CN101709271A - Saccharomyces cerevisiae and application thereof in preparing (R)-(+)-beta-hydroxy-benzenepropanoic acid ethylester by microbial transformation - Google Patents
Saccharomyces cerevisiae and application thereof in preparing (R)-(+)-beta-hydroxy-benzenepropanoic acid ethylester by microbial transformation Download PDFInfo
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 38
- 230000009466 transformation Effects 0.000 title claims abstract description 27
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 25
- 230000000813 microbial effect Effects 0.000 title claims abstract description 12
- DVIBDQWVFHDBOP-SNVBAGLBSA-N ethyl (3r)-3-hydroxy-3-phenylpropanoate Chemical compound CCOC(=O)C[C@@H](O)C1=CC=CC=C1 DVIBDQWVFHDBOP-SNVBAGLBSA-N 0.000 title abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- 239000003054 catalyst Substances 0.000 claims abstract description 9
- 239000002609 medium Substances 0.000 claims description 59
- 239000007788 liquid Substances 0.000 claims description 38
- 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 32
- 239000008103 glucose Substances 0.000 claims description 32
- JACRWUWPXAESPB-QMMMGPOBSA-N (R)-tropic acid Chemical compound OC[C@H](C(O)=O)C1=CC=CC=C1 JACRWUWPXAESPB-QMMMGPOBSA-N 0.000 claims description 25
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 23
- IHVMGRBYERPDRR-UHFFFAOYSA-N CCOC(=O)C(C=C=O)C1=CC=CC=C1 Chemical compound CCOC(=O)C(C=C=O)C1=CC=CC=C1 IHVMGRBYERPDRR-UHFFFAOYSA-N 0.000 claims description 22
- 230000001954 sterilising effect Effects 0.000 claims description 22
- 238000004659 sterilization and disinfection Methods 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 18
- 239000000843 powder Substances 0.000 claims description 18
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 14
- 239000000872 buffer Substances 0.000 claims description 12
- 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 12
- 239000002953 phosphate buffered saline Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- 210000001082 somatic cell Anatomy 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 239000002054 inoculum Substances 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 238000000247 postprecipitation Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 244000005700 microbiome Species 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
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- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 230000008020 evaporation Effects 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
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- 230000036983 biotransformation Effects 0.000 abstract description 3
- 239000005515 coenzyme Substances 0.000 abstract description 3
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- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
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- 238000009629 microbiological culture Methods 0.000 abstract 1
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- AYOLELPCNDVZKZ-UHFFFAOYSA-N 3-hydroxy-3-phenylpropionic acid Chemical compound OC(=O)CC(O)C1=CC=CC=C1 AYOLELPCNDVZKZ-UHFFFAOYSA-N 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 12
- 239000000047 product Substances 0.000 description 7
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- 238000006722 reduction reaction Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- VHGCDTVCOLNTBX-QGZVFWFLSA-N atomoxetine Chemical compound O([C@H](CCNC)C=1C=CC=CC=1)C1=CC=CC=C1C VHGCDTVCOLNTBX-QGZVFWFLSA-N 0.000 description 5
- 229960002430 atomoxetine Drugs 0.000 description 4
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 229960002748 norepinephrine Drugs 0.000 description 3
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
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- DVIBDQWVFHDBOP-UHFFFAOYSA-N ethyl 3-hydroxy-3-phenylpropanoate Chemical compound CCOC(=O)CC(O)C1=CC=CC=C1 DVIBDQWVFHDBOP-UHFFFAOYSA-N 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
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- 238000000825 ultraviolet detection Methods 0.000 description 1
- -1 β-carbonyl phenylpropionic acid ethyl ester-beta-hydroxyphenyl propionic acid Chemical compound 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a new strain-Saccharomyces cerevisiae CGMCC NO.3361 and application thereof in preparing (R)-(+)-beta-hydroxy-benzenepropanoic acid ethylester by microbial transformation. The Saccharomyces cerevisiae was collected in China General Microbiological Culture Collection Center (CGMCC), the address of which is Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101. The collection date is October 23, 2009 and the collection number is CGMCC NO.3361. The beneficial effects of preparing (R)-(+)-beta-hydroxy-benzenepropanoic acid ethylester by the microbial transformation method in the invention are mainly as follows: (1) the biocatalyst is a microbial thallus, thus is lower in cost than the chemical catalysts; (2) the environment is friendly, the reaction conditions are mild and the transformation can be carried out smoothly under normal temperature and normal pressure; (3) the strain used in the production is safe and nontoxic; (4) the method is not influenced by seasons and is easy to realize large-scale industrial production; (5) the production operation is simple and convenient and the biotransformation rate is higher; and (6) coenzyme is unnecessary to be added in the whole reaction process.
