CN108823275A - A method of phytosterol bio-conversion prepares androstenedione in system containing quaternary ammonium salt ionic liquid - Google Patents

A method of phytosterol bio-conversion prepares androstenedione in system containing quaternary ammonium salt ionic liquid Download PDF

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CN108823275A
CN108823275A CN201810622695.5A CN201810622695A CN108823275A CN 108823275 A CN108823275 A CN 108823275A CN 201810622695 A CN201810622695 A CN 201810622695A CN 108823275 A CN108823275 A CN 108823275A
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关怡新
袁俊杰
肖霞
肖超
姚善泾
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Zhejiang University ZJU
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Abstract

The invention discloses the methods that phytosterol Whole Cell Bioconversion in a kind of system containing quaternary ammonium salt ionic liquid prepares androstenedione (AD).Mycobacteria Mycobacterium sp.NRRL B-3683 strain is subjected to seed liquor culture, seed liquor is inoculated into fermentation medium again and is cultivated to prepare resting cell, gained resting cell activation culture 7h in the Tris-HCl buffer containing glucose, then hydrophily lactic acid/carboxylic acid quaternary ammonium salt ionic liquid of substrate phytosterol and 0.1~1vol.% are put into as cosolvent, bioconversion reaction is carried out, AD is prepared.Quaternary ammonium salt ionic liquid used in the present invention is up to 8~565g L to the solubility of substrate phytosterol‑1, it is much higher than common commercial ionic liquid, substrate and product dissolution can be effectively facilitated, improve process transformation efficiency, and ionic liquid materials are few, is conducive to save production cost.

Description

Phytosterol bio-conversion prepares androstene two in one kind system containing quaternary ammonium salt ionic liquid The method of ketone
Technical field
The present invention relates to phytosterol Whole Cell Bioconversions in a kind of system containing quaternary ammonium salt ionic liquid to prepare androstene two The method of ketone.
Background technique
4-AD (AD) is important Steroid medicine intermediates, can be used for synthesizing hundreds of steroidal and swashs Plain class drug.Using phytosterol abundant in nature as substrate, before preparing AD with wide application by biotransformation method Scape.However, mycobacteria degrading plant sterol side chain obtain AD need to the 16 steps reaction by 11 kinds of function enzyme system concerted catalysis go through Journey can only be completed in full cell so far.There is two main problems：(1) low aqueous solubility of sterol substrates；(2) it dredges Aqueous product AD is after the inhibition intracellular to cell activity even toxicity, and precipitation to the inclusion of substrate particle and cell.It is logical Often, based on solvent engineering strategy addition cosolvent (cosolvent), substrate and product can be promoted to dissolve, improves process conversion effect Rate.
As the requirement that people develop green processes is more more and more urgent, a kind of novel green solvent --- ionic liquid The visual field of people is progressed into its unique physicochemical property.Ionic liquid (ionic liquid, IL) is by cation and yin The organic salt being in a liquid state under critical-temperature (generally 100 DEG C) that ion is constituted.Currently used ionic liquid is mainly by 9 Cationoid (alkyl imidazole, alkyl pyridine class, quaternary ammonium, quaternary phosphine, pyrrolidines, piperidines, morpholine, guanidine etc.) and 28 anionoids (halogen, hexafluorophosphoric acid type, tetrafluoro boric acid type, nitric acid type, sulfonic acid type, carboxylic acid type, imine etc.) is constituted, and is mainly used for organic The fields such as synthesis and catalysis, extraction and separation, electrochemistry, functional material, the ionic liquid of poor biocompatibility is directly used as giving birth to The problems such as object catalysis reaction medium can bring low catalytic efficiency, the wasting of resources, eco-toxicity.Therefore, for biocatalytic reaction The ionic liquid of process screening and synthesis specific structure is to improve one of process efficiency, the approach for realizing green catalysis.
