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A glutamic acid decarboxylase recombinant bacterium and its construction method and application

A technology of glutamic acid decarboxylase and recombinant bacteria, which is applied in the field of genetic engineering, can solve the problems of low enzyme activity of L-glutamic acid decarboxylase, difficult downstream separation and purification, complex extraction process, etc., and achieve short reaction time and high source of raw materials Broad and high enzyme activity

Active Publication Date: 2015-08-05
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage is that the enzyme activity of L-glutamic acid decarboxylase contained in the microorganisms used in the current biosynthesis method is low and the downstream separation and purification are difficult.
The common problems in the preparation of γ-GABA by using wild E. coli are as follows: 1) The enzyme activity is not high; 2) The reaction cycle is long; 3) The product concentration is low; 4) The extraction process is still relatively complicated
[0005] With the rise of molecular biology, the use of molecular cloning and exogenous expression technology can greatly increase the expression of the target enzyme in the host microorganism, and the enzyme catalytic efficiency of the genetically engineered strain constructed by this method is much higher than that of ordinary microorganisms; There are few domestic reports on the production of γ-GABA by recombinant bacteria

Method used

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  • A glutamic acid decarboxylase recombinant bacterium and its construction method and application
  • A glutamic acid decarboxylase recombinant bacterium and its construction method and application
  • A glutamic acid decarboxylase recombinant bacterium and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Extraction of Kodamaea ohmeri NH-9 genomic DNA.

[0035] The genomic DNA of Kodamaeaohmeri NH-9 in the logarithmic growth phase was extracted with the Genomic DNA Purification Kit (Takara, Dalian), and the obtained genomic DNA was detected by 1% (10g / L) agarose gel electrophoresis. The results are shown in figure 2 .

Embodiment 2

[0036] Example 2: Cloning of L-glutamic acid decarboxylase gene (gad gene) and construction of genetically engineered bacteria.

[0037] 2.1 PCR amplification of L-glutamic acid decarboxylase gene (gad gene).

[0038] According to the sequence of the L-glutamic acid decarboxylase gene from Kodamaea ohmeri NH-9 reported on Genbank, the following two primers were designed using Vector NTI software:

[0039] Primer 1: 5′-CG GGATCC ATGTTACACAGGCAC-3′ (the underline is the BamH I restriction site)

[0040] Primer 2: 5′-GCCG CTCGAG TCAACATGTTCCTCT-3′ (Xho I restriction site is underlined)

[0041] Using the genomic DNA of Kodamaea ohmeri NH-9 obtained in Example 1 as a template, the target gene fragment was amplified.

[0042] PCR system: 2 μL of genomic DNA, 2 μL of each primer 1 and primer 2, 4 μL of dNTP, 10× exTaq buffer (containing Mg 2+ ) 5 μL, exTaq enzyme 1 μL, ddH 2 O 34 μL;

[0043] PCR reaction program: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C...

Embodiment 3

[0059] Example 3: Induced expression of L-glutamic acid decarboxylase.

[0060] Prepare 100 mL of seed liquid, the medium is LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L), after 121 ° C autoclaving for 15 min, put it into a 500 mL wide-mouth Erlenmeyer flask. Insert a loop of genetically engineered strains into the seed liquid with an inoculation loop, and place it on a shaker at 37° C. at a speed of 200 rpm for overnight cultivation. Prepare 500 mL of fermentation medium containing 20 g / L of yeast powder (or 30 g / L of peptone), 8 g / L of lactose, 0.5 g / L of L-glutamic acid, and 10 g / L of sodium chloride, and distribute it in a wide-mouthed triangle with a capacity of 500 mL. In the bottle, the liquid volume of each bottle is 100mL; the above-mentioned fermentation culture is sterilized based on 121°C high-pressure damp heat for 15min. After the culture medium is cooled, add 1mL of the overnight cultured seed solution, place the Erlenmeyer flask on a shake...

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Abstract

The invention discloses a glutamic acid decarboxylase recombinant bacterium, which is an Escherichia coli introduced with L-glutamic acid decarboxylase GAD; and the L-glutamic acid decarboxylase GAD gene is from yeast Kodamaea ohmeri NH-9 and has a nucleotide sequence shown as a SEQ ID No:1. The invention also discloses the construction method and application of the above glutamic acid decarboxylase recombinant bacterium. The invention has the following advantages: (1) hydrochloric acid pyridoxal has a price half of that of 5 ' -phosphoric acid pyridoxal, so as to reduce technology cost; (2) the method has high enzyme activity, short reaction time, mild conditions, substrate concentration up to 510 g / L and concentration of a product gamma-aminobutyric acid up to 352 g / L; (3) the bacteria and the buffer after rotary evaporation can be reused, and the conversion rate is 100%, and the yield is no less than 99%; and (4) the technology is simple, green and can realize a product purity higher than 99%.

Description

technical field [0001] The invention relates to a glutamic acid decarboxylase recombinant bacterium and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] γ-GABA is the product of the decarboxylation of L-glutamic acid ( figure 1 ), its chemical name is γ-aminobutyric acid (abbreviated as γ-GABA), and its molecular formula is NH 2 CH 2 CH 2 CH 2 COOH is very soluble in water, and can exist in the form of variable molecular structure in the solution, which can be stretched into a line or form a ring like proline. γ-GABA is an amphiphilic molecule with an isoelectric point of 7.3 close to the physiological pH value. The finished product is colorless to white odorless needle crystal or crystalline powder. [0003] γ-GABA has a wide range of uses and can be used in food, feed, medicine and chemical industries. It has the following physiological functions: 1 soothe the nerves and antidepressant; 2...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/60C12N15/70C12P13/00C12R1/19
Inventor 徐虹许露顾峰詹伊婧李莎
Owner NANJING TECH UNIV
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