CN1132928C - Two-saccharomycetes strains and its usage in preparing optically pure 2-aryl propionic acid - Google Patents
Two-saccharomycetes strains and its usage in preparing optically pure 2-aryl propionic acid Download PDFInfo
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Abstract
The present invention discloses two yeast strains of citeromyces CGMCC No. 0573 and trichosporon brassicae CGMCC No. 0574. Racemic 2-aryl propionate carries out one time or two times of serial hydrolysis reaction by cells or esterase separated from the yeast strains, and (S)-2-aryl propionic acid with high optical purity and (R)-2-aryl propionic acid with high optical purity are prepared. The present invention can simultaneously prepare single enantiomer products of the (S)-2-aryl propionic acid with high optical purity and the (R)-2-aryl propionic acid with high optical purity, and satisfy the requirements of different purposes for medicine industry or pesticide industry, the 2-aryl ethyl propionate which serves as a reaction substrate is easily synthesized, the expensive or toxic reagent does not need to be used, and thus, the costs of raw materials can be reduced. In addition, the present invention saves the racemization and the cyclic utilization of the residual reaction ester; thus, the operation steps are simplified, and the utilization rates of raw materials are enhanced.
Description
Technical field
The present invention relates to yeast strain and adopt microorganism to prepare the method for optical purity 2-arylpropionic acid.
Technical background
(available general formula is expressed as Ar-C to the 2-arylpropionic acid
*H (CH
3)-COOH, wherein Ar represents to contain the substituting group of aromatic ring, C
*The expression chiral carbon atom), be the very important chipal compounds of a class, have purposes very widely in fields such as medicine and agricultural chemicals.Two kinds of enantiomers that have been found that the 2-arylpropionic acid exist evident difference on biological activity.[2-(3-benzoyloxy phenyl)-propionic acid] is example with Ketoprofen BP 93, its (S)-enantiomorph is a kind of non-steroidal anti-inflammatory agent efficiently, anti-inflammatory activity is than (R)-mapping height more than 100 times, (R)-and Ketoprofen BP 93 then has analgesia and prevents and treats osteoporotic effect.Other 2-aryl propionic non-steroid antiphlogistic (as Naproxen Base, Ibuprofen BP/EP, fenbufen or the like) and 2-aryloxy propionic acid serial herbicide in also have similar chirality effect.
By the synthetic various chirality 2-arylpropionic acids that obtain of traditional organic chemistry method generally all is (R) of equivalent and (S) racemic mixture of two kinds of enantiomers.The classical way of resolving chiral carboxylic acid is first and the salt of a kind of Chiral Amine of individual isomer formation diastereomer, utilizes the difference of solubleness that a kind of isomer is preferentially crystallized out from solution then.Because the diastereomer crystallization process need use expensive optical homochiral resolving agent, cost is higher relatively, process is more numerous and diverse, and can produce a large amount of waste water, therefore utilize microorganism or its enzymatic chipal compounds enantioselectivity to transform method for splitting in recent years, obtained increasing development and application.Many documents and patent have related to the technology of this respect, and following two aspects are comparatively typically arranged:
(1) the lipase preparation of commodity in useization (being mainly derived from mycocandida Candida sp.) is a catalyzer.Because commercial enzyme source easily, the bibliographical information of this respect is more, but particularly often stereoselectivity is not ideal enough during Ketoprofen BP 93 being used to split the 2-arylpropionic acid, so must carry out purifying or other extra processing to enzyme.For example: document J.Am.Chem.Soc.1990,112:1990-1995 also improve the selectivity of enzyme with deoxycholate salt and acetone treatment by the purifying of enzyme; Patent U.S.Pat.5 108 916, Apr.28, and 1992 carry out purifying by ion-exchange or isoelectrofocusing to thick enzyme, obtain the higher one-component of selectivity (CSC-1 or CSC-2); Document J.Org.Chem. directly handles the stereoselectivity of improving thick enzyme with the 2-propyl alcohol; Document J.Am.Chem.Soc.1995,117:6854 have then reported the effect of crosslinked enzyme crystal resolving chiral carboxylicesters.A common drawback of aforesaid method is that commercial enzyme preparation price is higher relatively, and extra treating processes is cumbersome, and causes the vigor loss of enzyme, thereby the cost of enzyme catalyst is increased considerably, and has influenced the practical feasibility of industrial applications.
