CN102286596A - Preparation method of 15alpha hydroxylated levorotatory ethyl sterene diketone - Google Patents
Preparation method of 15alpha hydroxylated levorotatory ethyl sterene diketone Download PDFInfo
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Abstract
The invention relates to a preparation method of 15alpha hydroxylated levorotatory ethyl sterene diketone. The preparation method comprises the following steps of: performing penicillium raistrickii (ATCC 10490) fermentation; and adding a substrate levorotatory ethyl sterene diketone to perform biotransformation. The preparation method is characterized in that: a biotransformation system consists of an ionic liquid phase and a buffer liquid phase; and the volume ratio of the ionic liquid phase to the buffer liquid phase is (0.25-1.25):1. In an ionic liquid/water double-liquid phase system, hydroxylase contained in the penicillium raistrickii (ATCC 10490) thalli is used for catalyzing the 15alpha position of the levorotatory ethyl sterene diketone to generate hydroxylation reaction to generate the 15alpha hydroxyl levorotatory ethyl sterene diketone, wherein an organic solvent is not required to be added in the preparation process, so use of the organic solvent with severe damage effect on the environment is reduced, the reaction condition is mild, a transformation device is simple, sources of the raw materials are sufficient, the preparation process is simple, production cost is low, damage of environmental pollution is small, and production cost can be reduced.
Description
Technical field
The invention belongs to biocatalysis field, especially a kind of preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione.
Background technology
Along with population growth's speed is more and more faster, the excessive increase that appears as effective control population of contraceptive steroid has been brought hope, gestodene is one of main component of contraceptive bian of new generation, it combines with progesterone receptor with prototype, have the avidity of height and almost do not have male sex hormone and estrogen activity, oral absorption fast and fully, be one of at present ideal oral contraceptive, 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione of catalytic preparation are " gestodene " synthetic key intermediates among the present invention.
General steroidal class material has very strong hydrophobicity, and (solubility range is 10
-5～10
-4Mol/L), and the steroidal saccharase belongs to intracellular enzyme, and substrate only diffuses into cell and could contact with enzyme and carry out conversion reaction, and this just causes substrate can not finely to contact with saccharase, causes transformation efficiency on the low side, and fermentation time prolongs.Therefore, improving the solubility property (being effecting reaction concentration) of substrate, quicken the transmission of substrate and product molecule, is one of gordian technique that improves steroidal conversion reaction efficient.Report, organic solvent can be applied to steroidal and transform, to promote that substrate dissolves effectively as chaotropic agent, reach the purpose that improves transformation efficiency, but the organic solvent pair cell has very big toxicity, and can bring pollution to environment, and its damaging effect can not look down upon.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the biological preparation method that utilizes the left-handed ethyl steroid of Leix mould catalysis steroidal compounds alkene diketone to generate 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione in a kind of ionic liquid/water biliquid phase system is provided, and this method has that target product transformation efficiency height, process are simple, the advantage of environmental protection.
The present invention realizes that the technical scheme of purpose is as follows:
A kind of preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione, comprise that Leix mould Penicillium raistrickii (ATCC 10490) fermentation, the left-handed ethyl steroid of adding substrate alkene diketone carry out bio-transformation, it is characterized in that: the system of described bio-transformation is made of ionic liquid and buffering liquid phase, and wherein ionic liquid is 0.25～1.25: 1 with the volume ratio that cushions liquid phase mutually.
And described ionic liquid comprises [BMIM] [PF6], [HMIM] [PF6], [BMIM] [NTF2], [HMIM] [NTF], [BMPL] [NTF], [HMPL] [NTF], [BMPL] [FAP], [HMIM] [FAP], [EWTMG] [FAP], [(E2OH) MIM] [NTF], [(EOE) MMO] [NTF], [(MOP) MPI] [NTF], [(P3OH) PYR] [NTF]; Described buffering liquid phase is the phosphoric acid buffer of pH5～7.3;
And the weight percent that the left-handed ethyl steroid of described substrate alkene diketone carries out the substrate charging capacity of bio-transformation is 2～7 ‰, and temperature is 25～30 ℃, and shaking speed is 160～200r/min, and transformation time is 90～100h.
