Carbonyl reductase, its gene and its use in the preparation of duloxetine chiral intermediate

A reductase and carbonyl technology, applied in the field of biocatalyst production, can solve the problems of low optical purity, large waste discharge, difficult to use pharmaceutical intermediates, etc., and achieves simple conversion process, stable coenzyme circulation system, and large industrial application prospects. Effect

Active Publication Date: 2016-06-01
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the (S)-configuration chiral alcohol intermediate is mainly obtained through chemical means, but there are many problems, such as chemical resolution requires the use of a large amount of resolving agent, long reaction steps, high energy consumption and large waste discharge, etc.; Based on the transition metal chiral ligand catalytic hydrogenation system, due to the low optical purity of the product and the residue of heavy metals, it is difficult to apply it to the production of pharmaceutical intermediates

Method used

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  • Carbonyl reductase, its gene and its use in the preparation of duloxetine chiral intermediate
  • Carbonyl reductase, its gene and its use in the preparation of duloxetine chiral intermediate
  • Carbonyl reductase, its gene and its use in the preparation of duloxetine chiral intermediate

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Embodiment 1

[0018] Embodiment 1 Amplification of carbonyl reductase (ChKRED15) gene

[0019] According to the whole genome sequencing information of Chryseobacteriumsp.CA49, a large number of carbonyl reductases were excavated, one of which can catalyze the formation of (S)-N from N-methyl-3-oxo-3-(2-thienyl)propionamide - The enzyme with the function of methyl-3-hydroxy-3-(2-thienyl)propanamide is the carbonyl reductase ChKRED15 involved in the present invention, and the gene acquisition process is a conventional operation method. Design the following primers: Forward: 5′-GCG GAATTC ATGAAAACAGTATTAATTACAGGCGCC-3′ (restriction site: EcoRI); reverse: 5′-GCG AAGCTT CTACCACGGACTGATTCCGG-3′ (restriction site: HindIII), using the genomic DNA of Chryseobacterium aureus CCTCCM2012484 as a template, was amplified by PCR to obtain the gene of carbonyl reductase ChKRED15.

[0020] The PCR program was: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, ...

Embodiment 2

[0023] Example 2 Induced expression of carbonyl reductase (ChKRED15)

[0024]Pick the single clone of the recombinant E.coli (pET28a-ChKRED15) constructed in Example 1, inoculate it in LB medium containing 50 μg / mL kanamycin, and culture it at 37°C and 180 rpm for 16 hours as a seed solution. % inoculum rate was transferred to fresh LB or TB medium (500mL capacity shake flask liquid 200mL medium), cultured with shaking at 37°C for 4h, and then added IPTG with a final concentration of 0.5mM to induce the expression of carbonyl reductase ChKRED15 gene. After induction at 30°C for 36h, cells were harvested by refrigerated centrifugation at 8000rpm for 10min at 4°C.

[0025] Bacteria can be used as biocatalysts or for protein purification.

Embodiment 3

[0026] Example 3 Separation and Purification of Carbonyl Reductase (ChKRED15)

[0027] The bacteria obtained in Example 2 were resuspended in the binding buffer (100mM, pH8.0 sodium phosphate buffer, containing 300mMNaCl, 5mM imidazole), crushed by a high-pressure homogenizer, centrifuged at 12000rpm for 15min, and the supernatant was mixed with the above-mentioned After incubation with the Ni affinity chromatography resin equilibrated with the binding solution, use the washing buffer (100mM, pH8.0 sodium phosphate buffer, containing 300mM NaCl, 10mM imidazole) to rinse until there is basically no foreign protein, and then wash with the elution buffer (100mM , pH8.0 sodium phosphate buffer, containing 300mM NaCl, 250mM imidazole) to elute and collect the target protein, after electrophoresis to identify the purity, combine the target protein and dialyze with dialysis buffer (100mM, pH8.0 potassium phosphate buffer) for 48h, ultrafiltration After concentration, the protein conc...

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Abstract

The invention discloses a carbonyl reductase (i)Ch ( / i) KRED15 derived from Chryseobacterium ((i) Chryseobacterium ( / i) (i) ( / i) sp. CA49), and a coding gene thereof; the invention also discloses application of the carbonyl reductase as a biocatalyst in preparing a Duloxetine chiral intermediate ((i) S ( / i))-N-methyl-3-hydroxy-3-(2-thienyl) propanamide. The enantiomer excess of products is greater than 99.9%. 20 g / L of substrate can be catalyzed by Ch( / i) KRED15 crude enzyme (2U / mL), and the conversion rate of over 99% can be obtained in 2 hours; a coenzyme circulation system is simple, and has large industrial application prospect.

Description

technical field [0001] The present invention relates to a new gene and its protein product, in particular to a novel carbonyl reductase ChKRED15 and its gene derived from Chryseobacterium sp. The invention relates to a chiral intermediate, which belongs to the field of applied microorganism and enzyme engineering. Background technique [0002] Duloxetine is the third-generation antidepressant, and is the most important antidepressant sold in the market, which can effectively inhibit the reuptake of 5-hydroxytryptamine and norepinephrine (WongDT, etal.LifeSci, 1988, 43: 2049-2057), high efficiency, safety, and few side effects, and it is also effective for other symptoms such as general pain and gastrointestinal disorders (BymasterFP, etal. Neuropsychopharmacology, 2001, 25:871-880). The drug is also approved for the treatment of nerve pain caused by diabetes and female urinary incontinence (Norton PA, et al. AmJ Obstet Gynecol, 2002, 187: 40-48). [0003] Duloxetine contai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/04C12P17/00C12R1/01
Inventor 吴中柳任志强刘艳汤脱险裴小琼
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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