
E. coli recombinant stain is induced using 14-22 DEG C of isopropylthiogalactoside, 8-12h is arranged in induction time,
Coli strain is set to generate gst fusion protein；
S4, bacterium solution are collected: centrifugally operated are carried out to the E. coli recombinant stain that induction is completed, in order to collect large intestine bar
Thallus in bacterium recombinant bacterial strain, centrifugal rotational speed are set as 10000-12000RPM, and centrifugation time is set as 2-5min and collects bacterium solution；
S5, broken wall: carrying out broken wall operation to bacterium solution using 300W ultrasonication equipment, and broken time is set as 4-6min,
It is clarified to bacterium solution；
S6, supernatant collection: centrifugally operated is carried out to the bacterium solution after clarification, centrifugal rotational speed is set as 14000-
16000RPM, centrifuging temperature are set as 3-7 DEG C, and centrifugation time is set as 8-12min, then collect the supernatant in bacterium solution；
S7, combination: by supernatant in conjunction with Glutathione Sepharose 4B, gst fusion protein is made to be incorporated into gluathione
In peptide Ago-Gel, combination temperature is set as 2-6 DEG C, and binding time is set as 1.5-3h；
S8, second level are rinsed: using the PBS solution of long-pending 0.25% polysorbas20 of the pentaploid of pre-cooling to containing gst fusion protein
Glutathione Sepharose flushes three times, and is then flushed three times using the pentaploid product PBS of pre-cooling；
S9, digestion: the glutathione-agarose using PreScissionProtease enzyme direct enzyme cutting containing gst fusion protein
Sugared gel, digestion temperature setting are 2-5 DEG C, and the digestion time is set as 6-12h, and the product after being centrifuged digestion obtains destination protein；
S10, synthesis: bear is synthesized using 3-5 β of Alpha-hydroxy-7- oxo of above-mentioned purpose proteins carry substrate-cholanic acid 7-KLCA
Deoxycholic aicd UDCA obtains ursodeoxycholic acid crude；
S11, finished product: ursodeoxycholic acid crude is added in organic solvent, alkali is then added into organic solvent
Property substance, using dissolution, flow back, be filtered to remove solid content and acidification separation after, obtain ursodesoxycholic acid finished product.
Technical effect and advantage of the invention: the present invention is de- to novel 7-beta- hydroxy steroid using Escherichia coli
Hydrogen enzyme is recombinated, and then generates gst fusion protein using E. coli recombinant stain, then mesh is made by gst fusion protein
Albumen, finally using destination protein catalysis substrate be made ursodesoxycholic acid, compared to the prior art, in actual implementation, together
Etc. under costs, available more destination proteins of the present invention, and then the promotion of the production cost of cholic acid is effectively reduced are more suitable
For industrialized production.
Detailed description of the invention
The present invention will be further described in detail with reference to the accompanying drawing.
Fig. 1 is cholic acid synthesis flow schematic diagram of the invention.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiments of the present invention, instead of all the embodiments；Based on the embodiments of the present invention, ordinary skill people
Member's every other embodiment obtained without creative efforts, shall fall within the protection scope of the present invention.
Embodiment 1
A kind of novel 7-beta- hydroxysteroid dehydrogenase, the novel 7-beta- hydroxysteroid dehydrogenase
12nd amino acids residue and the 86th amino acids residue mutate, and the 12nd amino acids residue sported by tryptophan it is bad
Propylhomoserin, the 86th amino acids residue sport isoleucine by leucine.
The novel specific preparation method of 7-beta- hydroxysteroid dehydrogenase the following steps are included:
S1: cultivating e. coli host cell in the medium, to give expression to the novel 7-beta- hydroxy kind
Sterol dehydrogenase；
S2: by the novel 7-beta- hydroxysteroid dehydrogenase by being separated in host cell.
The e. coli host cell contains carrier, and the carrier contains polynucleotide molecule, the polynucleotides point
Son encodes novel 7-beta- hydroxysteroid dehydrogenase described in claim 1.