(2) directly use the microorganism cells that contains esterase that racemic substrate is carried out the stereoselectivity bio-transformation.Because microorganism cells is easy to cultivation, simple to operate, with low cost, therefore have more advantage than commercial enzyme economically, key is to screen efficient single-minded bacterium producing multi enzyme preparation.The typical document of this respect has: United States Patent (USP) U.S.Pat.5 516 690, and May14,1996 disclose a trichosporon montevideense bacterium Trichosporon labbacchii, are to be sole carbon source with ethanol, separate obtaining from soil; Transforming the Ketoprofen BP 93 ethyl ester with its nutrient solution, can to obtain purity be (S)-Ketoprofen BP 93 of 93%, if be substrate with the Ketoprofen methyl esters then can obtain (R)-Ketoprofen BP 93, but enantiomeric purity only has 86%.If obtain more highly purified product (generally should reach more than 98%), also need to remove residual a small amount of enantiomeric impurity by diastereomer crystalline method as pharmaceuticals purity.Another piece United States Patent (USP) U.S.Pat.5 457 051, Oct.10,1995 disclose the muscardine Beauveria bassiana ATCC44860 that a strain is specifically designed to preparation (R)-Ketoprofen BP 93, transform the Ketoprofen BP 93 cholinesterase with its mycelia, the enantiomeric excess value of gained under the best-case (R)-acid also has only 93.6%ee (being equivalent to enantiomeric purity is 96.8%).Because the synthesis step of Ketoprofen cholinesterase is very complicated, and needs to use toxic reagent (as methyl-sulfate and methyl iodide), therefore brought very big difficulty to industrial applications.
In sum, when splitting the 2-arylpropionic acid with biological catalysis, the zymin price of commodity in useization is higher, selectivity is not ideal enough, need to increase extra treatment step, and the product of usually reaction be (S)-acid and (R)-ester, and the latter need pass through further chemical hydrolysis just can change (R)-acid into; Comparatively speaking, directly use the ester of the microorganism cells catalysis resolving chiral carboxylic acid that contains enzyme, technology is fairly simple, and cost is cheaper, therefore has better industrial application prospects.But the selectivity that also exists at present bacterium producing multi enzyme preparation in the disclosed microbiological transformation technology is not high enough, and can not obtain highly purified (S) and (R) two kinds of defectives such as enantiomorph simultaneously.Summary of the invention
The technical issues that need to address of the present invention are to disclose the higher and enantioselectivity complementary yeast strain of two strain stereospecificities, and use their cell or racemic 2-arylprop acid esters is carried out placed in-line once or twice hydrolysis reaction from isolated esterase wherein, with (S) of preparation high-optical-purity and (R)-method of 2-arylpropionic acid, not high enough to overcome the selectivity that prior art exists, and can not obtain highly purified (S) and (R) two kinds of defectives such as enantiomorph simultaneously.
Design of the present invention is such:
(1) natural microbe species is very abundant, diversified.Since nature exists (R) and (S) chiral ester of two kinds of configurations, can infer according to evolutional viewpoint so, occurring in nature must also exist to (R) and (S) two kinds of substrates have higher narrow spectrum esterase.Therefore, the contriver thinks that screening and separating obtains highly narrow spectrum esterase and produces bacterium dexterously, by the ester hydrolysis of height enantioselectivity, just can obtain highly optically pure single enantiomer 2-arylpropionic acid so as long as design certain method.
(2) microorganism and enzymatic enantiomorph split process thereof, come down to not to be all what is called " kinetic resolution " process of prerequisite with the speed speed of the competitive reaction of two kinds of enantiomorph substrates, relevant quantitative analysis and simulation curve are documented among the 104:7294 at document J.Am.Chem.Soc.1982.Can obtain following enlightenment according to this theoretical model: when the target enantiomorph is the product (acid) of reaction, at first will select high as far as possible cell or the enzyme of mapping selection rate (E value); Secondly, not under the situation of high especially (as E＜50) in the selection rate of cell or enzyme, should control the time of reaction, make the transformation efficiency (c) of reaction suitably low (for example about 40%); Once more, when the long response time enantiomorph in the substrate by after the enrichment to a certain degree, carry out twice transformation with the opposite another kind of enzyme of enantioselectivity again, the optical purity of then reacting products therefrom is than single optical purity height of products therefrom when transforming racemic substrate with a kind of enzyme one-step in back.
