CN1168822C - Immobile bacillus and method for resolving and preparing chiral cyclopentenylone using said bacillus - Google Patents
Immobile bacillus and method for resolving and preparing chiral cyclopentenylone using said bacillus Download PDFInfo
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- CN1168822C CN1168822C CNB021370192A CN02137019A CN1168822C CN 1168822 C CN1168822 C CN 1168822C CN B021370192 A CNB021370192 A CN B021370192A CN 02137019 A CN02137019 A CN 02137019A CN 1168822 C CN1168822 C CN 1168822C
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Abstract
The present invention discloses an immobile bacillus CGMCC0789 and a method for preparing chiral cyclopentenone by the resolution of the strain. In the method, microbe cells with esterase activity or esterase separated by the cells is used as a catalyst and the fatty acid ester of racemic alcohol ketone is used as a raw material for the conversion reaction to obtain a mixture of (R)-alcohol ketone and (S)-alcohol ketone fatty acid ester; sulfonylation and basic hydrolysis are then carried out, and the final product of (S)-alcohol ketone single enantiomer is collected from the hydrolysate product. The present invention has the advantages of simple technology process, mild reaction condition, high catalytic activity and stereoselectivity of the used strains (containing the intracellular esterase) and over 90% of the optical purity of the obtained (S)-alcohol ketone. The method has the advantages of simple and practical use, low cost and hopeful popularization and use in industrial production.
Description
Technical field
The invention belongs to biological chemical field, relate to a kind of acinetobacter calcoaceticus and adopt this microorganism catalysis to split the method for preparing the chipal compounds enantiomer, particularly split the method for preparing cyclopentenone type chiral alcohol enantiomorph.
Background technology
Cyclopentenone type chiral alcohol compounds is the important intermediate of producing pyrethroid insectide, the basic framework of cyclopentenone type alcohol compound involved in the present invention is: 4-hydroxy-3-methyl-ring penta-2-alkene-1-ketone, mainly comprise two kinds, chemical structural formula is as follows:
Wherein: formula (I) is 2-allyl group-3-methyl-4-hydroxycyclopent-2-alkene-1-ketone, hereinafter to be referred as the allyl alcohol ketone; Formula (II) is 2-propargyl-3-methyl-4-hydroxycyclopent-2-alkene-1-ketone, hereinafter to be referred as propargyl alcohol ketone; But the two general name " alcohol ketone ".According to the difference of three-dimensional chemical configuration, each alcohol ketone can be divided into R-and two kinds of enantiomers of S-again.
Pyrethrin and pyrethroid are multi-functional family expenses of a class or agricultural insecticide.Natural pyrethrin derives from the pistil of chrysanthemum, and it is extremely unstable in sunlight and air; Used sterilant is generally the pyrethroid of synthetic now, has a large amount in variety, as propine chrysanthemum ester, esbiothrin, methyl propine chrysanthemum ester, Fenvalerate, permethrin, Deltamethrin, cyfloxylate etc.(S)-the allyl alcohol ketone is the important intermediate of preparation esbiothrin (allethrin) sterilant, full name of esbiothrin (allethrin) sterilant is (R, S)-3-allyl group-2-methyl-4-oxo ring penta-2-thiazolinyl (R, S)-suitable, instead-2,2-dimethyl-3-(2-methyl isophthalic acid-propenyl) cyclopropanecarboxylcompound.Another kind of intermediate chrysanthemumic acid has how much and optical isomer, has four steric isomers.It is reported, French Russell-excellent Ke Fu company 1967 will (1R, 3R)-trans-chrysanthemate with (S, R)-the vinyl carbinol reactive ketone obtains biological allyl alcohol ketone (bioallethrin).Seventies France Russell-excellent Ke Fu company again with (1R, 3R)-trans-chrysanthemate is that the vinyl carbinol reactive ketone of major ingredient has obtained E with containing (S)-body
s-Esbiothrin (esbiothrin).Allyl alcohol ketone component S in this ester and the ratio of R isomer 72: 21.Rauch in 1972 etc. developed (1R, 3R)-trans-chrysanthemate and S-vinyl carbinol reactive ketone obtain S-EXTHIN (S-bioallethrin).The ratio of used S-allyl alcohol ketone is high more when the synthesis of pyrethrin ester, and the relative effectivenes of product is just high more.Therefore, exploitation S-allyl alcohol ketone can improve the insecticidal activity of product greatly, reduces dosage.About synthesizing of S-allyl alcohol ketone, conclude and get up to mainly contain following several method:
(1) asymmetric synthesis method: this method is made reductive agent with bis-naphthol lithium aluminium, is raw material with cyclenes (1,3) diketone, directly synthesizes the allyl alcohol ketone of single configuration, this method products obtained therefrom optical purity height, but the preparation of used reductive agent is very difficult, so be difficult to scale operation.
