A class of ICCG fusion proteins and their application in the degradation of polyethylene terephthalate

Abstract

The invention discloses a type of cutinase ICCG fusion protein and application thereof in degrading polyethylene terephthalate (PET); the invention provides a type of cutinase ICCG protein having a good degradation effect on PET, the fusion protein is obtained by sequentially connecting the cutinase ICCG, a connecting peptide and an anchoring peptide, wherein the connecting peptide includes (GGGGS)2, (GGGGS)4, GSA or 17X, the anchoring peptide includes αSP or TA2, and the anchoring peptide fusion can effectively improve the binding ability of the cutinase ICCG to a substrate, thereby improving the efficiency of degrading PET; the invention obtains the fusion protein capable of effectively degrading PET by screening different connecting peptides and anchoring peptides, and has a wide application prospect in degrading PET waste by enzymatic method.

Description

Technical field:

[0001] The invention relates to an ICCG fusion protein and application thereof in degrading polyethylene terephthalate (PET), belonging to the field of biotechnology. Background technology:

[0002] Polyethylene terephthalate (PET) is a polymer synthesized from terephthalic acid and ethylene glycol. Due to its good heat resistance, plasticity, toughness and other advantages, it is widely used in packaging, bottle blowing and production of textile products. In 2020 alone, the production of PET exceeded 70 million tons. The mass production and widespread use of PET products inevitably produce a large amount of post-consumer waste, posing a huge threat to our living environment and health.

[0003] At present, there are two ways to deal with PET plastics: physical methods (such as landfill and incineration) and chemical methods. However, these two methods have high processing costs and may cause secondary pollution to the environment. Biodegradation may provide a more environmentally friendly way for the depolymerization and recycling of PET waste. In the past decade, various ester hydrolases, such as lipase, carboxylesterase, esterase and cutinase, have shown the potential to degrade PET. However, PET is a polyester that is extremely difficult to hydrolyze, contains a high proportion of aromatic terephthalate, and has poor chain mobility. Its glass transition temperature is 65°C, so the higher the temperature, the more violent the fluctuation of the PET chain, which is more conducive to the hydrolysis of PET by enzymes, but most PET hydrolases lose their activity at high temperatures.

[0004] In 2012, a cutinase that can degrade PET at high temperature was isolated from leaf and branch compost using metagenomic methods. It is called leaf and branch compost cutinase (LCC), and its efficiency in degrading PET is at least 33 times that of other enzymes. However, at 65°C, LCC has a weak ability to depolymerize bottle-grade PET. The degradation reaction stops after three days, and the conversion rate is only 31%. In order to further improve the degradation performance of LCC, amino acid mutations were performed on LCC. The results showed that the T of the mutant F243I / D238C / S283C / Y127G (ICCG) was 1.3 times that of the mutant F243I / D238C / S283C / Y127G (ICCG). m The value increased by 9.8℃ compared with the wild-type LCC, and the enzymatic conversion rate of PET plastic by the mutant LCC was 1.5 times that of the wild-type within 20 hours. Although the amino acid mutation improved the degradation activity of LCC on PET to a certain extent, the degree of improvement was very limited. In order to make ICCG have a higher degradation ability for PET, the fusion of an anchor peptide with adhesion to PET with ICCG can promote the adhesion of the enzyme to the PET substrate and increase the degradation rate. Summary of the invention:

[0005] The purpose of the present invention is to provide an enzyme that can effectively degrade PET, and further proposes a method of fusing the ICCG gene to the N-terminus of the anchor peptide gene through a connecting peptide gene and constructing a genetically engineered strain ( Figure 1 ), so that the C-terminal fusion anchor peptide of the amino acid sequence of the expressed ICCG forms a fusion protein, the fusion protein includes ICCG, a connecting peptide and an anchor peptide, wherein the anchor peptide is a polypeptide with adhesion ability to PET, which can bind to the surface of PET, promote the adhesion of the fusion enzyme to the surface of PET, and then promote the degradation of PET by the fusion enzyme. The connecting peptide, as a functional separator, can prevent the direct series connection of the anchor peptide and the ICCG domain from adversely affecting the properties of the fusion protein, and reduce the folding interference between them. The length and amino acid composition of the connecting peptide will also affect the properties of the fusion protein. Therefore, the selection of appropriate anchor peptides and connecting peptides is crucial to improving the degradation ability of ICCG to PET.

[0006] The technical solution of the present invention is summarized as follows:

[0007] A class of ICCG fusion proteins, wherein the fusion protein is obtained by sequentially connecting cutinase ICCG, a connecting peptide and an anchoring peptide; the connecting peptide includes (GGGGS) 2 、(GGGGS) 4 , GSA or 17X; anchor peptides include αSP or TA2.

[0008] Fusion proteins include the following: ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 4 -αSP, ICCG-GSA-αSP, ICCG-17X-αSP, ICCG-(GGGGS) 2 -TA2, ICCG-(GGGGS) 4 -TA2, ICCG-GSA-TA2, ICCG-17X-TA2.

