CN112159478A

CN112159478A - Fusion protein and application thereof in polymer degradation

Info

Publication number: CN112159478A
Application number: CN202011040885.XA
Authority: CN
Inventors: 吴敬; 刘展志; 张颖
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2021-01-01
Anticipated expiration: 2040-09-28
Also published as: CN112159478B

Abstract

The invention discloses a fusion protein and application thereof in polymer degradation, belonging to the technical field of biology. The invention provides a fusion protein capable of degrading polymers such as polyethylene terephthalate and the like with good degradation effect, the fusion protein is obtained by sequentially connecting cutinase, connecting peptide and carbohydrate combination components, the fusion protein is used for degrading a PET film for 4d, so that the content of phthalic acid (TPA) in a PET degradation product can reach 116.83mg/L, the PET film is degraded by using unfused cutinase with equal enzyme activity under the same condition, no phthalic acid (TPA) is detected in the degradation product, and the degradation rate of polyacrylate by using the fusion protein is improved by 36% compared with that of the unfused cutinase.

Description

Fusion protein and application thereof in polymer degradation

Technical Field

The invention relates to a fusion protein and application thereof in polymer degradation, belonging to the technical field of biology.

Background

With the rapid development of economy, the consumption level of plastic products by people is remarkably improved, the annual plastic consumption of the world exceeds 3.2 hundred million tons, and the annual plastic consumption is increased at a speed of 4-6%. However, since plastics are difficult to degrade, the annual plastic product recycling rate is only 14% worldwide, which causes the plastic wastes to continuously accumulate in the environment and poses a serious ecological threat.

Polyethylene terephthalate (PET) is a linear macromolecule formed by the sequential connection of Ethylene Glycol (EG) and Terephthalic acid (TPA) via ester bonds. Currently, polyethylene terephthalate (PET) plastic products account for approximately 60% of all plastic products, and correspondingly, PET plastic waste accounts for a relatively high percentage of all plastic waste. Therefore, the degradation of PET is very key to the treatment of plastic waste.

At present, people still stay at the stage of degrading PET by using traditional chemical degradation methods such as acidolysis, alkaline hydrolysis or alcoholysis or physical degradation methods such as pyrolysis. However, chemical degradation methods require the use of large amounts of chemicals and physical degradation methods require high temperature and pressure equipment, which greatly increases the cost of PET remediation. In addition, a large amount of toxic and harmful substances can be generated in the process of degrading PET by using a chemical degradation method, and the toxic and harmful substances can generate serious negative effects on the ecological environment, so that the degradation of PET is not paid. Therefore, countries around the world are still actively exploring new techniques for degrading PET.

The biological enzyme degradation technology is a technology for degrading plastics by directly using biological enzyme capable of degrading plastics, and is gradually a research hotspot in the field of plastics degradation due to the advantages of green, no pollution and low cost. The cutinase can catalyze and hydrolyze ester bonds of insoluble polymer plant cutin, and can degrade long-chain and short-chain fatty acid esters, soluble synthetic esters, emulsified triglyceride and the like, and is a multifunctional lyase. The cutinase has effectEster bonds in polyester molecules such as PET, polyurethane, etc. are broken. However, compared with C — O bond linked bio-based plastics such as Polyhydroxyalkanoate (PHA) and polylactic acid (PLA), PET molecular chains contain a large amount of aromatic groups, which results in large steric hindrance of PET molecular chains and more hydrophobic surfaces, and thus the PET molecular chains are difficult to be bio-enzymatically hydrolyzed, especially at room temperature or lower temperature. For example,

ronkvist et al degraded PET with cutinase from Pseudomonas mendocina for 96h, with a PET degradation rate of only 5% (see in particular the reference: Ronkvist;)

M,Xie W,Lu W,et al.Cutinase-Catalyzed Hydrolysis of Poly(ethylene terephthalate).Macromolecules,2009,42(14):5128-5138)。

Therefore, the effect of degrading PET by using the existing biological enzyme degradation technology needs to be improved.

Disclosure of Invention

[ problem ] to

The technical problem to be solved by the invention is to provide an enzyme which has good degradation effect and can degrade polymers such as polyethylene terephthalate (PET).

[ solution ]

To solve the above technical problems, the present invention provides a fusion protein comprising a cutinase, a linker peptide and a carbohydrate-binding module (CBM).

In one embodiment of the invention, the fusion protein is formed by sequentially linking cutinase, a linker peptide and a carbohydrate binding module.

In one embodiment of the invention, the cutinase has an amino acid sequence as shown in SEQ ID No.1, SEQ ID No.2SEQ ID No.3, SEQ ID No.4 or SEQ ID No. 5.

In one embodiment of the invention, the amino acid sequence of the linker peptide is as shown in SEQ ID NO.6, SEQ ID NO.7SEQ ID NO.8 or SEQ ID NO. 9.

In one embodiment of the invention, the amino acid sequence of the carbohydrate-binding module is as shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO. 13.

The invention also provides a gene, and the gene codes the fusion protein.

The invention also provides a recombinant plasmid which carries the gene.

In one embodiment of the present invention, the expression vector of the recombinant plasmid is pET-5a (+) plasmid, pET-11b (+) plasmid, pET-14b (+) plasmid, pHY300PLK plasmid, pET-20b (+) plasmid, pET-21b (+) plasmid, pET-24a (+) plasmid, pET-28a (+) plasmid, pET-30c (+) plasmid, pET-32b (+) plasmid, pET-34b (+) plasmid, pET-40b (+) plasmid, pET-41b (+) plasmid, pET-42c (+) plasmid, pET-49b (+) plasmid, pET-50b (+) plasmid or pRDuet-1 plasmid.

The invention also provides a host cell, which carries the gene or the recombinant plasmid.

In one embodiment of the invention, the host cell is Escherichia coli (Escherichia coli) or Bacillus subtilis (Bacillus subtilis).

The invention also provides a method for degrading a polymer, which is to add the fusion protein into a degradation system containing the polymer for degradation.

In one embodiment of the invention, the polymer is polyethylene terephthalate or polyacrylate.

In one embodiment of the present invention, the temperature of the degradation is 20 to 70 ℃, the pH is 5.0 to 9.0, and the rotation speed is 100 to 200 rpm.

The invention also provides the application of the fusion protein or the gene or the recombinant plasmid or the host cell or the method in degrading the polymer.

