CN102071227A - Method for preparing S-(+)-3-methyl hydroxybutyrate through microbial transformation - Google Patents
Method for preparing S-(+)-3-methyl hydroxybutyrate through microbial transformation Download PDFInfo
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Abstract
The invention provides a method for preparing S-(+)-3-methyl hydroxybutyrate through microbial transformation, which comprises the following steps that: in phosphate buffer solution with the pH value being 5.0 to 8.0, methyl acetoacetate is used as substrates, enzyme-containing strains obtained through fermenting saccharomyces cerevisiae CGMCC No. 2266 are used as microbial catalysts, conversion region is carried out for 8 to 40h at 25 to 45 DEG C, after the reaction is completed, and conversion liquid is prepared into the S-(+)-3-methyl hydroxybutyrate through separation and purification. The method provided by the invention has the main beneficial effects that 1. produced strains are safe and innoxious and are easy to be cultured in a large scale; 2. a large number of biological catalysts can be obtained, and the cost is low; 3. the reaction conditions are mild, and the environmental-friendly effect is realized; 4. the operation is simple and convenient, and the addition of coenzymes with a high price is not needed in the reaction process; and 5. the large-scale industrialized production is easy to realize, and the mol conversion rate is high.
Description
(1) technical field
The present invention relates to the method that a kind of microbial transformation prepares S-(+)-3-beta-hydroxymethyl butyrate, being particularly related to a kind of is biological catalyst with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266, is the method that substrate prepares S-(+)-3-beta-hydroxymethyl butyrate with the methyl acetoacetate.
(2) background technology
S-(+)-3-beta-hydroxymethyl butyrate ((S)-(+)-methyl 3-hydroxybutyrate), CAS accession number: 53562-86-0, molecular formula C
5H
10O
3, molecular weight 118.13.Can be used for the poly-beta-hydroxy-butanoic acid ester P[(R of optical activity)-HB] synthetic, P[(R)-and HB] be the biological polyester of a class in the bacterial cell, has favorable biological degradability, its degradation production can all be a biological utilisation, environment there is not any pollution, its melt temperature is 175～180 ℃, is a kind of thermoplastics that can decompose fully.Can be used as chiral intermediate, to be used for medicine synthetic.
Resolution of racemates 3-beta-hydroxymethyl butyrate can obtain S-(+)-3-beta-hydroxymethyl butyrate, but splitting the efficient maximum has only 50%, and production efficiency is not high.With the methyl acetoacetate is that substrate adopts the carbonyl in chemical method and the microbial method asymmetric reduction substrate all can obtain S-(+)-3-beta-hydroxymethyl butyrate.The chemical method asymmetric reduction need prepare the chirality chemical catalyst, costs an arm and a leg, and preparation process is loaded down with trivial details.Adopt microbial method asymmetric reduction methyl acetoacetate can obtain higher optical purity S-(+)-3-beta-hydroxymethyl butyrate of the superfluous value of enantiomorph, the reaction conditions gentleness, environmental friendliness, with low cost, be easy to realize the in-situ regeneration of coenzyme, microorganism is easy to large scale culturing, is easy to suitability for industrialized production, is the green synthesis process of S-(+)-3-beta-hydroxymethyl butyrate.
A large amount of bibliographical informations adopts the asyininetric hydrogenation of various types of nickel catalyzator catalysis methyl acetoacetates to prepare S-(+)-3-beta-hydroxymethyl butyrate, and Preparation of catalysts is comparatively loaded down with trivial details in this process, and cost is higher.The technological process that adopts microbe transformation method asymmetric reduction methyl acetoacetate to prepare S-(+)-3-beta-hydroxymethyl butyrate is not seen patent and bibliographical information, is a kind of synthesis technique of S-(+)-3-beta-hydroxymethyl butyrate of efficient, energy-saving and environmental protection.
