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Application of nitrile hydratase from Sinorhizobium meliloti in preparation of amide pyrazine compound

一种中华根瘤菌、腈水合酶的技术，应用在生物化工领域，能够解决工艺繁琐、合成复杂、转化率和总收率低等问题，达到没有副反应、转化率高、绿色环保操作的效果

Active Publication Date: 2021-06-25
ZHEJIANG SANMEN HYGECON PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 from Sinorhizobium meliloti in preparation of amide pyrazine compound
  • Application of nitrile hydratase from Sinorhizobium meliloti in preparation of amide pyrazine compound
  • Application of nitrile hydratase from Sinorhizobium meliloti in preparation of amide pyrazine compound

Examples

Experimental program
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Effect test

preparation example Construction

[0047] 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, importing it into an expression host bacterium, and obtaining a cell 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.

[0048] In the present invention, the nu...

Embodiment 1

[0064] 1. Acquisition of nitrile hydratase gene

[0065] 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

[0085] Example 2 Genetically engineered bacteria catalyze 2-cyanopyrazine to generate 2-amide pyrazine

[0086] 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 from Sinorhizobium meliloti in preparation of an amide pyrazine compound and belongs to the technical field of biochemical engineering. The nitrile hydratase catalyzes a hydration reaction of a cyanopyrazine compound to generate an amide pyrazine compound; The nitrile hydratase is derived from the Sinorhizobium meliloti (Singorhizobium meliloti). The cyanopyrazine compound is taken as a substrate, in-vitro nitrile hydratase or cells for expressing the nitrile hydratase are taken as a catalyst, a hydration reaction is carried out, and the amide pyrazine compound can be obtained. The raw material conversion rate is high, no side reaction exists, and the product is easy to separate and purify and high in purity; Compared with the traditional chemical catalysis process, the process is green and environment-friendly, the operation is simple and convenient, and the yield exceeds 99%.

Description

[0001] This application is a divisional application with an application date of June 23, 2020, an application number of 202010580723.9, and an invention title of "Application of Nitrile Hydratase in Catalyzing the Hydration Reaction of Cyanopyrazine Compounds to Form Amidepyrazine Compounds". technical field [0002] The invention relates to the field of biochemical technology, in particular to the application of nitrile hydratase derived from Sinorhizobium meliloti in the preparation of amidopyrazine compounds. Background technique [0003] 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...

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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