CN101503712A - Method for preparing optical pure (R)-2-octanol by immobilized microorganism - Google Patents
Method for preparing optical pure (R)-2-octanol by immobilized microorganism Download PDFInfo
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- CN101503712A CN101503712A CNA2008101075955A CN200810107595A CN101503712A CN 101503712 A CN101503712 A CN 101503712A CN A2008101075955 A CNA2008101075955 A CN A2008101075955A CN 200810107595 A CN200810107595 A CN 200810107595A CN 101503712 A CN101503712 A CN 101503712A
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Abstract
The invention discloses a method for preparing high optical pure (R)-2-octanol by a microbiological method, and a microorganism thereof. An immobilized-cell synthesis method of the invention has the advantages that: 1, an immobilization process is simple, and immobilized yeast cells have certain poison resistance, have excellent catalytic effect and can be repeatedly used in a reaction system; 2, the immobilized cells prepared by taking non-polar macroporous absorbent resin as a carrier are good in elasticity, strong in tolerance to temperature and pH, high in mechanical strength, stable in enzyme activity and long in half-life period; 3, as reaction is carried out in two phases of organic solvent/buffer solution, the toxic action of the organic solvent on the cells can be reduced, and the catalytic activity of the organic solvent is improved; and 4, the preparation of the optical pure (R)-2-octanol through the immobilized yeast cells is simple in process, free from need for no expensive coenzyme, mild in condition, short in reaction time, nearly free from byproducts, up to 90.25 percent e.e. in optical purity, simple in subsequent treatment, easy in product separation and friendly to environment.
Description
Technical field
Microorganism prepares the method for optical purity (R)-sec-n-octyl alcohol, belongs to the technical field of the synthetic optical pure compound of microorganism asymmetry catalysis prochirality compound.
Background technology
Sec-n-octyl alcohol is called secondary octanol again, often is used to Minute Organic Synthesis, can be used for production chirality chemical material and spices.(R)-sec-n-octyl alcohol is the important chiral material of preparation high-performance liquid crystal and liquid crystal device, can increase substantially the liquid crystal quality, makes low-grade liquid crystal (no chirality) become high-grade liquid crystal (chirality).It also is the important intermediate of synthetic many optical activity medicines and agricultural chemicals simultaneously.The method for preparing the optical purity sec-n-octyl alcohol known today mainly contains three approach: (1) chemical method.Normally utilize chemical method to prepare the optical purity sec-n-octyl alcohol, existing needs to add costliness and has toxic chiral catalyst (vauqueline), complicated operation, it is bigger to split difficulty, and yield is low, causes not enough [Ma Hongyi such as environmental pollution, Henan chemical industry .2004,8:15-16; Chi Shujuan etc., Liaoning chemical industry .2007,19 (4): 196-198].(2) enzyme process.Utilize lipase-catalyzed resolution of racemates sec-n-octyl alcohol to prepare the optical purity sec-n-octyl alcohol, but the not reproducible use of catalyzer, by product is many, yield is low, the subsequent disposal complexity, and product is not easily separated, be subjected to certain restriction [Gao Hongjuan etc., fine chemistry industry .2008,25 (4): 338-341 in actual applications; Shi Xianai etc., South China Science ﹠ Engineering University's journal (natural science edition) .2006,34 (12): 30-34; Single sky etc., catalysis journal .2008,29 (4): 403-408; Zhu Jie etc., catalysis journal .1998,19 (3): 255-259; Dai Dazhang etc., SCI .2007,28 (12): 2307-2310; Jiang Juan etc., chemical industry progress .2006,25 (8): 947-962].(3) cell method.Because microorganism cells contains complete enzyme system, for example contain abundant oxydo-reductase system in the yeast cell, the reducing/regenerating of coenzyme be can realize, expensive cofactors, several no coupling products do not needed to add in addition, yield improves greatly, simplified subsequent processes, but the not reproducible use of cell, product separates still too complicated [Li Yongning etc., medicine biotechnology .2006,13 (6): 446-450; Yang Zhonghua etc., chemical industry journal .2004,55 (8): 1301-1305; Hu Jian etc., chemical industry journal .2006,57 (10): 2383-2387].
Summary of the invention
The method that the purpose of this invention is to provide a kind of immobilized microorganism catalytic preparation optical purity (R)-sec-n-octyl alcohol.