The present invention also provides a kind of novel 7-beta- hydroxysteroid dehydrogenases to be used for cholic acid synthetic method, tool
Body the following steps are included:
S1, recombination: using coli strain as recombinant expression carrier to the novel 7-beta- hydroxy steroid
Dehydrogenase is recombinated；
S2, culture: successful E. coli recombinant stain will be transferred to and be added in LB liquid medium according to 3% additive amount
It is cultivated, culture revolving speed is set as 180RPM, and cultivation temperature is set as 25 DEG C；
S3, induction: detection E. coli recombinant stain 600 nanometer wave strong points light absorption value, when light absorption value be 0.5 when,
E. coli recombinant stain is induced using 14 DEG C of isopropylthiogalactoside, 8h is arranged in induction time, makes large intestine
Bacillus strain generates gst fusion protein；
S4, bacterium solution are collected: centrifugally operated are carried out to the E. coli recombinant stain that induction is completed, in order to collect large intestine bar
Thallus in bacterium recombinant bacterial strain, centrifugal rotational speed are set as 10000RPM, and centrifugation time is set as 2min and collects bacterium solution；
S5, broken wall: carrying out broken wall operation to bacterium solution using 300W ultrasonication equipment, and broken time is set as 4min, until
Bacterium solution clarification；
S6, supernatant collection: carrying out centrifugally operated to the bacterium solution after clarification, and centrifugal rotational speed is set as 14000RPM, is centrifuged
Temperature setting is 3 DEG C, and centrifugation time is set as 8min, then collects the supernatant in bacterium solution；
S7, combination: by supernatant in conjunction with Glutathione Sepharose 4B, gst fusion protein is made to be incorporated into gluathione
In peptide Ago-Gel, combination temperature is set as 2 DEG C, and binding time is set as 1.5h；
S8, second level are rinsed: using the PBS solution of long-pending 0.25% polysorbas20 of the pentaploid of pre-cooling to containing gst fusion protein
Glutathione Sepharose flushes three times, and is then flushed three times using the pentaploid product PBS of pre-cooling；
S9, digestion: the glutathione-agarose using PreScissionProtease enzyme direct enzyme cutting containing gst fusion protein
Sugared gel, digestion temperature setting are 2 DEG C, and the digestion time is set as 6h, and the product after being centrifuged digestion obtains destination protein；
S10, synthesis: bear is synthesized using 3-5 β of Alpha-hydroxy-7- oxo of above-mentioned purpose proteins carry substrate-cholanic acid 7-KLCA
Deoxycholic aicd UDCA obtains ursodeoxycholic acid crude；
S11, finished product: ursodeoxycholic acid crude is added in organic solvent, alkali is then added into organic solvent
Property substance, using dissolution, flow back, be filtered to remove solid content and acidification separation after, obtain ursodesoxycholic acid finished product.
As can be seen from the above embodiments: cultivating revolving speed in the present embodiment and be set as 180RPM, cultivation temperature is set as 25 DEG C, lures
Leading the time is set as 8h, and broken time is set as 4min, and combination temperature is set as 2 DEG C, and binding time is set as 1.5h, digestion temperature
Degree is set as 2 DEG C, and the digestion time is set as 6h, after actual implementation, conversion ratio 95.4%, and weight yield 96.2%.
Embodiment 2
A kind of novel 7-beta- hydroxysteroid dehydrogenase, the novel 7-beta- hydroxysteroid dehydrogenase
12nd amino acids residue and the 86th amino acids residue mutate, and the 12nd amino acids residue sported by tryptophan it is bad
Propylhomoserin, the 86th amino acids residue sport isoleucine by leucine.
The novel specific preparation method of 7-beta- hydroxysteroid dehydrogenase the following steps are included:
S1: cultivating e. coli host cell in the medium, to give expression to the novel 7-beta- hydroxy kind
Sterol dehydrogenase；
S2: by the novel 7-beta- hydroxysteroid dehydrogenase by being separated in host cell.
The e. coli host cell contains carrier, and the carrier contains polynucleotide molecule, the polynucleotides point
Son encodes novel 7-beta- hydroxysteroid dehydrogenase described in claim 1.