[0009] The use of a class of ICCG fusion proteins of the present invention in degrading polyethylene terephthalate comprises the following steps:

[0010] 1) Overlap extension polymerase chain reaction was used to separate the ICCG genes through (GGGGS) 2 、(GGGGS) 4 The recombinant gene ICCG-(GGGGS) was obtained by fusing the GSA or 17X connecting peptide gene with the N-terminus of the anchor peptide αSP gene or TA2 gene. 2 -αSP, ICCG-(GGGGS) 4 -αSP, ICCG-GSA-αSP, ICCG-17X-αSP, ICCG-(GGGGS) 2-TA2, ICCG-(GGGGS) 4 -TA2, ICCG-GSA-TA2, ICCG-17X-TA2; 2) subcloning the recombinant gene obtained by fusion in step 1) between the restriction enzyme sites NdeⅠ and XhoⅠ of the plasmid vector (pColdⅡ), and transforming it into competent cells Escherichia coli BL21 (DE3) to obtain the genetic engineering strain of the above fusion protein;

[0011] 3) Inducing the genetically engineered strain expressing the fusion protein obtained above, ultrasonically disrupting and centrifuging to collect the cell lysate, purifying the recombinant protein using a nickel ion metal chelate affinity chromatography column, and finally eluting with a high concentration imidazole buffer to obtain the fusion protein ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 4 -αSP, ICCG-GSA-αSP, ICCG-17X-αSP, ICCG-(GGGGS) 2 -TA2, ICCG-(GGGGS) 4 -TA2, ICCG-GSA-TA2, ICCG-17X-TA2;

[0012] 4) The ICCG fusion proteins obtained were placed in K containing PET materials 2 HPO 4 / KH 2 PO 4 The reaction system was then placed in a water bath shaker to carry out the degradation reaction of PET.

[0013] The ICCG gene sequence is SEQ ID NO.1; the anchor peptide αSP gene sequence is SEQ ID NO.2; the anchor peptide TA2 gene sequence is SEQ ID NO.3; the connecting peptide (GGGGS) 2 The gene sequence is SEQ ID NO.6; connecting peptide (GGGGS) 4 The gene sequence is SEQ ID NO.8; the gene sequence of the connecting peptide NL is SEQ ID NO.9; the gene sequence of the connecting peptide GSA is SEQ ID NO.10; and the gene sequence of the connecting peptide 17X is SEQ ID NO.11.

[0014] The recombinant gene ICCG-(GGGGS) 2 -αSP sequence is SEQ ID NO.12; recombinant gene ICCG-(GGGGS) 4-αSP is SEQ ID NO.13; the sequence of the recombinant gene ICCG-GSA-αSP is SEQ ID NO.14; the sequence of the recombinant gene ICCG-17X-αSP is SEQ ID NO.15; the sequence of the recombinant gene ICCG-(GGGGS) 2 -TA2 sequence is SEQ ID NO.16; recombinant gene ICCG-(GGGGS) 4 -TA2 has a sequence of SEQ ID NO.17; the sequence of the recombinant gene ICCG-GSA-TA2 has a sequence of SEQ ID NO.18; and the sequence of the recombinant gene ICCG-17X-TA2 has a sequence of SEQ ID NO.19.

[0015] The recombinant gene is subcloned into the plasmid pColdⅡ, the sequence of which is SEQ ID NO.23, and the recombinant plasmid is transformed into competent cells of Escherichia coli BL21 (DE3).

[0016] The imidazole concentration in the eluent during the purification of the recombinant protein is 100-500 mM.

[0017] The PET materials include amorphous PET film with a crystallinity of 8.1%, amorphous PET film or particles with a crystallinity of 5.3%, high-crystallinity PET film, Coca-Cola film or particles (crystallinity of 32%), and Yibao film or particles (crystallinity of 32.8%); the concentration of the fusion protein in the PET reaction system is 100-300nM, the rotation speed of the water bath shaker is 0-200rpm, the reaction temperature is 50-70°C, and the reaction time is 3-144h.

[0018] The present invention fuses an anchor peptide structure gene with adhesion to PET to the C-terminus of ICCG through a connecting peptide structure to obtain an ICCG fusion protein, utilizes the adhesion of the anchor peptide to PET to promote the adhesion of the fusion protein to the surface of PET, thereby enhancing the degradation of PET by ICCG, and obtains a fusion protein that can effectively degrade PET by screening different anchor peptides and connecting peptides.

[0019] Experiments have shown that the degradation ability of ICCG on PET is enhanced after fusion with anchor peptide, and different connecting peptide structures have different effects on the activity of fusion protein. By optimizing the anchor peptide and connecting peptide, a fusion protein that can effectively degrade PET can be obtained. This fusion protein will have broad application prospects in the treatment of PET waste. Description of the drawings:

[0020] Figure 1 This is the plasmid map of the genetically engineered strain.

[0021] Figure 2Fusion protein ICCG-(GGGGS) 2 -TA2, ICCG-(GGGGS) 2 -αSP and ICCG-(GGGGS) 2 -Degradation of PET materials by LCI.

[0022] Figure 3 The fusion protein ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 4 -αSP, ICCG-NL-αSP and ICCG-GSA-αSP on the degradation of PET materials.

[0023] Figure 4 It is a fusion protein ICCG-17X-TA2 and ICCG-(GGGGS) 3 -Degradation of PET materials by DSI.

[0024] Figure 5 The fusion protein ICCG-17X-TA2 can degrade PET over a long period of time. Specific implementation method:

[0025] In order to better understand the present invention, the method provided by the present invention will be further described below in conjunction with specific examples.

[0026] The specific instructions are as follows:

[0027] 1) Construction of recombinant genes of αSP short peptides fused with ICCG proteins connected by different connecting peptides:

[0028] The ICCG gene was cloned by overlapping extension PCR (GGGGS) 2 、(GGGGS) 4 The GSA, 17X connecting peptide genes were fused to the N-terminus of the anchor peptide αSP gene to construct the recombinant gene ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 4 -αSP, ICCG-GSA-αSP, ICCG-17X-αSP.

[0029] 2) Construction of recombinant genes of TA2 short peptides fused with ICCG proteins connected by different connecting peptides:

[0030] The ICCG gene was cloned by overlapping extension PCR (GGGGS) 2 、(GGGGS) 4 , GSA, 17X connecting peptide genes were fused to the N-terminus of the anchor peptide TA2 gene to construct the recombinant gene ICCG-(GGGGS) 2-TA2, ICCG-(GGGGS) 4 -TA2, ICCG-GSA-TA2, ICCG-17X-TA2.

