Bacillus pumilus capable of highly producing tetramethylpyrazine

一种短小芽孢杆菌、四甲基吡嗪的技术，应用在高产四甲基吡嗪的短小芽孢杆菌领域，能够解决四甲基吡嗪低、提取成本高、设备要求较高等问题，达到反应条件温和、原料价廉、环境友好的效果

Active Publication Date: 2007-11-28
SHANGHAI KAIXIN BIOTECH +2
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  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The method for extracting tetramethylpyrazine from plants is limited due to the limited source of raw materials, and the content of tetramethylpyrazine in the raw material is too low, and the extraction cost is too high to realize industrialization; the chemical synthesis method generally has more serious environmental protection problems, and the reaction The conditions are generally severe, the requirements for equipment are high, and the product is not natural; the biosynthesis method has outstanding advantages such as green and natural products, low cost, mild reaction conditions, and little environmental pollution. It is the future research on tetramethylpyrazine synthesis. direction, has excellent development prospects, but screening tetramethylpyrazine-producing bacteria from nature is a very arduous task, and the yield of wild bacteria screened is often very low, and the starting point for using other microbial technologies is very low. Therefore, the technology for producing tetramethylpyrazine by biosynthesis in the world is not yet mature, and industrial production cannot be carried out.

Method used

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  • Bacillus pumilus capable of highly producing tetramethylpyrazine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Step 1: Mutagenesis screening of Bacillus pumilus RX3-17 CCTCC M 206043 strain:

[0022] Soil samples were obtained from apple orchards, vineyards, etc., suspended in saline and heated at 80°C for 10 minutes, then streaked and spread onto nutrient agar dishes, and cultured at 37°C. The formula of nutrient agar medium is: 1 liter of distilled water contains: 10 grams of peptone, 3 grams of beef extract, 5 grams of sodium chloride, and 18 grams of agar. The initial pH value of the culture medium was 7.2, the sterilization temperature was 121° C., and the sterilization time was 15 minutes.

[0023] After single colonies were picked out, they were inoculated in nutrient broth medium and cultured on a shaker at 120 rpm at 37°C. After growing to the mid-logarithmic growth phase, the bacteria were suspended in 0.05 mol / L Tris-maleic acid buffer (pH 6.0), and treated with 0.5 mg / L nitrosoguanidine for 30 minutes. After the mutagenesis process was completed by the dilution met...

Embodiment 2

[0031]Embodiment two: utilize the Bacillus pumilus RX3-17 CCTCCM 206043 bacterial strain that the method for above-mentioned embodiment one step 1 and 2 obtains, obtain mature tetramethylpyrazine fermentation liquid with the SV fermentation medium containing 180 grams / liter of glucose:

[0032] Same as step 3 of the above-mentioned embodiment 1, the difference is only that the glucose concentration is changed to 180 g / L. A mature fermentation broth was obtained at 104 hours, in which the concentration of tetramethylpyrazine reached 1.79±0.08 g / L.

Embodiment 3

[0033] Embodiment three: Utilize the Bacillus pumilus RX3-17 CCTCCM 206043 bacterial strain obtained by the method of above-mentioned embodiment one step 1 and 2, obtain mature tetramethylpyrazine fermentation broth with the SV fermentation medium containing 200 grams / liter of glucose:

[0034] Same as step 3 of Example 1, the only difference is that the glucose concentration is changed to 200 g / L. A mature fermentation broth was obtained at 112 hours, in which the concentration of tetramethylpyrazine reached 2.22±0.06 g / L.

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Abstract

The invention discloses tetramethylpyrazine bacillus pumilis with high yield, called Bacillus pumilus RX3-17 and preserved in China Center for Type Culture Collection on 19th, April, 2006, whose serial number is CCTCC M 206043. This bacterial stripe is separated from soil, which have shape of stick, length of 1.5-3.0 micron, and diameter of 0.6-0.7micron with white colony. The striple has typical characteristic of bacillus pumilis, whose sequence of 16S rDNA has 99% similarity as that of other bacillus pumilis. The invention exists in field of biology and technology. Tetramethylpyrazine can be produced by Bacillus pumilus RX3-17 using glucose as substrate, solving problems of low concentration of tetramethylpyrazine by microbic fermentation method.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a newly isolated high-yield tetramethylpyrazine bacillus pumilus. Background technique [0002] Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine) was first isolated from Ligusticum wallichii in 1957. Tetramethylpyrazine has aromas of cocoa, coffee, lactose, foreign wine, and bonbon, and is widely used in the fragrance and fragrance industries such as food, cigarettes, and alcohol. Tetramethylpyrazine is also an active alkaloid that plays a major role in the traditional Chinese medicine Chuanxiong for promoting blood circulation, promoting qi and removing blood stasis. In recent years, with the in-depth research on it, people have continuously discovered its valuable value in pharmacy, and it has now been used as a bulk drug for circulatory, central nervous, respiratory and other system-related diseases, and is widely used in cardiovascular and cerebrovascular diseases, respiratory s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/31C12P17/16C12P1/04C12R1/08
CPCC12P17/12C12R1/07Y10S435/832C12R2001/07C12N1/205
Inventor 许平肖梓军魏中浩杜毅
Owner SHANGHAI KAIXIN BIOTECH
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