CN101709322A - Method for synthesizing betulic acid by carrying out biocatalysis on betulin - Google Patents
Method for synthesizing betulic acid by carrying out biocatalysis on betulin Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
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Abstract
The invention discloses a method for synthesizing betulic acid by carrying out biocatalysis on betulin, comprising the following steps: (1) preparing armillaria luteo-uirens ZJUQH wet cells; (2) adding armillaria luteo-uirens ZJUQH wet cells into ionic liquid or a two-phase system for pre-culture to obtain a pre-culture system; (3) adding betulin solution in the pre-culture system for fermentation culture for 6-24 hours at 25-30 DEG C to obtain transformed culture solution; (4) processing the transformed culture solution to obtain betulic acid. The two-phase system is mixed solvent of ionic liquid and normal hexane, or mixed solvent of ionic liquid and phosphoric acid buffer solution. Compared with a conventional method, the inventive method has higher product yield, shorter catalytic reaction time, simple product separation, lower volatility of ionic liquid system, better biocompatibility and no pollution to the environment.
Description
Technical field
The present invention relates to biotechnology and microbial fermentation field, relate in particular to a kind of method of synthesizing betulic acid by carrying out biocatalysis on betulin.
Background technology
Biocatalysis is to utilize enzyme or microorganism cells catalysis to carry out the process of certain chemical reaction, has advantages such as specificity is strong, catalytic efficiency is high, environmental friendliness, operational condition gentleness.Compare with the biocatalytic reaction of aqueous phase, the nonaqueous phase biocatalysis has its unique advantages.But traditional organic solvent is understood activity and the selectivity of restriction enzyme usually, and can cause a large amount of industrial pollution.
Ionic liquid (Ionic liquids, ILs) as the novel green solvent, it is the salt that constitutes by organic cation and inorganic anion or organic anion, under room temperature or lower temperature (＜100 ℃), be in a liquid state, have insignificant vapour pressure, high thermal stability, high chemical stability and chemical compound lot had characteristics such as good solubility, reveal many high-performances as the biocatalytic reaction media table, cause extensive attention in recent years.
2000, reported first such as Cull ionic liquid 1-butyl-3-Methylimidazole hexafluorophosphate ([bmim] [PF
6])/the water diphasic system in Rhodococcus R312 cell catalysis 1, the hydrolysis of 3-dicyanobenzenes generates the reaction of 3-cyano group benzamide, and finds [bmim] [PF
6] toxicity of pair cell is far smaller than organic solvents such as toluene (Cull SG, et a1.Biotechnol Bioeng 2000,69 (2): 227-233).Subsequently, the report of biocatalytic reaction day by day increases in the relevant ionic liquid, and major part studies show that the whole cell of enzyme or microorganism has higher activity, selectivity and stability (Manpreet S in ionic liquid, et a1.J Mol Catal B:Enzym 2009,56:294-299; He JY, et al.Process Biochem2009,44:316-321).
Publication number is to disclose in the Chinese patent application of CN101319236A that a kind of to produce bacterium with the selectivity carbonyl reductase be starting strain, in water/ionic liquid two-phase system, with the prochiral ketone is the corresponding chiral alcohol of substrate reduction preparation, improve transformation efficiency, concentration and the enantiomeric excess value of reaction product, accelerated the process of reaction.
Betulinic acid (betulinic acid) is very valuable natural compounds, it has antimalarial, anti-inflammatory and anti-tumor activity, especially can be comparable aspect the active and anti-various tumor cell line cytotoxicities of anti-AIDS with some clinical applications, be regarded as the most potential new medicinal preparation.At present, the acquisition of Betulinic acid is many to be precursor with the abundant trochol of occurring in nature, synthetic through chemical process, though productive rate is higher, but problems such as ubiquity complicated operation, pollution is big, cost is high, security is low have limited its application in production practice.Microbial transformation is carried out structural modification by specificity katalaze enzyme system complicated in the microorganism whole cell to active skull cap components, is widely used in the fields such as asymmetric synthesis, new drug development, the prediction of drug metabolism external model of conversion, the biocatalysis of medicine precursor compound.Exploration is that precursor has become present research emphasis by microorganism catalysis approach synthesizing betulic acid with the trochol.