[0031] 3) Subcloning the recombinant genes in 1) and 2) above into the restriction sites of NdeⅠ and XhoⅠ of the plasmid vector (pColdⅡ), and transforming them into competent cells of Escherichia coli BL21 (DE3) to obtain the genetic engineering strain of the above fusion protein;

[0032] 4) Inducing the genetically engineered strain expressing the fusion protein obtained above, ultrasonically disrupting and centrifuging to collect the cell lysate, purifying the recombinant protein using a nickel ion metal chelate affinity chromatography column, and finally eluting with a high concentration imidazole buffer to obtain the fusion protein ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 4 -αSP, ICCG-GSA-αSP, ICCG-17X-αSP, ICCG-(GGGGS) 2 -TA2, ICCG-(GGGGS) 4 -TA2, ICCG-GSA-TA2, ICCG-17X-TA2;

[0033] In the above step 1), the ICCG gene sequence is SEQ ID NO.1; the αSP gene sequence is SEQ ID NO.2; the TA2 gene sequence is SEQ ID NO.3; (GGGGS) 2 The gene sequence is SEQ ID NO.6; (GGGGS) 4 The gene sequence is SEQ ID NO.8; the GSA gene sequence is SEQ ID NO.10; and the 17X gene sequence is SEQ ID NO.11.

[0034] In the above step 1), the recombinant gene ICCG-(GGGGS) 2 -αSP sequence is SEQ ID NO.12; recombinant gene ICCG-(GGGGS) 4 -αSP sequence is SEQ ID NO.13; the sequence of the recombinant gene ICCG-GSA-αSP is SEQ ID NO.14; the sequence of the recombinant gene ICCG-17X-αSP is SEQ ID NO.15; the sequence of the recombinant gene ICCG-(GGGGS) 2 -TA2 sequence is SEQ ID NO.16; recombinant gene ICCG-(GGGGS) 4-TA2 has a sequence of SEQ ID NO.17; the sequence of the recombinant gene ICCG-GSA-TA2 has a sequence of SEQ ID NO.18; the sequence of the recombinant gene ICCG-17X-TA2 has a sequence of SEQ ID NO.19.

[0035] In the above step 1), the recombinant gene was subcloned into plasmid pColdⅡ (SEQ ID NO.23), and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells.

[0036] In the above step 2), the imidazole concentration in the eluent during purification of the recombinant protein is 100-500 mM.

[0037] The ICCG fusion proteins were placed in K containing different PET materials. 2 HPO 4 / KH 2 PO 4 Buffer (100 mM) was added, and then the reaction system was placed in a water bath shaker to carry out the degradation reaction of PET.

[0038] The PET materials include amorphous PET film with a crystallinity of 8.1%, amorphous PET film or particles with a crystallinity of 5.3%, high crystallinity PET film, Coca-Cola film or particles (crystallinity of 32%), Yibao particles or films (crystallinity of 32.8%). The concentration of the fusion protein in the PET reaction system is 100-300nM, the diameter of the degraded film material is 6mm, the quantity is 1 piece, the mass of the degraded granular material is 10mg, the speed of the water bath shaker is 0-200rpm, the reaction temperature is 50-70℃, and the reaction time is 3-144h.

[0039] Example 1: Cutinase ICCG and fusion protein ICCG-(GGGGS) 2 -TA2, ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 2 Preparation of -LCI

[0040] 1) The ICCG gene was subcloned between the restriction enzyme sites NdeⅠ and XhoⅠ of the plasmid pColdⅡ (SEQ ID NO.23) to form a recombinant plasmid (ICCG-pColdⅡ), which was then transformed into Escherichia coli shuffle T7 competent cells after sequencing verification to obtain a genetically engineered strain;

[0041] 2) The ICCG gene (SEQ ID NO.1) was cloned into (GGGGS) by overlapping extension PCR technique. 2The connecting peptide gene (SEQ ID NO.6) was fused with the N-terminus of αSP (SEQ ID NO.2), TA2 (SEQ ID NO.3), and LCI (SEQ ID NO.4) genes to obtain ICCG-(GGGGS) with gene sequences of SEQ ID NO.12, SEQ ID NO.16, and SEQ ID NO.20, respectively. 2 -αSP, ICCG-(GGGGS) 2 -TA2, ICCG-(GGGGS) 2 -LCI recombinant gene, and then subclone the fused gene into the restriction enzyme sites of NdeⅠ and XhoⅠ of plasmid pColdⅡ (SEQ ID NO.23) to form a recombinant plasmid (ICCG-(GGGGS) 2 -TA2-pColdⅡ、ICCG-(GGGGS) 2 -αSP-pColdⅡ and ICCG-(GGGGS) 2 -LCI-pColdⅡ), which was verified by sequencing and then transformed into competent E. coli BL21 (DE3) cells to obtain a genetically engineered strain;

[0042] 3) The genetically engineered strains in steps 1) and 2) above were cultured in two stages, first grown at 37°C until OD 600 When the p-value reaches 0.6-0.8, IPTG (final concentration is 0.5 mM) is then added at 16°C to induce the expression of the target protein for 24 h. After the induction, the expression strain is collected by centrifugation (4000 g, 30 min) and stored at -20°C for later use.

[0043] 4) The obtained strain was resuspended in a lysis buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0), the strain was ultrasonically disrupted and the cell lysate was collected by centrifugation, and the recombinant protein was purified using a nickel ion metal chelate affinity chromatography column, and the target proteins ICCG and (GGGGS) were eluted with an eluent (20 mM Tris-HCl, 100 mM imidazole, 300 mM NaCl, pH 8.0). 2 -αSP, ICCG-(GGGGS) 2 -LCI,ICCG-(GGGGS) 2 The difference between the eluent of -TA2 fusion protein and other target proteins is that its imidazole concentration is 500 mM. The eluted protein is further purified by size exclusion chromatography, and finally the concentration of the obtained protein is detected by Bradford method.

