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Method for preparing S-3-dimethylamino-1-(2-thienyl)-1-propanol through biocatalysis

A dimethylamino, S-3- technology, applied in the field of biocatalytic preparation of S-3-dimethylamino-1--1-propanol, can solve the problems of increasing operation difficulty and cost, and achieve production cost reduction effect

Active Publication Date: 2020-03-27
NANJING LANGEN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Method for preparing S-3-dimethylamino-1-(2-thienyl)-1-propanol through biocatalysis
  • Method for preparing S-3-dimethylamino-1-(2-thienyl)-1-propanol through biocatalysis
  • Method for preparing S-3-dimethylamino-1-(2-thienyl)-1-propanol through biocatalysis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 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.

[0031] 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.

[0032]

[0033]

[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 procedures: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0035] The DNA fragment obtained by PCR was gel-cut and purified, and cloned into the NdeI / XhoI site of pET...

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 dNT Pmix (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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Abstract

The invention discloses a method for preparing S-3-dimethylamino-1-(2-thienyl)-1-propanol through biocatalysis and belongs to the technical field of biologics. 3-dimethylamino-1-(2-thienyl)-1-acetonehydrochloride is converted to generate S-3-dimethylamino-1-(2-thienyl)-1-propanol which has high alcohol dehydrogenase activity, and has one or more mutants of K49R, A68T, E101D, F147E, T152A, S169V and A235S. The whole system of the method has catalysis of a single enzyme, an alcohol is adopted for coenzyme circulation, the concentration of a substrate is as high as 100-160g / L, the chirality purity is greater than 99%, the amount of the substrate / amount of coenzymes is as high as 6000:1; the coenzyme circulation times are high, and the production cost of the S-3-dimethylamino-1-(2-thienyl)-1-propanol can be effectively reduced.

Description

technical field [0001] The invention relates to a method for preparing S-3-dimethylamino-1-(2-thienyl)-1-propanol by biocatalysis, 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 necessary to use catalysts with the required stereospecificity to synthesize optically active alcohols. [0003] Duloxetine is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/00C12N15/70C12N9/04C12N15/53C12N1/21C12R1/19
CPCC12N9/0006C12N15/70C12N2800/22C12P17/00C12Y101/01047
Inventor 丁雪峰李佳松
Owner NANJING LANGEN BIOLOGICAL SCI & TECH
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