Publication number is to have screened in the Chinese patent application of CN101457250A to obtain the yellowish green armillaria mellea of a strain (Armillaria luteo-virens Sacc) ZJUQH, energy catalysis synthesizing betulic acid from betulin through in aqueous phase system, and utilize the response surface method further to optimize fermentation condition.Because substrate solution solubleness in aqueous phase system of organic phase is not high, not only influence the katalysis of enzyme to substrate, and to the certain murder by poisoning of microorganism cells existence, cause the productive rate of bacterial strain that previous experiments obtains and fermentation condition catalysis synthesizing betulic acid from betulin through still lower, and conversion reaction is chronic.For this reason, the method for bio-transformation synthesizing betulic acid from betulin through is still waiting further improvement, in the hope of improving the conversion yield of Betulinic acid.
Summary of the invention
The invention provides a kind of method of synthesizing betulic acid by carrying out biocatalysis on betulin, utilize the two-phase system of green solvent ionic liquid or ionic liquid and other solvent composition to substitute the reaction medium of traditional aqueous phase system as yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid from betulin through, the trochol bio-transformation is become Betulinic acid, have that simple to operate, product is easy to separate, a conversion condition gentleness, transformation efficiency height, short, the safe characteristics of transformation time.
Armillaria luteo-virens used in the present invention (Armillaria luteo-virens Sacc.) ZJUQHCGMCC No.1884, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 08th, 2006, preserving number is CGMCC No.1884, and it obtains, cultural method and form have a detailed description in Chinese invention patent CN200610155189.7.
A kind of method of synthesizing betulic acid by carrying out biocatalysis on betulin may further comprise the steps:
(1) Armillaria luteo-virens ZJUQH is obtained seed culture fluid after overactivation and seed culture, obtain Armillaria luteo-virens ZJUQH wet cell through frozen centrifugation again;
(2), obtain pre-culture system at ionic liquid or contain in the ion liquid two-phase system and to add Armillaria luteo-virens ZJUQH wet cell and cultivate in advance;
(3) in pre-culture system, add trochol solution, the nutrient solution after 25 ℃～30 ℃ fermentation culture obtained transforming in 6 hours～24 hours;
(4) nutrient solution after the conversion obtains Betulinic acid through aftertreatment;
Wherein, described trochol consumption is counted 15mg～150mg whenever to lift away from sub-liquid or to contain ion liquid two-phase system.
Ionic liquid is as the solvent of biocatalytic reaction, and application model has usually: neat solvent system, as the cosolvent of water, form two-phase system or heterogeneous systems with other organic solvents.Need select suitable ionic liquid application model at concrete biocatalytic reaction, bio-transformation obtains the characteristics of the conversion reaction of Betulinic acid according to trochol in the present invention, selects suitable reaction system, improves transformation efficiency, shortens transformation time.
The described mixed solvent that ion liquid two-phase system is ionic liquid and normal hexane or the mixed solvent of ionic liquid and phosphate buffer solution, the mixed solvent of preferred ion liquid and normal hexane of containing.
Described phosphate buffer solution adopts the compound method of this area routine to prepare and gets final product.
Suitable ionic liquid concentration can strengthen the activity and the selectivity of enzyme in the two-phase system, and therefore, the described concentration expressed in percentage by volume that contains ion liquid two-phase system intermediate ion liquid is preferably 3%～98%, and more preferably 40%～60%, most preferably 50%.
The catalysis of oxydo-reductase needs coenzyme usually, but the multiple desaturase and the coenzyme that contain the catalytic oxidation-reduction reaction in the microorganism cells, can save the purification procedures of enzyme with the whole cell of microorganism as biological catalyst, not need to add simultaneously the regenerating coenzyme system.Yet the coenzyme content in the microorganism cells is limited, adds the recycling that co-substrate helps coenzyme.Therefore, can add co-substrate in described two-phase system, the addition that whenever lifts away from sub-liquid or contain co-substrate in the ion liquid two-phase system is 0.05mol～0.1mol.
Wherein, described co-substrate is selected from a kind of in glucose, ethanol, propyl carbinol, glycerine, sucrose, the Virahol.
Described ionic liquid is selected from 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIM] BF
4), 1-butyl-3-Methylimidazole hexafluorophosphate ([BMIM] PF
6), 1-ethyl-3-methyl imidazolium tetrafluoroborate ([EMIM] BF
4), 1-octyl group-3-Methylimidazole hexafluorophosphate ([OMIM] PF
6) in a kind of.Can directly adopt the commercially available prod, as above the ionic liquid of purity 〉=99% of the prompt chemical company limited of marine origin production.
In the step (3), described trochol solution is the dimethyl sulphoxide solution of trochol, and promptly solvent is a dimethyl sulfoxide (DMSO), and trochol concentration wherein is preferably 7.5mg/mL; The trochol consumption is preferably counted 75mg whenever to lift away from sub-liquid or to contain ion liquid two-phase system.
