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Method for improving apparent catalytic activity of glutamic acid decarboxylase recombinant engineering bacteria

A technology of glutamic acid decarboxylase and recombinant engineering bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, and adding compounds to stimulate growth, etc., can solve the problem of increasing production costs, increasing multi-step process steps, and large-scale Unfavorable production and other problems, to achieve the effect of vigorous growth, low sensitivity, avoiding investment in steps and related equipment operation

Inactive Publication Date: 2014-05-07
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Treating the cultured cells with permeabilization reagents such as organic solvents or surfactants, or physical methods such as ultrasound and freeze-thawing can effectively improve the apparent catalytic activity of the bacteria, but the treatment of the cultured cells will undoubtedly cause Increase multi-step process steps, increase production costs, especially unfavorable for large-scale production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Pick the monoclonal cells from the activated LB solid medium and insert into 5mL containing kanamycin (50μg·mL -1 ) in LB liquid medium at 37°C with 200r / min shaking overnight to obtain seed liquid. The seed solution was inoculated into the seed solution containing kanamycin (50 μg·mL) according to the volume fraction of 1% inoculum -1 ) in LB medium, cultured at 37°C, 200r / min until OD 600 Add IPTG at 0.6-0.8, so that the final concentration of IPTG is 0.5μM, and at the same time add the final concentration of 3μg·mL -1 ampicillin, 30°C, 150r / min induction culture for 6h. At the same time, cultured cells without ampicillin were used as blank control.

[0031]After the cultivation, a certain amount of bacterial liquid was collected at 4°C and centrifuged at 10,000 g for 5 minutes to collect the bacterial cells, and the bacterial cells were washed once with 200 mM acetic acid buffer (pH 4.8), and the apparent catalytic activity of the bacterial cells was determined t...

Embodiment 2

[0033] Pick the monoclonal cells from the activated LB solid medium and insert into 5mL containing kanamycin (50μg·mL -1 ) in LB liquid medium at 37°C with 200r / min shaking overnight to obtain seed liquid. The seed solution was inoculated into the seed solution containing kanamycin (50 μg·mL) according to the volume fraction of 1% inoculum -1 ) in LB medium, cultured at 37°C, 200r / min until OD 600 Add IPTG at 0.6-0.8, so that the final concentration of IPTG is 0.5μM, and at the same time add the final concentration of 5μg·mL -1 ampicillin, 30°C, 150r / min induction culture for 6h. At the same time, cultured cells without ampicillin were used as blank control.

[0034] After the cultivation, a certain amount of bacterial liquid was collected at 4°C and centrifuged at 10,000 g for 5 minutes to collect the bacterial cells, and the bacterial cells were washed once with 200 mM acetate buffer (pH 4.8), and the apparent catalytic activity of the bacterial cells was determined to b...

Embodiment 3

[0036] Pick the monoclonal cells from the activated LB solid medium and insert into 5mL containing kanamycin (50μg·mL -1 ) in LB liquid medium at 37°C with 200r / min shaking overnight to obtain seed liquid. The seed solution was inoculated into the seed solution containing kanamycin (50 μg·mL) according to the volume fraction of 1% inoculum -1 ) in LB medium, cultured at 37°C, 200r / min until OD 600 Add IPTG at 0.6-0.8, so that the final concentration of IPTG is 0.5μM, and at the same time add the final concentration of 10μg·mL -1 CTAB, 30°C, 150r / min induction culture for 6h. At the same time, the cultured cells without CTAB were used as blank control.

[0037] After the cultivation, a certain amount of bacterial liquid was collected at 4°C and centrifuged at 10,000 g for 5 minutes to collect the bacterial cells, washed with 200 mM acetic acid buffer (pH 4.8) once, and the apparent catalytic activity of the bacterial cells was measured to be 1.40 U / mg. Compared with the ba...

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Abstract

The invention discloses a method for improving apparent catalytic activity of glutamic acid decarboxylase recombinant engineering bacteria. The method comprises the steps of inoculating the glutamic acid decarboxylase recombinant engineering bacteria into a culture medium for shake cultivation so as to obtain a seed solution; inoculating the seed solution into the culture medium for shake cultivation, adding an inducer for induction cultivation when OD600 is 0.6-0.8 hour, and simultaneously adding a cell wall synthesis inhibitor or surfactant into the culture medium so as to interfere in synthesis of recombinant bacteria cell walls or (and) cell membranes to achieve the purposes of enhancing the permeability of the bacteria and improving the apparent catalytic activity of the bacteria; and after induction cultivation, collecting the bacteria so as to obtain the glutamic acid decarboxylase recombinant engineering bacteria with the apparent catalytic activity improved. The method for improving apparent catalytic activity of glutamic acid decarboxylase recombinant engineering bacteria is free from multiple processing steps for permeability treatment after cultivation of conventional bacteria and is simple, convenient and efficient, and low in cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for improving the apparent catalytic activity of glutamic acid decarboxylase recombinant engineering bacteria. technical background [0002] γ-aminobutyric acid (γ-aminobutyric acid, GABA) is an important inhibitory neurotransmitter in the mammalian central nervous system. Various physiological functions such as protecting the liver and benefiting the kidney. In addition, GABA can be used as a precursor substance to synthesize chemical products such as biodegradable material polyamide-4 and environmentally friendly plastic solvent N-pyrrolidone. Therefore, GABA has broad application value in the fields of food, medicine and chemical industry. [0003] The methods for preparing GABA mainly fall into two categories: chemical synthesis and biosynthesis, and biosynthesis includes plant enrichment and microbial fermentation. Biological synthesis of GABA uses glutamate decarbox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/38C12R1/19
Inventor 梅乐和赵伟睿黄俊胡升雷引林金志华姚善泾
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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