CN105907828A - Substrate treatment method for phytosterol side chain degradation reaction - Google Patents
Substrate treatment method for phytosterol side chain degradation reaction Download PDFInfo
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- CN105907828A CN105907828A CN201610270775.XA CN201610270775A CN105907828A CN 105907828 A CN105907828 A CN 105907828A CN 201610270775 A CN201610270775 A CN 201610270775A CN 105907828 A CN105907828 A CN 105907828A
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- plant sterol
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- attapulgite
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/005—Degradation of the lateral chains at position 17
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/02—Dehydrogenating; Dehydroxylating
Abstract
The invention belongs to the field of microbial pharmaceutics and provides a substrate treatment method for phytosterol side chain degradation reaction. The method is applied to phytosterol side chain degradation reaction for AD (androstenedione) production. Phytosterol and attapulgite are modified prior to feeding of phytosterol, and according to a substrate feeding quantity and an attapulgite addition amount, a mass ratio of the phytosterol to the attapulgite is controlled to be 1:0.1-5, and concentration of fed co-modified phytosterol and attapulgite is 1-40g/L. The substrate treatment method has advantages that dispersity of a substrate in an aqueous solution is effectively improved, the utilization rate and the reaction rate of the substrate are increased, and substrate and product inhibition is relieved to some extent to increase the conversion rate of products; in addition, the method is simple in process, economical, practical, low in environment pollution and significant to biotransfomation based steroid medicine production.
Description
Technical field
The invention belongs to field of microbial pharmacy, refer more particularly to a kind of substrate processing method for the reaction of plant sterol Side chain cleavage.
Background technology
Steroid hormone class medicine is the important class of China's field of medicaments, and androstenedione (Androstenedion, AD) is the steroid hormone irreplaceable intermediate of class medicine.China is oil seed production big country, contains extremely abundant plant sterol resource in oil prodution industry by-product.Microbial selective degrading plant sterol (phytosterol, PS) side chain generates AD, complicated multi-step chemical synthetic method can be substituted, and alleviate at present owing to diosgenin is the resource scarcity that raw material causes, to Appropriate application China steroidal plant resources, the development important in inhibiting of promotion pharmaceutical industry.
Microbial selective degrading plant sterol side chain is to carry out under the Steroid Transformation enzyme effect that microbial cell produces, the topmost problem run into during plant sterol Side chain cleavage generates AD is that plant sterol has the strongest hydrophobicity, dissolubility in water is extremely low, easily it is gathered in aqueous phase surface, this allows for substrate and can not contact well with microbial cell, causes conversion ratio on the low side and the series of problems such as transformation time prolongation.
Attapulgite, also known as attapulgite clay (attapulgite, AT), the chain layered crystal structure of its uniqueness, imparts AT many character, mainly includes adsorptivity, carrier-mediated, catalytic, dispersibility and rheological characteristic etc..With thermal activation, be acidified, alkalize, former attapulgite clay is modified making it loose porous by organically-modified, mixing method, specific surface area increases, absorption property improves therewith, also becomes a kind of preferably carrier, thus its modification is had higher Social benefit and economic benefit.Substrate plant sterol is modified in advance together with attapulgite clay, makes plant sterol absorption carriage to loose porous attapulgite clay, be preferably distributed in aqueous solution culture medium with attapulgite clay so that substrate better contacts with microbial cell, and then improve the conversion ratio of reaction.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of substrate processing method generating androstenedione reaction for plant sterol Side chain cleavage.
The technical solution used in the present invention is: a kind of substrate processing method for the reaction of plant sterol Side chain cleavage, adds and plant sterol and attapulgite clay carry out before plant sterol reacts common modified formation modified attapulgite-plant sterol altogether in advance.
Preferably, the one during modified attapulgite-plant sterol is acid modified attapulgite-plant sterol altogether, silane coupler modified attapulgite-plant sterol altogether, alkali modified attapulgite-plant sterol altogether and surfactant modified attapulgite-plant sterol altogether altogether.