Described trochol can select for use according to publication number be put down in writing in the CN101328201A Chinese patent application from Japanese birch bark, extract the method obtain trochol and the trochol that extracts voluntarily, purity is generally 90%, compare purchase high purity trochol standard model and have more realistic meaning as catalytic substrate, not only can reduce production costs, can also realize the comprehensive utilization of resource.
In the step (3), described fermentation culture temperature is preferably 28 ℃, and fermented incubation time is preferably 18 hours, generally can cultivate on the shaking table of rotating speed 180～220r/min (preferred 200r/min).
In the step (1), activating used slant medium is potato dextrose agar, and its raw materials quality per-cent consists of: potato 20%, glucose 2%, agar 3%; Described activation temperature is 25 ℃～30 ℃, and soak time is 4 days～6 days.
The used seed culture medium of seed culture is the potato glucose liquid nutrient medium, and its raw materials quality per-cent consists of: potato 20%, and glucose 2%, surplus is a water, the pH nature; Described seed culture temperature is 25 ℃～30 ℃, and incubation time is 3 days, generally cultivates in rotating speed is the shaking table of 120r/min.
Armillaria luteo-virens ZJUQH after the activation is made Armillaria luteo-virens ZJUQH spore suspension insert seed culture medium, Armillaria luteo-virens ZJUQH spore suspension miospore concentration is 1 * 10
6Individual/milliliter～1 * 10
7Individual/milliliter, the volume ratio of the Armillaria luteo-virens ZJUQH spore suspension of seed culture medium and access is 10～30.
In the step (2), the consumption of described yellowish green armillaria mellea ZJUQH wet cell is counted 50g～400g whenever to lift away from sub-liquid or to contain ion liquid two-phase system, preferably counts 300g whenever to lift away from sub-liquid or to contain ion liquid two-phase system; Described pre-culture condition is to cultivate 5 minutes～15 minutes at 25 ℃～30 ℃, cultivates 10min for preferred 28 ℃, generally can cultivate on rotating speed 180～220r/min (preferred 200r/min) shaking table.
In the step (4), described aftertreatment comprises: the nutrient solution after will transforming carries out centrifugal treating and obtains supernatant liquor, regulates pH to 3～4, obtains extraction liquid behind the ethyl acetate extraction, obtains Betulinic acid through concentrating.Can adopt general acid, alkali when regulating the pH value, as sodium hydroxide, hydrochloric acid etc.
Trochol, Betulinic acid content synchronization detecting method are in the inventive method: the nutrient solution after transforming in the step (4) is through aftertreatment, in the 10ml volumetric flask, adopt reversed-phased high performace liquid chromatographic (RP-HPLC) to detect the content of trochol, Betulinic acid in the gains with methanol constant volume.The RP-HPLC chromatographic condition: chromatographic column is Diamonsil C18 (250mm * 4.6mm, 5 μ m); Moving phase is acetonitrile/water (volume ratio)=91/9; Flow velocity 1.0mL/min; Detect wavelength 210.1nm; 25 ℃ of column temperatures; Sample size 10 μ l; Run sample time 30～45min.
The present invention has following beneficial effect:
The present invention selects ionic liquid or contains ion liquid two-phase non-aqueous system to be used for microorganism intact cell catalysis synthesizing betulic acid from betulin through, compare with employing ortho-water phase system, have the following advantages: (1) ionic liquid or contain ion liquid two-phase system and help improving transformation efficiency and efficiency of pcr product, the productive rate of Betulinic acid can reach more than 12.17%, compares the ortho-water phase system and has improved 30.58%; (2) ionic liquid or contain ion liquid two-phase system and shortened the catalyzed reaction time greatly, transformation time is no more than 24 hours; (3) ionic liquid or contain in the ion liquid two-phase system product and separate simply, ionic liquid is easy to recycling use; (4) ionic liquid or to contain ion liquid two-phase system not volatile, good biocompatibility, free from environmental pollution, for the raising of trochol biocatalysis synthesizing betulic acid productive rate provides new approaches, provide theoretical foundation for the application of ion liquid system in Betulinic acid catalysis is synthetic simultaneously.
Description of drawings
Fig. 1 is the schematic flow sheet of the inventive method.