And described Leix mould Penicillium raistrickii (ATCC 10490) fermentation step is:
(1) preparation of spore suspension: Leix mould Penicillium raistrickii (ATCC 10490) is inoculated on the slant medium, after cultivating, use sterilized water, content is the Tween-80 aqueous solution of mass percent 1 ‰, and slant pore is washed, and prepares spore suspension;
(2) mycelium culture: the spore suspension that will prepare is linked in the fermention medium, and 28～32 ℃ of culture temperature, inoculum size are 10
8～10
11Individual/ml, shaking speed is 160～200r/min, and incubation time is 24～72h; Cultivate the spherical mycelium of ripe back gained and obtain the bacterium cake through suction filtration.
And the bacterium ball weight that described bacterium cake is put into the aqueous phase of ionic liquid/aqueous two phase system accounts for 5～8% of water quality.
And, described slant medium wt%: glucose 1.5, maltose 2, peptone 0.1, agar 2, pH nature; Described fermention medium wt%: glucose 3, corn steep liquor 1, NaNO
30.2, KH
2PO
40.1, MgSO
40.05, FeSO
40.002 KCl 0.05, K
2HPO
40.2 pH 7.2～7.5.
And the described bacterium cake that suction filtration is obtained is put in the buffering liquid phase, adds ionic liquid again, and the mode that feeds intake with dry powder is put into substrate in ionic liquid/damping fluid phase system and carried out microbial transformation simultaneously.
Advantage of the present invention and positively effect are as follows:
1, the inventive method is in ionic liquid/water biliquid phase system, utilize contained hydroxylase in Leix mould Penicillium raistrickii (ATCC10490) thalline, hydroxylating takes place in 15 α positions of catalyzing levorotatory ethyl steroid alkene diketone, generate the left-handed ethyl steroid of 15 α hydroxyls alkene diketone, need not to add organic solvent in the preparation process, reduced the use that environment is had the organic solvent of havoc effect, the reaction conditions gentleness, conversion system is simple, sufficient raw, preparation process is simple, and production cost is low, environmental pollution harm is little, can reduce production costs simultaneously.
2, the bright technology that 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione preparation in ionic liquid/water biliquid phase system is provided, and ionic liquid/water biliquid phase system is designed for whole-cell biological catalysis, it is low in aqueous phase solubleness to have solved substrate and product, organic solvent has problems such as toxicity for cell, under 4 ‰ charging capacity situations, its transformation efficiency reaches as high as more than 80%, operating procedure is easy, the characteristics of the times that both met environmental protection, reached the purpose that reduces production costs again, have certain industrial applications prospect, have great importance for the enforcement of China's pharmaceutical industry and family planning policy.
3, the biocatalysis preparation method of the present invention's key intermediate 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione of synthesizing steroid medicine " gestodene " in ionic liquid/water biliquid phase system, 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione are the key intermediates of synthetic " gestodene ", have great importance for the enforcement of China's pharmaceutical industry and family planning policy.
4, the used ionic liquid of the present invention is the ionic system that is in a liquid state under room temperature (or temperature of a little higher than room temperature), by the method that makes up a large amount of organic cations and a large amount of inorganic anions ion liquid kind is increased, more possible synthetic kind makes ionic liquid be called as " programmable solvent ", simultaneously, ionic liquid has electroconductibility, non-volatile, characteristics such as do not burn, the toxicity of pair cell is less even do not have, and eliminated and used potential safety hazard that volatile organic solvent brings and the pollution of environment, thereby obtained the address of " green " solvent, be widely used in the biocatalytic reaction.
Embodiment
Below in conjunction with embodiment technology contents of the present invention is described further; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The whole bag of tricks and raw material that the application is used are as follows:
1 preparation materials and methods
1.1 material
1.1.1 bacterial classification: bacterial classification source: Leix mould (Penicillium raistrickii) ATCC 10490 (available from U.S. typical case DSMZ).
1.1.2 instrument and analysis condition
Highly effective liquid phase chromatographic system: chromatographic column is the ODS-2HPERSI1C18 post: mobile phase methanol: water=80: 20; 20 ℃ of column temperatures; Detect wavelength 241nm, content calculates with the external standard peak area method.
