Preparation method of 5-methylpyrazine-2-carboxylic acid

A technology of methylpyrazine and dimethylpyrazine, which is applied in the field of bioengineering, can solve problems such as affecting production efficiency and poor bacterial growth, and achieve high comprehensive reaction yield, simple purification steps, and reduced production costs Effect

Inactive Publication Date: 2018-01-05
DISHA PHARMA GRP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This patent discloses the shake flask technology to produce bacteria, directly adding xylene as an inducer. The test found that the growth of bacteria in this technology is not good, and the fermentation liquid is relatively clear, which affects production efficiency.

Method used

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  • Preparation method of 5-methylpyrazine-2-carboxylic acid
  • Preparation method of 5-methylpyrazine-2-carboxylic acid
  • Preparation method of 5-methylpyrazine-2-carboxylic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Implementation Example 1: Preparation of Primary Seed Solution

[0047] Will Arthrobacter woluwensis Seeds were inoculated into LB medium at 150 r min -1 , Cultivate at 30° C. for 19 hours to obtain seed solution. The components of the LB seed medium used here are peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, and the solvent is water.

Embodiment 2

[0048] Implementation example 2: preparation of secondary seed solution

[0049] Will Arthrobacter woluwensis The seed liquid was transferred to 100 mL of fermentation medium with an inoculum of 2%, under the induction of p-xylene, the shaking table rotated at 150 r min -1 , pH 7.0, cultured at 30°C for 20 h, and prepared the secondary seed solution.

[0050] The fermentation medium uses p-xylene as an inducer, and other nutrients and trace elements are added. Secondary fermentation medium in the present invention consists of: yeast powder 0.6 g / L; peptone 1.2 g / L; ammonium sulfate 1.5 g / L; sodium bicarbonate 1.3 g / L; potassium dihydrogen phosphate 1.2 g / L; Sodium 0.8 g / L; Magnesium chloride 0.1 g / L; Calcium chloride 0.2 g / L; Zinc sulfate 0.02 g / L; Manganese chloride 0.05 g / L; Copper chloride 0.03 g / L; Nickel chloride 0.01 g / L ; EDTA Na 2 2H 2 O0.8 g / L; p-xylene 0.3 mL / L, the solvent is water.

Embodiment 3

[0051] Implementation Example 3: Tank Fermentation and Biotransformation

[0052] Will Arthrobacter woluwensis The secondary seed solution was connected to a 10 L fermenter with a 6% inoculation amount, and p-xylene was injected into the fermenter in the form of steam brought by compressed air. Under the induction of p-xylene, it was cultivated at 400 rpm, 30°C, and pH 7.0 After 6 hours, when the bacterial concentration reached 1.0, 20 g of raw material 2,5-dimethylpyrazine was added into the fermenter. And according to liquid phase detection (column: C 18 Column, acetonitrile: water: trifluoroacetic acid = 12:88:0.5, flow rate 1 mL min -1 , detection wavelength 270 nm, column temperature 25°C) product concentration and substrate concentration in the fermentation broth, when the product concentration exceeds 50%, add 20 g of raw material 2,5-dimethylpyrazine to the fermentation broth in due course . The conversion process took 360 h and 15 days, and a total of 14 additiona...

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Abstract

The invention belongs to the technical field of bioengineering, in particular to a method for preparing and purifying 5-methylpyrazine-2-carboxylic acid by microbial fermentation. The technical scheme of the present invention uses 2,5-dimethylpyrazine as a raw material, uses Arthrobacter woluwensis HW-1 strain with xylene monooxygenase activity as a catalyst, and uses compressed air to bring in xylene vapor as a catalyst. inducer, and add raw materials in batches in due time, and ferment and transform for a period of time to obtain a fermentation broth containing 5-methylpyrazine-2-carboxylic acid. The invention makes up for the deficiencies in the process of chemically synthesizing 5-methylpyrazine-2-carboxylic acid, overcomes the defect of low conversion rate of shake flask fermentation, and provides a method for purifying 5-methylpyrazine-2-carboxylic acid in fermentation broth for the first time method.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for preparing and purifying 5-methylpyrazine-2-carboxylic acid by microbial fermentation. Background technique [0002] 5-Methylpyrazine-2-carboxylic acid (5-Methylpyrazine-2-carboxylic acid) is a off-white solid crystal, with a CAS number of 5521-55-1 and a molecular formula of C 6 h 6 N 2 o 2 , molecular weight 138.12, melting point 166-169°C, has a pungent odor, will slowly oxidize when exposed to air, and changes from brown oil to brown-black viscous solid, which needs to be stored in a vacuum seal. [0003] [0004] 5-Methylpyrazine-2-carboxylic acid structure [0005] 5-Methylpyrazine-2-carboxylic acid is mainly used in the synthesis of the hypoglycemic drug glipizide in the pharmaceutical industry. At present, the market demand for this hypoglycemic drug of glipizide is huge, so looking for a method for preparing 5-methylpyrazine The green and envir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/12C12R1/06
Inventor 李广生杨涛曲欣欣牛占芃李靖万刚强
Owner DISHA PHARMA GRP
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