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Method for producing high-purity (R)-(-)-3-hydroxybutyric acid by adopting enzymatic method

A hydroxybutyric acid, high-purity technology, applied in the field of bioengineering, can solve the problems of simple process and inability to achieve efficient production of 3-hydroxybutyric acid monomer, and achieve high chemical purity, single optical activity, and high purity.

Inactive Publication Date: 2020-03-24
ZHEJIANG ENMAT BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to solve the technical problem that the prior art cannot realize efficient production of high-purity (R)-(-)-3-hydroxybutyric acid monomer, and the process is simple, to provide a kind of enzymatic production of high-purity (R) )-(-)-3-Hydroxybutanoic acid method

Method used

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  • Method for producing high-purity (R)-(-)-3-hydroxybutyric acid by adopting enzymatic method
  • Method for producing high-purity (R)-(-)-3-hydroxybutyric acid by adopting enzymatic method

Examples

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Effect test

Embodiment 1

[0035] Embodiment 1: Activation seed solution

[0036] The bacterial strain Diaphorobacterpolyhydroxybutyrativorans preserved in the lyophilized tube in Example 1 was inoculated in LB slant medium (yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, agar 20g / L), 35°C, static Cultured for 12h. Pick a single colony of EMD bacteria, inoculate it in LB liquid medium (yeast powder 5g / L, peptone 10g / L, NaCl 10g / L), culture at 35°C, 180rpm for 12h with shaking, and prepare a first-grade seed solution. According to the inoculation amount of 10%, inoculate the primary seed solution in the enzyme production medium (PHB 1g / L, KNO 3 2g / L, KH 2 PO 4 1g / L, Na 2 HPO 4 3g / L, (NH 4 ) 2 SO 4 1g / L, MgCl 2 ·6H 2 O 0.2g / L, NaCl 0.2g / L, yeast powder 0.15g / L, CaCl 2 0.036g / L, ferric ammonium citrate 0.01g / L), 35°C, 180rpm shaking culture for 12h, to prepare secondary seed liquid.

Embodiment 2-1

[0041] Example 2-1: Enzyme Production by Fermentation: Enzyme Production Activity of PHB as the Only Carbon Source and Inducer

[0042] The secondary seed liquid of Example 1 was cultured and fermented with a 250mL shake flask, the filling volume was 100mL, and the medium components were: PHB 1g / L, KNO 3 2g / L, KH 2 PO 4 1g / L, Na 2 HPO 4 3g / L, (NH 4 ) 2 SO 4 1g / L, MgCl 2 ·6H 2 O 0.01g / L, NaCl 0.2g / L, yeast powder 0.15g / L, CaCl 2 0.02g / L, ferric ammonium citrate 0.01g / L. The culture temperature is 35°C, the pH value is about 7.2, the rotation speed is 180 rpm, and the culture time is 18 hours. The fermentation time was 18 hours, the cells were removed by centrifugation, and the supernatant was taken to measure the enzyme activity. The total enzyme activity was 210U.

Embodiment 2-2 to 2-4

[0043] Embodiment 2-2 to 2-4: Enzyme production by fermentation

[0044] The secondary seed solution of Example 1 of 500 mL was inoculated in a 7 L fermenter, and the filling volume was 5 L. The components of the fermentation medium are listed in Table 1. The culture temperature is 35°C, the pH value is about 7.2, the dissolved oxygen constant is controlled at 20-25%, and the ventilation rate and stirring speed are flexibly adjusted according to the dissolved oxygen constant. Feed PHB was fed, and the concentration of PHB was controlled at 0.5g / L throughout the fermentation enzyme production process. The total fermentation time is 18h.

[0045] Table 1 embodiment 2-2 to 2-4 adopts the fermentation medium total enzyme activity of different content components

[0046]

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Abstract

The invention discloses a method for producing high-purity (R)-(-)-3-hydroxybutyric acid by adopting an enzymatic method. Compared with a chemical method and a whole-cell fermentation method, the method disclosed by the invention has the advantages of mild reaction conditions, less waste liquid generation, high chemical purity of (R)-(-)-3-hydroxybutyric acid, single optical activity and the like.As the components of a culture medium for producing (R)-(-)-3-hydroxybutyric acid by the enzyme method are much simpler than those of the culture medium produced by a whole-cell fermentation method,the product separation cost is greatly reduced, and the purity is higher. According to the method, Diaphorobacter polyhydroxybutyrativorans bacteria are adopted to generate the PHB depolymerizing enzyme in a specific culture environment, and meanwhile, the PHB is used as a carbon source and an inducer for enzymolysis, so that the catalytic efficiency of the enzyme is extremely high.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a method for producing high-purity (R)-(-)-3-hydroxybutyric acid by an enzymatic method, in particular to a method for depolymerizing poly-3-hydroxybutyrate produced by microbial fermentation Enzyme, and a method for producing high-purity (R)-(-)-3-hydroxybutyric acid monomer by using the depolymerase to degrade poly-3-hydroxybutyrate. Background technique [0002] (R)-(-)-3-Hydroxybutanoic acid ((R)-(-)-3-Hydroxybutanoic acid) is the R-configuration isomer in the racemate of 3-hydroxybutanoic acid, and is a An optically active chiral compound with a CAS number of 625-72-9. [0003] In the human body, (R)-(-)-3-hydroxybutyrate is usually synthesized in the liver, the raw material is acetyl-CoA, this reaction is by (R)-(-)-3-hydroxybutyrate dehydrogenase Catalyzed. (R)-(-)-3-Hydroxybutyric acid can be used as a backup energy source for the brain during hypoglycemia, and this compoun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N9/18C12R1/01
CPCC12N9/18C12P7/42C12Y301/01075
Inventor 刘倩倩高俊飞王刚朱世扬文鹏徐佳雄李瑞瑞何其昌赵欣园陈学军
Owner ZHEJIANG ENMAT BIOLOGICAL TECH CO LTD
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