[ advantageous effects ]

The invention provides a fusion protein capable of degrading polymers such as polyethylene terephthalate and the like with good degradation effect, the fusion protein is obtained by sequentially connecting cutinase, connecting peptide and carbohydrate combination components, the fusion protein is used for degrading a PET film for 4d, so that the content of phthalic acid (TPA) in a PET degradation product can reach 116.83mg/L, the PET film is degraded by using unfused cutinase with equal enzyme activity under the same condition, no phthalic acid (TPA) is detected in the degradation product, and the degradation rate of polyacrylate by using the fusion protein is improved by 36% compared with that of the unfused cutinase.

Drawings

FIG. 1: liquid phase diagram of terephthalic acid (TPA) standard.

FIG. 2: and (3) a liquid phase diagram of degradation products obtained by degrading the PET film by the fusion protein.

Detailed Description

pET-24a (+) plasmid, pET-20b (+) plasmid, pET-32b (+) plasmid, pET-14b (+) plasmid, and pET-40b (+) plasmid, which are referred to in the following examples, were purchased from TaKaRa bioengineering, Inc.; coli (Escherichia coli) BL21 referred to in the examples below was purchased from Youbao; the PET films referred to in the examples below were purchased from Goodfellow; the Phthalic Acid (TPA) standards referred to in the examples below were purchased from sigma.

The reagents and media involved in the following examples are as follows:

LB liquid medium: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract.

LB solid medium: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract and 20g/L of agar.

TB liquid medium: 10g/L of peptone, 24g/L of yeast powder and 5g/L, K of glycerol₂HPO₄·3H₂O 16.43g/L、KH₂PO₄2.31 g/L。

The detection methods referred to in the following examples are as follows:

determination of cutinase enzyme activity:

continuous spectrophotometry was used. The reaction system is 1.5mL and comprises 30 mu L of crude enzyme solution of cutinase, 30 mu L of 50mmol/L p-nitrobenzyl butyrate (pNPB) and 1440 mu L of 10mM Tris-HCl buffer solution (pH 8.0); the reaction system was reacted at 37 ℃ and the absorbance of the reaction solution at 405nm was measured by an ultraviolet spectrophotometer during the reaction to record the production rate of p-nitrophenol (see specifically references: Ruoyu Hong, Yirong Sun, Lingqai Su, et al. high-level expression of Humicola insolens enzymes in Pichia pastoris with out carbon stability and use in a cotton biological combustion. journal of Biotechnology,2019,304: 10-15).

The cutinase enzyme activity is defined as: the enzyme amount for catalyzing the hydrolysis of p-nitrobenzyl butyrate to generate 1 mu mol of p-nitrophenol per minute is an enzyme activity unit (1U) at 37 ℃.

Determination of phthalic acid content in degradation products:

centrifuging the degradation product to obtain a supernatant, mixing the supernatant with methanol according to a volume ratio of 6:4, filtering the mixed solution with a filter membrane of only 0.22 mu M, and injecting the filtered mixed solution into a liquid bottle by using an injector for HPLC detection;

the HPLC chromatographic conditions are as follows: agilent 1200HPLC chromatograph, Agilent autosampler, Athena C18-WP,

4.6X 250mm, 5 μm column chromatography, Agilent UV detector, 60% (v/v) 1% acetic acid solution and 40% methanol solution as mobile phase, column temperature set at 30 deg.C, flow rate of 0.5mL min^-1。

Example 1: preparation of cutinase HiC and fusion protein HiC-TrCBM

The method comprises the following specific steps:

synthesizing a gene HiC (the amino acid sequence is shown as SEQ ID NO. 1) of coding cutinase HiC, the nucleotide sequence of which is shown as SEQ ID NO. 14; the gene HiC encoding cutinase and the pET-32b (+) plasmid were ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via Megaprimer PCR of white Plasmids, Methods enzymol.2011; 499: 399-; the recombinant plasmid pET-32b (+) -HiC is transformed into E.coli BL21 to obtainTo the conversion product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and correctly sequencing to obtain the recombinant bacterium E.coli BL21/pET-32b (+) -HiC.

Synthesizing a gene (the amino acid sequence is shown as SEQ ID NO. 6) of the coding connecting peptide, the nucleotide sequence of which is shown as SEQ ID NO. 19; synthesizing a gene TrCBM (amino acid sequence shown as SEQ ID NO. 10) of which the nucleotide sequence is shown as SEQ ID NO.23 and which codes a CBM component; sequentially connecting the gene HiC for coding cutinase, the gene for coding connecting peptide and the gene TrCBM for coding a CBM component by MEGAWHOP and connecting the genes with a pET-32b (+) plasmid to obtain a recombinant plasmid pET-32b (+) -HiC-TrCBM; transforming the recombinant plasmid pET-32b (+) -HiC-TrCBM to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting a plasmid, sequencing, and obtaining the recombinant bacterium E.coli BL21/pET-32b (+) -HiC-TrCBM after verification.

Respectively scribing recombinant bacteria E.coli BL21/pET-32b (+) -HiC and recombinant bacteria E.coli BL21/pET-32b (+) -HiC-TrCBM on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernatant, wherein the cell disruption supernatant is crude enzyme liquid of cutinase HiC and fusion protein HiC-TrCBM.

Example 2: HiC and HiC-TrCBM degradation of polyethylene terephthalate (PET)

The method comprises the following specific steps:

sequentially incubating a PET membrane with SDS solution with concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase HiC and the fusion protein HiC-TrCBM (40U) of example 1 were added to a solution containing 1X 1cm²Degrading the PET film in a glass test tube for 4 days in a constant-temperature water bath shaker at 50 ℃ and 150rpm to obtain a degradation product.

The content of phthalic acid in the degradation product is detected (the liquid phase diagram of the TPA standard product is shown in figure 1, and the liquid phase diagram of the degradation product obtained by degrading the PET film by the fusion protein is shown in figure 2), and the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein HiC-TrCBM is up to 116.83mg/L, while TPA is not detected in the degradation product obtained by degrading the PET film by the unfused cutinase HiC.

Example 3: preparation of cutinase HiC and fusion protein HiC-LCI

The method comprises the following specific steps:

synthesizing a gene HiC (the amino acid sequence is shown as SEQ ID NO. 1) of coding cutinase HiC, the nucleotide sequence of which is shown as SEQ ID NO. 14; the gene HiC encoding cutinase and the pET-14b (+) plasmid were ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via Megaprimer PCR of white Plasmids, Methods enzymol.2011; 499: 399-; transforming the recombinant plasmid and pET-14b (+) -HiC to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and correctly sequencing to obtain the recombinant bacterium E.coli BL21/pET-14b (+) -HiC.

