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Carbonyl reductase gene, codase, vector, engineering bacterium and application thereof

A technology of genetically engineered bacteria and reductase, applied in genetic engineering, oxidoreductase, application, etc.

Active Publication Date: 2015-05-20
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, the use of carbonyl reductases from Burkholderia gladioli strains in the asymmetric reduction of these pharmaceutical chiral intermediates has not been seen

Method used

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  • Carbonyl reductase gene, codase, vector, engineering bacterium and application thereof
  • Carbonyl reductase gene, codase, vector, engineering bacterium and application thereof
  • Carbonyl reductase gene, codase, vector, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Amplification of the carbonyl reductase gene adh5

[0041] According to the whole genome sequencing information of Burkholderia gladioli (Burkholderia gladioli) ZJB-12126, a large number of carbonyl reductases were excavated, one of which has the ability to catalyze 2-benzamidomethyl-3-ketobutyrate, (R)-tert-butyl 6-cyano-5-hydroxy-3-carbonylhexanoate and ethyl 4,4,4-trifluoroacetoacetate generate (2S,3R)-2-benzamidomethyl- 3-Hydroxybutyrate, tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, and (S)-4,4,4-trifluoro-3-hydroxybutyrate-functional enzymes are The carbonyl reductase BgADH5 involved in the present invention.

[0042] Using FastDNA from MPBio The Spin kit was used to extract the total genomic DNA of Burkholderia gladioli ZJB12126 cells. Using the genomic DNA as a template, primer 1 (5'-ATGGCAGACGTCAACAGCCTGTTC-3'), primer 2 (5' -TCAGACCGTGCTGGTGAGGCC-3') for PCR amplification. PCR reaction system (total volume 50 μL): 5 μL of 10×Pfu DNA Polymerase...

Embodiment 2

[0045] Embodiment 2: Construction of recombinant Escherichia coli BL21(DE3) / pET28a-adh5

[0046] Design primer 3 (5'- CCATGG CAGACGTCAACAGCCTGTTC-3'), primer 4 (5'- CTCGAG GAC

[0047] CGTGCTGGTGAGGCC-3'), and NcoI and XhoI restriction enzyme sites (underlined) were introduced into primer 3 and primer 4, respectively. Under the priming of primer 3 and primer 4, high-fidelity PfuDNA polymerase was used to amplify to obtain a 774bp carbonyl reductase gene sequence (its nucleotide sequence is shown in SEQ ID NO: 1), and after sequencing, Nco The amplified fragment was treated with I and Xho I restriction enzymes (TaKaRa) and ligated with the commercial vector pET28a (Invitrogen) treated with the same restriction enzymes using T4 DNA ligase (TaKaRa) , construct the expression vector pET28a-adh5. The constructed expression vector pET28a-adh5 was transformed into Escherichia coli BL21(DE3) (Invitrogen), spread on LB plates containing kanamycin resistance at a final concentratio...

Embodiment 3

[0048] Embodiment 3: Recombinant carbonyl reductase (BgADH5) wet thallus

[0049] The recombinant Escherichia coli E.coliBL21(DE3) / pET28a-adh5 bacterial cell containing the expression recombinant plasmid pET28a-adh5 obtained in Example 2 was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, 37 Cultivate at 200rpm for 12h, then inoculate with 1% inoculum (v / v) into fresh LB liquid medium containing kanamycin resistance at a final concentration of 50μg / ml, and cultivate at 37°C at 150rpm until the bacteria Body OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mM, induce culture at 28°C for 12h, centrifuge at 5000rpm at 4°C for 5min, discard the supernatant, collect the precipitate, and obtain the recombinant Escherichia coli BL21( DE3) / pET28a-adh5 wet cells. The bacterium can be used directly as a biocatalyst or for protein purification.

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Abstract

The invention discloses a recombinant carbonyl reductase gene derived from Burkholderia gladioli ZJB-12126 and a codase thereof, a recombinant vector containing the gene, a recombinant gene engineering bacterium converted from the recombinant vector and application in catalyzing asymmetric reduction of prochiral carbonyl compound. 2-benzoylaminomethyl-3-one butyrate, tert-butyl (R)-6-cyano-5-hydroxy-3-carbonyl hexanoate and ethyl 4,4,4-trifluoro acetoacetate used as substrates are subjected to biological catalytic reaction to prepare high-optical-purity (2S,3R)-2-benzoylaminomethyl-3-hydroxy-butyrate, tert-butyl 6-cyano-(3R,5R)-dihydroxy hexanoate and ethyl (S)-4,4,4-trifluoro-3-hydroxy-butanoate. The recombinant Escherichia coli can be used as a biocatalyst to perform the biological catalytic reaction, thereby providing an alternative new enzyme source for biological catalytic synthesis of the drug chiral intermediate.

Description

(1) Technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a carbonyl reductase gene derived from Burkholderia gladioli ZJB-12126, an encoding enzyme, a recombinant vector containing the gene, and transformation of the recombinant vector The obtained recombinant genetically engineered bacteria and its application in catalytic asymmetric reduction of prochiral carbonyl compounds. (2) Background technology [0002] Carbonyl reductase (Carbonyl reducatase, E.C.1.1.1.x) is a class of enzymes that can catalyze the bidirectional reversible redox reaction between alcohols and aldehydes / ketones, and requires the coenzyme NAD(H) (nicotinamide adenine dinucleotide ) or NADP(H) (nicotinamide adenine dinucleotide phosphate) as hydrogen transporter. NADH and NADPH participate in the reduction reaction as electron donors, and NAD and NADP participate in the oxidation reaction as electron acceptors. At present, most of the carbonyl reducta...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/70C12N1/21C12P7/62C12R1/01
Inventor 柳志强郑裕国陈翔王亚军沈寅初
Owner ZHEJIANG UNIV OF TECH
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