CN102226213A - Method for synthesizing betulinic acid by catalyzing betulin with laccase - Google Patents
Method for synthesizing betulinic acid by catalyzing betulin with laccase Download PDFInfo
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Abstract
The invention discloses a method for synthesizing betulinic acid by catalyzing betulin with laccase, which comprises the following steps: adding a betulin solution, laccase and a mediator into an Na2HPO4-citric acid buffer solution of which the pH value is 2.0-5.0; uniformly mixing to obtain a reaction solution; reacting at 25-30 DEG C for 8-28 hours on a shaking table at a rotation speed of 60-180rpm; and separating and purifying to obtain the betulinic acid, wherein measured by every liter of reaction solution, the addition of the betulin solution is 1-20mL, the addition of the laccase is 7.667-23U, and the addition of the mediator is 0.015-0.067mmol; and the mediator is one or more of diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate), 5-hydroxyiminobarbituric acid, 2-acetaminophenol and 1-hydroxybenzotriazole. The method for synthesizing betulinic acid by catalyzing betulin has the characteristics of high synthesis efficiency, short cycle, high safety and reliability and the like, and is simple to operate and convenient to control.
Description
Technical field
The present invention relates to zymin and natural product and modify the field, relate in particular to a kind of method of laccase catalysis synthesizing betulic acid from betulin through.
Background technology
(Laccase EC1.10.3.2), is the cupric polyphenoloxidase of a class degradable xylogen to laccase, has characteristic absorption, as far back as 1883, is at first found in lacquer tree by Japanese scholar Yoshida.Studies show that the catalytic oxidation of laccase needs oxygen as intermediate, laccase is in the catalysis in aerobic condition oxidation substrates, and molecular oxygen is reduced to water simultaneously.Laccase is a kind of effect substrate enzyme widely, but the multiple bio-transformation with aromatic cycle compound of catalysis according to the preliminary statistics, can have been reached 250 by the substrate type of laccase catalyzed oxidation, and also has the trend that increases.According to the structure of substrate, substrate can be reduced six classes: (1) phenol and derivative thereof: account for laccase substrate sum half, mainly be polyphenol and derivatives thereof such as neighbour, Resorcinol; (2) arylamine and derivative thereof: its constructional feature is similar to the phenols substrate, mainly is polyamino benzene and derivative thereof; (3) carboxylic acid and derivative thereof: refer generally in the aromatic acid substrate, the ortho para of aromatic ring carboxyl is connected with hydroxyl, alkoxyl group or amino; In non-aromatic carboxylic acid, be connected with phenol or arylamine class group on the carbochain.As P-coumaric acid, coffic acid etc.; (4) steroid hormone and biochrome: in organic solvent and phosphate buffered saline buffer, but fungal laccase catalyzed oxidation estradiol, estriol etc.; (5) organometallics: as ferrocene (Fc-H) and derivative FcR (R=CH thereof
2OH, COO-, CH
2Fc etc.); (6) other non-phenol substrates: except the yellow prussiate of potash of early discovery, xitix etc., also found the derivative of 1-phenyl-2-(3, the 4-Dimethoxyphenyl) hexylene glycol and indoles etc. in recent years.
Because the redox-potential of laccase is lower, only can direct oxidation in catalytic process the degrade substrate of some suboxide reduction potentials, therefore add some amboceptors in the laccase reactions system of being everlasting, these amboceptors are compounds of some suboxide reduction potentials, in the laccase catalytic oxidation process as the carrier of electron transport.Amboceptor can form active height and the intermediate of certain stability is arranged under the effect of enzyme, physical efficiencys electron gain from oxygen molecule passes to substrate in the middle of these, thereby reaches the purpose of oxidation substrates.