Synthesizing a gene (amino acid sequence is shown as SEQ ID NO) of the coding connecting peptide with the nucleotide sequence shown as SEQ ID NO. 20.7) is shown in the specification; synthesizing a gene LCI (amino acid sequence shown as SEQ ID NO. 11) of the CBM component with a nucleotide sequence shown as SEQ ID NO. 24; sequentially connecting the gene HiC for coding cutinase, the gene for coding a connecting peptide and the gene LCI for coding a CBM component by MEGAWHOP and connecting the genes with a pET-14b (+) plasmid to obtain a recombinant plasmid pET-14b (+) -HiC-LCI; transforming the recombinant plasmid pET-14b (+) -HiC-LCI into E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, sequencing, and obtaining the recombinant bacterium E.coli BL21/pET-14b (+) -HiC-LCI after verification.

Respectively scribing recombinant bacteria E.coli BL21/pET-14b (+) -HiC and recombinant bacteria E.coli BL21/pET-14b (+) -HiC-LCI on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernatant, wherein the cell disruption supernatant is crude enzyme liquid of cutinase HiC and fusion protein HiC-LCI.

Example 4: degradation of PET by HiC and HiC-LCI

The method comprises the following specific steps:

sequentially incubating a PET membrane with SDS solution with concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase HiC and the fusion protein HiC-LCI (50U) from example 3 were added to a solution containing 1X 1cm²Degrading the PET film in a glass test tube for 4 days in a constant-temperature water bath shaker at 50 ℃ and 150rpm to obtain a degradation product.

And (3) detecting the content of phthalic acid in the degradation product, wherein the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein HiC-LCI is as high as 93.5mg/L, while TPA is not detected in the degradation product obtained by degrading the PET film by the unfused cutinase HiC.

Example 5: preparation of cutinase FsC and fusion protein FsC-LCI

The method comprises the following specific steps:

synthesizing a gene FsC (the amino acid sequence is shown as SEQ ID NO. 2) of coding cutinase FsC, the nucleotide sequence of which is shown as SEQ ID NO. 15; the gene FsC encoding cutinase and the pET-20b (+) plasmid were post-ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via Megaprimer PCR of white Plasmids, Methods enzymol.2011; 499: 399-; transforming the recombinant plasmid pET-20b (+) -FsC to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and correctly sequencing to obtain the recombinant bacterium E.coli BL21/pET-20b (+) -FsC.

Synthesizing a gene (the amino acid sequence is shown as SEQ ID NO. 8) of the coding connecting peptide, the nucleotide sequence of which is shown as SEQ ID NO. 21; synthesizing a gene LCI (amino acid sequence shown as SEQ ID NO. 11) of the CBM component with a nucleotide sequence shown as SEQ ID NO. 24; sequentially connecting a gene FsC for coding cutinase, a gene for coding a connecting peptide and a gene LCI for coding a CBM assembly through MEGAWHOP, and then connecting the genes with pET-20b (+) plasmid to obtain recombinant plasmid pET-20b (+) -FsC-LCI; transforming the recombinant plasmid pET-20b (+) -FsC-LCI into E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting transformants, inoculating the transformants into an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ under the condition of 180rpmAnd extracting the plasmid for sequencing, and obtaining the recombinant bacterium E.coli BL21/pET-20b (+) -FsC-LCI after verification is correct.

Respectively scribing recombinant bacteria E.coli BL21/pET-20b (+) -FsC and recombinant bacteria E.coli BL21/pET-20b (+) -FsC-LCI on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernatant, wherein the cell disruption supernatant is crude enzyme liquid of cutinase FsC and fusion protein FsC-LCI.

Example 6: degradation of PET by FsC and FsC-LCI

The method comprises the following specific steps:

sequentially incubating a PET membrane with SDS solution with the concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase FsC and the fusion protein FsC-LCI (50U) from example 5 were added to a solution containing 1X 1cm²Degrading the PET film in a glass test tube for 4 days in a constant-temperature water bath shaker at 40 ℃ and 150rpm to obtain a degradation product.

And (3) detecting the content of phthalic acid in the degradation product, wherein the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein FsC-LCI is up to 107.3mg/L, while TPA is not detected in the degradation product obtained by degrading the PET film by the unfused cutinase FsC.

Example 7: preparation of cutinase FsC and fusion protein FsC-TrCBM

The method comprises the following specific steps:

synthesizing a gene FsC (the amino acid sequence is shown as SEQ ID NO. 2) of coding cutinase FsC, the nucleotide sequence of which is shown as SEQ ID NO. 15; gene FsC encoding cutinase and pET-24a (+) Plasmid were used to prepare a Plasmid of white Plasmid by Megaprimer PCRs (MEGAWHOP) (see the literature: Miyazaki K, MEGAWHOP cloning: a method of cloning and random mutagenesis via a primer PCR of plasmid, Methods enzymol.2011; 499: 399-; transforming the recombinant plasmid pET-24a (+) -FsC to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and correctly sequencing to obtain the recombinant bacterium E.coli BL21/pET-24a (+) -FsC.

Synthesizing a gene (the amino acid sequence is shown as SEQ ID NO. 6) of the coding connecting peptide, the nucleotide sequence of which is shown as SEQ ID NO. 19; synthesizing a gene TrCBM (amino acid sequence shown as SEQ ID NO. 10) of which the nucleotide sequence is shown as SEQ ID NO.23 and which codes a CBM component; sequentially connecting the gene FsC for coding cutinase, the gene for coding connecting peptide and the gene TrCBM for coding a CBM component by MEGAWHOP and connecting the genes with a pET-24a (+) plasmid to obtain a recombinant plasmid pET-24a (+) -FsC-TrCBM; transforming the recombinant plasmid pET-24a (+) -FsC-TrCBM to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting a plasmid, sequencing, and obtaining the recombinant bacterium E.coli BL21/pET-24a (+) -FsC-TrCBM after verification.

Respectively scribing the recombinant bacterium E.coli BL21/pET-24a (+) -FsC and the recombinant bacterium E.coli BL21/pET-24a (+) -FsC-TrCBM on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain a single colony; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernatant, wherein the cell disruption supernatant is crude enzyme liquid of cutinase FsC and fusion protein FsC-TrCBM.

