CN104357526A - Method for preparing cholestenone by using eutectic mixture as solubilization promoting agent through resting cell transformation - Google Patents
Method for preparing cholestenone by using eutectic mixture as solubilization promoting agent through resting cell transformation Download PDFInfo
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- CN104357526A CN104357526A CN201410522163.6A CN201410522163A CN104357526A CN 104357526 A CN104357526 A CN 104357526A CN 201410522163 A CN201410522163 A CN 201410522163A CN 104357526 A CN104357526 A CN 104357526A
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Abstract
The invention relates to a method for preparing cholestenone by using a eutectic mixture as a solubilization promoting agent through resting cell transformation. The method comprises the steps of (1) performing centrifugation after resting cells are cultivated in a fermentation medium, and adding a sterile phosphate buffer solution for re-suspension so as to be used as transformation liquid, wherein the resting cells have the capacity of preparing cholestenone by oxidizing cholesterol; (2) feeding cholesterol into the transformation liquid, adding the eutectic mixture serving as the solubilization promoting agent, extracting the transformation liquid by ethyl acetate, and detecting the yield of a product 4-cholesten-3-one by an HPLC (high performance liquid chromatography) method; (3) separating the substrate cholesterol from the product cholestenone through a column chromatography separation method, and purifying to obtain a pure cholestenone product. According to the method, the eutectic mixture (DES) replaces an organic solvent such as methyl alcohol and serves as a green additive; after the eutectic mixture is added, the dispersion rate of the substrate is increased, contact of the substrate cholesterol and bacteria is effectively promoted, and the transformation efficiency of the resting cells is effectively improved; in adding and transforming processes, the operation conditions are mild, and the steps are simple.
Description
Technical field
The invention belongs to microbiological transformation technology field, especially a kind of conversion of resting cells that utilizes using eutectic mixture as chaotropic agent prepares the method for cholestenone.
Technical background
Courage Gona-4-en-3-one (4-Cholesten-3-one), has another name called cholestenone, and molecular formula is C
27h
44o, being one of important products of obtaining through the means such as chemical synthesis, microbe transformation method of cholesterol, is the oxidation products of cholesterol.Cholestenone can be used for treating hepatopathy, as fat-reducing promotor, can have effects such as preventing skin keratin.In addition, the ad hoc structure of cholestenone as the intermediate of steroid drugs, can be widely used in pharmaceutical industries.
Once the reaping hook spore bacterium having bibliographical information to screen from soil sample, optimizes substratum and the fermentation condition of strain fermentation process, cholesterol is changed into cholestenone.Also having patent CN1328133A to report adopts a kind of tyrothricin (Brevibacterium sp.) mutant strain DGCDC-82 to be bacterium producing multi enzyme preparation, using glucose and yeast extract paste as most suitable carbon source and nitrogenous source, using ammonium acetate as product enzyme promotor, using cholesterol as product enzyme inducer and thalli growth nutritional factor, biosynthesizing born of the same parents external form rCO, and then obtain courage Gona-4-en-3-one by fermentable cultivation.
Cholesterol is also cholesterol, belongs to steroidal drug.The solubleness of this class substrate in water is very low, and its scope is 10
-5~ 10
-6between mol/L, what this insoluble made steroidal and biological enzyme effectively contacts very difficult, has a strong impact on speed and the productive rate of Steroid Transformation reaction.Therefore, in the process of steroidal bio-transformation, add some chaotropic agents and can be conducive to substrate and contact with the effective of biological enzyme, improve speed of response or augmenting response speed.In the process of Steroid Synthesis by Microbial, great majority adopt the organic solvents such as methyl alcohol, acetone, dimethyl sulfoxide (DMSO) as chaotropic agent.The chemical property of eutectic mixture is similar to ionic liquid, and be liquid state under room temperature, they are without vapour pressure, can not pollute air.Compared with traditional organic solvent, eutectic mixture has non-inflammability, adds its security in operation.Therefore, study eutectic mixture replace organic solvent carry out microbial transformation experiment there is prior meaning.
Summary of the invention
The invention provides and utilize conversion of resting cells to prepare the method for cholestenone with a kind of using eutectic mixture as chaotropic agent, and thalline is adopted resting cell culture, carry out the conversion of cholesterol, in conversion solvent, add eutectic mixture, effectively improve transformation efficiency.
