CN101210258A - A kind of microbial transformation prepares the method for (R)-3-hydroxybutyrate ethyl ester - Google Patents

Abstract

The invention provides a method for the preparation of ethyl (R)-3-hydroxybutyrate by microbial transformation. The method uses ethyl acetoacetate as a substrate and the fermentation of Pichia membraneef aciens Hansen The product is an enzyme source, and undergoes an asymmetric reduction reaction catalyzed by microorganisms. After the reaction, the conversion liquid is separated and purified to obtain the (R)-3-hydroxybutyric acid ethyl ester. In the present invention, Pichia membranoidis is used as the enzyme-producing strain through self-screening, and the yield of (R)-EHB generated by catalyzing the asymmetric reduction of EOB in a single aqueous phase system reaches 95%, and the enantiomeric excess value (e.e.) reaches 99. %, that is, the obtained product has high optical purity and high concentration, and has great application prospects.

Description

A kind of method of microbial transformation preparation (R)-ethyl 3-hydroxybutanoate

(1) technical field

The present invention relates to a kind of method of microbial transformation preparation (R)-ethyl 3-hydroxybutanoate, belong to the biocatalysis technology field.

(2) background technology

Ethyl 3-hydroxybutanoate (ethy-3-hydroxybutyrate, EHB) be a kind of important pharmaceutical intermediate, contain multi-functional group in the molecule, the single enantiomer of its chirality (R)-EHB and (S)-the EHB 3-hydroxyethyl butyrate all is very promising important chirality building blocks, for example (S)-EHB is the chiral source of lavandulol, sulcatol, caryosphere shell bacterium, natural products such as lattice Kazakhstan rhzomorph, carbomycin and griseoviridin precursor, (R)-and EHB also is the important intermediate of chiral drugs such as carbapenem antibiotic such as synthetic L-carnitine, imipenum.

(ethyl 3-oxobutanoate/ethyl acetoacetate EOB) is easy to syntheticly and cheap, and obtaining chirality EHB with its asymmetric reduction reaction that is substrate carries out microorganism catalysis is very cost-effective preparation approach because methyl aceto acetate.And reaction product (R)-EHB is difficult for being the microbial metabolism utilization, and (R)-EHB is highly beneficial with the preparation of micro-organisms living cell catalysis process.

The method that is reduced to chiral alcohol from EOB known today mainly contains three paths:

(1) chemical method.What chemical method adopted mainly is that transition metal complex generates product as catalyst C=O base hydrogenation.Chemical method can obtain higher productive rate, but main drawback be the reaction stereoselectivity on the low side, and the preparation of chiral catalyst relatively the difficulty.

(2) directly utilize alcoholdehydrogenase catalysis methyl aceto acetate.This method not only will have zymoprotein to participate in reaction, but also will add expensive cofactors, is subjected to certain restriction in actual applications.

(3) microbe whole-cell biotransformation method (Biotransformation).Promptly the stereoselectivity biocatalysis by complete microorganism cells (as bread yeast) realizes, microorganism cells contains complete enzyme system, for example contain alcoholdehydrogenase system in the yeast cell, can realize the in-situ regeneration of coenzyme, do not need to add in addition expensive cofactors, but the existing known bacterial classification that can be used for methyl aceto acetate asymmetric reduction preparation (R)-EHB (as rhodotorula, produce dirty candiyeast, active dry yeast candiyeast etc.), transformation efficiency generally lower (being lower than 50%).

(3) summary of the invention

The present invention promptly is for the method for high microbial transformation preparation (the R)-ethyl 3-hydroxybutanoate of a kind of productive rate height, products therefrom optical purity height, production concentration is provided.

For reaching goal of the invention the technical solution used in the present invention be:

A kind of method of microbial transformation preparation (R)-ethyl 3-hydroxybutanoate, described method is to be substrate with the methyl aceto acetate, with Pichia membranaefaciens (Pichia membranaef aciens Hansen) cell is the enzyme source, carry out the microorganism catalysis asymmetric reduction reaction, reaction finishes the back conversion fluid and obtains described (R)-ethyl 3-hydroxybutanoate through separation and purification.

Fermented liquid that described Pichia membranaefaciens cell obtains through fermentation culture from: Pichia membranaefaciens or the wet thallus that obtains by filtering fermentation liquor.Wet thallus can be added and contain in the solution of substrate, or directly measure on demand substrate is added in the fermented liquid, as reaction system.