(2) chemical resolution method: this method with (1R, 4R, 5S)-4-hydroxyl-6,6-dimethyl-3-oxabicyclo (3,1,0) hexanaphthene-2-ketone is resolving agent, form the ether enantiomorph with racemic allyl alcohol ketone, through separating, make the allyl alcohol ketone of R and S configuration respectively after the hydrolysis again.This method complex synthetic route, total recovery is low, be difficult for making, thereby production cost of products is very high.
(3) enzymatic Split Method: because the imperfection of asymmetric synthesis method and chemical resolution method makes that people begin to consider to use enzyme catalysis method to split the racemic modification of allyl alcohol ketone.U.S. Pat Patent No.4571436 (1986) has reported that SUMITOMO CHEMICAL company screens and has obtained a kind of allyl alcohol ketone acetic ester lytic enzyme, this enzyme selective hydrolysis (R)-allyl alcohol ketone acetic ester, its ee from commercial enzyme
SAnd ee
PAll be higher than 90%.But this commercial enzyme preparation price is higher relatively, and the reaction back is difficult for reclaiming to be reused, and because the emulsifying effect of enzyme, make extraction separation process relatively more difficult, and cause product losses, thus production cost is significantly increased, influenced the feasibility of its industrial applications to a certain extent.(wherein enzyme is in cell interior with the microorganism whole cell, be equivalent to natural immobilized enzyme to a certain extent) method of direct hydrolysis (R)-pure keto fatty esters, saved the complex steps of enzyme purification, greatly reducing production cost, is a kind of method of new hydrolysis (R)-pure keto fatty esters.Technological line disclosed in the summary of " Shanghai chemical industry " the 24th volume the 10th phase 7-8 page or leaf, but also there is the on the low side and not high defective of optical purity of reaction conversion ratio in this bacterial classification.
Summary of the invention
One of technical issues that need to address of the present invention are to disclose the acinetobacter calcoaceticus that esterase is produced in a strain;
Two of the technical issues that need to address of the present invention are that the described bacterial classification of open employing carries out the method that the catalysis fractionation prepares the chirality cyclopentenone, to overcome higher or used bacterial activity of commercial enzyme preparation price and the lower defective of selectivity that prior art exists.
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, as long as the contriver thinks the design certain method, screening and separating obtains highly narrow spectrum esterase and produces bacterium dexterously, so by the processes such as ester hydrolysis of height enantioselectivity, just can obtain highly optically pure single enantiomer (S)-alcohol ketone or (R)-alcohol ketone.
(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 (alcohol) of reaction generation, 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 suitable reaction times, make the enantiomeric excess value ee of the product (alcohol) of residual substrate (ester) and generation
SAnd ee
PAll reach high value, i.e. ee
SAnd ee
PEquate, can obtain the higher final product of optical purity like this.
In view of the above, the contriver at first screens the bacterial strain of producing esterase from soil, and the esterase that utilizes this bacterial strain to produce carries out enantioselective hydrolysis to the vinyl carbinol keto fatty esters, obtains the higher R-alcohol ketone of optical purity; Then with p-methyl benzene sulfonic chloride with its sulfonylation, the configuration reversal by following in chemical hydrolysis and the sulphonate hydrolytic process can be converted into the S-alcohol ketone with R-alcohol ketone sulphonate again.Meanwhile, after originally residual S-alcohol keto fatty esters passes through chemical hydrolysis in the enzymically hydrolyse reaction, then keep original steric configuration, be converted into the S-alcohol ketone.
The microorganism of using:
Be used for (R, S)-microorganism that pure keto fatty esters carries out enantioselective hydrolysis is a kind of bacterial classification that belongs to acinetobacter, acinetobacter calcoaceticus (Acinetobacter sp.) YQ231 is the bacterial classification with this ability, hereinafter to be referred as YQ231, this bacterial classification on August 16th, 2002 in China Committee for Culture Collection of Microorganisms's preservation, preserving number is: CGMCC0789.