Preferably, the mass ratio being total to the plant sterol input by modified attapulgite-plant sterol and attapulgite clay is 1:0.1～5.
Wherein, the modification procedure of acid modified attapulgite-plant sterol altogether is: joining in acid solution by plant sterol and attapulgite clay according to mass ratio 1:0.1～5, acid solution is the one in sulphuric acid, hydrochloric acid, nitric acid or phosphoric acid, sonic oscillation, centrifugal water is washed till neutrality, grinds to form fine powder after drying.
Wherein, the modification procedure of silane coupler modified attapulgite-plant sterol altogether is: join in the aqueous acetic acid of pH 3.5 by plant sterol and attapulgite clay according to mass ratio 1:0.1～5, add silane coupler, silane coupler is APTES KH550, sonic oscillation, centrifugal water is washed till without silane coupler, grinds to form fine powder after drying.
Wherein, the modification procedure of alkali modified attapulgite-plant sterol altogether is: joining in aqueous slkali by plant sterol and attapulgite clay according to mass ratio 1:0.1～5, aqueous slkali is sodium hydroxide solution or potassium hydroxide solution, sonic oscillation, centrifugal water is washed till neutrality, grinds to form fine powder after drying.
Wherein, the modification procedure of surfactant modified attapulgite-plant sterol altogether is: join in surfactant-modified solution by plant sterol and attapulgite clay according to mass ratio 1:0.1～5, surfactant is cetyl trimethylammonium bromide, after sonic oscillation, centrifugal water is washed till surfactant-free, grinds to form fine powder after drying.
Preferably, the suitableeest adding proportion being total to the plant sterol input by modified attapulgite-plant sterol and attapulgite clay is 1:1～3.
The application in plant sterol Side chain cleavage reacts of a kind of substrate processing method for the reaction of plant sterol Side chain cleavage.
Preferably, modified attapulgite-plant sterol puts into concentration altogether is 1～40g/L, and the plant sterol input by modified attapulgite-plant sterol is preferably 1:1～3 with the mass ratio of attapulgite clay altogether, and modified attapulgite-plant sterol the suitableeest interpolation concentration is 1.5～10g/L altogether.
The present invention has the advantage that with good effect: by being modified with attapulgite clay by plant sterol in advance before adding plant sterol and converting, substrate dispersion in aqueous can be effectively improved, substrate is better contacted with microbial cell, and then improves the conversion ratio of reaction.Without adding organic solvent in preparation process, reaction condition is gentle, and reforming unit is simple, sufficient raw, and preparation process is simple, and production cost is low, and environmental pollution harm is little；The inventive method improves substrate conversion rate and conversion ratio, has easy and simple to handle, environmental protection, cheap advantage.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, and following example are illustrative, is not determinate, it is impossible to limit protection scope of the present invention with this.
Abbreviation implication:
AT attapulgite clay
PS plant sterol
AD androstenedione (4-AD, Androstenedione)
A kind of substrate processing method for the reaction of plant sterol Side chain cleavage that the present invention relates to, add and in advance plant sterol and attapulgite clay are carried out before plant sterol reacts common modified formation modified attapulgite-plant sterol altogether, wherein it is total to method of modifying to include, acid modification altogether, silane coupler modification altogether, alkali is the most modified and surfactant is the most modified, input common modified attapulgite-plant sterol concentration is 1～40g/L, it is 1:0.1～5 that generation is total to the mass ratio of the plant sterol input by modified attapulgite-plant sterol and attapulgite clay, wherein, attapulgite clay concentration is 0.5～10g/L, plant sterol concentration is 0.5～30g/L；It is 1:1～3 that generation is total to the suitableeest adding proportion of the plant sterol input by modified attapulgite-plant sterol and attapulgite clay.