1.2 implementation method
1.2.1 substratum
Slant medium (M1, wt%): glucose 1.5, maltose 2, peptone 0.1, agar 2, the pH nature adds above-mentioned substance in the 100g water, the quality percentage composition.
Seed culture medium (M2, wt%): glucose 3, corn steep liquor 1, NaNO
30.2, KH
2PO
40.1, MgSO
40.05, FeSO
40.002 KCl 0.05, K
2HPO
40.2 pH 7.2～7.5, add above-mentioned substance in the 100g water, the quality percentage composition.
1.2.2 culture condition
Inclined-plane: 28～32 ℃ of temperature, growth cycle 5～7d.
Hypha fermentation liquid: 28～32 ℃ of temperature, inoculum size 10
8～10
11Individual/ml, rotating speed 160～200r/min, incubation time 24～48h.
Conversion condition: 28～32 ℃ of temperature, rotating speed 160～200r/min.
1.2.3 preparation process
(1) preparation of spore suspension: Leix mould Penicillium raistrickii (ATCC 10490) is inoculated on the slant medium, after cultivating, slant pore is washed, prepare spore suspension with sterilized water (content is 1 ‰ the Tween-80 aqueous solution);
(2) mycelium culture: the spore suspension that will prepare is linked in the fermention medium, and inoculum size is 10
8～10
11Individual/ml, culture temperature is 28～32 ℃, and incubation time 24～48h cultivates the spherical mycelium of ripe back gained and obtains the bacterium cake through suction filtration;
Used biological catalyst is that incubation time is that 18～72h diameter is the spherical mycelium of Leix mould of 0.9～3.0mm; The bacterium ball weight of putting into the aqueous phase of ionic liquid/aqueous two phase system accounts for 5～8% of water quality.
(3) bio-transformation in the biliquid phase system: the bacterium cake that suction filtration is obtained is put into the aqueous phase of ionic liquid/aqueous two phase system, the mass ratio of bacterium cake and water is at least 5%, add ionic liquid again, the mode that adopts dry powder to feed intake simultaneously joins the left-handed ethyl steroid of substrate alkene diketone (granularity is 14～16 μ) in ionic liquid/water biliquid phase system carries out microbial transformation, charging capacity is 2 ‰～4 ‰, temperature is 25～30 ℃, shaking speed is 160～200r/min, and transformation time is 48～96h;
(4) determination and analysis of product yield: after substrate is transformed fully, sampling, and by its product yield of high-performance liquid chromatogram determination.
1.2.4 the method for calculation of product yield
After substrate conversion is finished, sample in the two-phase is handled respectively, wherein the liquid phase sample preparation method of aqueous phase is: the conversion fluid 500 μ l that get aqueous phase respectively are in centrifuge tube, add the equal-volume ethyl acetate, extraction 30min, centrifugal 5min under the 12000r/min gets supernatant 100 μ l and volatilizes ethyl acetate, add the 1ml dissolve with methanol, again by the content of high effective liquid chromatography for measuring purpose product at aqueous phase; The liquid phase sample preparation method of ionic liquid in mutually is: after substrate conversion is finished, get the conversion fluid 100 μ Ls of ionic liquid in mutually, add 900 μ L methyl alcohol, directly dilution detects by HPLC and gets final product.
The calculation formula of the purpose product amount in the whole system is: the middle mutually purpose product observed value (g) of the product observed value (g) of catalogue=aqueous phase purpose product observed value (g)+ionic liquid.
The calculation formula of the left-handed ethyl steroid of product 15 α hydroxyls alkene diketone productive rate is: the product observed value (g) of productive rate (%)=catalogue/purpose product theoretical value (g) * 100%.
Adopt highly effective liquid phase chromatographic system, chromatographic column is an ODS-2HPERSIL C18 post, and moving phase is methyl alcohol: water=80: 20, column temperature are 20 ℃, and the detection wavelength is 241nm, and content calculates with the external standard peak area method.
The concrete exemplifying embodiment that transforms
The used ionic liquid of this patent is water-insoluble ionic liquid, can form immiscible two-phase with water.
