CN101519674A - Method for preparing (S)-1-(3,5-bis(trifluoromethyl)) phenylethanol by microbial transformation - Google Patents
Method for preparing (S)-1-(3,5-bis(trifluoromethyl)) phenylethanol by microbial transformation Download PDFInfo
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- CN101519674A CN101519674A CN200910096423A CN200910096423A CN101519674A CN 101519674 A CN101519674 A CN 101519674A CN 200910096423 A CN200910096423 A CN 200910096423A CN 200910096423 A CN200910096423 A CN 200910096423A CN 101519674 A CN101519674 A CN 101519674A
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- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-phenylethanol Chemical compound OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 38
- 230000009466 transformation Effects 0.000 title claims abstract description 12
- 230000000813 microbial effect Effects 0.000 title claims abstract description 10
- 241000222178 Candida tropicalis Species 0.000 claims abstract description 37
- 239000000758 substrate Substances 0.000 claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- 238000006722 reduction reaction Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims description 40
- KRIOVPPHQSLHCZ-UHFFFAOYSA-N propiophenone Chemical compound CCC(=O)C1=CC=CC=C1 KRIOVPPHQSLHCZ-UHFFFAOYSA-N 0.000 claims description 36
- 229940067107 phenylethyl alcohol Drugs 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 22
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 17
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 17
- 239000000872 buffer Substances 0.000 claims description 17
- 239000006228 supernatant Substances 0.000 claims description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 14
- 238000004659 sterilization and disinfection Methods 0.000 claims description 14
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 13
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 13
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 10
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 239000002953 phosphate buffered saline Substances 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 9
- 238000011081 inoculation Methods 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 2
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 238000003825 pressing Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 241000223678 Aureobasidium pullulans Species 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 239000005515 coenzyme Substances 0.000 description 4
- 238000004817 gas chromatography Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000223252 Rhodotorula Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
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- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 241000235062 Pichia membranifaciens Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 description 1
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 1
- 108010020056 Hydrogenase Proteins 0.000 description 1
- 102000002002 Neurokinin-1 Receptors Human genes 0.000 description 1
- 108010040718 Neurokinin-1 Receptors Proteins 0.000 description 1
- 240000005373 Panax quinquefolius Species 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 241000187561 Rhodococcus erythropolis Species 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000202227 [Candida] mogii Species 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
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- 229910052703 rhodium Inorganic materials 0.000 description 1
- 239000010948 rhodium Substances 0.000 description 1
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for preparing (S)-1-(3,5-bis (trifluoromethyl)) phenylethanol by microbial transformation. The (S)-1-(3,5-bis (trifluoromethyl)) phenylethanol is prepared according to the following steps: 1-(3,5-bis (trifluoromethyl)) phenylethanol functioning as substrate is catalyzed by fermentation products of Candida tropicalis 104 functioning as enzyme source so as to be reduced asymmetrically at 28-34 DEG C, and transformation solution is separated and purified after the asymmetric reduction reaction. The Candida tropicalis 104 is chosen to be used as enzyme-producing strain after optimum strain seeking, and 1-(3,5-bis (trifluoromethyl)) phenylethanol is catalyzed to be reduced asymmetrically in a single-phase aqueous system so as to generate (S)-1-(3,5-bis (trifluoromethyl)) phenylethanol which has an enantiomeric excess value of over 99 percent and can be well applied.
Description
(1) technical field
The present invention relates to the Biotransfer process for preparing of a kind of important chiral drug intermediate (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol, belong to the biocatalysis technology field.
(2) background technology
(S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol is a kind of important chiral drug intermediate, can be used for synthetic nk 1 receptor antagonist, this medicine can be treated depression and other mental disorderes effectively, has good potential curative effect in a series of maincenters of treatment and peripheral nervous system inhibition.
