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A kind of short-chain dehydrogenase and its application

A short-chain dehydrogenase and reaction technology, applied in the field of bioengineering, can solve the problem of low yield of ring-opening reaction

Active Publication Date: 2022-07-15
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the ring-opening reaction yield of oxidation is very low (20%-30%) (patent WO9920607A1, CN103896872A, US6346532B1)

Method used

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  • A kind of short-chain dehydrogenase and its application
  • A kind of short-chain dehydrogenase and its application
  • A kind of short-chain dehydrogenase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Cloning of short-chain dehydrogenase gene

[0061] According to the sequence of the Bacillus subtilis (Bacillus subtilis) short-chain dehydrogenase gene (NCBI accession number: WP_013058339) recorded by NCBI, the PCR primers were designed as follows:

[0062] BsSDR10 f:5ˊ-CGGGATCCGATGAAGTACACAGTTATTACAGGA-3ˊ;

[0063] BsSDR10r:5ˊ-CCGCTCGAGCATCTCTAGAATCGGATAGATTTGA-3ˊ.

[0064] The underlined part of the upstream primer is the BamHI restriction site, and the underlined part of the downstream primer is the XhoI restriction site.

[0065] PCR amplification was performed using the genomic DNA of Bacillus subtilis CGMCC 1.3358 as a template. The PCR system is as follows:

[0066]

[0067] Amplification program: 94°C for 10 min, 35 cycles of (94°C for 30s, 50°C for 30s, 72°C for 60s), 72°C for 10 min, and cooling to 4°C.

[0068] The PCR product was purified by agarose gel electrophoresis, and the target band of about 750bp was recovered using a DNA recovery ...

Embodiment 2

[0069] Example 2 Construction of recombinant expression vector plasmid and preparation of recombinant expression transformant

[0070] The DNA fragment of the short-chain dehydrogenase gene in Example 1 was double-digested with restriction enzymes BamHI and XhoI at 37°C for 12 h, the product was purified by agarose gel electrophoresis, and the target fragment was recovered using a DNA recovery kit. Under the action of T4 DNA ligase, the target fragment was ligated with the plasmid pET-28b-MBP which was also double digested with restriction enzymes BamHI and XhoI at 16°C overnight to obtain the recombinant expression transformant pET-28b -MBP-BsSDR10. Plasmid construction map such as Figure 5 shown.

[0071] The above recombinant expression vector plasmid pET-28b-MBP-BsSDR10 was transformed into Escherichia coli (E.coli) BL21 competent cells. Screen the positive recombinants on the plate, pick a single colony, and verify the positive clones by PCR. figure 2 shown.

Embodiment 3

[0072] Example 3 Expression of short-chain dehydrogenase BsSDR10

[0073] The recombinant Escherichia coli obtained in Example 2 was inoculated into the LB medium (PH 7.0) containing kanamycin, and incubated overnight at 37°C with shaking. In a 250ml Erlenmeyer flask of culture medium, place it in a shaker at 37°C and 180rpm for shaking culture. When the OD of the culture medium is 600 When reaching 0.6, IPTG with a final concentration of 0.25 mM was added as an inducer, induced at 20 °C for 12 h, the culture medium was centrifuged, cells were resuspended with 3 ml of phosphate buffer solution (pH 6.0), and transferred to EP tubes. The supernatant was collected by centrifugation at low temperature (4°C), which was the crude enzyme solution of recombinant short-chain dehydrogenase. The crude enzyme solution and the precipitate were analyzed by polyacrylamide gel electrophoresis, such as image 3 shown.

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Abstract

The invention belongs to the field of biotechnology, and discloses a short-chain dehydrogenase gene, a short-chain dehydrogenase BsSDR10, and an engineered bacteria excavated from Bacillus subtilis (Bacillus subtilis). Carbonyl compounds to prepare optically active chiral alcohols. The enzyme has the advantages of stereospecificity, mild reaction conditions and simple operation, and has a good application prospect in the production of chiral pharmaceutical intermediates.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a short-chain dehydrogenase and its gene, a recombinant expression vector and a recombinant expression transformant containing the gene, a preparation method of the recombinant enzyme, and the short-chain dehydrogenase as a catalyst Application in asymmetric reduction of prochiral carbonyl compounds to prepare optically active chiral alcohols. Background technique [0002] Chiral alcohols are important intermediates for many popular pharmaceuticals and chiral chemicals. For example (R)-2-chloro-1-phenethyl alcohol (molecular formula C 8 H 9 ClO, molecular weight 156.61, CAS number: 56751-12-3) is an important intermediate in the synthesis of β3-adrenergic receptor agonist mirabegron. Mirabegron acts on the β3 adrenergic receptors of the detrusor smooth muscle of the bladder to relax the bladder, promote bladder filling and increase urine storage, prolong the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/22C12P7/62
CPCC12N9/0006C12N15/70C12P7/22C12P7/62C12Y101/01184
Inventor 游松秦斌刘亚林秦凤玉郭继阳张文鹤祝天慧张飞霆
Owner SHENYANG PHARMA UNIVERSITY
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