1 preparation being total to modified attapulgite-plant sterol:
(1) acid altogether modified attapulgite-plant sterol: in the acid solution of the 4mol/L that plant sterol and attapulgite clay are joined according to mass ratio 1:0.1～5 100mL, wherein the gross mass of plant sterol and attapulgite clay is less than 5g, acid solution is the one in sulphuric acid, hydrochloric acid, nitric acid or phosphoric acid, sonic oscillation 30min, centrifugal water is washed till neutrality, 60 DEG C of dry 8h, grind to form fine powder.
(2) silane coupler altogether modified attapulgite-plant sterol: plant sterol and attapulgite clay are joined in the aqueous acetic acid of pH 3.5 of 100mL according to mass ratio 1:0.1～5, the gross mass of plant sterol and attapulgite clay is less than 5g, add the silane coupler of 0.1mL, silane coupler is APTES KH550, sonic oscillation 30min, centrifugal water is washed till without silane coupler, 60 DEG C of dry 8h, grinds to form fine powder.
(3) alkali altogether modified attapulgite-plant sterol: in the aqueous slkali of the 4mol/L that plant sterol and attapulgite clay are joined according to mass ratio 1:0.1～5 100mL, the gross mass of plant sterol and attapulgite clay is less than 5g, aqueous slkali is sodium hydroxide solution or potassium hydroxide solution, sonic oscillation 30min, centrifugal water is washed till neutrality, 60 DEG C of dry 8h, grind to form fine powder.
(4) surfactant modified attapulgite-plant sterol altogether: plant sterol and attapulgite clay are joined 100mL according to mass ratio 1:0.1～5 and contains in the surfactant-modified solution of 0.01g, the gross mass of plant sterol and attapulgite clay is less than 5g, surfactant is cetyl trimethylammonium bromide, after sonic oscillation, centrifugal water is washed till surfactant-free, 60 DEG C of dry 8h, grind to form fine powder.
2 common modified attapulgite-plant sterols are applied to plant sterol Side chain cleavage and react:
Common modified attapulgite-plant sterol is added in mycobacteria fermentation medium and converts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, and ferric citrate presses 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, altogether modified attapulgite-plant sterol 1～40g/L, pH 7.2.
In order to verify common modified attapulgite-plant sterol to preparing the facilitation effect of androstenedione AD,
Set up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS (in addition modified attapulgite together-plant sterol fermentation system, the content of plant sterol is identical), pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids, uses mole production rate of the androstenedione AD of high performance liquid chromatography (HPLC) detection reaction generation.
Embodiment 1: silane coupler modified attapulgite-plant sterol altogether
The modification of 1 substrate:
1g plant sterol PS and 3g attapulgite clay AT is joined in 100mL acetum (pH3.5), add 0.1mL APTES KH550, sonic oscillation 30min, 5000r/min is centrifuged 10min, deionized water is washed till without KH550,60 DEG C of dry 8h, grind to form fine powder, are silane coupler modified attapulgite-plant sterol KH550-AT-PS altogether.
2 plant sterol Side chain cleavage reactions
Silane coupler modified attapulgite-plant sterol KH550-AT-PS altogether is added in mycobacteria fermentation medium and reacts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, silane coupler modified attapulgite-plant sterol KH550-AT-PS4g/L, pH 7.2 altogether.
In order to verify that common modified attapulgite-plant sterol, to preparing the facilitation effect of androstenedione AD, sets up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS 1g/L, pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids.
3AD mole of production rate comparison result:
Take two groups of fermentation liquids respectively, use high performance liquid chromatography (HPLC) detection.
With the ethyl acetate ultrasonic extraction fermentation liquid 0.5h of 1mL, centrifugal (12000 × g, 2min), take ethyl acetate phase 0.1mL in 1.5mL eppendorf pipe, add the flowing phase of 1mL after natural air drying, after centrifugal (12000 × g, 2min), carry out HPLC analysis.Chromatographic condition: Phenomenex C18 post, flowing is methanol mutually: water (4:1), flow velocity is 1mL/min, and column temperature is 30 DEG C, and detection wavelength is 254nm.