Preparation method known today mainly contains three paths:
(1) chemical method.Mainly be to be synthetic (S)-1-(3, the 5-two trifluoromethyls) phenylethyl alcohol of catalyzer with rare metal (rhodium, ruthenium etc.), reaction yield and ee value are all higher, but costing an arm and a leg of rare metal catalyzer is not suitable for suitability for industrialized production.
(2) enzyme transforming process.Enzyme (as carbonyl reductase) behind the employing extraction purifying carries out the asymmetric reduction of 1-(3,5-two trifluoromethyls) phenyl ethyl ketone, and advantage is that stereoselectivity is good, and by product is few.But need in the reaction process to solve the regenerating coenzyme problem, add costing an arm and a leg of coenzyme.Adopt from synthetic (S)-1-(3, the 5-two trifluoromethyls) phenylethyl alcohol of the alcoholdehydrogenase enzyme process of rhodococcus erythropolis Rhodococcus erythropolis, enantiomeric excess value as David etc. 99.9%, transformation efficiency〉98%.But (formate dehydrogenase FDH) to realize the in-situ regeneration of cofactor, is subjected to certain restriction in actual applications also to need to add hydrogenlyase in needing the alcoholdehydrogenase enzyme catalysis reduction process of cofactor NADH.
(3) microorganism cells biotransformation method.Promptly adopt complete microorganism cells to carry out the asymmetric reduction of 1-(3,5-two trifluoromethyls) phenyl ethyl ketone as biological catalyst.Microorganism cells contains complete enzyme system, and this method does not need intracellular enzyme is purified, and does not also need the extra coenzyme that adds costliness in the catalyzed reaction, thereby has reduced operation and reduced cost.But the existing known bacterial classification that can be used for biological asymmetric reduction preparation (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol (as rhodotorula etc.), enantiomeric excess value is no more than 88.5%.
(3) summary of the invention
The present invention seeks to for a kind of high-optical-purity is provided, technology is simple and be easy to the biological preparation method of (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol of suitability for industrialized production.
For reaching goal of the invention the technical solution used in the present invention be:
A kind of microbial transformation preparation (S)-1-(3,5-two trifluoromethyls) method of phenylethyl alcohol, described method comprises: with 1-(3,5-two trifluoromethyls) the phenyl ethyl ketone is substrate, is in the reaction system in enzyme source with the tunning of candida tropicalis (Candida tropicalis) 104, under 28～34 ℃, carry out asymmetric reduction reaction, reaction finishes the back conversion fluid and obtains described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol through separation and purification; Described candida tropicalis (Candida tropicalis) 104 is preserved in Chinese typical culture collection center, the address: Chinese Wuhan Wuhan University, 430072, deposit number: CCTCC No:M 209034, preservation date: on February 27th, 2009; Described tunning is fermented liquid or the wet thallus that candida tropicalis 104 obtains through fermentation; Wet thallus can be added in the buffered soln contain 1-(3,5-two trifluoromethyls) phenyl ethyl ketone substrate as reaction system, or directly measure on demand substrate is added in candida tropicalis 104 fermented liquids as reaction system.
The substrate starting point concentration is 10～90mmol/L in the described reaction system, and the tunning add-on is counted 50～400g/L with the weight in wet base of its mycetome.Weight in wet base genealogy of law unit volume culture is weighed wet thallus after centrifugal.
The maltose that also is added with final concentration in the described reaction system and is 10～100g/L is as cosubstrate, to promote the carrying out of reaction.
Preferably, described dissolvant of reaction system is 0.1M, and the phosphoric acid buffer of pH 8.0 adds substrate and wet thallus in the damping fluid, as reaction system.
The temperature of reaction of described asymmetric reduction reaction is 28～34 ℃, and the reaction times is 16～40 hours.The conversion fluid of gained carries out chirality gas-chromatography (GC) assay determination and shows that the main ingredient of converted product is (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol with this understanding.
Described tunning is prepared by following method: candida tropicalis 104 is seeded to fermention medium, after 28～34 ℃ of temperature are cultivated 16～30 hours down, gets fermented liquid, perhaps filtering fermentation liquor is obtained wet thallus.Described fermention medium is the conventional liquid fermentation medium that is applicable to candida tropicalis.
