CN102719497A - Method for preparing methyl (S)-(+)-mandelate by microbial transformation of methyl benzoylformate - Google Patents
Method for preparing methyl (S)-(+)-mandelate by microbial transformation of methyl benzoylformate Download PDFInfo
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- CN102719497A CN102719497A CN2012102134463A CN201210213446A CN102719497A CN 102719497 A CN102719497 A CN 102719497A CN 2012102134463 A CN2012102134463 A CN 2012102134463A CN 201210213446 A CN201210213446 A CN 201210213446A CN 102719497 A CN102719497 A CN 102719497A
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- 238000000034 method Methods 0.000 title claims abstract description 34
- ITATYELQCJRCCK-QMMMGPOBSA-N methyl (2s)-2-hydroxy-2-phenylacetate Chemical compound COC(=O)[C@@H](O)C1=CC=CC=C1 ITATYELQCJRCCK-QMMMGPOBSA-N 0.000 title claims abstract description 31
- YLHXLHGIAMFFBU-UHFFFAOYSA-N methyl phenylglyoxalate Chemical compound COC(=O)C(=O)C1=CC=CC=C1 YLHXLHGIAMFFBU-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 230000009466 transformation Effects 0.000 title claims abstract description 11
- 230000000813 microbial effect Effects 0.000 title claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 239000003054 catalyst Substances 0.000 claims abstract description 27
- 239000000758 substrate Substances 0.000 claims abstract description 21
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 19
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 claims abstract description 17
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 238000000926 separation method Methods 0.000 claims abstract description 8
- 238000000746 purification Methods 0.000 claims abstract description 6
- 239000002245 particle Substances 0.000 claims description 59
- 210000001822 immobilized cell Anatomy 0.000 claims description 41
- 210000004027 cell Anatomy 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 23
- 239000011259 mixed solution Substances 0.000 claims description 17
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- 239000000661 sodium alginate Substances 0.000 claims description 14
- 235000010413 sodium alginate Nutrition 0.000 claims description 14
- 229940005550 sodium alginate Drugs 0.000 claims description 14
- 239000012530 fluid Substances 0.000 claims description 8
- 210000001082 somatic cell Anatomy 0.000 claims description 7
- 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 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- 239000013028 medium composition Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000011426 transformation method Methods 0.000 abstract description 2
- 239000011942 biocatalyst Substances 0.000 abstract 1
- 230000003287 optical effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 21
- 230000036983 biotransformation Effects 0.000 description 19
- 239000000047 product Substances 0.000 description 15
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 10
- 238000006722 reduction reaction Methods 0.000 description 6
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 5
- 238000007792 addition Methods 0.000 description 5
- 229910001424 calcium ion Inorganic materials 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- ITATYELQCJRCCK-UHFFFAOYSA-N Mandelic Acid, Methyl Ester Chemical compound COC(=O)C(O)C1=CC=CC=C1 ITATYELQCJRCCK-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 230000000452 restraining effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000007810 chemical reaction solvent Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 description 1
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000007244 Zea mays Nutrition 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- OLVCFLKTBJRLHI-AXAPSJFSSA-N cefamandole Chemical compound CN1N=NN=C1SCC1=C(C(O)=O)N2C(=O)[C@@H](NC(=O)[C@H](O)C=3C=CC=CC=3)[C@H]2SC1 OLVCFLKTBJRLHI-AXAPSJFSSA-N 0.000 description 1
- 229960003012 cefamandole Drugs 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- WZHCOOQXZCIUNC-UHFFFAOYSA-N cyclandelate Chemical compound C1C(C)(C)CC(C)CC1OC(=O)C(O)C1=CC=CC=C1 WZHCOOQXZCIUNC-UHFFFAOYSA-N 0.000 description 1
- 229960000729 cyclandelate Drugs 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- ITATYELQCJRCCK-MRVPVSSYSA-N methyl (2r)-2-hydroxy-2-phenylacetate Chemical compound COC(=O)[C@H](O)C1=CC=CC=C1 ITATYELQCJRCCK-MRVPVSSYSA-N 0.000 description 1
- WRGLZAJBHUOPFO-UHFFFAOYSA-N methyl 3-oxo-3-phenylpropanoate Chemical compound COC(=O)CC(=O)C1=CC=CC=C1 WRGLZAJBHUOPFO-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 229940124549 vasodilator Drugs 0.000 description 1
- 239000003071 vasodilator agent Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides a method for preparing methyl (S)-(+)-mandelate by the microbial transformation of methyl benzoylformate. The method comprises the following steps of: performing transformation reaction at the temperature of 20 to 35 DEG C for 8 to 96 hours in dibutyl phthalate by taking methyl benzoylformate as a substrate, and enzyme-containing thallus cells obtained by fermenting a Saccharomyces cerevisiae strain CGMCC No.2230 as a biocatalyst, and separating and purifying a transformation solution to obtain a product, namely the methyl (S)-(+)-mandelate. Reaction conditions are mild, the microbial transformation method is environment-friendly and is suitable for industrialized production, the product has high optical purity, the substrate is high in transformation rate, the separation and purification process is simple, and the catalyst can be recycled.
