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Method for optimizing and improving biotransformation efficiency of (R)-carbonyl reductase by mRNA two-stage structure

A gene and substrate technology, applied in the field of mRNA secondary structure optimization to improve the biotransformation efficiency of (R)-carbonyl reductase, can solve the problems of low expression level and catalytic efficiency

Active Publication Date: 2010-06-09
JIANGNAN UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

The former catalyzes the reversible reaction between (R)-phenylethylene glycol and 2-hydroxyacetophenone; the latter uses the intermediate 2-hydroxyacetophenone as a substrate to produce (S)-phenylethylene glycol; Since the expression level and catalytic efficiency of (R)-carbonyl reductase are far lower than those of (S)-carbonyl reductase, Candida parapsilosis can only synthesize (S)-phenylethylene glycol

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  • Method for optimizing and improving biotransformation efficiency of (R)-carbonyl reductase by mRNA two-stage structure
  • Method for optimizing and improving biotransformation efficiency of (R)-carbonyl reductase by mRNA two-stage structure

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Embodiment 1

[0034] The optimization of embodiment 1 gene:

[0035] On the basis of reducing the stability or free energy (ΔG) of the secondary structure, the 13 nucleotides in the mRNA translation initiation region of the (R)-carbonyl reductase gene rcr, that is, the 9th, 16th, 17th, and 18th , 21, 52, 54, 57, 58, 60, 61, 63 and 66 positions t, a, g, c, c, c, t, g, t, g, a, a, t are mutated to A, T , C, A, A, T, A, A, C, A, C, T, C.

Embodiment 2

[0036] The acquisition of embodiment 2mrcr gene:

[0037] The recombinant plasmid pETRCR was used as a PCR reaction template, and mRCR_F containing a NcoI restriction site and mRCR_R containing a XhoI restriction site were used as primers:

[0038] mRCR_F: 5'-GACCATGGGATCAATACCATCATCATCACAATACGGATTCGTATTCAATAAGCAATCAGGATTAAAACTACGTAACGATTTG-3',

[0039] mRCR_R: 5'-TGACTCTCGAGTGGATTAAAAACAACTCTACCTTCATAAGCATTGTT-3',

[0040] PCR reaction system: ddH 2 O 37.5μL, 10×Reaction Buffer 5μL, 25mmol / L dNTP4μL, 50pmol / μL primers mRCR_F and mRCR_R 1μL each, recombinant plasmid pETRCR 1μL, 5U / μL Taq DNA polymerase 0.5μL; PCR reaction conditions: 95℃5min; 94℃ 1min, 57°C 1min, 72°C 1min, 30 cycles; 72°C 10min; PCR reaction to obtain the mrcr gene.

Embodiment 3

[0041] The acquisition of embodiment 3 recombinant plasmid pET32a-mrcr:

[0042] The gene mrcr and pET32a (the pET32a plasmid is from Novagen) were subjected to double digestion treatment with restriction endonucleases NcoI and XhoI respectively. After the treatment, the DNA fragments were ligated through cohesive ends to obtain the recombinant plasmid pET32a-mrcr with the gene mrcr. Plasmid pET32a-mrcr was extracted using the plasmid extraction kit Mini-PlasmidRapid Isolation Kit (purchased from Beijing Broadtech Biogene Technology Co., Ltd.).

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Abstract

The invention relates to a method for optimizing and improving biotransformation efficiency of (R)-carbonyl reductase by an mRNA two-stage structure, belonging to the technical field of biocatalysis asymmetric transformation. The invention optimizes an mRNA translation start area two-stage structure of (R)-carbonyl reductase and constructs a corresponding mutation recombination strain which is classified and named Escherichia coli BL21 / pET32a-mrcr with the CCTCC preservation No. M209289. The mutation strain not only realizes the efficient expression of the (R)-carbonyl reductase but also effectively improves the zymoprotein activity and biotransformation efficiency, and provides novel research idea and approach for efficiently preparing the (R)-phenylglycol in high substrate concentration.

Description

technical field [0001] mRNA secondary structure optimization is a method to improve the biotransformation efficiency of (R)-carbonyl reductase, by optimizing the secondary structure of the mRNA translation initiation region, overcoming the steric hindrance of protein translation initiation, and promoting the high-efficiency expression of the target protein and its space The method and application of correct folding of structure, effective improvement of enzyme protein activity and biocatalytic function belong to the technical field of biocatalytic asymmetric transformation. Background technique [0002] Chiral alcohols have special photoelectromagnetic properties and physiological activities, and are ideal chiral drug intermediates and liquid crystal material admixtures. Especially optically pure phenyl glycol is an important chiral module compound in medicine, pesticide and chemical materials. The preparation of optical chiral alcohols by using recombinant oxidoreductases ...

Claims

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Application Information

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