Among the present invention, described fermention medium final concentration is composed as follows: glucose 40～60g/L, yeast powder 10～15g/L, ammonium chloride 2～5g/L, potassium primary phosphate 0.5～2g/L, dipotassium hydrogen phosphate 0.5～2g/L, sal epsom 0.1～1g/L, solvent are water, pH value 6～8.5.
Described product separation purification method is as follows: conversion fluid is centrifugal, and the supernatant liquor ethyl acetate extraction obtains described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol in extraction liquid.
Concrete, described method is as follows:
(1) slant culture: get the slant medium that routine is applicable to candida tropicalis, inoculation Candida tropicalis 104 bacterial strains (CCTCC No:M 209034), 30 ℃ of slant culture 3～5 days are as the inclined-plane seed;
(2) seed culture: get the seed culture medium that routine is applicable to candida tropicalis, insert the inclined-plane seed, 28～34 ℃, shaking speed 100～250r/min cultivated 10～30 hours, as seed liquor;
(3) fermentation culture: seed liquor is seeded to the fermention medium that routine is applicable to candida tropicalis with 5～10% volume ratio inoculum sizes, and 28～34 ℃, shaking speed 100～250r/min cultivated 16～30 hours, gets fermented liquid;
(4) microbial transformation: fermented liquid is centrifugal, collect wet thallus, use 0.1M, after the phosphate buffered saline buffer washing of pH 8.0, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH 8.0, add substrate 1-(3,5-two trifluoromethyls) phenyl ethyl ketone simultaneously, making the substrate mass concentration is 10～90mmol/L, cell concentration is 50～400g/L, and to add final concentration be that the maltose of 10～100g/L is as cosubstrate, in 28～34 ℃, 100～250r/min reaction 16～40 hours; After reaction finishes, the centrifugal thalline of removing, the supernatant liquor ethyl acetate extraction obtains described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol in extraction liquid.
Preferably, described method is as follows:
(1) slant culture: the slant medium final concentration consists of: glucose 10g/L, yeast powder 3g/L, peptone 5g/L, agar 20g/L, solvent are water, pH 6.5, sterilized 20 minutes sterilization postcooling, bevel, inoculation Candida tropicalis 104 bacterial strains for 121 ℃, cultivated 3～5 days for 30 ℃, as the inclined-plane seed;
(2) seed culture: the seed culture medium final concentration consists of: glucose 50g/L, yeast powder 13g/L, NH
4Cl 3g/L, KH
2PO
41g/L, K
2HPO
41g/L, MgSO
47H
2O 0.4g/L, solvent are water, and 6.5,121 ℃ of pH sterilization 20 minutes, inserts the inclined-plane seed at the sterilization postcooling, cultivate 10～30 hours in 28～34 ℃, 100～250r/min, as seed liquor;
(3) fermentation culture: the fermention medium final concentration consists of: glucose 50g/L, yeast powder 13g/L, NH
4Cl 3g/L, KH
2PO
41g/L, K
2HPO
41g/L, MgSO
47H
2O 0.4g/L, solvent are water, 6.5,121 ℃ of pH sterilization 20 minutes, the sterilization postcooling, with 5～10% volume ratio inoculum sizes inoculation seed liquor, 28～34 ℃, 100～250r/min were cultivated 16～30 hours, fermented liquid;
(4) microbial transformation: fermented liquid is centrifugal, collection thalline, use 0.1M, after the phosphate buffered saline buffer washing of pH 8.0, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH 8.0, add substrate 1-(3 simultaneously, 5-two trifluoromethyls) phenyl ethyl ketone, making the substrate mass concentration is 10～90mmol/L, cell concentration is 50～400g/L, and add final concentration be the maltose of 10～100g/L as cosubstrate, 28～34 ℃, 100～250r/min reaction 16～40 hours; After reaction finishes, the centrifugal thalline of removing, the supernatant liquor ethyl acetate extraction obtains described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol in extraction liquid.