Trochol (betulin, lup-20 (29)-ene-3 β, 28-diol) and Betulinic acid (betulinicacid, 3 β-hydroxy-lup-20 (29)-en-28-oic acid) belong to lupane type triterpene compound, in nature, extensively exist, especially in the white birch (Betula spp.).In recent years scientific research finds that trochol and Betulinic acid show high pharmacologically active, particularly show huge potential at aspects such as anti-HIV and cancer therapy, and demonstrates and the different mechanism of action of medicine in the past.Therefore, the research of pure and mild Betulinic acid of dialogue pine gum and derivative thereof has become the focus of natural in recent years organic drug research.Especially Betulinic acid, biology and pharmacological activity with wide region, have antimalarial, anti-inflammatory and anti-tumor activity,, be regarded as the most potential new medicinal preparation comparing with some clinical applications aspect the active and anti-various tumor cell line cytotoxicities of anti-AIDS.In China, white birch has a very wide distribution, and plant resources is abundant, but research and development are very few.
The source of Betulinic acid in actual production mainly contains two classes: a class is to extract from natural phant; Another kind of is chemosynthesis.Betulinic acid is distributed widely in the various plants, as Araliaceae (Radix Et Caulis Acanthopanacis Senticosi), Betulaceae (white birch crust), Rhamnaceae (Spina Date Seed), Ebenaceae (black in vain persimmon leaf) etc.Therefore, early stage Betulinic acid extracts employing direct extraction method mostly.Yet the content of Betulinic acid in plant is extremely low, and the content of Betulinic acid only is 0.025% in the Japanese birch bark according to the literature.Direct extraction method raw material consumption amount is big, and cost height, resulting Betulinic acid foreign matter content be than higher, and is difficult for removing, and can't satisfy commercial needs.At present, with the trochol is precursor, prepares Betulinic acid through chemical synthesis and is applied in the production practice morely, though synthetic effect is better, but problems such as having complicated operation, pollute big, synthetic cost height, security is low has limited its application in practice.
Utilize the biotransformation method synthesizing betulic acid to draw attention gradually in recent years, a kind of method of synthesizing betulic acid from betulin through microbial cell bioconversion is disclosed among the patent of invention CN200810163820.7, comparing chemical synthesis has easy and simple to handle, be easy to control, with low cost, characteristics such as safe and reliable, but this method utilizes microbe whole-cell by direct growth method or resting cell method or microemulsion system method the trochol substrate to be carried out catalyzed conversion, need to cultivate in advance thalline or preparation resting cell etc. in the operating process, step is more loaded down with trivial details, and the transformation period is longer.
Summary of the invention
The invention provides a kind of method of laccase catalysis synthesizing betulic acid from betulin through, utilize laccase that the bio-transformation of trochol substrate is become Betulinic acid, this method is easy and simple to handle, and the transformation period is short, the transformation efficiency height.
A kind of method of laccase catalysis synthesizing betulic acid from betulin through comprises:
Trochol solution and laccase are added in the buffered soln, mix and obtain reaction solution, under 25~30 ℃ of conditions, react 8~28h after separation and purification obtains Betulinic acid.
Described trochol can be commercial goods or self-control product, and purity is 90%.Publication number CN101328201A Chinese patent discloses the trochol preparation method of extract of purity about 90%, adopts this method self-control trochol to have more realistic meaning as substrate of the present invention, can reduce production costs and utilize resources synthetically.
Described trochol solution is the dimethyl sulphoxide solution of trochol, and promptly the solvent of this solution is a dimethyl sulfoxide (DMSO).Dimethyl sulfoxide (DMSO) is important polar aprotic solvent, it can dissolve each other with many organic solvents and water, some reaction had effects such as acceleration, catalysis, raising yield, reform product performance, the present invention adopts the solvent of dimethyl sulfoxide (DMSO) as the trochol substrate, more helps contacting of substrate and enzyme.
Trochol concentration is 7~9mg/mL in the described trochol solution, preferred 8mg/mL; The add-on of trochol solution is counted 1~20mL with every liter of reaction solution, preferred 10mL.Select this interpolation concentration can guarantee the abundant catalysis of laccase, can effectively reduce the negative impact of dimethyl sulfoxide (DMSO) and trochol again laccase catalytic activity in the water react liquid to substrate.