The microorganism of using:
---yeast that Citeromycesbaodingensis Citeromyces matriensis CGMCC No.0573 and a kind of Trichosporon of belonging to belong to---the Trichosporon brassicae Trichosporon brassicae CGMCC No.0574, Citeromycesbaodingensis CGMCCNo.0573 that be the yeast that a kind of Citeromyces of belonging to belongs to that is used for that the present invention prepares optical purity 2-arylpropionic acid is unique kind during Citeromyces belongs to; Trichosporon brassicae CGMCC No.0574 and Trichosporon laibacchii are not of the same race in the same genus.These two kinds of bacterial strains in 05 month 09 day calendar year 2001 in China Committee for Culture Collection of Microorganisms's preservation, preserving number is respectively CGMCC No.0573 and CGMCC No.0574.
Above-mentioned bacterial classification has following character:
Morphological specificity:
Citeromycesbaodingensis CGMCC No.0573: this bacterium in malt juice liquid medium 30 ℃ cultivate after 3 days, vegetative cell spherical in shape, not maternity leave silk contains a thecaspore in each ascus, nitrate reduction test is positive, not urease-producing.This bacterium energy glucose fermentation, sucrose and maltose, unfermentable lactose.This bacterium can assimilate succsinic acid, lactic acid and ethanol, can not assimilate methyl alcohol, to assimilating a little less than the citric acid.
Trichosporon brassicae CGMCC No.0574: cultivate after 3 days for 25 ℃ in glucose yeast cream peptone liquid nutrient medium, fungal filament and arthrospore are arranged, it is rare to go out sprout cell; The cell size is (3-4.5) * (5-20) μ m, has mycoderm to form, and throw out is cotton-shaped.Cultivate after one month for 25 ℃ in glucose yeast cream peptone nutrient agar, the streak culture canescence has Zou Wen, and the goose down shape is strong but pliable in texture, and the edge has mycelia to form.When on Corn Meal Agar Dalmau flat board, cultivating, there are a large amount of fungal filaments to produce.The unfermentable carbohydrate of this bacterium.
According to document: " The yeasts:a taxonomic study " (Kurtzman et al, 1998) and " saccharomycetic characteristic and evaluation " (Barnett et al, 1983:Yeasts-characteristics and identification, press of Qingdao Marine University) authentication method that provides and above-mentioned test-results, show that Citeromycesbaodingensis CGMCC0573 belongs to the Citeromycesbaodingensis Pseudomonas, and have only kind of Citeromyces matriensis in this yeast monoid; Trichosporon brassicae CGMCC0574 belongs to the Trichosporon brassicae kind in the Trichosporon, and this bacterial classification is named (" No. the 119th, 2001 little searchings ") May 9 calendar year 2001 by Institute of Microorganism, Academia Sinica.By the fermentation process of routine, can cultivate above-mentioned Citeromycesbaodingensis CGMCC No.0573 and Trichosporon brassicae CGMCC No.0574 bacterial classification.
Above-mentioned bacterial classification obtains in the area, Shanghai, and screening method is as follows:
Get the 1g fresh soil sample, be added in the 50ml screening culture medium and (contain 0.2% (NH
4)
2SO
4, 0.2%K
2HPO
4, 0.05%NaCl and 0.05%MgSO
47H
2O), other adds the tween-80 emulsion of a small amount of Ketoprofen BP 93 ethyl ester or ethanolic soln as sole carbon source, carries out the two-wheeled enrichment culture; Carrying out thin plate layer after the enrichment analyses, get the nutrient solution of ester hydrolysate (Ketoprofen), dilution is coated with flat board (substratum is formed the same) and cultivates, get single bacterium colony of transparent circle, (for example: glucose 1% shaking the Guan Zhongyong rich medium, peptone 0.5%, yeast extract paste 0.5%) behind the cultivation 24h, the emulsion (10mM) that adds the Ketoprofen BP 93 ethyl ester, after transforming 24h, go out product acid and residual ester with equal volume of ethyl acetate, making HPLC analyzes and (to use the chiral chromatographic column ChiracelOJ of Japanese Daicel company, φ 0.46cm * 25cm, moving phase is normal hexane/Virahol/acetate=90: 10: 0.1), calculate the transformation efficiency of substrate and the enantiomeric excess value (ee of product
p).From the candidate of strain more than 500 bacterial strain, select the respectively microorganism of selective hydrolysis (R)-and (S)-Ketoprofen BP 93 ethyl ester of two strains at last: Citeromycesbaodingensis Citeromyces matriensis CGMCCNo.0573 and Trichosporon brassicae Trichosporon brassicae CGMCC No.0574.
