CN102071231B - Method for preparing S-(+)-3-hydroxy tetrahydrofuran through microbial conversion - Google Patents

Abstract

The invention discloses a method for preparing S-(+)-3-hydroxytetrahydrofuran by microbial transformation: in a phosphate buffer solution with a pH of 5.0 to 8.0, 3-ketotetrahydrofuran is used as a substrate and Saccharomyces cerevisiae CGMCC No. The enzyme-containing bacterial cells obtained by 2266 fermentation are used as biocatalysts, and the transformation reaction is carried out at 25-45°C for 8-40 hours. After the reaction is completed, the transformation liquid is separated and purified to obtain the S-(+)-3-hydroxytetrahydrofuran; this The beneficial effects of the invention are mainly reflected in: (1) the production strain is safe and non-toxic, easy to cultivate on a large scale, and the cost is low; (2) the operation is simple, no expensive coenzyme needs to be added in the reaction process, and the yield is high; (3) it is easy to realize Large-scale industrial production; (4) mild reaction conditions and environmental friendliness.

Description

A method for preparing S-(+)-3-hydroxytetrahydrofuran by microbial conversion

(1) Technical field

The invention relates to a method for preparing S-(+)-3-hydroxytetrahydrofuran by microbial transformation, in particular to a method for preparing S-(+)-3-hydroxytetrahydrofuran by using Saccharomyces cerevisiae CGMCC No.2266 as a biocatalyst and using 3-ketotetrahydrofuran as a substrate A new method for (S)-(+)-3-hydroxytetrahydrofuran.

(2) Background technology

(S)-(+)-3-Hydroxytetrahydrofuran ((S)-(+)-3-Hydroxytetrahydrofuran), CAS accession number: 86087-23-2, molecular formula C ₄ H ₈ O ₂ , molecular weight 88.11. (S)-(+)-3-Hydroxytetrahydrofuran is an important intermediate in the synthesis of AIDS drug Amprenavir. After introducing (S)-(+)-3-hydroxytetrahydrofuran group into the diphenyl ether herbicide pesticide, the herbicidal activity and selectivity of the diphenyl ether herbicide can be obviously improved.

In literature reports, the synthesis of (S)-(+)-3-hydroxytetrahydrofuran can be obtained by asymmetric hydroboration or hydrosilation of dihydrofuran, which requires a chiral catalyst. The preparation process of the chiral catalyst is cumbersome and expensive; it can be obtained by 4 -Preparation by asymmetric hydrogenation-reduction-intramolecular etherification of chloro-3-carbonyl butyrate, the method has high cost and is not suitable for industrial production; it can be prepared by enzymatic resolution of 3-hydroxytetrahydrofuran racemate, enzyme The resolution efficiency of method resolution can only reach 50% at the highest, and production efficiency is lower; It can be synthesized by reduction-intramolecular etherification of malic acid, but this method needs to reduce malic acid diester with expensive lithium aluminum hydride, and Protection of the secondary hydroxyl group is required to prevent racemization. In most cases, the optical purity of the product in the above-mentioned preparation method is insufficient, or there are factors unfavorable for industrial production such as separation and extraction difficulties.

Optically pure (S)-(+)-3-hydroxytetrahydrofuran with high enantiomeric excess value can be obtained by asymmetric reduction of 3-ketotetrahydrofuran by microbial transformation method, and the transformation can be realized at normal temperature and pressure, and the reaction conditions are mild. Environmentally friendly and low cost. Microbial cells contain coenzyme hydrogen donors needed in the reduction process, and microbial cells are rich in enzymes, which is conducive to the in situ regeneration of coenzymes by adding cheap auxiliary substrates (such as sucrose or glucose) to the reaction solution, greatly Improve the conversion efficiency of the substrate, theoretically the substrate can be 100% converted into (S)-(+)-3-hydroxytetrahydrofuran. Microorganisms are easy to be cultivated on a large scale and industrialized. Therefore, the microbial conversion method is a green synthetic process for preparing (S)-(+)-3-hydroxytetrahydrofuran.