Description
(1) technical field
The present invention relates to the new bacterial strain of a strain---yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3361, and the application in microbial transformation preparation (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.
(2) background technology
(R)-(+)-beta-hydroxyphenyl propionic acid ethyl (Benzenepropanoic acid, β-hydroxy, ethylester, (β R)-), molecular formula C
11H
14O
3, molecular weight 194.23, CAS number: 72656-47-4.(R)-(+)-beta-hydroxyphenyl propionic acid ethyl is the important intermediate of synthetic (R)-tomoxetine.Tomoxetine is a kind of inhibitor that norepinephrine absorbs that suppresses.Tomoxetine and norepinephrine absorb the affinity that there is height the position.The concentration that can keep neural norepinephrine reaches certain level, can improve people's endurance, antidepressant.(R)-drug effect of tomoxetine is 2 times of raceme tomoxetine drug effect, is 9 times of (S)-tomoxetine drug effect.
(R)-(+)-route of synthesis of beta-hydroxyphenyl propionic acid ethyl mainly contains two kinds of chemical method and biotransformation methods.Under the effect of chemical catalyst or biological catalyst, reduction β-carbonyl phenylpropionic acid ethyl ester can obtain (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.Adopt chemical method asymmetric reduction β-carbonyl phenylpropionic acid ethyl ester just can finish under the catalysis of chiral catalyst, chiral catalyst costs an arm and a leg in the chemical reduction process, preparation process is loaded down with trivial details.Microorganism cells asymmetric reduction β-carbonyl phenylpropionic acid ethyl ester that employing contains carbonyl reductase has reaction conditions gentleness, characteristics that stereoselectivity is good, with low cost, the green synthesis techniques of the chipal compounds that is otherwise known as.
Adopt microbe transformation method reduction β-carbonyl phenylpropionic acid ethyl ester preparation (R)-(+)-beta-hydroxyphenyl propionic acid ethyl not see patent report, the article report that has only minority to publish is as follows:
2008, Chinese researcher Jin Cui etc. utilized bread yeast to transform β-carbonyl phenylpropionic acid ethyl ester preparation (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.The superfluous value of enantiomorph that obtains product is 75%, productive rate 30%.
Report and research from synthetic (R)-(+) of present microbial method-beta-hydroxyphenyl propionic acid ethyl, the productive rate of product and enantio-selectivity are all undesirable, need find the higher bacterial strain of a kind of catalytic efficiency, and the selection appropriate reaction conditions, improve the superfluous value of the productive rate of product and enantiomorph to greatest extent.
(3) summary of the invention
The purpose of this invention is to provide a kind of is the microorganism novel bacterial yeast saccharomyces cerevisiae of (R)-(+)-beta-hydroxyphenyl propionic acid ethyl for bio-transformation β-carbonyl phenylpropionic acid ethyl ester, and utilizes this bacterial strain to transform the method for synthetic (R)-(+) of β-carbonyl phenylpropionic acid ethyl ester-beta-hydroxyphenyl propionic acid ethyl.
The technical solution used in the present invention is:
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3361, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: Datun Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, 100101, preservation date on October 23rd, 2009, deposit number CGMCC No.3361.