The gas-chromatography (GC) of substrate 1-(3,5-two trifluoromethyls) phenyl ethyl ketone and product (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol is measured: reaction with an amount of ethyl acetate extraction, is used the GC-2014 gas chromatographic analysis after finishing.Testing conditions: detector is FID, and the chromatographic column model is HP Chira110% Cyclodextrin (30m * 0.25mm * 0.25 μ m), and carrier gas is a nitrogen, chromatogram column temperature: 60 ℃-160 ℃, sampler and detector temperature are respectively 220 ℃ and 250 ℃.
Product (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol enantiomer excessive value (e.e.) measuring method is as follows:
The enantiomeric excess value of product is calculated by following formula:
e.e.＝(G
S-C
R)/(C
S+C
R)×100％
In the formula, C
SBe the concentration of (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol, G
RConcentration for (R)-1-(3,5-two trifluoromethyls) phenylethyl alcohol.
The inventive method has the following advantages with respect to traditional chemical asymmetric synthesis method or enzyme transforming process: 1. (the S)-1-of Sheng Chenging (3,5-two trifluoromethyls) phenylethyl alcohol enantiomeric excess value height reaches more than 99%; 2. biological catalyst is a microbial cells, fermentative production voluntarily, and steady quality, with low cost; 3. in whole asymmetric reduction reaction process, do not need to add coenzyme; 4. reaction conditions gentleness, environmental friendliness.
The present invention is preferred by bacterial classification, determine with candida tropicalis (Candida tropicalis) 104 as bacterium producing multi enzyme preparation, catalytic asymmetric reduction 1-(3 in single aqueous phase system, 5-two trifluoromethyls) the phenyl ethyl ketone generates (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol, the enantiomeric excess value of products therefrom has good application prospects more than 99%.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
The laboratory preservation strain has been carried out bacterial screening.Conversion condition is: at 20mL 0.1M, in the sodium phosphate buffer of pH 8.0, add substrate l-(3,5-two trifluoromethyls) phenyl ethyl ketone concentration is 50mmol/L, the cosubstrate maltose concentration is 50g/L, wet thallus concentration 300g/L, 30 ℃, transform 30h under the shaking speed 200r/min condition, the results are shown in Table 1.
Table 1: the asymmetric reduction result of different strain catalysis 1-(3,5-two trifluoromethyls) phenyl ethyl ketone
| Bacterial classification | Concentration of substrate/(mmol/L) | Maltose concentration/(g/L) | Time/h | Productive rate/% | E.e. value/% |
| Pichia membranaefaciens (Pichia membranaefaciens Hansen) | 50 | 50 | 30 | 46.8 | 98.2(S) |
| Rhodotorula (Rhodotorula qlutis) 2.102 | 50 | 50 | 30 | 31.7 | 80.3(S) |
| Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) B5 | 50 | 50 | 30 | 35.3 | 91.4(S) |
| Lattice candiyeast (Candida mogii) IFFI 01257 not | 50 | 50 | 30 | 39.5 | 97.5(S) |
| Aureobasidium pullulans (Aureobasidium pullulans) ACCC 30142 | 50 | 50 | 30 | 44.5 | 51.6(S) |
| Aureobasidium pullulans (Aureobasidium pullulans) ACCC 30156 | 50 | 50 | 30 | 40.7 | 52.3(S) |
| Candida tropicalis (Candida tropicalis) 104 | 50 | 50 | 30 | 70.3 | >99.9(S) |
Conclusion: candida tropicalis Candida tropicalis 104 (CCTCC No:M 209034) catalysis 1-(3,5-two trifluoromethyls) the phenyl ethyl ketone generates (S)-1-(3,5-two trifluoromethyls) productive rate and the enantiomeric excess value of phenylethyl alcohol are higher, with it as preferred bacterial classification.