The temperature of reaction of the catalytic asymmetric reduction reaction of described microorganism cells is 20～50 ℃, and the reaction times is 1～40 hour.The conversion fluid of gained adopts gas chromatographic analysis (GC) to measure production concentration with this understanding, and the polarimetry in conjunction with product obtains product e.e. value again, shows that the main ingredient that has transformed is (R)-EHB.

Described substrate mass concentration is 1～50%, and it is 0.3～6g/g substrate that the adding tunning makes the wet thallus amount, and the temperature of reaction of described asymmetric reduction reaction is 20～50 ℃, and the reaction times is 1～40 hour.

Described reaction system also can be added glucose, and addition is 0.1～25g/L.

Preferably, described asymmetric reduction reaction carries out in the phosphoric acid buffer of pH 8.0 at 0.1M.

Described tunning makes as follows: inoculation Pichia membranaefaciens bacterial classification in fermention medium, and inoculum size volume ratio 5～10%, 20～40 ℃ of temperature were cultivated after 1～5 day, get fermented liquid or filtering fermentation liquor is got described wet thallus.

Each component final concentration of used fermention medium is among the present invention: maltose 5～50g/L, yeast extract paste 100～500g/L, NH ₄Cl 1～10g/L, KH ₂PO ₄0.5～5g/L, K ₂HPO ₄0.5～5g/L differs from K ⁺Metal ion 0.1～1g/L, pH value 4～9.The described K that differs from ⁺Metal ion be meant the trace metal ion that can be applicable to substratum usually, as Al ³⁺, Ba ²⁺, Cu ²⁺, Co ²⁺, Fe ³⁺, Li ⁺, Mn ²⁺, Na ⁺, Sn ²⁺, Zn ²⁺Deng, select for use usually: AlCl ₃6H ₂O, BaCl ₂2H ₂O, CuCl ₂2H ₂O, CoCl ₂6H ₂O, Fe ₂(SO ₄) ₃6H ₂O, Li ₂SO ₄H ₂O, MnSO ₄7H ₂O, NaMoO ₄2H ₂O, MgSO ₄7H ₂O, SnCl ₂2H ₂O, ZnSO ₄7H ₂O etc. are preferably CoCl ₂6H ₂O, MnSO ₄7H ₂O, NaMoO ₄2H ₂O most preferably is MnSO ₄7H ₂O.

Described conversion fluid purification procedures is: use ethyl acetate extraction, get ethyl acetate layer dehydration, decolouring after, reclaim solvent, obtain describedly (R)-ethyl 3-hydroxybutanoate.

Concrete, described method is carried out as follows:

(1) slant culture: inoculation Pichia membranaefaciens bacterial strain, the inclined-plane is in 28 ℃ of cultivations 1～7 day, as the slant activation seed;

(2) seed culture: insert the slant activation seed, 20～40 ℃, shaking speed 100～250r/min cultivated 1～5 day, as seed liquor;

(3) fermentation culture: the inoculation seed liquor, inoculum size 5～10% volume ratios, 20～40 ℃, shaking speed 100～250r/min cultivated 1～5 day, got fermented liquid;

(4) microbial transformation: with the wet thallus of fermented liquid or the centrifugal collection of fermented liquid, use 0.1M, after the phosphate buffered saline buffer of pH8.0 cleans, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH 8.0, adding the substrate methyl aceto acetate simultaneously, to make the substrate mass concentration be 1～50%, the wet thallus consumption is 0.3～6g/g substrate, and adds the glucose of final concentration 0.1～25g/L, 20～50 ℃, 100～250r/min reaction 1～40 hour, reaction finishes, the centrifugal thalline of removing, supernatant liquor ethyl acetate extraction, dehydration again, decolouring, solvent is reclaimed in evaporation, gets described (R)-ethyl 3-hydroxybutanoate.

Used slant medium, seed culture medium, fermention medium all can adopt the conventional substratum that is suitable for the yeast growth.