(3) Contents of the invention

The object of the present invention is to provide a method for preparing S-(+)-3-hydroxytetrahydrofuran by microbial transformation, which has high catalytic efficiency, mild reaction conditions, environment-friendly, low cost and easy industrial production.

The technical scheme adopted in the present invention is:

A method for preparing S-(+)-3-hydroxytetrahydrofuran by microbial transformation, the method is to use 3-ketotetrahydrofuran as a substrate, and use the enzyme-containing bacterium obtained by fermentation of Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266 The cells are biocatalysts, and the transformation reaction is carried out to prepare the S-(+)-3-hydroxytetrahydrofuran.

The method of the present invention is carried out under the following conditions: in a phosphate buffer solution with a pH of 5.0 to 8.0, 3-ketotetrahydrofuran is used as a substrate, and enzyme-containing bacterial cells obtained by fermentation of Saccharomyces cerevisiae CGMCC No.2266 are used as organisms. Catalyst, conversion reaction at 25-45°C for 8-40 hours, after the reaction, the conversion solution is separated and purified to obtain the S-(+)-3-hydroxytetrahydrofuran.

The initial concentration of the 3-ketotetrahydrofuran in the phosphate buffer solution is 1-10 mmol/L.

The dosage of the enzyme-containing bacterial cells is 1 to 70 g/g 3-ketotetrahydrofuran substrate in terms of dry cell weight; the dry weight of the enzyme-containing bacterial cells is determined by centrifuging the fermentation broth and discarding the supernatant , dry the wet cells at 120°C for 48 hours to constant weight, and measure the weight of the dry cells; take a part of the wet cells obtained by centrifuging in the fermentation broth to measure the dry weight of the cells, calculate the ratio of dry cells in the unit enzyme-containing bacterial cells in the fermentation broth, and then Use this ratio to calculate the amount of enzyme-containing somatic cell fermentation broth required for quantitative cell dry weight.

Glucose with a final concentration of 10-150 g/L is added to the phosphate buffer as an auxiliary substrate, which is beneficial to improving the molar conversion rate of the substrate.

The enzyme-containing bacterial cells are prepared according to the following method: inoculate Saccharomyces cerevisiae CGMCC No.2266 into the fermentation medium, the shaker speed is 150-200r/min, culture at 26-35°C for 18-30h, and centrifuge the fermentation liquid. The enzyme-containing bacterial cells are prepared.

The S-(+)-3-hydroxytetrahydrofuran separation and purification method of the present invention is as follows: after the reaction is finished, the transformation solution is centrifuged at 4000r/min for 20 minutes, the bacterial cell precipitation is discarded, and the supernatant is continuously purified with an equal volume of ethyl acetate. Extract 3 times, combine the ethyl acetate extracts, add anhydrous sodium sulfate to the ethyl acetate extracts to remove water, filter with suction, distill the filtrate under reduced pressure to remove ethyl acetate, and obtain the (S)-(+)- 3-Hydroxytetrahydrofuran.