The present invention realizes in the following way:
A kind of immobilized microorganism prepares the method for optical purity (R)-sec-n-octyl alcohol, and its feature may further comprise the steps:
(1), the nonpolar macroporous adsorption resin pre-treatment: with nonpolar macroporous adsorption resin with washed with de-ionized water after with containing the NaOH solution soaking 24-48h of 0.5-5.0%, with washed with de-ionized water to neutral, oven dry;
(2), bread yeast immobilization: the conventional bread yeast DX213 Saccharomyces cerevisiaeDX213 CCTCC NO:M207082 that cultivates, get bacterium liquid, nonpolar macroporous adsorption resin is soaked in the bacterium liquid, 25-35 ℃ of bacterium liquid temp, the pH value is 6.0-8.0, shake a bottle revolution 100-400rpm, fixing bacterial classification 24-48h, elimination bacterium liquid, and wash with physiological saline, with immobilized cell thermal treatment 10-60min, make nonpolar macroporous adsorption resin immobilization bread yeast;
(3), immobilization bread yeast catalysis methyln-hexyl ketone prepares optical purity (R)-sec-n-octyl alcohol: immobilized cell is dissolved in the Tris-HCl damping fluid of 0.1mol/L, making its concentration is 0.2-0.5g/mL, pour organic solvent tetrahydrofuran into, methylene dichloride, normal hexane, toluene, among hexane or sherwood oil a kind of, making its organic solvent volume ratio is 5/30-35/0, divide 2-5 batch to add methyln-hexyl ketone then, make its mass concentration 24-60mmoL/L, with glucose, sucrose, 1, the 2-propylene glycol, a kind of in methyl alcohol or the citric acid is cosubstrate, addition is 0.5-3.0g, pH value of buffer solution is 3.0-10.0 in the reaction system, temperature of reaction 26-36 ℃, reaction times 2-72h gets optical purity (R)-sec-n-octyl alcohol.
The conventional substratum of cultivating consists of the contained component of every L nutrient solution in g: glucose 20.0-60.0, yeast extract paste 1.0-10.0, peptone 1.0-10.0, K
2HPO
40.2-4.0, KH
2PO
40.5-5.0, MgSO
40.02-2.0.
With respect to traditional chemical method, enzyme process and cell method, immobilized cell synthesis method of the present invention has following advantage: 1, immobilization technology is simple, and fixed yeast cell has certain poison resistance in reaction system, and excellent catalytic effect is reusable.2, nonpolar macroporous adsorption resin is the immobilized cell of preparing carriers, and good springiness is strong to the tolerance of temperature, pH value, the physical strength height, and enzyme is lived stable, long half time.3, in the organic solvent two-phase, react, can reduce the toxic action of organic solvent pair cell, improve its catalytic activity.4, fixed yeast cell catalytic preparation optical purity (R)-sec-n-octyl alcohol, technology is simple, need not add expensive coenzyme, mild condition, the reaction times is short, several no coupling products, optical purity is up to 90%e.e., and subsequent disposal is simple, and product is easily separated, environmental friendliness.
Embodiment
The present invention will be further described below in conjunction with embodiment:
The present invention is to be in the organic solvent two-phase system of 5/30-35/0 in volume ratio, initial substrate methyln-hexyl ketone 24-60mmoL/L, and the cosubstrate that adds 0.5-3.0g reacts 2-72h in 26-36 ℃; Product obtains optical purity (R)-sec-n-octyl alcohol after separation and purification.Bread yeast DX213 (Saccharomycescerevisiae DX213) bacterial classification is at applicant's application " production method of a kind of bread yeast and catalyzed production biodiesel thereof " patent (application number: 200710035224.6, the applying date: in the time of 2007.6.27), Chinese Wuhan Wuhan University Chinese typical culture collection center, deposit number CCTCC NO:M207082 have been deposited in on June 18th, 2007.
Embodiment 1
D-101 type nonpolar macroporous adsorption resin is repeatedly soaked 48h with 0.5% NaOH in the back with washed with de-ionized water, to neutral, dry with washed with de-ionized water;
In substratum, insert bread yeast and carry out routine cultivation, substratum consist of the contained component of every L nutrient solution in g: glucose 20.0, yeast extract paste 1.0, peptone 5.0, K
2HPO
41.0, KH
2PO
41.0, MgSO
40.2, make bacterium liquid, D-101 type nonpolar macroporous adsorption resin is soaked in the bacterium liquid, 30 ℃ of bacterium liquid temps, the pH value is 6.5, shake a bottle revolution 150rpm, fixing bacterial classification 28h, elimination bacterium liquid, and wash with physiological saline, with immobilized cell thermal treatment 15min, make nonpolar macroporous adsorption resin immobilization bread leaven matricyte.