Bacterium source: the microbial bacteria Accharomyces cerevisiae CGMCC No.3361 described in the present invention screens near the brew-house of the West Lake, Hangzhou the soil to obtain.Be used to transform β-carbonyl phenylpropionic acid ethyl ester with separating the bacterial strain that obtains in the soil, obtain yeast saccharomyces cerevisiae CGMCC No.3361 and have the ability that good conversion β-carbonyl phenylpropionic acid ethyl ester is produced (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.
The colony characteristics of described yeast saccharomyces cerevisiae CGMCC No.3361: on nutrient agar, present oyster white, glossy, smooth, neat in edge, moistening, smooth surface, the uniform colonial morphology of quality.
The invention still further relates to the application of described yeast saccharomyces cerevisiae CGMCC No.3361 in β-carbonyl phenylpropionic acid ethyl ester microbial transformation preparation (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.
Concrete, described being applied as: with β-carbonyl phenylpropionic acid ethyl ester is substrate, the enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae CGMCC No.3361 fermentation is a biological catalyst, in phosphate buffered saline buffer, in 25～45 ℃ of conversion reactions 8～40 hours, conversion fluid obtained product (R)-(+)-beta-hydroxyphenyl propionic acid ethyl through separation and purification.
Substrate β-the starting point concentration of carbonyl phenylpropionic acid ethyl ester in phosphate buffered saline buffer is 3.6～29.0mmol/L.For improving transformation efficiency, also can add 50～150g/L glucose in the described phosphate buffered saline buffer as cosubstrate.
The described enzyme somatic cells consumption that contains is 3～50g/mmol substrate (with the somatic cells dry weight basis).
The described enzyme somatic cells that contains is prepared by following method:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No.3361 is inoculated into slant medium, cultivates for 26～35 ℃ and got the thalline inclined-plane in 4～6 days; Described slant medium is prepared by following composition: wort 5～15g/L, and yeast powder 2～4g/L, peptone 4～6g/L, glucose 7～12g/L, agar 15～25g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel;
(2) seed culture: get a ring thalline from slant medium and be transferred to seed culture medium, 26～35 ℃, shaking speed is 150～200r/min, cultivates 18～26h and gets seed liquor; Described seed culture medium is prepared by following composition: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
2O0.5～1.5g/L, KH
2PO
40.6～1.5g/L, natural pH value, solvent is a water;
(3) fermentation culture: get seed liquor, be inoculated in the fermention medium with the inoculum size of volume ratio 10～20%, culture temperature is 26～35 ℃, and shaking speed is 150～200r/min, cultivates 18～30h and obtains containing the fermented liquid that contains the enzyme somatic cells; Described fermention medium is prepared by following composition: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
2O 0.5～1.5g/L, KH
2PO
40.6～1.5g/L, natural pH value, solvent is a water.
Described separation purification method is as follows: with conversion fluid centrifugation somatic cells, get clear liquid and boil and discard the post precipitation n-hexane extraction, the extraction liquid evaporation separates with silica gel column chromatography after reclaiming solvent, product (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.
The pure product of (R)-(+)-beta-hydroxyphenyl propionic acid ethyl that obtain detect with gas chromatograph-mass spectrometer determines degree of purity of production and molecular weight.
The superfluous value of molar yield and product (R)-(+)-beta-hydroxyphenyl propionic acid ethyl enantiomorph (ee%) adopts Agilent 1200 liquid chromatograph analyzing and testing.Chromatographic column is a chiral column, and model is DaicelOB-H; Moving phase is normal hexane/Virahol (80/20); The ultraviolet detection wavelength is 254nm.Chiral column can detect the content of (R)-(+)-beta-hydroxyphenyl propionic acid ethyl and (S)-(-)-two kinds of enantiomorphs of beta-hydroxyphenyl propionic acid ethyl, further calculates the superfluous value (ee%) of enantiomorph of the molar yield of reaction and (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.