Example 8: degradation of PET by FsC and FsC-TrCBM

Sequentially incubating a PET membrane with SDS solution with the concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase FsC and the fusion protein FsC-TrCBM (45U) of example 7 were added to a solution containing 1X 1cm²Degrading the PET film in a glass test tube for 4 days in a constant-temperature water bath shaker at 40 ℃ and 150rpm to obtain a degradation product.

And (3) detecting the content of phthalic acid in the degradation product, wherein the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein FsC-TrCBM is up to 36.28mg/L, while TPA is not detected in the degradation product obtained by degrading the PET film by the unfused cutinase FsC.

Example 9: preparation of cutinase TfC and fusion protein BaCBM2-TfC

The method comprises the following specific steps:

synthesizing a gene TfC (the amino acid sequence is shown as SEQ ID NO. 3) of a cutinase TfC with a nucleotide sequence shown as SEQ ID NO. 16; the gene TfC encoding cutinase and the plasmid pET-24a (+) were ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via Megaprimer PCR of white Plasmids, Methods enzymol.2011; 499: 399-; converting the recombinant plasmid pET-24a (+) -TfC into E.coli BL21 to obtain a conversion product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and obtaining the recombinant bacterium E.coli BL21/pET-24a (+) -TfC after sequencing is correct.

Synthetic nucleotide sequencesA gene (the amino acid sequence is shown as SEQ ID NO. 7) for coding the connecting peptide shown as SEQ ID NO. 20; synthesizing a gene BaCBM2 (the amino acid sequence is shown as SEQ ID NO. 12) of which the nucleotide sequence is shown as SEQ ID NO.25 and which codes a CBM component; sequentially connecting a gene BaCBM2 for encoding a CBM component, a gene for encoding a connecting peptide and a gene TfC for encoding cutinase through MEGAWHOP and connecting the genes with a pET-24a (+) plasmid to obtain a recombinant plasmid pET-24a (+) -BaCBM 2-TfC; converting the recombinant plasmid pET-24a (+) -BaCBM2-TfC to E.coli BL21 to obtain a conversion product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting a plasmid, sequencing, and obtaining a recombinant bacterium E.coli BL21/pET-24a (+) -BaCBM2-TfC after verification.

Respectively scribing the recombinant bacterium E.coli BL21/pET-24a (+) -TfC and the recombinant bacterium E.coli BL21/pET-24a (+) -BaCBM2-TfC on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain a single colony; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernatant, wherein the cell disruption supernatant is crude enzyme liquid of cutinase TfC and fusion protein BaCBM 2-TfC.

Example 10: degradation of PET by TfC and BaCBM2-TfC

The method comprises the following specific steps:

sequentially incubating a PET membrane with SDS solution with the concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase TfC and the fusion protein BaCBM2-TfC (50U) obtained in example 9 were added to a solution containing 1X 1cm²Degrading the PET film in a glass test tube for 4 days in a constant-temperature water bath shaker at 50 ℃ and 150rpm to obtain a degradation product.

Detecting the TPA content in the degradation product, wherein the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein BaCBM2-TfC is as high as 30mg/L, while TPA is not detected in the degradation product obtained by not fusing the cutinase TfC and degrading the PET film.

Example 11: preparation of PET degrading enzyme PETase and fusion protein BaCBM2-PETase

The method comprises the following specific steps:

synthesizing gene PETase (amino acid sequence is shown as SEQ ID NO. 4) of coding cutinase PETase, wherein the nucleotide sequence is shown as SEQ ID NO. 17; the genes PETase encoding cutinase and pET-40b (+) plasmid were ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via Megaprimer PCR of white Plasmids, Methods enzymol.2011; 499: 399-; transforming the recombinant plasmid pET-40b (+) -PETase to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and correctly sequencing to obtain the recombinant bacterium E.coli BL21/pET-40b (+) -PETase.

Synthesizing a gene (the amino acid sequence is shown as SEQ ID NO. 8) of the coding connecting peptide, the nucleotide sequence of which is shown as SEQ ID NO. 21; synthesizing a gene BaCBM2 (the amino acid sequence is shown as SEQ ID NO. 12) of which the nucleotide sequence is shown as SEQ ID NO.25 and which codes a CBM component; sequentially connecting a gene BaCBM2 for coding a CBM component, a gene for coding a connecting peptide and a gene PETase for coding cutinase through MEGAWHOP and connecting the genes with a pET-40b (+) plasmid to obtain a recombinant plasmid pET-40b (+) -BaCBM 2-PETase; transforming the recombinant plasmid pET-40b (+) -BaCBM2-PETase to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), incubation at 37 deg.CCarrying out inverted culture in a box for 8-12 h to obtain a transformant; selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids for sequencing, and obtaining a recombinant bacterium E.coli BL21/pET-40b (+) -BaCBM after verification₂-PETase。

Coli BL21/pET-40b (+) -PETase and E.coli BL21/pET-40b (+) -BaCBM₂Respectively streaking PETase on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernate, wherein the cell disruption supernate is crude enzyme liquid of cutinase PETatase and fusion protein BaCBM2-PETase respectively.

Example 12: degradation of PET by PETase and BaCBM2-PETase

The method comprises the following specific steps:

sequentially incubating a PET membrane with SDS solution with the concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase PETase and the fusion protein BaCBM2-PETase (50U) from example 11 were added to a mixture containing 1X 1cm²Degrading the PET film in a glass test tube for 4 days in a constant-temperature water bath shaker at 40 ℃ and 150rpm to obtain a degradation product.

Detecting the TPA content in the degradation product, wherein the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein BaCBM2-PETase is as high as 28mg/L, while TPA is not detected in the degradation product obtained by degrading the PET film by the unfused cutinase PETase.

Example 13: preparation of cutinase FsC and fusion protein FsC-LCI

The method comprises the following specific steps:

synthesis of nucleosidesA gene FsC (the amino acid sequence is shown as SEQ ID NO. 2) of coding cutinase FsC, the sequence of which is shown as SEQ ID NO. 15; the gene FsC encoding cutinase and the pET-14b (+) plasmid were ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via Megaprimer PCR of white Plasmids, Methods enzymol.2011; 499: 399-; transforming the recombinant plasmid pET-14b (+) -FsC to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and correctly sequencing to obtain the recombinant bacterium E.coli BL21/pET-14b (+) -FsC.