Preferably, described method is carried out as follows:

(1) slant culture: each component final concentration of substratum is: wort 10g/L, yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, pH 6.5, sterilized 20 minutes for 121 ℃, sterilization postcooling, bevel, inoculation Pichia membranaefaciens bacterial strain were cultivated 1～7 day for 20～40 ℃, as the slant activation seed;

(2) seed culture: each component final concentration of substratum is: maltose 30g/L, yeast extract paste 2g/L, NH ₄Cl 5g/L, KH ₂PO ₄1g/L, K ₂HPO ₄1g/L, MnSO ₄7H ₂O 0.5g/L, 6.5,120 ℃ of sterilizations of pH 20 minutes, sterilization postcooling, access slant activation seed, 20～40 ℃, shaking speed 100～250r/min cultivated 1～5 day, as seed liquor;

(3) fermentation culture: each component final concentration of substratum is: maltose 30g/L, yeast extract paste 2g/L, NH ₄Cl 5g/L, KH ₂PO ₄1g/L, K ₂HPO ₄1g/L, MnSO ₄7H ₂O 0.5g/L, 6.5,120 ℃ of sterilizations of pH 20 minutes, sterilization postcooling, inoculation seed liquor, inoculum size 5～10% volume ratios, 20～40 ℃, shaking speed 100～250r/min cultivated 1～5 day, got fermented liquid;

(4) microbial transformation: fermented liquid is centrifugal, collect thalline, use 0.1M, after the phosphate buffered saline buffer of pH 8.0 cleans, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH 8.0, adding the substrate methyl aceto acetate simultaneously, to make the substrate mass concentration be 1～50%, the wet thallus consumption is 0.3～6g/g substrate, and adds the glucose of final concentration 0.1～25g/L, 20～50 ℃, 100～250r/min reaction 1～40 hour, reaction finishes, the centrifugal thalline of removing, supernatant liquor ethyl acetate extraction, dehydration again, decolouring, solvent is reclaimed in evaporation, gets described (R)-ethyl 3-hydroxybutanoate.

The gas-chromatography of substrate EOB and product EHB (GC) is measured: reaction with an amount of ethyl acetate extraction, is used the GC112A gas chromatographic analysis after finishing.Testing conditions: detector is FID, chromatographic column (HP-19091Z-233) size: 30m * 0.25mm * 1.0nm, filler is a dimethyl polysiloxane, carrier gas is a nitrogen, carrier gas flux, air flow quantity and hydrogen flowing quantity are respectively 4.0 (11.21ml/min), 6.5 (304ml/min) with 4.5 (29.25ml/min), the temperature of chromatographic column, sampler and detector is respectively 70 ℃, 220 ℃ and 250 ℃.With n-dodecane is interior mark.Sample introduction 0.2 μ l, interior mark retention time is: 8.90min.

Product EHB enantiomer excessive value (e.e.) measuring method is as follows:

Reaction extracts reaction solution 20ml after finishing, and adds 5ml ethyl acetate extraction twice, and centrifugal again (4500r/min) 10min makes two phase stratification; Organic phase is carefully moved into polarization tube be full of, measure specific rotation A value.The 1ml that takes a sample from the organic phase of measuring optical value adds 2 μ l dodecanes (internal standard substance), gets 1 μ l behind the mixing and carries out gas chromatographic analysis.The concentration C that peak-to-peak relative correction factor high and that measure in advance calculates 3-EHB in the polarization tube that goes out according to 3-EHB, dodecane.The specific rotatory power of 3-EHB is calculated by following formula:

$\left[\mathrm{\α}\right]=\frac{\mathrm{\α}}{l\·C}\×100$

In the formula, 1 is the length (dm) of polarization tube, and C is the concentration (in per 100 milliliters contained gram number) of 3-EHB.The ee value of 3-EHB is calculated as follows:

ee＝([α]/[α] ₀)×100％

The inventive method is with respect to traditional chemical asymmetric synthesis method, bread yeast oxide-reduction method or with adding coenzyme NAD H/NAD ⁺Enzyme catalysis asymmetric reduction method have the following advantages: 1. (R)-EHB enantiomeric excess value height of Sheng Chenging reaches more than the 99%e.e.; 2. biological catalyst is a microbial cells, fermentative preparation voluntarily, and steady quality, with low cost; 3. in entire reaction course, do not need to add coenzyme; 4. reaction conditions gentleness, environmental friendliness.

The present invention passes through from row filter, adopt Pichia membranaefaciens (Pichia membranaef aciensHansen) as bacterium producing multi enzyme preparation, (R)-EHB enantiomeric excess value that catalytic asymmetric reduction EOB generates in single aqueous phase system reaches 99%e.e. and obtains product optical purity height, concentration height, has the major application prospect.