History of the ionic liquid for Whole Cell Biocatalysis process can trace back to (Biotechnology in 2000 and Bioengineering.2000,69:227-233).But application of the ionic liquid in steroid drugs bioconversion is also It is less.(the Biotechnology and Bioprocess Engineering.2011,16 such as Wang in 2011:852-857) will 2% hydrophily lactate ions liquid [BMIM] [Lac] is added in toluene/buffer two-phase system as cosolvent, uses In the dehydrogenation reaction of 11 beta-hydroxy Medroxyprogesterone of Arthrobacter simplex whole-cell catalytic, so that conversion ratio is from original 56.4% is increased to 83.2%.2012, author (Applied Biochemistry and Biotechnology.2012, 167:It 2131-2143) is catalyzed -16 Beta-methyl of 17 Alpha-hydroxy-pregnant steroid -4 in Arthrobacter simplex immobilized cell again, 9 (11)-diene -3,20- diketone C1The hydrophilic ionic-liquid [EMIM] [Lac] of addition 0.3% in the dehydrogenation reaction of position, will turn Rate is increased to 93.4%.Recently, (the Journal of Chemical Technology and such as Shen Biotechnology.2018,93:426-431) also reporting can in Arthrobacter simplex whole-cell catalytic acetic acid Loose C1[PrMIM] [BF of addition 0.5% in the dehydrogenation reaction of position4]、[PrMIM][PF6] plasma liquid as cosolvent, and Various ionic liquids are had studied in detail to growth of microbial cells, cell activity, permeability of cell membrane, cell composed structure and are urged The influence for changing transformation efficiency etc., has understanding more profound to whole-cell catalytic conversion process in the presence of ionic liquid.But this A little researchs all concentrate on the C of steroid drugs1In the dehydrogenation reaction of position, it yet there are no and be used successfully to sterol for ionic liquid as cosolvent The report of Side chain cleavage reaction.In view of commercialization ionic liquid to sterol dissolubility difference and to cell biological compatibility still not Enough ideal situation (ACS Sustainable Chemistry&Engineering.2017,5:10702-10709), of the invention The acid and full cell of the hydrophobicity and hydrogen bond of marisone is as this two major features of catalyst, with good biocompatibility, hydrogen bond alkali Property strong biomolecule lactic acid/carboxylic acid be anionic donor, biocompatibility and the preferable quaternary ammonium salt of solubility property be sun from Sub- donor synthesizes ionic liquid, as the cosolvent for promoting substrate and product dissolution, to improve phytosterol whole-cell biological Conversion process efficiency.
Summary of the invention
The object of the present invention is to provide phytosterol Whole Cell Bioconversion systems in a kind of system containing quaternary ammonium salt ionic liquid The method of standby AD.
Provide a kind of side that AD is prepared containing mycobacteria resting cell degrading plant sterol side chain in ion liquid system Method includes the following steps：
(a) it with mycobacteria Mycobacterium sp.NRRL B-3683 (bacterium numbering ATCC 29472) for strain, carries out Seed liquor culture；Wherein strain is commercially available from https://www.atcc.org/Search_Results.aspx？DsNav= Ntk:PrimarySearch%7c29472%7c3%7c, Ny:True,Ro:0,N:1000552&searchTerms= 29472&redir=1.The strain is also disclosed report in CN1507489A.
(b) seed liquor obtained by step (a) is inoculated into fermentation medium and is cultivated, prepare resting cell, freezen protective is spare；
(c) it by resting cell 2~12h of activation culture in the Tris-HCl buffer containing glucose obtained by step (b), then throws Enter 1~30g L-1The hydrophily of substrate phytosterol and 0.1~1vol.% lactic acid/carboxylic acid quaternary ammonium salt ionic liquid are as molten altogether Agent carries out bioconversion reaction, prepares AD.
Preferably, bead is added in the seed culture medium with cell dispersion, seed liquor cultivation temperature is 28 DEG C, shaking table Revolving speed is 180rpm, and incubation time is 24~48h.
Preferably, the fermented and cultured is in the 500mL conical flask that bottom is machined with 2 sizes about 15 × 8 × 2mm baffle It carries out, 1~2g L is added in fermentation medium-1The phytosterol for using 80 aqueous solution of tween pre-dispersed is inoculated with as inducer Amount is 8~12vol.%, and fermented and cultured temperature is 30 DEG C, shaking speed 200rpm, and incubation time is 48~60h.Fermentation training After the completion of supporting, thalline were collected by centrifugation, is washed with the phosphate buffer of pH 7.0 to get the resting cell.
