CN102719497B - 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 PDF

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CN102719497B
CN102719497B CN2012102134463A CN201210213446A CN102719497B CN 102719497 B CN102719497 B CN 102719497B CN 2012102134463 A CN2012102134463 A CN 2012102134463A CN 201210213446 A CN201210213446 A CN 201210213446A CN 102719497 B CN102719497 B CN 102719497B
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mandelate
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CN102719497A (en
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欧志敏
南颖康
严琴英
李仁玮
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Zhejiang University of Technology ZJUT
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    • 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
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    • 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
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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

A kind of method of microbial transformation methyl benzoylformate preparation (S)-(+)-methyl mandelate
(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 chirality pharmaceutical intermediate compound, can be widely used for synthesis of chiral acid, chiral alcohol, Chiral Amine compounds, chiral amino alcohol and chirality thiol etc.The chipal compounds prepared by (S)-(+)-methyl mandelate can be widely used in the preparation of chiral drug.For example, after (S)-(+)-methyl mandelate hydrolysis, (S)-(+) of preparation-amygdalic acid can be used for the intermediate 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 just can complete under the catalysis of chiral catalyst, in the chemical reduction process, chiral catalyst is expensive, 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 synthetic method.
Biotransformation can carry out in different reaction systems.The most traditional reaction system is aqueous phase system, and the catalytic activity of carrying out the bio-transformation microorganism in aqueous phase system is higher, but biotransformation efficiency can sharply descend during the organic substrates excessive concentration.In order to solve the restraining effect of high concentration of substrate to catalytic activity of cells, people consider to adopt the organic solvent of good biocompatibility for biotransformation, biocompatibility preferably in organic solvent microorganism can keep catalytic activity preferably, organic substrates can be scattered in 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 bio-transformation catalytic activity of Cell protection largely, be conducive to the separation and Extraction of product simultaneously, be conducive to the recycling of microorganism cells, enhance productivity.
(3) summary of the invention
The object of the invention is to provide a kind of method of utilizing yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2230 asymmetric reduction methyl benzoylformate preparation (S)-(+)-methyl mandelate.
The technical solution used in the present invention is:
A kind of method in methyl benzoylformate microbial transformation preparation (S)-(+)-methyl mandelate, described method is as follows:
Take methyl benzoylformate as substrate, what yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the CGMCC No.2230 of take fermentation obtained is biological catalyst containing the enzyme somatic cells, in dibutyl phthalate, carry out conversion reaction 8 ~ 96 hours under 20 ~ 35 ℃, conversion fluid obtains described (S)-(+)-methyl mandelate through separation and purification; In dibutyl phthalate, the starting point concentration of substrate methyl benzoylformate is preferably 30 ~ 150mmol/L of 1 ~ 300mmol/L().
In the present invention, yeast saccharomyces cerevisiae CGMCC No.2230 used is that near soil Hangzhou West Lake brew-house, screening obtains, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms preservation center, preservation date on October 24th, 2007, in CN 101205523A, disclose.This bacterial strain colony characteristics: present oyster white, glossy, smooth, neat in edge, moistening, smooth surface on nutrient agar, homogeneous colonial morphology.
Describedly containing the enzyme somatic cells, can be directly with the fermented liquid form, participate in reaction, also can participate in reaction with the wet thallus cells form after filtering, or participate in reacting as biological catalyst through immobilization after.
Preferably, described containing the enzyme somatic cells through immobilization after as biological catalyst, its consumption is: the biological catalyst contained in every L dibutyl phthalate is equivalent to entrapping method 1.0 ~ 4.0g(with dry weight basis) containing after the immobilization of enzyme somatic cells again through the immobilized cell particle of multiplication culture (preferably 72 hours) acquisition in 16 ~ 72 hours.
Described biological catalyst preparation method is as follows: the fermented liquid that the cell concentration that Wine brewing yeast strain CGMCC No.2230 fermentation culture is obtained is 3.0 ~ 10.0g/L mixes with the sodium alginate soln of equal-volume mass concentration 1 ~ 5%, stir and obtain mixed solution, mixed solution is dropwise splashed in the calcium chloride solution that enough mass concentrations are 2.5 ~ 4.0%, being fixed of continuously stirring suspension, be positioned under 35 ~ 38 ℃ of conditions and solidify, the immobilization particle obtained is with after the stroke-physiological saline solution washing, be scattered in fermention medium the immobilized cell particle carried out after multiplication culture 16 ~ 72h obtains multiplication culture, be biological catalyst.The diameter of described immobilized cell particle can be controlled by the needle sizes of syringe, is recommended as 1 ~ 5mm, most preferably 2mm.The concentration of sodium alginate is preferably 2%.
Described fermention medium is composed 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 water.