The method for preparing S-(+)-3-hydroxytetrahydrofuran by microbial transformation is recommended to be carried out in accordance with the following steps: (1) Slant culture: Inoculate Saccharomyces cerevisiae CGMCC No.2266 into the slant medium, and cultivate it at 26-35°C for 4 Bacterial slant was obtained in ~6 days; the final concentration of the slant medium was composed of: wort juice 5-15g/L, yeast powder 2-4g/L, peptone 4-6g/L, glucose 7-12g/L, agar 15～25g/L, natural pH value, solvent is water; (2) Seed culture: take an inoculation loop from the slope of the bacteria and transfer the bacteria to the seed medium, at 26～35°C, the speed of the shaker is 150～200r/ min, cultivated for 18-26 hours to obtain seed liquid; the final concentration of the seed medium is composed of: glucose 26-32g/L, yeast powder 2-4g/L, ammonium sulfate 3-6g/L, anhydrous MgSO ₄ 0.2- 0.4g/L, K ₂ HPO ₄ 3H ₂ O 0.5～1.5g/L, KH ₂ PO ₄ 0.6～1.5g/L, use NaOH or HCl solution to adjust the pH value of the liquid medium to 7.0, and the solvent is water (3) Fermentation culture: take the seed liquid, inoculate it in the fermentation medium with an inoculation amount of 10-20% by volume fraction, the culture temperature is 26-35°C, the shaking table speed is 150-200r/min, and cultivate for 18-30h , centrifuging the fermentation broth to separate enzyme-containing bacterial cells; the final concentration of the fermentation medium consists of: glucose 26-32g/L, yeast powder 2-4g/L, ammonium sulfate 3-6g/L, anhydrous MgSO ₄ 0.2～0.4g/L, K ₂ HPO ₄ 3H ₂ O 0.5～1.5g/L, KH ₂ PO ₄ 0.6～1.5g/L, use NaOH or HCl solution to adjust the pH value of the liquid medium to 7.0, solvent (4) Biotransformation: Add 1-10mmol/L 3-ketotetrahydrofuran to a phosphate buffer solution with a pH of 5.0-8.0, add 10-150g/L glucose as an auxiliary substrate, and cell stem Enzyme-containing bacterial cells whose weight is 1 to 50 times that of 3-ketotetrahydrofuran are transformed at 25 to 45°C for 8 to 40 hours, and the transformation liquid is obtained after the reaction is completed; (5) Separation and purification: the transformation liquid is 4000r/ Centrifuge for 20 minutes, discard the bacterial precipitate, extract the supernatant with an equal volume of ethyl acetate three times continuously, combine the ethyl acetate extracts, add anhydrous sodium sulfate to the ethyl acetate extracts to remove a small amount of water, and pump The filtrate was distilled off under reduced pressure to remove ethyl acetate to obtain the (S)-(+)-3-hydroxytetrahydrofuran.

Saccharomyces cerevisiae CGMCC No.2266, preserved in the General Microorganisms Preservation Center of China Committee for the Collection of Microorganisms, located in the Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, preservation number CGMCC No.2266, preservation date November 26, 2007 , has been protected as a new strain in the previously granted patent 200810059686.

Strain source: The microbial strain Saccharomyces cerevisiae CGMCC No.2266 described in the present invention is screened from the soil near Hangzhou West Lake Brewery. The strains isolated from the soil were used to transform 3-ketotetrahydrofuran, and the obtained Saccharomyces cerevisiae CGMCC No.2266 had a good ability to transform 3-ketotetrahydrofuran to produce (S)-(+)-3-hydroxytetrahydrofuran.

The colony characteristics of the Saccharomyces cerevisiae CGMCC No.2266: on the agar medium, it presents a milky white, shiny, flat, neat edge, moist, smooth surface, and uniform colony morphology.

Specifically, the method for preparing S-(+)-3-hydroxytetrahydrofuran by microbial transformation in the present invention is carried out according to the following steps:

(1) Slant culture: Inoculate Saccharomyces cerevisiae CGMCC No.2266 into the slant medium, and cultivate it at 26-35°C for 4-6 days to obtain the slant; the slant medium is prepared as follows: wort juice 5-15g/ L, yeast powder 2～4g/L, peptone 4～6g/L, glucose 7～12g/L, agar 15～25g/L, natural pH value, solvent is water; sterilize at 121℃ for 20min, cool down after sterilization beveled;

(2) Seed culture: take an inoculation ring from the slant of the thalline and transfer it to the seed medium, at 26-35° C., with a shaker speed of 150-200 r/min, and cultivate for 18-26 hours to obtain the seed liquid; the seeds The medium is prepared according to the following composition: glucose 26~32g/L, yeast powder 2~4g/L, ammonium sulfate 3~6g/L, anhydrous MgSO ₄ 0.2~0.4g/L, K ₂ HPO ₄ 3H ₂ 00.5~ 1.5g/L, KH ₂ PO ₄ 0.6～1.5g/L, use NaOH or HCl solution to adjust the pH value of the liquid medium to 7.0, and the solvent is water;

