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Method for preparing (R)-styrene glycol by changing coenzyme specificity and stereoselectivity via site-directed mutagenesis

A technology of phenylethylene glycol and its construction method, which is applied in the field of biocatalytic asymmetric transformation, and can solve the problems of consumption, insufficient substrate concentration, and low optical purity of products, etc.

Active Publication Date: 2009-04-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Biological conversion to prepare optically pure (R)-phenylethylene glycol, usually using microbial whole cells or enzymes as catalysts, mainly including Bakers' yeast asymmetric reduction of 2-hydroxyacetophenone method, using epoxide hydrolase to catalyze Hydrolysis of racemic styrene oxide method, or the use of naphthalene dioxygenase (NDO) selective oxidation of styrene method, etc., the above methods have low optical purity of the product, the concentration of the substrate is not high enough, or the reaction process needs to consume expensive NADPH as a coenzyme to optimize reaction conditions and other shortcomings

Method used

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  • Method for preparing (R)-styrene glycol by changing coenzyme specificity and stereoselectivity via site-directed mutagenesis

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Effect test

Embodiment 1

[0091] Cultivation of the recombinant strain E.coli / pETCPADH: The recombinant strain E.coli / pETCPADH has been published in [Applied and Environmental Microbiology 200773(11):3759-3764]. LB medium was used, and its composition was: tryptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0. Ampicillin (100 μg / mL) was added during cultivation. 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 100 μg / mL ampicillin, and cultivate overnight at 37°C with shaking at 200 rpm. On the next day, take 1 mL of the culture solution and transfer it to 50 mL of LB liquid medium containing 100 μg / mL ampicillin, culture at 37°C, 200 rpm for about 12 hours, until the OD 600 is 0.6.

Embodiment 2

[0093] Obtaining the pETCPADH plasmid from the recombinant strain E.coli / pETCPADH: centrifuge the cell culture solution at 5,000rpm for 10min, collect the cell, and extract the plasmid using the plasmid extraction kit Mini-Plasmid Rapid Isolation Kit (Beijing Broadtech Biogene Technology Co., Ltd.) pETCPADH.

Embodiment 3

[0095] Use the recombinant plasmid pETCPADH containing (S)-specific carbonyl reductase as the PCR amplification reaction template, use primer 1 containing the BamHI restriction enzyme site, primer 2 containing the XhoI restriction enzyme site, and the middle two Segment Primer 3 and Primer 4,

[0096] Primer 1: 5'-ATC GGATCC GATGGGCGAAATCGAATCTTATTG-3',

[0097] Primer 2: 5'-TGACTCTCGAGTGGACACGTGTATCCACCGTC-3',

[0098] Primer 3: 5'-CCATTTGGTACAACGATGATCCAGC-3',

[0099] Primer 4: 5'-CTCATCAGCTGGATCATCGTTGTAC-3',

[0100] The scr6768 gene was amplified by SOE-PCR reaction, and its full length was 837bp;

[0101] Acquisition of upstream cDNA fragments and downstream cDNA fragments: use the recombinant plasmid pETCPADH as a template, and perform PCR reactions with primers 1 and 4, and primers 2 and 3, respectively. PCR reaction system: ddH 2 O 37μL, 10×Reaction Buffer 5μL, 25mmol / L dNTP 0.5μL, 50pmol / μL primers 1 and 4, or primers 2 and 3 each 1μL, recombinant plasmid pETCPA...

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Abstract

The invention discloses a (R)-styrene glycol preparation method that utilizes site-directed mutagenesis to alter coenzyme specificity and stereoselectivity, belongs to the technical field of biocatalytic asymmetric transformation, and provides a recombinant Escherichia coli strain BL21 / pETSCR6768 with the preservation CCTCC NO of M208079, and an asymmetric transforming preparation method of (R)-styrene glycol. The preparation method leads Ser in position 67 and His in position 68 of a (S)-specific carbonyl reductase to mutate to Asp, constructs recombinant plasmid pETSCR6768, and feeds the Escherichia coli, thus obtaining the recombinant strain E.coli BL21 / pETSCR6768 which leads the coenzyme specificity of (S)-specific carbonyl reductase to change from NADPH to NADH; furthermore, the stereoselectivity of the product is altered from (S)-styrene glycol to (R)-styrene glycol, and the preparation method provides an effective way for preparing (R)-styrene glycol in high efficiency and low cost and has significant meaning for recognizing the stereoselectivity of functional zymoprotein in the molecule and protein level.

Description

technical field [0001] A method for preparing (R)-phenylethylene glycol by changing coenzyme specificity and stereoselectivity by site-directed mutagenesis. The present invention relates to the use of protein engineering technology to transform amino acids at key sites in the enzyme protein structure, and to construct recombinant strains by means of genetic engineering. The method for efficiently preparing (R)-phenylethylene glycol and its application belong to the technical field of biocatalytic asymmetric conversion. Background technique [0002] The important role of chiral compounds in medicine, agriculture, industry and life has attracted more and more attention. Since the two enantiomers are different in pharmacology, toxicology and functional effects, the preparation of optically pure The chiral modular compounds are of great significance. [0003] The chemical structure of phenyl glycol is: [0004] [0005] Optically pure (R)-phenylethylene glycol is not only a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/22C12N9/02C12N15/53C12N15/70C12R1/19
Inventor 张荣珍徐岩
Owner JIANGNAN UNIV
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