Application of nitrile hydratase in catalyzing hydration reaction of cyanopyrazine compound to generate amide pyrazine compound

A technology for catalyzing cyanopyrazine and nitrile hydratase, which is applied in the direction of enzymes, lyases, and carbon-oxygen lyases, can solve the problems of complex synthesis, low conversion rate and total yield, cumbersome process, etc., and achieve high conversion rate , no side effects, the effect of green environmental protection operation

Active Publication Date: 2020-09-15
ZHEJIANG SANMEN HYGECON PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these known chemical synthesis preparation methods have the following disadvantages: the synthesis is relatively complicated, the process is loaded down with trivial details, and the conversion rate and total yield are all low
More importantly, there is no bibliographical report that can catalyze the nitrile hydratase of cyanopyrazine compounds (such as Favipiravir) directly to prepare amide pyrazine compounds

Method used

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  • Application of nitrile hydratase in catalyzing hydration reaction of cyanopyrazine compound to generate amide pyrazine compound
  • Application of nitrile hydratase in catalyzing hydration reaction of cyanopyrazine compound to generate amide pyrazine compound
  • Application of nitrile hydratase in catalyzing hydration reaction of cyanopyrazine compound to generate amide pyrazine compound

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preparation example Construction

[0046] In the present invention, the preparation method of the cell (genetically engineered bacterium) expressing nitrile hydratase in vitro comprises: constructing the gene expressing nitrile hydratase into a target plasmid vector, introducing it into the expression host bacterium, and obtaining the cell (genetic engineering bacterium) expressing nitrile hydratase in vitro cells; the target plasmid vector is preferably pET-30a(+), pET-21a(+), pET-22b(+), pET-28a(+), pETDuet-1 or pACYCDuet-1, but not limited to the These vectors are preferably pET-28a(+), and the enzyme cutting sites are preferably EcoRI / BamHI and HindIII; the expression host bacteria are preferably Escherichia coli, more preferably E.coliBL21(DE3). The present invention has no special limitation on the specific method for constructing the gene expressing nitrile hydratase into the target plasmid vector and introducing it into the expression host bacterium, and conventional methods in the art can be used.

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Embodiment 1

[0063] 1. Acquisition of nitrile hydratase gene

[0064] The amino acid sequences of the reported cobalt-type nitrile hydratases were collected, and the ClustalW2 method (http: / / www.ebi.ac.uk / Tools / msa / clustalw2) was used to perform multiple sequence alignments on the nitrile hydratases to determine the the conservative region. The amino acid sequence of the conserved region was used as a molecular probe for the subsequent search of the nitrile hydratase gene in the gene information database. By comparing the reported amino acid sequences of nitrile hydratase, a conservative amino acid sequence in its α subunit -CTLCSC- was selected as a molecular probe, using BasicLocalAlignmentSearchTool (http: / / blast.ncbi.nlm.nih.gov) Homologous sequences were searched in the GenBank bioinformatics database (https: / / www.ncbi.nlm.nih.gov / genbank / ). Genes that have been reported and annotated as nitrile hydratase after whole-genome sequencing were selected. The amino acid sequences of the ...

Embodiment 2

[0083] Example 2 Genetically engineered bacteria catalyze 2-cyanopyrazine to generate 2-amide pyrazine

[0084] Get the fermented liquid of the engineered bacterium E.coliBL21(DE3) / pET-28a(+)-NH16 that 25ml embodiment 1 constructs, 12000rpm, 10min centrifugal collection thalline, then reconstitute with the buffer solution of 250ml50mM Tris-HCl (pH8.0) Suspend the collected bacteria, and the enzyme activity of the resuspended enzyme solution is 10U / ml. Add 1.0 g of 2-cyanopyrazine to the resuspension, and carry out hydration reaction at 20°C for 24 hours. Then liquid chromatography was used to detect the content of 2-cyanopyrazine, 2-amide pyrazine and 2-carboxypyrazine in the reaction system. The conversion rate of the substrate is greater than 99%, the yield of 2-amide pyrazine is greater than 92%, and no generation of 2-carboxypyrazine is found in the reaction system.

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Abstract

The invention provides an application of nitrile hydratase in catalyzing a hydration reaction of a cyanopyrazine compound to generate an amide pyrazine compound, and belongs to the technical field ofbiochemical engineering. The nitrile hydratase is derived from Agrobacterium tumefaciens, Stappia aggregata IAM 12614 or Sinorhizobium meliloti. The cyanopyrazine compound is used as a substrate, in-vitro nitrile hydratase or a cell expressing the nitrile hydratase is used as a catalyst, the hydration reaction is carried out, and the amide pyrazine compound can be obtained. The applied raw material is high in conversion rate and free of side reaction, and the product is easy to separate and purify and is relatively high in purity; and compared with a traditional chemical catalysis process, theprocess is environmentally friendly and easy and convenient to operate, and the yield exceeds 99%.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to the application of nitrile hydratase in catalyzing the hydration reaction of cyanopyrazine compounds to generate amide pyrazine compounds. Background technique [0002] Amidepyrazine (or pyrazinamide) is an important anti-tuberculosis drug, which has a good effect on tuberculosis. In recent years, it has been found that amidopyrazine has a good bactericidal effect on recalcitrant bacteria, so it is widely used in clinic; and amidopyrazine can be used as an intermediate for the preparation of various antibacterial and antiviral drugs. For example, derivatives of amide pyrazine: 6-fluoro-3-hydroxy-2-amide pyrazine, also known as Favipiravir (English: Favipiravir, also known as Avigan or favilavir), is an antiviral drug, produced by Professor Koyasu Shiraki of the Faculty of Medicine of Toyama University in Japan and Toyama Chemical Industry (now Fujifilm Toyama Chemical), a s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/12C12N9/88
CPCC12P17/12C12N9/88C12Y402/01084Y02A50/30
Inventor 姚红倪泉明王伟文黄晓飞陈文斌
Owner ZHEJIANG SANMEN HYGECON PHARMA CO LTD
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