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A kind of permeable glutamic acid decarboxylase engineering bacteria and preparation method thereof

A technology of glutamic acid decarboxylase and engineering bacteria, which is applied in the biological field, can solve the problems of downstream product purification adverse effects, high safety protection requirements, excessive cell lysis, etc., to improve apparent catalytic activity, low equipment investment requirements, and equipment simple effect

Active Publication Date: 2016-08-17
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Preparation of permeabilized cells by using permeabilization reagents such as organic solvents or surfactants or physical methods such as ultrasound and repeated freezing and thawing can effectively improve the apparent catalytic activity of the bacteria, but there are also some disadvantages
For example, the addition of chemical permeabilization reagents will often cause excessive lysis of cells, and the addition of organic solvents will cause residues of toxic reagents. At the same time, due to the volatility of organic solvents, the safety requirements for industrial production are high, and the addition of surfactants Can adversely affect the purification of downstream products
The freeze-thaw method and ultrasonic treatment method have problems such as high investment in equipment and difficulty in scaling up.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Strains: engineering bacteria E.coli BL21(DE3) / pET28a-gadB (see "Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain Lactobacillus brevis CGMCC1306, Annals of microbiology, 2012, 62 (2): 689 -698"), wherein the glutamic acid decarboxylase gene (gadB) is derived from Lactobacillus brevis (Lactobacillus brevis) with the preservation number CGMCC NO.1306.

[0030] Pick the engineered bacteria of glutamic acid decarboxylase (E.coliBL21(DE3) / pET28a-gadB) from the activated LB solid medium -1 In LB liquid medium with kanamycin, cultivate overnight at 37° C. with shaking at 200 r / min 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 liquid medium, cultured at 37°C, 200r / min until OD 600 At 0.6-0.8, add IPTG to make the final concentration of IPTG 0.5 μM, induce culture at 30°C, 150r / min for 6h.

Embodiment 2

[0032] Take 1 mg (dry weight of cells) of the engineered bacteria induced in Example 1, collect the bacteria by centrifugation at 10,000 g at 4°C for 5 minutes, wash the bacteria once with acetic acid buffer (0.2M, pH 4.8), and then buffer with acetic acid solution (0.2M, pH4.8) to suspend the bacteria, shake at 50°C for 30 minutes, centrifuge at 10,000×g for 1 minute, and collect the bacteria, which are GAD engineering bacteria with improved cell wall and cell membrane permeability (permeable GAD engineering bacteria) bacteria).

[0033]A unit of enzyme activity is defined as the amount of enzyme required to generate 1 μmol GABA in 1 min at 37°C (1U=1 μmol GABA in1min at 37°C). The specific activity of GAD is defined as the unit of enzyme activity per mg of dry weight cells (U·mg -1 cells, dry weight). It was determined that the apparent catalytic activity of the permeabilized GAD engineered bacteria was 1.59 U / mg, which was 2.49 times higher than the apparent catalytic act...

Embodiment 3

[0035] Take 1 mg (dry weight of cells) of the engineered bacteria induced and cultivated in Example 1, collect the bacteria by centrifugation at 10,000 g at 4°C for 5 minutes, wash the bacteria once with acetate buffer (0.2M, pH 4.8), wash the bacteria once with acetate buffer (0.2M, pH4.8) suspended bacteria, shaken at 70°C for 10min, centrifuged at 10000×g for 1min, collected the bacteria, which were GAD engineering bacteria with improved cell wall and cell membrane permeability.

[0036] Enzyme activity unit definition and specific activity definition are the same as embodiment 2. It was determined that the apparent catalytic activity of the permeabilized GAD engineering bacteria was 6.37 U / mg, which was 9.96 times higher than that of the GAD engineering bacteria without permeabilization treatment under the same culture conditions (not The apparent catalytic activity of the permeabilized GAD engineering bacteria was 0.64U / mg), and no enzyme leakage was found in the GAD acti...

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Abstract

The invention discloses a permeable glutamate decarboxylase engineering bacterium and a preparation method thereof. The preparation method comprises the steps: suspending a glutamate decarboxylase engineering bacterium body expressed with glutamate decarboxylase in water or a buffer solution, and after treating for 5-40 min at a temperature of 50-70 DEG C, collecting thallus to obtain the permeable glutamate decarboxylase engineering bacterium, wherein the permeable glutamate decarboxylase engineering bacterium comprises a host cell and a glutamate decarboxylase gene transferred into the host cell, and the host cell is escherichia coli. The invention further provides the permeable glutamate decarboxylase engineering bacterium prepared by adopting the preparation method. The preparation method disclosed by the invention is simple and convenient, low in cost, easy to operate, and capable of effectively improving the appearance catalytic activity of the glutamate decarboxylase of the bacterium body.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a permeable glutamic acid decarboxylase engineering bacterium and a preparation method thereof. technical background [0002] γ-aminobutyric acid (γ-aminobutyric acid, GABA) is an important inhibitory neurotransmitter in the human central nervous system. Liver and kidney and other physiological functions. Therefore, GABA has broad application value in food and medicine. 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. [0003] Currently, GABA can be prepared by chemical synthesis and biocatalysis. Compared with the chemical synthesis method, the biological preparation method using glutamate decarboxylase (Glutamate decarboxylase, GAD; EC4.1.1.15) to catalyze the decarboxylation of L-glutamic acid to generate GABA has abundant raw ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12R1/19C12R1/24
Inventor 梅乐和赵伟睿胡升黄俊雷引林姚善泾
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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