Preferably, the resting cell concentration is 50g L-1, concentration of glucose is 2g L-1, Tris-HCl buffer is pH 7.5,0.1M, the phytosterol is white powder, and main component is cupreol (45%), stigmasterol (26.2%), rape oil Sterol (23.5%) and brassicasterol (3.2%), purity >=95%.
Preferably, the cation of the hydrophilic quaternary ammonium salt ionic liquid is tetrabutylammonium (N4,4,4,4 +) or choline (Ch+), Anion is lactate or the carboxylate radical containing 8 carbon.Synthesis step is：By tetrabutylammonium hydroxide or choline and equimolar amounts Lactic acid/carboxylic acid mixing is stirred to react at 45 DEG C to complete neutralization, then by heating stirring and with air in round-bottomed flask Purging is until constant weight obtains ionic liquid to remove solvent.
Preferably, the bioconversion reaction carries out in the conical flask that bottom is machined with 2 baffles, reaction temperature 30 DEG C, shaking speed 200rpm.
Advantage of the invention is that：Hydrophily lactic acid/carboxylic acid quaternary ammonium salt ionic liquid as cosolvent is to substrate plant The solubility of sterol is up to 8~565g L-1, it is much higher than common commercial ionic liquid；Its low concentration aqueous solution is to substrate and production The solvability of object is also superior to the system that organic solvent participates in, and viscosity is no more than 1.6cP, and cell and substrate dispersion situation are good Good, cell membrane integrity is hardly influenced by ionic liquid.Therefore, these ionic liquids can effectively facilitate bottom as cosolvent Object and product dissolution, improve process transformation efficiency, and ionic liquid materials are few, are conducive to save production cost.
Detailed description of the invention
Fig. 1 is ionic liquid tetrabutylammonium the caprylate ([N of synthesis4,4,4,4][C7H15COO])1H NMR spectra；
Fig. 2 is ionic liquid tetrabutylammonium the caprylate ([N of synthesis4,4,4,4][C7H15COO])13C NMR(500MHz, DMSO-d6) spectrogram.
Specific embodiment
Specific embodiment below expands detailed description to the present invention, but the present invention is not restricted to following implementation Example.
Embodiment 1：Tetrabutylammonium octanoic acid ionic liquid ([N4,4,4,4][C7H15COO]) in-buffer cosolvent system Phytosterol bio-conversion prepares AD, specific as follows：
Step 1：Prepare ionic liquid [N4,4,4,4][C7H15COO].Specifically, by tetrabutylammonium hydroxide ([N4,4,4,4] [OH]) the octanoic acid of 25wt.% aqueous solution and equimolar amounts mix, 12h is stirred to react at 45 DEG C in round-bottomed flask to complete It neutralizes, then by heating stirring and with air purging until constant weight, to remove solvent, obtains transparent liquid at room temperature Ionic liquid [N4,4,4,4][C7H15COO]。
Use nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13[the N of C NMR characterization synthesis4,4,4,4][C7H15COO]。1H NMRδ/ppm (500MHz,DMSO-d6):0.84-0.87(3H,t；COO-C6-CH3),0.92-0.95(12H,t；N-C3-CH3),1.14-1.37 (18H,m；N-C2-CH2and COO-C-(CH2)5),1.54-1.60(8H,m；N-C-CH2),1.70-1.73(2H,t；COO- CH2),3.16-3.19(8H,m；N-CH2).13C NMRδ/ppm(500MHz,DMSO-d6):13.46(s；N-C4),13.95(s； COO-C8),19.18(s；N-C3),22.13(s；COO-C7),23.06(s；N-C2),26.80(s；COO-C6),28.90(s； COO-C5),29.58(s；COO-C4),31.41(s；COO-C3),39.17(s；COO-C2),57.45-57.50(t；N-C1), 174.83(s；COO-C1).Spectrogram is as illustrated in fig. 1 and 2.Analysis shows the ionic liquid structure of synthesis is errorless and with higher Purity.
Step 2：Seed liquor culture.Aseptically, by test tube slant mycobacteria Mycobacterium Sp.NRRL B-3683 strain inoculated is into the 250mL conical flask equipped with 50mL seed culture medium and 15 beades, in constant temperature 28 DEG C in shaken cultivation case, 48h is cultivated under the conditions of 180rpm.