The method that adopts above-mentioned microorganism to prepare optical purity 2-arylpropionic acid in turn includes the following steps:
(1) adopt conventional method to carry out the cultivation of bacterial classification and cell:
The preparation of bacterial classification: respectively with Citeromycesbaodingensis Citeromyces matriensis CGMCC No.0573 and Trichosporon brassicae Trichosporon brassicae CGMCC No.0574 (121 ℃ of the bacterium of going out, rich medium 20-40min) (for example: glucose 1%, peptone 0.5%, yeast extract paste 0.5%, agar 1.5%) line on the flat board, in 25-30 ℃ leave standstill cultivate about 2 days after picking list bacterium colony, carry out slant culture (culture condition is the same) as seed, be stored in 4 ℃ of refrigerators standby after about 2 days.
The cultivation of cell: the seed of the above-mentioned slant culture of picking, be inoculated into and the 20mL liquid nutrient medium is housed (forms the same, but do not add agar) 100mL shake in the bottle for a short time, (20-50 ℃ of proper temperature, preferably 25-40 ℃) and rotating speed (60-300r/min for example, general 120-180r/min) after cultivating 12-18h on the shaking table, being inoculated in the 500mL that contains the 100mL growth medium by suitable proportion (such as 5%) shakes in the bottle greatly, on shaking table (30 ℃, 150r/min) take out behind the cultivation 24-30h, at 4 ℃, the centrifugal removal supernatant liquor of 8000r/min, gained cell are with physiological saline (0.85%NaCl) washing once.
(2) conversion of substrate: with preserving number is the bacterial strain institute cultured cells (born of the same parents include S-specificity esterase) of CGMCC No.0574, concentration with 10g～200g (weight in wet base)/L is suspended in the potassium phosphate buffer, the concentration of potassium phosphate buffer is 20～100mmol/L, pH is 6-11, be preferably pH=8-9, the emulsion that adds the substrate ester that contains emulsifying agent, preferred solvent is a tween-80, a kind of and composition thereof in polyvinyl alcohol or the polyoxyethylene nonylphenol ether, add-on is 1～20g/L, after 1～5 day time of oscillatory reaction on 20～50 ℃ the shaking table, with reaction solution with hcl acidifying to pH be about 2～3, add equal volume of ethyl acetate again, leave standstill, the organic phase that contains acid and ester is collected in phase-splitting.Do the chiral high performance liquid chromatography analysis from the organic phase sampling, calculate the transformation efficiency of substrate and the enantiomorph content (ee of product
p);
Said substrate ester is 2-arylpropionic acid and C
1～C
8Fatty Alcohol(C12-C14 and C12-C18) forms a kind of in the ester.
(3) separation of product: add the sig water back extraction in the acid and the organic extract liquid of ester in above-mentioned containing, the organic phase that can obtain to contain the water of (S)-acid and contain (R)-ester.
Sig water comprises NaOH or/and NaHCO
3, its concentration is 0.01～1.0mol/L, add-on so that final pH be 10～12 o'clock comparatively suitable;
Adding hcl acidifying to aqueous phase is 2-3 to pH, and the standing over night after-filtration can obtain the crystal product of the single enantiomer 2-arylpropionic acid of optically pure (S)-type.
To contain the organic phase heating evaporation of (R)-ester, the place to go desolventizes, and promptly obtains (R)-ester.