Described laccase is the commercial goods, derives from white-rot fungi and belongs to variable color bolt bacterium (Trametesversicolor).The definition that enzyme is lived: under 25 ℃ of conditions, the Na of pH 3.0
2HPO
4In-the citrate buffer solution, per minute makes 1 μ mol 2, and the needed enzyme amount of 2 '-Lian nitrogen-two (3-ethyl benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) oxidation is an enzyme unit alive.The add-on of laccase is counted 7.667U~23U with every liter of reaction solution among the present invention, preferred 15.33U.
Described buffered soln is the Na of pH 2.0~5.0
2HPO
4-citrate buffer solution, the Na of preferred pH3.0
2HPO
4-citrate buffer solution.PH value of reaction system has than remarkably influenced laccase activity, and the present invention is under selected buffer conditions, and the laccase catalytic activity is the highest.The Na of described pH 2.0~5.0
2HPO
4-citrate buffer solution adopts the compound method of this area routine to prepare and gets final product.
Amboceptor is the compound of some suboxide reduction potentials, can be in the laccase catalytic oxidation process as the carrier of electron transport.The ideal amboceptor must be the good substrates of laccase, and its oxidation state must be stable with going back ortho states, and does not influence the reaction of laccase again; In addition, the redox conversion of amboceptor must be a round-robin.Amboceptor commonly used is some phenol type compound and heterocycles, as porphyrins etc.These amboceptor materials have derives from secondary fungus metabolite or lignin degradation product, as syringaldehyde, acetovanillon and Syringylethanone etc.; What have comes from the artificial-synthetic compound, as 2, and 2 '-Lian nitrogen-two (3-ethyl benzothiazole-6-sulfonic acid) di-ammonium salts, I-hydroxybenzotriazole, violuric acid, N-hydroxy-n-ethanoyl aniline, N-hydroxybutyranilide etc.Among the present invention, in reaction solution, add amboceptor, described amboceptor is selected from 2,2 '-Lian nitrogen-two (3-ethyl benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), murexide, in 2-acetaminophenol and the I-hydroxybenzotriazole one or more can directly adopt the commercially available prod, as the related products of Sigma company production.The add-on of every liter of reaction solution mesosome is 0.015~0.067mmol.
The preferred murexide of described amboceptor, the add-on of murexide helps laccase catalysis synthesizing betulic acid from betulin through most in every liter of preferred 0.067mmol of reaction solution under this condition.
Described temperature of reaction is preferably 28 ℃; The described reaction times is preferably 8h.
Described reaction solution places rotary shaking table to react, and shaking speed is 60~180rpm, and preferred 120rpm is convenient to substrate in the reaction process, laccase and amboceptor three's thorough mixing.
Described separation and purification process comprises: reacted reaction solution is carried out centrifugal treating, get supernatant liquor, use ethyl acetate extraction, extraction liquid obtains Betulinic acid through concentrating.Adopt method of extraction that product is carried out separation and purification, simple to operation, and can from reaction solution, effectively isolate institute's synthetic Betulinic acid product, be convenient to follow-up quantitative analysis.
The detection method of trochol and Betulinic acid is with reference to disclosed relevant portion among the patent of invention CN200810163820.7 in the inventive method.The trochol quality (mg) * 100% of the Betulinic acid quality (mg) of Betulinic acid yield (%)=generation/adding.
Compared with prior art, the present invention has following beneficial effect: the inventive method is utilized laccase catalysis synthesizing betulic acid from betulin through, have the combined coefficient height, simple to operate, be convenient to control, advantage such as the cycle is short, safe and reliable, for the acid of industrial production of white pine gum provides theoretical foundation and technology prerequisite, the while also provides new approaches for the structural modification of pentacyclic triterpene natural active matter.