The present invention also provides a kind of novel 7-beta- hydroxysteroid dehydrogenases to be used for cholic acid synthetic method, tool
Body the following steps are included:
S1, recombination: using coli strain as recombinant expression carrier to the novel 7-beta- hydroxy steroid
Dehydrogenase is recombinated；
S2, culture: successful E. coli recombinant stain will be transferred to and be added in LB liquid medium according to 4% additive amount
It is cultivated, culture revolving speed is set as 220RPM, and cultivation temperature is set as 33 DEG C；
S3, induction: detection E. coli recombinant stain 600 nanometer wave strong points light absorption value, when light absorption value be 0.5 when,
E. coli recombinant stain is induced using 18 DEG C of isopropylthiogalactoside, 10h is arranged in induction time, makes large intestine
Bacillus strain generates gst fusion protein；
S4, bacterium solution are collected: centrifugally operated are carried out to the E. coli recombinant stain that induction is completed, in order to collect large intestine bar
Thallus in bacterium recombinant bacterial strain, centrifugal rotational speed are set as 11000RPM, and centrifugation time is set as 4min and collects bacterium solution；
S5, broken wall: carrying out broken wall operation to bacterium solution using 300W ultrasonication equipment, and broken time is set as 5min, until
Bacterium solution clarification；
S6, supernatant collection: carrying out centrifugally operated to the bacterium solution after clarification, and centrifugal rotational speed is set as 15000RPM, is centrifuged
Temperature setting is 5 DEG C, and centrifugation time is set as 10min, then collects the supernatant in bacterium solution；
S7, combination: by supernatant in conjunction with Glutathione Sepharose 4B, gst fusion protein is made to be incorporated into gluathione
In peptide Ago-Gel, combination temperature is set as 4 DEG C, and binding time is set as 2h；
S8, second level are rinsed: using the PBS solution of long-pending 0.25% polysorbas20 of the pentaploid of pre-cooling to containing gst fusion protein
Glutathione Sepharose flushes three times, and is then flushed three times using the pentaploid product PBS of pre-cooling；
S9, digestion: the glutathione-agarose using PreScissionProtease enzyme direct enzyme cutting containing gst fusion protein
Sugared gel, digestion temperature setting are 3 DEG C, and the digestion time is set as 9h, and the product after being centrifuged digestion obtains destination protein；
S10, synthesis: bear is synthesized using 3-5 β of Alpha-hydroxy-7- oxo of above-mentioned purpose proteins carry substrate-cholanic acid 7-KLCA
Deoxycholic aicd UDCA obtains ursodeoxycholic acid crude；
S11, finished product: ursodeoxycholic acid crude is added in organic solvent, alkali is then added into organic solvent
Property substance, using dissolution, flow back, be filtered to remove solid content and acidification separation after, obtain ursodesoxycholic acid finished product.
As can be seen from the above embodiments: cultivating revolving speed in the present embodiment and be set as 220RPM, cultivation temperature is set as 33 DEG C, lures
Leading the time is set as 10h, and broken time is set as 5min, and combination temperature is set as 4 DEG C, and binding time is set as 2h, digestion temperature
Degree is set as 3 DEG C, and the digestion time is set as 9h, after actual implementation, conversion ratio 98.3%, and weight yield 98.4%.
Embodiment 3
A kind of novel 7-beta- hydroxysteroid dehydrogenase, the novel 7-beta- hydroxysteroid dehydrogenase
12nd amino acids residue and the 86th amino acids residue mutate, and the 12nd amino acids residue sported by tryptophan it is bad
Propylhomoserin, the 86th amino acids residue sport isoleucine by leucine.
The novel specific preparation method of 7-beta- hydroxysteroid dehydrogenase the following steps are included:
S1: cultivating e. coli host cell in the medium, to give expression to the novel 7-beta- hydroxy kind
Sterol dehydrogenase；
S2: by the novel 7-beta- hydroxysteroid dehydrogenase by being separated in host cell.
The e. coli host cell contains carrier, and the carrier contains polynucleotide molecule, the polynucleotides point
Son encodes novel 7-beta- hydroxysteroid dehydrogenase described in claim 1.