(3) summary of the invention
The object of the invention provides the method that a kind of microbial transformation prepares S-(+)-3-beta-hydroxymethyl butyrate, and this method reaction conditions gentleness is easy and simple to handle, environmental friendliness, and the product yield height is easy to suitability for industrialized production.
The technical solution used in the present invention is:
A kind of microbial transformation prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, described method is to be substrate with the methyl acetoacetate, the enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266 fermentation is a biological catalyst, carries out conversion reaction and makes described S-(+)-3-beta-hydroxymethyl butyrate.
The method that microbial transformation of the present invention prepares S-(+)-3-beta-hydroxymethyl butyrate is: in the phosphate buffered saline buffer of pH5.0～8.0, the enzyme somatic cells that contains that is substrate with the methyl acetoacetate, obtains with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266 fermentation is a biological catalyst, in 25～45 ℃ of following conversion reactions 8～40 hours, after reaction finished, conversion fluid obtained described S-(+)-3-beta-hydroxymethyl butyrate through separation and purification.
The initial final concentration of described methyl acetoacetate in phosphate buffered saline buffer is 0.01～0.10mol/L.
Further, the glucose that also is added with final concentration in the phosphate buffered saline buffer of the present invention and is 10～150g/L helps improving the molar yield of substrate as cosubstrate.
The described enzyme somatic cells consumption that contains is counted 1～70g/g substrate with dry cell weight, and the amount of substrate described here is the quality of methyl acetoacetate; The described mensuration that contains enzyme somatic cells dry weight is with after the fermented liquid centrifugation, and abandoning supernatant is dried 48 hours to constant weight with wet cell at 120 ℃, measures the weight of stem cell; Get in the part fermented liquid centrifugal gained wet cell and measure dry cell weight, calculate that unit contains stem cell ratio in the enzyme somatic cells in the fermented liquid, calculate the required enzyme somatic cells fermented liquid consumption that contains of quantitative dry cell weight with this ratio again.
The described enzyme somatic cells that contains prepares in accordance with the following methods: yeast saccharomyces cerevisiae CGMCC No.2266 is seeded in the fermention medium, and shaking speed is 150～200r/min, cultivates 18～30h down for 26～35 ℃, and fermented liquid is centrifugal, makes to contain the enzyme somatic cells.
Described S-(+)-3-beta-hydroxymethyl butyrate separation purification method is as follows: after reaction finishes, with centrifugal 20 minutes of conversion fluid 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid adds anhydrous sodium sulphate and removes moisture, suction filtration in acetic acid ethyl acetate extract, ethyl acetate is removed in the filtrate distillation, promptly gets described S-(+)-3-beta-hydroxymethyl butyrate.
S-of the present invention (+)-3-beta-hydroxymethyl butyrate preparation method recommends to carry out according to following steps: (1) slant culture: yeast saccharomyces cerevisiae CGMCC No.2266 is inoculated into slant medium, cultivates for 26～35 ℃ and got the thalline inclined-plane in 4～6 days; Described slant medium final concentration consists of: 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; (2) seed culture: get a transfering loop thalline from the thalline inclined-plane and be transferred to seed culture medium, shaking speed is 150～200r/min, cultivates 18～26h for 26～35 ℃ and gets seed liquor; Described seed culture medium final concentration consists of: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
2O 0.5～1.5g/L, KH
2PO
40.6～1.5g/L, the pH value of adjusting liquid nutrient medium with NaOH or HCl solution is 7.0, and solvent is a water; (3) fermentation culture: get seed liquor, be inoculated in the fermention medium with the inoculum size of volume fraction 10～20%, shaking speed is 150～200r/min, cultivates 18～30h, the fermented liquid centrifugation is obtained the described enzyme somatic cells that contains for 26～35 ℃; Described fermention medium final concentration consists of: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
2O 0.5～1.5g/L, KH
2PO
40.6～1.5g/L, the pH value of adjusting liquid nutrient medium with NaOH or HCl solution is 7.0, and solvent is a water; (4) bio-transformation: in the phosphate buffered saline buffer of pH 5.0～8.0, the glucose that adds 10～150g/L is as cosubstrate, the methyl acetoacetate that adds 0.01～0.10mol/L, and the dry cell weight quality is the somatic cells of 1～50 times of methyl acetoacetate, 25～45 ℃ of following conversion reactions 8～40 hours, conversion fluid; (5) separation and purification: after reaction finishes, with conversion fluid centrifugal 20 minutes in 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid adds anhydrous sodium sulphate and removes moisture, suction filtration in acetic acid ethyl acetate extract, ethyl acetate is removed in the filtrate distillation, promptly gets described S-(+)-3-beta-hydroxymethyl butyrate.