Description
(1) technical field
The present invention relates to utilize the method for yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2230 asymmetric reduction methyl benzoylformate preparation (S)-(+)-methyl mandelate.
(2) background technology
(S)-(+)-methyl mandelate (Methyl (S)-(+)-mandelate), CAS accession number: 21210-43-5, molecular formula C
9H
10O
3, molecular weight 166.1739.It is a kind of important chiral drug midbody, can be widely used for synthesis of chiral acid, chiral alcohol, Chiral Amine compounds, the pure and mild chirality thiol of chiral amino etc.Chipal compounds by (S)-(+)-methyl mandelate preparation can be widely used in the preparation of chiral drug.For example, (S)-(+)-(S)-(+)-racemic melic acid for preparing after the methyl mandelate hydrolysis can be used for the midbody of cefadole, vasodilator cyclandelate, the Hydrobenzole of putting drops in one's eyes etc., also can make sanitas in medicine industry.
(S)-(+)-route of synthesis of methyl mandelate mainly contains chemical method and biotransformation method.Under the effect of chemical catalyst or biological catalyst, the reduction methyl benzoylformate can obtain (S)-(+)-methyl mandelate.Adopt chemical method asymmetric reduction methyl benzoylformate under the catalysis of chiral catalyst, could accomplish, chiral catalyst costs an arm and a leg in the chemical reduction process, the preparation process is loaded down with trivial details.The microorganism cells that employing contains carbonyl reductase is that biological catalyst asymmetric reduction methyl benzoylformate has reaction conditions gentleness, characteristics that stereoselectivity is good, with low cost, is a kind of green synthesis techniques of chipal compounds.Utilizing the microorganism cells transformation technology to produce (S)-(+)-methyl mandelate is a kind of science, economy, eco-friendly compound method.
Biotransformation can carry out in different reaction systems.The most traditional reaction system is an aqueous phase system, and the catalytic activity of in aqueous phase system, carrying out the bio-transformation mikrobe is higher, but bio-transformation efficient can sharply descend during the organic substrates excessive concentration.In order to solve the restraining effect of high concentration of substrate pair cell catalytic activity; People consider to adopt the organic solvent of good biocompatibility to be used for biotransformation; Mikrobe can keep catalytic activity preferably in the biocompatibility preferred organic solvents; Organic substrates can be scattered in the reaction solvent well, thereby avoids substrate and product partial concn heterogeneity and the phenomenon that causes catalytic efficiency (to reduce in reaction solvent.Adopt immobilized cell to carry out the catalytic activity that bio-transformation can be protected cell largely, help the separation and Extraction of product simultaneously, help the recycling of microorganism cells, enhance productivity.
(3) summary of the invention
The object of the invention provides a kind of method of utilizing yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2230 asymmetric reduction methyl benzoylformate preparation (S)-(+)-methyl mandelate.
