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Method for preparing (S)-3-hydroxyl-3-(2-thienyl)-propionitrile by microbial transformation

A microbial transformation, thiophene-based technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of material transfer resistance, toxicity, and reduced reaction rate

Inactive Publication Date: 2011-09-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] During the asymmetric reduction reaction catalyzed by microorganisms, both the substrate 3-carbonyl-3-(2-thienyl)-propionitrile and the product (S)-3-hydroxyl-3-(2-thienyl)-propionitrile As a water-insoluble compound, the process of microbial cells converting 3-carbonyl-3-(2-thienyl)-propionitrile in the water phase has resistance to the transfer of substances at the phase interface, and at the same time, organic substrates will have certain resistance to microbial cells. slow reaction rate

Method used

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  • Method for preparing (S)-3-hydroxyl-3-(2-thienyl)-propionitrile by microbial transformation
  • Method for preparing (S)-3-hydroxyl-3-(2-thienyl)-propionitrile by microbial transformation
  • Method for preparing (S)-3-hydroxyl-3-(2-thienyl)-propionitrile by microbial transformation

Examples

Experimental program
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Effect test

Embodiment 1

[0046] 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, sterilized at 121°C for 20min to make slant spare;

[0047] Preparation of seed medium: glucose 30g / L, yeast powder 3g / L, ammonium sulfate 5g / L, anhydrous MgSO 4 0.25g / L, K 2 HPO 4 ·3H 2O 1g / L, KH 2 PO 4 1g / L, natural pH value, solvent is water, sterilized at 121°C for 20min, ready for use;

[0048] The preparation of the fermentation medium is the same as that of the seed medium.

[0049] Slant culture: Saccharomyces cerevisiae CGMCC No.2266 strain was inoculated into the slant medium, cultured at 30° C. for 4 to 6 days, and the slant of the bacteria was obtained.

[0050] Seed culture: 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 seed medium, and cultivate it at 30°C and 180 r / min for 24 hours to obtain a seed liqu...

Embodiment 2

[0057] Saccharomyces cerevisiae CGMCC No.2266 was cultivated according to the method in Example 1 to obtain a bacterial cell fermentation liquid. Mix four parts of 100mL fermentation broth with a cell concentration of 4.3g / L with an equal volume of 2% sodium alginate aqueous solution, and fill the sodium alginate mixture containing bacteria into needles with a size of 2mm, 3mm, and 4mm respectively. and 5mm syringe, drop 500mL 3.5% (w / v) CaCl 2 Form immobilized cell particles in aqueous solution, the diameters of the formed immobilized cell particles are 2mm, 3mm, 4mm and 5mm respectively, solidify at 37°C for 30min, and the obtained immobilized particles are washed with sterile normal saline to remove excess calcium ions and uncaptured cells, the obtained immobilized cell particles were continued to proliferate and culture in 500mL fermentation medium at 30°C for 24h to obtain proliferation cultured immobilized cell particles. The particle size after the proliferation cultur...

Embodiment 3

[0062] Saccharomyces cerevisiae CGMCC No.2266 was cultivated according to the method in Example 1 to obtain a bacterial cell fermentation broth. Four parts of 100mL of fermentation broth with a bacterial cell concentration of 4.3g / L were mixed with an equal volume of 2% sodium alginate aqueous solution, and the sodium alginate mixed solution containing the bacterial cell was respectively put into a syringe with a diameter of 2 mm, and dripped into 500mL 3.5% (w / v) CaCl 2 Form immobilized particles in an aqueous solution, the diameter of the formed immobilized particles is 2 mm, solidify at 37 ° C for 30 min, and the obtained immobilized particles are washed with sterile physiological saline to remove excess calcium ions and uncaptured cells, and the obtained 4 The immobilized cell granules were respectively cultured in 500mL fermentation medium at 30°C for 0h, 24h, 48h and 72h to obtain immobilized cell granules after proliferation.

[0063] Add the above 4 portions of immobi...

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Abstract

The invention discloses a method for preparing (S)-3-hydroxyl-3-(2-thienyl)-propionitrile by microbial transformation, which comprises the following steps of: forming a biological transformation system with 3-hydroxyl-3-(2-thienyl)-propionitrile as a substrate, with an immobilized cell granule prepared from an enzyme-containing bacteroid cell obtained by fermenting a saccharomyces cerevisiae strain with the preservation number of CGMCC (China General Microbiological Culture Collection) No.2266 as a biocatalyst, and with phthalandione as a solvent; carrying out conversion reaction at 20-40 DEG C for 8-72h; and separating and purifying a transformation liquid to obtain a product, i.e., the (S)-3-hydroxyl-3-(2-thienyl)-propionitrile. The (S)-3-hydroxyl-3-(2-thienyl)-propionitrile is prepared by adopting a microbial transformation method which has the advantages of mild reaction condition and environmental friendliness; microorganisms are easy to culture on a large scale and low in cost; the produced strains are safe and toxicity-free; the product is easy to extract; the transform amount of the substrate and the production efficiency are higher; and the method is easy to realize the large-scale industrial production.

Description

(1) Technical field [0001] The invention relates to a method for preparing (S)-3-hydroxyl-3-(2-thienyl)-propionitrile by microbial transformation, in particular to a method for regenerating enzyme-containing bacterial cells obtained by fermentation of Saccharomyces cerevisiae CGMCC No.2266 The prepared immobilized cell particles are used as biocatalysts, and 3-carbonyl-3-(2-thienyl)-propionitrile is used as a substrate to prepare (S)-3-hydroxyl-3-(2-thienyl)-propionitrile method. (2) Background technology [0002] (S)-3-hydroxy-3-(2-thienyl)-propanenitrile ((S)-3-hydroxy-3-(2-thienyl)propanenitrile), molecular formula C 7 h 7 NOS, molecular weight 153. (S)-3-Hydroxy-3-(2-thienyl)-propionitrile is one of the key chiral intermediates for the synthesis of antidepressant duloxetine. Duloxetine is a new type of antidepressant. It is a dual inhibitor of the reuptake of two important neurotransmitters serotonin and norepinephrine in the human brain. It can effectively treat the...

Claims

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Application Information

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IPC IPC(8): C12P17/00C12N11/10C12R1/865
CPCY02P20/50
Inventor 欧志敏沈文和李仁玮南颖康刘拥
Owner ZHEJIANG UNIV OF TECH
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