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Engineering bacterium and application thereof in preparation of atorvastatin drug intermediate

An engineering bacteria and purpose technology, applied in the field of biopharmaceuticals, can solve the problem of low activity of the substrate halohydrin, and achieve the effects of simple operation, mild reaction conditions, and improved conversion rate and purity.

Active Publication Date: 2015-05-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the enzymatic activity of such enzymes against long-chain C5 and C6 substrate halohydrins is not high

Method used

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  • Engineering bacterium and application thereof in preparation of atorvastatin drug intermediate
  • Engineering bacterium and application thereof in preparation of atorvastatin drug intermediate
  • Engineering bacterium and application thereof in preparation of atorvastatin drug intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Construction of embodiment 1 plasmid pET28-HheA

[0049] The halohydrin dehalogenase gene is the hhea gene, which is a fully synthetic gene and connected to the pMD18-HHEA plasmid. (The gene is derived from Arthrobacter sp.AD2, and the Genebank accession number is AF397297)

[0050] The HheA gene was cloned with primers F_HheA / R_HheA to obtain a 735bp HheA gene (SEQ ID No.3). Nucleic acid electrophoresis to verify gene size, such as figure 1 .

[0051] The sequence of primer F_HheA is:

[0052] 5'-CGCGGATCCATGGTCATCGCACTGGTGACGCA (SEQ ID No. 1);

[0053] The sequence of primer R_HheA is:

[0054] 5'-CCCAAGCTTTTACGGCAGATAGCCGCCGGTAAA (SEQ ID No. 2).

[0055] BamHI and HindIII double-enzyme digested HheA gene, recovered the gene band after digestion, BamHI and HindIII double-enzyme digested pET-28a(+) plasmid, recovered the plasmid band after digestion, and digested HheA gene and enzyme The excised pET-28a(+) plasmid was ligated with ligase and transformed into the...

Embodiment 2

[0056] Construction and induced expression of embodiment 2 genetically engineered bacteria

[0057] The plasmid pET28-HheA constructed in Example 1 was used to transform the expression host Escherichia coli BL21(DE3). Use primers F_HheA / R_HheA to do colony PCR to verify the transformed recombinants. The verified genetically engineered bacteria is Eco HheA. Inoculate Eco HheA into 3-5mL liquid LB test tube medium containing kanamycin resistance, activate it on a shaking table at 37°C for 12 hours, and transfer the culture obtained after activation to 1% transfer amount containing In the kanamycin-resistant liquid LB shake flask medium, the fermentation medium was shaken and cultured at a constant temperature for 3 hours, and the culture conditions were 37° C. and 200 rpm. When the cell concentration grows to OD 600 When =0.8, add 0.5mM IPTG (final concentration), induce at 18°C ​​for 13h, centrifuge at 10,000g for 5min to collect the cells, wash once with pH=7.0 HEPES buffer...

Embodiment 3

[0058] Construction and induced expression of embodiment 3 genetically engineered bacteria

[0059] The plasmid pET28-HheA constructed in Example 1 was used to transform the expression host Escherichia coli BL21(DE3). Use primers F_HheA / R_HheA to do colony PCR to verify the transformed recombinants. The verified genetically engineered bacteria is Eco HheA. Inoculate Eco HheA into 3-5mL liquid LB test tube medium containing kanamycin resistance, activate it on a shaking table at 35°C for 12 hours, and transfer the culture obtained after activation to 1% transfer amount containing In the kanamycin-resistant liquid LB shake flask medium, the fermentation medium was shaken at constant temperature for 3 hours, and the culture conditions were 35° C. and 200 rpm. When the cell concentration grows to OD 600 When = 1.2, add 0.1mM IPTG (final concentration), induce at 22°C for 16h, centrifuge at 10,000g for 5min to collect the cells, wash once with pH=9.0 HEPES buffer, discard the su...

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Abstract

The invention discloses an engineering bacterium and an application thereof in preparation of an atorvastatin drug intermediate. The engineering bacterium comprises a host cell and a target gene transferred into the host cell, wherein the target gene is a halohydrin dehalogenase gene of which a base sequence is shown in SEQ ID NO.3. The engineering bacterium constructed by introducing the halohydrin dehalogenase gene shown in SEQ ID NO.3 into the host cell is capable of expressing halohydrin dehalogenase HheA; and catalysis of tert-butyl (S)-6-chloro-5-hydroxy-3-carbonyl hexanoate into tert-butyl (R)-6-cyano-5-hydroxy-3-carbonyl hexanoate and catalysis of tert-butyl (3R,5S)-6-chloro-3,5-dyhydroxy hexanoate into tert-butyl (3R,5R)-6-cyano-3,5-dyhydroxy hexanoate can be realized through the catalysis action of enzyme. The reaction conditions are mild; the operation is simple and convenient; and the conversion rate and the purity of a product are improved.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to an engineering bacterium and its application in the preparation of atorvastatin pharmaceutical intermediates. Background technique [0002] Statins are a class of competitive inhibitors of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, and are widely used clinically to lower blood lipids. HMG-CoA reductase catalyzes the reduction of HMG-CoA to 3-methyl-3,5-dihydroxyvaleric acid, which is the biosynthesis pathway of cholesterol. Statins can inhibit cholesterol in vivo by inhibiting the synthesis of HMG-CoA reductase It can reduce the level of low-density lipoprotein cholesterol (LDL-C), and has a good curative effect on hyperlipidemia and coronary heart disease mainly caused by elevated cholesterol. Structurally, statins usually consist of a hydrophobic rigid planar core and (3R,5S / R)-bishydroxyhexanoate with a double chiral center. Among them, Atorvastatin (Atorvastatin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/00C12R1/19
CPCC12N9/88C12P13/002
Inventor 杨立荣王金玉陈少云吴坚平徐刚
Owner ZHEJIANG UNIV
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