Described separation purification method is as follows: conversion fluid is removed by filter to immobilized cell particle, filtrate decompression (0.07 ~ 0.09MPa) is distilled, the cut of collecting separates through silica gel column chromatography, product (S)-(+) of acquisition high purity (purity is more than 98%)-methyl mandelate.
The present invention adopts immobilized cell particle as catalyzer, and the bio-transformation mechanism that methyl benzoylformate is reduced to (S)-(+)-methyl mandelate is as follows:
Figure BDA00001797295500041
The present invention can obtain fermented liquid as follows:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No.2230 thalline is inoculated into to slant medium, cultivates the thalline that obtains slant culture in 4 ~ 6 days for 26 ~ 35 ℃; Described slant medium forms: 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 water; 121 ℃ of sterilizing 20min, cooling bevel after sterilizing;
(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 obtains seed liquor; Described seed culture medium forms: 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 water; 121 ℃ of sterilizings 20 minutes, cooling after sterilizing, obtain seed culture medium;
(3) inoculum size fermentation culture: get seed liquor, with 10 ~ 20%(v/v) is inoculated in fermention medium, and 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.
Concrete recommendation obtains biological catalyst in accordance with the following steps: the resulting fermented liquid in front is mixed mutually with the sodium alginate soln of equal-volume 2%, described fermented liquid cell concentration is 4.50g/L, stir and obtain mixed solution, mixed solution is dropwise splashed in the calcium chloride solution that mass concentration is 3.5%, continuously stirring, the sodium alginate mixed solution that contains thalline solidify to form the gel beads immobilization particle, the immobilization suspension obtained is positioned under 37 ℃ of conditions and solidifies, the immobilization particle obtained washs by stroke-physiological saline solution, obtains immobilized cell particle; The immobilized cell particle obtained is scattered in fermention medium and carries out multiplication culture 24h, obtains the immobilized cell particle after multiplication culture, and the immobilized cell particle of take after multiplication culture is biological catalyst.
Concrete recommend described method to be: the immobilized cell particle of usining after multiplication culture is as biological catalyst, take methyl benzoylformate as substrate, in dibutyl phthalate, conversion reaction 72h under 30 ℃, 180r/min condition, conversion fluid obtains product (S)-(+)-methyl mandelate through separation and purification; In dibutyl phthalate, the starting point concentration of substrate methyl benzoylformate is 5 ~ 40mmol/L, and the dry cell weight contained in every L dibutyl phthalate is equivalent to take the dry cell weight that fermented liquid that entrapping method is 4.5g/L by the 0.330L cell concentration is made the immobilized cell particle after the immobilized cell particle multiplication culture that multiplication culture obtained after 24 hours in fermention medium.Reaction removes by filter immobilized cell particle by conversion fluid after finishing, and by the conversion fluid underpressure distillation, the cut of collection separates through silica gel column chromatography, obtains highly purified product (S)-(+)-methyl mandelate.
(S)-(+) obtained-methyl mandelate sterling is detected purity and the molecular weight of determining product with gas chromatograph-mass spectrometer.
Determining 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 chiral column, and model is Chiral CYCLODEX-B(0.25mm * 30m * 0.25 μ m), carrier gas is nitrogen, flow velocity is 1ml/min.Chiral column can detect the content of (R)-(-)-methyl mandelate and (S)-(+)-two kinds of enantiomorphs of methyl mandelate, further calculates the superfluous value (ee%) of enantiomorph of the molar yield of reaction and (S)-(+)-methyl mandelate.
The immobilized cell particle that filtration obtains is reusable in the reaction of microbial transformation preparation (S)-(+)-methyl mandelate.
Utilize (S)-(+) that the inventive method produces-superfluous value of methyl mandelate enantiomorph to be greater than 99.0%, 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 bacterial strain safety non-toxic used.2. production operation is easy, and biological transformation ratio is higher.3. biological catalyst is more with low cost than chemical catalyst.4. be not subject to seasonal effect, be easy to realize large-scale industrial production.5. environmental friendliness, the reaction conditions gentleness, can be transformed under normal temperature and pressure smoothly.6. immobilized cell is conducive to realize the separation and Extraction of product as biological catalyst.7. immobilized cell is conducive to realize the recycling of catalyzer as biological catalyst, cost-saving.8. be easy to realize large-scale industrial production.9. dibutyl phthalate 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 is seeded in to slant medium, cultivates 4～6 days for 30 ℃.Slant medium forms: wort 10g/L, and yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, natural pH value, solvent is water, 121 ℃ of sterilizing 20min, cooling bevel after sterilizing.
