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Actinobacillus succinogenes capable of producing succinic acid

A technology of actinobacillus and succinic acid, applied in the biological field, can solve the problems of high cost and unreachable, and achieve the effect of reducing production cost

Active Publication Date: 2012-07-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the succinic acid production level of these strains is mostly less than 70g / L, and the production intensity is below 1.3 g / (L h), so the cost of applying to industrial production is relatively high

Method used

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  • Actinobacillus succinogenes capable of producing succinic acid
  • Actinobacillus succinogenes capable of producing succinic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Mutagenesis with acriflavine, ultraviolet light, ultraviolet-diethyl sulfate and nitrosoguanidine A. succinogenes f 3-21, 1056 single colonies were obtained; these single bacteria were subjected to "96-well plate culture-HPLC concentrated detection-anaerobic bottle re-screening" to obtain 11 mutant strains (Ⅲ-9-H, Ⅳ -7-A, Ⅳ-7-C, Ⅴ-12-B, Ⅵ-10-C, Ⅶ-11-H, Ⅷ-10-H, Ⅸ-2-C, Ⅹ-8-E, Ⅺ-8 -B and Ⅻ-7-B), the average shake flask produced succinic acid more than F 3 -21 improved by 13.9%. The 11 mutant strains were used as the starting bacterial library.

Embodiment 2

[0025] 11 mutagenized high-yielding strains were used as the starting bacterial library for the first round of fusion. The enzymatic hydrolysis conditions of the 11 strains were 0.25 mg / mL enzyme concentration, 60 min enzymatic hydrolysis time, double inactivation by ultraviolet light and heat killing, PEG After fusion, the selective regeneration double-layer plate was coated, and a total of more than 360 single colonies grew on the plate, and then these single colonies were picked into 96-well plates for flux screening. The first round of genome shuffling resulted in 4 strains of F 1 Generation of high-yielding recombinant bacteria (F 1 -Ⅰ-3-F, F 1 -Ⅱ-7-B, F 1 -Ⅲ-2-E and F 1 -IV-9-D), whose average shake flask produced succinic acid more than F 3 -21 increased by 20.2% to F 1 The second round of shuffling was carried out with the first-generation shuffled bacteria as the female parent, and 4 strains of F 2 Generation of high-yielding recombinant bacteria (F 2 -Ⅰ-5-B, F...

Embodiment 3

[0027] Put the F3-ZK seed liquid into a 5L fermenter with 3.5L fermentation liquid according to the inoculation amount of 10%, and ferment anaerobically at 37°C with a stirring speed of 200 r / min. During the fermentation process, the sugar concentration is maintained by supplementing glucose syrup 10-30 g / L, and use alkaline salt to control the pH to maintain between 5.8-6.4. Among them, seed medium (per L): glucose 5 g, yeast extract 5 g, K 2 HPO 4 ·3H 2 O 1.0 g, NaH 2 PO 4 2H 2 O 1.0 g, pH 7.0. Incubate anaerobically at 37°C for 16 hours. Fermentation medium (per L): sweet sorghum juice syrup (total reducing sugar) 20-80 g, yeast extract 0-15 g, corn steep liquor 20 g, MgCl 2 0.2 g, Na 2 HPO 4 12H 2 O 1.5 g, NaH 2 PO 4 2H 2 O 1.5 g, pH 6.5. Anaerobic culture at 37°C for 48 hours.

[0028] Fermentation curve as figure 1 . 95.6 g / L of succinic acid was produced in 48 hours.

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Abstract

The invention discloses actinobacillus succionogenes capable of producing succinic acid with high yield and a method for screening and producing the succinic acid by a fermentation method. The actinobacillus succinogenes takes CGMCC1593 as the original strain and is obtained by blending a plurality of turns of protoplast in a progressive way through a method of '96-pore plate culture, HPLC concentration detection and then anaerobic bottle re-screening'. The actinobacillus succinogenes has already been preserved on February 26, 2012 in China Center for Type Culture Collection with the preservation number CCTCCNO: M2012036. The actinobacillus succinogenes adopts fed-batch culture in a 5 to 15 L fermentation tank, 95.6 g / L succinic acid is produced in 48 hours, the production intensity is 1.99 g / (L.h), and the saccharic acid transformation rate is 0.71 g / g. Compared with other bacterial strains at home and abroad, the actinobacillus succinogenes has the advantages of high yield and lowerproduction cost.

Description

technical field [0001] The invention relates to the breeding of a microbial strain (F3-ZK) fermenting high-yielding succinic acid under anaerobic conditions and the process of fermenting and producing succinic acid, belonging to the field of biotechnology. Background technique [0002] Succinic acid, scientific name succinic acid, got its name because it was first found in amber. As an important C4 platform compound, it is widely used in medicine, food, degradable plastics and chemical industries. Succinic acid is mainly obtained by hydrolysis of maleic anhydride derived from petrochemical raw materials. Due to the non-renewable petrochemical resources and the negative impact of the petrochemical industry on the environment, the production of succinic acid by fermentation, as an emerging green process, has become a research hotspot in recent years. [0003] The production of succinic acid by fermentation has the characteristics of low carbon, clean and environmental protec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/01C12P7/46C12R1/01
Inventor 郑璞张坤坤
Owner JIANGNAN UNIV
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