As shown in table 1, after 72h, the AD mole of production rate adding KH550-AT-PS is 66.76 ± 1.41%, is 5.13 times of matched group (13.01 ± 0.41%)；After 120h, the AD mole of production rate adding KH550-AT-PS is 74.06 ± 0.64%, is 2.86 times of matched group (25.87 ± 0.19%)；After 168h, the AD mole of production rate adding KH550-AT-PS is 74.97 ± 0.35%, is 2.65 times of matched group (28.34 ± 0.04%).
AD mole of production rate of table 1
Embodiment 2: with acid modified attapulgite-plant sterol altogether
The modification of 1 substrate:
In the sulfuric acid solution of the 4mol/L that 1g plant sterol PS and 3g attapulgite clay AT is joined 100mL, sonic oscillation 30min, 5000r/min are centrifuged 10min, are washed with deionized water to neutrality, 60 DEG C of dry 8h, grind to form fine powder, are sulphuric acid modified attapulgite-plant sterol H altogether2SO4-AT-PS。
2 plant sterol Side chain cleavage reactions
By sulphuric acid modified attapulgite-plant sterol H altogether2SO4-AT-PS adds in mycobacteria fermentation medium and reacts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, sulphuric acid modified attapulgite-plant sterol H altogether2SO4-AT-PS 4g/L, pH 7.2.
In order to verify that common modified attapulgite-plant sterol, to preparing the facilitation effect of androstenedione AD, sets up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS 1g/L, pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids.
3AD mole of production rate comparison result:
Take two groups of fermentation liquids respectively, use high performance liquid chromatography (HPLC) detection.
With the ethyl acetate ultrasonic extraction fermentation liquid 0.5h of 1mL, centrifugal (12000 × g, 2min), take ethyl acetate phase 0.1mL in 1.5mL eppendorf pipe, add the flowing phase of 1mL after natural air drying, after centrifugal (12000 × g, 2min), carry out HPLC analysis.Chromatographic condition: Phenomenex C18 post, flowing is methanol mutually: water (4:1), flow velocity is 1mL/min, and column temperature is 30 DEG C, and detection wavelength is 254nm.
As shown in table 2, after 72h, add H2SO4The AD mole of production rate of-AT-PS is 50.77 ± 0.38%, is 3.90 times of matched group (13.01 ± 0.41%)；After 120h, add H2SO4The AD mole of production rate of-AT-PS is 59.34 ± 0.83%, is 2.30 times of matched group (25.87 ± 0.19%)；After 168h, add H2SO4The AD mole of production rate of-AT-PS is 64.11 ± 0.25%, is 2.26 times of matched group (28.34 ± 0.04%).
AD mole of production rate of table 2
Embodiment 3: with acid modified attapulgite-plant sterol altogether
The modification of 1 substrate:
In the hydrochloric acid solution of the 4mol/L that 1g plant sterol PS and 5g attapulgite clay AT is joined 100mL, sonic oscillation 30min, 5000r/min are centrifuged 10min, it is washed with deionized water to neutrality, 60 DEG C of dry 8h, grind to form fine powder, are hydrochloric acid modified attapulgite-plant sterol HCl-AT-PS altogether.
2 plant sterol Side chain cleavage reactions
Hydrochloric acid modified attapulgite-plant sterol HCl-AT-PS altogether is added in mycobacteria fermentation medium and converts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, hydrochloric acid modified attapulgite-plant sterol HCl-AT-PS 6g/L, pH 7.2 altogether.
In order to verify that common modified attapulgite-plant sterol, to preparing the facilitation effect of androstenedione AD, sets up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS 1g/L, pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids.
3AD mole of production rate comparison result:
Take two groups of fermentation liquids respectively, use high performance liquid chromatography (HPLC) detection.