Embodiment 2:(S)-preparation of 1-(3,5-two trifluoromethyls) phenylethyl alcohol
Slant culture: each component final concentration of substratum is: glucose 10g/L, yeast powder 3g/L, peptone 5g/L, agar 20g/L, solvent are water, pH 6.5, sterilized 20 minutes sterilization postcooling, bevel, inoculation candida tropicalis Candida tropicalis 104 bacterial strains (CCTCC No:M 209034) for 121 ℃, cultivated 3～5 days for 30 ℃, as the inclined-plane seed.
Seed culture: each component final concentration of substratum is: glucose 50g/L, yeast powder 13g/L, NH
4Cl 3g/L, KH
2PO
41g/L, K
2HPO
41g/L, MgSO
47H
2O 0.4g/L, solvent are water, 6.5,121 ℃ of sterilizations of pH 20 minutes, and sterilization postcooling, access inclined-plane seed, 30 ℃, shaking speed 200r/min cultivated 20 hours, as seed liquor;
Fermentation culture: each component final concentration of substratum is: glucose 50g/L, yeast powder 13g/L, NH
4Cl 3g/L, KH
2PO
41g/L, K
2HPO
41g/L, MgSO
47H
2O 0.4g/L, solvent are water, and pH6.5 sterilized 20 minutes for 121 ℃, sterilization postcooling, inoculation seed liquor, and inoculum size 10% volume ratio, 30 ℃, shaking speed 200r/min cultivated 24 hours, got fermented liquid.
Fermented liquid is centrifugal, collect thalline, use 0.1M, after the phosphate buffered saline buffer washing of pH 8.0, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH 8.0, adding substrate 1-(3,5-two trifluoromethyls) phenyl ethyl ketone simultaneously, to make the substrate mass concentration be 50mmol/L, the wet thallus dosage is 300g/L, and add final concentration be the maltose of 50g/L as cosubstrate, 30 ℃, 200r/min reaction 30 hours, reaction finishes, the centrifugal thalline of removing, the supernatant liquor ethyl acetate extraction gets described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol, productive rate is 70.3%, and enantiomeric excess value reaches more than 99%.
Embodiment 3～7:
With candida tropicalis Candida tropicalis 104 bacterium (CCTCC No:M 209034), after pressing embodiment 2 method fermentation culture, 20mL 0.1M is equipped with in the wet thallus adding, in the 250mL triangular flask of pH 8.0 phosphoric acid buffers (the wet thallus final concentration is 300g/L), and add final concentration be the maltose of 50g/L as cosubstrate, substrate 1-(3,5-two trifluoromethyls) phenyl ethyl ketone concentration is 10～90mmol/L, in 30 ℃, 200r/min transforms 30h.After reaction finished, the centrifugal thalline of removing got supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying after the filtration, adopts gas chromatographic analysis (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol content and enantiomeric excess value, the results are shown in Table 2.
Table 2: different concentration of substrate are to the influence of productive rate and enantiomeric excess value
| Embodiment | Concentration of substrate/(mmol/L) | Productive rate/% | E.e. value/% |
| 3 | 10 | 50.9 | 100.0 |
| 4 | 30 | 53.1 | 100.0 |
| 5 | 50 | 70.3 | 100.0 |
| 6 | 70 | 55.3 | 100.0 |
| 7 | 90 | 45.3 | 100.0 |
As known from Table 2, the preferred concentration of substrate 1-(3,5-two trifluoromethyls) phenyl ethyl ketone is 50mmol/L.
Embodiment 8～13:
With Candida tropicalis 104 bacterium (CCTCC No:M 209034), after pressing example 2 method fermentation culture, get wet thallus in 20mL 0.1M is housed, in the 250mL triangular flask of pH 8.0 phosphoric acid buffers (wet thallus final concentration 300g/L), concentration of substrate is 50mmol/L, and the different cosubstrates of interpolation final concentration 50g/L, in 30 ℃, transform 30h under the 200r/min.After reaction finished, the centrifugal thalline of removing got supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying is filtered laggard promoting the circulation of qi analysis of hplc (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol content and enantiomeric excess value, the results are shown in Table 3.
