CN102071231A - Method for preparing S-(+)-3-hydroxy tetrahydrofuran through microbial conversion - Google Patents
Method for preparing S-(+)-3-hydroxy tetrahydrofuran through microbial conversion Download PDFInfo
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Abstract
The invention discloses a method for preparing S-(+)-3-hydroxy tetrahydrofuran through microbial conversion. The method comprises the following steps of: performing conversion reaction at the temperature of between 25 and 45 DEG C for 8 to 40 hours in phosphate buffer with pH of 5.0 to 8.0 by taking 3-keto-tetrahydrofuran as a substrate, and an enzyme-containing bacteroid cell obtained through the fermentation of saccharomyces cerevisiae CGMCC No.2266 as a biocatalyst, and after the reaction is finished, separating and purifying conversion liquid to obtain the S-(+)-3-hydroxy tetrahydrofuran. The method has the advantages that: (1) production strains are safe and non-toxic, can be cultured in a large scale easily and are low in cost; (2) the method is easy to operate, coenzyme which is high in price is not needed to be added in the reaction process and the yield is high; (3) large-scale industrial production is easy to realize; and (4) reaction conditions are mild and the method is environmental-friendly.
Description
(1) technical field
The present invention relates to the method that a kind of microbial transformation prepares S-(+)-3-hydroxyl tetrahydrofuran, particularly a kind ofly be biological catalyst, be the novel method of substrate preparation (S)-(+)-3-hydroxyl tetrahydrofuran with 3-ketone group tetrahydrofuran (THF) with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266.
(2) background technology
(S)-(+)-3-hydroxyl tetrahydrofuran ((S)-(+)-3-Hydroxytetrahydrofuran), CAS accession number: 86087-23-2, molecular formula C
4H
8O
2, molecular weight 88.11.(S)-(+)-the 3-hydroxyl tetrahydrofuran is an important intermediate of synthetic treatment AIDS-treating medicine amprenavir.In the diphenyl ether herbicide agricultural chemicals, can obviously improve the weeding activity and the selectivity of diphenyl ether herbicide behind introducing (S)-(+)-3-hydroxyl tetrahydrofuran group.
The synthetic of (S)-(+) in the bibliographical information-3-hydroxyl tetrahydrofuran can obtain by the asymmetric hydroboration or the hydrogen silication of dihydrofuran, needs chiral catalyst, and the chiral catalyst preparation process is loaded down with trivial details, costs an arm and a leg; Can be by the asymmetric hydrogenation-reduction-intramolecularly etherificate preparation of 4-chloro-3-carbonyl butyric ester, this method cost is higher, is unsuitable for suitability for industrialized production; Can split preparation by the enzyme process of 3-hydroxyl tetrahydrofuran raceme, the fractionation that enzyme process splits is most effective can only to reach 50%, and production efficiency is lower; Reduction-intramolecularly etherificate that can be by oxysuccinic acid is synthetic, but this method need be reduced malic acid diester with the lithium aluminum hydride of costliness, and the secondary hydroxyl that needs protection is to prevent racemization.The above-mentioned preparation method in most cases optical purity of product is not enough, perhaps exists the factor that separation and Extraction difficulty etc. is unfavorable for suitability for industrialized production.
Adopt microbe transformation method asymmetric reduction 3-ketone group tetrahydrofuran (THF) can obtain higher optical purity (S)-(+)-3-hydroxyl tetrahydrofuran of the superfluous value of enantiomorph, can realize under the normal temperature and pressure transforming, the reaction conditions gentleness, environmental friendliness, with low cost.Contain the coenzyme hydrogen donor that needs in the reduction process in the microorganism cells, containing abundant enzyme system simultaneously in the microorganism cells helps by add the in-situ regeneration that cheap cosubstrate (as sucrose or glucose) is realized coenzyme in reaction solution, improve the transformation efficiency of substrate greatly, substrate can be converted into (S)-(+)-3-hydroxyl tetrahydrofuran in 100% ground in theory.Microorganism is easy to large scale culturing, is easy to suitability for industrialized production.Therefore, microbe transformation method is the green synthesis process of preparation (S)-(+)-3-hydroxyl tetrahydrofuran.
(3) summary of the invention
The object of the invention provides the method that a kind of microbial transformation prepares S-(+)-3-hydroxyl tetrahydrofuran, and this method catalytic efficiency height, reaction conditions gentleness, environmental friendliness, with low cost are 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-hydroxyl tetrahydrofuran, described method is to be substrate with 3-ketone group tetrahydrofuran (THF), 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-hydroxyl tetrahydrofuran.
