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Chondroitinase AC mutant, encoding gene, vector, engineering bacterium and preparation method of chondroitinase AC mutant

A technology for chondroitinase sulfate and encoding gene, applied in the field of genetic engineering, can solve the problems of low efficiency and limited catalytic activity, and achieve the effects of expanding the binding pocket, improving the activity, and reducing the binding steric hindrance

Active Publication Date: 2020-02-11
BEIJING POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current way to obtain chondroitinase AC is through Arthrobacter aurescens obtained by fermentation and purification, but Arthrobacter aurescens The derived chondroitin sulfate AC has problems such as limited catalytic activity and low efficiency in the cleavage reaction of polysaccharides such as chondroitin sulfate or hyaluronic acid

Method used

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  • Chondroitinase AC mutant, encoding gene, vector, engineering bacterium and preparation method of chondroitinase AC mutant
  • Chondroitinase AC mutant, encoding gene, vector, engineering bacterium and preparation method of chondroitinase AC mutant
  • Chondroitinase AC mutant, encoding gene, vector, engineering bacterium and preparation method of chondroitinase AC mutant

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Embodiment 1

[0028] Example 1 Chondroitinase AC mutant protein

[0029] This embodiment provides an AC mutant of chondroitinase, which has the amino acid sequence shown in SEQ ID NO:2.

[0030] This embodiment also provides a gene encoding the above-mentioned chondroitinase AC mutant, which has the nucleotide sequence shown in SEQ ID NO:3.

Embodiment 2

[0031] Example 2 Construction of wild-type chondroitinase AC recombinant vector

[0032] Using the wild-type chondroitinase AC shown in SEQ ID NO:1 as a template, the original primers were designed for PCR amplification, and NdeI and BamHI restriction enzyme sites were introduced, and then respectively connected to the plasmid vector for gene recombination construction to obtain wild-type chondroitinase AC. Type chondroitinase AC recombinant vector, and its N-terminus is linked to a his tag for subsequent isolation and purification of the chondroitinase AC mutant. The plasmid vector can be pET15b vector, PMAL-C2X or pGEX-4T1. In this example, the plasmid vector is the pET15b vector, and the wild-type chondroitinase AC recombinant vector is named pET-15b-AachAC. For a schematic diagram, see figure 1 .

[0033] Wherein, the original upstream primer F: 5 ’ -gggaaacatatgGAAGCTGAGCCAGGTGCAGCTG-3 ’ (Among them, the lowercase letters are mainly used for modification, and the upp...

Embodiment 3

[0035] Example 3 Construction of chondroitinase AC mutant and its recombinant vector

[0036] Using the wild-type chondroitinase AC recombinant vector pET-15b-AachAC provided in Example 2 as a template, the 170th DNA sequence in the amino acid sequence shown in SEQ ID NO: 1 is designed into the upstream primer, so that The 170th position is mutated to glycine, and then reverse PCR amplification is performed to obtain the PCR product. The PCR product does not need to be recovered by gel, and is digested with FD DpnI endonuclease at 37°C for 1 h to obtain the recombinant chondroitinase AC mutant. The vector was named pET-15b-AachAC(170G).

[0037] Wherein, the upstream primer F-170G: 5 ’ -GATCCGTGGCTGCAAGGTCCGCCTAAACGT-3 ’ (SEQ ID NO: 4);

[0038] The downstream primer R-170G: 5 ’ -TTGCAGCCACGGATCTGGGACGAAG-3 ’ (SEQ ID NO: 5).

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Abstract

The invention provides a chondroitinase AC mutant and a preparation method thereof. The mutant has an amino acid sequence as shown in SEQ ID NO: 2. The invention provides an encoding gene of the chondroitinase AC mutant. The encoding gene has a nucleotide sequence shown as shown in SEQ ID NO: 3. The invention further provides a recombinant vector and engineering bacterium of the chondroitinase ACmutant. Meanwhile, the invention also discloses a preparation method of the chondroitinase AC mutant. The 170th site in the amino acid sequence SEQ ID NO: 1 of wild chondroitinase AC 1 is mutated intoglycine to construct the chondrosulphatase AC mutant, so that the steric hindrance of the combination of the chondrosulphatase AC mutant and a polysaccharide substrate is reduced, the combination pocket of the chondrosulphatase AC mutant and polysaccharide is enlarged, the chondrosulphatase AC mutant can be more easily combined with the polysaccharide substrate, and the activity of chondrosulphatase AC is improved.

Description

technical field [0001] The invention belongs to genetic engineering, and in particular relates to a chondroitinase AC mutant, a coding gene, a recombinant vector, an engineering bacterium and a preparation method thereof. Background technique [0002] Chondroitin sulfate lyase (chondroitinase or chondroitin sulfate lyase) is a kind of lyase that can degrade glycosaminoglycans such as chondroitin sulfate, chondroitin and hyaluronic acid into unsaturated disaccharides (ΔDi and oligosaccharides). According to the differences of their substrates, they are mainly divided into chondroitinase ABC, chondroitinase AC, chondroitinase B and chondroitinase C. [0003] At present, there is more and more attention to chondroitinase AC in the world, and studies have shown that chondroitinase AC has many important uses. In basic research, it is used to construct gene libraries of disaccharides and oligosaccharides; research on glycosamines Polysaccharide structure and properties; in the cl...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12R1/19
CPCC12N9/88C12N15/70C12Y402/02005
Inventor 李晔周朝袁其朋陈亮邢丽楠张晓辉
Owner BEIJING POLYTECHNIC
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