Embodiment
Yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid from betulin through in embodiment 1～8 different two-phase systems
(1) (its raw materials quality per-cent consists of: potato 20% Armillaria luteo-virens ZJUQH to be inserted the potato dextrose agar of sterilizing, glucose 2% and agar 3%), at 28 ℃, activate and make Armillaria luteo-virens ZJUQH spore suspension after 4 days, (its raw materials quality per-cent consists of: potato 20% to insert the potato glucose liquid nutrient medium then, glucose 2%, surplus is a water, the pH nature), in 28 ℃, rotating speed are the rotary shaking table of 120r/min, cultivated 3 days, obtain seed culture fluid.Armillaria luteo-virens ZJUQH spore suspension miospore concentration is 1 * 10
6Individual/milliliter, the volume ratio of the Armillaria luteo-virens ZJUQH spore suspension of potato glucose liquid nutrient medium and access is 30.
Seed culture fluid through 3000r/min frozen centrifugation 30 minutes, is removed fermented liquid after the sterilized water washing 3 times and obtained Armillaria luteo-virens ZJUQH wet cell.
(2) add the mixed solvent of 5mL ionic liquid and phosphoric acid buffer or the mixed solvent of ionic liquid and normal hexane in the tool plug test tube and form two-phase system, the volumetric concentration of two-phase system intermediate ion liquid is 50%, aseptic condition adds above-mentioned yellowish green armillaria mellea ZJUQH wet cell down, the consumption of yellowish green armillaria mellea ZJUQH wet cell is counted 200g with every liter of two-phase system, in 28 ℃, the pre-10min that cultivates obtains pre-culture system on the rotary shaking table of rotating speed 200r/min.
(3) adding 0.1mL concentration in above-mentioned pre-culture system is the dimethyl sulphoxide solution of 7.5mg/mL trochol, continues at 28 ℃, and the nutrient solution after 18h obtains transforming is cultivated in the rotary shaking table top fermentation of rotating speed 200rpm.
(4) nutrient solution after will transforming through centrifugal, transfer aftertreatments such as pH to 3, ethyl acetate extraction and rotary evaporation to obtain Betulinic acid.
With [BMIM] BF
4, [BMIM] PF
6, [EMIM] BF
4, [OMIM] PF
6Four kinds of ionic liquids and phosphoric acid buffer, two kinds of solvents of normal hexane are formed two-phase system respectively to carry out the catalysis of Betulinic acid synthetic, the results are shown in Table 1:
Table 1
| Embodiment | Two-phase reaction system | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) |
| ??1 | ??[BMIM]BF 4/ phosphoric acid buffer | ??93.64 | ??2.81 |
| ??2 | ??[BMIM]PF 6/ phosphoric acid buffer | ??66.36 | ??2.71 |
| ??3 | ??[EMIM]BF 4/ phosphoric acid buffer | ??76.10 | ??0.99 |
| ??4 | ??[OMIM]PF 6/ phosphoric acid buffer | ??43.42 | ??4.05 |
| Embodiment | Two-phase reaction system | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) |
| ??5 | ??[BMIM]BF 4/ normal hexane | ??68.67 | ??2.86 |
| ??6 | ??[BMIM]PF 6/ normal hexane | ??46.23 | ??4.77 |
| ??7 | ??[EMIM]BF 4/ normal hexane | ??48.20 | ??4.81 |
| ??8 | ??[OMIM]PF 6/ normal hexane | ??44.95 | ??4.60 |
As can be seen from Table 1, [EMIM] BF
4Yellowish green armillaria mellea ZJUQH good catalytic activity in the/normal hexane system, the productive rate of synthesizing betulic acid is higher relatively.
Yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid in the two-phase system of embodiment 9～14 different ionic liquid concentration
(1) preparation of Armillaria luteo-virens ZJUQH wet cell is with embodiment 1.
(2) mixed solvent of adding 5mL ionic liquid or ionic liquid and normal hexane forms two-phase system in the tool plug test tube, the volumetric concentration of two-phase system intermediate ion liquid is 3%～100%, aseptic condition adds above-mentioned yellowish green armillaria mellea ZJUQH wet cell down, the consumption of yellowish green armillaria mellea ZJUQH wet cell is counted 200g with every liter of two-phase system, in 28 ℃, the pre-10min that cultivates obtains pre-culture system on the rotary shaking table of rotating speed 200r/min.
(3) adding 0.05mL concentration in above-mentioned pre-culture system is the dimethyl sulphoxide solution of 7.5mg/mL trochol, continues at 28 ℃, and the nutrient solution after 18h obtains transforming is cultivated in the rotary shaking table top fermentation of rotating speed 200rpm.