With the ethyl acetate ultrasonic extraction fermentation liquid 0.5h of 1mL, centrifugal (12000 × g, 2min), take ethyl acetate phase 0.1mL in 1.5mL eppendorf pipe, add the flowing phase of 1mL after natural air drying, after centrifugal (12000 × g, 2min), carry out HPLC analysis.Chromatographic condition: Phenomenex C18 post, flowing is methanol mutually: water (4:1), flow velocity is 1mL/min, and column temperature is 30 DEG C, and detection wavelength is 254nm.
As shown in table 3, after 72h, the AD mole of production rate adding HCl-AT-PS is 55.14 ± 0.72%, is 4.23 times of matched group (13.01 ± 0.41%)；After 120h, the AD mole of production rate adding HCl-AT-PS is 63.79 ± 0.84%, is 2.47 times of matched group (25.87 ± 0.19%)；After 168h, the AD mole of production rate adding HCl-AT-PS is 66.32 ± 0.61%, is 2.34 times of matched group (28.34 ± 0.04%).
AD mole of production rate of table 3
Embodiment 4: with acid modified attapulgite-plant sterol altogether
The modification of 1 substrate:
In the phosphoric acid solution of the 4mol/L that 3g plant sterol PS and 5g attapulgite clay AT is joined 100mL, sonic oscillation 30min, 5000r/min are centrifuged 10min, are washed with deionized water to neutrality, 60 DEG C of dry 8h, grind to form fine powder, are phosphoric acid modified attapulgite-plant sterol H altogether3PO4-AT-PS。
2 plant sterol Side chain cleavage reactions
By phosphoric acid modified attapulgite-plant sterol H altogether3PO4-AT-PS adds in mycobacteria fermentation medium and reacts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, phosphoric acid modified attapulgite-plant sterol H altogether3PO4-AT-PS 8g/L,
pH 7.2。
In order to verify that common modified attapulgite-plant sterol, to preparing the facilitation effect of androstenedione AD, sets up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS 3g/L, pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids.
3AD mole of production rate comparison result:
Take two groups of fermentation liquids respectively, use high performance liquid chromatography (HPLC) detection.
With the ethyl acetate ultrasonic extraction fermentation liquid 0.5h of 1mL, centrifugal (12000 × g, 2min), take ethyl acetate phase 0.1mL in 1.5mL eppendorf pipe, add the flowing phase of 1mL after natural air drying, after centrifugal (12000 × g, 2min), carry out HPLC analysis.Chromatographic condition: Phenomenex C18 post, flowing is methanol mutually: water (4:1), flow velocity is 1mL/min, and column temperature is 30 DEG C, and detection wavelength is 254nm.
As shown in table 4, after 72h, add H3PO4The AD mole of production rate of-AT-PS is 45.32 ± 0.58%, is 5.38 times of matched group (8.43 ± 0.41%)；After 120h, add H3PO4The AD mole of production rate of-AT-PS is 54.85 ± 0.17%, is 4.24 times of matched group (12.94 ± 0.19%)；After 168h, add H3PO4The AD mole of production rate of-AT-PS is 59.17 ± 1.12%, is 3.71 times of matched group (15.97 ± 0.04%).
AD mole of production rate of table 4
Embodiment 5: with acid modified attapulgite-plant sterol altogether
The modification of 1 substrate:
In the salpeter solution of the 4mol/L that 5g plant sterol PS and 3g attapulgite clay AT is joined 100mL, sonic oscillation 30min, 5000r/min are centrifuged 10min, it is washed with deionized water to neutrality, 60 DEG C of dry 8h, grind to form fine powder, are nitric acid modified attapulgite-plant sterol HNO altogether3-AT-PS。
2 plant sterol Side chain cleavage reactions
By nitric acid modified attapulgite-plant sterol HNO altogether3-AT-PS adds in mycobacteria fermentation medium and reacts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, nitric acid modified attapulgite-plant sterol H altogether3PO4-AT-PS 8g/L, pH 7.2.