Table 3: different cosubstrates are to the influence of productive rate and enantiomeric excess value
| Embodiment | Cosubstrate | Productive rate/% | E.e. value/% |
| 8 | Glucose | 60.2 | 100.0 |
| 9 | Sucrose | 47.5 | 100.0 |
| 10 | Maltose | 70.3 | 100.0 |
| 11 | Glycerine | 41.1 | 100.0 |
| 12 | Ethanol | 13.6 | 100.0 |
| 13 | Contrast | 1.7 | 100.0 |
Conclusion: as can be seen from Table 3, preferable cosubstrate is a maltose.
Embodiment 14～18:
With candida tropicalis Candida tropicalis 104 bacterium (CCTCC No:M 209034), after pressing embodiment 2 method fermentation culture, 20mL 0.1M is equipped with in the wet thallus adding, in the 250mL triangular flask of pH 8.0 phosphoric acid buffers (the wet thallus final concentration is 300g/L), substrate 1-(3,5-two trifluoromethyls) phenyl ethyl ketone concentration is 50mmol/L, and to add final concentration be that the maltose of 10～100g/L is as cosubstrate, in 30 ℃, 200r/min transforms 30h.After reaction finished, the centrifugal thalline of removing got supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying after the filtration, adopts gas chromatographic analysis (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol content and enantiomeric excess value, the results are shown in Table 4.
Table 4: different concentration of substrate are to the influence of productive rate and enantiomeric excess value
| Embodiment | Maltose concentration/(mmol/L) | Productive rate/% | E.e. value/% |
| 14 | 10 | 51.4 | 100.0 |
| 15 | 30 | 65.1 | 100.0 |
| 16 | 50 | 70.3 | 100.0 |
| 17 | 70 | 70.0 | 100.0 |
| 18 | 100 | 70.2 | 100.0 |
As known from Table 4, the preferred concentration of cosubstrate maltose is 50g/L.
Embodiment 14～21:
With Candida tropicalis 104 bacterium (CCTCC No:M 209034), after pressing example 2 method fermentation culture, get wet thallus in 20mL 0.1M is housed, (the wet thallus final concentration is 50～400g/L) in the 250mL triangular flask of pH 8.0 phosphoric acid buffers, concentration of substrate is 50mmol/L, and the maltose that adds 50g/L in 30 ℃, transforms 30h under the 200r/min condition as cosubstrate.Reaction finishes the centrifugal thalline of removing in back, gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying is filtered laggard promoting the circulation of qi analysis of hplc (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol content and enantiomeric excess value, the results are shown in Table 5.
Table 5: different cell concentrations are to the influence of productive rate and enantiomeric excess value
| Embodiment | Cell concentration/(g/L) | Productive rate/% | E.e. value/% |
| 19 | 50 | 29.6 | 100.0 |
| 20 | 100 | 33.3 | 100.0 |
| 21 | 150 | 43.4 | 100.0 |
| 22 | 200 | 61.6 | 100.0 |
| 23 | 250 | 68.5 | 100.0 |
| 24 | 300 | 70.3 | 100.0 |
| 25 | 350 | 69.8 | 100.0 |
| 26 | 400 | 70.1 | 100.0 |
Conclusion: as can be seen from Table 4, preferable cell concentration is 300g/L.
Embodiment 27:
With Candida tropicalis 104 bacterium (CCTCC No:M 209034), after pressing example 2 method fermentation culture, get the 300g/L wet thallus in 20mL 0.1M is housed, in the 250mL triangular flask of pH 8.0 phosphoric acid buffers, concentration of substrate is 50mmol/L, and the maltose that adds 50g/L in 30 ℃, transforms 2～48h as cosubstrate under the 200r/min.Reaction finishes the centrifugal thalline of removing in back, gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying is filtered laggard promoting the circulation of qi analysis of hplc (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol content and enantiomeric excess value.Conclusion: preferable transformation time is 30h.
