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High activity pet hydrolase mutant and its application

A hydrolytic enzyme and activity technology, applied in the field of genetic engineering, can solve the problem of low enzyme activity, achieve the effect of improving activity, good industrial application prospects, and improving degradation efficiency

Active Publication Date: 2021-09-07
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although PETase can degrade PET at 30°C, the enzyme activity is still low

Method used

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  • High activity pet hydrolase mutant and its application
  • High activity pet hydrolase mutant and its application
  • High activity pet hydrolase mutant and its application

Examples

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Comparison scheme
Effect test

Embodiment 1

[0063] Example 1, Preparation, expression purification and activity detection of PET hydrolase mutants

[0064] In order to increase the industrial application value of PET hydrolase, the present invention synthesized the gene of PET hydrolase derived from Ideonella sakaiensis, and expressed and purified it. After studying the structure of the PET hydrolase, its active region participated in the substrate The interacting amino acids were mutated to increase the activity of the enzyme towards the substrate PET.

[0065] 1. Construction of wild-type PET hydrolase recombinant plasmid and its mutant recombinant plasmid

[0066] 1. Construction of wild-type PET hydrolase recombinant plasmid

[0067] The wild-type PET hydrolase is PETase derived from Ideonella sakaiensis. The nucleotide sequence of the wild-type PET hydrolase is sequence 1 in the sequence listing, and the encoded amino acid sequence is sequence 2 in the sequence listing.

[0068] Insert the nucleotides of the wild...

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Abstract

The invention discloses a highly active PET hydrolase mutant and its application. The present invention provides a protein, which is any one of the following 1)-11): 1) The protein shown in 1) is that the asparagine at the 204th position of the wild-type PET hydrolase is mutated into alanine, and the asparagine at the 251st position is mutated into Arginine is mutated to alanine, and the amino acid residues at other positions remain unchanged, resulting in a protein with PET hydrolase activity; the present invention utilizes structural analysis and site-directed mutagenesis to mutate the original PET hydrolase (PETase) to obtain 8 This kind of mutant can change the problem of low activity of the original PET hydrolase, effectively improve the activity of PETase to degrade PET, and improve the degradation effect of PETase. PET is an insoluble high polymer that is difficult to degrade, and the eight mutants of the invention greatly improve the degradation efficiency of PET plastics, and have good industrial application prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to highly active PET hydrolase mutants and applications thereof. Background technique [0002] Plastic products have brought convenience to human life, but at the same time, the white pollution caused has seriously threatened the global ecosystem. In 2015 alone, 320 million tons of plastic were produced globally. Among them, polyethylene terephthalate (polyethyleneterephthalate, PET) plastic accounts for 18% of the total polymer in the world, and is an important source of white pollution. PET is polymerized from terephthalic acid (TPA) and ethylene glycol (EG) through ester bonds. It is stable and difficult to decompose. It is often used in mineral water bottles, polyester clothes and blister packaging. [0003] At present, the treatment methods of plastic PET waste mainly include: landfill, incineration and recycling. Although landfill and incineration are simple, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12P7/44
CPCC12N9/18C12P7/44
Inventor 韩旭刘卫东郑迎迎高健商娜李倩韦泓丽黄建文陈纯琪郭瑞庭
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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