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Carbonyl reductase, gene and applications of carbonyl reductase in asymmetric reduction of prochiral carbonyl compound

A carbonyl reductase and carbonyl compound technology, applied in the field of recombinant expression vectors and recombinant expression transformants, can solve problems such as poor substrate tolerance, insufficient product concentration, and difficulty in product separation and purification

Active Publication Date: 2013-01-16
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is that in the existing biocatalytic asymmetric reduction of prechiral carbonyl compounds to prepare chiral alcohols, the catalytic activity of carbonyl reductase is low, the substrate tolerance is poor, and the product concentration is not high enough. As well as problems such as long reaction time and difficulty in product separation and purification, the application of a carbonyl reductase as a catalyst in the preparation of chiral alcohols by catalyzing asymmetric reduction of prechiral carbonyl compounds, the gene and protein of the carbonyl reductase and its mutants are provided , and the recombinant expression vector and recombinant expression transformant comprising the carbonyl reductase gene or gene mutant

Method used

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  • Carbonyl reductase, gene and applications of carbonyl reductase in asymmetric reduction of prochiral carbonyl compound
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  • Carbonyl reductase, gene and applications of carbonyl reductase in asymmetric reduction of prochiral carbonyl compound

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Experimental program
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Effect test

Embodiment 1

[0059] Cloning of Example 1 Carbonyl Reductase Gene

[0060] According to the gene sequence (Genebank accession number: CAR23611.1) recorded in Genbank and predicted as Kluyveromyces thermotolerans reductase, the PCR primers were designed as follows:

[0061] Upstream primer: 5'-CCG GAATTC ATGCCCAAGACTATCGCCACA-3';

[0062] Downstream primer: 5'-CCG CTCGAG TCAGATGCTGCTGTACCCGC-3'.

[0063] Among them, the underlined part of the upstream primer (nucleotide sequence as shown in SEQ ID NO:5 in the sequence listing) is the EcoRI restriction site, and the downstream primer (nucleotide sequence as shown in SEQ ID NO:6 in the sequence listing) is underlined Part of it is the XhoI restriction site.

[0064] The genomic DNA of Kluyveromyces thermotolerans CGMCC 2.1492 was used as a template for PCR amplification. PCR system: 10 μl of 2×Taq PCR MasterMix, 1 μl of upstream primer and downstream primer (0.3 μmol / L), 1 μl of DNA template (0.1 μg) and ddH 2 O7 μl. The PCR amplificat...

Embodiment 2

[0065] Example 2 Preparation of Carbonyl Reductase Recombinant Plasmid and Recombinant Expression Transformant

[0066] The reductase gene DNA fragment and pET28a empty plasmid obtained in Example 1 were double-digested with restriction endonucleases EcoRI and XhoI at 37°C for 12 h, and purified by agarose gel electrophoresis ( image 3 , Figure 4 ), using the agarose gel DNA recovery kit to recover the target fragment. The target fragment was ligated overnight at 4°C under the action of T4 DNA ligase to obtain the recombinant expression plasmid pET-KtCR.

[0067] Transform the above-mentioned recombinant expression plasmid into Escherichia coli (E.coli) DH5α competent cells, screen the positive recombinants on the resistance plate containing kanamycin, pick a single clone, and the colony PCR verification is positive clone( Figure 5 ). Cultivate the recombinant bacteria, extract the plasmid after the plasmid is amplified, retransform into Escherichia coli (E. Inverted o...

Embodiment 3

[0068] Embodiment 3 Expression of carbonyl reductase

[0069] The recombinant Escherichia coli obtained in Example 2 was inoculated into LB medium containing kanamycin (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0), cultured with shaking at 37°C overnight, and Inoculum of 1% (v / v) was transferred into a 500ml Erlenmeyer flask containing 100ml LB medium, placed on a shaker at 37°C and 180rpm for shaking culture, when the OD of the culture medium 600 When it reached 0.6, IPTG with a final concentration of 0.5 mmol / L was added as an inducer, and after induction at 25°C for 12 hours, the culture medium was centrifuged to collect cells and washed twice with saline to obtain resting cells. The resulting resting cells were suspended in a pH 7.0 buffer, ultrasonically disrupted in an ice bath, and the supernatant was collected by centrifugation, which was the crude enzyme solution of the recombinant carbonyl reductase. The obtained crude enzyme solution was analyzed by polya...

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Abstract

The invention discloses applications of carbonyl reductase being as a catalyst in the preparation of chiral alcohol by asymmetric reduction of a prochiral carbonyl compound, and a gene, protein and a mutant of the carbonyl reductase. The invention also provides a recombinant expression vector and a recombinant expression transformant containing the gene of the carbonyl reductase. When the carbonyl reductase disclosed by the invention is utilized for catalyzing the asymmetric reduction reaction of the prochiral carbonyl compound, the reaction conditions of the asymmetric reduction are moderate, the operation process is simple and convenient, and the amplification is easy; and the chiral alcohol product prepared by the asymmetric reduction reaction is high in concentration and optical purity. Therefore, the carbonyl reductase has a good industrial application and development prospect in the preparation of the optically pure chiral alcohol.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to the application of a carbonyl reductase as a catalyst in the preparation of chiral alcohols by catalyzing the asymmetric reduction of prechiral carbonyl compounds, and the protein and gene mutants of the carbonyl reductase, including the The recombinant expression vector and recombinant expression transformant of the carbonyl reductase gene and its gene mutant. Background technique [0002] (S)-2-chloro-1-phenylethanol (molecular formula is C 6 h 5 CH(OH)CH 2 Cl, molecular weight 156.61, CAS number: 70111-05-6) has three active functional groups (chlorine, phenyl and optically pure hydroxyl), and is an important chiral building block for the synthesis of medicines, pesticides and other fine chemicals. Researchers have developed a variety of methods for the synthesis of optically active chiral alcohols, including kinetic resolution and asymmetric synthesis. A...

Claims

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Application Information

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IPC IPC(8): C12P7/22C12P13/00C12P17/12C12P17/00C12P7/62C12N9/04C12N15/53C12R1/72C12R1/645
Inventor 许建和许国超郁惠蕾潘江
Owner EAST CHINA UNIV OF SCI & TECH
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