Embodiment
The Betulinic acid yield relatively under the different amboceptor conditions of embodiment 1-4
To 30mL, pH is 3.0 Na
2HPO
4(concentration is 8mg/mL to add the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution successively, trochol purity is 90%), (Sigma company produces 50 μ L laccase solution, derive from white-rot fungi and belong to variable color bolt bacterium (Trametesversicolor), down together, concentration is 4.6U/mL) and the different types of amboceptor solution of 200 μ L (concentration is 5mmol/L), wherein used amboceptor is respectively 2,2 '-Lian nitrogen-two (3-ethyl benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), murexide, 2-acetaminophenol or I-hydroxybenzotriazole mix and obtain reaction solution.Reaction solution reacts 8h in 28 ℃ on the rotary shaking table of rotating speed 120rpm, and reacted reaction solution is got supernatant liquor through 3000rpm frozen centrifugation 30 minutes, uses ethyl acetate extraction, and extraction liquid concentrates through rotary evaporation and obtains Betulinic acid.Employing RP-HPLC method is carried out quantitative synchronous detection to the content of trochol and Betulinic acid in the gained sample, the results are shown in Table 1.
The Betulinic acid yield relatively under the different amboceptor conditions of table 1
Sequence number | Amboceptor | Betulinic acid yield (%) |
Embodiment 1 | ABTS | 7.29 |
Embodiment 2 | Murexide | 7.35 |
Embodiment 3 | The 2-acetaminophenol | 7.02 |
Embodiment 4 | I-hydroxybenzotriazole | 6.63 |
As can be seen from Table 1, add the efficiency of pcr product difference that different types of amboceptor obtains.When selecting for use murexide to be amboceptor, the yield of product Betulinic acid is the highest; When selecting for use I-hydroxybenzotriazole to be amboceptor, yield is then lower.
The Betulinic acid yield relatively under the different laccase add-ons of embodiment 5-7
To 30mL, pH is 3.0 Na
2HPO
4(concentration is 8mg/mL to add the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution successively, trochol purity is 90%), laccase solution (concentration is 4.6U/mL) and 200 μ L amboceptor murexide solution (concentration is 5mmol/L), wherein laccase solution add-on is respectively 50 μ L, 100 μ L, 150 μ L mix and obtain reaction solution.Reaction solution reacts 8h in 28 ℃ on the rotary shaking table of rotating speed 120rpm, and reacted reaction solution is got supernatant liquor through 3000rpm frozen centrifugation 30 minutes, uses ethyl acetate extraction, and extraction liquid concentrates through rotary evaporation and obtains Betulinic acid.Employing RP-HPLC method is carried out quantitative synchronous detection to the content of trochol and Betulinic acid in the gained sample, the results are shown in Table 2.
The Betulinic acid yield relatively under the different laccase add-ons of table 2
Sequence number | Laccase add-on (U) | Betulinic acid yield (%) |
Embodiment 5 | ?0.23 | 7.35 |
Embodiment 6 | ?0.46 | 12.84 |
Embodiment 7 | ?0.69 | 7.94 |
As can be seen from Table 2, the laccase add-on has certain influence to the Betulinic acid yield, and when adding laccase amount was 0.46U in the 30mL reaction solution, it is maximum that the Betulinic acid yield reaches.
The Betulinic acid yield relatively under the different amboceptor add-ons of embodiment 8-10
To 30mL, pH is 3.0 Na
2HPO
4(concentration is 8mg/mL to add the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution successively, trochol purity is 90%), 100 μ L laccase solution (concentration is 4.6U/mL) and amboceptor murexide solution (concentration is 5mmol/L), its mesosome murexide solution add-on is respectively 0 μ L, 200 μ L, 400 μ L mix and obtain reaction solution.Reaction solution reacts 8h in 28 ℃ on the rotary shaking table of rotating speed 120rpm, and reacted reaction solution is got supernatant liquor through 3000rpm frozen centrifugation 30 minutes, uses ethyl acetate extraction, and extraction liquid concentrates through rotary evaporation and obtains Betulinic acid.Employing RP-HPLC method is carried out quantitative synchronous detection to the content of trochol and Betulinic acid in the gained sample, the results are shown in Table 3.
The Betulinic acid yield relatively under the different amboceptor add-ons of table 3
Sequence number | Amboceptor add-on (mmol) | Betulinic acid yield (%) |
Embodiment 8 | 0 | 5.21 |
Embodiment 9 | 0.001 | 12.84 |
Embodiment 10 | 0.002 | 13.40 |
As can be seen from Table 3, laccase catalysis synthesizing betulic acid ability significantly strengthens behind the adding amboceptor, and simultaneously, the amboceptor add-on also has certain influence to the Betulinic acid yield, when adding amboceptor amount is 0.002mmol in the 30mL reaction solution, and Betulinic acid conversion yield maximum.