(3) Fermentation culture: take the seed liquid, inoculate it in the fermentation medium with an inoculation amount of 10-20% by volume, the culture temperature is 26-35°C, the shaking table speed is 150-200r/min, and cultivate for 18-30h to obtain The fermentation broth containing enzyme-containing bacterial cells is centrifuged to obtain the enzyme-containing bacterial cells; the fermentation medium is prepared according to the following composition: glucose 26-32g/L, yeast powder 2-4g/L, ammonium sulfate 3- 6g/L, anhydrous MgSO ₄ 0.2～0.4g/L, K ₂ HPO ₄ 3H ₂ 00.5～1.5g/L, KH ₂ PO ₄ 0.6～1.5g/L, adjust the liquid medium with NaOH or HCl solution The pH value is 7.0, and the solvent is water;

(4) Biotransformation: Add 1-10mmol/L 3-ketotetrahydrofuran to a phosphate buffer solution with a pH of 5.0-8.0, and add 10-150g/L glucose as an auxiliary substrate, which is beneficial to increase the mole of the substrate Transformation rate, and enzyme-containing bacterial cells whose dry weight of cells is 1 to 50 times that of 3-ketotetrahydrofuran, the transformation reaction is carried out at 25 to 45°C for 8 to 40 hours;

(5) Separation and purification: after the reaction, the transformation solution was centrifuged at 4000r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extracts were combined, and the Anhydrous sodium sulfate was added to the ester extract to remove a small amount of water, and after suction filtration, the obtained ethyl acetate solution was distilled off under reduced pressure to remove the ethyl acetate to obtain the (S)-(+)-3-hydroxytetrahydrofuran.

The pure product of (S)-(+)-3-hydroxytetrahydrofuran in the present invention can be detected by gas chromatography-mass spectrometry to determine the purity and molecular weight of the product.

Determination of the molar conversion rate of the present invention and the enantiomeric excess value (ee%) of product (S)-(+)-3-hydroxytetrahydrofuran:

It was detected by gas chromatography. The chromatographic column is Varian CP Chirasil-DEX chiral column. This chiral column can detect the content of (R)-(-)-3-hydroxytetrahydrofuran and (S)-(+)-3-hydroxytetrahydrofuran two enantiomers, and further calculate the molar conversion rate and ( Enantiomeric excess (ee%) of S)-(+)-3-hydroxytetrahydrofuran.

In the present invention, microbial cells are used to asymmetrically reduce 3-ketotetrahydrofuran to prepare (S)-(+)-3-hydroxytetrahydrofuran, and a product with high enantiomeric excess value can be obtained, and glucose with a final concentration of 10-150 g/L is added As an auxiliary substrate, it is beneficial to increase the molar conversion rate of the substrate.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

Compared with chemical synthesis and enzymatic catalysis, the microbial conversion method used in the present invention to prepare (S)-(+)-3-hydroxytetrahydrofuran has the following advantages: (1) the production strain is safe and non-toxic, and the microbial cells are easy to cultivate on a large scale. A large number of biocatalysts can be obtained, and the cost is lower than that of chemical catalysts; (2) the operation is simple, no expensive coenzymes need to be added in the reaction process, and the conversion efficiency of the substrate can be greatly improved by adding the auxiliary substrate glucose, and the yield is high; (3) It is easy to realize large-scale industrial production; (4) The biotransformation reaction can be realized under normal temperature and pressure, the reaction conditions are mild, and the environment is friendly, which is a clean synthesis process of (S)-(+)-3-hydroxytetrahydrofuran.

(4) Specific implementation methods

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

Preparation of slant medium: wort juice 10g/L, yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, natural pH value, solvent is water; into a bevel.