The method of the invention is carried out under the following conditions: in the phosphate buffered saline buffer of pH 5.0～8.0, with 3-ketone group tetrahydrofuran (THF) is substrate, the enzyme somatic cells that contains that obtains with yeast 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-hydroxyl tetrahydrofuran through separation and purification.
The starting point concentration of described 3-ketone group tetrahydrofuran (THF) in phosphate buffered saline buffer is 1～10mmol/L.
The described enzyme somatic cells consumption that contains is counted 1～70g/g3-ketone group tetrahydrofuran (THF) substrate with dry cell weight; 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 glucose that is added with final concentration in the described phosphate buffered saline buffer and is 10～150g/L helps improving the molar yield of substrate as cosubstrate.
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 for 26～35 ℃, and fermented liquid is centrifugal, makes to contain the enzyme somatic cells.
S-of the present invention (+)-3-hydroxyl tetrahydrofuran 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 decompression distillation, promptly gets described (S)-(+)-3-hydroxyl tetrahydrofuran.
Described microbial transformation prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, 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, 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
2O0.5～1.5g/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%, culture temperature is 26～35 ℃, and shaking speed is 150～200r/min, cultivates 18～30h, and fermented liquid is centrifugal, separates obtaining containing the enzyme somatic cells; 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 3-ketone group tetrahydrofuran (THF) that adds 1～10mmol/L, the glucose that adds 10～150g/L is as cosubstrate, and the dry cell weight quality be 1～50 times of 3-ketone group tetrahydrofuran (THF) contain the enzyme somatic cells, in 25～45 ℃ of following conversion reactions 8～40 hours, reaction finished to make conversion fluid; (5) separation and purification: 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 small amount of moisture, suction filtration, ethyl acetate is removed in the filtrate decompression distillation, promptly gets described (S)-(+)-3-hydroxyl tetrahydrofuran.
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 3-ketone group tetrahydrofuran (THF) with separating the bacterial strain that obtains in the soil, obtain yeast saccharomyces cerevisiae CGMCC No.2266 and have the ability that good conversion 3-ketone group tetrahydrofuran (THF) is produced (S)-(+)-3-hydroxyl tetrahydrofuran.
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.
Concrete, the present invention prepares the method for S-(+)-3-hydroxyl tetrahydrofuran with microbial transformation, carries 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 is prepared by following composition: wort 5～15g/L, and yeast powder 2～4g/L, peptone 4～6g/L, glucose 7～12g/L, agar 15～25g/L, natural pH value, solvent is a water; 121 ℃ of sterilization 20min, sterilization postcooling bevel;
(2) seed culture: get a 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 is prepared by following composition: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
200.5～1.5g/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, inoculum size with volume ratio 10～20% is inoculated in the fermention medium, and culture temperature is 26～35 ℃, and 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 is prepared by following composition: glucose 26～32g/L, yeast powder 2～4g/L, ammonium sulfate 3～6g/L, anhydrous MgSO
40.2～0.4g/L, K
2HPO
43H
20 0.5～1.5g/L, KH
2PO
40.6～1.5g/L, 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 3-ketone group tetrahydrofuran (THF) that adds 1～10mmol/L, the glucose that adds 10～150g/L helps improving the molar yield of substrate as cosubstrate, and the dry cell weight quality be 1～50 times of 3-ketone group tetrahydrofuran (THF) contain the enzyme somatic cells, under 25～45 ℃, carry out conversion reaction 8～40 hours;
(5) separation and purification: 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 small amount of moisture, behind the suction filtration, the ethyl acetate solution underpressure distillation that obtains is removed ethyl acetate and is promptly got described (S)-(+)-3-hydroxyl tetrahydrofuran.
The available gas chromatograph-mass spectrometer of the present invention (S)-(+)-pure product of 3-hydroxyl tetrahydrofuran detects determines degree of purity of production and molecular weight.
Determining of the superfluous value of the enantiomorph of molar yield of the present invention and product (S)-(+)-3-hydroxyl tetrahydrofuran (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-hydroxyl tetrahydrofuran and (S)-(+)-two kinds of enantiomorphs of 3-hydroxyl tetrahydrofuran, further calculates the superfluous value (ee%) of enantiomorph of the molar yield of reaction and (S)-(+)-3-hydroxyl tetrahydrofuran.
