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Incision-type sodium alginate lyase as well as encoding gene and application thereof

A technology of alginate lyase and coding gene, which is applied in the field of endo-type alginate lyase and its coded gene and application, and can solve the problems of poor activity, rare commercial alginate, low yield of natural alginate, etc. problem, to achieve the effect of stabilizing the properties of the enzyme

Active Publication Date: 2015-01-21
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Limited by the low yield of natural alginate lyase, transgenic production of alginate lyase is easy to increase the yield, but most enzymes are affected by comprehensive factors such as water solubility and poor activity. Compared with similar products such as susuzyme, there are few widely used commercial alginate lyases

Method used

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  • Incision-type sodium alginate lyase as well as encoding gene and application thereof
  • Incision-type sodium alginate lyase as well as encoding gene and application thereof
  • Incision-type sodium alginate lyase as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, Extraction of Flammeovirga yaeyamensis MY04 strain genomic DNA

[0030] 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.

[0031] The components per liter of the above-mentioned liquid medium YT04 are as follows:

[0032] Tryptone 10g, yeast extract 5.0g, sodium chloride 30g, water to 1L, pH7.2.

[0033] 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 1.0...

Embodiment 2

[0034] Example 2. Genome scanning and sequence analysis of Flammeovirga yaeyamensis MY04 strain.

[0035]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).

[0036] The results analyzed by the above-mentioned biological software show that the genomic DNA of the Flammeovirga yaeyamensis MY04 strain carries a coding factor algL-5 of alginate lyase, and the coding region of the gene algL-5 is 1701bp long, and the nucleotide sequence is as follows: Shown in SEQ ID NO.1. The alginate lyase AlgL-5 encoded by the gene algL...

Embodiment 3

[0037] Embodiment 3, the recombinant expression of gene algL-5 in Escherichia coli BL21 (DE3) bacterial strain

[0038] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0039] Forward primer AlgL5-F: 5, -C GGATCC GATGAACAACCAGTACAATG-3' (BamH I);

[0040] Reverse primer AlgL5-R: 5'-G CTCGAG GTGACTTACTTTTAGGTAAC-3' (Xho I);

[0041] The underlined mark in the forward primer AlgL5-F is the restriction endonuclease BamH I site, and the underlined mark in the reverse primer AlgL5-R is the restriction endonuclease Xho 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.

[0042] 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 110s, 30 cyc...

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Abstract

The invention relates to incision-type sodium alginate lyase as well as an encoding gene and application thereof. The amino acid sequence of the incision-type sodium alginate lyase AlgL-5 is as shown in SEQ ID NO.2. A gene for encoding the incision-type sodium alginate lyase AlgL-5 is as shown in SEQ ID NO.1. Main oligosaccharide products generated in a process of degrading sodium alginate by using the incision-type sodium alginate lyase AlgL-5 include unsaturated disaccharide, unsaturated trisaccharide, unsaturated tetrasccharide and unsaturated pentasaccharide. Pentasaccharide is the smallest unsaturated oligosaccharide substrate of lyase, and disaccharide is the smallest unsaturated oligosaccharide product of lyase. The recombinant enzyme is stable in property and has certain industrial application potential.

Description

technical field [0001] The invention relates to an endo-type alginate lyase 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 (Mannuronic acid, M) linked by glycosidic bonds. Poly M segment, poly G segment and M / G mixed segment are arranged alternately. Usually, alginate is processed from edible brown algae such as kelp and sargassum. Microorganisms such as Pseudomonas aeruginosa and brown nitrogen-fixing bacteria can secrete alginate with acetyl modification. [0003] Alginate derived from macroalgae, together with agar gum and carrageenan, is the three most productive and widely used marine polysaccharides, and is widely used in chemical, pharmaceutical and food industries. The latest research shows that the poly-M segment oligosaccharide drug "971" prepared from algin can inhibit the aggregation and cyt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/21C12P19/12C12P19/00
CPCC12N9/88C12P19/00C12P19/12
Inventor 李福川韩文君方玉春程媛媛古静燕
Owner SHANDONG UNIV
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