In order to verify common modified attapulgite-plant sterol to preparing the facilitation effect of androstenedione AD,
Set up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS 5g/L, pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids.
3AD mole of production rate comparison result:
Take two groups of fermentation liquids respectively, use high performance liquid chromatography (HPLC) detection.
With the ethyl acetate ultrasonic extraction fermentation liquid 0.5h of 1mL, centrifugal (12000 × g, 2min), take ethyl acetate phase 0.1mL in 1.5mL eppendorf pipe, add the flowing phase of 1mL after natural air drying, after centrifugal (12000 × g, 2min), carry out HPLC analysis.Chromatographic condition: Phenomenex C18 post, flowing is methanol mutually: water (4:1), flow velocity is 1mL/min, and column temperature is 30 DEG C, and detection wavelength is 254nm.
As shown in table 5, after 72h, add HNO3The AD mole of production rate of-AT-PS is 23.07 ± 0.32%, is 3.55 times of matched group (6.49 ± 0.31%)；After 120h, add HNO3The AD mole of production rate of-AT-PS is 37.20 ± 1.42%, is 3.61 times of matched group (10.29 ± 0.12%)；After 168h, add HNO3The AD mole of production rate of-AT-PS is 39.08 ± 1.52%, is 2.59 times of matched group (15.07 ± 0.15%).
AD mole of production rate of table 5
Embodiment 6: with alkali modified attapulgite-plant sterol altogether
The modification of 1 substrate:
In the sodium hydroxide solution of the 4mol/L that 1g plant sterol PS and 5g attapulgite clay AT is joined 100mL, sonic oscillation 30min, 5000r/min are centrifuged 10min, it is washed with deionized water to neutrality, 60 DEG C of dry 8h, grind to form fine powder, are sodium hydroxide modified attapulgite-plant sterol NaOH-AT-PS altogether.
2 plant sterol Side chain cleavage reactions
Sodium hydroxide modified attapulgite-plant sterol NaOH-AT-PS altogether is added in mycobacteria fermentation medium and reacts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, sodium hydroxide modified attapulgite-plant sterol NaOH-AT-PS 6g/L, pH 7.2 altogether.
In order to verify that common modified attapulgite-plant sterol, to preparing the facilitation effect of androstenedione AD, sets up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS 1g/L, pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids.
3AD mole of production rate comparison result:
Take two groups of fermentation liquids respectively, use high performance liquid chromatography (HPLC) detection.
With the ethyl acetate ultrasonic extraction fermentation liquid 0.5h of 1mL, centrifugal (12000 × g, 2min), take ethyl acetate phase 0.1mL in 1.5mL eppendorf pipe, add the flowing phase of 1mL after natural air drying, after centrifugal (12000 × g, 2min), carry out HPLC analysis.Chromatographic condition: Phenomenex C18 post, flowing is methanol mutually: water (4:1), flow velocity is 1mL/min, and column temperature is 30 DEG C, and detection wavelength is 254nm.
As shown in table 6, after 72h, the AD mole of production rate adding NaOH-AT-PS is 50.45 ± 0.25%, is 3.88 times of matched group (13.01 ± 0.41%)；After 120h, the AD mole of production rate adding NaOH-AT-PS is 57.22 ± 0.47%, is 2.21 times of matched group (25.87 ± 0.19%)；After 168h, the AD mole of production rate adding NaOH-AT-PS is 60.31 ± 0.94%, is 2.13 times of matched group (28.34 ± 0.04%).
AD mole of production rate of table 6
Embodiment 7: with alkali modified attapulgite-plant sterol altogether
The modification of 1 substrate:
In the potassium hydroxide solution of the 4mol/L that 2g PS and 3g AT is joined 100mL, sonic oscillation 30min, 5000r/min are centrifuged 10min, it is washed with deionized water to neutrality, 60 DEG C of dry 8h, grind to form fine powder, are potassium hydroxide modified attapulgite-plant sterol KOH-AT-PS altogether.