Yeast saccharomyces cerevisiae CGMCC No.2266; be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms preservation center; be positioned at Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica; preserving number CGMCC No.2266; preservation date on November 26th, 2007, formerly protected as new bacterial strain in the granted patent 200810059686.
Bacterium source: the microbial bacteria Accharomyces cerevisiae CGMCC No.2266 described in the present invention screens near the brew-house of the West Lake, Hangzhou the soil to obtain.Be used to transform methyl acetoacetate with separating the bacterial strain that obtains in the soil, obtain yeast saccharomyces cerevisiae CGMCC No.2266 and have the ability that good conversion methyl acetoacetate is produced S-(+)-3-beta-hydroxymethyl butyrate.
The colony characteristics of described yeast saccharomyces cerevisiae CGMCC No.2266: on nutrient agar, present oyster white, glossy, smooth, neat in edge, moistening, smooth surface, the uniform colonial morphology of quality.
The available gas chromatograph-mass spectrometer of described S-(+)-pure product of 3-beta-hydroxymethyl butyrate detects, and determines degree of purity of production and molecular weight.
Determining of the superfluous value of the enantiomorph of molar yield and product S-(+)-3-beta-hydroxymethyl butyrate (ee%):
The employing gas chromatographic analysis detects.Chromatographic column is a Varian CP Chirasil-DEX chiral column.This chiral column can detect the content of R-(-)-3-beta-hydroxymethyl butyrate and S-(+)-two kinds of enantiomorphs of 3-beta-hydroxymethyl butyrate, further calculates the superfluous value (ee%) of enantiomorph of the molar yield and S-(+)-3-beta-hydroxymethyl butyrate of reaction.
Adopt microorganism cells asymmetric reduction methyl acetoacetate to prepare S-(+)-3-beta-hydroxymethyl butyrate among the present invention, can obtain the product of the superfluous value of high antimer, the glucose that adds 10～150g/L helps improving the molar yield of substrate as cosubstrate.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the microbe transformation method that the present invention adopts prepares S-(+)-3-beta-hydroxymethyl butyrate and compares with chemical synthesis, enzyme catalysis method and have the following advantages: (1) produces the bacterial strain safety non-toxic, and microbial cells is easy to large scale culturing; (2) can obtain a large amount of biological catalysts, more with low cost than chemical catalyst; (3) reaction conditions gentleness, environmental friendliness; (4) easy and simple to handle, do not need to add expensive coenzyme in the reaction process; (5) be easy to realize large-scale industrial production, the molar yield height is the green synthesis process of S-(+)-3-beta-hydroxymethyl butyrate.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
The slant medium preparation: wort 10g/L, 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 and fermention medium preparation: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L, solvent are water, and the pH value of adjusting liquid nutrient medium with NaOH or HCl solution is 7.0,121 ℃ of sterilization 20min.
Embodiment 1
Yeast saccharomyces cerevisiae CGMCC No.2266 bacterial classification inoculation to slant medium, is cultivated for 30 ℃ and made the thalline inclined-plane in 4～6 days.Get a transfering loop thalline from the thalline inclined-plane 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.10mL seed liquor (inoculum size is a liquid nutrient medium volumetric usage 10%) is inoculated in the 250mL triangular flask that contains the 100mL liquid nutrient medium, cultivates 24h and obtain fermented liquid under 30 ℃, the condition of 180r/min, fermented liquid is centrifugal, must contain the enzyme somatic cells.