This bacterial classification is to obtain by following method screening:
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 a small amount of allyl alcohol ketone acetic ester (0.1%), carries out enrichment culture; Get a little nutrient solution after the enrichment, dilution is coated with flat board (substratum is formed the same) and cultivates, picking list bacterium colony (for example: glucose 1% is shaking the Guan Zhongyong rich medium again, peptone 0.5%, yeast extract paste 0.5%) behind the cultivation 24h, add allyl alcohol ketone acetic ester (1%), after transforming 24h, go out the pure and mild residual ester of product with equal volume of ethyl acetate, use the chirality gas chromatographic column to analyze (160 ℃ on post, sampler and detector temperature are 280 ℃), calculate the transformation efficiency of substrate and the enantiomeric excess value (ee of product
P).From 370 strain candidate bacterial strains, select the microorganism of strain specificity hydrolysis (R)-allyl alcohol ketone acetic ester at last: acinetobacter calcoaceticus YQ231.
The YQ231 bacterial classification has following character:
(1) morphological specificity: Gram-negative, shaft-like or spherical (the stable growth phase is spherical) reaches a plurality of chains that are arranged in pairs, and size is 1.0-1.5 * 1.5-2.5 μ m, no gemma, atrichia;
(2) cultural characteristic: the YQ231 bacterial strain is cultivated 12h on 30 ℃ of flat boards can form little bacterium colony, forms thickness, moistening white colony behind the 36h, and the edge is smooth, middle sinking.A small amount of filament is arranged in culture, aerobic;
(3) physiological and biochemical property: the physiological and biochemical test of bacterial strain YQ231 the results are shown in Table 1.
The physiological and biochemical test result of table 1:YQ231 bacterial strain
Characteristic Properties is Results as a result
Catalase Catalase+
V.P test V.P test-
M.R test M.R test-
Glucose produce sour Acid from glucose+
Fructose produce sour Acid from fructose+
Lactose fermentation lactose fermentation-
Starch hydrolysis Hydrolysis of starch-
Gelatin hydrolysis Hydrolysis of gelatin+
Hydrolyzed pectin Hydrolysis of pectin-
Citrate trianion utilize Utilization of citrate+
Oxidation of ethanol Alcohol oxidate-
Oxydase Oxidizing enzyme-
Urase Urease-
Generation indoles Production of indole-
Produce H
2S Production of H
2S+
Nitrate reduction Nitrate reduction-
Fat hydrolysis Fat hydrolysis+
Sucrose utilize Utilization of sucrose-
Glucose utilization Utilization of glucose-
N.F,USP MANNITOL utilize Utilization of mannitol-
Lactose utilization Utilization of lactose-
Galactose utilization Utilization of galactose+
Inositol utilize Utilization of creatol-
Fructose utilize Utilization of fructose-
Sorbyl alcohol utilize Utilization of sorbitol-
Morphological specificity, cultural characteristic and physiological and biochemical property according to bacterial strain YQ231, this bacterial strain and " the outstanding Bacteria Identification handbook of uncle (the 8th edition, press of Shandong University in December, 1988) characteristic of division of acinetobacter (Acinetobacter sp.) conforms to most in, so judge that bacterial strain YQ231 is an acinetobacter calcoaceticus, name and be acinetobacter calcoaceticus (Acinetobacter sp.) YQ231.
Method of the present invention comprises the steps:
(1) will contain the microorganism cells of esterase activity or be catalyzer by its isolating esterase, fatty acid ester with the racemize alcohol ketone is a raw material, at 20-50 ℃, pH=5-10, preferably carry out conversion reaction under 25-40 ℃ and the pH=6-8.5 condition, to certain transformation efficiency, general 40-50%, best stopped reaction during 45-50%, the reaction times is generally 2-48h.(R)-alcohol ketone and remaining (the S)-pure keto fatty esters of reaction that generates with the ethyl acetate extraction hydrolysis then adds evaporative removal organic solvent behind the anhydrous sodium sulfate drying, obtains the mixture of (R)-alcohol ketone and (S)-pure keto fatty esters.