(4) secondary splits: with (R)-ester that above-mentioned extracting and separating obtains, add a kind of and composition thereof in emulsifying agent such as tween-80, polyvinyl alcohol or the polyoxyethylene nonylphenol ether, the emulsifying agent add-on is 1～20g/L, makes the emulsion of (R)-ester.With preserving number is that the bacterial strain institute cultured cells (containing R-specificity esterase) of CGMCC No.0573 is made catalyzer, concentration with 10g～200g (weight in wet base)/L is suspended in the potassium phosphate buffer, the concentration of potassium phosphate buffer is 20～100mmol/L, and pH is 6-11, is preferably pH=8-9.The emulsion of (R)-ester is mixed with the suspension equal-volume of cell, in oscillatory reaction on 20～50 ℃ the shaking table after 1～5 day, with hcl acidifying to pH be about 2～3, add equal volume of ethyl acetate again, leave standstill, the organic phase that contains acid and ester is collected in phase-splitting.Do the chiral high performance liquid chromatography analysis from the organic phase sampling, calculate the transformation efficiency of substrate and the enantiomeric excess value (ee of product
p);
Add sig water (the control final pH is at 10-12) back extraction, the organic phase that can obtain to contain the water of (R)-acid and contain (S)-ester in the acid and the organic extract liquid of ester to above-mentioned containing.Aqueous phase after separating adds hydrochloric acid to pH=2-3, and standing over night is also filtered then, can obtain the crystal of optical purity (R)-arylpropionic acid.
According to the present invention, also racemic 2-arylprop acid esters the 30-40% transformation efficiency be can be hydrolyzed into the cell catalysis of the Citeromycesbaodingensis CGMCC No.0573 that contains R-specificity esterase earlier, higher (R)-type 2-arylpropionic acid of optical purity and lower (the S)-2-arylprop acid esters of optical purity be gone out through extracting and separating; And then with the ester of remaining (the S)-enantiomorph of the cell catalysis hydrolysis reaction part enrichment of the Trichosporon brassicae CGMCC No.0574 that contains S-specificity esterase, again through alkali lye extraction and acidizing crystal, optically pure (S)-type 2-arylpropionic acid crystal to obtain.
According to the present invention, preferably will cultivate the cell that obtained oscillation treatment 1～15 hour in the buffered soln that contains acetone or Virahol, be used further to above-mentioned catalyzed reaction.
Adopt the said bio-conversion process of the present invention, not only can obtain optical purity very high (S) and (R)-type 2-arylpropionic acid single enantiomer product simultaneously, satisfy the medicine of different purposes or the needs of pesticide industry, and be easy to synthesize as the 2-arylpropionic acid ethyl ester of reaction substrate, need not to use expensive or deleterious reagent, thereby reduced the cost of raw material; In addition, also save the racemization and the recycle of reaction residue ester, thereby simplified operation steps, and improved utilization ratio of raw materials.
Embodiment
Below will be described in detail the specific embodiment of the present invention by embodiment.
Embodiment 1～6
3L growth medium (the glucose 1% of in the 5L fermentor tank, packing into, peptone 0.5%, yeast extract paste 0.5%), CGMCC No.0574 seed culture fluid is inserted by 5% inoculum size in the sterilization back, in temperature is that 30 ℃, mixing speed are that 600r/min and Ventilation Rate are under the condition of 0.4vvm, glucose exhausts substantially behind about 12h, and cell density reaches 8.5g (dry weight)/L, and esterase activity reaches 10.3U/L.At this moment, add glucose to 5g/L, the continued growth and produce enzyme again of this bacterium, bacterium is dense when 24h is put jar improves 60%, and enzyme is lived and is improved about 50%.
Respectively get the above-mentioned nutrient solution of 100mL, centrifugal collection thalline, after the physiological saline washing, be suspended in (50mM in the 20mL phosphoric acid buffer, pH=7.0), add the 0.1g tween-80, add the ester (20mM) that Ketoprofen BP 93 and different alcohol synthesize gained again, insulation reaction on the shaking table of 30 ℃ and 120r/min the results are shown in following table.
Table 1 Trichosporon brassicae CGMCC No.0574 is to the different esterolytic results of Ketoprofen BP 93.