The present invention also provides a kind of novel 7-beta- hydroxysteroid dehydrogenases to be used for cholic acid synthetic method, tool
Body the following steps are included:
S1, recombination: using coli strain as recombinant expression carrier to the novel 7-beta- hydroxy steroid
Dehydrogenase is recombinated；
S2, culture: successful E. coli recombinant stain will be transferred to and be added in LB liquid medium according to 5% additive amount
It is cultivated, culture revolving speed is set as 260RPM, and cultivation temperature is set as 42 DEG C；
S3, induction: detection E. coli recombinant stain 600 nanometer wave strong points light absorption value, when light absorption value be 0.5 when,
E. coli recombinant stain is induced using 22 DEG C of isopropylthiogalactoside, 12h is arranged in induction time, makes large intestine
Bacillus strain generates gst fusion protein；
S4, bacterium solution are collected: centrifugally operated are carried out to the E. coli recombinant stain that induction is completed, in order to collect large intestine bar
Thallus in bacterium recombinant bacterial strain, centrifugal rotational speed are set as 12000RPM, and centrifugation time is set as 5min and collects bacterium solution；
S5, broken wall: carrying out broken wall operation to bacterium solution using 300W ultrasonication equipment, and broken time is set as 6min, until
Bacterium solution clarification；
S6, supernatant collection: carrying out centrifugally operated to the bacterium solution after clarification, and centrifugal rotational speed is set as 16000RPM, is centrifuged
Temperature setting is 7 DEG C, and centrifugation time is set as 12min, then collects the supernatant in bacterium solution；
S7, combination: by supernatant in conjunction with Glutathione Sepharose 4B, gst fusion protein is made to be incorporated into gluathione
In peptide Ago-Gel, combination temperature is set as 6 DEG C, and binding time is set as 3h；
S8, second level are rinsed: using the PBS solution of long-pending 0.25% polysorbas20 of the pentaploid of pre-cooling to containing gst fusion protein
Glutathione Sepharose flushes three times, and is then flushed three times using the pentaploid product PBS of pre-cooling；
S9, digestion: the glutathione-agarose using PreScissionProtease enzyme direct enzyme cutting containing gst fusion protein
Sugared gel, digestion temperature setting are 5 DEG C, and the digestion time is set as 12h, and the product after being centrifuged digestion obtains destination protein；
S10, synthesis: bear is synthesized using 3-5 β of Alpha-hydroxy-7- oxo of above-mentioned purpose proteins carry substrate-cholanic acid 7-KLCA
Deoxycholic aicd UDCA obtains ursodeoxycholic acid crude；
S11, finished product: ursodeoxycholic acid crude is added in organic solvent, alkali is then added into organic solvent
Property substance, using dissolution, flow back, be filtered to remove solid content and acidification separation after, obtain ursodesoxycholic acid finished product.
As can be seen from the above embodiments: cultivating revolving speed in the present embodiment and be set as 260RPM, cultivation temperature is set as 42 DEG C, lures
Leading the time is set as 12h, and broken time is set as 6min, and combination temperature is set as 6 DEG C, and binding time is set as 3h, digestion temperature
Degree is set as 5 DEG C, and the digestion time is set as 12h, after actual implementation, conversion ratio 94.2%, and weight yield 94.5%.
Following table is obtained by above-described embodiment 1-3:
As seen from the above table: be all made of in embodiment 1-3 Escherichia coli to novel 7-beta- hydroxysteroid dehydrogenase into
Then row recombination generates gst fusion protein using E. coli recombinant stain, then destination protein is made by gst fusion protein,
Ursodesoxycholic acid finally is made using destination protein catalysis substrate, compared to the prior art, in actual implementation, same cost
Under, the available more destination proteins of the present invention, and then the promotion of the production cost of cholic acid is effectively reduced, it is more applicable for work
The production of industry；Meanwhile all data all has higher conversion ratio and weight yield in embodiment 1-3, wherein to implement
Conversion ratio and weight yield in example 2 is higher.
Claims (4)
1. a kind of novel 7-beta- hydroxysteroid dehydrogenase, it is characterised in that: the novel 7-beta- hydroxy kind is solid
12nd amino acids residue of alcohol dehydrogenase and the 86th amino acids residue mutate, and the 12nd amino acids residue is by color ammonia
Acid mutation is lysine, and the 86th amino acids residue sports isoleucine by leucine.
2. the novel 7-beta- hydroxysteroid dehydrogenase of one kind according to claim 1, it is characterised in that: described new
The specific preparation method of 7-beta- hydroxysteroid dehydrogenase of type the following steps are included:
S1: cultivating e. coli host cell in the medium, to give expression to the novel 7-beta- hydroxy steroid
Dehydrogenase；
S2: by the novel 7-beta- hydroxysteroid dehydrogenase by being separated in host cell.
3. the novel 7-beta- hydroxysteroid dehydrogenase of one kind according to claim 1, it is characterised in that: described big
Enterobacteria host cell contains carrier, and the carrier contains polynucleotide molecule, and the polynucleotide molecule encodes claim 1
The novel 7-beta- hydroxysteroid dehydrogenase.