Embodiment 1
A kind of preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione, it is that 5.8 phosphoric acid buffer is as water that present embodiment adopts pH, ionic liquid is chosen ionic liquid 1-butyl-3 Methylimidazole hexafluorophosphate mutually, i.e. [BMIM] [PF6], V (ionic liquid phase): V (water)=0.25: 1, the total liquid amount of biliquid phase system is 30ml, and step is:
(1) preparation of spore suspension: Leix mould Penicillium raistrickii (ATCC 10490) is inoculated on the slant medium, after cultivating, slant pore is washed, prepare spore suspension with sterilized water (content is the Tween-80 aqueous solution of mass percent 1 ‰);
(2) mycelium culture: the spore suspension that will prepare is linked in the fermention medium, and inoculum size is 10
9Individual/ml, culture temperature is 28 ℃, and incubation time 36h cultivates the spherical mycelium of ripe back gained and obtains the bacterium cake through suction filtration;
(3) bio-transformation in the biliquid phase system: the bacterium cake that suction filtration is obtained is put into the aqueous phase of ionic liquid/aqueous two phase system, and the bacterium cake of input and the mass ratio of water are at least 5%; Add ionic liquid again, the mode that adopts dry powder to feed intake simultaneously joins the left-handed ethyl steroid of substrate alkene diketone (granularity is 14～16 μ) in ionic liquid/water biliquid phase system carries out microbial transformation, the weight percent of charging capacity is 3 ‰, temperature is 28 ℃, shaking speed is 180r/min, and transformation time is 96h;
(4) determination and analysis of product yield: after substrate conversion is finished, sample in the two-phase is handled respectively, wherein the liquid phase sample preparation method of aqueous phase is: the conversion fluid 500 μ l that get aqueous phase respectively are in centrifuge tube, add the equal-volume ethyl acetate, extraction 30min, centrifugal 5min under the 12000r/min gets supernatant 100 μ l and volatilizes ethyl acetate, add the 1ml dissolve with methanol, again by the content of high effective liquid chromatography for measuring purpose product at aqueous phase; The liquid phase sample preparation method of ionic liquid in mutually is: after substrate conversion is finished, get the conversion fluid 100 μ Ls of ionic liquid in mutually, add 900 μ L methyl alcohol, directly dilution detects and gets final product, and detects by the HPLC method.
The calculation formula of the purpose product amount in the whole system is: the middle mutually purpose product observed value (g) of the product observed value (g) of catalogue=aqueous phase purpose product observed value (g)+ionic liquid.
The calculation formula of the left-handed ethyl steroid of product 15 α hydroxyls alkene diketone productive rate is: the product observed value (g) of productive rate (%)=catalogue/purpose product theoretical value (g) * 100%.
Assay determination transformation efficiency, transformation efficiency are 82%.
Embodiment 2
A kind of preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione, it is that 5.8 phosphoric acid buffer is as water that present embodiment adopts pH, ionic liquid is chosen ionic liquid 1-hexyl-3-Methylimidazole hexafluorophosphate mutually, i.e. [HMIM] [PF6], V (ionic liquid phase): V (water)=0.5: 1, the total liquid amount of biliquid phase system is 30ml, and step is:
(1) preparation of spore suspension: Leix mould Penicillium raistrickii (ATCC 10490) is inoculated on the slant medium, after cultivating, slant pore is washed, prepare spore suspension with sterilized water (content is the Tween-80 aqueous solution of 1 ‰ (m/m));
(2) mycelium culture: the spore suspension that will prepare is linked in the fermention medium, and inoculum size is 10
9Individual/ml, culture temperature is 28 ℃, and incubation time 36h cultivates the spherical mycelium of ripe back gained and obtains the bacterium cake through suction filtration;
(3) bio-transformation in the biliquid phase system: the bacterium cake that suction filtration is obtained is put into the aqueous phase of ionic liquid/aqueous two phase system, add ionic liquid again, the mode that adopts dry powder to feed intake simultaneously joins the left-handed ethyl steroid of substrate alkene diketone (granularity is 14～16 μ) in ionic liquid/water biliquid phase system carries out microbial transformation, charging capacity is 4 ‰ (m/m), temperature is 28 ℃, shaking speed is 180r/min, and transformation time is 72h;
(4) determination and analysis of product yield: after substrate conversion is finished, sample in the two-phase is handled respectively, wherein the liquid phase sample preparation method of aqueous phase is: the conversion fluid 500 μ l that get aqueous phase respectively are in centrifuge tube, add the equal-volume ethyl acetate, extraction 30min, centrifugal 5min under the 12000r/min gets supernatant 100 μ l and volatilizes ethyl acetate, add the 1ml dissolve with methanol, again by the content of high effective liquid chromatography for measuring purpose product at aqueous phase; The liquid phase sample preparation method of ionic liquid in mutually is: after substrate conversion is finished, get the conversion fluid 100 μ Ls of ionic liquid in mutually, add 900 μ L methyl alcohol, directly dilution detects and gets final product, and detects by the HPLC method.
