Chondroitin sulfate ABC lyase mutant with high thermal stability and application thereof

A chondroitin sulfate, mutant technology, applied in the field of bioengineering, can solve the problems of low stability, mixed chondroitin sulfate impurities, limited application and the like

Active Publication Date: 2021-03-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the low stability limits its application in industry and medicine
[0007] In addition, in the current commercialized heparin, because heparin is mostly extracted from pig small intestine, chondroitin sulfate impurities are inevitably mixed in the process of separation and purification, which will greatly affect the clinical application of heparin as an anticoagulant drug. application and efficacy

Method used

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  • Chondroitin sulfate ABC lyase mutant with high thermal stability and application thereof
  • Chondroitin sulfate ABC lyase mutant with high thermal stability and application thereof
  • Chondroitin sulfate ABC lyase mutant with high thermal stability and application thereof

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Experimental program
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Effect test

Embodiment 2

[0059] The primers used in Table 2 Example 2

[0060]

[0061]

[0062] Primers used in table 3 embodiment 3

[0063]

[0064] Primers used in table 4 embodiment 4

[0065]

[0066]

Embodiment 1

[0067] Embodiment 1: the preparation method of chondroitin sulfate ABC lyase truncation mutant

[0068] Synthesize chondroitin sulfate ABC lyase derived from Proteus vulgaris, the original amino acid sequence of the chondroitin sulfate ABC lyase is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.4. PCR was performed using the primers shown in Table 1. The truncated gene was connected to the XhoI and NdeI restriction sites of the pET-28a plasmid, introduced into Escherichia coliBL21 (DE3), cultured at 37°C until a single clone was grown, and the single clone was picked for sequencing to verify that it was correct The positive transformant is the recombinant Escherichia coli strain expressing the chondroitin sulfate ABC lyase mutant.

[0069] The obtained recombinant strain was inoculated in LB medium, cultured overnight at 37°C, transferred to a 250mL Erlenmeyer flask filled with LB medium, cultured at 30°C for 1 hour, induced by adding IPTG with a fina...

Embodiment 3

[0084] Embodiment 3: Preparation of chondroitin sulfate ABC lyase saturation mutant

[0085] Saturation mutation of NΔ5: using the recombinant plasmid contained as a template, through saturation mutation primers (see Table 4 for the sequence of saturation mutation primers), amplify the entire plasmid of the target sequence to obtain a circular plasmid with a cross gap, and digest it with Dpn I fast cutting enzyme The template plasmid and the digestion solution were directly transformed into E.coli BL21 (DE3) competent cells, and the single clone was extracted and verified by plasmid sequencing.

[0086] The recombinant Escherichia coli strains with correct sequencing were inoculated in LB medium, cultured overnight at 37°C, then transferred to a 250mL Erlenmeyer flask filled with LB medium, cultured at 30°C for 1 hour, and induced by adding IPTG with a final concentration of 0.5mM, Placed at 27°C for 20h. After the cultivation is completed, centrifuge the fermentation broth a...

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Abstract

The invention discloses a chondroitin sulfate ABC lyase mutant with high thermal stability and application thereof, and belongs to the technical field of bioengineering. The chondroitin sulfate ABC lyase derived from proteus vulgaris is subjected to truncation mutation, site-specific mutagenesis is carried out on the basis of the truncation mutant, the mutant with obviously improved thermal stability is obtained, the half-life period of the obtained mutant at 37 DEG C is prolonged by 247 times compared with that of a wild type, the recombinant bacteria expressing the mutant ferment protein inbatches in a 3-L tank, and the protein expression quantity can reach 1.7 g / L. A new method is provided for industrial production of chondroitin sulfate and dermatan sulfate, and can also be used for eliminating mixed chondroitin sulfate in heparin preparation.

Description

technical field [0001] The invention relates to a chondroitin sulfate ABC lyase mutant with high thermal stability and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate is a type of glycosaminoglycan covalently linked to proteins to form proteoglycans. Chondroitin sulfate is widely distributed in the extracellular matrix and cell surface of animal tissues. The sugar chain is composed of alternating glucuronic acid and N-acetylgalactosamine (also known as N-acetylgalactosamine) disaccharide units. The linking region is linked to a serine residue of the core protein. [0003] Chondroitin sulfate exhibits high biological activities, such as anti-cancer, anti-atherosclerosis, anti-inflammation, immune regulation, anti-oxidation, etc., not only plays a role in biological processes such as cell migration, differentiation, proliferation, recognition, and tissue formation Important role, also used in medic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P19/26C08B37/00C12R1/19
CPCC12N9/88C12N15/70C12P19/26C08B37/0075C12Y402/02004
Inventor 康振堵国成陈坚王浩张琳刘平
Owner JIANGNAN UNIV
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