4. a kind of novel 7-beta- hydroxysteroid dehydrogenase is used for cholic acid synthetic method, which is characterized in that specifically include
Following steps:
S1, recombination: using coli strain as recombinant expression carrier to the novel 7-beta- hydroxysteroid dehydrogenase
Enzyme is recombinated；
S2, culture: will be transferred to successful E. coli recombinant stain according to 3-5% additive amount be added LB liquid medium in into
Row culture, culture revolving speed are set as 180-260RPM, and cultivation temperature is set as 25-42 DEG C；
S3, induction: detection E. coli recombinant stain uses when light absorption value is 0.5 in the light absorption value of 600 nanometer wave strong points
14-22 DEG C of isopropylthiogalactoside induces E. coli recombinant stain, and 8-12h is arranged in induction time, makes big
Enterobacteria bacterial strain generates gst fusion protein；
S4, bacterium solution are collected: centrifugally operated are carried out to the E. coli recombinant stain that induction is completed, in order to collect Escherichia coli weight
Thallus in group bacterial strain, centrifugal rotational speed are set as 10000-12000RPM, and centrifugation time is set as 2-5min and collects bacterium solution；
S5, broken wall: broken wall operation is carried out to bacterium solution using 300W ultrasonication equipment, broken time is set as 4-6min, until bacterium
Liquid clarification；
S6, supernatant collection: carrying out centrifugally operated to the bacterium solution after clarification, and centrifugal rotational speed is set as 14000-16000RPM, from
Heart temperature setting is 3-7 DEG C, and centrifugation time is set as 8-12min, then collects the supernatant in bacterium solution；
S7, combination: by supernatant in conjunction with Glutathione Sepharose 4B, gst fusion protein is made to be incorporated into glutathione fine jade
In sepharose, combination temperature is set as 2-6 DEG C, and binding time is set as 1.5-3h；
S8, second level are rinsed: using the PBS solution of long-pending 0.25% polysorbas20 of the pentaploid of pre-cooling to the paddy Guang containing gst fusion protein
Sweet peptide Ago-Gel flushes three times, and is then flushed three times using the pentaploid product PBS of pre-cooling；
S9, digestion: the glutathione agarose using PreScissionProtease enzyme direct enzyme cutting containing gst fusion protein is solidifying
Glue, digestion temperature setting are 2-5 DEG C, and the digestion time is set as 6-12h, and the product after being centrifuged digestion obtains destination protein；
S10, synthesis: using 3-5 β of Alpha-hydroxy-7- oxo of above-mentioned purpose proteins carry substrate-cholanic acid 7-KLCA synthesis bear deoxidation
Cholic acid UDCA obtains ursodeoxycholic acid crude；
S11, finished product: ursodeoxycholic acid crude is added in organic solvent, basic species are then added into organic solvent
Matter obtains ursodesoxycholic acid finished product after dissolution, flowing back, being filtered to remove solid content and acidification separation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114807070A (en) * | 2022-06-01 | 2022-07-29 | 重庆第二师范学院 | Thermophilic 7 alpha-hydroxysteroid dehydrogenase and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2327790A1 (en) * | 2009-11-30 | 2011-06-01 | Pharmazell GmbH | New 7ß-hydroxy steroid dehydrogenases and their use |
| CN102827847A (en) * | 2012-07-25 | 2012-12-19 | 上海凯宝药业股份有限公司 | Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene |
| CN108034643A (en) * | 2017-12-18 | 2018-05-15 | 重庆大学 | 7alpha-Hydroxysteroid dehydrogenase and its encoding gene and application |
-
2019
- 2019-04-10 CN CN201910284340.4A patent/CN109897835A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2327790A1 (en) * | 2009-11-30 | 2011-06-01 | Pharmazell GmbH | New 7ß-hydroxy steroid dehydrogenases and their use |
| CN102827847A (en) * | 2012-07-25 | 2012-12-19 | 上海凯宝药业股份有限公司 | Codon-optimized 7 beta-hydroxy steroid dehydrogenase gene |
| CN108034643A (en) * | 2017-12-18 | 2018-05-15 | 重庆大学 | 7alpha-Hydroxysteroid dehydrogenase and its encoding gene and application |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114807070A (en) * | 2022-06-01 | 2022-07-29 | 重庆第二师范学院 | Thermophilic 7 alpha-hydroxysteroid dehydrogenase and preparation method thereof |
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Application publication date: 20190618 |