The technical scheme that the present invention adopts is:
Method in a kind of methyl benzoylformate microbial transformation preparation (S)-(+)-methyl mandelate, said method is following:
With the methyl benzoylformate is substrate; The enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2230 fermentation is a biological catalyst; In Witcizer 300; Under 20 ~ 35 ℃, carry out conversion reaction 8 ~ 96 hours, conversion fluid obtains said (S)-(+)-methyl mandelate through separation and purification; The starting point concentration of substrate methyl benzoylformate is 1 ~ 300mmol/L (preferred 30 ~ 150mmol/L) in Witcizer 300.
Used yeast saccharomyces cerevisiae CGMCC No.2230 is that screening obtains near the soil the brew-house of the West Lake, Hangzhou among the present invention; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms preservation center; Preservation date on October 24th, 2007, in CN 101205523A, disclose.This bacterial strain colony characteristics: on nutrient agar, demonstrate oyster white, glossy, smooth, neat in edge, moistening, smooth surface, the uniform colonial morphology of quality.
The said enzyme somatic cells that contains can directly be participated in reaction with the fermented liquid form, also can participate in reaction with the wet thallus cells form after filtering, perhaps after immobilization, participates in reaction as biological catalyst.
Preferably; Said contain the enzyme somatic cells after immobilization as biological catalyst, its consumption is: the biological catalyst that contains in every L Witcizer 300 is equivalent to contain enzyme somatic cells immobilization after the again immobilized cell particle that through multiplication culture 16 ~ 72 hour (preferred 72 hour) obtain of entrapping method with 1.0 ~ 4.0g (with dry weight basis).
Said biological catalyst preparation method is following: the cell concentration that Wine brewing yeast strain CGMCC No.2230 fermentation culture is obtained is that the fermented liquid of 3.0 ~ 10.0g/L mixes with the sodium alginate soln of equal-volume mass concentration 1 ~ 5%; Stir and obtain mixed solution; It is in 2.5 ~ 4.0% the calcium chloride solution that mixed solution is dropwise splashed into the capacity mass concentration; Continuously stirring obtains immobilization suspension-s, is positioned under 35 ~ 38 ℃ of conditions to solidify, after the immobilization particle that obtains washs with SPSS; Be scattered in the immobilized cell particle that carries out in the fermention medium after multiplication culture 16 ~ 72h obtains multiplication culture, be biological catalyst.The diameter of described immobilized cell particle can be controlled through the needle sizes of syringe, is recommended as 1 ~ 5mm, most preferably 2mm.The concentration of sodium-alginate is preferably 2%.
Said fermention medium is formed as follows: glucose 26 ~ 32g/L, yeast powder 2 ~ 4g/L, ammonium sulfate 3 ~ 6g/L, anhydrous MgSO
40.2 ~ 0.4g/L, K
2HPO
43H
200.5 ~ 1.5g/L, KH
2PO
40.6 ~ 1.5g/L, the pH nature, solvent is a water.
Said separation purification method is following: conversion fluid is removed by filter immobilized cell particle; With filtrate decompression (0.07 ~ 0.09MPa) distillation; The cut of collecting separates through silica gel column chromatography, obtains product (S)-(+)-methyl mandelate of high purity (purity is more than 98%).
The present invention adopts immobilized cell particle as catalyzer, and the bio-transformation mechanism that methyl benzoylformate is reduced to (S)-(+)-methyl mandelate is following:
The present invention can obtain fermented liquid as follows:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No.2230 thalline is inoculated into slant medium, cultivates the thalline that obtained slant culture in 4 ~ 6 days for 26 ~ 35 ℃; Described slant medium is formed: 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 formed: glucose 26 ~ 32g/L, yeast powder 2 ~ 4g/L, ammonium sulfate 3 ~ 6g/L, anhydrous MgSO
40.2 ~ 0.4g/L, K
2HPO
43H
200.5 ~ 1.5g/L, KH
2PO
40.6 ~ 1.5g/L, the pH nature, solvent is a water; Sterilized 20 minutes for 121 ℃, the sterilization postcooling promptly gets seed culture medium;
(3) fermentation culture: get seed liquor, be inoculated in the fermention medium with the inoculum size of 10 ~ 20% (v/v), culture temperature is 26 ~ 35 ℃, and shaking speed is 150 ~ 200r/min, cultivates 18 ~ 30h and obtains fermented liquid; The composition of described fermention medium is with the seed substratum.
