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Method for producing D-alpha-hydroxybutyric acid

A technology of hydroxybutyric acid and hydroxyacid dehydrogenase, which is applied in the field of producing D-α-hydroxybutyric acid and α-ketobutyric acid, can solve the problems of low enantiomeric excess rate and achieve simple separation and low cost Inexpensive, easy-to-get results

Active Publication Date: 2013-04-24
上海肆芃科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] Aiming at the low problem of product D-alpha-hydroxybutyric acid output and enantiomeric excess rate value in the reported preparation method, the problem to be solved in the present invention is to utilize the complete cell of microorganism containing NAD-independent hydroxyacid dehydrogenase or crude Separation of racemic α-hydroxybutyrate by enzyme solution to produce D-α-hydroxybutyrate

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  • Method for producing D-alpha-hydroxybutyric acid
  • Method for producing D-alpha-hydroxybutyric acid
  • Method for producing D-alpha-hydroxybutyric acid

Examples

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Embodiment 1

[0055] (1) Preparation of complete microbial cells containing NAD-independent hydroxyacid dehydrogenase: Pseudomonas putida ATCC12633 was selected for routine culture, and NAD-independent hydroxyacid dehydrogenase was detected by high-performance liquid chromatography (HPLC). Hydrogenase activity, until its activity reaches 140 units / liter, stop the fermentation culture; separate and collect the thalline, wash the thalline twice with pH 7.4 potassium phosphate buffer, suspend the thalline resuspension in deionized water, make the thalline concentration reach 200 grams of wet cells / liter to obtain a complete cell suspension containing NAD-independent hydroxyacid dehydrogenase, stored at 4°C for future use;

[0056] (2) Heat denaturation inactivates NAD-independent D-hydroxyacid dehydrogenase: take the complete cell suspension containing NAD-independent hydroxyacid dehydrogenase prepared in step (1) and place it in a 60°C water bath for treatment 10 minutes, store at 4°C for lat...

Embodiment 2

[0064] (1) Preparation of complete microbial cells containing NAD-independent hydroxyacid dehydrogenase: Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC 10145 was selected and cultured routinely. Dependent on the activity of hydroxyacid dehydrogenase, when the activity reaches 160 units / liter, stop the fermentation culture; separate and collect the bacteria, wash the bacteria twice with pH 7.4 potassium phosphate buffer, and resuspend the bacteria in deionized water. Make the bacterial cell concentration reach 200 g wet cells / liter, obtain the complete cell suspension containing NAD-independent hydroxyacid dehydrogenase, store at 4°C for later use;

[0065] (2) Heat denaturation to inactivate NAD-independent D-hydroxyacid dehydrogenase: take the complete cell suspension containing NAD-independent hydroxyacid dehydrogenase prepared in step (1) and place it in a 55°C water bath for treatment 15 minutes, store at 4°C for later use;

[0066] (3) Resolution of racemic α-hydro...

Embodiment 3

[0073] (1) Preparation of crude enzyme solution containing NAD-independent L-hydroxyacid dehydrogenase: Pseudomonas stutzeri (Pseudomonas stutzeri) ATCC 17588 was selected and cultured routinely, during which the cells were detected by high performance liquid chromatography (HPLC) NAD-independent L-hydroxyacid dehydrogenase activity, when the activity reaches 170 units / liter, stop the fermentation culture; separate and collect the bacteria, wash the bacteria twice with pH 7.4 potassium phosphate buffer, and resuspend the bacteria in In deionized water, the concentration of bacteria reached 200 g wet cells / liter to obtain a complete cell suspension containing NAD-independent L-hydroxyacid dehydrogenase, and the complete cell suspension was ultrasonically crushed to obtain a crude enzyme solution, 4° C is stored for later use;

[0074] (2) Heat denaturation to inactivate NAD-independent D-hydroxyacid dehydrogenase: take the crude enzyme solution containing NAD-independent hydrox...

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Abstract

The invention discloses a method for producing D-alpha-hydroxybutyric acid, which comprises the following steps: (1) preparing complete cell suspension containing NAD independent hydroxy acid dehydrogenase or coarse enzyme liquid thereof with pseudomonas; (2) carrying out thermotropy on the complete cell suspension or the coarse enzyme liquid thereof prepared by the thermal denaturation step (1) to deactivate the NAD independent D-hydroxy acid dehydrogenase so as to obtain a biocatalyst only containing the NAD independent L-hydroxy acid dehydrogenase activity; (3) separating the racemic alpha-hydroxybutyric acid by the biocatalyst prepared in the step (2) to obtain the conversion fluid containing D-alpha-hydroxybutyric acid sodium salt and alpha-ketobutyric acid sodium salt; (4) pre-treating the conversion fluid; (5) acidifying the pre-treated conversion fluid; (6) separating the D-alpha-hydroxybutyric acid form the alpha ketobutyric acid; and (7) refining the D-alpha-hydroxybutyric acid. The method of the invention has the characteristics of simple components of culture medium, short growth period, low cost, easy subsequent separation and extraction, high enantiomeric excess of the product D-alpha-hydroxybutyric acid and the like and lays a foundation for efficient production of the D-alpha-hydroxybutyric acid.

Description

technical field [0001] The present invention relates to a method for producing D-α-hydroxybutyric acid and α-ketobutyric acid, in particular to the use of NAD (nicotinamide adenine dinucleotide)-independent L-hydroxyacid dehydrogenase A method for splitting racemic α-hydroxybutyric acid into D-α-hydroxybutyric acid and α-ketobutyric acid with complete microbial cells or crude enzyme solution. Background technique [0002] α-Hydroxybutyric acid is an intermediate in the synthesis of isoleucine and some drugs. High-purity chiral α-hydroxybutyric acid monomer can be used to synthesize high polymer poly α-hydroxybutyric acid [P(2HB)]. This polymer is biodegradable and can be used in biomedical, pharmaceutical and environmental aspects 【1】 . In addition, D-α-hydroxybutyric acid can be used to prepare azinothricin family anticancer antibiotics 【2】【3】 . [0003] There are two kinds of production of high-purity D-α-hydroxybutyric acid reported in the literature, one is enzymati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P41/00C12P7/42C12P7/40C12R1/38C12R1/385C12R1/40
Inventor 马翠卿高超张文许平
Owner 上海肆芃科技有限公司
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