CN102226213B - 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 Catalyzed synthesizing betulic acid from betulin through.
Background technology
Laccase (Laccase, EC1.10.3.2) is the cupric polyphenoloxidase of a class degradable xylogen, has characteristic absorption, and as far back as 1883, Yoshida at first found in lacquer tree by Japanese scholars.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 widely enzyme of a kind of effect substrate, 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 summarized as six classes: (1) phenol and derivative thereof: account for laccase substrate sum half, mainly be polyphenol and the derivatives thereof such as neighbour, Resorcinol; (2) arylamine and derivative thereof: its constructional feature is similar to the phenols substrate, mainly is the polyamino benzene and its derivative; (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.Such 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: such 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 in recent years the derivative of 1-phenyl-2-(3,4-Dimethoxyphenyl) hexylene glycol and indoles etc.
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, the carrier that transmits as electronics in the Laccase Catalyzed oxidising process.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 the mechanism of action different from medicine in the past.Therefore, the research of the pure and mild Betulinic acid of dialogue pine gum and derivative thereof has become the in recent years focus of natural organic drug research.Especially Betulinic acid, biology and pharmacological activity with wide region, have antimalarial, anti-inflammatory and anti-tumor activity, comparing with some clinical applications aspect anti-HIV activity and the anti-various tumor cell line cytotoxicities, be regarded as most potential new medicinal preparation.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, such as Araliaceae (Radix Et Caulis Acanthopanacis Senticosi), Betulaceae (white birch crust), Rhamnaceae (Spina Date Seed), Ebenaceae (in vain black persimmon leaf) etc.Therefore, early stage Betulinic acid extracts and mostly adopts direct extraction method.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 large, and cost is high, and resulting Betulinic acid foreign matter content is higher, and is difficult for removing, and can't satisfy commercial needs.At present, take trochol as precursor, prepare Betulinic acid through chemical synthesis and more be applied in the production practice, although synthetic effect is better, but there is complicated operation, pollutes the problems such as large, synthetic cost is high, security is low, limited its application in practice.
Utilize in recent years the biotransformation method synthesizing betulic acid to draw attention gradually, 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, the characteristics such as safe and reliable, but the 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 preculture 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 Catalyzed synthesizing betulic acid from betulin through, utilize laccase that the bio-transformation of trochol substrate is become Betulinic acid, the method is easy and simple to handle, and the transformation period is short, and transformation efficiency is high.
A kind of method of Laccase Catalyzed 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 and obtain Betulinic acid by separation and purification.
Described trochol can be commercial goods or self-control product, and purity is 90%.Publication number CN101328201A Chinese patent discloses the preparation method of the trochol 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 namely the solvent of this solution is 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 the effects such as acceleration, catalysis, raising yield, reform product performance, the present invention adopts dimethyl sulfoxide (DMSO) as the solvent of trochol substrate, more is conducive to 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 laccase to the abundant catalysis of substrate, can effectively reduce again dimethyl sulfoxide (DMSO) and trochol to the negative impact of Laccase Catalyzed activity in the water react liquid.
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 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 Catalyzed 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, the carrier that can transmit as electronics in the Laccase Catalyzed oxidising process.Desirable amboceptor must be the good substrates of laccase, and its oxidized and reduced must be stable, and does not affect again the reaction of laccase; In addition, the redox conversion of amboceptor must circulate.Amboceptor commonly used is some phenol type compound and heterocycles, such as porphyrins etc.These amboceptor materials have derives from secondary fungus metabolite or lignin degradation product, such as syringaldehyde, acetovanillon and Syringylethanone etc.; What have comes from the artificial-synthetic compound, such 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, such 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 is conducive to the Laccase Catalyzed synthesizing betulic acid from betulin through in every liter of preferred 0.067mmol of reaction solution most 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 abundant mixing.
Described separation and purification process comprises: reacted reaction solution is carried out centrifugal treating, get supernatant liquor, use ethyl acetate extraction, extraction liquid is through the concentrated Betulinic acid that obtains.Adopt the method for extraction that product is carried out separation and purification, simple to operation, and can from reaction solution, go out the Betulinic acid product that is synthesized by effective separation, 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 the Laccase Catalyzed synthesizing betulic acid from betulin through, have the combined coefficient height, simple to operate, be convenient to control, the 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 successively the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution, 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), together lower, 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 is through the concentrated Betulinic acid that obtains of rotary evaporation.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 murexide to be amboceptor, the yield of product Betulinic acid is the highest; When selecting 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 successively the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution, 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 is through the concentrated Betulinic acid that obtains of rotary evaporation.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 successively the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution, 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 is through the concentrated Betulinic acid that obtains of rotary evaporation.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 Catalyzed 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, and when adding amboceptor amount was 0.002mmol in the 30mL reaction solution, the Betulinic acid conversion yield was 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 successively the dimethyl sulphoxide solution of trochol in the-citrate buffer solution, 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 is through the concentrated Betulinic acid that obtains of rotary evaporation.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, and when adding the trochol solution 300 μ L of concentration 8mg/mL in the 30mL reaction solution, the Betulinic acid conversion yield is 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 successively the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution, 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 respectively 60rpm, 120rpm, 180rpm, reacted reaction solution was through 3000rpm frozen centrifugation 30 minutes, get supernatant liquor, use ethyl acetate extraction, extraction liquid is through the concentrated Betulinic acid that obtains of rotary evaporation.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 successively the dimethyl sulphoxide solution of 300 μ L trochols in the-citrate buffer solution, 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 respectively 8h in 28 ℃ 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 is through the concentrated Betulinic acid that obtains of rotary evaporation.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 (6)
1. the method for a Laccase Catalyzed 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 and obtain Betulinic acid by separation and purification;
Wherein, described trochol solution is the dimethyl sulphoxide solution that trochol concentration reaches 7～9mg/mL; In every liter of reaction solution, the add-on of trochol solution is 1～20mL;
The add-on of described laccase is counted 7.667U～23U with every liter of reaction solution;
Described buffered soln is the Na of pH2.0～5.0
2HPO
4-citrate buffer solution;
Described reaction solution places shaking table to react, and shaking speed is 60～180rpm.
2. method according to claim 1, it is characterized in that: the add-on of described laccase is counted 15.33U with every liter of reaction solution.
3. 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 and obtain Betulinic acid by separation and purification 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-acetoamidophenol and the I-hydroxybenzotriazole.
4. method according to claim 3, it is characterized in that: the add-on of described amboceptor is counted 0.015～0.067mmol with every liter of reaction solution.
5. method according to claim 3, it is characterized in that: described amboceptor is murexide.
6. method according to claim 1, it 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 is through the concentrated Betulinic acid that obtains.
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