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Fusion protein and application of fusion protein in degrading polymers

A technology of fusion protein and carbohydrate, applied in fusion protein and its application field in degrading polymer, can solve problems such as effect to be improved

Active Publication Date: 2021-01-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the effect of the existing bio-enzyme degradation technology to degrade PET needs to be improved

Method used

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  • Fusion protein and application of fusion protein in degrading polymers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Preparation of cutinase HiC and fusion protein HiC-TrCBM

[0042] Specific steps are as follows:

[0043] Synthetic nucleotide sequence is as shown in SEQ ID NO.14 the gene HiC of coding cutinase HiC (amino acid sequence is as shown in SEQ ID NO.1); The gene HiC of coding cutinase and pET-32b (+) plasmid pass Megaprimer PCRofWhole Plasmids (MEGAWHOP) (specific references: Miyazaki K, MEGAWHOP cloning: amethod of creating random mutagenesis via megaprimer PCR of whole plasmamids, Methods Enzymol.2011; 499:399-406) was ligated to obtain the recombinant plasmid pET-32b ( +)-HiC; transform the recombinant plasmid pET-32b(+)-HiC into E.coli BL21 to obtain the transformation product; spread the transformation product on LB solid medium (containing 100 μg·mL -1 ampicillin) and cultured upside down in a constant temperature incubator at 37°C for 8-12 hours to obtain transformants; pick the transformants and inoculate them into LB liquid medium, shake the flask at...

Embodiment 2

[0046] Example 2: Degradation of polyethylene terephthalate (PET) by HiC and HiC-TrCBM

[0047] Specific steps are as follows:

[0048] The PET film was incubated with SDS solution with a concentration of 0.1% (v / v) at 50°C for 30 minutes, then with ultra-clean water at 50°C for 5 minutes, then with absolute ethanol at 50°C for 5 minutes, and then dried at 50°C to obtain the treated PET film; respectively add cutinase HiC and fusion protein HiC-TrCBM (40U) in the embodiment 1 containing 1 * 1cm 2 In a glass test tube of PET film, degrade on a constant temperature water bath shaker at 50° C. and 150 rpm for 4 days to obtain a degradation product.

[0049] Detect the content of phthalic acid in the degradation product (the liquid phase diagram of the TPA standard is shown in figure 1 , the liquid phase diagram of the degradation product obtained by the fusion protein degraded PET film is shown in figure 2 ), the test results were: the content of TPA in the degradation produc...

Embodiment 3

[0050] Embodiment 3: Preparation of cutinase HiC and fusion protein HiC-LCI

[0051] Specific steps are as follows:

[0052] Synthetic nucleotide sequence is as the gene HiC (amino acid sequence is as shown in SEQ ID NO.1) of the coding cutinase HiC shown in SEQ ID NO.14; The gene HiC and pET-14b (+) plasmid of coding cutinase are passed Megaprimer PCRofWhole Plasmids (MEGAWHOP) (specific references: Miyazaki K, MEGAWHOP cloning: amethod of creating random mutagenesis via megaprimer PCR of whole plasmamids, Methods Enzymol.2011; 499:399-406) was ligated to obtain the recombinant plasmid pET-14b ( +)-HiC; transform the recombinant plasmid and pET-14b(+)-HiC into E.coli BL21 to obtain the transformation product; spread the transformation product on LB solid medium (containing 100 μg·mL -1 ampicillin) and cultured upside down in a constant temperature incubator at 37°C for 8-12 hours to obtain transformants; pick the transformants and inoculate them into LB liquid medium, shake ...

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Abstract

The invention discloses a fusion protein and application of the fusion protein in degrading polymers, and belongs to the technical field of biology. The invention provides the fusion protein capable of degrading polymers such as polyethylene glycol terephthalate with good degradation effect. The fusion protein is formed by sequentially connecting a cutinase, a connecting peptide and a carbohydratebinding assembly, and when the fusion protein is used for degrading a PET film for 4 d, the content of phthalic acid (TPA) in a PET degradation product can reach up to 116.83 mg / L. When an unfused cutinase with the equal enzyme activity is used for degrading the PET film under the same condition, no phthalic acid (TPA) is detected in the degradation product, and the degradation rate of polyacrylate degraded by the fusion protein is improved by 36% compared with the degradation rate of the unfused cutinase.

Description

technical field [0001] The invention relates to a fusion protein and its application in degrading polymers, belonging to the field of biotechnology. Background technique [0002] With the rapid development of the economy, people's consumption of plastic products has increased significantly. The global annual plastic consumption exceeds 320 million tons, and this plastic consumption is increasing at a rate of 4-6% per year. However, because plastics are difficult to degrade, the global annual recycling rate of plastic products is only 14%, which makes plastic waste continue to accumulate in the environment and poses a serious threat to the ecology. [0003] Polyethylene terephthalate (PET) is a linear macromolecule formed by sequentially connecting ethylene glycol (EG) and terephthalic acid (TPA) with ester bonds. At present, polyethylene terephthalate (PET) plastic products account for about 60% of all plastic products. Correspondingly, PET plastic waste also accounts for a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N9/18C12N15/70C12N15/75C12P7/44C12R1/125C12R1/19
CPCC12N9/18C12Y301/01074C12N15/70C12N15/75C12P7/44C07K2319/01Y02W30/62
Inventor 吴敬刘展志张颖
Owner JIANGNAN UNIV
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