Carbonyl reductase gene, enzyme, carrier, engineering bacteria and its application in asymmetric reduction of prochiral carbonyl compounds

A technology of carbonyl compounds and reductases, which is applied in genetic engineering, oxidoreductases, and the introduction of foreign genetic material using vectors, etc.

Active Publication Date: 2018-06-26
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the carbonyl reductase in Burkholderiagladioli strain has not been used in the asymmetric reduction of these pharmaceutical chiral intermediates

Method used

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  • Carbonyl reductase gene, enzyme, carrier, engineering bacteria and its application in asymmetric reduction of prochiral carbonyl compounds
  • Carbonyl reductase gene, enzyme, carrier, engineering bacteria and its application in asymmetric reduction of prochiral carbonyl compounds
  • Carbonyl reductase gene, enzyme, carrier, engineering bacteria and its application in asymmetric reduction of prochiral carbonyl compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Amplification of the carbonyl reductase gene adh1

[0043] 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 can catalyze 4-chloroacetoacetate ethyl, (R)-6-cyano -tert-butyl 5-hydroxy-3-carbonylhexanoate and (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxo Oxazolidinone generates (2S,3R)-2-benzamidomethyl-3-hydroxybutyrate, (S)-4-chloro-3-hydroxybutyrate ethyl ester, 6-cyano-(3R, 5R)-tert-butyl dihydroxyhexanoate and (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxaza The functional enzyme of heterocyclopentane-2-one is the carbonyl reductase BgADH1 involved in the present invention.

[0044] Using MPBio's 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'-ATGGGTCGTTCGATCAATCTGGAAGGCAAGG-3'), primer 2 (5'...

Embodiment 2

[0047] Embodiment 2: Construction of recombinant Escherichia coli BL21(DE3) / pET28a-adh1

[0048] Design primer 3 (5'- CATATG GGTCGTTCGATCAATCTGGAAGGCAAGG-3'), primer 4 (5'- CTCGAG TGCGAGCCCGAATCCGTCGTCG-3'), and introduced NdeI and XhoI restriction enzyme sites (underlined marks) in primer 3 and primer 4, respectively. Under the priming of primer 3 and primer 4, the high-fidelity Pfu DNA polymerase was used to amplify to obtain a 777bp carbonyl reductase gene sequence (its nucleotide sequence is shown in SEQ ID NO: 1), and after sequencing, use The amplified fragment was treated with Nde I and Xho I restriction endonucleases (TaKaRa), and the fragment was treated with the commercial vector pET28a (Invitrogen) treated with the same restriction enzymes using T4 DNA ligase (TaKaRa). Connected to construct the expression vector pET28a-adh1. The constructed expression vector pET28a-adh1 was transformed into Escherichia coli BL21(DE3) (Invitrogen), spread on LB plates containing...

Embodiment 3

[0049] Embodiment 3: Recombinant carbonyl reductase (BgADH1) wet thallus

[0050] The recombinant Escherichia coli E.coli BL21(DE3) / pET28a-adh1 thallus obtained in Example 2 containing the expression recombinant plasmid pET28a-adh1 was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, Cultivate at 37°C for 12 hours at 200rpm, then inoculate with 1% inoculum size (v / v) into fresh LB liquid medium containing kanamycin resistance at a final concentration of 50 μg / ml, and cultivate at 37°C at 150rpm until Cell 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 recombinant Escherichia coli BL21( DE3) / pET28a-adh1 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 sourced from Burkholderia gladioli ZJB-12126 as well as an encoding gene of the recombinant carbonyl reductase, a recombinant vector containing the gene, a recombinant gene engineering bacterium obtained by transforming the recombinant vector, and an application of the recombinant carbonyl reductase in asymmetrically reducing prochiral carbonyl compounds. According to the recombinant carbonyl reductase disclosed by the invention, by respectively taking 2-benzamidomethyl-3-ketone butyrate, 4-chloroacetoacetic acid ethyl ester (COBE), (R)-6-cyano-5-hydroxyl-3-carbonyl caproate tert-butyl ester and (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone as substrates, (2S, 3R)-2-benzamidomethyl-3-polyhydroxybutyrate, (S)-4-chloro-3-ethyl hydroxybutyrate, 6-cyano-(3R, 5R)-dihydroxyhexanoic acid tert-butyl ester and (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxyl valeryl]-4-phenyl-1,3-oxo-azacyclopentane-2-ketone with high optical purity are prepared by virtue of biocatalytic reaction, wherein the biocatalytic reaction can be performed by using recombinant escherichia coli as a biocatalyst, and thus optional new enzyme sources can be provided for biocatalytic synthesis of chiral intermediates of medicines.

Description

(1) Technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a carbonyl reductase derived from Burkholderia gladioli (Burkholderia gladioli) ZJB-12126, a gene, a recombinant vector containing the gene, and a product obtained by transforming the recombinant vector. Recombinant genetically engineered bacteria and its application in catalyzing asymmetric reduction of prochiral carbonyl compounds to prepare chiral alcohols. (2) Background technology [0002] Carbonyl reductase (Carbonyl reducatase, E.C.1.1.1.x) belongs to the oxidoreductase system, which 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 its reduction reaction as electron donors, NAD + and NADP + It acts as an ele...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/70C12N1/21C12P13/02C12P7/62C12P13/00C12P17/14
Inventor 柳志强郑裕国陈翔王亚军沈寅初
Owner ZHEJIANG UNIV OF TECH
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