The configuration mapping selection rate of Ketoprofen BP 93 relative rate transformation efficiency product
Various esters (%) (%) and content (%) (E value) embodiment 1 methyl esters 211 49 R (93) 34 embodiment 2 ethyl esters 100 45 S (96) 54 embodiment 3 chloroethene esters 67 49 S (80) 7.0 embodiment 4 isopropyl esters 38 44 R (85) 9.7 embodiment 5 positive butyl ester 47 37 S (70) 2.9 embodiment 6 n-octyls 26 23 S (65) 2.0
Embodiment 7
Citeromycesbaodingensis CGMCC No.0573 is inoculated in contains 4mL growth medium (glucose 1%, peptone 0.5%, shaking in pipe yeast extract paste 0.5%), after 30 ℃, 120r/min were cultivated 42h, adding 20 μ l concentration was the ethanolic soln of the Ketoprofen BP 93 ethyl ester of 2M, after transforming 48h, behind the 4mL ethyl acetate extraction, carry out HPLC and analyze, the result has obtained (R)-Ketoprofen BP 93, enantiomeric purity is 96.8%, and productive rate is 27%.
When substrate Ketoprofen BP 93 ethyl ester added in the conversion reaction system with the tween-80 emulsion, speed of response was obviously accelerated, and transformation efficiency reaches 31% during 16h, and the enantiomeric purity of product (R)-Ketoprofen BP 93 is 96.5%.
Embodiment 8
Respectively get the Trichosporon brassicae CGMCC No.0574 nutrient solution 10mL that cultivates behind the 24h with test tube, the gained thalline is collected in centrifugal, washing, again be suspended in 2mL and contain (50mM in the phosphoric acid buffer of 1% acetone or Virahol, pH7.0), in oscillation treatment on the shaking table after 12 hours, centrifugal, flush away reagent treatment are added with insulation reaction in the 10mM substrate emulsion phosphoric acid buffer of (containing 0.5% tween-80) at 2mL again, measure esterolytic relative rate and enantioselectivity.The result compares with the new fresh cell of not doing penetratingization processing, the initial velocity of the cell response of crossing with acetone treatment has improved 61%, the speed of response of the cell of handling with Virahol then is increased to 2.35 times, the enantioselectivity of reaction does not then have considerable change, and the enantiomeric purity of product remains on 96-97%.
When the damping fluid of above-mentioned penetrating processing was replaced with the glycine of pH=10-NaOH damping fluid by the phosphoric acid salt of pH=7.0, speed of response had improved 75% again.Virahol with 1% in the damping fluid of pH=10 is handled different time, and the result is the best with the effect of 10h.With this understanding, the treatment effect of 2% Virahol is better than 1%, and speed of response is more than 3 times of control group.When the fixing concentration of Virahol is 2% when changing the concentration of cell, the effect of handling also there is tangible influence, be the best with the cell concentration of 30g (weight in wet base)/L.
Embodiment 9
The bottle that shakes greatly with 500mL is cultivated the cell (condition is the same) of CGMCC No.0573 and CGMCC No.0574 respectively, and is centrifugal, the washing back is standby.
The cell of getting 4g Trichosporon brassicae CGMCC No.0574 is at (30g/L cell, 2% Virahol, pH=10) after handling 10h under, be suspended in (100mM in the phosphoric acid buffer of 50mL, pH8.0), add 1.4g Ketoprofen BP 93 ethyl ester (100mM) and 0.25g tween-80 (0.5%), behind 30 ℃, 120r/min insulation reaction 36h, addend drips concentrated hydrochloric acid and is acidified to pH=2, the Ketoprofen acid and the remaining ester of reaction that generates with the 50mL ethyl acetate extraction then, sampling 1mL makes HPLC and analyzes, the productive rate of result (S)-acid is 30%, and enantiomeric purity is 95.5%.Remaining extraction liquid obtains the salts solution of (S)-Ketoprofen BP 93 with the back extraction of 0.1NNaOH solution, with the precipitation that promptly obtains (S)-Ketoprofen behind the hcl acidifying, passes through further crystallization (NaHCO again
3/ HAc) after, optical purity is brought up to more than 98%.
With the organic phase evaporation concentration that obtains after the above-mentioned back extraction, obtain (R)-Ketoprofen ethyl ester (enantiomorph content is 78%).Get this ester 0.46g and be suspended in 50mL phosphoric acid buffer (100mM, pH=7.0) in, the wet cell that adds 0.25g tween-80 and 4g Citeromycesbaodingensis CGMCC No.0574, at 30 ℃, 120r/min insulation reaction 90h, the productive rate that records (R)-Ketoprofen BP 93 is 40%, and enantiomeric purity is 97.8%; After separation and crystallization, the content of (R)-enantiomorph surpasses 99% in the product.