(4) nutrient solution after will transforming through centrifugal, transfer aftertreatments such as pH to 4, ethyl acetate extraction and rotary evaporation to obtain Betulinic acid, the results are shown in Table 2:
Table 2
| Embodiment | Ionic liquid [EMIM] BF 4Volumetric concentration (%) | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) |
| ??9 | ??3 | ??63.79 | ??3.77 |
| ??10 | ??10 | ??86.02 | ??4.16 |
| ??11 | ??30 | ??58.31 | ??5.57 |
| ??12 | ??50 | ??71.76 | ??8.39 |
| ??13 | ??80 | ??81.54 | ??4.32 |
| ??14 | ??100 | ??54.09 | ??6.84 |
As can be seen from Table 2, ionic liquid [EMIM] BF
4In/normal hexane the two-phase system, yellowish green armillaria mellea ZJUQH transforms the reaction of trochol, at ionic liquid [EMIM] BF
4Concentration is that 50% o'clock Betulinic acid productive rate is the highest.
Yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid in embodiment 15～21 different co-substrates
(1) preparation of Armillaria luteo-virens ZJUQH wet cell is with embodiment 1.
(2) add 5mL ionic liquid [EMIM] BF in the tool plug test tube
4Form two-phase system with the mixed solvent of normal hexane, the volumetric concentration of two-phase system intermediate ion liquid is 50%, adds co-substrate simultaneously in two-phase system, and the addition of co-substrate is 0.1mol in every liter of two-phase system.Aseptic condition adds above-mentioned yellowish green armillaria mellea ZJUQH wet cell down, and the consumption of yellowish green armillaria mellea ZJUQH wet cell is counted 200g with every liter of two-phase system, and in 28 ℃, the pre-10min that cultivates obtains pre-culture system on the rotary shaking table of rotating speed 200r/min.
(3) adding 0.05mL concentration in above-mentioned pre-culture system is the dimethyl sulphoxide solution of 7.5mg/mL trochol, continues at 28 ℃, and the nutrient solution after 18h obtains transforming is cultivated in the rotary shaking table top fermentation of rotating speed 200rpm.
(4) nutrient solution after will transforming through centrifugal, transfer aftertreatments such as pH to 3.5, ethyl acetate extraction and rotary evaporation to obtain Betulinic acid.
Co-substrate is chosen glucose, ethanol, propyl carbinol, glycerine, sucrose or Virahol, compares with the reaction system of not adding co-substrate, and experimental result sees Table 3.
Table 3
| Embodiment | Co-substrate | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) |
| ??15 | Do not add | ??71.76 | ??8.39 |
| ??16 | Glucose | ??63.90 | ??6.41 |
| ??17 | Ethanol | ??93.66 | ??6.57 |
| ??18 | Propyl carbinol | ??54.13 | ??14.21 |
| ??19 | Glycerine | ??62.29 | ??5.22 |
| ??20 | Sucrose | ??56.84 | ??8.31 |
| ??21 | Virahol | ??62.14 | ??8.60 |
As can be seen from Table 3, ionic liquid [EMIM] BF
4In/normal hexane the two-phase system, add the 0.1mol/L propyl carbinol effectively improves yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid as cobasis mass-energy ability.
Embodiment 22～24 different concentration of substrate are to the influence of yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid reaction
(1) preparation of Armillaria luteo-virens ZJUQH wet cell is with embodiment 1.
(2) add 5mL ionic liquid [EMIM] BF in the tool plug test tube
4Form two-phase system with the mixed solvent of normal hexane, the volumetric concentration of two-phase system intermediate ion liquid is 50%, adds propyl carbinol simultaneously in two-phase system, and the addition of propyl carbinol is 0.1mol in every liter of two-phase system.Aseptic condition adds above-mentioned yellowish green armillaria mellea ZJUQH wet cell down, and the consumption of yellowish green armillaria mellea ZJUQH wet cell is counted 200g with every liter of two-phase system, and in 28 ℃, the pre-10min that cultivates obtains pre-culture system on the rotary shaking table of rotating speed 200r/min.
(3) in above-mentioned pre-culture system, add 0.01mL, 0.05mL respectively, 0.1mL concentration is the dimethyl sulphoxide solution of 7.5mg/mL trochol, continues at 28 ℃, the nutrient solution after 18h obtains transforming is cultivated in the rotary shaking table top fermentation of rotating speed 200rpm.
(4) nutrient solution after will transforming through centrifugal, transfer aftertreatments such as pH to 3.5, ethyl acetate extraction and rotary evaporation to obtain Betulinic acid, the results are shown in Table 4.