The object of the invention is to be achieved through the following technical solutions:
Utilize conversion of resting cells to prepare the method for cholestenone using eutectic mixture as chaotropic agent, step is as follows:
(1) after resting cell is cultivated in the fermentation medium, centrifugal, it is resuspended as conversion fluid to add sterile phosphate buffer; Described resting cell has ability cholesterol oxidation being generated cholestenone;
(2) in conversion fluid, drop into cholesterol, and to add eutectic mixture be chaotropic agent, extraction into ethyl acetate conversion fluid, adopt HPLC method to detect the productive rate of product courage Gona-4-en-3-one;
(3) substrate cholesterol is separated with product cholestenone by the method that uses column chromatography, and purifying obtains cholestenone sterling.
And described resting cell is Arthrobacter simplex (Arthrobacter simplex).
And the slant medium of described Arthrobacter simplex is g/L: glucose 8 ~ 10, corn steep liquor 8 ~ 9, peptone 4 ~ 5, KH
2pO
42, agar 20, pH7.0 ~ 7.2,115 DEG C of autoclaving 20min; Fermention medium is g/L: glucose 8 ~ 10, corn steep liquor 8 ~ 9, peptone 4 ~ 5, KH
2pO
42, pH7.0 ~ 7.2,115 DEG C of autoclaving 20min.
And, the cultural method of described Arthrobacter simplex is for scrape a ring in sterilized water by Arthrobacter simplex, vibration mixing, the concentration being 0.440 ~ 0.520 according to OD has been calculated ratio and has been joined in 50ml/250ml shaking flask and react, strain fermentation incubation time is 18h ~ 22h, temperature is 25 DEG C ~ 35 DEG C, and shaking speed is 150rpm ~ 200rpm.
And the pH of described sterile phosphate buffer is 7.0 ~ 7.2, the phosphoric acid buffer that concentration is.
And, add sterile phosphate buffer resuspended after its cell concentration of conversion fluid be 0.35-0.45g
dW/ mL.
And, the described eutectic mixture one that to be choline chloride 60/ethylene glycol mixing mol ratio be in 1:2,1:1,2:1, or choline chloride 60/glycerine mixing mol ratio is the one in 1:2,1:1,2:1, or choline chloride/urea mixing mol ratio is the one in 1:2,1:1,2:1.
And the interpolation concentration of described eutectic mixture is 1% ~ 5%.
And after the feed concentrations of described substrate cholesterol, adopt invert point to be 25 DEG C ~ 35 DEG C, shaking speed is 150rpm ~ 200rpm, and the reaction times is that the mode of 48 ~ 72h carries out microbial transformation cultivation.
And described column chromatography for separation method is separated substrate product, adopting sherwood oil: the ratio of ethyl acetate=10:1V:V is separated, adopting vacuum rotary steam method to carry out the purified pool of product by collecting the liquid obtained.
Advantage of the present invention and positively effect:
1, the present invention adopts eutectic mixture (DES) to replace the organic solvents such as methyl alcohol as green additive, after adding eutectic mixture, improve the scatter coefficient of substrate, the contact of effective promotion substrate cholesterol and thalline, effectively improve the transformation efficiency of resting cell, adding and operational condition gentleness in conversion process, step is simple.
2, the invention solves steroidal substrate solubility in microbial transformation experiment low, difficult effectively contact with biological enzyme carries out conversion reaction, add organic solvent toxicity large, volatile, to problems such as thalline toxicity are large, production for courage Gona-4-en-3-one opens new approach, simultaneously provide a new approach for the environmental protection of steroid drugs, High-efficient Production, industrialization is significant.
Accompanying drawing explanation
Fig. 1 is that inventive substrate cholesterol detects under 205nm, and retention time is 25.455min;
Fig. 2 is that product cholestenone of the present invention detects under 240nm, and retention time is 22.989min;
Fig. 3 is the transformation efficiency comparison diagram of the different DES percentage composition of the present invention;
Fig. 4 is the transformation efficiency comparison diagram that inventive substrate concentration is different;
Fig. 5 is the changing conditions figure of transformation efficiency of the present invention with thalline addition.