The concrete recommendation obtains biological catalyst according to following steps: the resulting fermented liquid in front is mixed with the sodium alginate soln of equal-volume 2% mutually; Described fermented liquid cell concentration is 4.50g/L; Stirring obtains mixed solution, and it is in 3.5% the calcium chloride solution that mixed solution is dropwise splashed into mass concentration, continuously stirring; The sodium-alginate mixed solution that contains thalline solidify to form the gel beads immobilization particle; The immobilization suspension-s that obtains is positioned under 37 ℃ of conditions solidifies, the immobilization particle that obtains washs with SPSS, obtains immobilized cell particle; The immobilized cell particle that obtains is scattered in and carries out multiplication culture 24h in the fermention medium, obtains the immobilized cell particle behind the multiplication culture, is biological catalyst with the immobilized cell particle behind the multiplication culture.
Concrete recommend described method to be: with the immobilized cell particle behind the multiplication culture as biological catalyst; With the methyl benzoylformate is substrate; In Witcizer 300; Conversion reaction 72h under 30 ℃, 180r/min condition, conversion fluid obtains product (S)-(+)-methyl mandelate through separation and purification; In Witcizer 300; The starting point concentration of substrate methyl benzoylformate is 5 ~ 40mmol/L, and it is the dry cell weight that the fermented liquid of 4.5g/L is processed the immobilized cell particle behind the immobilized cell particle multiplication culture that multiplication culture obtained after 24 hours in fermention medium with the 0.330L cell concentration that the dry cell weight that contains in every L Witcizer 300 is equivalent to entrapping method.Reaction removes by filter immobilized cell particle with conversion fluid after finishing, and with the conversion fluid underpressure distillation, the cut of collection separates through silica gel column chromatography, obtains highly purified product (S)-(+)-methyl mandelate.
The pure article of (S)-(+)-methyl mandelate that obtain detect with gas chromatograph-mass spectrometer confirms degree of purity of production and molecular weight.
Confirming of the superfluous value of molar yield and product (S)-(+)-methyl mandelate enantiomorph (ee%):
Adopt Agilent 7820 gas chromatograph analyzing and testing.Chromatographic column is a chiral column, and model is Chiral CYCLODEX-B (0.25mm * 30m * 0.25 μ m), and carrier gas is a nitrogen, and flow velocity is 1ml/min.Chiral column can detect (R)-(-)-methyl mandelate with (S)-(+)-content of two kinds of enantiomorphs of methyl mandelate, further calculate the molar yield of reaction with (S)-(+)-the superfluous value (ee%) of enantiomorph of methyl mandelate.
The immobilized cell particle that filtration obtains is reusable in the reaction of microbial transformation preparation (S)-(+)-methyl mandelate.
The superfluous value of (S)-(+)-methyl mandelate enantiomorph that utilizes the inventive method to produce is greater than 99.0%, and the substrate molar yield can reach 99.0%, and product purity reaches 98.0%, and immobilized cell particle can reuse 12 times.
Microbe transformation method preparation (S)-(+)-methyl mandelate of the present invention has the following advantages: 1. produce used bacterial strain safety non-toxic.2. production operation is easy, and biological transformation ratio is higher.3. biological catalyst is more with low cost than chemical catalyst.4. do not receive seasonal effect, be easy to realize large-scale industrial production.5. environmental friendliness, reaction conditions is gentle, can transform smoothly under the normal temperature and pressure.6. immobilized cell helps realizing the separation and Extraction of product as biological catalyst.7. immobilized cell helps realizing the recycling of catalyzer as biological catalyst, practices thrift cost.8. be easy to realize large-scale industrial production.9. Witcizer 300 can dissolve a large amount of substrates and product, can reduce organic substrates and the product restraining effect to the biocatalytic reaction process, improves the inversion quantity of substrate, improves the production efficiency of bio-transformation.