Table 4
| Embodiment | Substrate consumption (mL) | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) |
| ??22 | ??0.01 | ??85.81 | ??6.16 |
| ??23 | ??0.05 | ??59.02 | ??11.04 |
| ??24 | ??0.1 | ??48.19 | ??4.81 |
As can be seen from Table 4, ionic liquid [EMIM] BF
4In/normal hexane the two-phase system, when the trochol substrate solution that adds was 0.05mL, the catalysis that helps Betulinic acid most was synthetic.
Embodiment 25～29 DIFFERENT WET cell concns are to the influence of yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid reaction
(1) preparation of Armillaria luteo-virens ZJUQH wet cell is with embodiment 1.
(2) add 5mL ionic liquid [EMIM] BF in the tool plug test tube
4Form two-phase system with the mixed solvent of normal hexane, the volumetric concentration of two-phase system intermediate ion liquid is 50%, adds propyl carbinol simultaneously in two-phase system, and the addition of propyl carbinol is 0.1mol in every liter of two-phase system.Aseptic condition adds above-mentioned yellowish green armillaria mellea ZJUQH wet cell down, the consumption of yellowish green armillaria mellea ZJUQH wet cell is respectively 50g, 100g, 200g, 300g, 400g in every liter of two-phase system, in 28 ℃, the pre-10min that cultivates obtains pre-culture system on the rotary shaking table of rotating speed 200r/min.
(3) adding 0.05mL concentration in above-mentioned pre-culture system is the dimethyl sulphoxide solution of 7.5mg/mL trochol, continues at 28 ℃, and the nutrient solution after 18h obtains transforming is cultivated in the rotary shaking table top fermentation of rotating speed 200rpm.
(4) nutrient solution after will transforming through centrifugal, transfer aftertreatments such as pH to 3.5, ethyl acetate extraction and rotary evaporation to obtain Betulinic acid, the results are shown in Table 5.
Table 5
| Embodiment | Wet cell consumption (g/L) | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) |
| ??25 | ??50 | ??71.99 | ??4.74 |
| Embodiment | Wet cell consumption (g/L) | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) |
| ??26 | ??100 | ??67.57 | ??9.77 |
| ??27 | ??200 | ??59.02 | ??11.04 |
| ??28 | ??300 | ??67.35 | ??12.17 |
| ??29 | ??400 | ??72.19 | ??8.87 |
As can be seen from Table 5, ionic liquid [EMIM] BF
4In/normal hexane the two-phase system, when the yellowish green armillaria mellea ZJUQH wet cell amount of adding was 300g/L, the productive rate of Betulinic acid was the highest.
The transformation time process of embodiment 30～33 yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid reactions
(1) preparation of Armillaria luteo-virens ZJUQH wet cell is with embodiment 1.
(2) add 5mL ionic liquid [EMIM] BF in the tool plug test tube
4Form two-phase system with the mixed solvent of normal hexane, the volumetric concentration of two-phase system intermediate ion liquid is 50%, adds propyl carbinol simultaneously in two-phase system, and the addition of propyl carbinol is 0.1mol in every liter of two-phase system.Aseptic condition adds above-mentioned yellowish green armillaria mellea ZJUQH wet cell down, and the consumption of yellowish green armillaria mellea ZJUQH wet cell is counted 200g with every liter of two-phase system, and in 28 ℃, the pre-10min that cultivates obtains pre-culture system on the rotary shaking table of rotating speed 200r/min.
(3) add the dimethyl sulphoxide solution that 0.05mL concentration is the 7.5mg/mL trochol in above-mentioned pre-culture system, continue at 28 ℃, 6～24h is cultivated in the rotary shaking table top fermentation of rotating speed 200rpm, and the nutrient solution after obtaining transforming is got sample one time every 6h.
(4) nutrient solution after will transforming through centrifugal, transfer aftertreatments such as pH to 3.5, ethyl acetate extraction and rotary evaporation to obtain Betulinic acid, the results are shown in Table 6.
Table 6
| Embodiment | Transformation time (h) | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) |
| ??30 | ??6 | ??63.71 | ??3.75 |
| ??31 | ??12 | ??68.54 | ??5.87 |
| ??32 | ??18 | ??59.02 | ??11.04 |
| ??33 | ??24 | ??57.39 | ??4.55 |
As can be seen from Table 6, ionic liquid [EMIM] BF
4In/normal hexane the two-phase system, yellowish green armillaria mellea ZJUQH catalyzed conversion trochol 18h can obtain higher Betulinic acid productive rate.
Yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid in embodiment 34 two-phase systems
(1) preparation of Armillaria luteo-virens ZJUQH wet cell is with embodiment 1.
(2) add 5mL ionic liquid [EMIM] BF in the tool plug test tube
4Form two-phase system with the mixed solvent of normal hexane, the volumetric concentration of two-phase system intermediate ion liquid is 50%, adds propyl carbinol simultaneously in two-phase system, and the addition of propyl carbinol is 0.1mol in every liter of two-phase system.Aseptic condition adds above-mentioned yellowish green armillaria mellea ZJUQH wet cell down, and the consumption of yellowish green armillaria mellea ZJUQH wet cell is counted 300g with every liter of two-phase system, and in 28 ℃, the pre-10min that cultivates obtains pre-culture system on the rotary shaking table of rotating speed 200r/min.
(3) add the dimethyl sulphoxide solution that 0.05mL concentration is the 7.5mg/mL trochol in above-mentioned pre-culture system, continue at 28 ℃, the nutrient solution after 18h obtains transforming is cultivated in the rotary shaking table top fermentation of rotating speed 200rpm.
(4) nutrient solution after will transforming through centrifugal, transfer aftertreatments such as pH to 3.5, ethyl acetate extraction and rotary evaporation to obtain Betulinic acid, the results are shown in Table 7.
Yellowish green armillaria mellea ZJUQH catalysis synthesizing betulic acid in Comparative Examples 1 aqueous phase system
(1) activation of Armillaria luteo-virens ZJUQH is with embodiment 1, Armillaria luteo-virens ZJUQH after the activation is made spore suspension insert 30mL potato liquid nutrient medium (potato mass concentration 20%, pH5.0, tween 80 mass concentration 0.57%, surplus is a water), place 28 ℃ of temperature, cultivated 3 days on the rotary shaking table of rotating speed 120r/min, obtain seed culture fluid.
(2) adding concentration under the aseptic condition in above-mentioned seed culture fluid is the dimethyl sulphoxide solution of 7.5mg/mL trochol, the consumption of the dimethyl sulphoxide solution of trochol is counted 15mL with every liter of seed culture fluid, continue at 28 ℃, the nutrient solution after obtaining transforming in 3 days is cultivated in the rotary shaking table top fermentation of rotating speed 120r/min.
(3) nutrient solution after will transforming through centrifugal, transfer aftertreatments such as pH to 3.5, ethyl acetate extraction and rotary evaporation to obtain Betulinic acid, the results are shown in Table 7.
Table 7
| Reaction system | Trochol transformation efficiency (%) | Betulinic acid productive rate (%) | |
| Embodiment 34 | ??[EMIM]BF 4/ normal hexane system | ??67.35 | ??12.17 |
| Comparative Examples 1 | Aqueous phase system | ??66.63 | ??9.32 |
As can be seen from Table 7, compare ionic liquid [EMIM] BF with the ortho-water phase system
4/ normal hexane two-phase system more helps the synthetic trochol of yellowish green armillaria mellea ZJUQH catalysis.
Claims (10)
1. the method for a synthesizing betulic acid by carrying out biocatalysis on betulin may further comprise the steps:
(1) Armillaria luteo-virens ZJUQH is obtained seed culture fluid after overactivation and seed culture, obtain Armillaria luteo-virens ZJUQH wet cell through frozen centrifugation again;
(2), obtain pre-culture system at ionic liquid or contain in the ion liquid two-phase system and to add Armillaria luteo-virens ZJUQH wet cell and cultivate in advance;
(3) in pre-culture system, add trochol solution, the nutrient solution after 25 ℃～30 ℃ fermentation culture obtained transforming in 6 hours～24 hours;
(4) nutrient solution after the conversion obtains Betulinic acid through aftertreatment;
Wherein, described trochol consumption is counted 15mg～150mg whenever to lift away from sub-liquid or to contain ion liquid two-phase system;
The described mixed solvent that ion liquid two-phase system is ionic liquid and normal hexane or the mixed solvent of ionic liquid and phosphate buffer solution of containing.
2. the method for claim 1, it is characterized in that: the concentration expressed in percentage by volume of described two-phase system intermediate ion liquid is 3%～98%.
3. method as claimed in claim 2 is characterized in that: the concentration expressed in percentage by volume of described two-phase system intermediate ion liquid is 40%～60%.
