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M-preferred monosaccharide exo-algin lyase Aly-6 as well as coding gene and application thereof

A technology of alginate lyase and coding gene, which is applied in the direction of lyase, carbon-oxygen lyase, enzyme, etc., can solve the problem that there is no relevant report on the production characteristics of oligosaccharide products, and achieve the effect of stable physical and chemical properties and high activity

Inactive Publication Date: 2018-12-21
JINAN ENLIGHTEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, there are no relevant reports on the M-prone bifunctional monosaccharide exonuclease and its related substrate degradation mechanism and the resulting oligosaccharide product formation characteristics.

Method used

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  • M-preferred monosaccharide exo-algin lyase Aly-6 as well as coding gene and application thereof
  • M-preferred monosaccharide exo-algin lyase Aly-6 as well as coding gene and application thereof
  • M-preferred monosaccharide exo-algin lyase Aly-6 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1, Extraction of Flammeovirga yaeyamensis MY04 strain genomic DNA

[0061] 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, pH 8.0) to suspend the bacterial , and centrifuged at 12,000rmp for 15min to collect the cell pellet.

[0062] The above-mentioned liquid medium YT04 has the following components per liter:

[0063] Tryptone 10g, yeast extract 5.0g, sodium chloride 30g, dissolved in water and adjusted to 1L, pH 7.2.

[0064]Add 6.0 mL of lysozyme buffer solution (purchased from Shanghai Sangon Bioengineering Co., Ltd.) to each tube to obtain about 7.0 mL of bacterial liquid, add 280 μL of lysozyme solution with a concentration of 20 mg / mL, Make the final c...

Embodiment 2

[0065] Example 2. Genome scanning and sequence analysis of Flammeovirga yaeyamensis MY04 strain.

[0066] 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).

[0067] The results analyzed with the above-mentioned biological software show that the genomic DNA of the Flammeovirga yaeyamensis MY04 strain carries an encoding gene aly-6 of alginate lyase, and the coding region of the gene aly-6 is 2238bp long, and the nucleotide sequence is as follows: Shown in SEQ ID NO.1. The recombinant alginate lyase rAly-6 encoded by...

Embodiment 3

[0069] Embodiment 3, the recombinant expression of gene aly-6 and truncated body aly-6Lm, aly-6Hpm in Escherichia coli BL21 (DE3) bacterial strain

[0070] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0071] Forward primer for aly-6 amplification Aly6-F: 5'-gg CATATG CAGAACGGAAATTTAATTACTTCCG-3' (NdeI);

[0072] Reverse primer Aly6-R for aly-6 amplification: 5'-gc TCTAGA AATTTCTTCCAATAGGTAAACCCC-3'(XbaI);

[0073] Forward primer for aly-6Lm amplification Aly6Lm-F: 5'-gg CATATG CAGAACGGAAATTTAATTACTTCCGAG-3' (Nde I);

[0074] Reverse primer for aly-6Lm amplification Aly6Lm-R: 5'-gc TCTAGA CAAACTTCTTTTTTGGTAAGTCGTTGC-3' (Xba I);

[0075] Forward primer for aly-6Hpm amplification Aly6Hpm-F: 5'-gg CATATG TACTATTCAAGAGAATTGAAACTAGCC-3' (Nde I);

[0076] Reverse primer for aly-6Hpm amplification Aly6Hpm-R: 5'-gc TCTAGA AATTTCTTCCAATAGGTAAACCCC-3' (Xba I);

[0077] The underlined f...

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Abstract

The invention relates to an M-preferred monosaccharide exo-algin lyase Aly-6 as well as a coding gene and application thereof. An amino acid sequence of the M-preferred monosaccharide exo-algin lyaseAly-6 is shown in SEQ ID NO. 2; and a nucleotide sequence for encoding the Aly-6 is shown in SEQ ID NO. 1. The invention discloses for the first time that an M-preferred monosaccharide exo-algin lyaseAly-6 is obtained from a genome of the flammeovirga yaeyamensis MY04. The enzyme is a monosaccharide exo-algin lyase and has an obvious M-preferred feature; the enzyme Aly-6 can continuously degradethe guluronic acid fragment from the algin in a monosaccharide excision manner, and can also continuously degrade the mannuronic acid fragment in a monosaccharide excision manner; and the enzyme Aly-6is easier to degrade the mannosolic acid fragment. The sequence characteristics, substrate degradation pattern and oligosaccharide formation characteristics of the enzyme are significantly differentfrom the sequence characteristics, substrate degradation pattern and oligosaccharide formation characteristics of the existing known algin lyase. The physical and chemical properties are stable, the activity is high, and the potential for industrial application is appreciable.

Description

technical field [0001] The invention relates to an M-prone monosaccharide exo-type alginate lyase Aly-6 and its coding gene and application, belonging to the technical field of genetic engineering. Background technique [0002] Alginate is a linear polysaccharide composed of α-L-guluronic acid (G) and β-D-mannuronic acid (Mannuronicacid, M) two sugar units. Segments alternate with MG or GM hybrid segments [1] . Alginate is usually processed from large seaweeds such as kelp, sargassum, and macroalgae. The latest research shows that the class I candidate drug "GV971" prepared with poly-M-segment alginate oligosaccharides can inhibit the aggregation and cytotoxicity of β-amyloid cells, and can be used for the treatment of mild to moderate Alzheimer's disease [2] , has passed Phase III clinical research; saturated poly-G oligosaccharides can synergistically inhibit multidrug-resistant pathogenic bacteria with antibiotics [3] . This shows that algin oligosaccharides with a s...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12P19/00C12P19/04
CPCC12N9/88C12P19/00C12P19/04C12Y402/02
Inventor 韩文君程媛媛李俊鸽王延辉古静燕李新卫洁
Owner JINAN ENLIGHTEN BIOTECH CO LTD
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