Preparation of seed culture and fermentation medium: both seeds and fermentation medium are liquid medium, prepared according to the following composition: glucose 30g/L, yeast powder 3g/L, ammonium sulfate 5g/L, anhydrous MgSO ₄ 0.25g/L , K ₂ HPO ₄ ·3H ₂ O 1g/L, KH ₂ PO ₄ 1g/L, the solvent is water, the pH of the liquid medium is adjusted to 7.0 with NaOH or HCl solution, and sterilized at 121°C for 20min.

Example 1

Inoculate the slant medium with CGMCC No.2266 strain, and culture at 30°C for 4-6 days to obtain the slant. Use an inoculation needle to take an inoculation loop from the slant of the bacterium and inoculate it into a 250mL Erlenmeyer flask containing 100mL of liquid medium, and incubate at 30°C and 180r/min for 24h to obtain a seed solution. Inoculate 10 mL of seed solution (inoculum volume fraction 10% of the medium) into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, cultivate it for 24 hours at 30°C and 180 r/min to obtain a fermented liquid, centrifuge the fermented liquid, Obtain enzyme-containing bacterial cells.

The determination of the dry weight of the bacteria is to take a small part of the wet bacteria containing the enzyme cells after centrifuging the fermentation broth, and dry it at 120°C for 48 hours to a constant weight, measure the weight of the dry cells, and calculate the unit content of the fermentation broth. The ratio of dry cells in the enzyme bacterium cells, and then calculate the amount of fermented liquid containing enzyme bacterium cells required for the quantitative dry weight of the cells. The dry weight of enzyme-containing bacterium cells contained in every liter of fermented liquid of the present embodiment is 50 grams.

In 10 portions of Erlenmeyer flasks containing 100 mL of pH7.0 phosphate buffer solution, respectively add the wet cells obtained by centrifuging 200 mL of the above-mentioned fermented broth, wherein the dry weight of cells containing enzymes is 10 g, and respectively add 3-keto Tetrahydrofuran, so that the final concentration of 3-ketotetrahydrofuran is 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L and 10mmol /L, placed in a shaker at 30°C and 180r/min for 24h. After the reaction, the transformation solution was centrifuged at 4000r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extracts were combined and added to the ethyl acetate extract. Add anhydrous sodium sulfate to remove a small amount of water, filter with suction, and distill the filtrate under reduced pressure to remove ethyl acetate to obtain the (S)-(+)-3-hydroxytetrahydrofuran. The initial concentration of the substrate is relative to S-(+)-3 Table 1 shows the influence of the molar conversion rate of methyl-hydroxybutyrate and the enantiomeric excess value (ee%).

Table 1: Effect of initial substrate concentration on conversion and (S)-(+)-3-hydroxytetrahydrofuran

It can be seen from Table 1 that as the substrate concentration increases gradually, the conversion rate decreases gradually. When the substrate concentration is 1 and 2mmol/L, the product is in the R configuration, and when the substrate concentration is greater than or equal to 3mmol/L, the product is in the S configuration, indicating that the enantiometry of the product (S)-(+)-3-hydroxytetrahydrofuran The enantiomeric excess value of (S)-(+)-3-hydroxytetrahydrofuran has a great change with different substrate concentrations, and the increase of substrate concentration is beneficial to increase the enantiomeric excess value of (S)-(+)-3-hydroxytetrahydrofuran.

Example 2:

Inoculate the slant medium with CGMCC No.2266 strain, and culture at 30°C for 4-6 days to obtain the slant. Use an inoculation needle to take an inoculation loop from the slant of the bacterium and inoculate it into a 250mL Erlenmeyer flask containing 100mL of liquid medium, and incubate at 30°C and 180r/min for 24h to obtain a seed solution. Inoculate 10 mL of seed solution (the inoculation amount is based on 10% of the volume of the medium used) in a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, cultivate it for 24 hours at 30°C and 180 r/min to obtain a fermentation liquid, and centrifuge the fermentation liquid to obtain Enzyme-containing bacterium cells, the dry weight of enzyme-containing bacterium cells per liter of fermentation broth is 50 grams.