2 plant sterol Side chain cleavage reactions
Potassium hydroxide modified attapulgite-plant sterol KOH-AT-PS altogether is added in mycobacteria fermentation medium and reacts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, potassium hydroxide modified attapulgite-plant sterol KOH-AT-PS 5g/L, pH 7.2 altogether.
In order to verify that common modified attapulgite-plant sterol, to preparing the facilitation effect of androstenedione AD, sets up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS 2g/L, pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids.
3AD mole of production rate comparison result:
Take two groups of fermentation liquids respectively, use high performance liquid chromatography (HPLC) detection.
With the ethyl acetate ultrasonic extraction fermentation liquid 0.5h of 1mL, centrifugal (12000 × g, 2min), take ethyl acetate phase 0.1mL in 1.5mL eppendorf pipe, add the flowing phase of 1mL after natural air drying, after centrifugal (12000 × g, 2min), carry out HPLC analysis.Chromatographic condition: Phenomenex C18 post, flowing is methanol mutually: water (4:1), flow velocity is 1mL/min, and column temperature is 30 DEG C, and detection wavelength is 254nm.
As shown in table 7, after 72h, the AD mole of production rate adding KOH-AT-PS is 47.22 ± 0.34%, is 4.70 times of matched group (10.05 ± 0.41%)；After 120h, the AD mole of production rate adding KOH-AT-PS is 53.09 ± 0.53%, is 3.60 times of matched group (14.77 ± 0.19%)；After 168h, the AD mole of production rate adding KOH-AT-PS is 57.83 ± 0.64%, is 2.86 times of matched group (20.22 ± 0.24%).
AD mole of production rate of table 7
Embodiment 8: with surfactant modified attapulgite-plant sterol altogether
The modification of 1 substrate:
2g PS and 1g AT is joined 100mL and contains in the surfactant-modified solution of 0.01g in (cetyl trimethylammonium bromide) solution, sonic oscillation 30min, 5000r/min is centrifuged 10min, it is washed with deionized water to neutrality, 60 DEG C of dry 8h, grind to form fine powder, be surfactant modified attapulgite-plant sterol CTAB-AT-PS altogether.
2 plant sterol Side chain cleavage reactions
Surfactant modified attapulgite-plant sterol CTAB-AT-PS altogether is added in mycobacteria fermentation medium and reacts.
Wherein fermentation medium components is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, pH 7.2.
Mycobacteria strain inoculum concentration is 7% (v/v).
The fermentation system adding modified attapulgite-plant sterol altogether is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, surfactant modified attapulgite-plant sterol CTAB-AT-PS3g/L, pH 7.2 altogether.
In order to verify that common modified attapulgite-plant sterol, to preparing the facilitation effect of androstenedione AD, sets up matched group to test simultaneously:
Matched group fermentation system is: glucose 10g/L, citric acid 2g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, diammonium phosphate 3.5g/L, plant sterol PS 2g/L, pH 7.2.
Being positioned over together under 29 DEG C of environment by two groups of fermentation systems, 140r/min shaken cultivation 168h obtains two groups of fermentation liquids.
3AD mole of production rate comparison result:
Take two groups of fermentation liquids respectively, use high performance liquid chromatography (HPLC) detection.
After 72h, the AD mole of production rate adding CTAB-AT-PS is 40.77 ± 0.12%, is 4.06 times of matched group (10.05 ± 0.41%)；After 120h, the AD mole of production rate adding CTAB-AT-PS is 48.69 ± 1.97%, is 3.29 times of matched group (14.77 ± 0.19%)；After 168h, the AD mole of production rate adding CTAB-AT-PS is 53.84 ± 0.72%, is 2.66 times of matched group (20.22 ± 0.24%).
AD mole of production rate of table 1
Above the section Example of the present invention is described in detail, but described content has been only presently preferred embodiments of the present invention, it is impossible to be considered the practical range for limiting the present invention.All impartial changes made according to the present patent application scope and improvement etc., within all should still belonging to the patent covering scope of the present invention.