(4) description of drawings

Fig. 1 is the different influence of biomass to transforming;

Fig. 2 is the different influence of glucose dosage to transforming;

Fig. 3 is the transformation time curve.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1: the screening of producing the carbonyl reductase microorganism strains

Pichia membranaefaciens-218 (providing by Zhejiang Polytechnical University), rhodotorula-2.102 (being provided by Zhejiang Polytechnical University), Candida utilis 1257-2.12 (being provided by Zhejiang Polytechnical University), active dry yeast (the sharp yeast of Harbin horse company limited provides), candida tropicalis 104 (being provided by Zhejiang Polytechnical University) bacterial classification are provided, are cultivated as follows respectively and conversion reaction:

Slant culture: substratum consists of (final concentration): wort 10g/L, yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, pH 6.5, sterilized 20 minutes for 121 ℃, the inoculation of sterilization postcooling, bacterial classification is the various microorganism strains shown in the table 1, cultivated 2 days for 28 ℃, as the slant activation seed.

Seed culture: substratum consists of (final concentration): maltose 30g/L, yeast extract paste 2g/L, NH ₄Cl5g/L, KH ₂PO ₄1g/L, K ₂HPO ₄1g/L, MgSO ₄7H ₂O 0.4g/L, pH 6.5, and liquid amount is the bottled liquid 75ml of 250ml triangle, sterilizes 20 minutes for 120 ℃, and sterilization postcooling inoculation inclined-plane seed is put rotary shaking table, and 200rpm cultivated 1 day for 30 ℃, as seed liquor;

Fermentation culture: substratum consists of (final concentration): maltose 30g/L, yeast extract paste 2g/L, NH ₄Cl5g/L, KH ₂PO ₄1g/L, K ₂HPO ₄1g/L, MgSO ₄7H ₂O 0.4g/L, pH 6.5, and liquid amount is the bottled liquid 75mL of 250mL triangle, sterilizes 20 minutes for 120 ℃, and sterilization postcooling inoculation seed liquor (inoculum size 10% volume ratio) is put rotary shaking table, and 200rpm cultivated 1 day for 30 ℃, got fermented liquid;

The wet thallus amount is about 10g/100mL in the fermented liquid, centrifugal 10 minutes (5000 rev/mins) collect thalline, with sodium phosphate buffer (0.1M, pH 8.0) washed twice, thalline is changed in the damping fluid of 20ml same composition, add 0.6g substrate (EOB) simultaneously, in 30 ℃, 200 rev/mins of reactions down.Reaction finishes, and the centrifugal thalline of removing gets supernatant liquor.The supernatant liquor ethyl acetate extraction filters laggard promoting the circulation of qi analysis of hplc (R)-EHB content and enantiomeric excess value.Result such as table 1:

Table 1: the screening of producing the carbonyl reductase microorganism strains

Strain	Yield/％	e.e/％	R/S
				Pichia membranaefaciens-218 (Pichia membranaef aciens Hansen)	94.17	＞99	R
Rhodotorula-2.102 (Rhodotorula qlutis)	46.75	＞99	R
				Candida utilis 1257-2.12 (Candida utilis)	43.67	85.4	R
The sharp board active dry yeast of horse	20.37	34.7	R
				Candida tropicalis 104 (Candida pseudotropicalis)	14.12	67	S

Embodiment 2:

With Pichia membranaefaciens-218 (Pichia membranaef aciens Hansen-218), cultivate by example 1 method, contain substrate EOB amount in the 20mL reaction system for 0.6g, with MgSO in seed culture medium and the fermention medium ₄7H ₂O is replaced into other metal ions, and content is respectively 0.05g/L and 0.5g/L, and other conversion reaction conditions are taken a sample behind the reaction 2h with embodiment 1, analyze result such as table 2.

Table 2: the influence that the different metal ions enzyme is lived

Conclusion: metal ion CoCl ₂, MnSO ₄, NaMoO ₄Biosynthesizing to R-EHB has promoter action, particularly adds MnSO ₄, enzyme is lived improve 9.7%.Be unfavorable for producing enzyme on the contrary and add other metal ions.

Embodiment 3:

With Pichia membranaefaciens-218 (Pichia membranaef aciens Hansen-218), press example 1 method fermentation culture after 24 hours, the wet thallus amount is 10g/100mL in the fermenting enzyme liquid, take by weighing wet thallus and be added on the bottled 0.1M of 250mL triangle, among the pH 8.0 sodium phosphate salt damping fluid 20mL, making wet thallus concentration is 10～140g/L, (the wet thallus amount is 0.2～2.8g/ bottle to initial substrate EOB amount for the 0.6g/ bottle, i.e. 0.3～4.7g/g substrate), in 30 ℃, carry out conversion reaction under the 200rpm.Afterreaction finished in 16 hours, and the centrifugal thalline of removing gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO ₄Drying is filtered laggard promoting the circulation of qi analysis of hplc (R)-EHB content and enantiomeric excess value,

The results are shown in Figure 1.