Synthesizing a gene (the amino acid sequence is shown as SEQ ID NO. 7) of the coding connecting peptide, the nucleotide sequence of which is shown as SEQ ID NO. 20; synthesizing a gene LCI (amino acid sequence shown as SEQ ID NO. 11) of the CBM component with a nucleotide sequence shown as SEQ ID NO. 24; sequentially connecting the gene FsC for coding cutinase, the gene for coding a connecting peptide and the gene LCI for coding a CBM component by MEGAWHOP and connecting the genes with a pET-40b (+) plasmid to obtain a recombinant plasmid pET-14b (+) -FsC-LCI; transforming the recombinant plasmid pET-14b (+) -FsC-LCI into E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, sequencing, and obtaining the recombinant bacterium E.coli BL21/pET-14b (+) -FsC-LCI after verification.

Respectively scribing recombinant bacteria E.coli BL21/pET-14b (+) -FsC and recombinant bacteria E.coli BL21/pET-14b (+) -FsC-LCI on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernatant, wherein the cell disruption supernatant is crude enzyme liquid of cutinase FsC and fusion protein FsC-LCI.

Example 14: degradation of PEA by FsC and FsC-LCI

The method comprises the following specific steps:

cutinase FsC and fusion protein FsC-LCI (30U) from example 13 were added to 10mL of 0.1% (v/v) Polyacrylate (PEA) solution, respectively, to a final volume of 10 mL. The reaction was carried out at pH 7.0 and 40 ℃ and the turbidity change of the reaction system was measured by a spectrophotometer. The relative turbidity of FsC group was stabilized at 42.5%, and the relative turbidity of FsC-LCI fusion protein group was stabilized at 27.2%, which was 36% higher than the degradation efficiency of FsC group.

Example 15: preparation of cutinase LCC and fusion protein DSK-LCC

The method comprises the following specific steps:

synthesizing a gene LCC (amino acid sequence shown as SEQ ID NO. 4) of the cutinase LCC, wherein the nucleotide sequence is shown as SEQ ID NO. 17; the gene FsC encoding cutinase and the pET-24a (+) plasmid were ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via Megaprimer PCR of white Plasmids, Methods enzymol.2011; 499: 399-; transforming the recombinant plasmid pET-24a (+) -LCC to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and obtaining the recombinant bacterium E.coli BL21/pET-24a (+) -LCC after sequencing is correct.

The synthetic nucleotide sequence is shown as SEQ ID NO.22 (the amino acid sequence is shown as SEQ ID NO. 9); synthesizing a gene DSK (amino acid sequence shown as SEQ ID NO. 13) of the CBM component with a nucleotide sequence shown as SEQ ID NO. 26; sequentially connecting a gene DSK for encoding a CBM component, a gene for encoding a connecting peptide and a gene LCC for encoding cutinase through MEGAWHOP and connecting the genes with a pET-24a (+) plasmid to obtain a recombinant plasmid pET-24a (+) -LCC-DSK; converting the recombinant plasmid pET-24a (+) -LCC-DSK to E.coli BL21 to obtain a conversion product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, sequencing, and obtaining the recombinant bacterium E.coli BL21/pET-24a (+) -LCC-DSK after verification.

Respectively scribing recombinant bacteria E.coli BL21/pET-24a (+) -LCC and recombinant bacteria E.coli BL21/pET-24a (+) -LCC-DSK on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernatant, wherein the cell disruption supernatant is crude enzyme liquid of cutinase LCC and fusion protein DSK-LCC.

Example 16: degradation of PET by LCC and DSK-LCC

The method comprises the following specific steps:

sequentially incubating a PET membrane with SDS solution with the concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase LCC obtained in example 15 and the fusion protein DSK-LCC (30U) were added to a mixture containing 1X 1cm²In the glass test tube of the PET film of (1),degrading for 4d in a constant temperature water bath shaker at 50 ℃ and 200rpm to obtain a degradation product.

Detecting the TPA content in the degradation product, wherein the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein DSK-LCC is up to 38mg/L, while the content of TPA obtained by degrading the PET film by the unfused cutinase LCC is up to 5 mg/L.

Example 17: preparation of cutinase FsC and fusion protein FsC-BaCBM2

The method comprises the following specific steps:

synthesizing a gene FsC (the amino acid sequence is shown as SEQ ID NO. 2) of coding cutinase FsC, the nucleotide sequence of which is shown as SEQ ID NO. 15; the gene FsC encoding cutinase and the pET-14b (+) plasmid were ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via Megaprimer PCR of white Plasmids, Methods enzymol.2011; 499: 399-; transforming the recombinant plasmid pET-14b (+) -FsC to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and correctly sequencing to obtain the recombinant bacterium E.coli BL21/pET-14b (+) -FsC.

Synthesizing a gene (the amino acid sequence is shown as SEQ ID NO. 7) of the coding connecting peptide, the nucleotide sequence of which is shown as SEQ ID NO. 20; synthesizing a gene BaCBM2 (the amino acid sequence is shown as SEQ ID NO. 12) of which the nucleotide sequence is shown as SEQ ID NO.25 and which codes a CBM component; sequentially connecting a gene FsC for coding cutinase, a gene coding a connecting peptide and a gene BaCBM2 for coding a CBM component by MEGAWHOP and connecting the genes with a pET-14b (+) plasmid to obtain a recombinant plasmid pET-14b (+) -FsC-BaCBM 2; transforming the recombinant plasmid pET-14b (+) -FsC-BaCBM2 into E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. mu.g.mL)^-1Ampicillin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; selecting a transformant to inoculate the transformant into an LB liquid culture medium,carrying out shake flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids for sequencing, and obtaining the recombinant bacterium E.coli BL21/pET-14b (+) -FsC-BaCBM2 after verification.

Respectively scribing recombinant bacteria E.coli BL21/pET-14b (+) -FsC and recombinant bacteria E.coli BL21/pET-14b (+) -FsC-BaCBM2 on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernatant, wherein the cell disruption supernatant is crude enzyme liquid of cutinase FsC and fusion protein FsC-BaCBM 2.