Concrete embodiment
In order to understand the present invention, below in conjunction with embodiment, the invention will be further described: following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The resting cell that the present invention adopts is that Arthrobacter simplex carries out fermenting experiment, take cholesterol as substrate, and interpolation eutectic mixture is that the microbial transformation reaction formula of chaotropic agent is as follows:
The synthetic method of low melting point eutectic described in the application is as follows: be that 1 ~ 2:1 ~ 2 take choline chloride 60, urea in molar ratio, 2 ~ 3h is stirred in the oil bath of 95 ~ 105 DEG C, until mixture forms homogeneous, transparent liquid, be degree of depth congruent melting solvent (DES).In like manner, choline chloride 60/glycerine, choline chloride 60/ethylene glycol also synthesize according to the method described above.
Slant medium (g/L): glucose 8, corn steep liquor 8 ~ 9, peptone 4 ~ 5, KH
2pO
42, agar 20, pH7.0 ~ 7.2,115 DEG C of autoclaving 20min.
Fermention medium (g/L): glucose 8, corn steep liquor 8 ~ 9, peptone 4 ~ 5, KH
2pO
42, pH7.0 ~ 7.2,115 DEG C of autoclaving 20min.
The operation steps of present method is as follows:
(1) resting cell yeast culture: Arthrobacter simplex is scraped a ring in sterilized water, vibration mixing, the concentration being 0.440 according to OD has been calculated ratio and has been joined in 50ml/250ml shaking flask and react.Strain fermentation incubation time is 18 ~ 22h, and temperature is 30 ~ 32 DEG C, and shaking speed is 160rpm.
(2) microbe conversion experiment: by the yeast culture in 50ml/250ml shaking flask well after, 5000r, 10min collected by centrifugation thalline, adopts 20ml phosphoric acid buffer resuspended thalline, and adjustment cell concentration is 0.3 ~ 0.5 (g
dW/ mL) substrate cholesterol is dropped in conversion fluid, and add eutectic mixture and carry out microbial transformation reaction.Transformation time is 48 ~-72h, and temperature is 30 ~ 32 DEG C, and shaking speed is 180rpm.
(3) the mensuration of cholestenone productive rate: adopt high effective liquid chromatography for measuring, separator column is Waters C18 post (4.6 × 250mm, 5 μm), substrate determined wavelength is 205nm, retention time is 25.455min (as shown in Figure 1), and product determined wavelength is 240nm, and retention time is 22.989min (as shown in Figure 2), moving phase is methyl alcohol: water (V/V)=95:5, and flow velocity is 1mL/min.Area normalization method is adopted to calculate substrate conversion efficiency.Transformation efficiency is that the percentage result that the substrate peak area being converted into product in reaction process accounts for initial substrate total peak area represents.
(4) purifying reclaims: column chromatography for separation method is separated and purifying reclaims: adopt sherwood oil: the ratio of ethyl acetate (V:V)=10:1 is separated, and adopts vacuum rotary steam method to carry out the purified pool of product by collecting the liquid obtained.
The title of the thalline of the Arthrobacter simplex that the present invention uses: A.simplex1 56, deposit number is: IFO12069, buy from Osaka, Japan fermentation research institute, the present invention adopts other bacterial classifications with microbe conversion cholestenone all can realize, and is not limited only to bacterial classification provided by the invention.
Embodiment 1:
Utilize conversion of resting cells to prepare a method for cholestenone using eutectic mixture as chaotropic agent, the present embodiment, by changing DES blending ratio, detects the catalyzed conversion effect of its different sorts DES, specific as follows:
(1) Arthrobacter simplex is scraped a ring in sterilized water, vibration mixing, the concentration being 0.440 according to OD has been calculated ratio and has been joined in 50ml/250ml shaking flask and react.Strain fermentation incubation time is 18 ~-22h, and temperature is 30 DEG C, and shaking speed is 160rpm.