Conclusion: as can be seen from Figure 1, efficiency of pcr product increases along with the increase of cell concentration in the conversion fluid, and when cell concentration reaches 100g/L (being the 3.3g/g substrate), efficiency of pcr product can reach 94.8%, and the enantiomorph excess quantity also can reach 99%.When cell concentration continues to be increased to 140g/L (being the 4.7g/g substrate), efficiency of pcr product and enantiomorph excess quantity are respectively 95.0% and 99%, with cell concentration be that the conversion results of 100g/L is close, so we select for use 100g/L (being the 3.3g/g substrate) to be the cell concn of bioconversion reaction.

Embodiment 4:

With Pichia membranaefaciens-218 (Pichia membranaef aciens Hansen-218), press example 1 method fermentation culture after 24 hours, the wet thallus amount is 10g/100mL in the fermenting enzyme liquid, take by weighing wet thallus and add the 0.1M that 20ml is housed, in the 250ml triangular flask of pH 8.0 sodium phosphate buffers, making the wet thallus amount is 100g/L, substrate EOB amount is the 0.6g/ bottle, add the glucose of final concentration 0～25g/L,, carry out conversion reaction under the 200rpm in 30 ℃.Afterreaction finished in 16 hours, and the centrifugal thalline of removing gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO ₄Drying is filtered laggard promoting the circulation of qi phase chromatogram and polarimeter analysis (R)-EHB content and enantiomeric excess value, the results are shown in Figure 2.

Conclusion: as can be seen from Figure 2, the optimum concn of glucose is 10g/L.

Embodiment 5:

With Pichia membranaefaciens-218 (Pichia membranaef aciens Hansen-218), press the fermentation of example 1 method, contain substrate EOB amount in the 20ml reaction system and be 0.6g, other conversion reaction conditions are with embodiment 1.In the sampling of reaction different time, carry out gas chromatographic analysis (R)-EHB content and calculate conversion yield, measure specific rotation and calculate the e.e. value with polarimeter, the results are shown in Figure 3.

Conclusion: can find that from Fig. 3 substantially finished when reaction proceeds to the 16h left and right sides, productive rate can reach about 95%, the e.e. value remains on about 99%.Therefore determine that optimum reacting time is 16h.

Claims (10)

1. the method for microbial transformation preparation (R)-ethyl 3-hydroxybutanoate, described method is to be substrate with the methyl aceto acetate, with Pichia membranaefaciens (Pichia membranaef aciensHansen) cell is the enzyme source, carry out the microorganism catalysis asymmetric reduction reaction, reaction finishes the back conversion fluid and obtains described (R)-ethyl 3-hydroxybutanoate through separation and purification.

2. the method for microbial transformation as claimed in claim 1 preparation (R)-ethyl 3-hydroxybutanoate is characterized in that fermented liquid that described Pichia membranaefaciens cell obtains through fermentation culture from: Pichia membranaefaciens or the wet thallus that is obtained by filtering fermentation liquor.

3. the method for microbial transformation preparation (R)-ethyl 3-hydroxybutanoate as claimed in claim 1 or 2, it is characterized in that: described substrate mass concentration is 1～50%, it is 0.3～6g/g substrate that the adding tunning makes the wet thallus amount, the temperature of reaction of described asymmetric reduction reaction is 20～50 ℃, and the reaction times is 1～40 hour.

4. the method for microbial transformation preparation (R)-ethyl 3-hydroxybutanoate as claimed in claim 3, it is characterized in that: be added with glucose in the reaction system, addition is 0.1～25g/L.

5. the method for microbial transformation preparation (R)-ethyl 3-hydroxybutanoate as claimed in claim 4, it is characterized in that: described asymmetric reduction reaction carries out in the phosphoric acid buffer of pH8.0 at 0.1M.

6. the method for microbial transformation preparation (R)-ethyl 3-hydroxybutanoate as claimed in claim 1, it is characterized in that described tunning makes as follows: inoculation Pichia membranaefaciens bacterial classification in fermention medium, inoculum size volume ratio 5～10%, 20～40 ℃ of temperature, cultivate after 1～5 day, get fermented liquid or filtering fermentation liquor is got described wet thallus.