Add the wet cells obtained after centrifugation of the above 200 ml fermentation broth to ten portions of Erlenmeyer flasks each containing 100 mL pH7.0 phosphate buffer solution, wherein the dry weight of enzyme-containing bacterial cells is 10 g, and the final concentration of adding is 5 mmol/ L of 3-ketotetrahydrofuran, add the auxiliary substrate glucose, so that the final concentration of glucose is 10g/L, 30g/L, 50g/L, 70g/L, 100g/L, 150g/L, place at 30°C, 180r /min shaker reaction 24h. After the reaction, the transformation solution was centrifuged at 4000r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extracts were combined and added to the ethyl acetate extract. Add anhydrous sodium sulfate to remove a small amount of water, filter with suction, and distill the filtrate under reduced pressure to remove ethyl acetate to obtain the (S)-(+)-3-hydroxytetrahydrofuran. Table 2 shows the influence of the molar conversion rate of methyl-hydroxybutyrate and the enantiomeric excess value (ee%).

Table 2: Effect of glucose concentration on conversion and enantiomeric excess of (S)-(+)-3-hydroxytetrahydrofuran

It can be seen from Table 2 that the addition of glucose is beneficial to improve the conversion rate of the reaction. The optimal amount of glucose added is 50g/L. The addition of glucose is beneficial to realize the in situ regeneration of coenzymes, thereby increasing the conversion rate. The enantiomeric excess of the product (S)-(+)-3-hydroxytetrahydrofuran basically did not change with the amount of glucose added, and both remained at 100%.

Example 3:

Inoculate the Saccharomyces cerevisiae CGMCC No.2266 strain into the slant medium, and culture it at 30°C for 4-6 days to obtain the slant surface of the bacteria. Use an inoculation needle to take an inoculation loop from the slant of the bacterium and inoculate it into a 250mL Erlenmeyer flask containing 100mL of liquid medium, and incubate at 30°C and 180r/min for 24h to obtain a seed solution. Inoculate 10 mL of seed solution (the inoculation amount is based on 10% of the volume of the medium used) in a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, cultivate it for 24 hours at 30°C and 180 r/min to obtain a fermentation liquid, and centrifuge the fermentation liquid to obtain Enzyme-containing bacterium cells, the dry weight of enzyme-containing bacterium cells per liter of fermentation broth is 50 grams.

Add the wet cells obtained after centrifugation of the above-mentioned 200 milliliters of fermentation broth to five parts of each conical flask containing 100 mL of pH7.0 phosphate buffer solution, wherein the dry weight of the enzyme-containing bacterial cells is 10 g, and the final concentration of each addition is 5 mmol /L of 3-ketotetrahydrofuran, adding the auxiliary substrate glucose at a final concentration of 50g/L, were reacted at 25°C, 30°C, 35°C, 40°C, and 45°C for 24h in a shaker at 180r/min. After the reaction, the transformation solution was centrifuged at 4000r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extracts were combined and added to the ethyl acetate extract. Add anhydrous sodium sulfate to remove a small amount of water, filter with suction, and distill the filtrate under reduced pressure to remove ethyl acetate to obtain the (S)-(+)-3-hydroxytetrahydrofuran. The molar conversion of methyl butyrate and the influence of enantiomeric excess value (ee%) are shown in Table 3.

Table 3: Effect of reaction temperature on conversion and enantiomeric excess of (S)-(+)-3-hydroxytetrahydrofuran

It can be seen from Table 3 that the reaction conversion rate is affected by the conversion temperature, and the optimum reaction temperature is 30°C. Too high conversion temperature can promote the partial inactivation of the enzyme, which is not conducive to the conversion reaction to obtain a higher conversion efficiency. The enantiomeric excess of the product (S)-(+)-3-hydroxytetrahydrofuran basically does not change with the change of the conversion temperature, and both maintain 100%.

