Method for synthesizing (R)-1,3-butanediol with whole cells of microorganism

A technology of butanediol and whole cells, applied in the field of asymmetric transformation of biocatalysis, can solve the problems such as the limited scale of the catalytic system of biochemical transformation efficiency, the inability to meet the requirements of industrialization, and the optical purity of the product not reaching 99%.

Inactive Publication Date: 2020-05-05
JIANGNAN UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

There have been some reports on the synthesis of (R)-1,3-BDO by biotransformation methods. For example, Atsuyama et al. used K.lactis IFO 1267 and C.utilis IAM 4277 to achieve the substrate 4- Catalytic conversion of hydroxy-2-butanone (4H2B), the optical purity of the product reaches 93% and 94%, but the optical purity of the product does not reach 99%, and it needs complicated separation and purification before it can be used as a raw material for pharmaceutical intermediates; Zheng and Rao et al. used C.krusei ZJB-09162 and P.jadinii HBY61 to catalyze the conversion of 4H2B, the optical purity of the product reached 99%, but the substrate concentration could only reach 45g/L, and the yield was 83.9% and 85.1%; Yamamoto coli cells expr

Method used

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  • Method for synthesizing (R)-1,3-butanediol with whole cells of microorganism
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  • Method for synthesizing (R)-1,3-butanediol with whole cells of microorganism

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Effect test

Embodiment 1

[0032] The screening and identification of embodiment 1 highly efficient catalytic bacterial strain

[0033] In the strain screening process, 4H2B was used as the substrate and glucose was used as the auxiliary substrate to isolate and screen more than 200 strains of bacteria from the soil. After the screened strains were cultured in a 48-well plate for 48 hours, the bacteria were collected by centrifugation and placed in a 48-well plate. Add substrate and auxiliary substrate, react for 48 hours, detect and analyze the product after reaction, and use high product optical purity and yield as selection criteria to obtain a strain with good catalytic efficiency and high specificity for the substrate 4H2B, Finally, genetic identification was carried out. After identification, the strain was Pichia kudriazvii, and it was sent to the preservation institution for preservation with the preservation number CCTCC M 2019329.

Embodiment 2

[0034] Example 2 Preparation of whole cells of Pichia kudriavzevii CCTCC M 2019329

[0035] Pick a single colony of Pichia kudriavzevii CCTCC M 2019329 strain and inoculate it into a test tube containing 5mL of YPD liquid medium, cultivate it at 30°C and 200rpm for 16h, and transfer it to a 500mL Erlenmeyer shaker flask (containing 100mL YPD liquid medium), 30°C, 200rpm shaking culture for 48h, the fermentation broth was collected, centrifuged at 6000rpm for 10min, and washed with physiological saline for 3 times to collect the bacteria, which was used as a catalyst for the asymmetric transformation reaction.

Embodiment 3

[0036] Example 3 Effects of different pH on the asymmetric reduction of 4H2B catalyzed by Pichia kudriavzevii CCTCC M 2019329

[0037] The catalytic reaction system is 10mL, the concentration of the substrate 4H2B is 20g / L, the final concentration of the catalyst is 1g / 10mL, the temperature is 30°C, the rotation speed is 200rpm, the auxiliary substrate is glucose, and the reaction is 12h.

[0038]Under different pH conditions, the catalytic activities of enzymes show great differences. By comparing the best buffers of different buffer solutions, the optimal pH (2.0-10.0) range is divided into 4 gradients: 0.1mol / L citrate buffer (pH 2.0-6.0), 0.1mol / L phosphate buffer (pH 6.0~8.0), 0.1mol / L Tris-HCl buffer solution (pH 8.0~9.0) and 0.1mol / L carbonate buffer solution (pH 9.0~10.0).

[0039] like figure 1 Shown, when the pH of reaction is lower than 5, the productive rate of product is lower than 40%; When the pH of reaction is greater than 8, along with the increase of pH, pr...

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Abstract

The invention discloses a method for synthesizing (R)-1,3-butanediol with whole cells of a microorganism, and belongs to the technical field of bio-catalytic asymmetric conversion. According to the method, a microorganism, namely Pichia kudriavzevii CCTCC M 2019329, capable of realizing highly selective whole-cell synthesis of (R)-1,3-butanediol is obtained by screening, whole cells of the microorganism are used as a catalyst, 4-hydroxyl-2-butanone is used as a substrate, the reaction pH, temperature, revolution speed, and auxiliary substrate obtained after single-factor optimization are used,the yield of (R)-1,3-butanediol is up to 80% or more, the optical purity is up to 99% or more, and the product concentration is up to 100 g/L or more.

Description

technical field [0001] The invention relates to a method for synthesizing (R)-1,3-butanediol by whole cells of microorganisms, and belongs to the technical field of biocatalytic asymmetric transformation. Background technique [0002] The chemical structure of (R)-1,3-butanediol ((R)-1,3-BDO) is: [0003] [0004] (R)-1,3-BDO is an unnatural active alcohol and an important pharmaceutical intermediate. In industrial production, it is usually used as an intermediate in the synthesis of pheromones and pesticides, especially in the preparation of azetidinone derivatives. Azetidinone derivatives are the starting materials for the synthesis of penem and carbapenem antibiotics, and penem antibiotics are important β-lactam antibiotics, which have strong bactericidal activity, low toxicity, It has the advantages of wide clinical indications and excellent efficacy. As the demand for penem and carbapenem antibiotics expands in the global market, the demand for (R)-1,3-BDO also in...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12P7/18C12R1/84
CPCC12N1/16C12P7/18C12N1/165C12R2001/84
Inventor 聂尧徐岩祖晗
Owner JIANGNAN UNIV
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