The seed culture based formulas is：Glucose 5g L-1, beef extract 3g L-1, tryptone 5g L-1, NaCl 15g L-1, glycerol 1.5vol.%, pH 7.0；High pressure steam sterilization 30min at 115 DEG C.
Step 3：Fermented and cultured prepares resting cell.The seed liquor cultivated 2 days is transferred to the inoculum concentration of 12vol.% In 500mL tool baffle conical flask equipped with 100mL fermentation medium.Wherein, fermentation medium adds 1g L before sterilization-1With The pre-dispersed phytosterol of 80 aqueous solution of tween is to induce mycobacteria producing enzyme (to control 80 concentration of tween in final culture medium 0.05%), to be placed in constant-temperature shaking incubator, at 30 DEG C, cultivate under the conditions of 200rpm to mycobacterial cells logarithmic growth In latter stage (60h), cell concentration and active enzyme amount higher and intracellular are abundant at this time.It is centrifuged (4 DEG C, 8000rpm, 2min) collection bacterium Body, and washed 3 times with phosphate buffer, wet cell (i.e. resting cell, every gram of wet cell is containing about stem cell weight 0.2g) is obtained, It is spare in -20 DEG C of stored frozens.
The fermentative medium formula is：Glucose 8g L-1, citric acid 2g L-1, ferric citrate 0.05g L-1, MgSO4 0.5g L-1, K2HPO4 0.5g L-1, (NH4)2HPO4 6g L-1, pH 7.0；High pressure steam sterilization 30min at 115 DEG C.
Step 4：Phytosterol bio-conversion prepares AD in ionic liquid-buffer cosolvent system.Point of aerobic environment Branch bacillus resting cell catalysis phytosterol Side chain cleavage process carries out in 100mL tool baffle conical flask.Take out culture in advance And in the mycobacteria Mycobacterium sp.NRRL B-3683 resting cell of -20 DEG C of stored frozens as biocatalysis Agent.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g L-1Glucose is added to pH In 7.5 Tris-HCl buffer, 7h is activated under the conditions of 30 DEG C and 200rpm.3g L is added into system-1Phytosterol and Ionic liquid [the N of 0.1vol.%4,4,4,4][C7H15COO] it is used as cosolvent, bioconversion reaction is carried out, not add ion The buffer solution system of liquid is as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD space-time yield (conversion speed Rate) it is 116mg L-1h-1, much higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 2：Tetrabutylammonium octanoic acid ionic liquid ([N4,4,4,4][C7H15COO]) in-buffer cosolvent system Phytosterol bio-conversion prepares AD, specific as follows：Ionic liquid [N is prepared as described in Example 14,4,4,4][C7H15COO], And carry out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13The characterization of C NMR.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system Add 3g L-1Ionic liquid [the N of phytosterol and 0.4vol.%4,4,4,4][C7H15COO] it is used as cosolvent, carry out bioconversion Reaction, not add the buffer solution system of ionic liquid as compareing.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD Space-time yield (conversion rate) is 65mg L-1h-1, higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 3：Tetrabutylammonium lactate ions liquid ([N4,4,4,4] [Lac]) plant in-buffer cosolvent system Sterol bioconversion prepares AD, specific as follows：
Ionic liquid [N is prepared as described in Example 14,4,4,4] [Lac], and carry out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13C The characterization of NMR.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system Add 3g L-1Ionic liquid [the N of phytosterol and 0.4vol.%4,4,4,4] [Lac] be used as cosolvent, carry out bioconversion reaction, Not add the buffer solution system of ionic liquid as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD space-time Yield (conversion rate) is 72mg L-1h-1, higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 4：Tetrabutylammonium lactate ions liquid ([N4,4,4,4] [Lac]) plant in-buffer cosolvent system Sterol bioconversion prepares AD, specific as follows：
Ionic liquid [N is prepared as described in Example 14,4,4,4] [Lac], and carry out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13C The characterization of NMR.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system Add 3g L-1Ionic liquid [the N of phytosterol and 1vol.%4,4,4,4] [Lac] be used as cosolvent, carry out bioconversion reaction, with The buffer solution system of ionic liquid is not added as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate the production of AD space-time Rate (conversion rate) is 86mg L-1h-1, much higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 5：Phytosterol bio in choline lactate ions liquid ([Ch] [Lac])-buffer cosolvent system Conversion prepares AD, specific as follows：
Ionic liquid [Ch] [Lac] is prepared as described in Example 1, and carries out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13C NMR Characterization.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system Add 3g L-1The ionic liquid [Ch] [Lac] of phytosterol and 0.1vol.% are used as cosolvent, bioconversion reaction are carried out, with not The buffer solution system of ionic liquid is added as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD space-time yield (conversion rate) is 109mg L-1h-1, much higher than AD yield (the 54mg L in control buffer solution system-1h-1)。
Embodiment 6：Phytosterol bio in choline lactate ions liquid ([Ch] [Lac])-buffer cosolvent system Conversion prepares AD, specific as follows：
Ionic liquid [Ch] [Lac] is prepared as described in Example 1, and carries out nucleus magnetic hydrogen spectrum1H NMR and carbon spectrum13C NMR Characterization.