(4) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1:
Slant culture: CGMCC No.2230 bacterial classification inoculation at slant medium, was cultivated 4～6 days for 30 ℃.Slant medium is formed: 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, and composition is glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O1g/L, KH
2PO
41g/L, natural pH value, solvent is a water.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 liquor.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, be used for the immobilization of thalline with the fermented liquid that obtains.
With dry cell weight is that 100ml fermented liquid and the isopyknic concentration of 450mg is that 2% sodium alginate soln is mixed and made into mixed solution, and mixed solution is packed in the syringe, splashes into 3.5%CaCl
2Form immobilization particle in the solution (1000ml); The immobilization particle diameter that forms is 2mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in fermention medium with the immobilized cell particle that obtains.
The immobilized cell particle that multiplication culture is good joins respectively in 5 bottles of 300ml Witcizer 300s that contain 9.15mmol, 18.3mmol, 27.45mmol, 36.6mmol and 45.75mmol methyl benzoylformate; Carry out bio-transformation 72h under 30 ℃, the condition of 180r/min, the result is as shown in table 1.The result shows that the molar yield of reaction reduces along with the raising of the initial addition of substrate.There is restraining effect in the raising that the substrate addition is described to the bioconversion reaction of mikrobe.When the initial addition of substrate during less than 36.6mmol, the molar yield of reaction all is higher than 99.0%.The superfluous value of the enantiomorph of product (S)-(+)-methyl mandelate that in the bio-transformation of above-mentioned 5 kinds of different initial additions of substrate, obtains is all greater than 99.0%.
Table 1: immobilization CGMCC No.2230 transforms the methyl benzoylformate of different initial additions
Embodiment 2:
CGMCC No.2230 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With 4 bottles of dry cell weights 100ml fermented liquid that is 450mg is that 2% sodium alginate soln is mixed and made into mixed solution with isopyknic concentration respectively, and mixed solution is respectively charged in the syringe, splashes into 3.5%CaCl
2Form immobilization particle in the solution (1000ml); The immobilization particle diameter that forms is 2mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 0h, 24h, 48h and 72h in fermention medium respectively with 4 bottles of immobilized cell particles that obtain.
4 kinds of good immobilized cell particles of multiplication culture are added respectively in the 300ml Witcizer 300 that contains the 36.6mmol methyl benzoylformate; Carry out bio-transformation 72h under 30 ℃, the condition of 180r/min; After transforming end; Adopt the superfluous value of gas Chromatographic Determination product content and enantiomorph, the result is as shown in table 2.The result shows; The raising of substrate molar yield is grown and more helped to the multiplication culture time of immobilized cell particle; The immobilized cell particle that the multiplication culture time reaches 72h is used to transform the methyl benzoylformate molar yield and can reaches 99.0%, and the immobilized cell particle that obtains with above-mentioned 4 kinds of different multiplication culture times is that the superfluous value of enantiomorph of (S)-(+)-methyl mandelate of biological Preparation of Catalyst is all greater than 99.0%.
Table 2: the immobilized cell particle of different multiplication culture times transforms methyl benzoylformate
| The multiplication culture time (h) | 0 | 24 | 48 | 72 |
| Molar yield (%) | 0 | 80.2 | 86.5 | 99.0 |
Embodiment 3:
CGMCC No.2230 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With 4 bottles of dry cell weights 100ml fermented liquid that is 450mg is that 2% sodium alginate soln is mixed and made into mixed solution with isopyknic concentration respectively, and mixed solution is respectively charged in the different syringe of needle sizes, splashes into 3.5%CaCl
2(1000ml) form immobilization particle in the solution; The immobilization particle diameter that forms is respectively 2mm, 3mm, 4mm and 5mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in fermention medium with the immobilized cell particle that obtains.