Adopt microorganism cells asymmetric reduction β-carbonyl phenylpropionic acid ethyl ester to prepare (R)-(+)-beta-hydroxyphenyl propionic acid ethyl among the present invention, can obtain the superfluous value of high antimer, high molar yield, importantly, help improving transformation efficiency with the regenerating coenzyme process in the reaction.
Microbe transformation method preparation (R)-(+)-beta-hydroxyphenyl propionic acid ethyl that the present invention adopts is compared with chemical synthesis, enzyme catalysis method and had the following advantages: 1. biological catalyst is microbial cells, and is more with low cost than chemical catalyst; 2. environmental friendliness, the reaction conditions gentleness can transform under the normal temperature and pressure smoothly; 3. produce used bacterial strain safety non-toxic; 4. be not subjected to seasonal effect, be easy to realize large-scale industrial production; 5. production operation is easy, and biological transformation ratio is higher; 6. do not need to add coenzyme in the entire reaction course.
(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:
Slant culture: CGMCC No.3361 bacterial classification inoculation to slant medium, was cultivated 4～6 days for 30 ℃.Described slant medium is prepared by following composition: wort 10g/L, and yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel.
Seed culture and fermentation: seed and fermention medium all adopt liquid nutrient medium, prepare by following composition: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L, 121 ℃ of sterilization 20min.PH value with 1mol/LNaOH or 1mol/LHCl adjustment liquid nutrient medium is 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0 respectively.
From slant medium, get a ring thalline with inoculating needle and be seeded in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain seed.The inoculum size of seed liquor with volume ratio 10% is inoculated in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain fermented liquid, in the centrifugal 20min of 4500r/min, the gained thalline is used for microbial transformation with fermented liquid.
In the triangular flask that contains 100ml pH7.0 phosphate buffered saline buffer, add above-mentioned gained thalline 9g (dry weight), add 0.36mmol β-carbonyl phenylpropionic acid ethyl ester, place 30 ℃, react 24h in the shaking table of 200r/min as substrate.After reaction finished, the centrifugal thalline that discards, supernatant liquor boiled and discard post precipitation equal-volume n-hexane extraction 3 times, the superfluous value of its molar yield of liquid-phase chromatographic analysis and enantiomorph.
Table 1: initial pH value of medium transforms the influence of β-carbonyl phenylpropionic acid ethyl ester to yeast saccharomyces cerevisiae CGMCC No.3361
| The initial pH value of substratum | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| ??5 | ??52.9 | ??88.9 |
| ??5.5 | ??51.5 | ??88.5 |
| ??6 | ??52.6 | ??90.1 |
| The initial pH value of substratum | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| ??6.5 | ??51.9 | ??93.5 |
| ??7 | ??51.8 | ??95.2 |
| ??7.5 | ??50.5 | ??95.6 |
| ??8 | ??48.2 | ??95.5 |
| ??8.5 | ??46.5 | ??95 |
| ??9 | ??45 | ??95.2 |
Embodiment 2:
Slant culture: CGMCC No.3361 bacterial classification inoculation to slant medium, was cultivated 4～6 days for 30 ℃.Described slant medium is prepared by following composition: wort 10g/L, and yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel.
Seed culture and fermentation: seed and fermention medium all adopt liquid nutrient medium, prepare by following composition: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L, 121 ℃ of sterilization 20min.The pH value of adjusting liquid nutrient medium is 7.0.
From slant medium, get a ring thalline with inoculating needle and be seeded in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain seed.Seed liquor is inoculated in the 250ml triangular flask that contains the 100ml liquid nutrient medium with 10% inoculum size, under 30 ℃, the condition of 180r/min, cultivates 24h and obtain fermented liquid.
