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Mixed bacteria secondary fermentation technology and application thereof in industrial production of L-alanine

A secondary fermentation, alanine technology, applied in fermentation, microorganism-based methods, microorganisms, etc., can solve problems such as limitations, inability to dock industrialization, etc.

Pending Publication Date: 2020-05-12
BEIJING CITY UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cheng Chi et al. constructed a genetically engineered bacterium that simultaneously expressed L-aspartase and L-aspartic acid-β-decarboxylase to catalyze the reaction of fumaric acid, catalyzed by single-bacteria fermentation, and achieved catalysis with fumaric acid as the substrate for the first time Produce L-alanine (CN 105018405), but this method uses genetically engineered bacteria to ferment, which has restrictions on food industry industrialization projects, and currently cannot be industrialized

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  • Mixed bacteria secondary fermentation technology and application thereof in industrial production of L-alanine
  • Mixed bacteria secondary fermentation technology and application thereof in industrial production of L-alanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Embodiment 1 L-aspartase catalyzed pH range

[0011] The strain Escherichia coli CGMCC 9850 obtained by the previous screening and mutagenesis was fermented and cultured, and the fermentation medium was (g / L): corn steep liquor dry powder 13.0, magnesium sulfate 0.2, potassium dihydrogen phosphate 1.0, sodium chloride 1.5, fumaric acid 5.0, glucose 5.0, and ammonia water to adjust the pH to 6.5-7.0, collect the fermentation broth, and measure the enzyme activity under the conditions of pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, and the results are shown in the attached figure 1 As shown, according to the measured enzyme activity and the corresponding pH value, the fitting curve is drawn, and it can be seen that within the pH range of 6.0-8.0, there is no significant difference in the L-aspartase activity of Escherichia coli CGMCC 9850, and the pH range Synchronized with Comamonas testosteroni CGMCC 6083, laying the foundation for mixed fermentation and joint catalysis. ...

Embodiment 2

[0012] Embodiment 2 fermentation medium optimization

[0013] Select the key factors of fermentation, carbon source, nitrogen source and inducer, and use single factor analysis combined with orthogonal experiment method to screen glucose, sucrose, citric acid, fumaric acid, glutamic acid, peptone, beef extract, corn steep liquor dry powder, yeast Flour, hydrolyzed cottonseed protein and other carbon sources, nitrogen sources and inducers were comprehensively evaluated by bacterial growth and enzyme activity. The results showed that the best nitrogen sources were peptone and corn steep liquor powder, and the best carbon sources were sodium glutamate and rich For malic acid, further optimize the concentration to obtain a primary fermentation medium formula range (g / L): peptone 5-10, corn steep liquor powder 10-20, sodium glutamate 6-10, magnesium sulfate 0.1-0.3, dihydrogen phosphate Potassium 0.5-1.5, the pH is adjusted to 6.5-7.5 with NaOH, the final concentration of the secon...

Embodiment 3

[0014] Embodiment 3 50L tank fermentation and 20t tank fermentation

[0015] Pilot and trial production were carried out in 50L and 20t fermenters with the above-mentioned optimized optimal medium, and secondary fermentation was carried out in stages. Comamonas testosteroni CGMCC 6083 was fermented once, and Escherichia coli CGMCC 9850 was inoculated after culturing at 36°C for 8 hours. The fermentation was continued for 12 hours. At the end of the fermentation, the vigor of the two strains was the highest, reaching above 26000U / mL and 4900U / mL respectively. Since the activity of L-aspartic acid enzyme is high enough, and it is the rate-limiting step of co-production, although the activity of L-aspartic acid-β-decarboxylase in mixed-bacteria fermentation is slightly lower than that of single-bacteria fermentation, it has no effect on production efficiency. influences.

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Abstract

The invention discloses a mixed bacteria secondary fermentation technology and an application thereof in industrial production of L-alanine, and belongs to the field of microbial fermentation engineering. Escherichia coli and comamonas testosterone are used as starting strains, the composition of a mixed fermentation medium and a fermentation process are optimized, and then the mixed secondary fermentation method is established successfully. Under optimum fermentation conditions, enzyme activity of L-aspartase in a fermentation broth reaches 4900 U / mL, the pH tolerance and application range ofL-aspartase is 6.0-8.0, and activity of aspartase decarboxylase reaches 26000 U / mL. The fermentation broth is taken as an enzyme source to catalyze fumaric acid and ammonia water, double enzyme catalysis can be realized, and L-alanine can be obtained in one step, wherein the molar conversion rate reaches 98.6%, the catalytic time is 8 h, and catalytic efficiency is increased by 300%. The mixed bacteria secondary fermentation technology has an industrial application value.

Description

technical field [0001] The invention relates to a mixed bacteria secondary fermentation technology and its application, belonging to the field of microbial fermentation engineering. Background technique [0002] The mixed-bacteria fermentation technology has the advantage that pure-bred fermentation is difficult to achieve. It can make full use of the medium and equipment, and undergo the same process in the same fermentation tank to achieve fermentation, which can improve the quality and quantity of the target product or obtain two or more products. . However, the reaction mechanism of mixed bacteria fermentation is relatively complex, especially the culture conditions of different species of bacteria are very different, and it is difficult to unify. Therefore, industrial production is rarely used. At present, it is mainly used in traditional solid fermentation, such as liquor. In addition, the catalytic conversion of the fermentation broth prepared by mixed bacteria ferme...

Claims

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Application Information

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IPC IPC(8): C12P13/06C12R1/19C12R1/01
CPCC12P13/06
Inventor 张奇姜国政崔颖杨奕马秀亮
Owner BEIJING CITY UNIVERSITY
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