Said microorganism, be meant with racemic allyl alcohol ketone acetic ester be the sole carbon source separation screening arrive can specificity hydrolysis (R)-pure keto fatty esters esterase produce bacterium, for example acinetobacter calcoaceticus Acinetobacter sp.YQ231.
Said racemic alcohol keto fatty esters comprises vinyl carbinol keto fatty esters or propargyl alcohol keto fatty esters; Said ester is meant by allyl alcohol ketone or propargyl alcohol ketone and short chain (C
1-C
8) lipid acid ester, the especially acetic ester and the butyric ester that constitute.
Said catalyzer, be meant microorganism to be addressed, as acinetobacter calcoaceticus Acinetobacter sp.YQ231 is seed, utilize the biomaterial with above-mentioned esterase hydrolyzed vigor of fermentation engineering, enzyme engineering and genetic engineering technique preparation, comprise fermented liquid, viable cell, resting cell, the zymin of freeze drying cell, immobilized cell, cytoclasis liquid and extract, different purity and various forms of immobilized enzyme also comprise various " clones " and the expressed protein product thereof that contain above-mentioned esterase gene.
Because the substrate ester is difficult to dissolving in water, disperse bad and influence the functioning efficiency of enzyme, preferably in reactive system, add an amount of solubility promoter when therefore reacting,, perhaps add emulsifying agent such as tween-80, Triton, polyoxyethylene nonylphenol ether etc. as ethanol, acetone, methyl-sulphoxide etc.; Its concentration range can be preferably in 0.5-1.5% (w/v) within 0.1-5%.
According to the present invention, can preferably adopt the cell through the YQ231 that cultivate to obtain is catalyzer, and add-on is 5～20g (weight in wet base)/L, and is that 6～8.5 potassiumphosphate is a damping fluid with pH.
(2) processing of product: adopt prior art; as U.S. Pat Patent No.4571436 (1986) disclosed method; the mixture that contains (R)-alcohol ketone and (S)-pure keto fatty esters that is obtained is carried out sulfonylation and hydrolysis, from hydrolysate, collect final product (S)-alcohol ketone single enantiomer then.This method is summarized as follows: add acetone and triethyl ammonia in the mixture that contains (R)-alcohol ketone and (S)-pure keto fatty esters, drip p-methyl benzene sulfonic chloride/acetone soln then, stirring reaction, reaction mixture is poured in the dilute hydrochloric acid solution, from reaction product, collected the mixture of (R)-alcohol ketone sulphonate and (S)-pure keto fatty esters then; The concentration of dilute hydrochloric acid solution is 0.5～2wt%.
Add lime carbonate and water reflux then, pour in the sodium bicarbonate aqueous solution after the reactant cooling, use ethyl acetate extraction, anhydrous magnesium sulfate drying again, evaporation concentration is removed solvent and is promptly obtained final product (S)-alcohol ketone single enantiomer.
The culture of strains of being addressed may further comprise the steps:
(1) preparation of bacterial classification: with acinetobacter calcoaceticus YQ231 (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.
(2) 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 temperatures, best 25～40 ℃) and rotating speed (60～300r/min for example, after cultivating 12～18h on general 100～200r/min) 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 cultivation 24～30h, at 4 ℃, the centrifugal supernatant liquor of removing of 8000r/min, gained cell with physiological saline (0.85%NaCl) washing once place 4 ℃ of refrigerators storages standby.The cell of above-mentioned cultivation gained also can be without centrifugation, promptly directly adds an amount of substrate ester and the emulsifying agent resolution reaction that is hydrolyzed in the fermented liquid that contains cell.
Method of the present invention, technological process is fairly simple, and reaction conditions is relatively gentleer; The catalytic activity and the stereoselectivity of used bacterial classification (containing born of the same parents' lactonase) are higher, and (the S)-alcohol ketone optical purity that is obtained can reach more than the 90%ee.This method has advantage simple and practical, with low cost, is expected to apply on industrial production.
Embodiment
Embodiment 1
(contain sucrose 1%, tween-80 0.15%, soybean cake powder 2%, sulphur ammonium 0.2%, K at shaking greatly of the volume 1L 150mL growth medium of packing in the bottle
2HPO
40.2%, NaCl 0.1%, MgSO
40.02%, pH8.0), the YQ231 seed culture fluid is inserted by 6% inoculum size in sterilization back, cultivates 24h on temperature is 30 ℃, the shaking table of 140r/min.
