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Cutinase mutant capable of efficiently degrading polyethylene glycol terephthalate and application of cutinase mutant

A technology of polyethylene terephthalate and mutants, applied in the field of environmental science, can solve the problem of low efficiency of cutinase degradation of PET, and achieve the effects of improved efficiency, improved half-life and good industrial prospects

Active Publication Date: 2022-04-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the low efficiency of cutinase degradation PET, and provide new mutants and applications of cutinase

Method used

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  • Cutinase mutant capable of efficiently degrading polyethylene glycol terephthalate and application of cutinase mutant
  • Cutinase mutant capable of efficiently degrading polyethylene glycol terephthalate and application of cutinase mutant
  • Cutinase mutant capable of efficiently degrading polyethylene glycol terephthalate and application of cutinase mutant

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Experimental program
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preparation example Construction

[0103] Preparation of variants

[0104] Cutinase variants of the invention can be prepared using any mutagenesis procedure known in the art (eg, site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.).

[0105] Site-directed mutagenesis is a technique for introducing one or more (eg, several) mutations at one or more defined sites in the polynucleotide encoding the parent Cutinase.

[0106] Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis can also be performed by cassette mutagenesis, which involves cleavage by restriction enzymes at sites in the plasmid containing the polynucleotide encoding the parental cutinase and subsequent insertion of oligonucleotides containing the mutation into acid linkages in polynucleotides. Typically, the restriction enzymes that digest the plasmid and...

Embodiment 1

[0156] Embodiment 1: Construction of TfC mutant recombinant plasmid

[0157] (1) Insert wild-type cutinase (the amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 11) into the NdeI site and XhoI site of pET24a by restriction endonucleases point to obtain pET24a-TfC; then use site-directed mutagenesis (site-directed mutagenesis), with the pET24a-TfC plasmid as a template, carry out PCR with the primers shown in Table 1 respectively, and obtain the target gene mutation fragment (primers are shown in Table 1 ). The target fragment was ligated with the pET-24a expression vector by Megawhop PCR to obtain a recombinant plasmid. The constructed recombinant plasmid was transformed into Escherichia coli JM109 to obtain the transformation product; the transformation product was spread on LB solid medium (containing 40 μg / mL kanamycin), and cultured upside down in a constant temperature incubator at 37°C for 8 After ~12 hours, transformant...

Embodiment 2

[0164] Example 2: Performance of TfC mutants

[0165] The wild-type and TfC mutants were used to determine the enzyme activity, protein concentration and heat resistance.

[0166] As shown in Table 2, compared with the wild-type TfC, the activities of the nine TfC mutants have been significantly improved, such as the mutant M5 increased from 86.5U / mL to 150.4U / mL, an increase of 73.87%. At the same time, the expression of the mutants has also been improved to varying degrees, for example, the mutant M2 has increased from 0.10 mg / mL of the wild type to 0.59 mg / mL, which is 4.9 times higher than that of the wild type.

[0167] Compared with the wild-type TfC, the thermal stability of the nine TfC mutants at 60°C has been significantly improved, from 96h to 102-150h compared with the wild-type, and the half-life of the mutant M6 has been increased from 96h to 150h .

[0168] Table 2 Enzyme activity and concentration of TfC mutants

[0169] mutant Enzyme activity ...

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Abstract

The invention discloses a cutinase mutant capable of efficiently degrading polyethylene glycol terephthalate and application of the cutinase mutant, and belongs to the field of environmental science. The cutinase TfC derived from Thermobifida fusca is modified to obtain a series of mutants, the mutants are subjected to single-point or multi-point mutation near a substrate binding site of the TfC, the 65th site, the 92th site, the 184th site, the 209th site and the 213th site of the TfC are subjected to mutation, and nine mutants are constructed. Compared with wild TfC, the 9 TfC mutants (M1-M9) have the advantages that the enzyme activity and the half-life period at 60 DEG C are obviously improved, the PET degradation efficiency is obviously improved, and the industrial prospect is good.

Description

technical field [0001] The invention relates to a cutinase mutant capable of efficiently degrading polyethylene terephthalate and its application, belonging to the field of environmental science. Background technique [0002] Polyethylene terephthalate (PET) is a kind of plastic, because of its advantages of high mechanical strength, low gas permeability, light weight, low cost and price, it has been widely used all over the world . A large amount of PET waste cannot be effectively recycled and is difficult to degrade in the natural state, resulting in the accumulation of PET in the global ecosystem, causing the loss of land nutrients and soil compaction, and a large amount of plastic floating on the ocean will cause devastating damage to the marine ecosystem. hit. At present, landfill, incineration, physical and chemical recovery treatment methods cause energy waste and have extremely serious impact on the environment. Therefore, biodegradation is considered to be the mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/63A62D3/02A62D101/28
CPCY02W30/62
Inventor 吴敬颜正飞陈晓倩王蕾
Owner JIANGNAN UNIV
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