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Method for preparing optically pure 2-hydroxy-4-phenyl butyric acid with lactonase

A technology of phenylbutyric acid and dihydroxybutyric acid, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of low efficiency, achieve mild reaction conditions, wide substrate adaptability, good The effect of catalytic effect

Inactive Publication Date: 2012-01-11
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The asymmetric reduction of 2-carbonyl-4-phenylbutyric acid (and its ethyl ester) by the whole cell of microorganism or the reductase produced by microorganism or the pair of 2-hydroxy-4-phenylbutyric acid racemic ester catalyzed by hydrolase Enantioselective hydrolytic resolution is also a known method (Adv.Syn.Catal.2008, 350, 426), but generally requires the use of large amounts of microbial cells or enzymes, and they are not efficient

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The preparation of embodiment 1 biocatalyst

[0020] Prepare LB medium with the following components: yeast extract 5g / L, tryptone 10g / L, NaCl 10g / L. Separately prepare MLB medium, the composition is as follows: glucose 3g / L, tryptone 10g / L, NaCl 5g / L, K 2 HPO 4 5g / L, pH 7.0. Inoculate recombinant Escherichia coli JM109(DE3) or BL21(DE3) into 3ml LB medium, use kanamycin as antibiotic, carry out enrichment culture at 37°C, 160rpm / min for 12h; after enrichment culture, use as seeds Inoculate 50ml of LB medium with kanamycin as an antibiotic and carry out enrichment culture at 37°C and 160rpm / min for 4 hours; after enrichment culture, inoculate it into 3L MLB medium as seed liquid, and inoculate at 37°C , 160rpm / min under the fermentation growth of 8 ~ 12h, keep the pH 7.0 unchanged. Lower the temperature of the fermentation broth to 15°C, and add IPTG to induce expression for 12-24 hours. Finally, the cells were collected by centrifugation and washed three times wi...

Embodiment 2

[0022] Example 2 Catalytic Preparation of (2S)-4-Phenyl-2-Hydroxy-4-Butyrolactone Using Resting Cells

[0023] 1 g of recombinant Escherichia coli resting cells obtained by centrifugation was suspended in 100 ml of tap water, and acetonitrile (20%, v / v) and substrate 4-phenyl-2-hydroxyl-4-butyrolactone were added to make the final concentration of the substrate The pH of the reaction solution was 100 mM, and the pH of the reaction solution was controlled to 6.4 by an automatic potentiometric titrator. After stirring and reacting at 30° C. for 1 hour, the reaction solution was centrifuged at 12,000×g for 10 minutes to remove cells. NaCl was added to the supernatant until saturated, extracted with 50 ml of ethyl acetate, and repeated three times. The cells obtained by centrifugation were soaked with 20 ml of ethyl acetate, repeated twice, the two parts of ethyl acetate were combined, and then washed twice with saturated NaCl solution, 50 ml each time. The obtained ethyl acetate...

Embodiment 3

[0024] Example 3 Preparation of (2R)-4-phenyl-2-hydroxyl-4-butyrolactone by resting cells

[0025] 1 g of recombinant Escherichia coli resting cells obtained by centrifugation was suspended in 100 ml tap water, and acetonitrile (20%, v / v) and substrate 4-phenyl-2-hydroxyl-4-butyrolactone were added to make the final concentration 100mM, the pH of the solution was controlled to 6.4 with an automatic potentiometric titrator, and the reaction was shaken on a constant temperature shaker at 30°C and 160rpm until the conversion rate reached 30%, and the reaction solution was centrifuged at 12,000×g for 10min to remove cells. NaCl was added to the supernatant to saturation, extracted with 50 ml of ethyl acetate, and repeated eight times to completely remove the unwanted 2S-configuration lactone. The obtained aqueous phase was adjusted to pH 1.0-2.0 with hydrochloric acid, heated at 60°C for 1 h, and then extracted with ethyl acetate, repeated three times. The obtained extract was dri...

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Abstract

The invention discloses a novel method for preparing optically pure 2-hydroxy-4-phenyl butyric acid by chemical-enzymatic synthesis process. Recombined escherichia coli cells for expressing fusarium proliferatum lactonase genes are fermented in an optimum condition to produce enzyme, then the diastereoisomers of 4-phenyl-2-hydroxy-4-butyrolactone are selectively hydrolyzed in the same way under the catalysis, and the hydrolysate of the needed conformation is hydrogenized into the target compound under the catalysis of palladium / carbon (Pd / C) at normal temperature and pressure. The (R)-2-hydroxy-4-phenyl butyric acid has the enantiomeric excess value of 98 percent and the molar conversion ratio of 36 percent, and the (S)-2-hydroxy-4-phenyl butyric acid has the enantiomeric excess value of 99 percent and the molar conversion ratio of 46 percent. The biological catalyst has better catalytic effect and lower cost, the chemical-enzymatic synthesis process is easy and safe to implement, and the product of reaction is easy to purify and is environmental friendly and has industrial development and application prospect.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to a method for producing optically pure 2-hydroxy-4-phenylbutyric acid with lactone hydrolase. technical background [0002] Small molecule hydroxy acids, such as lactic acid, malic acid, tartaric acid, citric acid, and gluconic acid, contain two functional groups, hydroxyl and carboxyl, in their molecular structure, and are very useful intermediates in chemical synthesis. Important organic acids have special physiological functions in organisms, and are very useful functional additives in the food / feed industry. Some unnatural hydroxy acids, especially optically active chiral hydroxy acids, can also be used as "structural building blocks" for the chemical synthesis of many natural products and drugs. [0003] 2-Hydroxy-4-phenylbutyric acid, as an important member of the α-hydroxy acid family, is a key chiral intermediate for the synthesis of many "Pulley" angiotensin-converting ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/42C12R1/19
Inventor 许建和陈兵
Owner EAST CHINA UNIV OF SCI & TECH
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