[0044] Example 2: Cutinase ICCG and fusion protein ICCG-(GGGGS) 2 -TA2, ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 2 -LCI degradation of PET materials

[0045] The cutinase ICCG and fusion protein ICCG-(GGGGS) in Example 1 were used respectively. 2 -TA2, ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 2 -LCI degraded different PET materials. 20 μL of the above enzyme solution (final concentration of 100 nM) was added to centrifuge tubes containing amorphous PET film and particles with a crystallinity of 5.3%, Coca-Cola film and particles with a crystallinity of 32%, and Yibao film and Yibao particles with a crystallinity of 32.8% (the diameter of each film material was 6 mm, the number of films was 1 piece, and the particles were 10 mg, among which the particle size of amorphous PET particles, Yibao particles and Coca-Cola particles was ≤0.5 mm). Then 280 μL of reaction buffer was added, and the degradation was carried out at 60°C, 150 rpm constant temperature water bath shaker for 24 hours. The amount of degradation products generated was determined by high performance liquid chromatography. The experimental results are shown in the figure. Figure 2 As shown: The fusion of TA2 and αSP can improve the degradation ability of ICCG on different PET materials, among which ICCG-(GGGGS) 2 -αSP has a degradation capacity of 1.23, 1.90, 1.67, 1.30, 1.88 and 1.25 times that of ICCG on amorphous PET particles, amorphous PET films, Coca-Cola particles, Coca-Cola films, Yibao particles and Yibao films, respectively; ICCG-(GGGGS) 2 -TA2 has the ability to degrade amorphous PET particles, amorphous PET films, Coca-Cola particles, Coca-Cola films, Yibao particles and Yibao films, respectively, which are 1.11, 1.53, 1.03, 1.14, 1.20 and 1.08 times that of ICCG. Therefore, ICCG-(GGGGS) 2 -αSP has higher degradation activity on PET than ICCG-(GGGGS) 2 -TA2 degradation activity on PET, αSP fusion improved ICCG degradation performance more than TA2 fusion on ICCG, while ICCG-(GGGGS) 2 -LCI has a weaker degradation activity on PET than ICCG.

[0046] Example 3: Cutinase ICCG and fusion protein ICCG-(GGGGS) 2-αSP, ICCG-(GGGGS) 4 Preparation of -αSP, ICCG-NL-αSP, and ICCG-GSA-αSP

[0047] 1) The ICCG gene was subcloned between the restriction enzyme sites NdeⅠ and XhoⅠ of the plasmid pColdⅡ (SEQ ID NO.23) to form a recombinant plasmid (ICCG-pColdⅡ), which was then transformed into Escherichia coli shuffle T7 competent cells after sequencing verification to obtain a genetically engineered strain;

[0048] 2) The ICCG gene (SEQ ID NO.1) was cloned into 2 Gene (SEQ ID NO.6), (GGGGS) 4 The N-terminus of the αSP gene (SEQ ID NO.2) was fused to obtain ICCG-(GGGGS) with gene sequences of SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.21, respectively. 2 -αSP, ICCG-(GGGGS) 4 -αSP, ICCG-GSA-αSP and ICCG-NL-αSP recombinant genes, and then subcloned the fused genes into the restriction enzyme sites of NdeⅠ and XhoⅠ of plasmid pColdⅡ (SEQ ID NO.23) to form the recombinant plasmid ICCG-(GGGGS) 2 -αSP-pColdⅡ、ICCG-(GGGGS) 4 -αSP-pColdⅡ, ICCG-NL-αSP-pColdⅡ and ICCG-GSA-αSP-pColdⅡ, which were transformed into competent Escherichia coli BL21 (DE3) cells after sequencing verification to obtain genetically engineered strains;

[0049] 3) The genetically engineered strains in steps 1) and 2) above were cultured in two stages, first grown at 37°C until OD 600 When the p-value reaches 0.6-0.8, IPTG (final concentration is 0.5 mM) is then added at 16°C to induce the expression of the target protein for 24 h. After the induction, the expression strain is collected by centrifugation (4000 g, 30 min) and stored at -20°C for later use.

[0050] 4) The strain obtained above was resuspended in a lysis buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0), the strain was ultrasonically disrupted and the cell lysate was collected by centrifugation, the recombinant protein was purified using a nickel ion metal chelate affinity chromatography column, and the target protein was eluted with an eluent (20 mM Tris-HCl, 100 mM imidazole, 300 mM NaCl, pH 8.0). The eluted protein was further purified by size exclusion chromatography, and finally the concentration of the obtained protein was detected by the Bradford method.

[0051] Example 4: Cutinase ICCG and fusion protein ICCG-(GGGGS) 2 -αSP, ICCG-(GGGGS) 4 Degradation of PET materials by -αSP, ICCG-NL-αSP and ICCG-GSA-αSP