The mensuration of dry cell weight is little part to be got in the centrifugal back of fermented liquid dry 48 hours to constant weight at 120 ℃ from wet cell, measure the weight of stem cell, calculate that unit contains stem cell ratio in the enzyme somatic cells in the fermented liquid, calculate the required enzyme somatic cells fermented liquid consumption that contains of quantitative dry cell weight with this ratio again.
The dry weight that contains the enzyme somatic cells in every liter of fermented liquid of present embodiment is 50 grams.
In ten parts of triangular flasks that contain 100mL pH7.0 phosphate buffered saline buffer, add the wet cell that the centrifugal back of above-mentioned gained 200mL fermented liquid obtains, the dry weight that wherein contains the enzyme somatic cells is 10g, add methyl acetoacetate respectively, make the final concentration of methyl acetoacetate be respectively 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.04mol/L, 0.05mol/L, 0.06mol/L, 0.07mol/L, 0.08mol/L, 0.09mol/L, 0.10mol/L, all place 30 ℃, react 24h in the shaking table of 180r/min.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate and remove small amount of moisture, suction filtration, ethyl acetate is removed in the filtrate distillation, promptly get described S-(+)-3-beta-hydroxymethyl butyrate, the substrate starting point concentration sees Table 1 to the influence of the superfluous value of corresponding body (ee%) of molar yield and S-(+)-3-beta-hydroxymethyl butyrate.
Table 1 substrate starting point concentration is to the influence of transformation efficiency and S-(+)-3-beta-hydroxymethyl butyrate
Table 1 is as can be seen: along with the raising transformation efficiency of concentration of substrate reduces gradually.Concentration of substrate has bigger influence to the superfluous value of the enantiomorph of product S-(+)-3-beta-hydroxymethyl butyrate, when concentration of substrate is low be 0.01 and during 0.02mol/L product be the R configuration, when concentration of substrate during more than or equal to 0.03mol/L product be the S configuration, when concentration of substrate during more than or equal to 0.05mol/L the superfluous value of enantiomorph of S-(+)-3-beta-hydroxymethyl butyrate can reach 100%.The addition of adjusting substrate can obtain the product of desirable configuration.
Embodiment 2
Yeast saccharomyces cerevisiae CGMCC No.2266 bacterial classification inoculation to slant medium, is cultivated for 30 ℃ and got the thalline inclined-plane in 4～6 days.Get a transfering loop thalline from the thalline inclined-plane 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, cultivate 24h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells, and the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In five parts of triangular flasks that contain 100mL pH7.0 phosphate buffered saline buffer, add the wet cell that the centrifugal back of 200 milliliters of fermented liquids of above-mentioned gained obtains, the dry weight that wherein contains the enzyme somatic cells is 10g, adding methyl acetoacetate respectively, to make the methyl acetoacetate final concentration be 0.05mol/L, adding cosubstrate glucose separately makes the glucose final concentration be respectively 10g/L, 30g/L, 50g/L, 70g/L, 100g/L, 150g/L, place 30 ℃, react 24h in the shaking table of 180r/min.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid, add anhydrous sodium sulphate and remove small amount of moisture in acetic acid ethyl acetate extract, suction filtration is removed ethyl acetate with the filtrate distillation, promptly get described S-(+)-3-beta-hydroxymethyl butyrate, the cosubstrate glucose concn sees Table 2 to the influence of the superfluous value of corresponding body (ee%) of molar yield and S-(+)-3-beta-hydroxymethyl butyrate.