4 kinds of good immobilized cell particles of multiplication culture are joined respectively in the 300ml Witcizer 300 that contains the 36.6mmol methyl benzoylformate; Carry out bio-transformation 72h under 30 ℃, the condition of 180r/min; Every in the conversion process at a distance from the 8h sampling; Adopt the superfluous value of gas Chromatographic Determination product content and enantiomorph, the result is as shown in table 3.The result shows; The difference of immobilized cell particle diameter is influential to the molar yield of bio-transformation; When optimal fixation cell granulations diameter is 2mm; The molar yield of bio-transformation reaches 99.0%, with the immobilization particle of above-mentioned four kinds of diameters as the superfluous value of the enantiomorph of (S)-(+)-methyl mandelate of Preparation of Catalyst all greater than 99.0%.
Table 3: the immobilized cell particle of different diameter transforms methyl benzoylformate
Embodiment 4:
CGMCC No.2230 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With 5 bottles of dry cell weights 100ml fermented liquid that is 450mg is that 1%, 2%, 3%, 4% and 5% sodium alginate soln is mixed and made into mixed solution with isopyknic concentration respectively, and mixed solution is respectively charged into syringe, splashes into 3.5%CaCl
2Form immobilization particle in the solution (1000ml); The particle diameter size is 2mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in fermention medium with the immobilized cell particle that obtains.
5 kinds of good immobilized cell particles of multiplication culture are joined respectively in the 300ml Witcizer 300 that contains the 36.6mmol methyl benzoylformate; Carry out bio-transformation 72h under 30 ℃, the condition of 180r/min; Every in the conversion process at a distance from the 8h sampling; Adopt the superfluous value of gas Chromatographic Determination product content and enantiomorph, the result is as shown in table 4.The result shows; In the immobilization process; The concentration of sodium-alginate is influential to the bio-transformation ability of immobilized cell particle; When the concentration of sodium-alginate was optimum concn 2%, the molar yield of bio-transformation can reach 99.0%, and the superfluous value of the enantiomorph of resulting (S)-(+) of the immobilization CGMCC No.2230 particle conversion of substrate-methyl mandelate that under the sodium alginate to embed condition of 5 kinds of different concns, obtains is all greater than 99.0%.
Table 4: the sodium alginate soln immobilization CGMCC No.2230 of different concns transforms methyl benzoylformate
Embodiment 5:
CGMCC No.2230 is cultivated acquisition thalline fermented liquid according to the method in the instance 1.With dry cell weight is that 100mL fermented liquid and the equal-volume concentration of 450mg is that 2% sodium alginate soln is mixed and made into mixed solution, and mixed solution is packed in the syringe, splashes into 3.5%CaCl
2(1000ml) form immobilization particle in the solution; The immobilization particle diameter that forms is 2mm; Solidify 30min in 37 ℃; The immobilization particle that obtains washs with SPSS, and calcium ion that flush away is excessive and captured cell not continue multiplication culture 72h in fermention medium with the immobilized cell particle that obtains.
The immobilized cell particle that multiplication culture is good joins in the 300ml Witcizer 300 that contains the 36.6mmol methyl benzoylacetate; Carry out bio-transformation 72h under 30 ℃, the condition of 180r/min; Transform the end after-filtration and go out immobilized cell particle; Immobilized cell particle is suspended in the 300ml Witcizer 300 again; Carry out bio-transformation 72h under 30 ℃, the condition of 180r/min, so reuse immobilized cell particle 12 times, all adopt the superfluous value of liquid chromatogram measuring product content and enantiomorph at every turn.The result is as shown in table 5.The result shows; Immobilized cell particle can be re-used in the biosynthesizing of (S)-(+)-methyl mandelate well; The superfluous value of the enantiomorph of product (S)-(+)-methyl mandelate all greater than 99.0%, the 12 time molar yield be the first time molar yield 32.9%.
