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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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  • Summary
  • 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 magnetic field The chemical cells are easier to separate from the reaction solution, which is beneficial to the separation and extraction of products, solves the problem of low substrate treatment in the catalytic biotransformation process of free yeast cells, increases the number of utilization of yeast cells, and then improves the biotransformation efficiency

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
Effect test

Embodiment 1

[0035] Example 1: Preparation of Saccharomyces cerevisiae cells

[0036] (1) Slant culture: Saccharomyces cerevisiae CGMCC No.3361 was inoculated into the slant medium, and cultivated at 37°C for 24 hours to obtain a slant; the slant medium was prepared as follows: wort juice 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 20min, cool down after sterilization to make a slope;

[0037] (2) Seed culture: Take a ring of thalline from the thalline slope and transfer it to the seed culture medium, at 35°C, the shaking table speed is 150r / min, cultivate for 24h, and obtain the seed liquid; the described 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: get seed liquid, inoculate in...

Embodiment 2

[0039] Example 2: Preparation of magnetically immobilized cells

[0040] (1) Weigh 12.675g FeCl 2 Dissolved in 1000mL deionized water, configured as 0.1M FeCl 2 Solution, weigh 16.220g FeCl 3 Dissolved in 1000mL deionized water, configured as 0.1M FeCl 3 solution. FeCl 2 Solution 30mL, FeCl 3 Mix 60mL of the solution into a 250mL three-neck flask, add 20mL of deionized water, and stir at 1000rpm at a high speed, keep a water bath at 30-40°C, slowly add ammonia water dropwise to adjust the pH to 10, and the solution turns black and shiny. Raise the temperature to 60°C and react for 1h. Then raise the temperature to 80°C and ripen for 1h. After the reaction was finished, it was cooled to room temperature, and it was repeatedly washed with deionized water until it was colorless and transparent, and the pH of the supernatant was neutral, and collected by an external magnetic field to obtain a black magnetic particle precipitate (Fe 3 o 4 ), pre-frozen in -80°C refrigerato...

Embodiment 3

[0046] Embodiment 3: the influence of immobilization time on immobilization rate

[0047] (1) In five 50 mL small Erlenmeyer flasks, 3 g of the dry yeast prepared by the method in Example 1 were fully dissolved in 20 mL of phosphate buffered saline (pH 9), and 0.01 g of carrier Fe was added 3 o 4 -Arg, mixed evenly, no carrier was added to the other five Erlenmeyer flasks as a control group, fixed at 30°C for 2 h, 3 h, 4 h, 5 h, and 6 h in a shaker shaker at 150 r / min. After the reaction, the reaction solution was recovered by an external magnetic field, and the ultraviolet absorption value of the 10-fold diluted upper layer at a wavelength of 260 nm was used as A 1 value, the UV absorbance value of the control group diluted 10 times at the same time as A 0 , and the average immobilization rate is shown in Table 2. Immobilization rate AD = (A 0 -A 1 ) / A 0 ×100%.

[0048] (2) In five 50 mL small Erlenmeyer flasks, fully dissolve 3 g of the dry yeast prepared by the metho...

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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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