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Excision-type agarose, coding gene and application thereof

A technology that encodes a gene and agarase, which is applied in the field of genetic engineering, can solve problems such as the inability to achieve specific production of disaccharides, and achieve the effect of stabilizing the properties of the enzyme

Active Publication Date: 2014-11-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, after chemical methods such as acid degradation and alkali degradation, or physical methods such as ultrasonic degradation and microwave degradation, after degrading agar or agarose, the degree of polymerization of the oligosaccharide products is relatively diverse, and the specificity of disaccharides has not been achieved so far. Production

Method used

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  • Excision-type agarose, coding gene and application thereof
  • Excision-type agarose, coding gene and application thereof
  • Excision-type agarose, coding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1, Extraction of Flammeovirga yaeyamensis MY04 strain genomic DNA

[0029] Flammeovirga yaeyamensis MY04 was inoculated into liquid medium YT04, and cultured with shaking at 28°C and 200 rpm until the absorbance value at 600 nm (OD 600 ) is 1.2; take 10mL of cultured bacteria, centrifuge at 12,000×g (g, earth’s gravitational constant) for 15min, and collect the bacterial sediment; use 10mL of lysozyme buffer (10mM Tris-HCl, pH8.0) to suspend the bacteria The cells were centrifuged at 12,000rmp for 15min to collect the cell pellet.

[0030] The components per liter of the above-mentioned liquid medium YT04 are as follows:

[0031] Tryptone 10g, yeast extract 5.0g, sodium chloride 30g, water to 1L, pH 7.2.

[0032] Add 6.0mL of lysozyme buffer solution to each tube to obtain about 7.0mL of bacterial solution, and add 280μL of lysozyme solution with a concentration of 20mg / mL to make the final concentration of lysozyme 800μg / mL; Place in an ice-water bath for 1....

Embodiment 2

[0033] Example 2. Genome scanning and sequence analysis of Flammeovirga yaeyamensis MY04 strain.

[0034] Genomic DNA prepared in Example 1 was scanned and sequenced by Shanghai Meiji Biotechnology Co., Ltd. using pyrosequencing technology. The DNA sequencing results were analyzed with the online software of NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ) website. The analysis software used on the NCBI website is Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0035]The analysis results of the above-mentioned biological software show that the genomic DNA of the Flammeovirga yaeyamensis MY04 strain carries an agarase coding factor agaO, the coding region of the gene agaO is 2118bp long, and its nucleotide sequence is as shown in SEQ ID NO.1 shown. The agarase AgaO encoded by the gene agaO contains 705 amino acids in ...

Embodiment 3

[0036] Embodiment 3, the recombinant expression of gene agaO in Escherichia coli TOP10 bacterial strain

[0037] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0038] Forward primer BAgaO-F: 5'-GCCG CTCGAG TTTTTTTTAGCATTCAATCCGCC-3' (Xho I);

[0039] Reverse primer BAgaO-R: 5'-GCCG TCTAGA AAGTTATTCACATTACCCAAACGG-3' (Xba I);

[0040] The underlined mark in the forward primer BAgaO-F is the restriction endonuclease Xho I site, and the underlined mark in the reverse primer BAgaO-R is the restriction endonuclease Xba I site. The high-fidelity DNA polymerase Primerstar HS used was purchased from China Dalian Biotech Co., Ltd., and the PCR reaction reagents used were operated according to the product instructions provided by the company.

[0041] PCR reaction conditions: pre-denaturation at 95°C for 4min; denaturation at 94°C for 40s, annealing at 60°C for 40s, extension at 72°C for 140s, 35 ...

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Abstract

The invention relates to an excision-type agarose, coding gene and application thereof. The excision-type agarase AgaO has an amino acid sequence shown as SEQ ID NO.2. The gene coding the excision-type agarase AgaO has nucleotide sequence shown as SEQ ID NO.1. In the degradation process of the agarose AgaO, the oligosaccharide main product is always noagarobiose; and the agarose has stable property and the potential for industrial application.

Description

technical field [0001] The invention relates to an exo-type agarase and its coding gene and application, belonging to the technical field of genetic engineering. Background technique [0002] Agar (Agar) is mainly produced from edible red algae such as Gracilaria and Laver. Together with alginate and carrageenan, it is the three major marine polysaccharides with the largest output and widest application. It is widely used in food, medicine and chemical industries. Agarose (Agarose) is one of the main components of agarose, which is a neutral polysaccharide. ) is formed by repeating and alternately linking β1-4 glycosidic bonds. Agarase can catalyze the hydrolysis of glycosidic bonds in agarose molecules to produce water-soluble oligosaccharides and oligosaccharides, and is divided into α-agarase and β-agarase due to the different types of glycosidic bonds catalyzed. Enzyme; the corresponding oligosaccharide products have 3,6-inner ether-α-L-galactopyranose (A) and β-D-gala...

Claims

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Application Information

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IPC IPC(8): C12N9/38C12N15/56C12P19/14C12P19/12
CPCC12N9/2468C12Y302/01081
Inventor 李福川韩文君程媛媛方玉春古静燕高长健刘生云
Owner SHANDONG UNIV
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