[0052] The cutinase ICCG and fusion protein ICCG-(GGGGS) in Example 3 were used respectively. 2 -αSP, ICCG-(GGGGS) 4 -αSP, ICCG-NL-αSP, and ICCG-GSA-αSP were used to degrade Coca-Cola film (32% crystallinity) and amorphous PET film with high crystallinity. 100 μL of the above enzyme solution (final concentration of 300 nM) was added to centrifuge tubes containing a 6 mm diameter Coca-Cola film and amorphous PET film with high crystallinity, respectively. Then, 900 μL of reaction buffer was added. The degradation was carried out in a constant temperature water bath shaker at 60°C and 150 rpm for 24 h. The amount of degradation products was determined by high performance liquid chromatography. The experimental results are shown in Figure 2. Figure 3 Shown: Fusion proteins ICCG-GSA-αSP, ICCG-(GGGGS) 2 -αSP and ICCG-(GGGGS) 4 -αSP showed higher degradation activity on Coca-Cola film and amorphous PET film with high crystallinity than ICCG, among which ICCG-GSA-αSP, ICCG-(GGGGS) 2 -αSP and ICCG-(GGGGS) 4 -αSP has a degradation activity of 4.3, 4.3 and 3.6 times that of ICCG for Coca-Cola film, and 1.4, 2.1 and 2.0 times that of ICCG for amorphous PET film with high crystallinity. It can be seen that compared with GSA and (GGGGS) 4 Connector peptide, containing (GGGGS) 2The fusion protein had high degradation activity against Coca-Cola film and highly crystalline amorphous PET film, while the fusion of the anchor peptide αSP to the C-terminus of ICCG through the NL connecting peptide reduced the degradation activity of ICCG against Coca-Cola film and highly crystalline amorphous PET film.

[0053] Example 5: Cutinase ICCG and fusion proteins ICCG-17X-TA2, ICCG-(GGGGS) 3 -DSI preparation

[0054] 1) The ICCG gene was subcloned between the restriction enzyme sites NdeⅠ and XhoⅠ of the plasmid pColdⅡ (SEQ ID NO.23) to form a recombinant plasmid (ICCG-pColdⅡ), which was transformed into Escherichia coli shuffle T7 competent cells after sequencing verification to obtain a genetically engineered strain.

[0055] 2) The ICCG gene (SEQ ID NO.1) was cloned into 3 The N-terminal of the gene (SEQ ID NO.7) and 17X gene (SEQ ID NO.11) was fused with the DSI gene (SEQ ID NO.5) and TA2 gene (SEQ ID NO.3) to obtain ICCG-(GGGGS) with gene sequences of SEQ ID NO.22 and SEQ ID NO.18, respectively. 3 -DSI, ICCG-17X-TA2 recombinant gene, and then subclone the fused gene into the restriction enzyme sites of NdeⅠ and XhoⅠ of plasmid pColdⅡ (SEQID NO.23) to form the recombinant plasmid ICCG-(GGGGS) 3 -DSI-pColdⅡ and ICCG-17X-TA2-pColdⅡ were transformed into competent cells of Escherichia coli BL21 (DE3) after sequencing verification to obtain genetically engineered strains.

[0056] 3) The genetically engineered strains in steps 1) and 2) above were cultured in two stages, first grown at 37°C until OD 600 When the p-value reaches 0.6-0.8, IPTG (final concentration is 0.5 mM) is then added at 16°C to induce the expression of the target protein for 24 h. After the induction, the expression strain is collected by centrifugation (4000 g, 30 min) and stored at -20°C for later use.

[0057] 4) The strain obtained above was resuspended in a lysis buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0), the strain was ultrasonically disrupted and the cell lysate was collected by centrifugation, the recombinant protein was purified using a nickel ion metal chelate affinity chromatography column, and the target protein was eluted with an eluent (20 mM Tris-HCl, 100 mM imidazole, 300 mM NaCl, pH 8.0). The eluted protein was further purified by size exclusion chromatography, and finally the concentration of the obtained protein was detected by the Bradford method.

[0058] Example 6: Cutinase ICCG and fusion proteins ICCG-17X-TA2, ICCG-(GGGGS) 3 -Degradation of PET by DSI

[0059] The cutinase ICCG and fusion proteins ICCG-17X-TA2 and ICCG-(GGGGS) in Example 5 were used respectively. 3 -DSI degradation of amorphous PET film with a crystallinity of 8.1%, 20 μL of the above enzyme solution (final concentration of 100 nM) was added to a centrifuge tube containing a piece of amorphous PET film with a diameter of 6 mm, followed by 280 μL of reaction buffer, and degradation was carried out in a water bath at 50-70°C for 3 h, and the amount of degradation product generated was determined by high performance liquid chromatography. The experimental results are shown in Figure 4 As shown in the figure, it can be seen that the degradation activity of ICCG-17X-TA2 on PET at 50-70℃ is 1.33-1.51 times that of ICCG. Therefore, the fusion of TA2 increases the adsorption amount of ICCG on the PET surface, thereby improving the degradation activity of ICCG on PET, while ICCG-(GGGGS) 3 -DSI's degradation activity on PET is only about 20% of ICCG, so DSI is used (GGGGS) 3 The linker peptide was fused to the C-terminus of ICCG, which weakened the activity of ICCG.

[0060] Example 7: Long-term degradation of PET by cutinase ICCG and fusion protein ICCG-17X-TA2

[0061] The long-term degradation activity of the cutinase ICCG and the fusion protein ICCG-17X-TA2 in Example 5 on an amorphous PET film with a crystallinity of 8.1% was measured respectively. 20 μL of the above enzyme solution (final concentration of 100 nM) was added to a centrifuge tube containing a 6 mm diameter amorphous PET film, followed by 280 μL of reaction buffer, and the degradation was continued in a water bath at 50 and 60° C. for 6 days, and samples were taken every 24 hours, and the amount of degradation products was determined by high performance liquid chromatography. The experimental results are shown in FIG. Figure 5As shown, it can be seen that at 50 and 60°C, for 6 consecutive days, the degradation activity of ICCG-17X-TA2 on PET was always higher than that of ICCG. At 50°C, the degradation activity of ICCG-17X-TA2 on PET was up to 1.60 times that of ICCG, and at 60°C, the degradation activity of ICCG-17X-TA2 on PET was up to 1.57 times that of ICCG, indicating that TA2 still plays an adhesion role in the process of long-term degradation of PET, thereby improving the degradation activity of ICCG on PET.

