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 degradin

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
Comparison scheme
Effect test

Example Embodiment

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

[0029] Flammeovirga yaeyamensis MY04 was inoculated into liquid medium YT04, and cultured at 28°C and 200 rpm with shaking to 600nm absorbance (OD 600 ) Is 1.2; Take 10mL of the culture broth, centrifuge for 15min under the condition of 12,000×g (g, the earth's gravitational constant), collect the bacterial pellet; Suspend the bacteria with 10mL of lysozyme buffer (10mM Tris-HCl, pH8.0) The cells were centrifuged at 12,000 rpm for 15 min to collect the bacterial pellet.

[0030] The composition per liter of the above liquid medium YT04 is as follows:

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

[0032] Add 6.0 mL of lysozyme buffer solution to each tube to obtain about 7.0 mL of bacterial solution. Add 280 μL of lysozyme solution with a concentration of 20 mg / mL to make the final concentration of lysozyme 800 μg / mL; In an ice water bath for 1.0 h, then...

Example Embodiment

[0033] Example 2. Scanning and sequence analysis of the genome of Flammeovirga yaeyamensis strain MY04.

[0034] The genomic DNA prepared in Example 1 was scanned and sequenced using pyrosequencing technology, which was completed by Shanghai Meiji Biological Company. The online software of NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ) website was used to analyze the DNA sequencing results. The analysis software of the NCBI website used 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 Flammeovirga yaeyamensis strain MY04 carries an agarase code agaO, the coding region of the gene agaO is 2118 bp, and its nucleotide sequence is as SEQ ID NO.1 Shown. The agarase AgaO encoded by the gene agaO contains 705 amino acids in to...

Example Embodiment

[0036] Example 3. Recombinant expression of gene agaO in Escherichia coli TOP10 strain

[0037] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequence is 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 in the forward primer BAgaO-F is the restriction enzyme Xho I site, and the underlined in the reverse primer BAgaO-R is the restriction endonuclease Xba I site. The high-fidelity DNA polymerase Primerstar HS was purchased from Dalian Bao Biological Company, China, and the PCR reagents used were operated in accordance with the product instructions provided by the company.

[0041] PCR reaction conditions: 95°C pre-denaturation 4min; 94°C denaturation 40s, 60°C annealing 40s, 72°C extension 140s, 35 cycles; 72°C extension 5min; 4°C stable 15min.

[0042] The PCR product was doub...

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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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