In the triangular flask that contains 100ml pH7.0 phosphate buffered saline buffer, add above-mentioned gained thalline 9g (dry weight), add β-carbonyl phenylpropionic acid ethyl ester 0.36mmol as substrate, its concentration is 3.6mmol/L, add cosubstrate glucose, make its concentration be respectively 10g/L, 30g/L, 50g/L, 70g/L, 100g/L, 150g/L, place 30 ℃, react 24h in the shaking table of 200r/min.After reaction finished, the centrifugal thalline that discards, supernatant liquor boiled and discard post precipitation equal-volume n-hexane extraction 3 times, the superfluous value of its molar yield of liquid-phase chromatographic analysis and enantiomorph.
Table 2: add the influence of glucose to (R)-(+)-beta-hydroxyphenyl propionic acid ethyl transformation efficiency and the superfluous value of enantiomorph
| Cosubstrate | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| 10g/L glucose | ??45.8 | ??95.3 |
| Cosubstrate | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| 30g/L glucose | ??52.9 | ??95.8 |
| 50g/L glucose | ??61.3 | ??95.6 |
| 70g/L glucose | ??74.8 | ??95.1 |
| 100g/L glucose | ??90.5 | ??95.5 |
| 150g/L glucose | ??90.2 | ??96.1 |
Embodiment 3:
Slant culture: CGMCC No.3361 bacterial classification inoculation to slant medium, was cultivated 4～6 days for 30 ℃.Described slant medium is prepared by following composition: wort 10g/L, and yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel.
Seed culture and fermentation: seed and fermention medium all adopt liquid nutrient medium, prepare by following composition: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L, 121 ℃ of sterilization 20min.The pH value of adjusting liquid nutrient medium is 7.0.
From slant medium, get a ring thalline with inoculating needle and be seeded in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain seed.Seed liquor is inoculated in the 250ml triangular flask that contains the 100ml liquid nutrient medium with 10% inoculum size, under 30 ℃, the condition of 180r/min, cultivates 24h and obtain fermented liquid.
In the triangular flask that contains 100ml pH7.0 phosphate buffered saline buffer, add above-mentioned gained thalline 9g (dry weight), add β-carbonyl phenylpropionic acid ethyl ester 0.36mmol as substrate, its concentration is 3.6mmol/L, add cosubstrate glucose, making its concentration is 10%, reacts 24h down respectively at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ in the shaking table of 200r/min.After reaction finished, the centrifugal thalline that discards, supernatant liquor boiled and discard post precipitation equal-volume n-hexane extraction 3 times, the superfluous value of its molar yield of liquid-phase chromatographic analysis and enantiomorph.
Table 3: temperature of reaction is to the influence of (R)-(+)-beta-hydroxyphenyl propionic acid ethyl transformation efficiency and the superfluous value of enantiomorph
| Invert point (℃) | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| ??25 | ??85.7 | ??95.0 |
| ??30 | ??90.5 | ??95.5 |
| Invert point (℃) | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| ??35 | ??83.2 | ??95.9 |
| ??40 | ??78.5 | ??95.5 |
| ??45 | ??71.2 | ??96.0 |
Embodiment 4:
Slant culture: CGMCC No.3361 bacterial classification inoculation to slant medium, was cultivated 4～6 days for 30 ℃.Described slant medium is prepared by following composition: wort 10g/L, and yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel.
Seed culture and fermentation: seed and fermention medium all adopt liquid nutrient medium, prepare by following composition: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L, 121 ℃ of sterilization 20min.The pH value of adjusting liquid nutrient medium is 7.0.
From slant medium, get a ring thalline with inoculating needle and be seeded in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain seed.Seed liquor is inoculated in the 250ml triangular flask that contains the 100ml liquid nutrient medium with 10% inoculum size, under 30 ℃, the condition of 180r/min, cultivates 24h and obtain fermented liquid.