Table 2: add the influence of glucose to transformation efficiency and the superfluous value of S-(+)-3-beta-hydroxymethyl butyrate enantiomorph
Table 2 is as can be seen: the adding of glucose helps improving transformation efficiency.Contain a large amount of Hexose phosphate dehydrogenases in the microorganism cells, the Hexose phosphate dehydrogenase transforming glucose generates gluconic acid, simultaneously hydrogen is passed to NADP and generate NADPH, the adding of glucose helps realizing the in-situ regeneration of coenzyme NADP 11, for reaction process provides a large amount of hydrogen donors, thereby improve the transformation efficiency of reaction.Best glucose addition is 50g/L.The superfluous value of the enantiomorph of S-(+)-3-beta-hydroxymethyl butyrate does not change with the change of glucose addition, all remains on 100%.
Embodiment 3
Yeast saccharomyces cerevisiae CGMCC No.2266 bacterial classification inoculation to slant medium, is cultivated for 30 ℃ and got the thalline inclined-plane in 4～6 days.Get a transfering loop thalline from the thalline inclined-plane 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, cultivate 24h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells, and the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In five parts of triangular flasks that contain 100mL pH7.0 phosphate buffered saline buffer, add the wet cell that the centrifugal back of 200 milliliters of fermented liquids of above-mentioned gained obtains, the dry weight that wherein contains the enzyme somatic cells is 10g, add methyl acetoacetate separately and make methyl acetoacetate final concentration 0.05mol/L, add cosubstrate glucose, making the glucose final concentration is 50g/L, in the shaking table of 180r/min respectively at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ following conversion reaction 24h.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate and remove small amount of moisture, suction filtration, ethyl acetate is removed in the filtrate distillation, promptly get described S-(+)-3-beta-hydroxymethyl butyrate, temperature of reaction sees Table 3 to the influence of the superfluous value of corresponding body (ee%) of molar yield and S-(+)-3-beta-hydroxymethyl butyrate.
Table 3 temperature of reaction is to the influence of transformation efficiency and the superfluous value of S-(+)-3-beta-hydroxymethyl butyrate enantiomorph
Table 3 is as can be seen: temperature of reaction has considerable influence to transformation efficiency.The too high part inactivation that can cause enzyme of temperature of reaction, thereby transformation efficiency reduces.Preferable invert point is 30 ℃.The superfluous value of the enantiomorph of S-(+)-3-beta-hydroxymethyl butyrate does not change with the change of invert point, all remains on 100%.
Embodiment 4:
Yeast saccharomyces cerevisiae CGMCC No.2266 bacterial classification inoculation to slant medium, is cultivated for 30 ℃ and got the thalline inclined-plane in 4～6 days.Get a transfering loop thalline from the thalline inclined-plane 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, cultivate 24h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells, and the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In five parts of triangular flasks that contain 100mL pH7.0 phosphate buffered saline buffer, add the wet cell that the centrifugal back of 200 milliliters of fermented liquids of above-mentioned gained obtains, the dry weight that wherein contains the enzyme somatic cells is 10g, add methyl acetoacetate, making the methyl acetoacetate final concentration is 0.05mol/L, add cosubstrate glucose, making the glucose final concentration is 50g/L, places 30 ℃, reacts 8h, 16h, 24h, 32h, 40h in the shaking table of 180r/min respectively.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate and remove small amount of moisture, suction filtration, filtrate heating up in a steamer removed ethyl acetate, promptly get described S-(+)-3-beta-hydroxymethyl butyrate, the influence of reaction times to the superfluous value of corresponding body (ee%) of molar yield and S-(+)-3-beta-hydroxymethyl butyrate sees Table 4.
Table 4: the reaction times is to the influence of transformation efficiency and the superfluous value of S-(+)-3-beta-hydroxymethyl butyrate enantiomorph
Table 4 is as can be seen: along with the prolongation transformation efficiency of transformation time improves gradually.Transformation efficiency can reach 100% basically after reacting 24 hours, and therefore best transformation time is 24 hours.The superfluous value of the enantiomorph of S-(+)-3-beta-hydroxymethyl butyrate does not change with the change of transformation time, all remains on 100%.
