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PET degradation biocatalyst and its application

A catalyst and enzyme-degrading technology, applied in biochemical cleaning devices, enzyme production/bioreactors, biochemical equipment and methods, etc., can solve the problems of difficult separation and recycling of blended fibers, secondary pollution of the environment, expensive catalysts, etc., to achieve Significant economy and high efficiency, low reaction temperature, and reduced process cost

Active Publication Date: 2021-12-31
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem of this type of method is: secondary pollution to the environment, or the use of expensive catalysts, the economic benefit is low
In addition, since PET is a common textile material, it is often used to synthesize polyester, and the blended fabric of polyester and cotton—polyester cotton is widely used, but the blended fiber is difficult to separate and recycle, so its waste treatment is also a problem that needs to be solved for environmental protection. serious problem

Method used

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  • PET degradation biocatalyst and its application
  • PET degradation biocatalyst and its application
  • PET degradation biocatalyst and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Plasmid expresses PET degrading enzyme

[0037] The genes encoding PET degrading enzymes such as LCC (SEQ NO.1), Tcur (serial number ACY96861.1), PETase (serial number GAP38373.1), Tcut (serial number WP_011291330) were selected for expression in Clostridium thermocellum DSM1313, Construction of PET degradation biocatalyst. The specific steps are:

[0038] 1) Connect the signal peptide sequence (SEQ NO. 2) to the 5' end of the above gene by seamless cloning.

[0039] 2) Connect the sequence obtained in step 1) to the 3' end of the promoter I of the pHK plasmid using the seamless cloning method. Wherein pHK is a shuttle plasmid with replicons of Escherichia coli and Clostridium at the same time, with chloramphenicol and thiamphenicol resistance genes; the promoter I sequence is SEQ NO. 3, and the obtained PET degrading enzyme expression plasmid is shown in figure 1 . pLCC is a plasmid expressing LCC; pTcur is a plasmid expressing Tcur; pPETase is a pla...

Embodiment 2

[0042] Example 2: Genomic expression of PET degrading enzymes

[0043] (1) Using the method of seamless cloning, sequentially connect the nucleic acid sequences of promoter I, signal peptide, LCC and MHETase in SEQ NO.3, SEQ NO.2, SEQ NO.1 and SEQ NO.9 to form a fusion expression box, wherein the stop codon of LCC is removed.

[0044] (2) Select the 16SrRNA gene sequence as the target site, and clone the above fusion expression cassette as the target gene into the homologous recombination plasmid pHK-HR (Reference 1) to obtain the genomic recombination plasmid pHK-HR-PET ( figure 2 ). Using the scarless knockout and screening method (document 1), it was integrated into the genome of Clostridium thermocellum, and strains expressing LCC and MHETase were obtained by genome fusion.

[0045] (3) After the mutant strain is obtained, the enzyme activity of the extracellular protein with pNPB as the substrate is determined to confirm whether the PET degrading enzyme is successfully...

Embodiment 3

[0046] Embodiment 3: express PET degradation body:

[0047] (a) The plasmid pLa-Mf co-transcriptionally expressing LCC and MHETase was constructed, and the two enzymes were respectively fused with assembly modules from Clostridium acetobutylicum and Clostridium flavum. Specifically: the expression assembly module DocCa (SEQ NO.4) was fused at the 3' end of the LCC coding gene of the above pLCC plasmid to obtain plasmid pLa; the RBS sequence (AGGAGG) and the coding gene of MHETase ( SEQ NO.9) and the gene of the assembly module DocCf (SEQ NO.5) to obtain the plasmid pLDa-MDc.

[0048] (b) Cloning the co-transcriptional expression cassette in pLDa-MDf as the target gene into the homologous recombination plasmid pHK-HR (Reference 1), selecting the 16S rRNA gene sequence as the target site, and using the scarless knockout and screening method (Reference 1) 1), integrated into the genome of Clostridium thermocellum, to obtain strains co-transcribing and expressing LCC and MHETase ...

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Abstract

Aiming at the problems existing in the degradation of PET plastics in the prior art, the invention provides a novel PET degradation biocatalyst and a construction method thereof. The PET-degrading whole-bacteria catalyst is obtained by expressing PETase in a heat-resistant strain; the PETase has a sequence of SEQ NO.1 or a sequence with a sequence similarity of 99% to SEQ NO.1; the heat-resistant strain is Clostridium thermocellum. The method for constructing the PET-degrading whole bacterium catalyst includes ① expressing the PET-degrading enzyme in a plasmid, ② expressing the PET-degrading enzyme in a genome, and ③ expressing a PET-degrading body. The PET-degrading whole-bacteria catalyst overcomes the problem of feedback inhibition, not only has a degradation efficiency significantly higher than that of known whole-bacteria biodegradation systems, but also as an anaerobic microorganism, its culture conditions do not require aeration and stirring, thereby significantly reducing the process cost. In addition, this application also uses a one-pot method to realize the simultaneous degradation of fibers and PET in blended fabrics; not only the reaction temperature is low, no pre-separation of fibers is required, and no additional carbon source is required during the degradation process, which has significant economical and Efficiency.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a biocatalyst, in particular to a biocatalyst for degrading PET plastics and its application in the degradation of PET-containing materials. Background technique [0002] Plastic products have brought great convenience to human life, but due to the lack of effective means of recycling and regeneration, it has caused global white pollution. Polyethylene terephthalate (PET) is one of the most used plastic raw materials in the world; PET plastic has a wide range of applications in the packaging field, such as packaging films, rolls and beer bottles. PET plastic bottles are not only widely used in packaging carbonated beverages, drinking water, fruit juices, enzymes and tea beverages, but also in many fields such as food, chemical and pharmaceutical packaging. Tens of billions of PET plastic bottles are produced in China every year, but when these materials are discarded, they have to face...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12M1/40C12M1/38C12M1/02C12M1/00C08J11/10C12R1/145C08L67/02
CPCC12N9/18C12N15/74C12M21/18C12M23/58C12M25/18C12M27/02C12M41/24C08J11/105C12Y301/01C07K2319/02C08J2367/02Y02W30/62
Inventor 崔球刘亚君颜飞韦韧
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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