Example 4:

Inoculate the Saccharomyces cerevisiae CGMCC No.2266 strain into the slant medium, and culture it at 30°C for 4-6 days to obtain the slant surface of the bacteria. Use an inoculation needle to take an inoculation loop from the slant of the bacterium and inoculate it into a 250mL Erlenmeyer flask containing 100mL of liquid medium, and culture it at 30°C and 180r/min for 24h to obtain a seed solution. Inoculate 10 mL of seed solution (the inoculation amount is based on 10% of the volume of the medium used) in a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, cultivate it for 24 hours at 30°C and 180 r/min to obtain a fermentation liquid, and centrifuge the fermentation liquid to obtain Enzyme-containing bacterium cells, the dry weight of enzyme-containing bacterium cells per liter of fermentation broth is 50 grams.

Add the above-mentioned 200 milliliters of fermented liquid centrifugation to five portions of conical flasks containing 100 mL of pH 7.0 phosphate buffer respectively, wherein the dry weight of enzyme-containing bacterial cells is 10 g, and the final concentration of each addition is 5 mmol. /L of 3-ketotetrahydrofuran, and then add the auxiliary substrate glucose with a final concentration of 50g/L, and place them in a shaker at 30°C and 180r/min for 8h, 16h, 24h, 32h, and 40h respectively. After the reaction, the transformation solution was centrifuged at 4000r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extracts were combined and added to the ethyl acetate extract. Add anhydrous sodium sulfate to remove a small amount of water, filter with suction, and distill the filtrate under reduced pressure to remove ethyl acetate to obtain the (S)-(+)-3-hydroxytetrahydrofuran. The molar conversion of methyl butyrate and the influence of enantiomeric excess value (ee%) are shown in Table 4.

Table 4: Effect of reaction time on conversion and enantiomeric excess of (S)-(+)-3-hydroxytetrahydrofuran

It can be seen from Table 4 that the reaction conversion rate increases with the prolongation of the reaction time, the optimum conversion time is 24 hours, and the short reaction cycle is beneficial to industrial production to improve production efficiency. The enantiomeric excess of the product (S)-(+)-3-hydroxytetrahydrofuran does not change with the reaction time, and the enantiomeric excess is 100%.

Example 5:

Saccharomyces cerevisiae CGMCC No.2266 was inoculated into the slant medium, and cultured at 30°C for 4-6 days to prepare the slant. Use an inoculation needle to take a ring of bacteria from the slant of the bacteria and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, and culture it at 30°C and 180 r/min for 24 hours to obtain a seed solution. Inoculate 10 mL of seed solution (the inoculation amount is based on 10% of the volume of the medium used) in a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, cultivate it for 24 hours at 30°C and 180 r/min to obtain a fermentation liquid, and centrifuge the fermentation liquid to obtain Enzyme-containing bacterium cells, the dry weight of enzyme-containing bacterium cells per liter of fermentation broth is 50 grams.

Wet cells obtained after centrifugation of 40 ml, 100 ml, 200 ml, 300 ml and 400 ml of the fermentation broth obtained above were added to five Erlenmeyer flasks each containing 100 mL of pH7.0 phosphate buffer solution, which contained enzyme-containing bacteria The dry weights of somatic cells were 2g, 5g, 10g, 15g and 20g, respectively. Add the final concentration of 5mmol/L 3-ketotetrahydrofuran and the final concentration of 50g/L auxiliary substrate glucose to the above five Erlenmeyer flasks respectively, and place them in a shaker at 30°C and 180r/min for 24h. After the reaction, the transformation solution was centrifuged at 4000r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extract was combined, and added to the ethyl acetate extract Remove a small amount of water with anhydrous sodium sulfate, filter with suction, and distill the filtrate under reduced pressure to remove ethyl acetate to obtain the (S)-(+)-3-hydroxytetrahydrofuran. See Table 5 for the molar conversion of methyl butyrate and the effect of enantiomeric excess (ee%).

