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Cyclohexanone monooxygenase and application thereof in synthesis of esomeprazole

A technology of cyclohexanone monooxygenase and cyclohexanone monooxygenase, which is applied in the field of bioengineering, can solve the problems of low yield, high cost, difficult post-treatment, etc., and achieve the effect of high yield and low price

Active Publication Date: 2016-06-22
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In view of the low yield in the resolution process adopted in the synthesis of prazole drugs in the prior art, and the disadvantages of high cost, low yield and difficult post-treatment in the process of adopting noble metal catalysis, the invention provides a catalytic activity Asymmetric Oxidation of Cyclohexanone Monooxygenase with High Reaction Yield and Environment-Friendly Synthesis of Single-configuration Prazoles

Method used

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  • Cyclohexanone monooxygenase and application thereof in synthesis of esomeprazole
  • Cyclohexanone monooxygenase and application thereof in synthesis of esomeprazole
  • Cyclohexanone monooxygenase and application thereof in synthesis of esomeprazole

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Embodiment 1

[0047] Embodiment 1 screening cyclohexanone monooxygenase

[0048] Soil samples were collected from Shanghai Jinshan Chemical Industry Park and DNA was extracted (extraction method refers to ChromaSpinTE-1000, ClontechLaboratories, Inc., USA), partially digested with Sau3AI, and 0.5-3kb fragments were collected by electrophoresis, recovered and ligated to pWF-1 The BamHI site was used to obtain a plasmid library. Transform the library into E.coliDH5α and smear it on an LB plate containing 100 μg / mL ampicillin, select positive clones and inoculate them into a 96-deep-well plate containing 500 μg / mL LB (containing 100 μg / mL ampicillin), and incubate at 37°C for 4 hours Add 1mMIPTG for induction, and continue culturing overnight at 30°C; then take 50μL of each deep-well culture to a new 96-well plate added with 50mM sodium phosphate buffer (pH8.5) and 1mg / mL lysozyme, and incubate at 30°C for 30 minutes , down to -80°C and repeated freezing and thawing to lyse the bacteria.

[...

Embodiment 2

[0050] Expression and fermentation of embodiment 2 cyclohexanone monooxygenase

[0051] The cyclohexanone monooxygenase gene obtained in Example 1 was cloned into pET28a, and the restriction sites were NdeI-HindIII. The plasmid was transformed into E.coliBL21(DE3), positive colonies were screened on 50μg / mL kanamycin LB plate, inoculated into 200mL LB medium, and cultured at 37°C, 180-220rpm for 10-16h. The above-mentioned cultivated seeds were inoculated in a 3L upper tank fermentation medium (M9, containing glucose 4g / L, disodium hydrogen phosphate 12.8g / L, potassium dihydrogen phosphate 3g / L, Ammonium chloride 1g / L, sodium sulfate 0.5g / L, calcium chloride 0.0152g / L, magnesium chloride hexahydrate 0.41g / L), at 25~35℃, 300~800rpm, air flow 2~6L / min cultivated under conditions. After culturing for 6-10 hours, feed medium containing 60% glycerol was fed at a rate of 5-20 mL / h until the end of fermentation. Fed feed medium for several hours to OD 600 When reaching 20-40, add...

Embodiment 3

[0052] The assay method of embodiment 3 enzyme activity

[0053] The bacteria were resuspended in PBS buffer (pH9.0) at a ratio of 1:10, and after high-pressure crushing, 50 μL of crude enzyme solution was added to 1.8 mL of PBS buffer (pH9.0), and methanol containing 100 mM cyclohexanone was added to aid dissolution. , so that the final concentration of cyclohexanone is 10 mM; add NADPH 0.1 mM to start the reaction, and detect the change of ultraviolet absorption at a wavelength of 340 nm.

[0054] Calculation of enzyme activity: U=ΔA 340 ×1000 / (6220×20), that is, the specific enzyme activity per mL of lysate.

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Abstract

The invention provides application of a cyclohexanone monooxygenase characterized by high catalytic activity, high reaction yield, and environmental friendliness to asymmetric oxidation reaction to synthesize single-configuration prazole drugs. The invention also provides a gene of the cyclohexanone monooxygenase, a recombinant expression vector containing the gene, a recombinant expression transformant and an efficient preparation method of the cyclohexanone monooxygenase, and application of the cyclohexanone monooxygenase in catalysis of a prochiral thioether compound into single-configuration sulfoxide.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a cyclohexanone monooxygenase, the construction of a genetically engineered bacterium producing the cyclohexanone monooxygenase, and the production and application of the cyclohexanone monooxygenase. Background technique [0002] Proton pump inhibitors (protonpumpinhibitors, PPIs) are used in the treatment of acid-related diseases, which are widely used clinically and have the best curative effect in the past ten years. PPIs are H + / K + -ATPase inhibitor, which has strong acid suppression effect, high specificity and long duration. The final step in gastric acid secretion is the exchange of intracellular H+ with intratubular K+ by the proton pump in parietal cells. It is believed that PPIs in the blood accumulate in the strongly acidic secretory tubules after entering the gastric parietal cells, and are converted into active sulfenamide compounds, which are covalently ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P17/16
Inventor 罗煜丁时澄瞿旭东王海涛
Owner ABIOCHEM BIOTECH CO LTD
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