Yeast culture well after, collected by centrifugation thalline, adopts 20ml phosphoric acid buffer resuspended thalline, makes its concentration be 0.4g
dW/ mL.The substrate cholesterol of 5 ‰ is dropped in conversion fluid, and add eutectic mixture and carry out microbial transformation reaction, the eutectic mixture wherein added is choline chloride 60/ethylene glycol mixing mol ratio is 1:2,1:1,2:1, or choline chloride 60/glycerine mixing mol ratio is the one in 1:2,1:1,2:1, or choline chloride/urea mixing mol ratio is 1:2,1:1,2:1, totally nine kinds of prioritization schemes.Transformation time is 48 ~-72h, and temperature is 30 DEG C, and shaking speed is 180rpm.After conversion terminates, get 500 μ l samples and add 200 μ l ethyl acetate, oscillation extraction, after leaving standstill 2min, get 20 μ l and dry, add chromatogram methyl alcohol and cross film, treat that HPLC detects to obtain transformation efficiency.
(3) as shown in table 1, add choline chloride/urea, and mol ratio to be the conversion results of 1:1 better.Collecting conversion fluid, adopt sherwood oil: the ratio of ethyl acetate (V:V)=10:1 is separated, adopting vacuum rotary steam method to carry out the purified pool of product by collecting the liquid obtained.
The different DES transformation efficiency of table 1
Embodiment 2:
Utilize conversion of resting cells to prepare a method for cholestenone using eutectic mixture as chaotropic agent, the present embodiment is different by changing DES percentage composition, detects the catalyzed conversion effect of its different concns DES, specific as follows:
(1) Arthrobacter simplex is scraped a ring in sterilized water, vibration mixing, the concentration being 0.440 according to OD has been calculated ratio and has been joined in 50ml/250ml shaking flask and react.Strain fermentation incubation time is 18 ~-22h, and temperature is 30 DEG C, and shaking speed is 160rpm.
Yeast culture well after, collected by centrifugation thalline, makes its concentration be 0.4g
dW/ mL adopts the resuspended thalline of 20ml phosphoric acid buffer.The substrate cholesterol of 5 ‰ is dropped in conversion fluid, and add eutectic mixture choline chloride/urea (mol ratio is 1:1) reaction and transform, the percentage composition that wherein eutectic solvent accounts for DES/ phosphoric acid buffer liquid system is respectively 0%, 2%, 4%, 6%, 8%.Transformation time is 48 ~ 72h, and temperature is 30 DEG C, and shaking speed is 180rpm.After conversion terminates, get 500 μ l samples and add 200 μ l ethyl acetate, oscillation extraction, after leaving standstill 2min, get 20 μ l and dry, add chromatogram methyl alcohol and cross film, treat that HPLC detects to obtain transformation efficiency.
(3) when as shown in Figure 3, DES percentage composition is 2%, conversion results is higher.Collecting conversion fluid, adopt sherwood oil: the ratio of ethyl acetate (V:V)=10:1 is separated, adopting the outstanding steaming method of decompression to carry out the purified pool of product by collecting the liquid obtained.
Embodiment 3:
Utilize conversion of resting cells to prepare a method for cholestenone using eutectic mixture as chaotropic agent, the present embodiment, by changing concentration of substrate, detects the catalyzed conversion effect of its concentration of substrate change, specific as follows:
(1) Arthrobacter simplex is scraped a ring in sterilized water, vibration mixing, the concentration being 0.440 according to OD has been calculated ratio and has been joined in 50ml/250ml shaking flask and react.Strain fermentation incubation time is 18-22h, and temperature is 30 DEG C, and shaking speed is 160rpm.
Yeast culture well after, collected by centrifugation thalline, adopts 20ml phosphoric acid buffer resuspended thalline, makes its concentration be 0.4g
dW/ mL.Substrate cholesterol dropped in conversion fluid, wherein substrate addition is 1 ‰, 5 ‰, 1%, 2%, 3%, and the eutectic mixture adding 2% choline chloride/urea (mol ratio is 1:1) carries out microbial transformation reaction.Transformation time is 48 ~-72h, and temperature is 30 DEG C, and shaking speed is 180rpm.After conversion terminates, get 500 μ l samples and add 200 μ l ethyl acetate, oscillation extraction, leave standstill after 2min, get 20 μ l and dry, add chromatogram methyl alcohol and cross film, treat HPLC detect transformation efficiency as shown in Figure 5, when adopting 2% substrate addition, its transformation efficiency is the highest.
(3) collecting conversion fluid, adopt sherwood oil: the ratio of ethyl acetate (V:V)=10:1 is separated, adopting vacuum rotary steam method to carry out the purified pool of product by collecting the liquid obtained.