Adopt microorganism cells asymmetric reduction 3-ketone group tetrahydrofuran (THF) preparation (S)-(+)-3-hydroxyl tetrahydrofuran among the present invention, can obtain the product of the superfluous value of high antimer, the glucose that adds final concentration and be 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:
Microbe transformation method preparation (S)-(+)-3-hydroxyl tetrahydrofuran that the present invention adopts is compared with chemical synthesis, enzyme catalysis method and had the following advantages: (1) produces the bacterial strain safety non-toxic, microbial cells is easy to large scale culturing, can obtain a large amount of biological catalysts, more with low cost than chemical catalyst; (2) easy and simple to handle, do not need to add expensive coenzyme in the reaction process, by adding the transformation efficiency that cosubstrate glucose can increase substantially substrate, yield height; (3) be easy to realize large-scale industrial production; (4) normal temperature and pressure just can be realized bioconversion reaction down, the reaction conditions gentleness, and environmental friendliness is the clean synthesis process of (S)-(+)-3-hydroxyl tetrahydrofuran.
(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 preparation of slant medium: 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, cooling back bevel.
The preparation of seed culture and fermention medium: seed and fermention medium all adopt liquid nutrient medium, prepare by following composition: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO
40.25g/L, K
2HPO
43H
2O 1g/L, KH
2PO
41g/L, 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
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.10mL seed liquor (inoculum size is in culture volume mark 10%) is inoculated in the 250mL triangular flask that contains the 100mL liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivates 24h and obtain fermented liquid, 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 the wet thallus that contains the enzyme somatic cells, 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 10 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 respectively, the dry weight that wherein contains the enzyme somatic cells is 10g, add 3-ketone group tetrahydrofuran (THF) separately, make the final concentration of 3-ketone group tetrahydrofuran (THF) be respectively 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L and 10mmol/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 adds anhydrous sodium sulphate and removes small amount of moisture in acetic acid ethyl acetate extract, suction filtration, ethyl acetate is removed in the filtrate decompression distillation, promptly gets described (S)-(+)-3-hydroxyl tetrahydrofuran, and the substrate starting point concentration sees Table 1 to the molar yield of S-(+)-3-beta-hydroxymethyl butyrate and the influence of the superfluous value of corresponding body (ee%).
Table 1: the substrate starting point concentration is to the influence of transformation efficiency and (S)-(+)-3-hydroxyl tetrahydrofuran
Table 1 is as can be seen: along with the increase gradually of concentration of substrate, transformation efficiency reduces gradually.Concentration of substrate be respectively 1 and during 2mmol/L product be the R configuration, when concentration of substrate during more than or equal to 3mmol/L product be the S configuration, the superfluous value of enantiomorph that product (S)-(+)-3-hydroxyl tetrahydrofuran is described is along with the difference of concentration of substrate has bigger variation, and concentration of substrate strengthens the enantiomorph surplus value that helps improving (S)-(+)-3-hydroxyl tetrahydrofuran.
Embodiment 2:
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.10mL seed liquor (inoculum size is in culture volume consumption 10%) is inoculated in the 250mL triangular flask that contains the 100mL liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells, the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In ten parts of triangular flasks that contain 100mL pH7.0 phosphate buffered saline buffer separately, add the wet cell that the centrifugal back of above-mentioned 200 milliliters of fermented liquids obtains respectively, the dry weight that wherein contains the enzyme somatic cells is 10g, adding final concentration is the 3-ketone group tetrahydrofuran (THF) of 5mmol/L, add cosubstrate glucose, make 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, 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 decompression distillation, promptly get described (S)-(+)-3-hydroxyl tetrahydrofuran, the substrate glucose concn sees Table 2 to the molar yield of S-(+)-3-beta-hydroxymethyl butyrate and the influence of the superfluous value of corresponding body (ee%).
Table 2: glucose concn reaches (S)-(+)-the superfluous influence that is worth of 3-hydroxyl tetrahydrofuran enantiomorph transformation efficiency
Table 2 is as can be seen: being added with of glucose is beneficial to the transformation efficiency that improves reaction.The optimum addition of glucose is 50g/L.The adding of glucose helps realizing the in-situ regeneration of coenzyme, thereby improves transformation efficiency.The superfluous value of the enantiomorph of product (S)-(+)-3-hydroxyl tetrahydrofuran does not have to change with the addition of glucose substantially, all keeps 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.10mL seed liquor (inoculum size is in culture volume consumption 10%) is inoculated in the 250mL triangular flask that contains the 100mL liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells, 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 separately, add the wet cell that the centrifugal back of above-mentioned 200 milliliters of fermented liquids obtains respectively, the dry weight that wherein contains the enzyme somatic cells is 10g, adding final concentration separately is the 3-ketone group tetrahydrofuran (THF) of 5mmol/L, adding final concentration is 50g/L cosubstrate glucose, all reacts 24h down respectively at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ 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 adds anhydrous sodium sulphate and removes small amount of moisture in acetic acid ethyl acetate extract, suction filtration, ethyl acetate is removed in the filtrate decompression distillation, promptly gets described (S)-(+)-3-hydroxyl tetrahydrofuran, and temperature of reaction sees Table 3 to the molar yield of S-(+)-3-beta-hydroxymethyl butyrate and the influence of the superfluous value of corresponding body (ee%).