Example 18: FsC and FsC-BaCBM2 degradation of PET

The method comprises the following specific steps:

sequentially incubating a PET membrane with SDS solution with the concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase FsC and the fusion protein FsC-BaCBM2(50U) obtained in example 17 were added to a solution containing 1X 1cm²Degrading the PET film in a glass test tube for 4 days in a constant-temperature water bath shaker at 40 ℃ and 150rpm to obtain a degradation product.

Detecting the TPA content in the degradation product, wherein the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein FsC-BaCBM2 is up to 48mg/L, while TPA is not detected in the degradation product obtained by degrading the PET film by the unfused cutinase FsC.

Example 19: preparation of PET degrading enzyme PETase and fusion protein PETase-TrCBM

The method comprises the following specific steps:

synthesizing a gene PETase (amino acid sequence is shown as SEQ ID NO. 5) of coding cutinase PETase, wherein the nucleotide sequence is shown as SEQ ID NO. 18; will codeThe cutinase genes PETase and pET-40b (+) Plasmids were ligated by Megaprimer PCR of white Plasmids (MEGAWHOP) (see in particular Miyazaki K, MEGAWHOP cloning: a method of creating random mutagenesis via PCR of white Plasmids, Methods enzymol.2011; 499: 399-; transforming the recombinant plasmid pET-40b (+) -PETase to E.coli BL21 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, and correctly sequencing to obtain a recombinant bacterium E.coli BL21/pET40b (+) -PETase.

Synthesizing a gene (the amino acid sequence is shown as SEQ ID NO. 6) of the coding connecting peptide, the nucleotide sequence of which is shown as SEQ ID NO. 19; synthesizing a gene TrCBM (amino acid sequence shown as SEQ ID NO. 10) of which the nucleotide sequence is shown as SEQ ID NO.23 and which codes a CBM component; sequentially connecting a gene PETase for coding cutinase, a gene for coding a connecting peptide and a gene TrCBM for coding a CBM component by MEGAWHOP and connecting the genes with a pET-40b (+) plasmid to obtain a recombinant plasmid pET-40b (+) -PETase-TrCBM; transforming the recombinant plasmid into E.coli BL21 to obtain a transformed product; the transformed product was spread on LB solid medium (containing 50. mu.g.mL)^-1Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; and (3) selecting a transformant, inoculating the transformant to an LB liquid culture medium, performing shake-flask culture for 8-12 h at 37 ℃ and 180rpm, extracting plasmids, sequencing, and obtaining the recombinant bacterium E.coli BL21/pET-40b (+) -PETase-TrCBM after verification.

Respectively scribing E.coli BL21/pET-40b (+) -PETase and E.coli BL21/pET-40b (+) -PETase-TrCBM on an LB solid culture medium, and culturing for 8-12 h at 37 ℃ to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 8-10 hours at 37 ℃ to obtain seed liquid; inoculating the seed liquid into a TB liquid culture medium with the inoculation amount of 5% (v/v), culturing for 2h at 37 ℃ and 200rpm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 5mM into the TB liquid culture medium, and continuously performing induction culture for 24h at 25 ℃ and 200rpm to obtain a fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 10min, and collecting thallus; respectively carrying out ultrasonic cell disruption on the thalli to obtain cell disruption supernate, wherein the cell disruption supernate is crude enzyme liquid of cutinase PETase and fusion protein PETase-TrCBM respectively.

Example 20: degradation of PETase and PETase-TrCBM on PET

The method comprises the following specific steps:

sequentially incubating a PET membrane with SDS solution with concentration of 0.1% (v/v) at 50 ℃ for 30min, incubating with 50 ℃ ultra-clean water for 5min, incubating with 50 ℃ absolute ethyl alcohol for 5min, and drying at 50 ℃ to obtain a treated PET membrane; the cutinase PETase and the fusion protein PETase-TrCBM (20U) obtained in example 19 were added to a mixture containing 1X 1cm²Degrading the PET film in a glass test tube for 4 days in a constant-temperature water bath shaker at 30 ℃ and 200rpm to obtain a degradation product.

Detecting the TPA content in the degradation product, wherein the detection result is as follows: the content of TPA in the degradation product obtained by degrading the PET film by the fusion protein PETase-TrCBM is as high as 6.28mg/L, while TPA is not detected in the degradation product obtained by degrading the PET film by the unfused cutinase PETase.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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Arg Leu Ser Val Ser Gly Phe Gly Gly Gly Val Ile Tyr Tyr Pro Thr

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Ala Asp Ala Ser Ser Leu Ala Trp Leu Gly Arg Arg Leu Ala Ser His

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Gly Phe Val Val Leu Val Ile Asn Thr Asn Ser Arg Phe Asp Tyr Pro

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Thr Asn Ser Thr Leu Asp Gln Pro Ser Ser Arg Ser Ser Gln Gln Met

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Ala Ala Leu Arg Gln Val Ala Ser Leu Asn Gly Thr Ser Ser Ser Pro

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Met Gly Gly Gly Gly Ser Leu Ile Ser Ala Ala Asn Asn Pro Ser Leu

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Asn Ala Lys Gln Phe Leu Glu Ile Asn Gly Gly Ser His Ser Cys Ala

