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Glycosaminoglycan lyase and encoding gene and application thereof

A technology of glycosaminoglycans and coding genes, which is applied in the field of genetic engineering and achieves the effect of broad application prospects

Active Publication Date: 2014-07-16
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few high-activity glycosaminoglycan degrading enzymes with application value at present, so it is of great significance to find and identify new high-efficiency and stable glycosaminoglycan degrading enzymes

Method used

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  • Glycosaminoglycan lyase and encoding gene and application thereof
  • Glycosaminoglycan lyase and encoding gene and application thereof
  • Glycosaminoglycan lyase and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the acquisition of glycosaminoglycan lyase

[0038] Pick Vibrio sp. FC509 strain and inoculate it into 100mL of liquid medium, and culture it on a shaking table for 12 hours at a temperature of 28°C and a rotation speed of 200rpm, and add chondroitin sulfate to make the mass concentration reach 0.01%. The culture was continued for 48 hours; the culture medium was centrifuged to collect the supernatant medium, the supernatant was precipitated with ammonium sulfate with a final concentration of 80%, the precipitate was collected, and the precipitate was dialyzed with PBS buffer solution to remove ammonium sulfate to obtain glycosaminoglycan lyase.

[0039] The above-mentioned Vibrio sp. FC509 was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on March 12, 2014, with the preservation number CGMCC NO.8913, and address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing Institute of Mic...

Embodiment 2

[0043] Example 2, Extraction of Genomic DNA of Vibrio sp. FC509 Strain

[0044] Inoculate the Vibrio sp. FC509 strain into the liquid medium (same as Example 1), and culture it with shaking at 30°C and 200rpm to OD 600 =0.8; Take 40mL of the culture solution, centrifuge at 12,000rmp for 25min, collect the bacterial pellet, wash with 20mL of lysozyme buffer (10mM Tris-HCl pH8.0), centrifuge at 12,000rmp for 25min, collect the bacteria Body precipitation;

[0045] Add 12.0mL of lysozyme buffer solution to each tube to obtain about 14.0mL of bacterial solution in the above bacteria precipitation, add 560μL of lysozyme with a concentration of 20mg / mL respectively, and the final concentration is about 800μg / mL; after 1.0h in ice bath , incubate at 37°C for 2h until the solution is viscous; add 0.82mL of 10wt% SDS, 60μL of 100mg / mL proteinase K solution, bathe in water at 52°C for 1.0h; add Tris-balanced phenol / chloroform / isoamyl alcohol (volume ratio 25 :24:1) 15mL, gently invert...

Embodiment 3

[0046] Example 3 Genome scanning and sequence analysis of Vibrio sp. FC509 strain.

[0047] The large-molecular-weight genomic DNA prepared in Example 2 was sequenced (Meiji Biotechnology Co., Ltd.). The sequencing results were analyzed with the software on NCBI (National Center for Biotechnology Information, http: / / www.ncb1.nlm.nih.gov / ). The NCBI analysis software used is Open Reading Frame Finder (ORF Finder, http: / / www.ncb1.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast. ncb1.nlm.nih.gov / Blast.cgi).

[0048] NCBI analysis results showed that Vibrio sp.FC509 strain genome carried a glycosaminoglycan lyase gene hcdase, the hcdase gene coding region was 2436bp long, and its nucleotide sequence was shown in SEQ ID NO.1. It has 49% identity with 384 amino acids of hyaluronate lyase gene in the whole genome sequence of Vibrio fischeri ES114 (NCBI accession number: YP_206952.1).

[0049] The glycosaminoglycan lyase HCDase encoded by the ...

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Abstract

The invention relates to glycosaminoglycan lyase and an encoding gene and an application thereof. The amino acid sequence of the glycosaminoglycan lyase is as shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene of the glycosaminoglycan lyase is as shown in SEQ ID NO.1; the specific activity of the glycosaminoglycan lyase prepared in the invention to hyaluronic acid is 110,000 U / Mg, and the specific activity to chondroitin sulfate is 45, 000 U / Mg; the enzyme activity is tens to hundreds of times as large as that of the existing commercial glycosaminoglycan lyase (such as CSaseABC, CSaseAC, I and II and the like), and the glycosaminoglycan lyase can be used in such fields as medicine and cosmetics and the like, thereby having a broad application prospect.

Description

technical field [0001] The invention relates to a glycosaminoglycan lyase and its coding gene and application, belonging to the technical field of genetic engineering. Background technique [0002] Glycosaminoglycans (Proteoglycans, GAGs), also known as mucopolysaccharides, are linear polysaccharides composed of repeating disaccharide units. Mainly include hyaluronic acid (Hyaluronic Acid, HA), heparin / heparan sulfate (Hep / HS), chondroitin sulfate / dermatan sulfate (Chondroitin Sulfate / Dermatan Sulfate, CS / DS), keratan sulfate (Keratan Sulfate, KS). Except that the disaccharide unit of keratan sulfate is composed of neutral D-galactose and N-acetylglucosamine, the disaccharide units of other glycosaminoglycans are composed of hexuronic acid (D-glucuronic acid / L -iduronic acid) and hexosamine (N-acetylglucosamine / N-acetylgalactosamine). Among them, the structure of hyaluronic acid is relatively simple, and the disaccharide units composed of D-glucuronic acid and N-acetylglu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P19/26A61K47/42
CPCA61K47/42C12N9/88C12P19/26
Inventor 李福川韩文君王文爽赵梅
Owner SHANDONG UNIV
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