Table 5: immobilization CGMCC No.2230 transforms the recycling of methyl benzoylformate
Claims (5)
1. the method for microbial transformation methyl benzoylformate preparation (S)-(+)-methyl mandelate; Said method comprises: be substrate with the methyl benzoylformate; Is biological catalyst with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2230 at the enzyme somatic cells that contains that fermentation obtains; In Witcizer 300, under 20 ~ 35 ℃, carry out conversion reaction 8 ~ 96 hours, conversion fluid obtains said (S)-(+)-methyl mandelate through separation and purification; The starting point concentration of substrate methyl benzoylformate is 1 ~ 300mmol/L in Witcizer 300.
2. the method for claim 1; It is characterized in that said contain the enzyme somatic cells after immobilization as biological catalyst, its consumption is: the biological catalyst that contains in every L Witcizer 300 is equivalent to the contain immobilized cell particle that enzyme somatic cells immobilization after again through multiplication culture 16 ~ 72 hour obtain of entrapping method with 1.0 ~ 4.0g.
3. method as claimed in claim 2; It is characterized in that said biological catalyst preparation method is following: the cell concentration that Wine brewing yeast strain CGMCC No.2230 fermentation culture is obtained is that the fermented liquid of 3.0 ~ 10.0g/L mixes with the sodium alginate soln of equal-volume mass concentration 1 ~ 5%; Stir and obtain mixed solution; It is in 2.5 ~ 4.0% the calcium chloride solution that mixed solution is dropwise splashed into mass concentration; Continuously stirring obtains immobilization suspension-s, is positioned under 35 ~ 38 ℃ of conditions to solidify, after the immobilization particle that obtains washs with SPSS; Be scattered in the immobilized cell particle that carries out in the fermention medium after multiplication culture 16 ~ 72h obtains multiplication culture, be biological catalyst.
4. method as claimed in claim 3 is characterized in that said fermention medium composition as follows: glucose 26 ~ 32g/L, yeast powder 2 ~ 4g/L, ammonium sulfate 3 ~ 6g/L, anhydrous MgSO
40.2 ~ 0.4g/L, K
2HPO
43H
200.5 ~ 1.5g/L, KH
2PO
40.6 ~ 1.5g/L, the pH nature, solvent is a water.
5. the method for claim 1; It is characterized in that said separation purification method is following: reaction removes by filter biological catalyst with conversion fluid, with the conversion fluid underpressure distillation after finishing; Collect cut and separate, obtain product (S)-(+)-methyl mandelate through silica gel column chromatography.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101205548A (en) * | 2007-12-14 | 2008-06-25 | 浙江工业大学 | Use of saccharomyces cerevisiae in preparation of (S)-(-)-3-chlorine-1-phenylpropanol |
| CN101205523A (en) * | 2007-12-14 | 2008-06-25 | 浙江工业大学 | Saccharomyces cerevisiae and its application in preparation of beta-hydroxyphenyl propionic acid ethyl |
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| CN101205523A (en) * | 2007-12-14 | 2008-06-25 | 浙江工业大学 | Saccharomyces cerevisiae and its application in preparation of beta-hydroxyphenyl propionic acid ethyl |
Non-Patent Citations (2)
| Title |
|---|
| 张辉: "微生物全细胞转化法制备（R）-扁桃酸的研究", 《中国优秀硕士学位论文全文数据库》, 1 June 2006 (2006-06-01) * |
| 高炳学等: "酿酒酵母不对称合成(R)-(-)-扁桃酸甲酯", 《华东理工大学学报》, vol. 34, no. 3, 30 June 2008 (2008-06-30) * |
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| CN106086090A (en) * | 2016-07-19 | 2016-11-09 | 浙江工业大学 | A kind of method that two-step microbial conversion method prepares R mandelic acid |
| CN106086090B (en) * | 2016-07-19 | 2019-05-28 | 浙江工业大学 | A kind of method that two-step microbial conversion method prepares R-MA |
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