225 230 235 240

Asn Ser Gly Asn Ser Asn Gln Ala Leu Ile Gly Lys Lys Gly Val Ala

245 250 255

Trp Met Lys Arg Phe Met Asp Asn Asp Thr Arg Tyr Ser Thr Phe Ala

260 265 270

Cys Glu Asn Pro Asn Ser Thr Arg Val Ser Asp Phe Arg Thr Ala Asn

275 280 285

Cys Ser

290

<210> 6

<211> 17

<212> PRT

<213> Artificial sequence

<400> 6

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

Ala

<210> 7

<211> 10

<212> PRT

<213> Artificial sequence

<400> 7

Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

1 5 10

<210> 8

<211> 24

<212> PRT

<213> Artificial sequence

<400> 8

Pro Pro Gly Gly Asn Arg Gly Thr Thr Thr Thr Arg Arg Pro Ala Thr

1 5 10 15

Thr Thr Gly Ser Ser Pro Gly Pro

20

<210> 9

<211> 15

<212> PRT

<213> Artificial sequence

<400> 9

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 10

<211> 36

<212> PRT

<213> Artificial sequence

<400> 10

Thr Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys

1 5 10 15

Lys Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg

20 25 30

Tyr Tyr Val Gln

35

<210> 11

<211> 47

<212> PRT

<213> Artificial sequence

<400> 11

Ala Ile Lys Leu Val Gln Ser Pro Asn Gly Asn Phe Ala Ala Ser Phe

1 5 10 15

Val Leu Asp Gly Thr Lys Trp Ile Phe Lys Ser Lys Tyr Tyr Asp Ser

20 25 30

Ser Lys Gly Tyr Trp Val Gly Ile Tyr Glu Val Trp Asp Arg Lys

35 40 45

<210> 12

<211> 72

<212> PRT

<213> Artificial sequence

<400> 12

Ala Thr Phe Ser Val Thr Ser Asn Trp Gly Ser Gly Tyr Asn Phe Ser

1 5 10 15

Ile Val Ile Lys Asn Ser Gly Thr Thr Pro Ile Lys Asn Trp Lys Leu

20 25 30

Glu Phe Asp Tyr Asn Gly Asn Leu Thr Gln Val Trp Asp Ser Lys Ile

35 40 45

Ser Ser Lys Ile Asn Asn His Tyr Val Ile Thr Asn Ala Gly Trp Asn

50 55 60

Gly Glu Ile Pro Pro Gly Gly Ser

65 70

<210> 13

<211> 29

<212> PRT

<213> Artificial sequence

<400> 13

Gly Leu Trp Ser Thr Ile Lys Gln Lys Gly Lys Glu Ala Ala Ile Ala

1 5 10 15

Ala Ala Lys Ala Ala Gly Gln Ala Ala Leu Gly Ala Leu

20 25

<210> 14

<211> 585

<212> DNA

<213> Artificial sequence

<400> 14

caattgggtg ctattgaaaa cggacttgaa tcaggatcag ctaacgcctg tcctgatgcc 60

attcttattt ttgccagagg ttcaactgaa cctggaaaca tgggaattac cgttggacca 120

gctttagcca acggtttaga atctcatatt cgtaacattt ggattcaagg tgttggaggt 180

ccatacgatg ccgccttagc tactaacttt cttcctcgtg gtacttcaca agccaacatt 240

gatgaaggaa agagattatt tgccttggcc aaccaaaagt gtccaaacac cccagttgtt 300

gcgggtggct actcacaagg ggccgcttta attgctgccg ccgtttccga attatccgga 360

gctgttaagg aacaagttaa gggagttgcc ttgtttggtt acactcaaaa cttgcaaaac 420

agaggtggta ttcctaacta ccctagagaa agaactaagg tattctgtaa cgttggtgac 480

gctgtttgta ccggaacttt aattattact cctgctcatc tttcatacac cattgaagcc 540

cgtggagaag ccgctagatt tcttcgtgat cgtattcgtg cttaa 585

<210> 15

<211> 693

<212> DNA

<213> Artificial sequence

<400> 15

atgaaattct tcgctctcac cacacttctc gccgccacgg cttcggctct gcctacttct 60

aaccctgccc aggagcttga ggcgcgccag cttggtagaa caactcgcga cgatctgatc 120

aacggcaata gcgcttcctg ccgcgatgtc atcttcattt atgcccgagg ttcaacagag 180

acgggcaact tgggaactct cggtcctagc attgcctcca accttgagtc cgccttcggc 240

aaggacggtg tctggattca gggcgttggc ggtgcctacc gagccactct tggagacaat 300

gctctccctc gcggaacctc tagcgccgca atcagggaga tgctcggtct cttccagcag 360

gccaacacca agtgccctga cgcgactttg atcgccggtg gctacagcca gggtgctgca 420

cttgcagccg cctccatcga ggacctcgac tcggccattc gtgacaagat cgccggaact 480

gttctgttcg gctacaccaa gaacctacag aaccgtggcc gaatccccaa ctaccctgcc 540

gacaggacca aggtcttctg caatacaggg gatctcgttt gtactggtag cttgatcgtt 600

gctgcacctc acttggctta tggtcctgat gctcgtggcc ctgcccctga gttcctcatc 660

gagaaggttc gggctgtccg tggttctgct tga 693

<210> 16

<211> 783

<212> DNA

<213> Artificial sequence

<400> 16

gccaaccctt atgagcgtgg cccgaaccct accgatgccc tgctggaagc aagcagcggt 60

ccgttcagcg tgagcgagga aaatgtgagc cgcttaagtg ccagcggctt tggcggtggc 120

accatttact atccgcgcga gaataacacc tatggtgccg tggcaattag cccgggctat 180

accggcaccg aagcaagcat tgcatggctg ggtgaacgca tcgcaagtca tggcttcgtg 240

gtgatcacca tcgataccat caccaccctg gatcagccgg atagccgtgc agaacagctg 300

aacgccgccc tgaatcacat gattaatcgt gccagcagca ccgtgcgtag ccgcattgac 360

agtagccgcc tggccgtgat gggccatagt atgggtggtg gcggtaccct gcgcttagcc 420

agccaacgcc ctgatctgaa agccgcaatc ccgctgaccc cgtggcatct gaacaaaaac 480

tggagcagcg tgaccgtgcc gaccctgatt attggcgccg atctggatac aattgccccg 540

gttgccaccc acgccaaacc tttctacaat agcctgccga gcagcattag caaggcctat 600

ctggaactgg atggtgccac ccattttgcc ccgaatatcc cgaacaagat tattggcaaa 660

tatagcgtgg cctggctgaa gcgctttgtg gacaacgaca cccgctacac ccagtttctg 720

tgccctggcc ctcgtgatgg tctgttcggc gaagtggagg aataccgcag cacctgcccg 780

ttt 783

<210> 17

<211> 882

<212> DNA

<213> Artificial sequence

<400> 17

atggacggag ttctctggcg agtgcgaacc gcggcgctca tggccgcgct gctcgccctc 60

gcagcctggg cgctggtctg ggcttcgccc agcgtcgagg ctcaatccaa cccgtaccag 120

cgcggtccca accccacgcg gagcgcgctc acggccgacg ggccgttctc ggtggcaacc 180

