Magnetic immobilized yeast cell and application thereof to synthesis of ethyl R-4-chloro-3-hydroxybutyrate

A technology of ethyl hydroxybutyrate and immobilized yeast, which is applied in the direction of immobilized on or in the inorganic carrier, based on microorganism methods, fermentation, etc., can solve the problem of low substrate processing capacity, improve transformation efficiency, and facilitate Recycling and reuse, improving the effect of production

Active Publication Date: 2020-03-31
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Magnetically immobilized yeast cells can be evenly dispersed in the reaction solution under the action of a magnetic field in an alternating magnetic field, and can fully contact with the substrate, and repeated use can improve the efficiency of the catalytic reaction; the reaction conditions are mild, and they are magnetically immobilized under the action of a

Method used

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  • Magnetic immobilized yeast cell and application thereof to synthesis of ethyl R-4-chloro-3-hydroxybutyrate
  • Magnetic immobilized yeast cell and application thereof to synthesis of ethyl R-4-chloro-3-hydroxybutyrate
  • Magnetic immobilized yeast cell and application thereof to synthesis of ethyl R-4-chloro-3-hydroxybutyrate

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0035] Example 1: Preparation of Saccharomyces cerevisiae cells

[0036] (1) Slant culture: Saccharomyces cerevisiae CGMCC No.3361 was inoculated into a slant medium, cultured at 37°C for 24h to obtain a bacterial slant; the slant medium was prepared as follows: wort 10g / L, yeast Powder 3g / L, peptone 5g / L, glucose 10g / L, agar 20g / L, natural pH value, solvent is water; sterilize at 121°C for 20 minutes, and cool to make a slope after sterilization;

[0037] (2) Seed culture: Take a loop of bacteria from the sloping surface of the bacteria and transfer it to the seed culture medium, 35℃, the shaker speed is 150r / min, culture for 24h, to obtain the seed liquid; the seed culture medium is prepared according to the following composition :Glucose 30g / L, yeast powder 3g / L, ammonium sulfate 5g / L, anhydrous MgSO 4 0.3g / L, K 2 HPO 4 ·3H 2 O 1.0g / L, KH 2 PO 4 1.0g / L, natural pH value, solvent is water;

[0038] (3) Fermentation culture: Take the seed liquid and inoculate it in 100 mL ferment...

Example Embodiment

[0039] Example 2: Preparation of magnetically immobilized cells

[0040] (1) Weigh 12.675g FeCl 2 Dissolve in 1000mL deionized water and configure to 0.1M FeCl 2 Solution, weigh 16.220g FeCl 3 Dissolve in 1000mL deionized water and configure to 0.1M FeCl 3 Solution. Take FeCl 2 Solution 30mL, FeCl 3 Mix 60 mL of the solution into a 250 mL three-necked flask, add 20 mL of deionized water, and maintain a 30-40°C water bath under high-speed stirring at 1000 rpm. Add ammonia slowly to adjust the pH to 10, and the solution will turn black and shiny. The temperature was raised to 60°C and reacted for 1 hour. Then the temperature was raised to 80°C and matured for 1h. After the reaction is over, cool to room temperature, wash it repeatedly with deionized water until it is colorless and transparent, and the pH of the supernatant is neutral. Use an external magnetic field to collect and obtain black magnetic particle precipitation (Fe 3 O 4 ), pre-frozen in the refrigerator at -80℃ for 8...

Example Embodiment

[0046] Example 3: Influence of immobilization time on immobilization rate

[0047] (1) In five 50 mL small Erlenmeyer flasks, 3 g of dry yeast prepared by the method in Example 1 was fully dissolved in 20 mL of phosphate buffered saline solution (pH 9), and 0.01 g carrier Fe was added. 3 O 4 -Arg, mixed uniformly, the other five Erlenmeyer flasks were not added to the carrier as a control group, and they were fixed for 2 h, 3 h, 4 h, 5 h, 6 h in a shaker at 30°C and 150 r / min. After the reaction is over, the reaction solution is applied with an external magnetic field to recover the precipitate, and the ultraviolet absorption value of the upper layer diluted 10 times at a wavelength of 260 nm is taken as A 1 Value, the ultraviolet absorption value of the cell fluid diluted 10 times in the control group at the same time is taken as A 0 , The mean immobilization rate is shown in Table 2. Immobilization rate AD=(A 0 -A 1 ) / A 0 ×100%.

[0048] (2) In five 50 mL small conical flasks, 3 ...

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Abstract

The invention discloses a magnetic immobilized yeast cell and an application thereof to synthesis of ethyl R-4-chloro-3-hydroxybutyrate. The magnetic immobilized yeast cell is prepared through the steps of performing ultrasonic dispersion on Fe3O4 magnetic granules and an amino acid aqueous solution, performing recovery and precipitation with an external magnetic field, washing the precipitate, and then performing freeze-drying so as to obtain amino acid modified magnetic nanometer particles; and performing vibration and uniform mixing on the amino acid modified magnetic nanometer particles, yeast cell freeze-dried powder and a phosphoric acid buffer salt solution so as to obtain the magnetic immobilized yeast cell. According to the magnetic immobilized yeast cell disclosed by the invention, vibration and stirring are not needed to promote mass transfer, so that the reaction time can be significantly shortened, the COBE transformation efficiency can be greatly improved, and the magnetic immobilized cells can be repeatedly recycled. After the magnetic immobilized yeast cell is reused for 13 times in an alternating magnetic field, the conversion rate is still 99.8%, and compared withthe recycling number of times of the magnetic immobilized yeast cell in in a shaking table, the recycling number of times of the magnetic immobilized yeast cell in the alternating magnetic field is increased.

Description

[0001] (1) Technical field [0002] The present invention relates to the preparation of (R)-4-chloro-3-hydroxybutyric acid ethyl ester, in particular to the use of amino acid modified magnetic nanoparticles as a carrier to effectively immobilize yeast and use it for 4-chloro-3-carbonyl Preparation of ethyl (R)-4-chloro-3-hydroxybutyrate by asymmetric reduction of ethyl butyrate. [0003] (2) Background technology [0004] (R)-4-chloro-3-hydroxybutyrate (Ethyl R-4-chloro-3-hydroxybutyrate, R-CHBE), molecular formula C 6 h 11 ClO 3 , Molecular weight 166.6, Appearance is light yellow liquid, Density 1.19g / mL, Boiling point 94℃. R-CHBE is an important intermediate for the synthesis of a large number of chiral drugs. It can synthesize L-carnitine, macrolide A, and (R)-γ-amino-β-hydroxybutyric acid (GABOB), negative mycin ( negamicyn) and 2,5-cyclohexadiene synthetic fibers, asymmetric reduction of ethyl 4-chloro-3-oxobutyrate (COBE) can prepare R-CHBE. [0005] R-CHBE can be p...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N1/18C12P7/62C12R1/865
CPCC12N1/18C12N11/14C12P7/62
Inventor 欧志敏卢媛代洪倩唐岚杜理华徐敏
Owner ZHEJIANG UNIV OF TECH
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