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Method for preparing (R)-styrene glycol by employing asymmetric conversion of recombinant strain

A phenylethylene glycol, recombinant strain technology, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of not using auxiliary substrates and using a large amount of recombinant cells.

Active Publication Date: 2012-04-18
JIANGNAN UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] In the method for preparing (R)-phenylethylene glycol by biological methods, wild-type microbial cells or enzymes are usually used as catalysts, while the method for preparing (R)-phenylethylene glycol by catalytic asymmetric transformation using recombinant strains There are few reports, and Nie Yao et al [Chemical Progress, Volume 25, No. 10, 1231-1236, "Recombinant Escherichia coli asymmetric reduction of 2-hydroxyacetophenone to synthesize (R)-phenylethylene glycol"] once used Recombinant Escherichia coli was transformed to produce (R)-phenylethylene glycol, but the amount of recombinant cells was large, and no auxiliary substrate was used

Method used

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  • Method for preparing (R)-styrene glycol by employing asymmetric conversion of recombinant strain

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Experimental program
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Effect test

Embodiment 1

[0057] Induced expression culture: LB medium consists of tryptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0. If necessary, add ampicillin (50 μg / mL) before use, and add 1.5% agar powder to the solid medium. Pick a single colony of the positive clone and inoculate it in 3 mL of LB liquid medium containing 50 μg / mL ampicillin, and culture overnight at 37°C with shaking at 200 rpm. Transfer 1 mL of the culture solution to 50 mL of LB liquid medium containing 50 μg / mL ampicillin, and culture at 37°C with shaking at 200 rpm until OD 600 The inducer IPTG 0mmol / L was added to the culture at about 0.6, and the induction culture was carried out at a culture temperature of 30°C.

Embodiment 2

[0059] Induced expression culture: The composition of LB medium is the same as that in Example 1. Pick a single colony of the positive clone and inoculate it in 3 mL of LB liquid medium containing 50 μg / mL ampicillin, and culture overnight at 37°C with shaking at 200 rpm. Transfer 1 mL of the culture solution to 50 mL of LB liquid medium containing 50 μg / mL ampicillin, and culture at 37°C with shaking at 200 rpm until OD 600 About 0.6. The inducer IPTG 1mmol / L was added to the culture, and induction culture was carried out at a culture temperature of 37°C.

Embodiment 3

[0061] In 1mL 0.1mol / L Tris-HCl buffer (pH8.0), add 1g / L substrate 2-hydroxyacetophenone, 0.1g / mL recombinant Escherichia coli wet cells, and auxiliary substrate isopropanol 2.5%, after mixing, shake and react on a constant temperature shaker at 30°C for 48 hours. After the reaction, the mixture was centrifuged, and the supernatant was taken for extraction. The optical purity of the product (R)-phenylethylene glycol was 86.2% e.e., and the yield was 81.4%.

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Abstract

The invention relates to a method of using recombinant strain to asymmetrically transform and manufacture (R)-phenyl glycol, which belongs to the field of biological catalytic asymmetric transforming technique. The invention inserts the (R)-specific alcohol dehydrogenase gene rcr into the vector pET21c to construct a recombinant plasmid pET-RCR which is transformed to the E.coli to construct the recombinant plasmid E.coli BL21 / pET-RCR. The recombinant strain has the capability of asymmetrically catalyzing the 2-hydroxy phenyl ethyl ketone into (R)-phenyl ethylene glycol. Under optimized reaction condition, the 2.5-10 percent isopropanol is added to pH 8.0 Tris-HCL buffer as an auxiliary substrate, the 0.1-0.4g / ml recombinant cell solution is used for transforming the 0.5-5g / L 2-hydroxy phenyl ethyl ketone substrate for 48h, the product (R)-phenyl ethylene glycol with optical purity of 80-100 percent e.e. and productive rate of 70 to 100 percent is finally gained. The invention not only provides an effective way to manufacture (R)-phenyl ethylene glycol, but also makes sense for developing the biological catalyst for the chiral transformation.

Description

technical field [0001] The invention relates to a method for preparing (R)-phenylethylene glycol through asymmetric transformation of recombinant strains, which belongs to the technical field of biocatalytic asymmetric transformation. Background technique [0002] The chemical structure of phenyl glycol is: [0003] [0004] Optically pure (R)-phenylethylene glycol is not only an indispensable and important chiral additive in liquid crystal materials, but also has become an important intermediate for the preparation of optically active medicines, pesticides and functional materials. The study of the sub-method is very meaningful. [0005] Chiral compounds play an important role in people's lives. Since the two enantiomers are different in pharmacology, toxicology and functional effects, the preparation of optically pure chiral modular compounds is very important in medicine, agriculture, materials and Environmental protection and other fields are of great significance. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12P7/22C12R1/19C12R1/72
Inventor 聂尧徐岩
Owner JIANGNAN UNIV
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