Table 3: temperature of reaction reaches (S)-(+)-the superfluous influence that is worth of 3-hydroxyl tetrahydrofuran enantiomorph transformation efficiency
Table 3 is as can be seen: reaction conversion ratio is subjected to the influence of invert point, and best temperature of reaction is 30 ℃.Invert point is too high can impel enzyme part inactivation, is unfavorable for that conversion reaction obtains higher transformation efficiency.The superfluous value of the enantiomorph of product (S)-(+)-3-hydroxyl tetrahydrofuran does not change with the variation of invert point substantially, all keeps 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.10mL seed liquor (inoculum size is in culture volume consumption 10%) is inoculated in the 250mL triangular flask that contains the 100mL liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells, the dry weight that contains the enzyme somatic cells in every liter of fermented liquid is 50 grams.
In the triangular flask that contains the 100mLpH7.0 phosphate buffered saline buffer, add the wet cell that the centrifugal back of above-mentioned 200 milliliters of fermented liquids obtains respectively at each five parts, the dry weight that wherein contains the enzyme somatic cells is 10g, adding final concentration separately is the 3-ketone group tetrahydrofuran (THF) of 5mmol/L, adding final concentration more separately is 50g/L cosubstrate glucose, all place 30 ℃, react 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 adds anhydrous sodium sulphate and removes small amount of moisture in acetic acid ethyl acetate extract, suction filtration, ethyl acetate is removed in the filtrate decompression distillation, promptly gets described (S)-(+)-3-hydroxyl tetrahydrofuran, and the reaction times sees Table 3 to the molar yield of S-(+)-3-beta-hydroxymethyl butyrate and the influence of the superfluous value of corresponding body (ee%).
Table 4: the reaction times reaches (S)-(+)-the superfluous influence that is worth of 3-hydroxyl tetrahydrofuran enantiomorph transformation efficiency
Table 3 is as can be seen: reaction conversion ratio increases along with the prolongation in reaction times, and best transformation time is 24 hours, and reaction time is short to be helped suitability for industrialized production and enhance productivity.The superfluous value of the enantiomorph of product (S)-(+)-3-hydroxyl tetrahydrofuran does not change with the change in reaction times, and the superfluous value of enantiomorph is 100%.
Embodiment 5:
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 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.10mL seed liquor (inoculum size is in culture volume consumption 10%) is inoculated in the 250mL triangular flask that contains the 100mL liquid nutrient medium, under 30 ℃, the condition of 180r/min, cultivate 24h and obtain fermented liquid, fermented liquid is centrifugal, must contain the enzyme somatic cells, 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 100mL pH7.0 phosphate buffered saline buffer separately respectively, the dry weight that wherein contains the enzyme somatic cells is respectively 2g, 5g, 10g, 15g and 20g.Adding final concentration in above-mentioned five triangular flasks respectively is 5mmol/L 3-ketone group tetrahydrofuran (THF), and final concentration is a 50g/L cosubstrate glucose, 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 decompression distillation, promptly get described (S)-(+)-3-hydroxyl tetrahydrofuran, biomass sees Table 3 to the molar yield of S-(+)-3-beta-hydroxymethyl butyrate and the influence of the superfluous value of corresponding body (ee%).
Table 5: biomass reaches (S)-(+)-the superfluous influence that is worth of 3-hydroxyl tetrahydrofuran enantiomorph transformation efficiency
Table 5 is as can be seen: reaction conversion ratio improves along with increasing of biomass, and the increasing of biomass has not only improved the consumption of enzyme, has improved simultaneously to participate in the consumption of the coenzyme of reaction, thereby has helped improving transformation efficiency.The increasing of biomass all keeps 100% to the not influence of the superfluous value of the enantiomorph of product.
Claims (8)
1. a microbial transformation prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, it is characterized in that described method is is substrate with 3-ketone group tetrahydrofuran (THF), the enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCCNo.2266 fermentation is a biological catalyst, carries out conversion reaction and makes described S-(+)-3-hydroxyl tetrahydrofuran.
2. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, it is characterized in that described method is: in the phosphate buffered saline buffer of pH 5.0～8.0, with 3-ketone group tetrahydrofuran (THF) is substrate, the enzyme somatic cells that contains that obtains with yeast 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-hydroxyl tetrahydrofuran through separation and purification.
2, microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, it is characterized in that the starting point concentration of described 3-ketone group tetrahydrofuran (THF) in phosphate buffered saline buffer is 1～10mmol/L.
3. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, it is characterized in that the described enzyme somatic cells consumption that contains counts 1～70g/g substrate with dry cell weight.
4. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, 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-hydroxyl tetrahydrofuran, 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 for 26～35 ℃, fermented liquid is centrifugal, make and contain the enzyme somatic cells.
6. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, it is characterized in that described S-(+)-3-hydroxyl tetrahydrofuran 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 decompression distillation, promptly gets described (S)-(+)-3-hydroxyl tetrahydrofuran.
7. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, 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, 26～35 ℃, shaking speed is 150～200r/min, cultivates 18～26h 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%, culture temperature is 26～35 ℃, shaking speed is 150～200r/min, cultivate 18～30h, fermented liquid is centrifugal, separate obtaining the described enzyme somatic cells that contains; (4) bio-transformation: in the phosphate buffered saline buffer of pH 5.0～8.0, adding final concentration is the 3-ketone group tetrahydrofuran (THF) of 1～10mmol/L, the glucose that adds final concentration again and be 10～150g/L is as cosubstrate, and with the dry cell weight be 1～50 times of 3-ketone group tetrahydrofuran (THF) quality contain the enzyme somatic cells, in 25～45 ℃ of following conversion reactions 8～40 hours, reaction finished to make conversion fluid; (5) separation and purification: 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 decompression distillation, promptly gets described (S)-(+)-3-hydroxyl tetrahydrofuran.
8. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, 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, 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, inoculum size with volume ratio 10～20% is inoculated in the fermention medium, and culture temperature is 26～35 ℃, and 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 3-ketone group tetrahydrofuran (THF) that adds 1～10mmol/L, the glucose that adds final concentration 10～150g/L again is as cosubstrate, and dry cell weight be 1～50 times of 3-ketone group tetrahydrofuran (THF) contain the enzyme somatic cells, in 25～45 ℃ of following conversion reactions 8～40 hours, reaction finished to make conversion fluid;
(5) separation and purification: 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 decompression distillation, promptly gets described (S)-(+)-3-hydroxyl tetrahydrofuran.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624227A (en) * | 2016-03-01 | 2016-06-01 | 苏州艾缇克药物化学有限公司 | Method for preparing (S)-3-hydroxytetrahydrofuran based on erythritol microorganisms |
| CN107904269A (en) * | 2017-12-29 | 2018-04-13 | 安徽联创生物医药股份有限公司 | A kind of method that engineering strain conversion prepares (S) (+) 3 hydroxyl tetrahydrofuran |
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|---|---|---|---|---|
| CN1887880A (en) * | 2006-07-20 | 2007-01-03 | 厦门大学 | Synthesis of S-(3)-hydroxy tetrahydrofuran |
| WO2007081065A1 (en) * | 2006-01-10 | 2007-07-19 | Sk Holdings Co., Ltd. | Method for preparing 3-hydroxytetrahydrofuran using cyclodehydration |
| CN101230319A (en) * | 2008-02-04 | 2008-07-30 | 浙江工业大学 | Saccharomyces cerevisiae CGMCC No.2266 and its application in preparation of (S)-(-)- beta-hydroxyphenyl propionic acid ethyl |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007081065A1 (en) * | 2006-01-10 | 2007-07-19 | Sk Holdings Co., Ltd. | Method for preparing 3-hydroxytetrahydrofuran using cyclodehydration |
| CN1887880A (en) * | 2006-07-20 | 2007-01-03 | 厦门大学 | Synthesis of S-(3)-hydroxy tetrahydrofuran |
| CN101230319A (en) * | 2008-02-04 | 2008-07-30 | 浙江工业大学 | Saccharomyces cerevisiae CGMCC No.2266 and its application in preparation of (S)-(-)- beta-hydroxyphenyl propionic acid ethyl |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624227A (en) * | 2016-03-01 | 2016-06-01 | 苏州艾缇克药物化学有限公司 | Method for preparing (S)-3-hydroxytetrahydrofuran based on erythritol microorganisms |
| CN107904269A (en) * | 2017-12-29 | 2018-04-13 | 安徽联创生物医药股份有限公司 | A kind of method that engineering strain conversion prepares (S) (+) 3 hydroxyl tetrahydrofuran |
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