Embodiment 5:
Yeast saccharomyces cerevisiae CGMCC No.2266 bacterial classification inoculation to slant medium, is cultivated for 30 ℃ and got the thalline inclined-plane in 4～6 days.Get a ring thalline from the thalline inclined-plane 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, cultivate 24h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells, and the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
Add the wet cell that the centrifugal back of fermented liquid of 40 milliliters of above-mentioned gained, 100 milliliters, 200 milliliters, 300 milliliters and 400 milliliters obtains in five parts of triangular flasks that contain the 100mLpH7.0 phosphate buffered saline buffer respectively, the dry weight that wherein contains the enzyme somatic cells is respectively 2g, 5g, 10g, 15g and 20g.Add methyl acetoacetate respectively in above-mentioned five triangular flasks, making the methyl acetoacetate final concentration is 0.05mol/L, and adding cosubstrate glucose more separately, to make the glucose final concentration be 50g/L, places 30 ℃, reacts 24h in the shaking table of 180r/min.After reaction finishes with conversion fluid centrifugal 20 minutes in 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate and remove small amount of moisture, suction filtration, ethyl acetate is removed in the filtrate distillation, promptly get described S-(+)-3-beta-hydroxymethyl butyrate, the somatic cells amount sees Table 5 to the influence of the superfluous value of corresponding body (ee%) of molar yield and S-(+)-3-beta-hydroxymethyl butyrate.
Table 5 biomass is to the influence of transformation efficiency and the superfluous value of S-(+)-3-beta-hydroxymethyl butyrate enantiomorph
Table 5 is as can be seen: along with the increase transformation efficiency of thalline consumption increases gradually.The increase of thalline consumption has not only improved the consumption of enzyme, has improved the consumption of coenzyme NADP 11 simultaneously, therefore helps improving reaction conversion ratio.The superfluous value of the enantiomorph of S-(+)-3-beta-hydroxymethyl butyrate does not change with the change of thalline add-on, all remains on 100%.
Claims (9)
1. a microbial transformation prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, it is characterized in that described method is is substrate with the methyl acetoacetate, the enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266 fermentation is a biological catalyst, carries out conversion reaction and makes described S-(+)-3-beta-hydroxymethyl butyrate.
2. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, it is characterized in that described method is: in the phosphate buffered saline buffer of pH 5.0～8.0, the enzyme somatic cells that contains that is substrate with the methyl acetoacetate, obtains with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266 fermentation is a biological catalyst, in 25～45 ℃ of following conversion reactions 8～40 hours, after reaction finished, conversion fluid obtained described S-(+)-3-beta-hydroxymethyl butyrate through separation and purification.
3. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, it is characterized in that the initial final concentration of described methyl acetoacetate in phosphate buffered saline buffer is 0.01～0.10mol/L.
4. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, and the glucose that it is characterized in that also being added with in the described phosphate buffered saline buffer final concentration and be 10～150g/L is as cosubstrate.
5. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, it is characterized in that the described enzyme somatic cells consumption that contains counts 1～70g/g substrate with dry cell weight.
6. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, it is characterized in that the described enzyme somatic cells that contains prepares in accordance with the following methods: yeast saccharomyces cerevisiae CGMCC No.2266 is seeded in the fermention medium, shaking speed is 150～200r/min, cultivate 18～30h down for 26～35 ℃, fermented liquid is centrifugal, make and contain the enzyme somatic cells.
7. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, it is characterized in that described S-(+)-3-beta-hydroxymethyl butyrate separation purification method is as follows: after reaction finishes, with centrifugal 20 minutes of conversion fluid 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate and remove moisture, suction filtration, ethyl acetate is removed in the filtrate distillation, promptly gets described S-(+)-3-beta-hydroxymethyl butyrate.
8. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-beta-hydroxymethyl butyrate, it is characterized in that described method carries out according to following steps: (1) slant culture: yeast saccharomyces cerevisiae CGMCC No.2266 is inoculated into slant medium, 26～35 ℃ cultivated 4～6 days the thalline inclined-plane; (2) seed culture: get a transfering loop thalline from the thalline inclined-plane and be transferred to seed culture medium, shaking speed is 150～200r/min, cultivates 18～26h for 26～35 ℃ and gets seed liquor; (3) fermentation culture: get seed liquor, be inoculated in the fermention medium with the inoculum size of volume fraction 10～20%, shaking speed is 150～200r/min, cultivates 18～30h, the fermented liquid centrifugation is obtained the described enzyme somatic cells that contains for 26～35 ℃; (4) bio-transformation: in the phosphate buffered saline buffer of pH5.0～8.0, the glucose that adds final concentration and be 10～150g/L is as cosubstrate, adding final concentration is the methyl acetoacetate of 0.01～0.10mol/L, and the dry cell weight quality be 1～50 times of methyl acetoacetate contain the enzyme somatic cells, 25～45 ℃ of following conversion reactions 8～40 hours, reaction finishes to make conversion fluid; (5) separation and purification: after conversion reaction finishes, with conversion fluid centrifugal 20 minutes in 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid adds anhydrous sodium sulphate and removes moisture, suction filtration in acetic acid ethyl acetate extract, ethyl acetate is removed in the filtrate distillation, promptly gets described S-(+)-3-beta-hydroxymethyl butyrate.
9. the method for preparing S-(+)-3-beta-hydroxymethyl butyrate as microbial transformation as described in the claim 2 is characterized in that described method carries out as follows:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No.2266 is inoculated into slant medium, cultivates for 26～35 ℃ and got the thalline inclined-plane in 4～6 days; Described slant medium final concentration consists of: 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;
(2) seed culture: get a transfering loop thalline from the thalline inclined-plane 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 final concentration consists of: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
2O 0.5～1.5g/L, KH
2PO
40.6～1.5g/L, the pH value of adjusting liquid nutrient medium with NaOH or HCl solution is 7.0, and solvent is a water;
(3) fermentation culture: get seed liquor, with volume fraction is that 10～20% inoculum size is inoculated in the fermention medium, culture temperature is 26～35 ℃, shaking speed is 150～200r/min, cultivate the fermented liquid that 18～30h obtains containing the enzyme somatic cells, centrifugation obtains the described enzyme somatic cells that contains; Described fermention medium final concentration consists of: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
2O 0.5～1.5g/L, KH
2PO
40.6～1.5g/L, the pH value of adjusting liquid nutrient medium with NaOH or HCl solution is 7.0, and solvent is a water;
(4) bio-transformation: in the phosphate buffered saline buffer of pH 5.0～8.0, the methyl acetoacetate that adds 0.01～0.10mol/L, the glucose that adds final concentration and be 10～150g/L is as cosubstrate, and with the dry cell weight quality be 1～50 times of methyl acetoacetate contain the enzyme somatic cells, in 25～45 ℃ of following conversion reactions 8～40 hours conversion fluid;
(5) separation and purification: after reaction finishes with conversion fluid centrifugal 20 minutes in 4000r/min, discard bacterial sediment, with supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, the combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate and remove moisture, suction filtration, ethyl acetate is removed in the filtrate distillation, promptly gets described S-(+)-3-beta-hydroxymethyl butyrate.
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| CN101824438A (en) * | 2010-02-09 | 2010-09-08 | 浙江工业大学 | Method for preparing (S)-3-hydroxy butyric acid ethyl ester through ethyl acetoacetate microbial conversion |
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| CN101824438A (en) * | 2010-02-09 | 2010-09-08 | 浙江工业大学 | Method for preparing (S)-3-hydroxy butyric acid ethyl ester through ethyl acetoacetate microbial conversion |
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| CN102703524A (en) * | 2012-05-31 | 2012-10-03 | 浙江工业大学 | Method for preparing (S)-(+)-1, 3-butanediol through microbial transformation |
| CN102703524B (en) * | 2012-05-31 | 2014-06-11 | 浙江工业大学 | Method for preparing (S)-(+)-1, 3-butanediol through microbial transformation |
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