In the triangular flask that contains 100ml pH7.0 phosphate buffered saline buffer, add above-mentioned gained thalline 9g (dry weight), add β-carbonyl phenylpropionic acid ethyl ester 0.36mmol as substrate, its concentration is 3.6mmol/L, add cosubstrate glucose, making its concentration is 100g/L, place 30 ℃, react 8h, 16h, 24h, 32h, 40h in the shaking table of 200r/min respectively.After reaction finished, the centrifugal thalline that discards, supernatant liquor boiled and discard post precipitation equal-volume n-hexane extraction 3 times, the superfluous value of its molar yield of liquid-phase chromatographic analysis and enantiomorph.
Table 4: the reaction times is to the influence of (R)-(+)-beta-hydroxyphenyl propionic acid ethyl transformation efficiency and the superfluous value of enantiomorph
| Transformation time (h) | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| ??8 | ??20.8 | ?95.2 |
| ??16 | ??47.5 | ?95.5 |
| ??24 | ??90.5 | ?95.5 |
| ??32 | ??90.8 | ?95.0 |
| Transformation time (h) | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| ??40 | ??89.2 | ?96.5 |
Embodiment 5:
Slant culture: CGMCC No.3361 bacterial classification inoculation to slant medium, was cultivated 4～6 days for 30 ℃.Described slant medium is prepared by following composition: wort 10g/L, and yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel.
Seed culture and fermentation: seed and fermention medium all adopt liquid nutrient medium, prepare by following composition: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L, 121 ℃ of sterilization 20min.The pH value of adjusting liquid nutrient medium is 7.0.
From slant medium, get a ring thalline with inoculating needle and be seeded in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain seed.Seed liquor is inoculated in the 250ml triangular flask that contains the 100ml liquid nutrient medium with 10% inoculum size, under 30 ℃, the condition of 180r/min, cultivates 24h and obtain fermented liquid.
In the triangular flask that contains 100ml pH7.0 phosphate buffered saline buffer, add above-mentioned gained thalline 9g (dry weight), making its concentration is 90g/L (dry weight/reaction volume), the concentration that adds β-carbonyl phenylpropionic acid ethyl ester is respectively 3.6mmol/L, 7.3mmol/L, 14.5mmol/L, 21.8mmol/L, 29.0mmol/L, add cosubstrate glucose, making its concentration is 10%, place 30 ℃, react 24h in the shaking table of 200r/min respectively.After reaction finished, the centrifugal thalline that discards, supernatant liquor boiled and discard post precipitation equal-volume n-hexane extraction 3 times, the superfluous value of its molar yield of liquid-phase chromatographic analysis and enantiomorph.
Table 5: the substrate starting point concentration is to the influence of the superfluous value of (R)-(+)-beta-hydroxyphenyl propionic acid ethyl enantiomorph
| Concentration of substrate (mmol/L) | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| ??3.6 | ??90.5 | ??95.5 |
| ??7.3 | ??62.5 | ??95 |
| ??14.5 | ??47.2 | ??94.5 |
| ??21.8 | ??30.7 | ??95.1 |
| ??29 | ??0 | ??- |
Embodiment 6:
Slant culture: CGMCC No.3361 bacterial classification inoculation to slant medium, was cultivated 4～6 days for 30 ℃.Described slant medium is prepared by following composition: wort 10g/L, and yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel.
Seed culture and fermentation: seed and fermention medium all adopt liquid nutrient medium, prepare by following composition: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L, 121 ℃ of sterilization 20min.The pH value of adjusting liquid nutrient medium is 7.0.
From slant medium, get a ring thalline with inoculating needle and be seeded in the 250ml triangular flask that contains the 100ml liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain seed.Seed liquor is inoculated in the 250ml triangular flask that contains the 100ml liquid nutrient medium with 10% inoculum size, under 30 ℃, the condition of 180r/min, cultivates 24h and obtain fermented liquid.