Embodiment 4:
Utilize conversion of resting cells to prepare a method for cholestenone using eutectic mixture as chaotropic agent, the present embodiment, by changing cell concentration, detects the catalyzed conversion effect of different cell concentration, specific as follows:
(1) Arthrobacter simplex is scraped a ring in sterilized water, vibration mixing, the concentration being 0.440 according to OD has been calculated ratio and has been joined in 50ml/250ml shaking flask and react.Strain fermentation incubation time is 18-22h, and temperature is 30 DEG C, and shaking speed is 160rpm.
Yeast culture well after, collected by centrifugation thalline, adopts 20ml phosphoric acid buffer resuspended thalline, makes cell concentration (g
dW/ mL) be 0.35,0.4,0.45,0.5.Substrate cholesterol is dropped in conversion fluid, and the eutectic mixture adding 2% choline chloride/urea (mol ratio is 1:1) carries out microbial transformation reaction.Transformation time is 48-72h, and temperature is 30 DEG C, and shaking speed is 180rpm.After conversion terminates, get 500 μ l samples and add 200 μ l ethyl acetate, oscillation extraction, leave standstill after 2min, go 20 μ l to dry, add chromatogram methyl alcohol and cross film, treat HPLC detect transformation efficiency as shown in Figure 5.
(3) collecting conversion fluid, adopt sherwood oil: the ratio of ethyl acetate (V:V)=10:1 is separated, adopting vacuum rotary steam method to carry out the purified pool of product by collecting the liquid obtained.
It should be noted that: this experiment utilizes conventional ion liquid as [PrMIM] [PF
6], [BMIM] [PF
6], [HMIM] [PF
6], [PrMIM] [NTf
2], [BMIM] [NTf
2], [HMIM] [NTf
2], [AMIM] [NTf
2] etc. carry out cholesterol dehydrogenation biocatalysis attempt, found that this ionic liquid is comparatively large to thalline toxicity, and transformation efficiency is 0.Different ionic liquid is also confirmed in the research of Zhejiang University Huang Jing for the toxicity of Arthrobacter simplex.
Claims (10)
1. utilize using eutectic mixture as chaotropic agent conversion of resting cells to prepare the method for cholestenone, it is characterized in that: step is as follows:
(1) after resting cell is cultivated in the fermentation medium, centrifugal, it is resuspended as conversion fluid to add sterile phosphate buffer; Described resting cell has ability cholesterol oxidation being generated cholestenone;
(2) in conversion fluid, drop into cholesterol, and to add eutectic mixture be chaotropic agent, extraction into ethyl acetate conversion fluid, adopt HPLC method to detect the productive rate of product courage Gona-4-en-3-one;
(3) substrate cholesterol is separated with product cholestenone by the method that uses column chromatography, and purifying obtains cholestenone sterling.
2. utilize using eutectic mixture as chaotropic agent conversion of resting cells to prepare the method for cholestenone according to claim 1, it is characterized in that: described resting cell is Arthrobacter simplex (Arthrobacter simplex).
3. utilize using eutectic mixture as chaotropic agent conversion of resting cells to prepare the method for cholestenone according to claim 2, it is characterized in that: the slant medium of described Arthrobacter simplex is g/L: glucose 8 ~ 10, corn steep liquor 8 ~ 9, peptone 4 ~ 5, KH
2pO
42, agar 20, pH7.0 ~ 7.2,115 DEG C of autoclaving 20min; Fermention medium is g/L: glucose 8 ~ 10, corn steep liquor 8 ~ 9, peptone 4 ~ 5, KH
2pO
42, pH7.0 ~ 7.2,115 DEG C of autoclaving 20min.
4. utilize conversion of resting cells to prepare the method for cholestenone according to claim 2 using eutectic mixture as chaotropic agent, it is characterized in that: the cultural method of described Arthrobacter simplex is for scrape a ring in sterilized water by Arthrobacter simplex, vibration mixing, the concentration being 0.440 ~ 0.520 according to OD has been calculated ratio and has been joined in 50ml/250ml shaking flask and react, strain fermentation incubation time is 18h ~ 22h, temperature is 25 DEG C ~ 35 DEG C, and shaking speed is 150rpm ~ 200rpm.
