A kind of arginine decarboxylase genetically engineered bacteria and its high-density fermentation culture method

A technology of arginine decarboxylase and genetically engineered bacteria, which is applied in the field of bioengineering, can solve the problem that the enzyme activity and transformation ability of recombinant arginine decarboxylase are different, affect the fermentation production capacity of genetically engineered bacteria, and the expression level of arginine decarboxylase. It can reduce the fermentation cost, improve the utilization rate, and achieve the effect of stable performance.

Active Publication Date: 2020-12-08
JINAN GUOLI BIOLOGICAL SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Based on prior art reports and combined with actual production experience, the inventors found that the recombinant arginine decarboxylase enzyme activity and conversion ability of the obtained recombinant arginine decarboxylase were significantly different in constructing genetically engineered bacteria with genes encoding arginine decarboxylase from different sources, and now The concentration of bacteria in the fermentation liquid obtained by the culture method in the prior art is not high, which leads to the low expression of arginine decarboxylase
At present, there are almost no reports on the use of the obtained recombinant bacteria for high-density fermentation to produce arginine decarboxylase
In addition, different fermentation media, different induction culture methods and different fermentation regulation methods will also affect the fermentation production capacity of genetically engineered bacteria

Method used

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  • A kind of arginine decarboxylase genetically engineered bacteria and its high-density fermentation culture method
  • A kind of arginine decarboxylase genetically engineered bacteria and its high-density fermentation culture method
  • A kind of arginine decarboxylase genetically engineered bacteria and its high-density fermentation culture method

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Embodiment 1

[0060] The construction of embodiment 1 engineering bacterium

[0061] Expressing the arginine decarboxylase derived from E.coliMG1655 strain, the base sequence of the arginine decarboxylase gene in the genetically engineered bacteria is shown in SEQ ID NO.1. The construction method of genetically engineered bacteria is as follows:

[0062] Since there is no intron in the adiA gene of E.coliMG1655, the bacterial genome was extracted using a bacterial genome extraction kit. And design primers according to the nucleotide sequence of the target gene. And the restriction enzyme cutting sites SacI and BamHI were respectively added in the forward primer and the reverse primer.

[0063] Forward primer F: 5'-CGAGCTCGAATGCGAAAGTGCGTGTATTG-3', as shown in the nucleotide sequence of SEQ ID NO.3;

[0064] Reverse primer R: 5'-CGGGATCCCGTACTTTCATAATTAACAAC-3', as shown in the nucleotide sequence of SEQ ID NO.4.

[0065] The PCR reaction system and conditions are as follows:

[0066] P...

Embodiment 2

[0076] Embodiment 2: Optimization of fermentation medium and fermentation conditions

[0077] (1) Optimization of fermentation medium

[0078] By screening, it is determined that the composition of the fermentation medium is:

[0079] Glycerin 5-52g / L, peptone 8-45g / L, yeast powder 2-36g / L, Na 2 HPO 4 12H 2 O 6~50g / L, K 2 HPO 4 ·3H 2 O 3-46g / L, potassium dihydrogen phosphate 3-35g / L, sodium chloride 0.5-18g / L, magnesium sulfate 0.5-15g / L, and the balance is water.

[0080] Using eleven factors and two levels L 12 (2 11 ) Orthogonal test method to further optimize the Escherichia coli fermentation medium, and take the enzyme activity of the unit fermentation broth as the response value, determine the optimal medium composition: glycerol 7g / L, peptone 15g / L, yeast powder 5g / L, Na 2 HPO 4 12H 2 O 18g / L, K 2 HPO 4 ·3H 2 O 13g / L, potassium dihydrogen phosphate 5g / L, sodium chloride 1g / L, magnesium sulfate 1g / L.

[0081] In order to verify the superiority of this cul...

Embodiment 3

[0089] Embodiment 3: the cultivation of seed liquid

[0090] (1) small test cultivation

[0091] Take the glycerol tube and inoculate it on the Petri dish plate to activate the strains, and cultivate for 24 hours; store the Petri dish plate in a refrigerator at 4°C; use an inoculation loop to dig out a ring of plate seeds under aseptic conditions and inoculate it in the seed medium (50mL / 500mL Erlenmeyer flask). The composition of the medium is: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, pH natural, sterilized at 121°C and 0.1Mpa pressure for 20min. Then the inoculated seed shake flask was cultured in a constant temperature air bath shaker at 37° C. and 180 rpm for 16 hours to obtain a seed culture solution for small-scale fermentation.

[0092] (2) Pilot test cultivation

[0093] Take the glycerol tube and inoculate it on the Petri dish plate to activate the strains, and cultivate for 24 hours; store the Petri dish plate in a refrigerator at 4°C; use an inoculation loo...

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Abstract

The present invention belongs to the technical field of bioengineering, relates to a high-density cell fermentation method, and particularly relates to an arginine decarboxylase genetically engineeredbacterium and a high-density fermentation culture method thereof. The genetically engineered bacterium uses E.coliK12 as a host and pET28A as a vector, and expresses an adiA gene. A nucleotide sequence of the adiA gene is shown in SEQ ID NO.1. The genetically engineered bacterium can secrete a highly active recombinant arginine decarboxylase, can be applied in pilot scale test high-density fermentation culture, and enables industrialized production of the recombinant arginine decarboxylase to be possible. The fermentation culture method optimizes fermentation culture medium screening and fermentation induction culture conditions, controls supplement time, supplement rate and dosage of glycerin and ammonia water, and supply of carbon source and nitrogen source, adjusts stirring rotating speed and ventilation amount to control oxygen supply, and thus realizes high-density growth of the bacterium and large expression of the target protease.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a high-density cell fermentation method, in particular to an arginine decarboxylase genetically engineered bacterium and a high-density fermentation culture method thereof. Background technique [0002] Arginine decarboxylase (Arginine decarboxylase, ADC) is a pyridoxal phosphate (PLP)-dependent decarboxylase, which uses L-arginine as a substrate to generate agmatine after decarboxylation (see figure 1 ). As an important biogenic amine, agmatine has great physiological functions and medical value. At present, enterprises mainly use chemical methods to produce agmatine, which is complicated in the production process and seriously pollutes the environment. The biological enzymatic method has the characteristics of environmental protection and high efficiency. How to use arginine decarboxylase to produce agmatine on a large scale has become a research hotspot. [0003] There ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/60C12N9/88C12P13/00C12R1/19
CPCC12N9/88C12P13/001C12Y401/01019
Inventor 袁建国张言慧高先岭
Owner JINAN GUOLI BIOLOGICAL SCI & TECH
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