Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof

A kind of technology of cyanopyrazine and pyrazinamide, applied in the field of biocatalysis 2-cyanopyrazine to produce pyrazinamide

Active Publication Date: 2009-07-15
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And utilize nitrile hydratase to produce pyrazinamide, there is no industrialization report both at home and abroad

Method used

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  • Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof
  • Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof
  • Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Screening of new bacterial strain ZJB-09104

[0028] The present invention obtains 10 parts of soil samples near a certain pharmaceutical factory in Hangzhou City, Zhejiang Province, and quickly screens strains from them within 12 hours; the specific method of screening is: first enrich and cultivate the samples in the enrichment medium for 24 hours, and then apply Distributed on a screening medium plate containing 2-cyanopyrazine; through plate screening, 5 strains that can biocatalyze 2-cyanopyrazine to produce pyrazinamide were isolated and obtained; the 5 strains were first divided into 5 After the % inoculum was put into the fermentation medium and cultured for 24 hours, the bacterial cells were obtained by centrifugation. Add 0.1 g of wet bacteria into the reaction solution of 2-cyanopyrazine with a final concentration of 10 g / L, and after reacting for 24 hours, measure the content of pyrazinamide respectively, and screen out the strain with the stronge...

Embodiment 2

[0032] Embodiment 2: Identification of new bacterial strain ZJB-09104

[0033] The bacterial strain ZJB-09104 obtained by screening in Example 1 was identified according to "Bergey's Bacteria Identification Handbook (Eighth Edition)" and "Common Bacteria System Identification Handbook" (see Table 1):

[0034] The colony of this strain is round, with neat edges, slightly protruding, white, opaque, dull, easy to pick, cultured at 30°C for 24 hours, the diameter of a single colony is about 1-2mm, and the bacteria are short rod-shaped;

[0035] On the basis of the above, the chromosomal DNA of the strain ZJB-09104 was further extracted according to the refined molecular biology experiment guide method, and the extracted total cell DNA was used as a template, using primers: p16S-8: 5′-aga gtt tga tcc tgg ctc ag-3′ and p16S-1541: 5′-aag gag gtg atc cag ccg ca-3′ amplifies the 16S rDNA gene of the strain, connects the gene product with the T vector, and entrusts Dalian Bao Biotechnol...

Embodiment 3

[0067] Example 3: Biocatalytic production of pyrazinamide by strain ZJB-09104

[0068] Follow these steps:

[0069] (1) Preparation of slant medium: sucrose 10g, yeast powder 10g, NaCl 5g, K 2 HPO 4 0.1g, MgSO 4 0.3g, 20g agar powder, make up to 1000mL with tap water, pH value 6; preparation of seed medium (g / L): sucrose 10, yeast powder 5, NaCl 1, K 2 HPO 4 0.5, MgSO 4 0.1, the solvent is water, and the pH value is 4.0-8.0.

[0070](2) Activation culture of strains: use the slant medium in step (1) to activate and cultivate the new Serratia marcescens (Serratia marcescens) strain ZJB-09104 CCTCC No.M208231 at 30°C for 18 hours, and set aside;

[0071] (3) Preparation of seed solution: insert the microorganisms activated in step (2) into the seed medium described in (1), and cultivate them at 30° C. and 200 r / min for 24 hours.

[0072] (4) Fermentation culture of strains: insert the strain liquid of step (3) into the fermentation medium according to the inoculum amou...

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Abstract

The invention provides a method of producing 2-cyanopyrazine by biocatalysis to produce pyrazinamide and a new strain, Serratia marcescens ZJB-09104, which is used in the preparation process. In the method, the pyrazinamide is obtained through the catalytic hydration reaction under the conditions that the pH is 6.0-8.0 and the temperature is 20-40 DEG C in a reaction system which uses 2-cyanopyrazine as a substrate and nitrile hydratase which is obtained by the culturing of the Serratia marcescens as a catalyst. The method applies the Serratia marcescens ZJB-09104 to the experiments producing pyrazinamide by biocatalysis. Results show that the strain has a wide application prospect in the field of producing pyrazinamide by biocatalysis as the strain can effectively produce the pyrazinamide by biocatalysis and the transformation ratio of the 2-cyanopyrazine.attains 83.8 percent within 12h.

Description

(1) Technical field [0001] The invention relates to a method for biocatalyzing 2-cyanopyrazine to produce pyrazinamide, and a new bacterial strain used in the preparation process—Serratia marcescens ZJB-09104. (2) Background technology [0002] Pyrazinamide is an important first-line anti-tuberculosis drug, mainly used for chronic tuberculosis, fever, cough, phlegm, etc. It has inhibitory effect on intracellular Mycobacterium tuberculosis, can enter human cells and penetrate the blood-brain barrier into human brain tissue to kill Mycobacterium tuberculosis; it has a unique sterilizing effect on semi-dormant bacteria that are slowly metabolized by macrophages, and is a short-term treatment for tuberculosis. One of the main drugs of chemotherapy. Because pyrazinamide has some advantages different from other anti-tuberculosis drugs, the research on pyrazinamide has gradually become a hot spot in recent years. [0003] In addition to being famous as an anti-tuberculosis drug, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/12C12N1/20C12R1/43
Inventor 郑裕国金利群柳志强薛亚平沈寅初
Owner ZHEJIANG UNIV OF TECH
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