A kind of chondroitin sulfate lyase and its encoding gene and application

A technology of chondroitin sulfate and coding genes, which is applied in the field of enzyme engineering to achieve the effects of high degradation activity, easy heterologous expression and purification, and stable physical and chemical properties

Active Publication Date: 2022-07-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult to prepare its highly pure and active form through purification and recombinant expression

Method used

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  • A kind of chondroitin sulfate lyase and its encoding gene and application
  • A kind of chondroitin sulfate lyase and its encoding gene and application
  • A kind of chondroitin sulfate lyase and its encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Obtaining of Photobacterium sp. QA16

[0043] Take sea mud leachate, add 1 mL of supernatant to 9 mL of sterile water, and dilute to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Then, by conventional dilution method or streaking method, it was inoculated on the sole carbon source solid medium, incubated at 30°C for 1 day, the colonies were counted, and then the colonies with obvious differences in colony morphology were selected, and the streaking was repeated. It was inoculated on the corresponding complete nutrient agar plate until a single colony was obtained after purification, and then transferred to the corresponding agar slant for later use.

[0044] The cultured strains were respectively inoculated onto the only carbon source liquid medium for cultivation. Incubate at 200rpm and 30°C for 72h, observe the turbidity of the bacterial liquid, and take the culture supernatant for carbazole reaction to detect the consumption of carbon source. The en...

Embodiment 2

[0049] Example 2. Extraction of genomic DNA of Photobacterium sp. QA16

[0050] Photobacterium sp. QA16 was inoculated into the liquid medium and cultured to OD with shaking at 30°C and 200rpm. 600 About 0.8; take 40 mL of cultured bacterial solution, centrifuge at 12,000 rpm for 25 min, collect the bacterial pellet, wash with 20 mL of lysozyme buffer (10 mM Tris-HCl pH 8.0), and centrifuge at 12,000 rpm for 25 min to collect bacteria. body precipitation;

[0051] In the above-mentioned cell precipitation, add 12.0 mL of lysozyme buffer (10 mM Tris-HCl pH 8.0) to each tube to obtain about 14.0 mL of bacterial liquid, respectively add 560 μL of lysozyme with a concentration of 20 mg / mL, and its final concentration is about 800 μg / mL; after ice bath for 1.0h, incubate at 37°C for 2h until the solution is viscous; add 0.82mL of 10wt% SDS (sodium dodecyl sulfonate), 60μL of 100mg / mL proteinase K solution, water bath at 52°C for 1.0h; Add 15mL of Tris-equilibrated phenol / chlorof...

Embodiment 3

[0052] Example 3. Genome scanning and sequence analysis of Photobacterium sp. QA16 strain

[0053] The large molecular weight genomic DNA obtained in Example 2 was sequenced (Beijing Yuanyi). The sequencing results were analyzed with 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 SearchTool (BLAST, http: / / blast.ncb1 .nlm.nih.gov / Blast.cgi).

[0054] The NCBI analysis results showed that the chondroitin sulfate lyase gene encsase carried on the genome of Photobacterium sp. QA16 strain has a length of 2943 bp in the gene coding region, and its nucleotide sequence is shown in SEQ ID NO.1. The chondroitin sulfate lyase enCSase encoded by the chondroitin sulfate lyase gene encsase consists of 980 amino acids, the amino acid sequence of which is shown in SEQ ID NO. 2, and the theoretical mol...

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Abstract

The present invention relates to a chondroitin sulfate lyase and its encoding gene and application. The chondroitin sulfate lyase enCSase in the present invention is isolated from Photobacterium sp. QA16, the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1. The chondroitin sulfate lyase enCSase in the present invention is a new type of CSase ABC enzyme, which has high enzymatic activity on chondroitin sulfate, dermatan sulfate and hyaluronic acid, and degrades chondroitin sulfate, dermatan sulfate and hyaluronic acid. It is in endonuclease mode; it can be used in the preparation of chondroitin sulfate oligosaccharide, dermatan sulfate oligosaccharide and hyaluronic acid oligosaccharide, and has broad application prospects in the fields of medicine and food.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and relates to a chondroitin sulfate lyase and its encoding gene and application. Background technique [0002] Chondroitin sulfate (CS) / dermatan sulfate (DS) is composed of β-D-glucuronic acid (GlcUA) or α-L-iduronic acid (IdoUA) and N-acetyl-D-galactosamine (GalNAc) ) is a linear anionic polysaccharide formed by repeated connection of disaccharide units. In animal tissues, it is widely distributed on the cell surface as well as in the extracellular matrix, usually as side chains of proteoglycans. As an important class of glycosaminoglycans, CS / DS are involved in various physiological and pathological processes, such as cell adhesion, cell differentiation, cell migration, cell proliferation, wound repair, nervous system development, signal transduction and immune response. During biosynthesis, the basic disaccharide unit GlcUAβ1-3GalNAc(O unit) that constitutes CS / DS glycans often u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N1/20C12N15/70C12N1/21C12P19/26C12R1/01C12R1/19
CPCC12N9/88C12Y402/0202C12N15/70C12P19/26C12R2001/01C12N1/205
Inventor 李福川张庆冬路丹荣魏琳
Owner SHANDONG UNIV
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