Chondrosulphatase B as well as coding gene and construction method thereof

A chondroitin sulfate enzyme and gene technology, applied in the field of genetic engineering, can solve problems such as limitations, limited thermostability, pH stability, etc.

Pending Publication Date: 2022-02-15
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In related technologies, chondroitin sulfate B has limited thermal stability and pH stability under acid-ba...

Method used

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  • Chondrosulphatase B as well as coding gene and construction method thereof
  • Chondrosulphatase B as well as coding gene and construction method thereof
  • Chondrosulphatase B as well as coding gene and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 In vitro expression of chondroitinase B

[0032] (1) Gene cloning

[0033] With primer F (5'-CGC GGATCC GCGGAATTCCTGGTGCACAACCAGC-3', BamHI site underlined, SEQ ID NO: 3) and primer R (5'-CCG CTCGAG ATTCGTGACTTCGTTATTGGCGAGG-3', XhoI site underlined, SEQ ID NO: 4), the chondroitinase B gene was amplified from the genome of Microbulbifer sp. The amplicon is 2181 bp in length and encodes mature chondroitinase B, excluding the signal peptide.

[0034] The PCR reaction system was as follows: 5 μL of 10×Taq DNA polymerase buffer, 4 μL of 10 mmol / L 4 kinds of dNTPs mixture, 1 μL of gene-specific upstream primers, 1 μL of gene-specific downstream primers, 0.5 μL of Primer STAR enzyme, and 2 μL of genome template , add sterile water to make up to 50 μL.

[0035] The PCR amplification program is: pre-denaturation (95°C, 5min); denaturation (94°C, 55s), annealing (55°C, 45s), extension (72°C, 90s), 30 cycles; extension (72°C, 10min) . The obtained PCR amplificat...

Embodiment 2

[0039] Example 2 Determination of the activity of chondroitinase B

[0040] Utilize 50mmol / L Na 2 HPO 4 -NaH 2 PO 42 mg / mL chondroitin sulfate B (Soiarbio, China) substrate solution was prepared in buffer (pH 8.0). Take 680 μL of substrate solution, add 15 μL of purified recombinant enzyme solution (0.6 mg / mL), react at 40° C. for 20 min, and stop the reaction in a boiling water bath for 10 min. After cooling the mixture to room temperature, the UV absorbance was measured at 232 nm. 1 unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmoL of unsaturated carbon bonds per minute under the above conditions.

Embodiment 3

[0041] The enzymatic property of embodiment 3 chondroitin sulfate enzyme B

[0042] (1) Substrate specificity of chondroitinase B

[0043] Using the activity assay method of Example 2, measure the enzymatic activity using different substrates respectively, including chondroitin sulfate A (CSA), chondroitin sulfate B (CSB), chondroitin sulfate C (CSC) and hyaluronic acid (HA) , to study the substrate specificity of enzymes. The result is as figure 2 As shown, the recombinant enzyme is active on CSB and HA, indicating that the enzyme belongs to chondroitinase B.

[0044] (2) Effects of temperature and pH on the activity and stability of chondroitinase B

[0045] The optimal reaction temperature of chondroitin sulfate B is determined in the range of 4-60°C, specific operation: adopt the method for measuring the activity of Example 2, under the conditions of 4, 10, 20, 30, 40, 50 and 60°C respectively reaction to measure enzyme activity. The result is as image 3 As shown, ...

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Abstract

The invention provides chondrosulphatase B, a coding gene and a construction method thereof, and the chondrosulphatase B has amino acid as shown in SEQ ID NO: 1. The chondrosulphatase B can effectively carry out enzymolysis on a chondroitin sulfate B substrate into disaccharide, the optimum pH is 8.0, and the optimum temperature is 40 DEG C; the heat stability is good, and the residual activity is about 50% after treatment at 60 DEG C for 2 h; the recombinase has good stability in a pH range of 4.0 to 10.0. The recombinase shows excellent stability to a non-ionic surface active agent (Triton X-100, Tween 20 and Tween 80) to be tested and a cationic surface active agent CTAB (Cetyltrimethyl Ammonium Bromide). Through enzymolysis of the chondroitin sulfate B, the oxidation resistance of the chondroitin sulfate B serving as the hydroxyl free radical scavenger is improved. The activity and stability of the enzyme make it competitive candidates for further fundamental research, medicine, industrial and biotechnology applications.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a chondroitinase B, its encoding gene and a construction method thereof. Background technique [0002] Chondroitin sulfate lyase (chondroitinase or chondroitin sulfate lyase, referred to as "ChSase") is a class of glycosaminoglycans that can degrade chondroitin sulfate, chondroitin, hyaluronic acid and other glycosaminoglycans into unsaturated disaccharides. Chondroitin sulfate B only specifically degrades chondroitin sulfate B and hyaluronic acid, and the enzyme has potential application value in basic research, biomedicine and other fields. [0003] In the related art, chondroitinase B has limited thermal stability and pH stability under acid-base conditions, which makes chondroitinase B limited in the process of chondroitin sulfate B biological resource development. Therefore, the discovery of a new type of heat-resistant, acid-alkali-resistant chondroitinase B is...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70A61P39/06
CPCC12N9/88C12N15/70A61P39/06C12Y402/02019
Inventor 朱艳冰黄天祥牟明静倪辉姜泽东杜希萍李志朋郑明静杨远帆李利君
Owner JIMEI UNIV
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