[0062] The results of the study on PET degradation (Examples 2, 4, 6 and 7) show that fusing an anchor peptide with adhesion to PET to the cutinase ICCG can improve the degradation ability of ICCG on PET materials, such as the fusion protein ICCG-(GGGGS) in the present invention. 2 -αSP, ICCG-(GGGGS) 2 -TA2 and ICCG-17X-TA2 have higher degradation activities on PET materials than ICCG, among which ICCG-(GGGGS) 2 -αSP has a degradation capacity of 1.23, 1.90, 1.67, 1.30, 1.88 and 1.25 times that of ICCG on amorphous PET particles, amorphous PET films, Coca-Cola particles, Coca-Cola films, Yibao particles and Yibao films, respectively; ICCG-(GGGGS) 2 -TA2 has the ability to degrade amorphous PET particles, amorphous PET films, Coca-Cola particles, Coca-Cola films, Yibao particles and Yibao films, respectively, which are 1.11, 1.53, 1.03, 1.14, 1.20 and 1.08 times that of ICCG; the degradation activity of ICCG-17X-TA2 on PET at 50-70°C is 1.33-1.51 times that of ICCG, and the fusion of TA2 can still improve the degradation activity of ICCG during the long-term degradation of PET; in addition, different connecting peptides will also affect the activity of the fusion protein, for example, in the present invention, compared with NL, GSA, (GGGGS) 4 Connector peptide, using (GGGGS) 2 The fusion protein ICCG-(GGGGS) was obtained by fusing the short peptide αSP to the C-terminus of ICCG using a connecting peptide. 2 -αSP has high degradation activity on both Coca-Cola film and highly crystalline amorphous PET film. These results show that the anchor peptide fusion protein of the present invention is very beneficial for the degradation of PET, which is very important for the enzymatic degradation of PET waste.

[0063] In summary, the fusion of the anchor peptide with adhesion to PET to the C-terminus of cutinase ICCG in the present invention can greatly improve the degradation ability of ICCG on PET, and the prepared fusion protein has high catalytic activity on PET, and has high practical application value in the enzymatic degradation of PET waste.