Claims (9)
1. a microbial transformation prepares (S)-1-(3,5-two trifluoromethyls) method of phenylethyl alcohol, described method comprises: with 1-(3,5-two trifluoromethyls) the phenyl ethyl ketone is substrate, is in the reaction system in enzyme source with the tunning of candida tropicalis (Candida tropicalis) 104, under 28～34 ℃, carry out asymmetric reduction reaction, reaction finishes the back conversion fluid and obtains described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol through separation and purification; Described tunning is fermented liquid or the wet thallus that candida tropicalis 104 obtains through fermentation; Described candida tropicalis (Candida tropicalis) 104 is preserved in Chinese typical culture collection center, the address: Chinese Wuhan Wuhan University, 430072, deposit number: CCTCC No:M 209034, preservation date: on February 27th, 2009.
2. the method for claim 1, it is characterized in that: the substrate starting point concentration is 10～90mmol/L in the described reaction system, and the tunning add-on is counted 50～400g/L with the weight in wet base of contained wet thallus.
3. method as claimed in claim 2 is characterized in that also being added with in the described reaction system maltose of final concentration 10～100g/L.
4. the method for claim 1, it is characterized in that: described dissolvant of reaction system is 0.1M, the phosphoric acid buffer of pH 8.0.
5. the method for claim 1, it is characterized in that described tunning is prepared by following method: candida tropicalis 104 is seeded to fermention medium, after 28～34 ℃ of temperature are cultivated 16～30 hours down, get fermented liquid, perhaps filtering fermentation liquor is obtained wet thallus.
6. method as claimed in claim 5, it is characterized in that described fermention medium final concentration is composed as follows: glucose 40～60g/L, yeast powder 10～15g/L, ammonium chloride 2～5g/L, potassium primary phosphate 0.5～2g/L, dipotassium hydrogen phosphate 0.5～2g/L, sal epsom 0.1～1g/L, solvent is a water, pH value 6～8.5.
7. the method for claim 1, it is characterized in that described separation purification method is as follows: conversion fluid is centrifugal, and the supernatant liquor ethyl acetate extraction obtains described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol in extraction liquid.
8. the method for claim 1 is characterized in that described method is as follows:
(1) slant culture: be applicable to slant medium inoculation Candidatropicalis 104 bacterial strains of candida tropicalis, 30 ℃ of slant culture 3～5 days are as the inclined-plane seed;
(2) seed culture: the seed culture medium that is applicable to candida tropicalis inserts the inclined-plane seed, and 28～34 ℃, shaking speed 100～250r/min cultivated 10～30 hours, as seed liquor;
(3) fermentation culture: seed liquor is seeded to the fermention medium that is applicable to candida tropicalis with 5～10% volume ratio inoculum sizes, and 28～34 ℃, shaking speed 100～250r/min cultivated 16～30 hours, gets fermented liquid;
(4) microbial transformation: fermented liquid is centrifugal, collect wet thallus, use 0.1M, after the phosphate buffered saline buffer washing of pH 8.0, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH 8.0, add substrate 1-(3,5-two trifluoromethyls) phenyl ethyl ketone simultaneously, making the substrate mass concentration is 10～90mmol/L, cell concentration is 50～400g/L, and to add final concentration be that the maltose of 10～100g/L is as cosubstrate, in 28～34 ℃, 100～250r/min reaction 16～40 hours; After reaction finishes, the centrifugal thalline of removing, the supernatant liquor ethyl acetate extraction obtains described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol in extraction liquid.
