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Ketoreductase mutant and application thereof in preparing duloxetine chiral alcohol intermediate and analogues thereof

A technology of reducing enzymes and mutants, which is applied in the fields of biological enzyme engineering and microbial applications, can solve the problems of unreported research on enzymes and their mutants, and achieve the effects of large-scale industrial application prospects, mild reaction conditions, and stable coenzyme cycle system

Pending Publication Date: 2019-09-13
南京趣酶生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there are no research reports on enzymes and their mutants that can be used for industrial production in China.

Method used

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  • Ketoreductase mutant and application thereof in preparing duloxetine chiral alcohol intermediate and analogues thereof
  • Ketoreductase mutant and application thereof in preparing duloxetine chiral alcohol intermediate and analogues thereof
  • Ketoreductase mutant and application thereof in preparing duloxetine chiral alcohol intermediate and analogues thereof

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Experimental program
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Embodiment 1

[0079] The construction of embodiment 1 prokaryotic expression system

[0080] The ketoreductase Rr Kred gene fragment whose nucleotide sequence is shown as SEQ ID NO: 2 was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and recombined into pET21a vector. The positive recombinant plasmid Rr Kred-pET21a (+) was transformed into the expression host strain BL21 (DE3) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the prokaryotic expression strain Rr Kred-pET21a (+) / BL21 (DE3) was obtained. Primary strains used as subsequent catalytic reactions.

[0081] Glucose dehydrogenase (BsGDH) (LOC111893255) and alcohol dehydrogenase (TbADH) (LOC101068320) genes for coenzyme regeneration were synthesized by Nanjing GenScript Co., Ltd. ) plasmids were constructed and transformed into BL21(DE3) to obtain BsGDH-pET21a(+) / BL21(DE3) and TbADH-pET21a(+) / BL21(DE3) expression strains respectively.

Embodiment 2

[0082] Fermentation preparation of embodiment 2 enzyme

[0083] The expression strains Rr Kred-pET21a(+) / BL21(DE3), BsGDH-pET21a(+) / BL21(DE3) and TbADH-pET21a(+) / BL21(DE3) constructed above were added with a final concentration of 100ug / mL Ampicillin in 5mL LB liquid medium [10g / L tryptone (OXIOD), 5g / L yeast powder (OXIOD), 10g / L sodium chloride (Sinopharm Reagent)] was shaken at 37°C and 200rpm overnight, then press 1% (V / V) ratio was inoculated in 500 mL LB liquid medium containing ampicillin with a final concentration of 100 ug / mL, and cultured with shaking at 37° C. and 200 rpm. When the OD600 was between 0.8-1.0, the inducer IPTG (isopropyl-β-D-thiogalactopyranoside, IPTG) was added at a final concentration of 0.1 mM, and induced overnight at 25°C. The bacteria were collected by centrifugation at 8000rpm, then suspended in 50mM pH7.0 sodium phosphate buffer, ultrasonically disrupted (200W, 3s / 5s, 10min), centrifuged at 12000rpm at 4°C for 20min, and the supernatant was ...

Embodiment 3

[0084] The catalytic activity detection of embodiment 3 enzyme

[0085] The enzyme solution obtained above is used for the substrate-catalyzed reaction.

[0086] Coenzyme regeneration using glucose dehydrogenase BsGDH: Dissolve 1g of the substrate (compound I) in 40mL of 50mM pH7.0 sodium phosphate buffer containing 10% isopropanol, then add 800mg of glucose, stir and dissolve, then add 5mg of NADP+ , 250 mg ketoreductase LfHSDH lyophilized powder, 100 mg BsGDH lyophilized powder, and supplement the volume to 50 mL with buffer. The reaction solution was placed in a constant temperature water bath at 37°C, and stirred by magnetic force for reaction. During the reaction, the pH of the system was maintained at 7.0 with 1M sodium hydroxide solution. After reacting for 24 hours, samples were taken and detected by HPLC. The conversion rate of the substrate reached 62%, and the chiral purity value of the product was >99%.

[0087] Coenzyme regeneration using alcohol dehydrogenase ...

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Abstract

The present invention provides a ketoreductase mutant and an application thereof, a nucleotide sequence encoding the mutant, a recombinant vector comprising the nucleotide sequence, and a recombinantstrain, and also provides the application of the ketoreductase mutant as a catalyst for preparing the S-configuration duloxetine chiral alcohol intermediate and the analogues thereof. The ketoreductase mutant can be used as the catalyst together with a coenzyme regeneration system to prepare the S-configuration duloxetine chiral alcohol intermediate and the analogues thereof, a reaction conversionrate can reach 90%, 95% or 99% or more, and a chiral purity value of the product can reach 90%, 95% or 99% or more. The reaction is mild in conditions and almost free of by-products, the coenzyme cycle system is stable, and the ketoreductase mutant has a relatively large industrial application prospect.

Description

technical field [0001] The invention relates to a ketoreductase mutant and its application in the preparation of duloxetine chiral alcohol intermediates and analogues thereof, belonging to the fields of biological enzyme engineering and microbial application. Background technique [0002] Duloxetine hydrochloride [Duloxetine hydrochloride, (S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)-1-propanamine hydrochloride] is produced by Eli Lilly and Company of the United States The new antidepressant developed can effectively inhibit the reuptake of 5-hydroxytryptamine and norepinephrine. It is highly efficient, safe, and has few side effects. It is also effective for other symptoms such as general pain and gastrointestinal disturbance. Approved for the treatment of nerve pain caused by diabetes and urinary incontinence in women. Among the 20 best-selling prescription drugs in the world in 2012, duloxetine ranked 9th with annual sales of US$4.994 billion. [0003] [0004] The s...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P17/00C12R1/19
CPCC12N9/0006C12N15/70C12P17/00Y02P20/584
Inventor 林涛徐明文蒋丽丽于丽珺
Owner 南京趣酶生物科技有限公司
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