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Genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and immobilization method

A technology of genetically engineered bacteria and Enterococcus faecium, applied in the direction of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problem of low GAD expression, inability to reuse and continuous production, free enzymes Difficult to recycle and other issues, to achieve the effect of cost reduction

Pending Publication Date: 2019-04-19
LINGNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The technical problem to be solved by the present invention is to overcome the generally low expression of GAD in wild strains, low activity, difficult recovery of free enzymes, difficulty in immobilization, short half-life, inability to reuse and continuous production, high cost, and difficulty in meeting GABA or DL-Glu industrial production needs to provide a genetically engineered bacterium for efficient immobilization of Enterococcus faecium GAD, and provide an immobilization method for efficiently producing GAD immobilized enzyme of Enterococcus faecium

Method used

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  • Genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and immobilization method
  • Genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and immobilization method
  • Genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and immobilization method

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Experimental program
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Embodiment 1

[0080] The construction of embodiment 1 genetically engineered bacteria Escherichia coli (DH5α-LNSF1)

[0081] S1. Insert Enterococcus faecium LNSF2 into PSB or MRS medium, culture at 37°C for 12 hours, take 5mL of the culture solution and centrifuge at 8500r / min, collect the bacterial precipitate, discard the supernatant, and follow the Kangwei Century Bacterial Genome Extraction Kit Instructions Extract genomic DNA and store at -20°C for later use; design efa-1 and efa-2 primers, the biological sequences of primers efa-1 and efa-2 are shown in Table 1, and entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize ;Using efa-1 and efa-2 as primers, using Enterococcus faecium LNSF2 genomic DNA as a template, and using full gold TransStart Fast Pfu DNA polymerase for PCR reaction;

[0082]The PCR reaction conditions are: 95°C for 2min; 95°C for 20s, 50°C for 30s, 72°C for 55s, cycle 33 times; 72°C for 5min;

[0083] After the PCR reaction was completed, 1 μL Taq DNA Pol...

Embodiment 2

[0093] Example 2 Preparation of phosphoric acid modified chitin immobilized insoluble carrier

[0094] SS1. Add distilled water 3 times the mass of chitin, mix thoroughly to prepare chitin slurry, stir rapidly and slowly add concentrated phosphoric acid that is 50 times the mass of chitin that has been pre-frozen at -7°C under ice bath conditions, Stir until the mixture becomes transparent, ice bath for 1h (stir occasionally);

[0095] SS2. Under rapid stirring, add ice water 500 times the mass of chitin to the solution obtained in step SS1 in 4 times, let it stand for 30 minutes to fully precipitate the chitin, centrifuge at 4°C and 8500r / min for 15 minutes, and collect the chitin precipitate;

[0096] SS3. Add ice water 20 times the weight of the chitin precipitate, stir to resuspend, then centrifuge at 4°C, 8500r / min for 15min, collect the chitin precipitate, repeat twice to wash away phosphoric acid;

[0097] SS4. Then add distilled water according to 20 times of the mass...

Embodiment 3

[0100] Example 3 Preparation of NaOH / urea mixed solution modified chitin immobilized insoluble carrier

[0101] SS1. Add NaOH / urea mixed solution 50 times its mass to chitin, stir and mix well, freeze at -30°C for 4 hours, thaw at room temperature and stir quickly, then freeze at -30°C for 4 hours, thaw at room temperature and stir quickly, repeat Freeze and thaw until the chitin dissolves into a transparent state;

[0102] SS2. Add ice water with 500 times the mass of chitin to the solution obtained in step SS1 while stirring, let it stand for 30 minutes to fully precipitate chitin, centrifuge at 4°C and 8500r / min for 15 minutes, remove the supernatant, and collect the chitin precipitate;

[0103] SS3. Repeat washing the chitin precipitate 4 times with distilled water 20 times of the chitin precipitation to remove NaOH and urea;

[0104] SS4. Add 20 times of distilled water to the chitin precipitation, stir and resuspend, use 1mol / L HCl to adjust the pH close to neutral, neu...

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Abstract

The invention discloses a genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and an immobilization method. The invention provides the genetically engineered bacterium, namely Escherichia coli (DH5alpha-LNSF1), for efficiently immobilizing enterococcus faecium GAD. The engineered bacterium is preserved in the Guangdong Microbial Culture Collection Center on September 18th, 2018, and the preservation number is GDMCC No.60445. The genetically engineered bacterium can be used for efficiently producing enterococcus faecium GAD immobilized enzyme. According to the method, a modified chitin affinity adsorption CBM-GAD fusion enzyme crude enzyme solution is used for obtaining the enterococcus faecium GAD immobilized enzyme. Modified chitin and CBM-GAD fusion enzyme which are used in the method are high in affinity and firm in combination, and the obtained GAD immobilized enzyme is high in activity and stability. The problems that GAD is difficult in immobilization, short in half-life period and high in cost are solved. In addition, the immobilization method is rapid and high in efficiency, the cost is greatly reduced, and the method has wide application prospects in the aspect of industrial production of efficient production of enterococcus faecium GAD immobilized enzyme.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and more specifically relates to a genetically engineered bacterium for efficient immobilization of Enterococcus faecium glutamic acid decarboxylase and an immobilization method. Background technique [0002] Glutamate decarboxylase (glutamate decarboxylase, GAD, EC4.1.1.15) specifically catalyzes the decarboxylation of L-glutamic acid (L-glutamic acid, L-Glu) α-carboxyl, in the γ-aminobutyric acid ( γ-aminobutyric acid, GABA) biosynthesis and chiral material DL-glutamic acid (DL-glutamic acid, DL-Glu) resolution has important applications (Brazilian Journal of Microbiology,2012,43(4):1230-1241 ; Amino Acids, 2016, 48(11): 2519-2531; Chemical and Biological Engineering, 2013, 30(11): 55-56.). Due to the good safety of lactic acid bacteria, it is easy to apply in fermented food, and lactic acid bacteria GAD has received great attention. Lactobacillus paracasei (Food Microbiol 2005,22:497-504), ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/88C12N11/10C07K19/00C12R1/19
CPCC12N9/88C12N11/10C12N15/70C12Y401/01015C07K2319/00
Inventor 杨胜远韦锦
Owner LINGNAN NORMAL UNIV
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