5. utilize using eutectic mixture as chaotropic agent conversion of resting cells to prepare the method for cholestenone according to claim 1, it is characterized in that: the pH of described sterile phosphate buffer is 7.0 ~ 7.2, the phosphoric acid buffer that concentration is.
6. utilize using eutectic mixture as chaotropic agent conversion of resting cells to prepare the method for cholestenone according to claim 1, it is characterized in that: add sterile phosphate buffer resuspended after its cell concentration of conversion fluid be 0.35 ~ 0.45gDW/mL.
7. utilize conversion of resting cells to prepare the method for cholestenone according to claim 1 using eutectic mixture as chaotropic agent, it is characterized in that: the described eutectic mixture one that to be choline chloride 60/ethylene glycol mixing mol ratio be in 1:2,1:1,2:1, or choline chloride 60/glycerine mixing mol ratio is the one in 1:2,1:1,2:1, or choline chloride/urea mixing mol ratio is the one in 1:2,1:1,2:1.
8. utilize using eutectic mixture as chaotropic agent conversion of resting cells to prepare the method for cholestenone according to claim 1, it is characterized in that: the interpolation concentration of described eutectic mixture is 1% ~ 5%.
9. utilize conversion of resting cells to prepare the method for cholestenone according to claim 1 using eutectic mixture as chaotropic agent, it is characterized in that: after the feed concentrations of described substrate cholesterol, invert point is adopted to be 25 DEG C ~ 35 DEG C, shaking speed is 150rpm ~ 200rpm, and the reaction times is that the mode of 48 ~ 72h carries out microbial transformation cultivation.
10. utilize conversion of resting cells to prepare the method for cholestenone according to claim 1 using eutectic mixture as chaotropic agent, it is characterized in that: described column chromatography for separation method is separated substrate product, adopting sherwood oil: the ratio of ethyl acetate=10:1V:V is separated, adopting vacuum rotary steam method to carry out the purified pool of product by collecting the liquid obtained.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106636287A (en) * | 2016-12-30 | 2017-05-10 | 山东宝利甾体生物科技有限公司 | Method for preparing 16,17[alpha]-epoxyprogesterone by virtue of biological process |
| CN107746849A (en) * | 2017-09-29 | 2018-03-02 | 天津科技大学 | A kind of high-efficiency screening method of steroidal '-hydroxylase gene |
| CN110079387A (en) * | 2019-05-28 | 2019-08-02 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of method that the extraction of ultrasonic wave added eutectic solvent removes cholesterol in lard |
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| CN1328133A (en) * | 2001-06-05 | 2001-12-26 | 江南大学 | Process for biologically synthesizing extracellular cholesterol oxidase from Brevibacterium and its transferred product, cholest-4-ene-3-one |
| US20090117628A1 (en) * | 2007-09-21 | 2009-05-07 | Gorke Johnathan T | Enzymatic processing in deep eutectic solvents |
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| CN1328133A (en) * | 2001-06-05 | 2001-12-26 | 江南大学 | Process for biologically synthesizing extracellular cholesterol oxidase from Brevibacterium and its transferred product, cholest-4-ene-3-one |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636287A (en) * | 2016-12-30 | 2017-05-10 | 山东宝利甾体生物科技有限公司 | Method for preparing 16,17[alpha]-epoxyprogesterone by virtue of biological process |
| CN106636287B (en) * | 2016-12-30 | 2021-01-05 | 山东宝利甾体生物科技有限公司 | Method for preparing 16,17 alpha-epoxy progesterone by biological method |
| CN107746849A (en) * | 2017-09-29 | 2018-03-02 | 天津科技大学 | A kind of high-efficiency screening method of steroidal '-hydroxylase gene |
| CN110079387A (en) * | 2019-05-28 | 2019-08-02 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of method that the extraction of ultrasonic wave added eutectic solvent removes cholesterol in lard |
| CN110079387B (en) * | 2019-05-28 | 2022-08-09 | 广东省农业科学院蚕业与农产品加工研究所 | Method for removing cholesterol in lard oil through ultrasonic-assisted eutectic solvent extraction |
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| CN104357526B (en) | 2018-05-18 |
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