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Chondroitin sulfate lyase and coding gene and application thereof

A technology of chondroitin sulfate and coding genes, applied in the field of enzyme engineering, to achieve broad application prospects, high degradation activity, and stable physical and chemical properties

Active Publication Date: 2021-01-22
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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  • Chondroitin sulfate lyase and coding gene and application thereof
  • Chondroitin sulfate lyase and coding gene and application thereof
  • Chondroitin sulfate lyase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the acquisition of Photobacterium sp. QA16

[0043] Take the sea mud leaching solution, add 1 mL of the supernatant to 9 mL of sterile water, and dilute to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Concentration gradient, then inoculate and spread on the only carbon source solid medium by conventional dilution method or streaking method, culture at 30°C for 1 day, count the colonies, then select the colonies with obvious differences in colony morphology, and repeat the streaking Inoculate on the corresponding full nutrient agar plate until a single colony is obtained after purification, and then transfer to the corresponding agar slant for later use.

[0044] The cultured strains were inoculated on the only carbon source liquid medium for cultivation. Cultivate at 200 rpm and 30°C for 72 hours, observe the turbidity of the bacterial solution, and take the culture supernatant for carbazole reaction to detect the consumption of carbon sources. The enzy...

Embodiment 2

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

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

[0051] Add 12.0mL of lysozyme buffer solution (10mM Tris-HCl pH 8.0) to each tube to obtain about 14.0mL of bacterial liquid, and add 560μL of lysozyme with a concentration of 20mg / mL respectively, and the final concentration is about 800μg. / mL; after ice bath for 1.0h, warm bath at 37°C for 2h until the solution becomes viscous; add 0.82mL of 10wt% SDS (sodium dodecylsulfonate), 60μL of 100mg / mL proteinase K solution, and bathe in water at 52°C for 1.0h; Add Tris-balanced phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) 15mL, gently invert and mix until fully e...

Embodiment 3

[0052] Example 3, Photobacterium sp. QA16 strain genome scanning and its sequence analysis

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

[0054] NCBI analysis results showed that the chondroitin sulfate lyase gene encsase carried on the genome of Photobacterium sp. QA16 strain has a coding region of 2943 bp, 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, its amino acid sequence is shown in SEQ ID NO.2, and the theoretical molecular weight of the protein...

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Abstract

The invention relates to chondroitin sulfate lyase and an encoding gene and application thereof. The chondroitin sulfate lyase enCSase is separated from Photobacterium sp. QA16, an amino acid sequenceis shown as SEQ ID NO.2, and a nucleotide sequence of the coding gene is shown as SEQ ID NO.1. The chondroitin sulfate lyase enCSase is a novel CSase ABC enzyme, has relatively high enzyme activity on chondroitin sulfate, dermatan sulfate and hyaluronic acid, and is in an incision enzyme mode when being used for degrading chondroitin sulfate, dermatan sulfate and hyaluronic acid; and the chondroitin sulfate lyase can be applied to preparation of chondroitin sulfate oligosaccharides, dermatan sulfate oligosaccharides and hyaluronic acid oligosaccharides, and has a wide application prospect inthe fields of medicines, foods and the like.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and relates to a chondroitin sulfate lyase, its coding gene and its 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 composed of repeated links of disaccharide units. In animal tissues, they are 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 the biosynthesis process, the basic disaccharide unit GlcUAβ1-3GalNAc (O unit) constituting CS / DS sugar ch...

Claims

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Application Information

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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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