Enzyme for preparing antidepressant drug duloxetine intermediate, biological catalysis method and application of the enzyme

A duloxetine and antidepressant technology, applied in the biological field, can solve the problems of increasing operation difficulty and cost, and achieve the effect of reducing production cost

Active Publication Date: 2020-04-07
NANJING LANGEN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] U.S. Patent No. 8,426,178 discloses an engineered ketoreductase with improved properties, and also provides a polynucleotide encoding the engineered ketoreductase, a host cell capable of expressing the engineered ketoreductase, and a method for synthesizing various chiral compounds using the engineered ketoreductase; Engineered ketoreductase polypeptides optimized to catalyze the conversion of N,N-dimethyl-3-keto-3-(2-thienyl)-1-ketopropylamine to (S)-N,N-dimethyl-3 -Hydroxy-3-(2-thienyl)-1-propanamine; this scheme uses isopropanol as the coenzyme cycle method, but the reaction requires suction filtration to form a negative pressure to remove the by-product acetone of the reaction, so that the reaction can be carried out effectively , it will increase the operation difficulty and cost when the production is enlarged

Method used

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  • Enzyme for preparing antidepressant drug duloxetine intermediate, biological catalysis method and application of the enzyme
  • Enzyme for preparing antidepressant drug duloxetine intermediate, biological catalysis method and application of the enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.

[0032] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C, and the primer length is controlled within 60base , and the obtained primers were dissolved in double-distilled water, and added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the first and last primers was 0.6 μM.

[0033] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH2O Bring the total volume of the reaction system to 50 μl

[0034] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according to the following p...

Embodiment 2

[0038] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.

[0039] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C, and the primer length is controlled within 60base , and the obtained primers were dissolved in double-distilled water, and added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the first and last primers was 0.6 μM.

[0040] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl ddH2O Bring the total volume of the reaction system to 50 μl

[0041] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according to the following p...

Embodiment 3

[0045] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.

[0046] Use Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C, and the primer length is controlled within 60base , and the obtained primers were dissolved in double-distilled water, and added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the first and last primers was 0.6 μM.

[0047] 2mM dNTP mix (2mM each dNTP) 5μl 10×Pfu buffer 5μl Pfu DNA polymerase (10U / μl) 0.5μl wxya 2 o

Bring the total volume of the reaction system to 50 μl

[0048] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according to the foll...

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PUM

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Abstract

The invention, which belongs to the technical field of biology, discloses an enzyme for preparing an antidepressant drug duloxetine intermediate, a biological catalysis method and application of the enzyme. 3-dimethylamino-1-(2-thienyl)-1-acetone hydrochloride can be transformed into S-3-dimethylamino-1-(2-thienyl)-1-propanol and high dehydrogenase activity is realized; and the enzyme has one or more mutations of K49R, A68T, E101D, F147E, T152A, S169V and A235S. The enzyme has obvious high specific enzyme activity that is improved by 2-10 times than that of wild type ketoreductase; and the enzyme can be used for biologically catalyzing transformation of 3-dimethylamino-1-(2-thienyl)-1-acetone hydrochloride to S-3-dimethylamino-1-(2-thienyl)-1-propanol; the reaction conditions are mild; equipment requirements are low; no high temperature or cooling in the production process is needed; energy consumption is low; purification is convenient; and the green and environment-friendly preparation process is realized.

Description

technical field [0001] The invention relates to an enzyme, a biocatalysis method and application thereof, in particular to an enzyme used for the preparation of an antidepressant drug duloxetine intermediate, a biocatalysis method and application thereof, and belongs to the field of biotechnology. Background technique [0002] Ketoreductases are versatile catalysts through the enantioselective reduction of aldehydes or ketones to the corresponding alcohols; (R)-specific ketoreductases have distinct properties from (S)-specific ketoreductases, and these catalysts are Optically active alcohols are used more and more frequently in the industrial synthesis; optical activity is a necessary condition for the selective action of many pharmaceutically and agrochemically active compounds, and in some cases one enantiomer has beneficial pharmaceutical However, enantiomers have genotoxic effects; therefore, in the synthesis of pharmaceutical and agricultural active compounds, it is nec...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P17/00C12R1/19
CPCC12N9/0006C12N15/70C12P17/00C12Y101/01
Inventor 丁雪峰李佳松
Owner NANJING LANGEN BIOLOGICAL SCI & TECH
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