The calculation formula of the purpose product amount in the whole system is: the middle mutually purpose product observed value (g) of the product observed value (g) of catalogue=aqueous phase purpose product observed value (g)+ionic liquid.
The calculation formula of the left-handed ethyl steroid of product 15 α hydroxyls alkene diketone productive rate is: the product observed value (g) of productive rate (%)=catalogue/purpose product theoretical value (g) * 100%.
Assay determination transformation efficiency, transformation efficiency are 84%.
Embodiment 3
The method of catalytic preparation 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione in a kind of ionic liquid/aqueous two phase system, it is that 5.8 phosphoric acid buffer is as water that present embodiment adopts pH, ionic liquid is chosen the two trifluoromethane sulfimide salt of ionic liquid 1-butyl-3-Methylimidazole mutually, i.e. [BMIM] [NTF], V (ionic liquid phase): V (water)=0.5: 1, the total liquid amount of biliquid phase system is 50ml, and step is:
(1) preparation of spore suspension: Leix mould Penicillium raistrickii (ATCC 10490) is inoculated on the slant medium, after cultivating, slant pore is washed, prepare spore suspension with sterilized water (content is 1 ‰ the Tween-80 aqueous solution);
(2) mycelium culture: the spore suspension that will prepare is linked in the fermention medium, and inoculum size is 10
9Individual/ml, culture temperature is 28 ℃, and incubation time 36h cultivates the spherical mycelium of ripe back gained and obtains the bacterium cake through suction filtration;
(3) bio-transformation in the biliquid phase system: the bacterium cake that suction filtration is obtained is put into the aqueous phase of ionic liquid/aqueous two phase system, add ionic liquid again, the mode that adopts dry powder to feed intake simultaneously joins the left-handed ethyl steroid of substrate alkene diketone (granularity is 14～16 μ) in ionic liquid/water biliquid phase system carries out microbial transformation, charging capacity is 3 ‰, temperature is 28 ℃, shaking speed is 180r/min, and transformation time is 72h;
(4) determination and analysis of product yield: after substrate conversion is finished, sample in the two-phase is handled respectively, wherein the liquid phase sample preparation method of aqueous phase is: the conversion fluid 500 μ l that get aqueous phase respectively are in centrifuge tube, add the equal-volume ethyl acetate, extraction 30min, centrifugal 5min under the 12000r/min gets supernatant 100 μ l and volatilizes ethyl acetate, add the 1ml dissolve with methanol, again by the content of high effective liquid chromatography for measuring purpose product at aqueous phase; The liquid phase sample preparation method of ionic liquid in mutually is: after substrate conversion is finished, get the conversion fluid 100 μ Ls of ionic liquid in mutually, add 900 μ L methyl alcohol, directly dilution detects and gets final product, and detects by the HPLC method.
The calculation formula of the purpose product amount in the whole system is: the middle mutually purpose product observed value (g) of the product observed value (g) of catalogue=aqueous phase purpose product observed value (g)+ionic liquid.
The calculation formula of the left-handed ethyl steroid of product 15 α hydroxyls alkene diketone productive rate is: the product observed value (g) of productive rate (%)=catalogue/purpose product theoretical value (g) * 100%.
Assay determination transformation efficiency, transformation efficiency are 92%.