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 water.Get a ring thalline with inoculating needle and be seeded in the 250ml triangular flask that contains the 100ml liquid nutrient medium from slant medium, cultivate 24h and obtain seed liquor under 30 ℃, the condition of 180r/min.Seed liquor is inoculated in the 250ml triangular flask that contains the 100ml liquid nutrient medium with 10% inoculum size, cultivates 24h and obtain fermented liquid, the immobilization with the fermented liquid obtained for thalline under 30 ℃, the condition of 180r/min.
The sodium alginate soln that the 100ml fermented liquid that is 450mg by dry cell weight and isopyknic concentration are 2% is mixed and made into mixed solution, and mixed solution is packed in syringe, splashes into 3.5%CaCl 2Form immobilization particle in solution (1000ml), the immobilization particle diameter formed is 2mm, in 37 ℃ of curing 30min, the immobilization particle obtained washs by stroke-physiological saline solution, wash away excessive calcium ion and the cell of not catching, the immobilized cell particle obtained is continued to multiplication culture 72h in fermention medium.
By multiplication culture, good immobilized cell particle joins respectively in 5 bottles of 300ml dibutyl phthalates 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, result is as shown in table 1.Result shows, 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 microorganism.When the initial addition of substrate is less than 36.6mmol, the molar yield of reaction is all higher than 99.0%.The superfluous value of the enantiomorph of product (S)-(+) obtained in the bio-transformation of above-mentioned 5 kinds of different initial additions of substrate-methyl mandelate all is greater than 99.0%.
Table 1: immobilization CGMCC No.2230 transforms the methyl benzoylformate of different initial additions
Figure BDA00001797295500071
Embodiment 2:
CGMCC No.2230 is cultivated and obtains the thalline fermented liquid according to the method in example 1.The 100ml fermented liquid that is 450mg by 4 bottles of dry cell weights is 2% with isopyknic concentration respectively sodium alginate soln is mixed and made into mixed solution, and mixed solution is respectively charged in syringe, splashes into 3.5%CaCl 2Form immobilization particle in solution (1000ml), the immobilization particle diameter formed is 2mm, in 37 ℃ of curing 30min, the immobilization particle obtained washs by stroke-physiological saline solution, wash away excessive calcium ion and the cell of not catching, 4 bottles of immobilized cell particles that obtain are continued respectively to multiplication culture 0h, 24h, 48h and 72h in fermention medium.
4 kinds of good immobilized cell particles of multiplication culture are added respectively in the 300ml dibutyl phthalate 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, result is as shown in table 2.Result shows, the more long raising that more is conducive to the substrate molar yield of the multiplication culture time of immobilized cell particle, the immobilized cell particle that the multiplication culture time reaches 72h can reach 99.0% for transforming the methyl benzoylformate molar yield, and the superfluous value of enantiomorph that the immobilized cell particle that above-mentioned 4 kinds of different multiplication culture times obtain of take is (S)-(+) prepared by biological catalyst-methyl mandelate all is 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 and obtains the thalline fermented liquid according to the method in example 1.The 100ml fermented liquid that is 450mg by 4 bottles of dry cell weights is 2% with isopyknic concentration respectively sodium alginate soln is mixed and made into mixed solution, and mixed solution is respectively charged in the syringe that needle sizes is different, splashes into 3.5%CaCl 2(1000ml) form immobilization particle in solution, the immobilization particle diameter formed is respectively 2mm, 3mm, 4mm and 5mm, in 37 ℃ of curing 30min, the immobilization particle obtained washs by stroke-physiological saline solution, wash away excessive calcium ion and the cell of not catching, the immobilized cell particle obtained is continued to multiplication culture 72h in fermention medium.
4 kinds of good immobilized cell particles of multiplication culture are joined respectively in the 300ml dibutyl phthalate that contains the 36.6mmol methyl benzoylformate, carry out bio-transformation 72h under 30 ℃, the condition of 180r/min, in conversion process, every 8h, sample, adopt the superfluous value of gas Chromatographic Determination product content and enantiomorph, result is as shown in table 3.Result shows, the difference of immobilized cell particle diameter is influential to the molar yield of bio-transformation, when best immobilized cell particle diameter is 2mm, the molar yield of bio-transformation reaches 99.0%, and the superfluous value of the enantiomorph of (S)-(+) prepared as catalyzer by the immobilization particle of above-mentioned four kinds of diameters of usining-methyl mandelate all is greater than 99.0%.
Table 3: the immobilized cell particle of different diameter transforms methyl benzoylformate
Figure BDA00001797295500091
Embodiment 4:
CGMCC No.2230 is cultivated and obtains the thalline fermented liquid according to the method in example 1.The 100ml fermented liquid that is 450mg by 5 bottles of dry cell weights is 1%, 2%, 3%, 4% and 5% with isopyknic concentration respectively sodium alginate soln is mixed and made into mixed solution, and mixed solution is respectively charged into to syringe, splashes into 3.5%CaCl 2Form immobilization particle in solution (1000ml), the particle diameter size is 2mm, in 37 ℃ of curing 30min, the immobilization particle obtained washs by stroke-physiological saline solution, wash away excessive calcium ion and the cell of not catching, the immobilized cell particle obtained is continued to multiplication culture 72h in fermention medium.
