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Fumaric acid producing strain and its mutagenesis screening method and application

A fumaric acid and bacteria-producing technology, which is applied to fumaric acid-producing strains, fermented fumaric acid, and mutagenesis screening fields, can solve the problem of unclear application prospects, backward fumaric acid fermentation technology, and biosynthesis of fumaric acid. Problems such as low production

Inactive Publication Date: 2008-01-09
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Later, due to reasons such as low yield, high cost, many by-products, and unclear application prospects of biosynthetic fumaric acid, further in-depth research was not possible, making the current fermentation technology of fumaric acid in my country seriously lagging behind the international level.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: compound mutagenesis

[0061] (1) Inoculate the Rhizopus oryzae with fumaric acid production ability screened out from the soil under forest trees by conventional methods on solid medium 1 (yeast extract juice 3g / L, malt extract juice 3g / L, peptone 3g / L , glycerol 20g / L, agar 20g / L), cultured at 35°C for 5 days, after the spores matured, the spores were eluted with pH6. In the triangular flask of beads, shake fully at 35°C and 130rpm for 20min to disperse the spores evenly, filter with sterilized absorbent cotton, the filtrate is the spore suspension 1, adjust the spore concentration to 10 6 pcs / ml spare.

[0062] (2) Mix 20ml of the above-mentioned spore suspension 1 with sterilized liquid medium 1 (yeast extract juice 3g / L, malt extract juice 3g / L, peptone 3g / L, glycerol) in a ratio of 1:1 (volume ratio). 20g / L), mixed and put into a 250ml Erlenmeyer flask, placed on a shaker at 35°C and 150rpm for 2 hours, centrifuged and washed to collect spores, and...

Embodiment 2

[0065] Embodiment 2: strain screening

[0066] (1) Preliminary screening: the spore suspension obtained after compound mutagenesis was spread and inoculated on solid medium 2 (adding bromocresol green and sodium deoxycholate to the PDA medium, so that the concentrations were 0.1g / L and 1.0g / L), the fumaric acid produced during the growth of Rhizopus oryzae can make the culture medium around the colony appear yellow discoloration circles. By measuring the diameter of the color change circle and the diameter of the colony, calculate the ratio between the two, and select the strain with a larger ratio for preservation for the second primary screening.

[0067] The larger culture medium of the yellow discoloration circle obtained by the primary screening for the first time was moved into solid medium 3 (KMnO 4 2g / L, KBr 2.5g / L, agar 20g / L) carry out the second primary screening. After culturing for 24 hours, the fumaric acid produced by the growth of the strain will be secreted...

Embodiment 3

[0073] Embodiment 3: Shake flask fermentation

[0074] (1) Inoculate the fumaric acid high-yield strain obtained by mutagenesis screening on slant medium (yeast extract juice 3g / L, malt extract juice 3g / L, peptone 3g / L, glycerol 20g / L, agar 20g / L), According to the cultivation method described in embodiment 2 (2), the preparation concentration is 10 6 The spore suspension per ml was used for later use.

[0075] (2) Insert the above-mentioned spore suspension into the seed culture medium (glucose 50g / L, urea 2g / L, KH 2 PO 4 1.0g / L, MgSO 4 .7H 2 O 0.5g / L, MnSO 4 .H 2 O0.05g / L, CuSO 4 .5H 2 (0.006g / L, pH3.0), placed on a shaker with a rotating speed of 220rpm, cultivated at 35°C for 48 hours to obtain a seed solution.

[0076] (3) Insert the above-mentioned seed liquid into the fermentation medium (glucose 120g / L, urea 1g / L, CaCO 3 80g / L, KH 2 PO 4 0.6g / L, MgSO 4 .7H 2 O 0.5g / L, ZnSO 4 .7H 2 O 0.02g / L, FeSO 4 .7H 2 (0.01g / L), placed 35 DEG C, rotating speed i...

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Abstract

A (Rhizopus Oryzae)ME-F11, its mutation screening method and method for fermenting fumaric acid are disclosed. The preserving code is CCTCC NO: M 207066. The fumaric acid concentration reaches to 90-120g / L, total yield reaches to 90-95 wt% . It's simple and can be used for industrial production.

Description

technical field [0001] The invention belongs to the field of bioengineering technology, especially the field of organic acid production by microbial fermentation, and specifically relates to a fumaric acid-producing bacterial strain, a mutagenesis screening method for obtaining the bacterial strain, and a method for producing fumaric acid by fermentation of the bacterial strain . Background technique [0002] Fumaric acid, also known as fumaric acid and fumaric acid, is an important intermediate metabolite of organisms and an important organic chemical raw material, widely used in food, medicine, materials and chemical industries. And as an important four-carbon platform compound, fumaric acid can also produce L-aspartic acid, malic acid, succinic acid, maleic acid, 1,4- Bulk chemicals such as butanediol (BDO), gamma-butyrolactone (GBL) and tetrahydrofuran (THF). [0003] At present, the production capacity of fumaric acid in the world is 340,000 tons per year, of which th...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N13/00C12N15/01C12P7/46C12R1/845
Inventor 黄和高振李霜张昆刘宁徐晴付永前韦萍
Owner NANJING UNIV OF TECH
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