Claims (7)
1. the preparation method of an alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione, comprise that Leix mould Penicillium raistrickii (ATCC 10490) fermentation, the left-handed ethyl steroid of adding substrate alkene diketone carry out bio-transformation, it is characterized in that: the system of described bio-transformation is made of ionic liquid and buffering liquid phase, and wherein ionic liquid is 0.25～1.25: 1 with the volume ratio that cushions liquid phase mutually.
2. the preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione according to claim 1 is characterized in that: described ionic liquid comprises [BMIM] [PF6], [HMIM] [PF6], [BMIM] [NTF2], [HMIM] [NTF], [BMPL] [NTF], [HMPL] [NTF], [BMPL] [FAP], [HMIM] [FAP], [EWTMG] [FAP], [(E2OH) MIM] [NTF], [(EOE) MMO] [NTF], [(MOP) MPI] [NTF], [(P3OH) PYR] [NTF]; Described buffering liquid phase is the phosphoric acid buffer of pH5～7.3.
3. the preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione according to claim 1, it is characterized in that: the weight percent that the left-handed ethyl steroid of described substrate alkene diketone carries out the substrate charging capacity of bio-transformation is 2～7 ‰, temperature is 25～30 ℃, shaking speed is 160～200r/min, and transformation time is 90～100h.
4. the preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione according to claim 1 is characterized in that: described Leix mould Penicillium raistrickii (ATCC 10490) fermentation step is:
(1) preparation of spore suspension: Leix mould Penicillium raistrickii (ATCC 10490) is inoculated on the slant medium, after cultivating, use sterilized water, content is the Tween-80 aqueous solution of mass percent 1 ‰, and slant pore is washed, and prepares spore suspension;
(2) mycelium culture: the spore suspension that will prepare is linked in the fermention medium, and 28～32 ℃ of culture temperature, inoculum size are 10
8～10
11Individual/ml, shaking speed is 160～200r/min, and incubation time is 24～72h; Cultivate the spherical mycelium of ripe back gained and obtain the bacterium cake through suction filtration.
5. the preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione according to claim 4 is characterized in that: the bacterium ball weight that described bacterium cake is put into the aqueous phase of ionic liquid/aqueous two phase system accounts for 5～8% of water quality.
6. the preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione according to claim 4 is characterized in that: described slant medium wt%: glucose 1.5, maltose 2, peptone 0.1, agar 2, pH nature; Described fermention medium wt%: glucose 3, corn steep liquor 1, NaNO
30.2, KH
2PO
40.1, MgSO
40.05, FeSO
40.002 KCl 0.05, K
2HPO
40.2 pH 7.2～7.5.
7. the preparation method of 15 alpha-Hydroxylation of 13-ethyl-estr-4-ene-3,17-dione according to claim 4, it is characterized in that: the described bacterium cake that suction filtration is obtained is put in the buffering liquid phase, add ionic liquid again, the mode that feeds intake with dry powder is put into substrate in ionic liquid/damping fluid phase system and is carried out microbial transformation simultaneously.
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| CN111454855A (en) * | 2020-05-13 | 2020-07-28 | 天津科技大学 | Recombinant pichia pastoris, construction method thereof and application thereof in efficient preparation of 15 α -levo-ethyl sterenedione |
| CN112980910A (en) * | 2021-03-19 | 2021-06-18 | 上海应用技术大学 | Method for microbial transformation of 11 alpha-hydroxy canrenone in phase transfer catalyst system |
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| CN103834712A (en) * | 2012-11-26 | 2014-06-04 | 复旦大学 | Optimization method for biotransformation of desogestrel intermediate nano-liposome |
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| CN111454855B (en) * | 2020-05-13 | 2022-10-14 | 天津科技大学 | Recombinant pichia pastoris, construction method thereof and application thereof in efficient preparation of 15 alpha-levo-ethyl sterene dione |
| CN112980910A (en) * | 2021-03-19 | 2021-06-18 | 上海应用技术大学 | Method for microbial transformation of 11 alpha-hydroxy canrenone in phase transfer catalyst system |
| CN112980910B (en) * | 2021-03-19 | 2024-01-05 | 上海应用技术大学 | Method for microbial conversion of 11 alpha-hydroxy canrenone in phase transfer catalyst system |
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