According to technical scheme disclosed by the invention and embodiment, relevant engineering technical personnel can be easily with said two saccharomycetes of the present invention and cultivation thereof and conversion process is used for the 2-arylpropionic acid (as Naproxen Base and Ibuprofen BP/EP etc.) of other type with drawing inferences about other cases from one instance and the enantiomorph of 2-aryloxy propionic acid splits.
Claims (10)
1. Citeromycesbaodingensis bacterial strain CGMCC No.0573.
2. Trichosporon brassicae bacterial strain CGMCC No.0574.
3. a method for preparing optical purity 2-arylpropionic acid is characterized in that, this method comprises the steps:
(1) adopt conventional method that the bacterial strain of claim 1 and claim 2 is cultivated;
(2) with preserving number be the bacterial strain institute cultured cells of CGMCC No.0574, at pH is in the damping fluid of 6-11, and with the emulsion oscillatory reaction of the substrate ester that contains emulsifying agent, then reaction solution being acidified to pH with acid is 2～3, use ethyl acetate extraction, collect the organic phase that contains acid and ester;
Said substrate ester is 2-arylpropionic acid and C
1～C
8Fatty Alcohol(C12-C14 and C12-C18) forms a kind of in the ester;
(3) separation of product: add the alkali lye back extraction in the acid and the organic phase of ester in above-mentioned containing, the organic phase that obtains to contain the water of (S)-acid and contain (R)-ester, the alkali lye add-on is so that final pH is 10～12;
Is 2-3 with aqueous phase as acidified to pH with acid, the single enantiomer 2-arylpropionic acid of collection optically pure (S)-type wherein;
The organic phase heating evaporation that will contain (R)-ester is removed solvent, obtains (R)-ester.
(4) secondary splits: with the preserving number be CGMCC No.0573 bacterial strain institute cultured cells pH be in the damping fluid of 6-11 with the emulsion oscillatory reaction that contains (the R)-ester of emulsifying agent, being acidified to pH with acid is 2～3, use ethyl acetate extraction, collect the organic phase that contains acid and ester;
Add alkali lye in organic phase, the control final pH is collected water that contains (R)-acid and the organic phase that contains (S)-ester at 10-12, to pH=2-3, collects optical purity (R)-2-arylpropionic acid wherein with the acidifying water.
4. method as claimed in claim 3 is characterized in that, said damping fluid is a potassium phosphate buffer, and concentration is 20～100mmol/L, and the cell in the damping fluid is counted 10g～200g/L with weight in wet base, and pH of buffer is 8-9.
5. method as claimed in claim 3 is characterized in that, said emulsifying agent is a kind of and composition thereof in tween-80, polyvinyl alcohol or the polyoxyethylene nonylphenol ether, and add-on is 1～20g/L.
6. method as claimed in claim 3 is characterized in that, said alkali comprises NaOH or/and NaHCO
3, concentration of lye is 0.01～1.0mol/L.
7. method as claimed in claim 3 is characterized in that, said acid is hydrochloric acid.
8. method as claimed in claim 3 is characterized in that, the oscillatory reaction temperature is 20～50 ℃, and the time is 1～5 day.
9. a method for preparing optical purity 2-arylpropionic acid is characterized in that, this method comprises the steps:
(1) adopt conventional method that the bacterial strain of claim 1 and claim 2 is cultivated;
(2) racemic 2-arylprop acid esters is hydrolyzed into the 30-40% transformation efficiency with the cell catalysis of Citeromycesbaodingensis CGMCC No.0573 earlier, go out higher (R)-type 2-arylpropionic acid of optical purity and lower (the S)-2-arylprop acid esters of optical purity through extracting and separating, and then with the ester of remaining (the S)-enantiomorph of the cell catalysis hydrolysis reaction part enrichment of Trichosporon brassicae CGMCC No.0574, through alkali lye extraction and acidizing crystal, obtain optically pure (S)-type 2-arylpropionic acid again.
10. as the arbitrary described method of claim 3～9, it is characterized in that, will cultivate the cell obtained oscillation treatment 1～15 hour in the buffered soln that contains acetone or Virahol.
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