Claims (10)
1. the substrate processing method for the reaction of plant sterol Side chain cleavage, it is characterised in that: throw
Add and in advance plant sterol and attapulgite clay are carried out before plant sterol reacts common modified formation change altogether
Property attapulgite clay-plant sterol.
The most according to claim 1 a kind of at the substrate of plant sterol Side chain cleavage reaction
Reason method, it is characterised in that: described altogether modified attapulgite-plant sterol is acid modified attapulgite altogether-plant
Thing sterol, silane coupler modified attapulgite-plant sterol altogether, alkali modified attapulgite-plant sterol altogether
With the one in surfactant altogether modified attapulgite-plant sterol.
The most according to claim 2 a kind of at the substrate of plant sterol Side chain cleavage reaction
Reason method, it is characterised in that: the described plant sterol being total to input by modified attapulgite-plant sterol
It is 1:0.1～5 with the mass ratio of attapulgite clay.
The most according to claim 3 a kind of at the substrate of plant sterol Side chain cleavage reaction
Reason method, it is characterised in that: the modification procedure of described acid modified attapulgite-plant sterol altogether is:
Plant sterol and attapulgite clay are joined in acid solution according to mass ratio 1:0.1～5, described acid solution
For the one in sulphuric acid, hydrochloric acid, nitric acid or phosphoric acid, sonic oscillation, centrifugal water is washed till neutrality,
Grind to form fine powder after drying.
The most according to claim 3 a kind of at the substrate of plant sterol Side chain cleavage reaction
Reason method, it is characterised in that: the modification of described silane coupler modified attapulgite-plant sterol altogether
Step is: according to mass ratio 1:0.1～5, plant sterol and attapulgite clay are joined the vinegar of pH 3.5
In aqueous acid, adding silane coupler, described silane coupler is 3-aminopropyl three second
TMOS KH550, sonic oscillation, centrifugal water is washed till without silane coupler, grinds after drying
Wear into fine powder.
The most according to claim 3 a kind of at the substrate of plant sterol Side chain cleavage reaction
Reason method, it is characterised in that: the modification procedure of described alkali modified attapulgite-plant sterol altogether is:
Plant sterol and attapulgite clay are joined in aqueous slkali according to mass ratio 1:0.1～5, described aqueous slkali
For sodium hydroxide solution or potassium hydroxide solution, sonic oscillation, centrifugal water is washed till neutrality, is dried
After grind to form fine powder.
The most according to claim 3 a kind of at the substrate of plant sterol Side chain cleavage reaction
Reason method, it is characterised in that: the modification of described surfactant modified attapulgite-plant sterol altogether
Step is: according to mass ratio 1:0.1～5, plant sterol and attapulgite clay are joined surfactant and changes
In property solution, described surfactant is cetyl trimethylammonium bromide, after sonic oscillation,
Centrifugal water is washed till surfactant-free, grinds to form fine powder after drying.
8. according to arbitrary described a kind of for the reaction of plant sterol Side chain cleavage in claim 4-7
Substrate processing method, it is characterised in that: planting input by described modified attapulgite-plant sterol altogether
Thing sterol is 1:1～3 with the suitableeest adding proportion of attapulgite clay.
9. utilize in claim 1-7 the most described a kind of for the reaction of plant sterol Side chain cleavage
The application in plant sterol Side chain cleavage reacts of the substrate processing method.
The most according to claim 9 a kind of at the substrate of plant sterol Side chain cleavage reaction
The application in plant sterol Side chain cleavage reacts of the reason method, it is characterised in that: described modification altogether
It is 1～40g/L that attapulgite clay-plant sterol puts into concentration, and described modified attapulgite-plant sterol altogether is thrown
The plant sterol entered is preferably 1:1～3, described modified attapulgite-plant altogether with the mass ratio of attapulgite clay
Sterol the suitableeest interpolation concentration is 1.5～10g/L.
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