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Novel biological preparation method of optically pure 2-hydroxy butyric acid

A technology of hydroxybutyric acid and a new method, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, lyase, etc., can solve the problems of high production cost, low optical purity, and low product concentration

Inactive Publication Date: 2016-06-22
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems of low concentration of (R)-2-hydroxybutyric acid or (S)-2-hydroxybutyric acid product, low optical purity and high production cost in the reported preparation method, the present invention proposes to use threonine dehydratase, lactic acid One-pot production of (R)-2-hydroxybutyrate or (S)-2-hydroxybutyrate from threonine catalyzed by dehydrogenase and formate dehydrogenase

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  • Novel biological preparation method of optically pure 2-hydroxy butyric acid

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Effect test

Embodiment 1

[0028] The preparation of the cultivation of implementation example 1 genetically engineered bacterium and resting cell

[0029] Pick a single colony on the plate and inoculate it into 5ml of fermentation medium containing corresponding antibiotics, cultivate it for about 15 hours as a seed solution, inoculate it into 600ml of fermentation medium according to the inoculation amount of 1%, and cultivate it on a shaker at 37°C and 200rpm to OD 600 =0.6~0.8, add IPTG with a final concentration of 0.1mM to induce at 25°C for more than 10h, and collect the bacteria by centrifuging the culture medium at 8000rpm.

Embodiment 2

[0030]Implementation example 2 Threonine dehydratase, formate dehydrogenase, D-lactate dehydrogenase recombinant bacteria resting cells one-pot synthesis of (R)-2-hydroxybutyric acid

[0031] In a 250mL three-neck flask, add 89mL water, 8.93g threonine, 3.58g disodium hydrogen phosphate, 15.6g sodium formate, 1mL10mMPLP and 50mgNAD in sequence + , adjust the pH to 7.0, and then add 0.6g of resting cells of threonine dehydratase engineering bacteria (wet weight, 240U), 10mL of formate dehydrogenase enzyme solution (10U) and 1.6g of D-lactate dehydrogenase engineering bacteria of static For living cells (wet weight, 1493U), use the pH online control system to add 2M HCl solution to the reaction system, control the pH of the reaction system to maintain around pH 7.0, and control the reaction temperature at 25-30°C. After 24 hours of reaction, high performance liquid chromatography showed that the reaction was complete (Agilent1200seriesHPLC, Acclaim120C18column (4.6×250mm, 5.0μm)...

Embodiment 3

[0032] Implementation Example 3 Threonine Dehydratase, Formate Dehydrogenase, L-Lactate Dehydrogenase Recombinant Bacteria Static Cells Catalyze Threonine One-Pot Synthesis of (S)-2-Hydroxybutyric Acid

[0033] Replace the resting cells of D-lactate dehydrogenase engineering bacteria in Example 2 with resting cells of 1.6gL-lactate dehydrogenase engineering bacteria (wet weight, 133U), and react for 36 hours to obtain light yellow oil The product (S)-2-hydroxybutyric acid was 7.57g, the yield was 97%, and the ee value was greater than 99%. 1 HNMR (400MHz, CDCl 3 ):δ=4.26(dd,J=6.7,4.5Hz,1H),1.84-1.97(m,1H),1.76(dquin,J=14.3,7.2,7.2,7.2,7.2Hz,1H),1.02(t ,J=7.5Hz,3H). 13 CNMR (100MHz, CDCl 3 ):δ=179.59,71.31,27.27,8.95.MS(ESI):m / z:calcdforC4H7O3 - :103.0395[M-H] - ,found: 103.0405.

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Abstract

The invention relates to a novel production and synthesis method of (R)-2-hydroxy butyric acid and (S)-2-hydroxy butyric acid, and particularly provides a method for producing (R)-2-hydroxy butyric acid and (S)-2-hydroxy butyric acid by catalyzing threonine through threonine dehydrase, lactic dehydrogenase and formate dehydrogenase in a one-pot mode. The (R)-2-hydroxy butyric acid and the (S)-2-hydroxy butyric acid can be widely applied to medicine synthesis, biodegradable material synthesis and fine chemicals synthesis. The method has the remarkable advantages that the reaction condition is mild, pollution is avoided, and the technological path is simple.

Description

technical field [0001] The invention belongs to the fields of biocatalytic preparation of pharmaceutical intermediates and green chemistry, and relates to a method for preparing optically pure 2-hydroxybutyric acid from threonine in one pot. The invention mainly relates to a new biological preparation method of (R)-2-hydroxybutyric acid and (S)-2-hydroxybutyric acid. Background technique [0002] Chiral 2-hydroxybutyric acid can be widely used in the synthesis of pharmaceuticals, biodegradable materials and fine chemicals. For example, (R)-2-hydroxybutyric acid can be used in the synthesis of antiepileptic drugs levetiracetam and buvaracetam, and (S)-2-hydroxybutyric acid can be used in the synthesis of PPARα antagonist (R)-K-13675 Synthesis with PPARγ antagonist MK-0533. In addition, optically pure 2-hydroxybutyric acid also has great application prospects in the field of biodegradable materials. [0003] It is reported in the literature that the production of optically ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N15/70
CPCC12P7/42C12N9/0006C12N9/88C12Y101/01027C12Y101/01028C12Y101/05006
Inventor 姚培圆崔云凤冯进辉吴洽庆朱敦明马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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