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High-activity PET hydrolase mutants and application thereof

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 increasing activity, improving degradation efficiency, and improving degradation effect

Active Publication Date: 2020-04-24
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • 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 mutants and application thereof
  • High-activity PET hydrolase mutants and application thereof
  • High-activity PET hydrolase mutants and application thereof

Examples

Experimental program
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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 high-activity PET hydrolase mutants and application thereof. A protein provided by the invention is any one of the following 1)-11): 1) the protein shown in the specification is a protein with PET hydrolase activity obtained by mutating asparagine at the 204th site of wild PET hydrolase into alanine and mutating arginine at the 251th site into alanine and keeping amino acidresidues at other positions unchanged. According to the invention, original PET hydrolase (PETase) is mutated by utilizing a structure analysis and site-directed mutagenesis technology to obtain eight mutants, so that the problem that the original PET hydrolase is relatively low in activity is solved, the PET degradation activity of PETase is effectively improved, and the degradation effect of PETase is improved. PET is an insoluble high polymer which is difficult to degrade, the eight mutants greatly improve the degradation efficiency of PET plastic, and the industrial application prospect is promising.

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 Applications(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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