D-101 type nonpolar macroporous adsorption resin immobilization bread leaven matricyte is dissolved in the Tris-HCl damping fluid of 0.1mol/L, and making its quality is 0.3g/mL, pours toluene into, and the volume ratio that makes toluene and buffered soln is 15/20.Then, divide two batches to add methyln-hexyl ketone in the mixed solution of toluene/buffered soln, make its mass concentration 30mmol/L, cosubstrate glucose addition is 0.7g.PH value of buffer solution is 7.0 in the reaction system, 28 ℃ of temperature of reaction, reaction times 38h.Separating catalyst, standing demix, the upper strata is for containing the mixed solution of optical purity (R)-sec-n-octyl alcohol, use the equal-volume ethyl acetate extraction, add anhydrous magnesium sulfate in acetic acid ethyl acetate extract, static spending the night, remove residual water-content, reclaim the residual acetic acid ethyl ester, get optical purity (R)-sec-n-octyl alcohol.The productive rate of optical purity (R)-sec-n-octyl alcohol is 16.56%, and optical purity is 30.57% (liquid chromatography).
Embodiment 2
H103 type nonpolar macroporous adsorption resin is repeatedly soaked 30h with 2.0% NaOH in the back with washed with de-ionized water, and washed with de-ionized water is dried to neutral;
In substratum, insert bread yeast and carry out routine cultivation, substratum consist of the contained component of every L nutrient solution in g: glucose 60.0, yeast extract paste 8.0, peptone 1.0, K
2HPO
42.0, KH
2PO
42.0, MgSO
40.5, make bacterium liquid, H103 type nonpolar macroporous adsorption resin is soaked in the bacterium liquid, 28 ℃ of bacterium liquid temps, the pH value is 7.0, shake a bottle revolution 200rpm, fixing bacterial classification 40h, elimination bacterium liquid, and wash with physiological saline, with immobilized cell thermal treatment 60min, make nonpolar macroporous adsorption resin immobilization bread leaven matricyte.
H103 type nonpolar macroporous adsorption resin immobilization bread leaven matricyte is dissolved in the Tris-HCl damping fluid of 0.1mol/L, and making its quality is 0.3g/mL, pours sherwood oil into, and making sherwood oil and volume of buffer solution ratio is 25/20.Then, time interpolation methyln-hexyl ketone makes its mass concentration 50mmol/L in the mixed solution of sherwood oil/buffered soln in four batches.Cosubstrate glucose addition is 2.0g.PH value of buffer solution is 6.5 in the reaction system, 35 ℃ of temperature of reaction, reaction times 42h.Separating catalyst, standing demix, the upper strata is for containing the mixed solution of optical purity (R)-sec-n-octyl alcohol, use the equal-volume ethyl acetate extraction, add anhydrous magnesium sulfate in acetic acid ethyl acetate extract, static spending the night, remove residual water-content, reclaim the residual acetic acid ethyl ester, get optical purity (R)-sec-n-octyl alcohol.The productive rate 86.46% of optical purity (R)-sec-n-octyl alcohol, optical purity are 90.25% (liquid chromatography).
Embodiment 3
HPD-300 type nonpolar macroporous adsorption resin is repeatedly soaked 24h with 4.0% NaOH in the back with washed with de-ionized water, and washed with de-ionized water is dried to neutral;
In substratum, insert bread yeast and carry out routine cultivation, substratum consist of the contained component of every L nutrient solution in g: glucose 30.0, yeast extract paste 4.0, peptone 6.0, K
2HPO
41.0, KH
2PO
42.0, MgSO
40.5, make bacterium liquid, HPD-300 type nonpolar macroporous adsorption resin is soaked in the bacterium liquid, 28 ℃ of bacterium liquid temps, the pH value is 7.0, shake a bottle revolution 180rpm, fixing bacterial classification 48h, elimination bacterium liquid, and wash with physiological saline, with immobilized cell thermal treatment 55min, make nonpolar macroporous adsorption resin immobilization bread leaven matricyte.