5 kinds of good immobilized cell particles of multiplication culture are joined respectively in the 300ml dibutyl phthalate that contains the 36.6mmol methyl benzoylformate, carry out bio-transformation 72h under 30 ℃, the condition of 180r/min, in conversion process, every 8h, sample, adopt the superfluous value of gas Chromatographic Determination product content and enantiomorph, result is as shown in table 4.Result shows, in immobilization process, the concentration of sodium alginate is influential to the bio-transformation ability of immobilized cell particle, when the concentration of sodium alginate is optimum concn 2%, the molar yield of bio-transformation can reach 99.0%, and the superfluous value of the enantiomorph of resulting (S)-(+) of immobilization CGMCC No.2230 particle conversion of substrate obtained under the sodium alginate to embed condition of 5 kinds of different concns-methyl mandelate all is greater than 99.0%.
Table 4: the sodium alginate soln immobilization CGMCC No.2230 of different concns transforms methyl benzoylformate
Figure BDA00001797295500101
Embodiment 5:
CGMCC No.2230 is cultivated and obtains the thalline fermented liquid according to the method in example 1.The sodium alginate soln that the 100mL fermented liquid that is 450mg by dry cell weight and equal-volume concentration are 2% is mixed and made into mixed solution, and mixed solution is packed in syringe, splashes into 3.5%CaCl 2(1000ml) form immobilization particle in solution, the immobilization particle diameter formed is 2mm, in 37 ℃ of curing 30min, the immobilization particle obtained washs by stroke-physiological saline solution, wash away excessive calcium ion and the cell of not catching, the immobilized cell particle obtained is continued to multiplication culture 72h in fermention medium.
By multiplication culture, good immobilized cell particle joins in the 300ml dibutyl phthalate that contains the 36.6mmol methyl benzoylacetate, carry out bio-transformation 72h under 30 ℃, the condition of 180r/min, filter out immobilized cell particle after transforming end, by the immobilized cell particle Eddy diffusion in the 300ml dibutyl phthalate, carry out bio-transformation 72h under 30 ℃, the condition of 180r/min, so the recycling immobilized cell particle is 12 times, all adopts the superfluous value of liquid chromatogram measuring product content and enantiomorph at every turn.Result is as shown in table 5.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 be greater than the molar yield of 99.0%, the 12 time be for 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, described method comprises: take methyl benzoylformate as substrate, what yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the CGMCC No.2230 of take obtained in fermentation is biological catalyst containing the enzyme somatic cells, in dibutyl phthalate, carry out conversion reaction 8 ~ 96 hours under 20 ~ 35 ℃, conversion fluid obtains described (S)-(+)-methyl mandelate through separation and purification; In dibutyl phthalate, the starting point concentration of substrate methyl benzoylformate is 1 ~ 300mmol/L.
2. the method for claim 1, it is characterized in that described containing the enzyme somatic cells through immobilization after as biological catalyst, its consumption is: the biological catalyst contained in every L dibutyl phthalate be equivalent to entrapping method by 1.0 ~ 4.0g containing after the immobilization of enzyme somatic cells again through the immobilized cell particle of multiplication culture acquisition in 16 ~ 72 hours.
3. method as claimed in claim 2, it is characterized in that described biological catalyst preparation method is as follows: the fermented liquid that the cell concentration that Wine brewing yeast strain CGMCC No.2230 fermentation culture is obtained is 3.0 ~ 10.0g/L mixes with the sodium alginate soln of equal-volume mass concentration 1 ~ 5%, stir and obtain mixed solution, mixed solution is dropwise splashed in the calcium chloride solution that mass concentration is 2.5 ~ 4.0%, being fixed of continuously stirring suspension, be positioned under 35 ~ 38 ℃ of conditions and solidify, the immobilization particle obtained is with after the stroke-physiological saline solution washing, be scattered in fermention medium the immobilized cell particle carried out after multiplication culture 16 ~ 72h obtains multiplication culture, be biological catalyst.
4. method as claimed in claim 3, is characterized in that described fermention medium is composed 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 water.
5. the method for claim 1, it is characterized in that described separation purification method is as follows: reaction removes by filter biological catalyst by conversion fluid, by the conversion fluid underpressure distillation after finishing, collect cut and separate through silica gel column chromatography, obtain product (S)-(+)-methyl mandelate.
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