Chondroitinase ac mutant and preparation method thereof

A chondroitin sulfate enzyme and mutant technology, applied in the field of genetic engineering, can solve problems such as low efficiency and limited catalytic activity

Active Publication Date: 2021-07-06
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 and preparation method thereof
  • Chondroitinase ac mutant and preparation method thereof
  • Chondroitinase ac mutant and preparation method thereof

Examples

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Embodiment 1

[0026] Example 1 Chondroitinase AC mutant protein

[0027] This embodiment provides an AC mutant of chondroitinase, the amino acid sequence of which is shown in SEQ ID NO:2.

[0028] This embodiment also provides a gene encoding the AC mutant of chondroitinase sulfate.

Embodiment 2

[0029] Example 2 Construction of wild-type chondroitinase AC recombinant vector

[0030] Using the DNA synthesis sequence corresponding to the amino acid sequence of 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 ligated to the plasmid The wild-type chondroitinase AC recombinant vector was constructed by carrying out gene recombination on the vector, and his tag was linked to its N-terminus to facilitate 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 .

[0031] Wherein, the original upstream primer F: 5 ’ -gggaaacatatgGAAGCTGAGCCAGGTGCAGCTG-3 ’ (Among them, the lowercase ...

Embodiment 3

[0033] Example 3 Construction of chondroitinase AC mutant and its recombinant vector

[0034] 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 was designed into the upstream primer, Make the 170th position mutated to glycine, and then carry out inverse PCR amplification 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 chondroitinase AC mutant body The group vector was named pET-15b-AachAC-170G.

[0035] Wherein, the upstream primer F-170G: 5 ’ -GATCCGTGGCTGCAAGGTCCGCCTAAACGT-3 ’ (SEQ ID NO: 3);

[0036] The downstream primer R-170G: 5 ’ -TTGCAGCCACGGATCTGGGACGAAG-3 ’ (SEQ ID NO: 4).

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Abstract

The present invention provides a chondroitinase AC mutant, the amino acid sequence of which is shown in SEQ ID NO:2. The present invention also provides a method for preparing the AC mutant of chondroitinase sulfate. The present invention constructs the chondroitinase AC mutant by mutating the 170th position in the wild-type chondroitinase AC amino acid sequence SEQ ID NO:1 to glycine, thus reducing the chondroitinase AC mutant and polysaccharide The steric hindrance of substrate binding expands its binding pocket with polysaccharides, making it easier to bind polysaccharide substrates, thereby increasing the activity of chondroitinase AC.

Description

technical field [0001] The invention belongs to genetic engineering, and in particular relates to a chondroitinase AC mutant 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 clinical field, it is used to treat some diseases. In addition, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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