In the triangular flask that contains 100ml pH7.0 phosphate buffered saline buffer, add above-mentioned gained thalline 9g, 17.2g, 25.8g, 34.4g, 43.0g (dry weight) respectively, make its concentration be respectively 90g/L, 172g/L, 258g/L, 344g/L, 430g/L (dry weight/reaction volume), the concentration that adds β-carbonyl phenylpropionic acid ethyl ester is respectively 21.8mmol/L, add cosubstrate glucose, making its concentration is 100g/L, place 30 ℃, react 24h in the shaking table of 200r/min.After reaction finished, the centrifugal thalline that discards, supernatant liquor boiled and discard post precipitation equal-volume n-hexane extraction 3 times, the superfluous value of its molar yield of liquid-phase chromatographic analysis and enantiomorph.
Table 6: biomass is to the influence of (R)-(+)-beta-hydroxyphenyl propionic acid ethyl transformation efficiency and the superfluous value of enantiomorph
| Somatic cells concentration (g/L) | Transformation efficiency (%) | (R)-(+)-the superfluous value of the enantiomorph of beta-hydroxyphenyl propionic acid ethyl (ee%) |
| ??90 | ??30.7 | ?95.1 |
| ??172 | ??45.7 | ?95 |
| ??258 | ??59.8 | ?94.6 |
| ??344 | ??79.9 | ?95.2 |
| ??430 | ??91.5 | ?94.7 |
Claims (8)
1. yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3361, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: Datun Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, 100101, preservation date on October 23rd, 2009, deposit number CGMCC No.3361.
2. the application of yeast saccharomyces cerevisiae CGMCC No.3361 as claimed in claim 1 in β-carbonyl phenylpropionic acid ethyl ester microbial transformation preparation (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.
3. application as claimed in claim 2, it is characterized in that described being applied as: with β-carbonyl phenylpropionic acid ethyl ester is substrate, the enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae CGMCC No.3361 fermentation is a biological catalyst, in phosphate buffered saline buffer, in 25～45 ℃ of conversion reactions 8～40 hours, conversion fluid obtained product (R)-(+)-beta-hydroxyphenyl propionic acid ethyl through separation and purification.
4. application as claimed in claim 3 is characterized in that the substrate β-starting point concentration of carbonyl phenylpropionic acid ethyl ester in phosphate buffered saline buffer is 3.6～29.0mmol/L.
5. application as claimed in claim 3 is characterized in that also being added with in the described phosphate buffered saline buffer 50～150g/L glucose as cosubstrate.
6. application as claimed in claim 3 is characterized in that the described enzyme somatic cells consumption that contains is 3～50g/mmol substrate.
7. application as claimed in claim 3 is characterized in that the described enzyme somatic cells that contains is prepared by following method:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No.3361 is inoculated into slant medium, cultivates for 26～35 ℃ and got the thalline inclined-plane in 4～6 days; Described slant medium is prepared by following composition: wort 5～15g/L, and yeast powder 2～4g/L, peptone 4～6g/L, glucose 7～12g/L, agar 15～25g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel;
(2) seed culture: get a ring thalline from slant medium and be transferred to seed culture medium, 26～35 ℃, shaking speed is 150～200r/min, cultivates 18～26h and gets seed liquor; Described seed culture medium is prepared by following composition: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
20 0.5～1.5g/L, KH
2PO
40.6～1.5g/L, natural pH value, solvent is a water;
(3) fermentation culture: get seed liquor, be inoculated in the fermention medium with the inoculum size of volume ratio 10～20%, culture temperature is 26～35 ℃, and shaking speed is 150～200r/min, cultivates 18～30h and obtains containing the fermented liquid that contains the enzyme somatic cells; Described fermention medium is prepared by following composition: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
20 0.5～1.5g/L, KH
2PO
40.6～1.5g/L, natural pH value, solvent is a water.
8. application as claimed in claim 3, it is characterized in that described separation purification method is as follows: with conversion fluid centrifugation somatic cells, getting clear liquid boils and discards the post precipitation n-hexane extraction, the extraction liquid evaporation separates with silica gel column chromatography after reclaiming solvent, gets product (R)-(+)-beta-hydroxyphenyl propionic acid ethyl.
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