Get the above-mentioned nutrient solution of 100mL, centrifugal collection thalline is after the physiological saline washing, be suspended in 100mL Veronal sodium-hydrochloride buffer that (100mM pH8.5), adds the 1.5g polyoxyethylene nonylphenol ether, adding concentration again is the allyl alcohol ketone acetic ester of 5wt%, and insulation reaction on the shaking table of 30 ℃ and 160r/min generates (R)-allyl alcohol ketone, the result is: when reaction proceeds to 2 hours, transformation efficiency promptly is higher than 30%, continues reaction to 14 hours, and reaction is near terminal point (50%), at this moment, ee
SAnd ee
PReach 80%, mapping selection rate (E value) is near 40.
Embodiment 2
Get the fermented liquid 50mL among the embodiment 1, centrifugal collection thalline after the physiological saline washing, is suspended in (100mM in 100mL Veronal sodium-hydrochloride buffer, pH8.5), add the 1.5g polyoxyethylene nonylphenol ether, add 2.0g allyl alcohol ketone acetic ester again, insulation reaction on the shaking table of 30 ℃ and 160r/min, the result is: reaction proceeds to 24 hours, the conversion reaction of allyl alcohol ketone acetic ester finishes substantially, at this moment, and ee
SAnd ee
PReach 90%, the E value is near 50.
Embodiment 3
The 3L growth medium of packing in the 5-L fermentor tank (contains sucrose 1%, tween-80 0.15%, soybean cake powder 2%, sulphur ammonium 0.2%, K
2HPO
40.2%, NaCl 0.1%, MgSO
40.02%, pH8.0), the YQ231 seed culture fluid is inserted by 5% inoculum size in sterilization back, is that 30 ℃, mixing speed are that 500r/min and Ventilation Rate are the condition bottom fermentation of 1.0vvm in temperature, and bacterium is dense when 24h is put jar is 3g/L (dry weight), and enzyme is lived and is 900U/L.
Get above-mentioned fermented liquid 50mL, potassium phosphate buffer with 0.1M, pH8.0 is diluted to 100mL, directly add 2g allyl alcohol ketone acetic ester and 1.5g polyoxyethylene nonylphenol ether, in 30 ℃ of magnetic agitation 3h, stopped reaction and use ethyl acetate extraction, boil off obtain behind the solvent enantiomeric purity all above (R)-allyl alcohol ketone of 94% with (S)-the allyl alcohol ketone acetic ester; Add 3g acetone again, in temperature be-add the 0.65g triethylamine under the 15-0 ℃ condition, under same temperature, drip p-methyl benzene sulfonic chloride (0.62g)/acetone (2g) solution then, after dripping off in about 10 minutes, continued stirring reaction 1.5 hours, reaction mixture is poured in the 30ml dilute hydrochloric acid solution (1%), used dichloromethane extraction, anhydrous sodium sulfate drying then, evaporation concentration obtains the mixture of (R)-alcohol ketone sulphonate and (S)-alcohol ketone acetic ester.Added 0.1g lime carbonate and 10mL water reflux then 2 hours, pour in 5% sodium bicarbonate aqueous solution after the reactant cooling, use ethyl acetate extraction, anhydrous magnesium sulfate drying again, evaporation concentration is removed solvent and is promptly obtained final product (S)-allyl alcohol ketone, optical purity is 91.0%, and yield is 85.0%.
Embodiment 4
Get the fermented liquid 100mL described in the embodiment 3, centrifugal collecting cell also is suspended in (20mmol/L in the 25mL Tris-HCl damping fluid again, pH7.5), adding 75mL contains the Tris-HCl damping fluid (20mmol/L of 2% (w/v) sodium alginate, pH7.5), the back that is uniformly dispersed is added drop-wise to the CaCl of 0.1mol/L with syringe
2In the solution, behind the spherical particle to be formed, place fresh CaCl
2Soaking in the solution fully hardened particle in 30 minutes.Collect immobilized cell particle then, again be suspended in (50mmol/L in the 100ml Tris-HCl damping fluid, pH8.0), add 2g allyl alcohol ketone acetic ester and 1.5g polyoxyethylene nonylphenol ether, oscillatory reaction 5h on 30 ℃ of shaking tables, use ethyl acetate extraction behind the stopped reaction, boil off and obtain enantiomeric purity behind the solvent and surpass (R)-allyl alcohol ketone of 93% and (S)-allyl alcohol ketone acetic ester.By the post-processing step in the foregoing description 3 (R)-alcohol ketone is carried out sulfonylation and two kinds of blended esters are carried out heating hydrolysis then, it is (S)-allyl alcohol ketone of 90.5% that the result obtains optical purity, and yield is 86%.After first reaction finishes, filter and collect immobilized cell, and (50mmol/L pH8.0) after the washing, drops into fresh reactant liquid and continues reaction, carries out the conversion and the separation and Extraction of product then by same step with the Tris-HCl damping fluid.After so repeating 5 batches, the vigor of enzyme does not have remarkable loss, and the optical purity of gained (S)-alcohol ketone also all remains on more than 90%, and average yield is 85%.