It carries out seed liquor culture as described in Example 1 and fermented and cultured prepares resting cell.
The mycobacteria resting cell catalysis phytosterol Side chain cleavage process of aerobic environment has baffle conical flask in 100mL Middle progress.Taking-up culture in advance is simultaneously quiet in the mycobacteria Mycobacterium sp.NRRL B-3683 of -20 DEG C of stored frozens Cell is ceased as biocatalyst.To improve catalytic rate, before carrying out bioconversion reaction, first by 50g L-1Wet cell and 2g L-1Glucose is added in the Tris-HCl buffer of pH 7.5, activates 7h under the conditions of 30 DEG C and 200rpm.Add into system Add 3g L-1The ionic liquid [Ch] [Lac] of phytosterol and 0.4vol.% are used as cosolvent, bioconversion reaction are carried out, with not The buffer solution system of ionic liquid is added as control.It is spaced 2h sampling and measures AD concentration with HPLC, calculate AD space-time yield (conversion rate) is 91mg L-1h-1, higher than AD yield (the 54mg L in control buffer solution system-1h-1)。

Claims (10)

1. a kind of method that phytosterol Whole Cell Bioconversion prepares androstenedione (AD) in system containing quaternary ammonium salt ionic liquid, Characterized by the following steps：
(a) it with mycobacteria Mycobacterium sp.NRRL B-3683 (bacterium numbering ATCC 29472) for strain, carries out Seed liquor culture；
(b) seed liquor obtained by step (a) is inoculated into fermentation medium and is cultivated, prepare resting cell, freezen protective is spare；
(c) it by resting cell 2~12h of activation culture in the Tris-HCl buffer containing glucose obtained by step (b), then throws Enter 1~30g L-1Substrate phytosterol uses hydrophily lactic acid/carboxylic acid quaternary ammonium salt ionic liquid of 0.1~1vol.% as altogether Solvent carries out bioconversion reaction, prepares AD.
2. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist Bead is added in seed culture medium in step (a) with cell dispersion.
3. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist Seed liquor cultivation temperature is 28 DEG C, shaking speed 180rpm in step (a), and incubation time is 24~48h.
4. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist 1~2g L is added in fermentation medium in step (b)-1The phytosterol for using 80 aqueous solution of tween pre-dispersed as induction Agent.
5. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist Bioconversion reaction is machined with 2 size 15 × 8 × 2mm baffles in bottom in fermented and cultured and step (c) in step (b) 500mL conical flask in carry out.
6. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist Seed liquor inoculum concentration is 8~12vol.% in step (b), and fermented and cultured temperature is 30 DEG C, shaking speed 200rpm, culture Time is 48~60h.
7. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist In step (b) after the completion of fermented and cultured, thalline were collected by centrifugation, is washed with the phosphate buffer of pH 7.0 to get described quiet Cease cell.
8. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist Resting cell concentration is 50g L in step (c)-1, concentration of glucose is 2g L-1, Tris-HCl buffer be pH 7.5, 0.1M。
9. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist The cation of hydrophilic quaternary ammonium salt ionic liquid is tetrabutylammonium or choline in step (c), and anion is for lactate or containing 8 The carboxylate radical of carbon.
10. the method that phytosterol Whole Cell Bioconversion prepares AD in cosolvent system as described in claim 1, feature exist Bioconversion reaction temperature is 30 DEG C in step (c), shaking speed 200rpm.
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Publication number Priority date Publication date Assignee Title
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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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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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