7. the method for microbial transformation preparation (R)-ethyl 3-hydroxybutanoate as claimed in claim 6 is characterized in that each component final concentration of described fermention medium is: maltose 5～50g/L, yeast extract paste 100～500g/L, NH ₄Cl 1～10g/L, KH ₂PO ₄0.5～5g/L, K ₂HPO ₄0.5～5g/L differs from K ⁺Metal ion 0.1～1g/L, pH value 4～9.

8. the method for microbial transformation preparation (R)-ethyl 3-hydroxybutanoate as claimed in claim 1, it is characterized in that described conversion fluid purification procedures is: use ethyl acetate extraction, after getting ethyl acetate layer dehydration, decolouring, reclaim solvent, obtain described (R)-ethyl 3-hydroxybutanoate.

9. the method for microbial transformation as claimed in claim 1 preparation (R)-ethyl 3-hydroxybutanoate is characterized in that described method carries out as follows:

(1) slant culture: inoculation Pichia membranaefaciens bacterial strain, the inclined-plane is in 28 ℃ of cultivations 1～7 day, as the slant activation seed;

(2) seed culture: insert the slant activation seed, 20～40 ℃, shaking speed 100～250r/min cultivated 1～5 day, as seed liquor;

(3) fermentation culture: the inoculation seed liquor, inoculum size 5～10% volume ratios, 20～40 ℃, shaking speed 100～250r/min cultivated 1～5 day, got fermented liquid or wet thallus;

(4) microbial transformation: with the wet thallus of fermented liquid or the centrifugal collection of fermented liquid, use 0.1M, after the phosphate buffered saline buffer of pH8.0 cleans, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH8.0, adding the substrate methyl aceto acetate simultaneously, to make the substrate mass concentration be 1～50%, the wet thallus consumption is 0.3～6g/g substrate, and adds the glucose of final concentration 0.1～25g/L, 20～50 ℃, 100～250r/min reaction 1～40 hour, reaction finishes, the centrifugal thalline of removing, supernatant liquor ethyl acetate extraction, dehydration again, decolouring, solvent is reclaimed in evaporation, gets described (R)-ethyl 3-hydroxybutanoate.

10. the method for microbial transformation as claimed in claim 1 preparation (R)-ethyl 3-hydroxybutanoate is characterized in that described method carries out as follows:

(1) slant culture: each component final concentration of substratum is: wort 10g/L, yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, pH6.5, sterilized 20 minutes for 121 ℃, sterilization postcooling, bevel, inoculation Pichia membranaefaciens bacterial strain were cultivated 1～7 day for 20～40 ℃, as the slant activation seed;

(2) seed culture: each component final concentration of substratum is: maltose 30g/L, yeast extract paste 2g/L, NH ₄Cl 5g/L, KH ₂PO ₄1g/L, K ₂HPO ₄1g/L, MnSO ₄7H ₂O0.5g/L, pH6.5 sterilized 20 minutes for 120 ℃, sterilization postcooling, access slant activation seed, 20～40 ℃, shaking speed 100～250r/min cultivated 1～5 day, as seed liquor;

(3) fermentation culture: each component final concentration of substratum is: maltose 30g/L, yeast extract paste 2g/L, NH ₄Cl 5g/L, KH ₂PO ₄1g/L, K ₂HPO ₄1g/L, MnSO ₄7H ₂O0.5g/L, pH6.5 sterilized 20 minutes for 120 ℃, sterilization postcooling, inoculation seed liquor, inoculum size 5～10% volume ratios, 20～40 ℃, shaking speed 100～250r/min cultivated 1～5 day, got fermented liquid;

(4) microbial transformation: fermented liquid is centrifugal, collect thalline, use 0.1M, after the phosphate buffered saline buffer of pH8.0 cleans, change wet thallus over to 0.1M, in the phosphate buffered saline buffer of pH8.0, adding the substrate methyl aceto acetate simultaneously, to make the substrate mass concentration be 1～50%, the wet thallus consumption is 0.3～6g/g substrate, and adds the glucose of final concentration 0.1～25g/L, 20～50 ℃, 100～250r/min reaction 1～40 hour, reaction finishes, the centrifugal thalline of removing, supernatant liquor ethyl acetate extraction, dehydration again, decolouring, solvent is reclaimed in evaporation, gets described (R)-ethyl 3-hydroxybutanoate.

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