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Recombined bacillus subtilis capable of performing extracellular secretion of inulin endonuclease and preparation method and application of recombined bacillus subtilis

A technology of Bacillus subtilis and endo-inulinase, which is applied in the field of genetic engineering and fermentation engineering, and can solve the problems of less research on extracellular endo-inulinase

Active Publication Date: 2018-08-31
YANCHENG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when studying the heterologous expression of endo-inulinase, it is only limited to the study of intracellular enzymes, and there are few studies on extracellular endo-inulinase

Method used

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  • Recombined bacillus subtilis capable of performing extracellular secretion of inulin endonuclease and preparation method and application of recombined bacillus subtilis
  • Recombined bacillus subtilis capable of performing extracellular secretion of inulin endonuclease and preparation method and application of recombined bacillus subtilis
  • Recombined bacillus subtilis capable of performing extracellular secretion of inulin endonuclease and preparation method and application of recombined bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of recombinant plasmids

[0051] 1. Acquisition of the target gene inuQ: through genome mining technology, comparison tools such as Genbank and blastn, it was determined that the source of endo-inulinase was Pseudomonas mucidolens (NCBI), and one-step cloning primers were designed and synthesized , upstream primer (SEQ ID NO: 2): aaaaaacgaaaaacagacctcgagATGCATAATACCGAAGATACCGGG, downstream primer: tcctgcaggtcgtcgactctAAGCTTTTATTTGGTTTGGACT (SEQ ID NO: 3), PCR amplification of the target gene inuQ;

[0052] PCR amplification system: 50 μL = 2 μL Pseudomonas mycotice genomic DNA + 2 μL upstream primer + 2 μL downstream primer + 25 μL pfuMasterMix + 19 μL ddH2O. Among them, pfuMasterMix (Shanghai Jijin Chemical Technology Co., Ltd., E006-01A) contains dNTP, buffer, MgSO4, pfu enzyme and other components. Genomic DNA extracted from Pseudomonas mucidolens donated by the professor and using the genome extraction kit (AXYGEN, AP-MN-BT-GDNA-50) purchased...

Embodiment 2

[0098] Example 2 Transformation of Bacillus subtilis WB800-R with recombinant plasmid to obtain recombinant Bacillus subtilis WB800-R-pP sacB -NMK-SP-inuQ

[0099] Since Bacillus subtilis is a Gram-positive bacterium, the method selected is a chemical transformation method, and the transformation efficiency is relatively high. Use chemical reagents to make Bacillus subtilis WB800-R competent, aliquot into 500 μL / tube, and verify the correct plasmids pPsacB-NMK-SPsacB-inuQ, pPsacB-NMK-SPsacC-inuQ, pPsacB-NMK obtained in Example 1 -SPbpr-inuQ, pPsacB-NMK-SPamye-inuQ or pPsacB-NMK-SPnprb-inuQ were respectively added to 500 μL of Bacillus subtilis WB800-R competent in different tubes, shaken at 200 rpm for 90 min at 37 ° C, and finally respectively Spread onto LB plates with a final concentration of Cannabis antibiotics of 20 μg / mL, culture them at 37°C, pick transformants, and inoculate them in liquid medium containing Cannabis antibiotics, shake the bacteria at 200 rpm for 12 h...

Embodiment 3

[0102] Recombinant Bacillus subtilis WB800-R-pP sacB -NMK-SP-inuQ fermented endo-inulinase:

[0103] First, the verified correct recombinant Bacillus subtilis WB800-R-pP prepared in Example 2 sacB -NMK-SPsacB-inuQ, WB800-R-pPsacB-NMK-SPsacC-inuQ, WB800-R-pPsacB-NMK-SPbpr-inuQ, WB800-R-pPsacB-NMK-SPamye-inuQ, and WB800-R-pPsacB-NMK -SPnprb-inuQ were respectively streaked into solid LB medium containing 25 μg / mL Cannabis antibiotics from glycerol tubes, cultured statically at 37°C for 12 hours, single colonies were inserted into liquid LB containing 25 μg / mL Cannabis antibiotics, 37°C, Shake the bacteria at 200rpm for 12h to activate, according to the optimized enzyme production medium (including 5.0-20.0g / L sucrose, 20.0-22.0g / L yeast powder, 15.0-16.5g / L NaCl, 0.5-0.8g / L MgSO 4 ·7H 2 O, 2.0~2.8g / L KH 2 PO 4 ), prepare the enzyme-producing medium, and the recombinant Bacillus subtilis WB800-R-pP containing different signal peptides sacB -NMK-SPsacB-inuQ, WB800-R-pPsacB-NM...

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Abstract

The invention discloses a recombined plasmid, stacking PCR is used to stack the CDS sequence inuQ of the inulin endonuclease and signal peptide, then the obtained signal peptide fragments and large intestine-subtilis shuttle plasmids pPsacB-NMK are connected, and the recombined bacillus subtilis is obtained. The invention further discloses the recombined bacillus subtilis which is prepared from recombined plasmid transformation bacillus subtilis WB800-R and capable of performing extracellular secretion of the inulin endonuclease. The invention further discloses a preparation method of the recombined plasmid and the recombined bacillus subtilis and application of recombined bacillus subtilis. According to the recombined bacillus subtilis, the potential safety hazard that the escherichia coli produces fructo-oligosaccharide doesn't exist, the recombined bacillus subtilis has higher advantages over other engineering bacteria on the aspect of producing the inulin endonuclease, the fructo-oligosaccharide with degrees of polymerization of DP3, DP4 and DP5 can be obtained, the fructo-oligosaccharide can be prepared with a one-step fermentation method, the time is saved, and the strict conditions of the enzymatic catalysis are reduced.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and fermentation engineering, in particular to a recombinant Bacillus subtilis capable of extracellularly secreting endo-inulinase and its preparation method and application Background technique [0002] Fructose-oligosaccharides are divided into cane fruit and fruit-type fructo-oligosaccharides. Cane-fruit-type fructo-oligosaccharides are fructo-oligosaccharides produced by combining 1 to 3 fructosyl groups with fructosyl groups in sucrose through β (2-1) glycosidic bonds. A mixture of sugar, kestetose and kestopentaose; the fruit-fruit fructo-oligosaccharides are hydrolyzed from inulin into fructo-oligosaccharides with different degrees of polymerization under the action of endo-inulinase. There are about 60-70 grams of inulin in 100 grams of dry weight Jerusalem artichoke. Inulin is a fructan connected by a linear β-2,1-glucoside chain, and its end is a sucrose group. Therefore, J...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N15/66C12N9/24C12P19/14C12P19/04C12R1/125
CPCC12N9/2402C12N15/75C12P19/04C12P19/14C12Y302/01007
Inventor 高健姜瑞凡徐虹黄巍巍张丽薛锋倪浩梅笛芬张红
Owner YANCHENG INST OF TECH