PET lytic enzyme mutant, coding gene and application thereof

A technology for mutant and decomposing enzymes, applied in the field of genetic engineering of enzymes, can solve the problems of low absolute degradation rate and inability to degrade hcPET quickly, and achieve the effect of improving enzyme activity

Inactive Publication Date: 2017-02-01
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, PETase has a good degradation effect on highly crystalline PET plastic (hcPET), and other PET-degradable enzymes such as TfH, LCC, and FsC cannot degrade hcPET quickly
[0005] Although the PETase has the above characteristics, the absolute degradation rate of the enzyme to PET is still low

Method used

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  • PET lytic enzyme mutant, coding gene and application thereof
  • PET lytic enzyme mutant, coding gene and application thereof
  • PET lytic enzyme mutant, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Construction and application of pET-21b-PET decomposing enzyme mutant J:

[0020] Chemically synthesize the pET-21bP nucleotide sequence shown in SEQ ID NO.1;

[0021] The PETase gene shown in SEQ ID NO.2 was excised from the pET-21bP nucleotide sequence and ligated into the pET-21b plasmid.

[0022] a) enzyme digestion system

[0023]

[0024] b) conditions

[0025] i. Digest the target gene for 1 hour at 37°C.

[0026] ii. Digest the plasmid for 2 hours at 37°C.

[0027] iii. After digestion, inactivate at 80°C for 5 minutes.

[0028] Linking the PETase gene shown in SEQ ID NO.2 into pET-21b to obtain the nucleotide sequence of pET-21b-PETase;

[0029] a) Connection system

[0030]

[0031] b) Conditions: 22°C, 2h.

[0032] Using OverlapPCR technology to mutate the nucleotide sequence of pET-21b-PETase;

[0033] Prepare Overlap PCR reaction system

[0034] a) Plasmid 10ng

[0035] b)Primer + 1μL

[0036] c) Primer- 1μL

[0037] d) 2xTransSt...

Embodiment 2

[0058] Example 2 Construction and application of pET-21b-PET decomposing enzyme mutant M:

[0059] Chemically synthesize the pET-21bP nucleotide sequence shown in SEQ ID NO.1;

[0060] The PETase gene shown in SEQ ID NO.2 was excised from the pET-21bP nucleotide sequence and ligated into the pET-21b plasmid.

[0061] a) enzyme digestion system

[0062]

[0063] b) conditions

[0064] i. Digest the target gene for 1 hour at 37°C.

[0065] ii. Digest the plasmid for 2 hours at 37°C.

[0066] iii. After digestion, inactivate at 80°C for 5 minutes.

[0067] Linking the PETase gene shown in SEQ ID NO.2 into pET-21b to obtain the nucleotide sequence of pET-21b-PETase;

[0068] c) Connection system

[0069]

[0070] d) Conditions: 22°C, 2h.

[0071] Using OverlapPCR technology to mutate the nucleotide sequence of pET-21b-PETase;

[0072] Prepare Overlap PCR reaction system

[0073] f) Plasmid 10ng

[0074] g)Primer + 1μL

[0075] h)Primer- 1μL

[0076] i) 2xTransSta...

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Abstract

The invention discloses a PET (polyethylene terephthalate) lytic enzyme mutant, a coding gene and application thereof. The PET lytic enzyme mutant is represented by an amino acid sequence SEQ ID NO.8 or SEQ ID NO.9 obtained by performing point mutation on an amino acid sequence SEQ ID NO.7. A lytic enzyme gene of escherichia coli is subjected to directed evolution; by site-specific mutagenesis, the PET lytic enzyme mutant is obtained. According to the amount of decomposition products within the same time, the enzyme activity of the PET lytic enzyme mutant is improved.

Description

technical field [0001] The invention belongs to the genetic engineering of enzymes, and in particular relates to mutants of PET decomposing enzymes, coding genes and applications thereof. Background technique [0002] PET (polyethylene terephthalate) has been industrially produced in large quantities for a long time due to its plasticity, transparency and durability. In 2013 alone, the world produced about 56 million tons of PET plastic. Due to the lack of enzymes or extremely low activity that can degrade PET in nature, PET plastics are difficult to be degraded naturally in nature, and become a kind of pollutant, which has a great impact on the environment. [0003] PET decomposing enzyme ISF6_4831 is a kind of cutinase. Cutinase is a decomposing enzyme that can catalyze the decomposition of ester groups in cutin and produce a large amount of fatty acid monomers. It is a member of the α / β decomposing enzyme family with a smaller molecular weight. The catalytic activity of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55
CPCC12N9/18
Inventor 王泽方杨海涛段胤凯蒋林芮陈卓芝牟钟毓刘曼琳胡睿行
Owner TIANJIN UNIV
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