4. the method for claim 1, it is characterized in that: be added with co-substrate in described ionic liquid or the two-phase system, the addition that whenever lifts away from co-substrate in sub-liquid or the two-phase system is 0.05mol～0.1mol;
Wherein, described co-substrate is selected from a kind of in glucose, ethanol, propyl carbinol, glycerine, sucrose, the Virahol.
5. the method for claim 1 is characterized in that: described ionic liquid is selected from a kind of in 1-butyl-3-methyl imidazolium tetrafluoroborate, 1-butyl-3-Methylimidazole hexafluorophosphate, 1-ethyl-3-methyl imidazolium tetrafluoroborate, the 1-octyl group-3-Methylimidazole hexafluorophosphate.
6. the method for claim 1, it is characterized in that: in the step (3), described trochol solution is the dimethyl sulphoxide solution of trochol, and the concentration of trochol wherein is 7.5mg/mL; The trochol consumption is counted 75mg whenever to lift away from sub-liquid or two-phase system.
7. the method for claim 1, it is characterized in that: in the step (3), described fermentation culture temperature is 28 ℃, and fermented incubation time is 18 hours.
8. the method for claim 1, it is characterized in that: in the step (1), activating used slant medium is potato dextrose agar; The used seed culture medium of seed culture is the potato glucose liquid nutrient medium; Described activation temperature is 25 ℃～30 ℃, and soak time is 4 days～6 days; Described seed culture temperature is 25 ℃～30 ℃, and incubation time is 3 days.
9. the method for claim 1, it is characterized in that: in the step (2), the consumption of described yellowish green armillaria mellea ZJUQH wet cell is counted 50g～400g whenever to lift away from sub-liquid or two-phase system; Described pre-culture condition is to cultivate 5 minutes～15 minutes at 25 ℃～30 ℃.
10. the method for claim 1, it is characterized in that: in the step (4), described aftertreatment comprises: the nutrient solution after will transforming carries out centrifugal treating and obtains supernatant liquor, regulates pH to 3～4, obtain extraction liquid behind the ethyl acetate extraction, obtain Betulinic acid through concentrating.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102286595A (en) * | 2011-09-07 | 2011-12-21 | 浙江大学 | Method for synthesizing betulinic acid through converting cunninghamella blakesleeana into betulin |
| CN104271550A (en) * | 2011-12-14 | 2015-01-07 | 葛兰素史克有限责任公司 | Propenoate derivatives of betulin |
| CN107573397A (en) * | 2017-09-13 | 2018-01-12 | 江苏耐雀生物工程技术有限公司 | A kind of method for preparing betulic acid |
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| CN1385410A (en) * | 2002-04-10 | 2002-12-18 | 中国科学院兰州化学物理研究所 | Method for preparing phenol by directly hydrocylating benzene |
| CN1187309C (en) * | 2002-11-07 | 2005-02-02 | 华东师范大学 | Method of esterifying in ion liquid [Hmim]+ BF4- |
| CN100453636C (en) * | 2006-12-13 | 2009-01-21 | 浙江大学 | Armillaria luteo-virens and its culture process and application |
| CN101319236A (en) * | 2008-07-01 | 2008-12-10 | 江南大学 | Method for biological catalysis of unsymmetrical reduction carbon based compound in water/ion liquid diphasic system |
| CN101457250B (en) * | 2008-12-25 | 2010-11-03 | 浙江大学 | Method for synthesizing betulic acid from betulin through microbial cell bioconversion |
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| CN102286595A (en) * | 2011-09-07 | 2011-12-21 | 浙江大学 | Method for synthesizing betulinic acid through converting cunninghamella blakesleeana into betulin |
| CN102286595B (en) * | 2011-09-07 | 2013-08-28 | 浙江大学 | Method for synthesizing betulinic acid through converting cunninghamella blakesleeana into betulin |
| CN104271550A (en) * | 2011-12-14 | 2015-01-07 | 葛兰素史克有限责任公司 | Propenoate derivatives of betulin |
| CN104271550B (en) * | 2011-12-14 | 2016-09-07 | 葛兰素史克有限责任公司 | The acrylate derivative of betulin |
| CN107573397A (en) * | 2017-09-13 | 2018-01-12 | 江苏耐雀生物工程技术有限公司 | A kind of method for preparing betulic acid |
| CN107573397B (en) * | 2017-09-13 | 2019-07-30 | 江苏耐雀生物工程技术有限公司 | A method of preparing betulic acid |
| CN109370916A (en) * | 2018-12-04 | 2019-02-22 | 昆明理工大学 | A kind of process for rapid activation of halimasch aging strain |
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