The Betulinic acid yield relatively under the different substrate add-ons of embodiment 11-13
To 30mL, pH is 3.0 Na
2HPO
4(concentration is 8mg/mL to add the dimethyl sulphoxide solution of trochol in the-citrate buffer solution successively, trochol purity is 90%), 100 μ L laccase solution (concentration is 4.6U/mL) and 200 μ L amboceptor murexide solution (concentration is 5mmol/L), wherein trochol solution add-on is respectively 37.5 μ L, 300 μ L, 562.5 μ L mixes and obtains reaction solution.Reaction solution reacts 8h in 28 ℃ on the rotary shaking table of rotating speed 120rpm, and reacted reaction solution is got supernatant liquor through 3000rpm frozen centrifugation 30 minutes, uses ethyl acetate extraction, and extraction liquid concentrates through rotary evaporation and obtains Betulinic acid.Employing RP-HPLC method is carried out quantitative synchronous detection to the content of trochol and Betulinic acid in the gained sample, the results are shown in Table 4.
The Betulinic acid yield relatively under the different substrate add-ons of table 4
Sequence number | Substrate solution add-on (μ L) | Betulinic acid yield (%) |
Embodiment 11 | ?37.5 | 5.21 |
Embodiment 12 | ?300 | 12.84 |
Embodiment 13 | ?562.5 | 7.94 |
Table 4 shows that the trochol concentration of substrate has certain influence to the Betulinic acid yield in the reaction solution, when adding the trochol solution 300 μ L of concentration 8mg/mL in the 30mL reaction solution, and Betulinic acid conversion yield maximum.
The Betulinic acid yield relatively under the embodiment 14-16 different rotating speeds condition
To 30mL, pH is 3.0 Na
2HPO
4(concentration is 8mg/mL to add the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution successively, trochol purity is 90%), 100 μ L laccase solution (concentration is 4.6U/mL) and 200 μ L amboceptor murexide solution (concentration is 5mmol/L) mix and obtain reaction solution.Reaction solution reacts 8h in 28 ℃ on the rotary shaking table of different rotating speeds, and its medium speed is chosen 60rpm respectively, 120rpm, 180rpm, reacted reaction solution was through 3000rpm frozen centrifugation 30 minutes, get supernatant liquor, use ethyl acetate extraction, extraction liquid concentrates through rotary evaporation and obtains Betulinic acid.Employing RP-HPLC method is carried out quantitative synchronous detection to the content of trochol and Betulinic acid in the gained sample, the results are shown in Table 5.
The Betulinic acid yield relatively under the table 5 different rotating speeds condition
Sequence number | Rotating speed (rpm) | Betulinic acid yield (%) |
Embodiment 14 | 60 | 12.11 |
Embodiment 15 | 120 | 12.84 |
Embodiment 16 | 180 | 10.67 |
As shown in Table 5, reaction solution reacts under the different rotating speeds condition, and the difference of Betulinic acid yield is little, and the Betulinic acid efficiency of pcr product is slightly high under the 120rpm rotating speed.
The Betulinic acid yield relatively under the embodiment 17-19 differential responses time conditions
To 30mL, pH is 3.0 Na
2HPO
4(concentration is 8mg/mL to add the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution successively, trochol purity is 90%), 100 μ L laccase solution (concentration is 4.6U/mL) and 200 μ L amboceptor murexide solution (concentration is 5mmol/L) mix and obtain reaction solution.Reaction solution reacts 8h in 28 ℃ respectively on the rotary shaking table of rotating speed 120rpm, 18h, and 28h, reacted reaction solution is got supernatant liquor through 3000rpm frozen centrifugation 30 minutes, uses ethyl acetate extraction, and extraction liquid concentrates through rotary evaporation and obtains Betulinic acid.Employing RP-HPLC method is carried out quantitative synchronous detection to the content of trochol and Betulinic acid in the gained sample, the results are shown in Table 6.
