Bacillus subtilis JG-1 capable of producing endo-inulinase as well as preparation method and application of bacillus subtilis JG-1

A technology of Bacillus subtilis and inulin endonuclease, applied in the fields of genetic engineering and fermentation engineering

Inactive Publication Date: 2017-09-29
YANCHENG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few studies on endoinuclease, which are mai

Method used

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  • Bacillus subtilis JG-1 capable of producing endo-inulinase as well as preparation method and application of bacillus subtilis JG-1
  • Bacillus subtilis JG-1 capable of producing endo-inulinase as well as preparation method and application of bacillus subtilis JG-1
  • Bacillus subtilis JG-1 capable of producing endo-inulinase as well as preparation method and application of bacillus subtilis JG-1

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0039] Example 1 Construction of recombinant plasmid vector pMA05-inu

[0040] Through gene mining technology, it is determined that the target gene is an endoinulinase from Pseudomonas mold, and a one-step cloning primer is designed. Upstream primer: aaaagagcgatttacatatgATGCACAACACAGAAGACACAGG Downstream primer: gagctcgactctagaggatccTTATTTTGTCTGCACGCCATCG; transfer the target gene by PCR and use restriction Dicer NdeI and BamHI double cut the plasmid vector pMA05. After recovery and purification, the concentration is calculated and the connection system is determined. According to the principle of homologous recombination, a one-step cloning method is used to connect on ice at 30°C, and the connected reaction solution is heat shocked. Transform into DH5α, spread it on a solid LB plate containing 100μg / ml ampicillin antibiotics, incubate overnight at 37°C for 12h, and select transformants (from the plasmid pMA05 gifted by Professor Xu Hong from Nanjing University of Technology) is...

Example Embodiment

[0041] Example 2: Transformation of Bacillsu subtilis with recombinant vector

[0042] Since the subtilis expression plasmid transformation method uses chemical transformation methods, the transformation efficiency of Bacillus subtilis is higher. First, make Bacillus subtilis competent, and finally aliquot into 200μl / tube. Take 5μl of the verified plasmid and add it directly to 200μl. In the competent Bacillus subtilis, shake the bacteria for 90min at 37℃, 200rpm, and finally spread the plate, the concentration of kanamycin antibiotic is 20μg / ml, cultivate overnight at 37℃, pick the transformants, and inoculate the liquid culture containing the kana antibiotic In the base, shake the bacteria for 12 hours at 37°C at 200 rpm, and then follow the steps in Example 1 for restriction enzyme digestion verification. The electrophoresis diagram is attached figure 2 b: Lane 1 is Maker, Lane 2 is single restriction digestion verification, and Lane 3 is dual restriction digestion verificatio...

Example Embodiment

[0043] Example 3: JG-1 fermentation enzyme production optimization

[0044] The positive clone Bacillsu subtilis JG-1 obtained from the screening was inoculated into 500ml enzyme-producing fermentation medium to ferment to produce endo-inulinase, and the initial maximum specific enzyme activity was about 1.2U / ml. The conditions of B.subtilis JG-1 for producing endoinulinase were optimized. JG-1 was inoculated into a 500ml shake flask and fermented at 32℃. Samples were taken every 4h. 200μl of fermentation broth and 800μl of 2% Inulin was reacted for 30 minutes, and the endoinulinase activity was measured. As the fermentation progressed, the cell concentration (OD600nm) of the recombinant strain JG-1 reached the maximum value of 2.5. Centrifuge at 9000 rpm at 4°C. After 10 minutes, the cells were obtained, and the cells were washed twice with 200 μl 0.02 mM phosphate buffered saline PBS (pH 7.4), and finally the cells were resuspended with the same volume (200 μl) of PBS, and the ...

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Abstract

The invention discloses bacillus subtilis JG-1 capable of producing endo-inulinase. According to the bacillus subtilis JG-1, a pMA05 escherichia coli-bacillus subtilis shuttle plasmid is used as a skeleton for constructing pMA05-inu, pMA05-inu is converted into bacillus subtilis 168 by virtue of a chemical conversion method, and a positive transformant, namely the bacillus subtilis JG-1, is screened by virtue of a kanamycin selection marker of the plasmid in bacillus subtilis. The invention further discloses a preparation method and application of the bacillus subtilis JG-1 capable of producing endo-inulinase. The bacillus subtilis is a recognized as food safety microorganism, so that the potential risk that escherichia coli is capable of producing fructo-oligosaccharide is avoided; the fructo-oligosaccharides with relatively high purities and polymerization degrees of DP3, DP4 and DP5 can be acquired; and the bacillus subtilis has relatively high superiorities than other engineering bacteria in the production of endo-inulinase. The maximum enzyme activity of endo-inulinase is 20.16U/ml.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and fermentation engineering, in particular to a bacillus subtilis JG-1 producing endo-inulinase and a preparation method and application thereof. Background technique [0002] Fructooligosaccharides, also known as fructooligosaccharides or sucrose trisaccharide oligosaccharides, are a type of bifidobacteria proliferation factor, with low heat, stability, safety and non-toxic properties. It is a non-digestible oligosaccharide that cannot be hydrolyzed by the digestive enzymes secreted by the animal itself. After reaching the intestinal tract, it can be selectively utilized by Bifidobacteria and Lactobacillus to produce organic acids and essential vitamins that are beneficial to animal growth. etc.; reduce intestinal pH, inhibit the proliferation of harmful bacteria (such as Escherichia coli, Clostridium and spoilage bacteria, etc.), reduce the generation of spoilage metabolites in the...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N9/24C12P19/14C12P19/00C12R1/125
CPCC12N9/2402C12N15/75C12P19/00C12P19/14C12Y302/01007
Inventor 高健姜瑞凡徐虹张丽薛锋倪浩黄巍巍
Owner YANCHENG INST OF TECH
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