tacaccgttt cgcggctctc ggtgagcggc ttcgggggcg gggtgatcta ctaccccaca 240

ggcacctcgc tgaccttcgg cgggatcgcc atgtcgccgg ggtacacggc cgacgccagt 300

tcgctggcgt ggctcgggcg gcggttggca tcgcacggct tcgtggtgct cgtcatcaac 360

acgaactcgc gattcgacta ccctgactcc cgcgcaagcc agctatcggc ggcgctgaac 420

tacctgcgga cgagcagccc ctcagccgta cgcgcccggc tcgatgcgaa ccgcctggcc 480

gttgcggggc attcgatggg cggcggcggg accctccgca tcgcagagca gaacccgtcg 540

ctgaaagcgg ccgtaccgct cacgccgtgg cacaccgaca agacgttcaa cacgtcggta 600

ccggtgctca tcgtgggagc ggaagcggac acggtcgcgc ccgtgagcca gcacgccatt 660

ccgttctacc agaacttgcc ctcgaccacg ccgaaggtgt acgtggagct cgacaatgcg 720

tcgcacttcg cgcccaacag caacaacgcg gcgatctccg tgtacaccat ctcgtggatg 780

aagctgtggg tggataacga cacccgctac cggcagttcc tctgcaacgt gaacgatccg 840

gcgctgagcg acttccggac gaacaaccgc cactgccagt ag 882

<210> 18

<211> 870

<212> DNA

<213> Artificial sequence

<400> 18

atgaattttc cccgcgcttc tcgtcttatg caggccgctg ttcttggagg tttaatggca 60

gtgtcggcag ccgcaaccgc gcagaccaat ccatacgctc gcggcccaaa tcccactgct 120

gcatcgttgg aggcgagcgc tggacccttt accgttcgtt catttacggt ttcccgccct 180

tctggatacg gcgccggaac tgtatattat ccgactaatg ctggcggcac ggtcggtgcc 240

attgccattg taccaggcta cactgcgcgt cagtcttcta ttaaatggtg gggaccacgc 300

ttggcgagtc acgggttcgt agtaattacc attgacacga atagcacgtt ggaccaaccg 360

tcttcccgtt cgtcccagca aatggctgcc cttcgtcagg tagctagcct taatggcaca 420

tcatcctcac cgatctacgg caaggtggac accgcccgca tgggagtcat gggttggtca 480

atgggtggcg ggggctcgtt aatcagcgcg gccaacaatc catctcttaa ggcagcagcg 540

cctcaagccc cgtgggatag tagtacgaac ttcagttccg tcaccgtgcc cactcttatc 600

tttgcatgtg aaaacgatag tatcgctccc gtaaattcta gtgcgcttcc gatttacgac 660

agtatgtcgc gtaacgctaa acagttcttg gaaattaatg gcggaagtca ttcgtgtgct 720

aatagcggga acagcaatca agccttgatt ggcaagaagg gcgtagcatg gatgaagcgc 780

tttatggata acgacacacg ttactccact ttcgcttgcg agaatccaaa ttctacacgc 840

gtgtctgact ttcgtacggc gaattgctcc 870

<210> 19

<211> 51

<212> DNA

<213> Artificial sequence

<400> 19

gcggaagcag ccgccaaaga agcagcagca aaagaagccg cagccaaggc t 51

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence

<400> 20

gccgcagctg ctgcggcggc tgccgcagcg 30

<210> 21

<211> 72

<212> DNA

<213> Artificial sequence

<400> 21

cccccaggtg gtaaccgcgg cacaacgaca acacgccgcc cagcaactac caccggtagt 60

agtccgggtc ca 72

<210> 22

<211> 45

<212> DNA

<213> Artificial sequence

<400> 22

gggggcggtg gttcaggagg cggtggatcg ggtggtggcg ggagc 45

<210> 23

<211> 108

<212> DNA

<213> Artificial sequence

<400> 23

accagctttt atggcccggg cagcagcttt accctggata ccaccaaaaa actgaccgtg 60

gtgacccagt ttgaaaccag cggcgcgatt aaccgctatt atgtgcag 108

<210> 24

<211> 141

<212> DNA

<213> Artificial sequence

<400> 24

gccattaagt tggtgcagag cccgaacggc aattttgctg cgagcttcgt tttggacggc 60

accaagtgga ttttcaaaag taagtattac gactccagca aaggctattg ggtggggatc 120

tacgaagtgt gggatcgtaa g 141

<210> 25

<211> 216

<212> DNA

<213> Artificial sequence

<400> 25

gccaccttct cagttactag taactggggt tctggttata atttctcaat tgttatcaag 60

aatagtggga cgacacccat caagaattgg aaattggagt tcgactataa cgggaattta 120

actcaggtct gggactcgaa gattagctct aagattaata atcattacgt tattactaat 180

gctgggtgga acggcgaaat tcctcccggt ggtagt 216

<210> 26

<211> 87

<212> DNA

<213> Artificial sequence

<400> 26

ggtttatgga gtacgattaa acagaaaggc aaagaggcag ctatcgctgc tgcaaaggcg 60

gcaggacaag ccgcccttgg agcactg 87

Claims

1. A fusion protein comprising a cutinase, a linker peptide and a carbohydrate-binding module.

2. The fusion protein of claim 1, wherein the fusion protein is formed by sequentially linking cutinase, a linker peptide and a carbohydrate binding module.

3. A gene encoding the fusion protein of claim 1 or 2.

4. A recombinant plasmid carrying the gene of claim 3.

5. The recombinant plasmid of claim 4, wherein the expression vector of the recombinant plasmid is pET-5a (+) plasmid, pET-11b (+) plasmid, pET-14b (+) plasmid, pHY300PLK plasmid, pET-20b (+) plasmid, pET-21b (+) plasmid, pET-24a (+) plasmid, pET-28a (+) plasmid, pET-30c (+) plasmid, pET-32b (+) plasmid, pET-34b (+) plasmid, pET-40b (+) plasmid, pET-41b (+) plasmid, pET-42c (+) plasmid, pET-49b (+) plasmid, pET-50b (+) plasmid, or pRSFDuet-1 plasmid.

6. A host cell carrying the gene of claim 3 or the recombinant plasmid of claim 4 or 5.

7. A host cell according to claim 6, wherein the host cell is Escherichia coli (Escherichia coli) or Bacillus subtilis (Bacillus subtilis).

8. A method for degrading a polymer, wherein the fusion protein of claim 1 or 2 is added to a degradation system containing a polymer to degrade the polymer.

9. The method of claim 5, wherein the degradation is at 20-70 ℃, pH is 5.0-9.0, and the rotation speed is 100-200 rpm.

10. Use of the fusion protein of claim 1 or 2 or the gene of claim 3 or the recombinant plasmid of claim 4 or 5 or the host cell of claim 6 or 7 or the method of claim 8 or 9 for degrading a polymer.