Table 5: Effects of bacterial mass on conversion rate and enantiomeric excess of (S)-(+)-3-hydroxytetrahydrofuran

It can be seen from Table 5 that the conversion rate of the reaction increases with the increase of the amount of bacteria, and the increase of the amount of bacteria not only increases the amount of enzymes, but also increases the amount of coenzymes participating in the reaction, thus helping to improve the conversion rate. The increase of the amount of bacteria has no effect on the enantiomeric excess value of the product, and both remain 100%.

Claims (9)

1. a microbial transformation prepares the method for S-(+)-3-hydroxyl tetrahydrofuran; It is characterized in that said method is is substrate with 3-ketone group 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 said 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 said method is: in the phosphate buffered saline buffer of pH 5.0 ~ 8.0; With 3-ketone group THF is substrate, and the enzyme somatic cells that contains that obtains with yeast saccharomyces cerevisiae CGMCC No.2266 fermentation was a biological catalyst, in 25 ~ 45 ℃ of following conversion reactions 8 ~ 40 hours; After reaction finished, conversion fluid obtained said S-(+)-3-hydroxyl tetrahydrofuran through separation and purification.

3. 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 said 3-ketone group THF in phosphate buffered saline buffer is 1 ~ 10mmol/L.

4. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, it is characterized in that the said enzyme somatic cells consumption that contains counts 1 ~ 70g/g substrate with dried cell weight.

5. microbial transformation as claimed in claim 2 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, and the glucose that it is characterized in that also being added with in the said phosphate buffered saline buffer final concentration and be 10 ~ 150g/L is as cosubstrate.

6. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran; It is characterized in that the said enzyme somatic cells that contains prepares according to following method: 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.

7. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran; It is characterized in that said S-(+)-3-hydroxyl tetrahydrofuran separation purification method is following: after reaction finishes,, discard bacterial sediment with centrifugal 20 minutes of conversion fluid 4000r/min; With supernatant with equal-volume ETHYLE ACETATE continuous extraction 3 times; The combined ethyl acetate extraction liquid adds SODIUM SULPHATE ANHYDROUS 99PCT and removes moisture, suction filtration in acetic acid ethyl acetate extract; ETHYLE ACETATE is removed in the filtrate decompression distillation, promptly gets said 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 said 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(tric)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 the said 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 THF of 1 ~ 10mmol/L; The glucose that adds final concentration again and be 10 ~ 150g/L is as cosubstrate; And dried cell weight be 1 ~ 50 times of 3-ketone group 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 with equal-volume ETHYLE ACETATE continuous extraction 3 times, combined ethyl acetate extraction liquid; In acetic acid ethyl acetate extract, add SODIUM SULPHATE ANHYDROUS 99PCT and remove moisture; Suction filtration, ETHYLE ACETATE is removed in the filtrate decompression distillation, promptly gets said S-(+)-3-hydroxyl tetrahydrofuran.

9. microbial transformation as claimed in claim 1 prepares the method for S-(+)-3-hydroxyl tetrahydrofuran, it is characterized in that said 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, using the pH value of NaOH or HCl solution adjustment liquid nutrient medium 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, spinning obtains the said enzyme somatic cells that contains; Said 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, using the pH value of NaOH or HCl solution adjustment liquid nutrient medium 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 THF that adds 1 ~ 10mmol/L; The glucose that adds final concentration 10 ~ 150g/L again is as cosubstrate; And dried cell weight be 1 ~ 50 times of 3-ketone group 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 with equal-volume ETHYLE ACETATE continuous extraction 3 times, combined ethyl acetate extraction liquid; In acetic acid ethyl acetate extract, add SODIUM SULPHATE ANHYDROUS 99PCT and remove moisture; Suction filtration, ETHYLE ACETATE is removed in the filtrate decompression distillation, promptly gets said S-(+)-3-hydroxyl tetrahydrofuran.