Constitutive expression genetically engineered bacterium and application thereof to produce L-alanine

A genetically engineered bacteria and constitutive expression technology, applied in the field of bioengineering, can solve the problems of high production cost and less by-products, and achieve the effects of easy operation, simple production process and simple process.

Inactive Publication Date: 2015-11-04
CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the widely used L-alanine production method is microbial conversion method, which catalyzes the decarboxylation reaction of L-aspartic acid by L-aspartic acid-β-decarboxylase to produce L-alanine, with reaction conditi

Method used

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  • Constitutive expression genetically engineered bacterium and application thereof to produce L-alanine
  • Constitutive expression genetically engineered bacterium and application thereof to produce L-alanine
  • Constitutive expression genetically engineered bacterium and application thereof to produce L-alanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1. Genetically engineered bacteria E . coli / pETP p - asd build

[0048] (1) Inoculation of activated E. coli CICC 11022S in LB medium 37 o C 200 rpm shaking culture overnight.

[0049] (2) 1% inoculum transfer E. coli 11022S in fresh LB medium, 37 o Shake at C 200 rpm for 2-4 h.

[0050] (3) Transfer the culture to a 1.5 mL sterile centrifuge tube and place on ice for 30 min, 4 o Centrifuge at 4000 rpm for 5 min, and discard the supernatant.

[0051] (4) Add 200 μL of pre-cooled 0.1 mol / L CaCl to the cell pellet 2 , ice bath 5 min, 4 o Centrifuge at 4000 rpm for 5 min, and discard the supernatant.

[0052] (5) Add 50 μL of pre-cooled 0.1mol / L CaCl to the cell pellet 2 and 1 μL vector pETPp- asd , mix well, and ice bath for 20-30 min.

[0053] (6) 42 o After heat shock for 30s at C, quickly ice-bath for 2-3 min, add 400 μL fresh LB medium, 37 o C 160 rpm shake recovery culture for 1 h.

[0054] (7) Take 100 μL of the recovery culture ...

Embodiment 2

[0055] Example 2. Strains E . coli / pETP p - asd fermentation culture

[0056] Streak the genetically engineered strain onto an LB medium plate containing 100 mg / L ampicillin and 1.5 – 2.0% agar, 30±1 o C cultured for 12±2 hours to obtain a single colony.

[0057] Under sterile conditions, use a sterilized toothpick to pick a single colony on the plate, inoculate the fermentation medium, 30±1 o C culture on a shaker for 12±2 hours to obtain seed liquid.

[0058] Inoculate the seed liquid into the fermentation medium with 0.1 – 5% inoculum, 30±1 o C culture on a shaker for 12±2 hours to obtain a fermentation broth.

[0059] Wherein, the formula of the LB medium is: 5 g of yeast powder, 10 g of peptone, 10 g of sodium chloride, 115 o C for 30 minutes.

[0060] The formula of the fermentation medium is: 10 g of fumaric acid, 10 g of corn steep liquor dry powder, 0.2 g of magnesium sulfate, 1.0 g of potassium dihydrogen phosphate, 1.5 g of sodium chloride, and ammonia w...

Embodiment 3

[0061] Example 3. E . coli / pETP p - asd High-density transformation of whole cells to produce L-alanine

[0062] Put fumaric acid into the conversion tank, add a certain amount of water, then add ammonia water while stirring, and measure the pH change of the solution with a pH meter. After the fumaric acid is completely dissolved, adjust the pH to 7.0-8.5 with ammonia water to control Fumaric acid final concentration 0.5 – 1.2 mol / L. cultured by fermentation E . coli / pETP p - asd The bacterial liquid was collected by centrifugation, suspended in normal saline, and added to the transformation tank until the bacterial cell density OD 600 = 10 – 20. Adjust temperature 40 – 50 o C, stirring at 50 – 200 rpm for transformation. Regular sampling, high performance liquid chromatography quantitatively detects the production of L-alanine (attached image 3 ). After 9 hours, the production of L-alanine can reach 112.7 g / L, the production rate is 12.5 g / (L·h), and the ...

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Abstract

The invention relates to a constitutive expression genetically engineered bacterium Escherichia coli CICC 11032S for producing L-alanine. The host is escherichia coli CICC 11022S (same as CGMCC No. 5450) producing L-aspartase, and contains L-aspartate beta-decarboxylase gene of comamonas testosterone CICC 11023S (same as CGMCC No. 6083). By utilizing the constructed engineered bacterium for whole-cell high-density catalysis, fumaric acid and ammonia water can be efficiently conversed for producing L-alanine, the output is 112.7 g/L, the production rate is 12.5 g/(L*h), and the conversion rate is 93.9%. The substrates are cheap, the production process is simple, by-products are less, the production cost is effectively reduced, and extremely good application value is provided.

Description

technical field [0001] The invention relates to a genetically engineered bacterium and its application, in particular to a genetically engineered bacterium producing L-alanine and its application, belonging to the technical field of bioengineering. Background technique [0002] L-alanine, also known as L-alpha-alanine (L-Ala), is one of the amino acids that are closely related to glucose metabolism in organisms and are in high demand. With the deepening of research and development, L-alanine has been widely used in food and pharmaceutical industries. [0003] Food industry: As an additive, L-alanine can significantly increase the utilization rate of protein in beverages. At the same time, because it can be directly absorbed by cells, it can quickly eliminate fatigue and boost spirits after drinking. L-alanine has a mild sweet taste, 70% of the sweetness of sucrose, and can increase the sweetness. It can be used to improve the taste of artificial sweeteners and the sour tast...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/06C12R1/19
Inventor 程池徐友强裴疆森张奇马玉岳姜增妍
Owner CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD
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