9. the method for claim 1 is characterized in that described method is as follows:
(1) slant culture: the slant medium final concentration consists of: glucose 10g/L, yeast powder 3g/L, peptone 5g/L, agar 20g/L, solvent are water, pH 6.5, sterilized 20 minutes sterilization postcooling, bevel, inoculation Candida tropicalis 104 bacterial strains for 121 ℃, cultivated 3～5 days for 30 ℃, as the inclined-plane seed;
(2) seed culture: the seed culture medium final concentration consists of: glucose 50g/L, yeast powder 13g/L, NH
4Cl 3g/L, KH
2PO
41g/L, K
2HPO
41g/L, MgSO
47H
2O 0.4g/L, solvent are water, and 6.5,121 ℃ of pH sterilization 20 minutes, inserts the inclined-plane seed at the sterilization postcooling, cultivate 10～30 hours in 28～34 ℃, 100～250r/min, as seed liquor;
(3) fermentation culture: the fermention medium final concentration consists of: glucose 50g/L, yeast powder 13g/L, NH
4Cl 3g/L, KH
2PO
41g/L, K
2HPO
41g/L, MgSO
47H
2O 0.4g/L, solvent are water, 6.5,121 ℃ of pH sterilization 20 minutes, the sterilization postcooling, with 5～10% volume ratio inoculum sizes inoculation seed liquor, 28～34 ℃, 100～250r/min were cultivated 16～30 hours, fermented liquid;
(4) microbial transformation: fermented liquid is centrifugal, collection thalline, use 0.1M, after the phosphate buffered saline buffer washing of pH 8.0, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH 8.0, add substrate 1-(3 simultaneously, 5-two trifluoromethyls) phenyl ethyl ketone, making the substrate mass concentration is 10～90mmol/L, cell concentration is 50～400g/L, and add final concentration be the maltose of 10～100g/L as cosubstrate, 28～34 ℃, 100～250r/min reaction 16～40 hours; After reaction finishes, the centrifugal thalline of removing, the supernatant liquor ethyl acetate extraction obtains described (S)-1-(3,5-two trifluoromethyls) phenylethyl alcohol in extraction liquid.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102102087A (en) * | 2010-10-28 | 2011-06-22 | 浙江工业大学 | Leifsonia xyli and use thereof in preparation of (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol |
| CN101724568B (en) * | 2009-12-25 | 2011-08-31 | 浙江工业大学 | Trichoderma asperellum and application thereof in synthesizing (R)-[3,5-dual (trifluoromethyl) phenyl] ethanol |
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| CN103773724A (en) * | 2014-01-17 | 2014-05-07 | 浙江工业大学 | Rhodococcus erythropolis XS1012 and application thereof in preparation of chiral alcohol |
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Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101302552B (en) * | 2007-05-10 | 2010-12-29 | 重庆博腾制药科技股份有限公司 | Method for preparing (R)-2-chlorin-1-(3-chlorphenyl) ethanol by microorganism catalysis |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724568B (en) * | 2009-12-25 | 2011-08-31 | 浙江工业大学 | Trichoderma asperellum and application thereof in synthesizing (R)-[3,5-dual (trifluoromethyl) phenyl] ethanol |
| CN102102087A (en) * | 2010-10-28 | 2011-06-22 | 浙江工业大学 | Leifsonia xyli and use thereof in preparation of (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol |
| CN102102087B (en) * | 2010-10-28 | 2012-02-29 | 浙江工业大学 | Leifsonia xyli and use thereof in preparation of (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol |
| CN103099832A (en) * | 2011-11-10 | 2013-05-15 | 冯淑琴 | Method for biotransformation of pseudo-ginseng by using Candida |
| CN103099832B (en) * | 2011-11-10 | 2014-12-03 | 冯淑琴 | Method for biotransformation of pseudo-ginseng by using Candida |
| CN103773724A (en) * | 2014-01-17 | 2014-05-07 | 浙江工业大学 | Rhodococcus erythropolis XS1012 and application thereof in preparation of chiral alcohol |
| CN112048538A (en) * | 2020-08-21 | 2020-12-08 | 浙江工业大学 | Method for preparing (S) - [3, 5-bis (trifluoromethyl) phenyl ] ethanol by using Verticillium terrestris |
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