According to technical scheme disclosed by the invention and embodiment, relevant engineering technical personnel can be easily with the said microbial strains of the present invention and cultivation and conversion process, and the enantiomorph that is used for the ester (as: allyl alcohol ketone butyric ester and propargyl alcohol ketone acetic ester) of other type splits with drawing inferences about other cases from one instance.
Claims (11)
1. acinetobacter calcoaceticus CGMCC0789.
2. one kind splits the method for preparing the chirality cyclopentenone, it is characterized in that comprising the steps:
(1) being catalyzer with the described acinetobacter calcoaceticus CGMCC0789 of claim 1, is that raw material carries out conversion reaction with the fatty acid ester of racemize alcohol ketone, the right mixture of collecting (R)-alcohol ketone and (S)-pure keto fatty esters;
(2) adopt conventional method that the mixture that contains (R)-alcohol ketone and (S)-pure keto fatty esters that is obtained is hydrolyzed, from hydrolysate, collect final product (S)-alcohol ketone single enantiomer then.
3. method according to claim 2 is characterized in that the racemic alcohol keto fatty esters comprises vinyl carbinol keto fatty esters or propargyl alcohol keto fatty esters.
4. method according to claim 3 is characterized in that said ester is by allyl alcohol ketone or propargyl alcohol ketone and short chain C
1-C
8The ester that lipid acid constitutes.
5. method according to claim 4 is characterized in that said ester is acetic ester and butyric ester.
6. according to each described method of claim 2～5, it is characterized in that conversion reaction at 20-50 ℃, carry out conversion reaction under the pH=5-10 condition, the reaction times is 2-48h.
7. method according to claim 6 is characterized in that conversion reaction carries out under 25-40 ℃ and pH=6-8.5 condition.
8. according to each described method of claim 2～5, in reactive system, add an amount of solubility promoter when it is characterized in that reacting.
9. method according to claim 8 is characterized in that solubility promoter comprises ethanol, acetone, methyl-sulphoxide or emulsifying agent.
10. method according to claim 9 is characterized in that solubility promoter concentration is 0.1-5%, w/v.
11. according to each described method of claim 2～5, it is characterized in that said catalyzer is the acinetobacter calcoaceticus CGMCC0789 through cultivating, add-on is 5～20g, weight in wet base/L, and be that 6～8.5 potassiumphosphate is a damping fluid with pH.
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| KR100803093B1 (en) * | 2005-10-07 | 2008-02-18 | 한국해양연구원 | Enantioselective epoxide hydlrolase and method for preparing an enantiopure epoxide using the same |
| CN101230327B (en) * | 2008-03-03 | 2010-06-16 | 中国热带农业科学院环境与植物保护研究所 | Plant pathogenic fungi antagonistic bacteria capable of generating siderophore and uses thereof |
| CN101591628B (en) * | 2009-06-11 | 2011-05-25 | 浙江工业大学 | Acinetobacter juni. X8 and application thereof in preparing algin lyase |
| CN107058453B (en) * | 2016-12-27 | 2020-02-21 | 长兴制药股份有限公司 | Method for splitting lisinopril hydride by biocatalysis |
| CN110272839B (en) | 2019-05-08 | 2020-10-30 | 江南大学 | Acinetobacter and application thereof in production of chiral 3-cyclohexene-1-formic acid |
| CN111778229B (en) * | 2020-07-23 | 2022-02-22 | 江南大学 | Cyclohexene carboxylate hydrolase, mutant thereof, encoding gene, expression vector, recombinant bacterium and application |
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