HPD-300 type macroporous adsorbent resin immobilization bread leaven matricyte is dissolved in the Tris-HCl damping fluid of 0.1mol/L, and making its quality is 0.3g/mL, pours methylene dichloride into, and making methylene dichloride and volume of buffer solution ratio is 30/8.Three batches are added methyln-hexyl ketone in the mixed solution of methylene dichloride/buffered soln then, make its mass concentration 25mmol/L, and cosubstrate glucose addition is 2.0g.PH value of buffer solution is 8.0 in the reaction system, 27 ℃ of temperature of reaction, reaction times 45h.Separating catalyst, standing demix, the upper strata is for containing the mixed solution of optical purity (R)-sec-n-octyl alcohol, use the equal-volume ethyl acetate extraction, add anhydrous magnesium sulfate in acetic acid ethyl acetate extract, static spending the night, remove residual water-content, reclaim the residual acetic acid ethyl ester, get (R)-sec-n-octyl alcohol.(R)-and the productive rate 86.46% of sec-n-octyl alcohol, optical purity is 90.25% (liquid chromatography).
Claims (2)
1, a kind of immobilized microorganism prepares the method for optical purity (R)-sec-n-octyl alcohol, and its feature may further comprise the steps:
(1), the nonpolar macroporous adsorption resin pre-treatment: with nonpolar macroporous adsorption resin with washed with de-ionized water after with containing the NaOH solution soaking 24-48h of 0.5-5.0%, with washed with de-ionized water to neutral, oven dry;
(2), bread yeast immobilization: the conventional bread yeast DX213 Saccharomyces cerevisiaeDX213 CCTCC NO:M 207082 that cultivates, get bacterium liquid, nonpolar macroporous adsorption resin is soaked in the bacterium liquid, 25-35 ℃ of bacterium liquid temp, the pH value is 6.0-8.0, shake a bottle revolution 100-400rpm, fixing bacterial classification 24-48h, elimination bacterium liquid, and wash with physiological saline, with immobilized cell thermal treatment 10-60min, make nonpolar macroporous adsorption resin immobilization bread yeast;
(3), immobilization bread yeast catalysis methyln-hexyl ketone prepares optical purity (R)-sec-n-octyl alcohol: immobilized cell is dissolved in the Tris-HCl damping fluid of 0.1mol/L, making its concentration is 0.2-0.5g/mL, pour organic solvent tetrahydrofuran into, methylene dichloride, normal hexane, toluene, among hexane or sherwood oil a kind of, making its organic solvent volume ratio is 5/30-35/0, divide 2-5 batch to add methyln-hexyl ketone then, make its mass concentration 24-60mmoL/L, with glucose, sucrose, 1, the 2-propylene glycol, a kind of in methyl alcohol or the citric acid is cosubstrate, addition is 0.5-3.0g, pH value of buffer solution is 3.0-10.0 in the reaction system, temperature of reaction 26-36 ℃, reaction times 2-72h gets optical purity (R)-sec-n-octyl alcohol.
2, a kind of immobilized microorganism according to claim 1 prepares the method for optical purity (R)-sec-n-octyl alcohol, it is characterized in that the conventional substratum of cultivating consists of the contained component of every L nutrient solution in g: glucose 20.0-60.0, yeast extract paste 1.0-10.0, peptone 1.0-10.0, K
2HPO
40.2-4.0, KH
2PO
40.5-5.0, MgSO
40.02-2.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342464A (en) * | 2014-10-30 | 2015-02-11 | 青岛科技大学 | Method for producing chiral phenyl methanol employing catalysis of tarlaromyces flavus |
| CN104388490A (en) * | 2014-10-30 | 2015-03-04 | 青岛科技大学 | Method for producing chiral pyrazinyl ethanol by biological catalysis |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5112750A (en) * | 1985-06-25 | 1992-05-12 | Asama Chemical Co., Ltd. | Immobilized cells and culture method utilizing the same |
| US6451587B1 (en) * | 1999-09-29 | 2002-09-17 | Pfizer Inc. | Microbial asymmetric reduction of 2-chloro-1-[-6-(2,5-dimethyl-pyrrol-1-yl)-pyridin-3-yl]-ethanone |
| CN1285729C (en) * | 2004-07-19 | 2006-11-22 | 江南大学 | Process for preparation optical pure (R)-2- octanol by microorganism and its special microorganism |
| CN101230320B (en) * | 2007-06-27 | 2010-04-14 | 湘潭大学 | Baker's yeast and catalytic production method of biodiesel thereof |
| CN101314787A (en) * | 2008-06-24 | 2008-12-03 | 江苏华荣生物科技有限公司 | Method for preparing optical activity chiral secondary alcohol by using rhodotorula reductase preparation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342464A (en) * | 2014-10-30 | 2015-02-11 | 青岛科技大学 | Method for producing chiral phenyl methanol employing catalysis of tarlaromyces flavus |
| CN104388490A (en) * | 2014-10-30 | 2015-03-04 | 青岛科技大学 | Method for producing chiral pyrazinyl ethanol by biological catalysis |
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