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Construction method and fermenting method of antibiotic-resistance-free recombinant bacillus subtilis for expressing glutamate decarboxylase

A technology of Bacillus subtilis and glutamic acid decarboxylase, which is applied in the field of bioengineering, can solve the problems of endotoxin production, low enzyme production efficiency, and long culture period, and achieve the effects of simple process, environmental friendliness, and strong protein expression ability

Inactive Publication Date: 2017-07-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Fermentation of wild bacteria such as lactic acid bacteria to produce GABA faces disadvantages such as long cultivation period, low enzyme production efficiency, and complicated separation and purification steps; although the enzyme activity of exogenous glutamic acid decarboxylase is greatly improved by Escherichia coli, due to Escherichia coli is a Gram-negative bacterium that produces endotoxin during fermentation, so the use of Escherichia coli as a production strain to produce GABA cannot be used in the food industry

Method used

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  • Construction method and fermenting method of antibiotic-resistance-free recombinant bacillus subtilis for expressing glutamate decarboxylase
  • Construction method and fermenting method of antibiotic-resistance-free recombinant bacillus subtilis for expressing glutamate decarboxylase
  • Construction method and fermenting method of antibiotic-resistance-free recombinant bacillus subtilis for expressing glutamate decarboxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: D-alanine-deficient Bacillus subtilis WB600 ( dal - ) construction

[0033]

[0034] Using the plasmid p7S6 as a template, the primer pair P3 / P4 will lox 71- spc - lox 66 antibiotic resistance gene fragments were amplified and recovered. D-alanine racemase gene on chromosome of Bacillus subtilis WB600 dal The fragments with a length of 800-900 bp on both sides were selected as homologous regions, and the homologous regions at both ends were amplified and recovered with primer pairs P1 / P2 and P5 / P6 respectively. Use the primer pair P1 / P6 to combine the homology arm fragments at both ends with antibiotics lox 71- spc - lox 66 were fused together by PCR technology.

[0035] PCR reaction system: Add the following reagents in order in 0.2mL PCR tube: 1.5μL each of upstream and downstream primers; 5μL Husion HF buffer (5×); 2μL 10mM dNTP mix (2.5mM each); 2μL upstream homology arm; 0.5μL lox 71- spc - lox 66; 2 μL downstream homology ar...

Embodiment 2

[0040] Embodiment 2: Construction of food-grade safety plasmid pUB-HpaII-P43-gad-dal

[0041]

[0042] Materials and reagents: DNA polymerase PrimeSTAR Max DNA Polymerase (2×) and DL 5000 DNA Marker were purchased from Dalian Bao Biological Engineering Co., Ltd.; SanPrep column type plasmid DNA mini-extraction kit and SanPrep column type DNA gel recovery kit were purchased from From Sangon Bioengineering (Shanghai) Co., Ltd.; primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; plasmid pET-22b-gad was synthesized by Shanghai Jierui Bioengineering Co., Ltd.; other common reagents were purchased from Sinopharm Chemicals Reagents Ltd.

[0043] Using P7 and P8 as primers and pET-22b-gad as a template, the gad gene was amplified by PCR,

[0044] reaction system:

[0045] pET-22b-gad 1 μL

[0046] P7 1.25 μL

[0047] P8 1.25 μL

[0048] wxya 2 O 21.5 μL

[0049] PrimeSTAR Max DNA Polymerase (2×) 25μL

[0050] Reaction conditions: pre-denaturation at 98°...

Embodiment 3

[0067] Example 3 Fermentative expression of glutamic acid decarboxylase by recombinant bacillus subtilis

[0068] Pick a single colony on the plate and inoculate it in 4 mL of LB medium, and incubate at 37°C for 12 h at 200 r / min; add the seed solution to 50 mL of fermentation medium at 37°C at 200 r / min according to the volume fraction of 2%. R / min is cultivated to 21h, the OD600 of measuring fermented liquid and enzyme activity ( image 3 ).

[0069] Media formulations involved:

[0070] LB medium: tryptone 10 g / L; yeast extract 5 g / L; sodium chloride 10 g / L; prepared with deionized water, sterilized at 121°C for 20 min.

[0071] Fermentation medium: soybean peptone 25 g / L; lactose 5 g / L; Na 2 HPO 4 12H 2 O 3g / L; MnSO 4 ·H 2 O0.1g / L; Prepared with deionized water, sterilized at 115°C for 30min.

[0072] Definition of fermentation enzyme activity: under the reaction conditions of 50°C and pH 4.5, the amount of enzyme required to produce 1 μmol of GABA per minute is de...

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Abstract

The invention relates to a construction method and a fermenting method of antibiotic-resistance-free recombinant bacillus subtilis for expressing glutamate decarboxylase and belongs to the technical field of bioengineering. The construction method includes taking bacillus subtilis WB600 as an original strain, and knocking out a D-alanine racemase gene on a chromosome of the bacillus subtilis WB600 so as to obtain D-alanine deficient WB600 (dal); fusing an optimized gad gene with an antibiotic-resistance-free expression vector pUB110 (a antibiotics resistance gene is replaced by the D-alanine racemase gene) through an overlap extension PCR technology to obtain a polymer, transforming the polymer into competence of the bacillus subtilis WB600, and enabling the polymer to recombine in a host so as to obtain a recombinant plasmid Pub-HpaII-P43-gad-dal, which is named as bacillus subtilis SK44.001 with the preservation number being CCTCC NO:M 2016774. The fermentation liquor enzyme activity of the antibiotic-resistance-free recombinant bacillus subtilis can be up to 8.6 U / mL, and accordingly the antibiotic-resistance-free recombinant bacillus subtilis has significant industrial application value.

Description

technical field [0001] The invention relates to a method for constructing and fermenting non-antibiotic-resistant recombinant Bacillus subtilis expressing glutamic acid decarboxylase, belonging to the technical field of bioengineering. Background technique [0002] Gamma aminobutyric acid (GABA) is a naturally occurring non-protein amino acid that is widely distributed in animals and plants. Studies have found that GABA can improve cardiovascular, treat neurological diseases, improve liver and kidney functions and regulate endocrine System and other important roles, so it has broad application prospects in drug therapy and functional food. [0003] The methods for preparing GABA mainly include chemical methods, animal and plant enrichment methods and microbial synthesis methods. The chemical synthesis method uses toxic chemical reagents in production, and the GABA produced cannot be added to food; while the content of GABA in animals and plants is very low, GABA is obtained...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/61C12N9/88C12N15/75C12P13/00C12R1/125
CPCC12N9/88C12N15/75C12P13/005C12Y401/01015
Inventor 江波沐万孟丁汪洋张涛
Owner JIANGNAN UNIV
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