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Cloning, expression and application of Lactobacillus brevis glutamate decarboxylase gene

A bacterial glutamic acid decarboxylase, glutamic acid decarboxylase technology, applied in the field of preparation of gamma-aminobutyric acid, can solve the problems of increasing production costs, and achieve high production efficiency, high product purity, and low production costs Effect

Inactive Publication Date: 2011-06-01
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This requires complex separation and purification of the product to obtain a product that meets the quality requirements, which increases the production cost

Method used

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  • Cloning, expression and application of Lactobacillus brevis glutamate decarboxylase gene
  • Cloning, expression and application of Lactobacillus brevis glutamate decarboxylase gene
  • Cloning, expression and application of Lactobacillus brevis glutamate decarboxylase gene

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Experimental program
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Embodiment 1

[0031] Cloning of embodiment 1 Lactobacillus breve glutamic acid decarboxylase gene

[0032] Pick the preserved strain Lactobacillus brevis (Lactobacillus brevis) with the preservation number CGMCC NO.1306, activate it on the agar slant medium at 30°C for 24h, then inoculate it on 25ml GYP liquid medium, and place it in a 250ml Erlenmeyer flask Cultivate statically at 30°C until the bacteria reach the mid-exponential growth stage (about 24 hours), then take 4.5ml of the bacterial solution and centrifuge at 10,000rpm for one minute, discard the supernatant, and use the genome extraction kit produced by Shanghai Sangong, according to the instructions Genome extraction. It was verified by electrophoresis that the extracted product was a single band, which was comparable in size to the genome, without RNA contamination.

[0033] The existing bacterial GAD gene sequences in the GenBank database were searched, and after analysis and comparison, five GAD gene sequences of lactic aci...

Embodiment 2

[0043] The construction of embodiment 2 object gene prokaryotic expression vector

[0044] Since the restriction endonuclease cutting sites (BamH I and EcoR I) have been introduced at both ends of the cloning primer when cloning the target gene, when constructing the expression vector, as long as the required plasmid is selected, BamH I and EcoR I respectively perform double enzyme digestion on the target gene and the carrier plasmid, and then connect the target gene and the carrier plasmid according to the conventional double enzyme digestion ligation method to obtain a vector that can be used for expression. Specifically, in this example, pET-28a(+) was used as the carrier plasmid, and the target gene and pET-28a(+) were subjected to double digestion according to the double digestion conditions described in Table 2.

[0045] Table 2 Double enzyme digestion reaction conditions

[0046]

[0047] Note: Enzyme digestion temperature is 35°C.

[0048] The plasmid was digested...

Embodiment 3

[0055] Expression of embodiment 3 Lactobacillus brevis glutamic acid decarboxylase in Escherichia coli engineering bacteria

[0056] Get the constructed plasmid pET-28a(+)-gad expressing glutamic acid decarboxylase to transform Escherichia coli BL21(λDE3) competent cells, and obtain recombinant engineering bacteria expressing Lactobacillus brevis glutamate decarboxylase (BL21(λDE3)- pET-28a(+)-gad). Pick the cells and activate them on the LB solid medium plate with a final concentration of 30 μg / ml kanamycin at 37°C for 10 hours, then pick a single colony and inoculate 20 ml of LB liquid medium containing 30 μg / ml kanamycin After cultivating on a shaking table at 37°C and 200rpm overnight, inoculate 50ml of LB liquid medium containing 30μg / ml kanamycin in a 250ml Erlenmeyer flask with a 2% inoculum amount, shake at a constant temperature of 37°C and a rotation speed of 200rpm Incubate for 2-3 hours. When the cell concentration in the fermentation broth (OD 600 ) reaches 0.6...

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Abstract

The invention discloses cloning, expression and application of a Lactobacillus brevis glutamate decarboxylase gene. The gene is derived from Lactobacillus brevis CGMCC No.1306, and is obtained by amplifying a Lactobacillus brevis genome DNA through PCR (polymerase chain reaction); and the full length of the gene is 1407bp. BamH I and EcoRI restriction enzyme recognition sequences are respectively added to both ends of the gene; and the gene is connected with pET-28a(+) which is digested by the same restriction enzymes, and is converted into colibacillus expression host bacteria BL21 (DE3), thereby realizing the recombinant expression in the colibacillus; and the molecular weight of the expression product glutamate decarboxylase is 53538.6Da. The recombinant glutamate decarboxylase, or the engineering bacteria for expressing the recombinant glutamate decarboxylase can convert L-glutamic acid or salt thereof into gamma-aminobutyric acid by decarboxylation, and has the advantages of high conversion efficiency, high product purity and high production efficiency.

Description

technical field [0001] The invention relates to the cloning, expression and application of a Lactobacillus breve glutamic acid decarboxylase gene, belonging to the technical field of bioengineering. More specifically, the present invention relates to a new glutamic acid decarboxylase gene, its expressed glutamic acid decarboxylase gene, its expressed glutamic acid decarboxylase, and the enzyme or the engineering bacteria expressing the enzyme in the preparation Application of gamma-aminobutyric acid. Background technique [0002] γ-aminobutyric acid (GABA for short), also known as aminobutyric acid, is an important non-protein amino acid. It is not only an important neurotransmitter of the central nervous system, but also has a variety of beneficial physiological functions for the human body. A large number of literatures have reported that GABA has the functions of lowering blood pressure and cholesterol, treating epilepsy, calming the nerves, relieving depression symptom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/88C12P13/04
Inventor 梅乐和胡升黄俊
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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