The Betulinic acid yield relatively under the table 6 differential responses time conditions
Sequence number | Reaction times (h) | Betulinic acid yield (%) |
Embodiment 17 | 8 | 13.39 |
Embodiment 18 | 18 | 12.84 |
Embodiment 19 | 28 | 12.34 |
As can be seen from Table 6, along with the growth in reaction times, the Betulinic acid yield is on a declining curve, and the Betulinic acid efficiency of pcr product is the highest when reaction 8h.
Claims (10)
1. the method for a laccase catalysis synthesizing betulic acid from betulin through comprises:
Trochol solution and laccase are added in the buffered soln, mix and obtain reaction solution, under 25~30 ℃ of conditions, react 8~28h after separation and purification obtains Betulinic acid.
2. method according to claim 1 is characterized in that: described trochol solution reaches the dimethyl sulphoxide solution of 7~9mg/mL for trochol concentration; In every liter of reaction solution, the add-on of trochol solution is 1~20mL.
3. method according to claim 1 is characterized in that: the add-on of described laccase is counted 7.667U~23U with every liter of reaction solution.
4. method according to claim 3 is characterized in that: the add-on of described laccase is counted 15.33U with every liter of reaction solution.
5. method according to claim 1 is characterized in that: described buffered soln is the Na of pH2.0~5.0
2HPO
4-citrate buffer solution.
6. method according to claim 1 is characterized in that: trochol solution, laccase and amboceptor are added in the buffered soln, mix and obtain reaction solution, react 8~28h after separation and purification obtains Betulinic acid under 25~30 ℃ of conditions;
Wherein, described amboceptor is 2,2 '-Lian nitrogen-two (3-ethyl benzothiazole-6-sulfonic acid) di-ammonium salts, murexide, one or more in 2-acetaminophenol and the I-hydroxybenzotriazole.
7. method according to claim 6 is characterized in that: the add-on of described amboceptor is counted 0.015~0.067mmol with every liter of reaction solution.
8. method according to claim 6 is characterized in that: described amboceptor is a murexide.
9. method according to claim 1 is characterized in that: described reaction solution places shaking table to react, and shaking speed is 60~180rpm.
10. method according to claim 1 is characterized in that: described separation and purification comprises: reacted reaction solution is carried out centrifugal treating, get supernatant liquor, use ethyl acetate extraction, extraction liquid obtains Betulinic acid through concentrating.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102643886A (en) * | 2012-04-13 | 2012-08-22 | 东华大学 | Method for preparing polymerized rutin by laccase mediator system catalysis |
CN110882515A (en) * | 2019-11-07 | 2020-03-17 | 桂林理工大学 | Method for improving degradation rate of diethylstilbestrol degraded by laccase by using acetosyringone and application |
CN111302499A (en) * | 2019-11-07 | 2020-06-19 | 桂林理工大学 | Method for rapidly catalyzing and degrading diethylstilbestrol |
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CN101457250A (en) * | 2008-12-25 | 2009-06-17 | 浙江大学 | Method for synthesizing betulic acid from betulin through microbial cell bioconversion |
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CN101457250A (en) * | 2008-12-25 | 2009-06-17 | 浙江大学 | Method for synthesizing betulic acid from betulin through microbial cell bioconversion |
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CHEN QI-HE ET AL: "The betulinic acid production from betulin through biotransformation by fungi", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102643886A (en) * | 2012-04-13 | 2012-08-22 | 东华大学 | Method for preparing polymerized rutin by laccase mediator system catalysis |
CN110882515A (en) * | 2019-11-07 | 2020-03-17 | 桂林理工大学 | Method for improving degradation rate of diethylstilbestrol degraded by laccase by using acetosyringone and application |
CN111302499A (en) * | 2019-11-07 | 2020-06-19 | 桂林理工大学 | Method for rapidly catalyzing and degrading diethylstilbestrol |
CN111302499B (en) * | 2019-11-07 | 2022-04-08 | 桂林理工大学 | Method for rapidly catalyzing and degrading diethylstilbestrol |
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