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Recombinant bacterium obtained by in-situ expression of (R)-carbonyl reductase in candida parapsilosis and method using same to produce (R)-phenyl glycol efficiently

A phenylethylene glycol, Candida technology, applied in microorganism-based methods, oxidoreductases, botanical equipment and methods, etc., can solve the problem of unsatisfactory catalytic function of recombinant bacteria, low expression and enzyme activity, etc. question

Inactive Publication Date: 2015-08-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But due to ( R )-carbonyl reductase expression level and enzyme activity are lower, causing the catalytic function of recombinant bacteria unsatisfactory, biotransformation ( R )-phenylglycol with only 61% and 81% efficiency and optical purity

Method used

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  • Recombinant bacterium obtained by in-situ expression of (R)-carbonyl reductase in candida parapsilosis and method using same to produce (R)-phenyl glycol efficiently

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Candida parapsilosis ( C. parapsilosis ) CCTCC NO: M203011 (disclosed in the patent number: CN 03132140.2) cell culture, the composition of the growth medium YPD: glucose 2%, yeast extract 1%, peptone 2%. Candida parapsilosis was inoculated into 5 mL test tubes and cultured at 30 °C with shaking at 200 r / min for 24 h.

Embodiment 2

[0076] Candida parapsilosis ( C. parapsilosis ) CCTCC NO: M203011 Genome Extraction: Centrifuge the thalline cultured in Example 1 at 8,000 r / min for 10 min, wash twice with normal saline, and collect the cells. Genomic DNA Mini Preparation Kit (Takara Corporation) was used to extract the genomic DNA. ) to extract the genome.

Embodiment 3

[0078] Cloning the target gene from the genome of Candida parapsilosis rcr .

[0079] Synthetic primers at both ends:

[0080] RCR_1: 5'-ctacaactat tccgcgggag ctcatgtcaa ttccatca-3'( Sac I),

[0081] RCR_2: 5'-cactcggtac cctagtggtg gtggtggtgg tgtggattaa aa-3' ( Kpn I),

[0082] PCR reaction system: ddH 2 O 33.5 μL, DNA polymerase 0.5 μL, RCR_1 1 μL, RCR_2 1 μL, 5×buffer 10 μL, dNTP 4 μL;

[0083] PCR conditions: heat denaturation at 98°C for 10 s; 10 s at 98°C, 15 s at 52°C, and 1 min at 72°C. After 30 cycles, a final extension was performed at 72°C for 10 min. DNA fragments were purified using 3S Spin Agarose Gel DNA Purification Kit (Shanghai Shenergy Biotechnology Co., Ltd.). get rcr Gene, rcr The full length of the gene is 1011 bp.

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Abstract

The invention discloses a recombinant bacterium obtained by in-situ expression of (R)-carbonyl reductase in candida parapsilosis and a method using the same to produce (R)-phenyl glycol efficiently, and belongs to the technical field of bio-catalytic asymmetric conversion. The invention provides a strain of candida parapsilosis pCP-rcr, which has been preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC No. M2015106. The expression plasmid pCP of foreign protein in candida parapsilosis is established, then (R)-carbonyl reductase is cloned to the plasmid to obtain recombinant plasmid pCP-rcr, and finally the recombinant plasmid pCP-rcr is electrically converted into candida parapsilosis to obtain the recombinant strain candida parapsilosis pCP-rcr. The recombinant bacterium can be used to catalyze 2-hydroxy acetophenone, by optimizing the biological conversion reaction conditions, the optical purity of the product namely (R)-phenyl glycol can reach 99.9%, the yield can reach 99.6%, the biological conversion time is shortened from 48 hours to 13 hours, and an efficient and low-cost method is provided for preparing (R)-phenyl glycol.

Description

technical field [0001] Utilize the expression plasmid of the Candida parapsilosis designed, realize ( R )-expression of carbonyl reductase, efficient preparation of ( R )-phenyl glycol method, the present invention relates to the construction of genetic engineering means ( R )-carbonyl reductase in situ expression recombinant bacteria in Candida parapsilosis, and its high-efficiency preparation ( R )-phenyl glycol, belongs to the technical field of biocatalytic asymmetric conversion. Background technique [0002] Chiral alcohols are precursors for the synthesis of many high-value chiral compounds, and are widely used in the pharmaceutical industry, fine chemical industry and information industry. 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. R )-Phenylgly...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N9/04C12P7/22C12N15/81C12R1/72
Inventor 张荣珍徐岩
Owner JIANGNAN UNIV
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