[0064] The present invention proposes to improve the degradation ability of ICCG to PET by using anchor peptide fusion, which has been described through the preferred embodiments in the field. It is obvious that relevant technical personnel can modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to realize the technology of the present invention. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention. Sequence Listing <110> Tianjin University <120> A class of ICCG fusion proteins and their application in the degradation of polyethylene terephthalate <160> twenty three <170> SIPOSequenceListing 1.0 <210> 1 <211> 774 <212> DNA <213> Artificial Sequence <400> 1 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggcggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag cccgggctat 180 accgcggacg cgagcagcct ggcgtggtta ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggcgc tgaactatct gcgcactagc agtccgtcgg ccgtgcgcgc gcgccttgac 360 gcgaaccgcc ttgcggtagc gggccatagc atgggcggcg gcggcaccct gcgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaaca accgccattg tcag 774 <210> 2 <211> 48 <212> DNA <213> Artificial Sequence <400> 2 tacgaaatgc cgagcgaaga aggctatcaa gattatgaac cggaagcg 48 <210> 3 <211> 132 <212> DNA <213> Artificial Sequence <400> 3 tatagtcgtt gtcagctgca aggctttaac tgcgtggtgc gcagctatgg cctgccgacc 60 attccgtgct gccgcggcct gacctgccgc agctattttc cgggcagcac ctatggccgc 120 tgtcagcgct at 132 <210> 4 <211> 141 <212> DNA <213> Artificial Sequence <400> 4 gccattaaac tggtgcagag cccgaacggc aactttgcgg cgagctttgt gctggatggc 60 accaaatgga tttttaaaag caaatattat gatagcagca aaggctattg ggtgggcatt 120 tatgaagtgt gggatcgcaa a 141 <210> 5 <211> 87 <212> DNA <213> Artificial Sequence <400> 5 ggcctgtgga gcaccattaa acagaaaggc aaggaagccg ccatcgcggc ggccaaagcg 60 gccggccaag ccgcgttagg cgcctta 87 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 ggcggtggcg gcagcggtgg tggcggtagc 30 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <400> 7 ggcggtggcg gtagcggcgg cggtggcagc ggcggcggtg gtagc <210> 8 <211> 61 <212> DNA <213> Artificial Sequence <400> 8 ggcggtggcg gtagtggtgg tggtggcagc ggtggcggtg gcagttggcg gtggcggcag t 61 <210> 9 <211> 48 <212> DNA <213> Artificial Sequence <400> 9 gcgggctatg gcaaagcggg cggcaccgtg accccgaccc cgaacacc <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <400> 10 30. ggcagcgcgg gcagcgcggc gggcagcggc <210> 11 <211> 51 <212> DNA <213> Artificial Sequence <400> 11 gcggaagcgg ccgcgaaaga agcggcggcc aaagaagccg cggcgaaagc g <210> 12 <211> 852 <212> DNA <213> Artificial Sequence <400> 12 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag tccgggttac 180 accgcggatg cgagcagcct ggcgtggtta ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggcgc tgaactatct gcgtacgagc agcccgagcg cggttcgcgc gcgtctggac 360 gcgaatcgcc tggcggtggc cggccatagc atgggcggtg gcggcacgct gcgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaaca accgccattg ccaaggcggt 780 ggcggtagtg gcggtggcgg cagctacgaa atgccgagcg aagaaggcta tcaagattat 840 gaaccggaag cg 852 <210> 13 <211> 883 <212> DNA <213> Artificial Sequence <400> 13 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag tccgggttac 180 accgcggatg cgagcagcct ggcgtggtta ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggcgc tgaactatct gcgtacgagc agcccgagcg cggttcgcgc gcgtctggac 360 gcgaatcgcc tggcggtggc cggccatagc atgggcggtg gcggcacgct gcgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaaca accgccattg ccaaggcggt 780 ggcggtagtg gtggtggtgg cagcggtggc ggtggcagtt ggcggtggcg gcagttacga 840 aatgccgagc gaagaaggct atcaagatta tgaaccggaa gcg 883 <210> 14 <211> 851 <212> DNA <213> Artificial Sequence <400> 14 gcaacccgta tcagcgcggc ccgaacccga cccgcagcgc gctgaccgcg gatggcccgt 60 ttagcgtggc gacctatacc gtgagccgcc tgagcgtgag cggctttggc ggtggcgtga 120 tttattatcc gaccggcacg agcctgacct ttggcggcat tgcgatgagc ccgggctata 180 cggccgacgc gagtagttta gcgtggctgg gccgccgcct ggcgagccat ggctttgtgg 240 tgctggtgat taacaccaac agccgctttg atggcccgga tagccgcgcg agtcagctga 300 gcgcggcgct gaattatctg cgcacgagca gtccgagcgc ggtgcgcg cgcttagatg 360 cgaaccgcct ggcggtggcg ggccatagca tgggcggcgg tggcacgtta cgcattgcgg 420 aacagaaccc gagcctgaaa gcggcggtgc cgctgacccc gtggcatacc gataaaacct 480 ttaacacgag cgtgccggtg ctgattgtgg gcgcggaagc ggataccgtg gcgccggtga 540 gtcagcatgc gattccgttt tatcagaacc tgccgagcac caccccgaaa gtgtatgtgg 600 aactgtgcaa cgcgagccat attgcgccga acagcaacaa cgcggcgatt agcgtgtata 660 ccattagctg gatgaaactg tgggtggata acgatacccg ctatcgtcag tttctgtgca 720 acgtgaacga tccggcgctg tgcgattttc gcaccaataa ccgtcattgt caaggcagcg 780 cgggcagcgc ggcgggcagc ggctatgaaa tgccgagcga agaaggctat caagattatg 840 aaccggaagc g 851 <210> 15 <211> 873 <212> DNA <213> Artificial Sequence <400> 15 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag tccgggttac 180 accgcggatg cgagcagcct ggcgtggtta ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggcgc tgaactatct gcgtacgagc agcccgagcg cggttcgcgc gcgtctggac 360 gcgaatcgcc tggcggtggc cggccatagc atgggcggtg gcggcacgct gcgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaaca accgccattg ccaagcggaa 780 gcggccgcga aagaagcggc ggccaaagaa gccgcggcga aagcgtacga aatgccgagc 840 gaagaaggct atcaagatta tgaaccggaa gcg 873 <210> 16 <211> 936 <212> DNA <213> Artificial Sequence <400> 16 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag cccgggctat 180 accgcggatg cgagcagctt agcgtggtta ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggcgt taaattatct gcgcacgagc agcccgagcg cggttcgcgc gcgcttagat 360 gcgaaccgcc tggccgtggc gggccatagc atgggcggtg gcggcacctt acgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcacgaaca accgccattg ccaaggcggt 780 ggcggcagcg gtggtggcgg tagctatagt cgttgtcagc tgcaaggctt taactgcgtg 840 gtgcgcagct atggcctgcc gaccattccg tgctgccgcg gcctgacctg ccgcagctat 900 tttccgggca gcacctatgg ccgctgtcag cgctat 936 <210> 17 <211> 967 <212> DNA <213> Artificial Sequence <400> 17 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag tccgggttac 180 accgcggatg cgagcagcct ggcgtggtta ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggcgc tgaactatct gcgtacgagc agcccgagcg cggttcgcgc gcgtctggac 360 gcgaatcgcc tggcggtggc cggccatagc atgggcggtg gcggcacgct gcgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaaca accgccattg ccaaggcggt 780 ggcggtagtg gtggtggtgg cagcggtggc ggtggcagtt ggcggtggcg gcagttatag 840 tcgttgtcag ctgcaaggct ttaactgcgt ggtgcgcagc tatggcctgc cgaccattcc 900 gtgctgccgc ggcctgacct gccgcagcta ttttccgggc agcacctatg gccgctgtca 960 gcgctat 967 <210> 18 <211> 936 <212> DNA <213> Artificial Sequence <400> 18 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag tccgggttac 180 accgcggatg cgagcagcct ggcgtggtta ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggcgc tgaactatct gcgtacgagc agcccgagcg cggttcgcgc gcgtctggac 360 gcgaatcgcc tggcggtggc cggccatagc atgggcggtg gcggcacgct gcgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaaca accgccattg ccaaggcagc 780 gcgggcagcg cggcgggcag cggctatagt cgttgtcagc tgcaaggctt taactgcgtg 840 gtgcgcagct atggcctgcc gaccattccg tgctgccgcg gcctgacctg ccgcagctat 900 tttccgggca gcacctatgg ccgctgtcag cgctat 936 <210> 19 <211> 957 <212> DNA <213> Artificial Sequence <400> 19 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag cccgggctat 180 accgcggacg cgagtagctt agcgtggctg ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agtgcggcgc tgaactatct gcgtacgagc agtccgagcg cggtgcgc gcgcctggac 360 gccaaccgtc tggcggttgc gggccatagc atgggcggtg gcggcaccct gcgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaaca accgccattg ccaagcggaa 780 gcggccgga aagaagcggc ggccaaagaa gccgcggcga aagcgtatag ccgctgtcag 840 ctgcaaggct ttaactgcgt ggtgcgcagc tatgcctgc cgaccattc gtgctgccgc 900 ggcctgacct gccgcagcta ttttccgggc agcacctatg gccgctgtca acgttat 957 <210> 20 <211> 945 <212> DNA <213> Artificial Sequence <400> 20 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag cccgggctat 180 accgcggacg cgagcagcct ggcgtggtta ggtcgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgccgcgc tgaattatct gcgcacgagc agcccgagcg cggtgcgcgc ccgtttagat 360 gcgaaccgcc tggcggtggc gggccatagc atgggtggcg gtggtacctt acgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaaca accgccattg ccaaggcggt 780 ggcggcagcg gcggcggtgg cagcgccatt aaactggtgc agagcccgaa cggcaacttt 840 gcggcgagct ttgtgctgga tggcaccaaa tggattttta aaagcaaata ttatgatagc 900 agcaaaggct attgggtggg catttatgaa gtgtgggatc gcaaa 945 <210> 21 <211> 870 <212> DNA <213> Artificial Sequence <400> 21 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag cccgggctat 180 acggccgacg cgagtagttt agcgtggctg ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggcgc tgaattatct gcgcacgagc agtccgagcg cggtgcgcgc gcgcttagat 360 gcgaaccgcc tggcggtggc gggccatagc atgggcggcg gtggcacgtt acgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcaccaata accgtcattg tcaagcgggc 780 tatggcaaag cgggcggcac cgtgaccccg accccgaaca cctatgaaat gccgagcgaa 840 gaaggctatc aagattatga accggaagcg 870 <210> 22 <211> 906 <212> DNA <213> Artificial Sequence <400> 22 agcaacccgt atcagcgcgg cccgaacccg acccgcagcg cgctgaccgc ggatggcccg 60 tttagcgtgg cgacctatac cgtgagccgc ctgagcgtga gcggctttgg cggtggcgtg 120 atttattatc cgaccggcac gagcctgacc tttggcggca ttgcgatgag cccgggctat 180 acggcggatg cgagcagctt agcgtggctg ggccgccgcc tggcgagcca tggctttgtg 240 gtgctggtga ttaacaccaa cagccgcttt gatggcccgg atagccgcgc gagtcagctg 300 agcgcggccc tgaattatct gcgcacgagc agcccgagcg cggtgcgtgc gcgtctggat 360 gcgaaccgct tagccgtggc gggccatagc atgggtggcg gcggtacgct gcgcattgcg 420 gaacagaacc cgagcctgaa agcggcggtg ccgctgaccc cgtggcatac cgataaaacc 480 tttaacacga gcgtgccggt gctgattgtg ggcgcggaag cggataccgt ggcgccggtg 540 agtcagcatg cgattccgtt ttatcagaac ctgccgagca ccaccccgaa agtgtatgtg 600 gaactgtgca acgcgagcca tattgcgccg aacagcaaca acgcggcgat tagcgtgtat 660 accattagct ggatgaaact gtgggtggat aacgataccc gctatcgtca gtttctgtgc 720 aacgtgaacg atccggcgct gtgcgatttt cgcacgaaca atcgccattg tcaaggcggt 780 ggcggcagcg gcggtggcgg tagcggtggc ggtggcagcg gtctgtggag caccattaaa 840 cagaaaggca aagaagcggc gattgcggcc gcgaaagcgg cgggccaagc ggcgctgggc 900 gcgctg 906 <210> 23 <211> 4392 <212> DNA <213> Artificial Sequence <400> 23 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa 60 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat 120 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt 180 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt 240 aagaggtaat acaccatgaa tcacaaagtg catcatcatc atcatcatat ggagctcggt 300 accctcgagg gatccgaatt caagcttgtc gacctgcagt ctagataggt aatctctgct 360 taaaagcaca gaatctaaga tccctgccat ttggcgggga tttttttatt tgttttcagg 420 aaataaataa tcgatcgcgt aataaaatct attattattt ttgtgaagaa taaatttggg 480 tgcaatgaga atgcgcaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct 540 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag 600 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc 660 ggcatcagag cagattgtac tgagagtgca ccataaaatt gtaaacgtta atattttgtt 720 aaaattcgcg ttaaattttt gttaaatcag ctcattttt aaccaatagg ccgaaatcgg 780 caaaatccct tataaatcaa aagaatagcc cgagataggg ttgagtgttg ttccagtttg 840 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 900 tcagggcgat ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg 960 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacgggggaaa 1020 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct 1080 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct 1140 acagggcgcg tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta 1200 aggagaaaat accgcatcag gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 1260 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 1320 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 1380 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 1440 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 1500 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 1560 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 1620 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 1680 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 1740 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 1800 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 1860 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 1920 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 1980 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 2040 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 2100 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 2160 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 2220 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 2280 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 2340 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 2400 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 2460 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 2520 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 2580 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 2640 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 2700 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 2760 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 2820 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 2880 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 2940 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 3000 ggttcctggc cttttgctgg ccttttgctc acatagtcat gccccgcgcc caccggaagg 3060 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga 3120 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 3180 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc 3240 agggtggtt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg 3300 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt 3360 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact 3420 accgagatat ccgcaccaac gcgcagccg gactcggtaa tggcgcgcat tgcgcccagc 3480 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc 3540 atggttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga 3600 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa 3660 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg 3720 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag 3780 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg 3840 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc 3900 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc 3960 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga 4020 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg 4080 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa 4140 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct 4200 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg 4260 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg 4320 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag 4380 caccgccgcc gc 4392

Claims

1. A class of ICCG fusion proteins, characterized in that The fusion protein is obtained by sequentially connecting cutinase ICCG, a connecting peptide and an anchoring peptide, wherein the connecting peptide is selected from (GGGGS)2 or GSA, and the anchoring peptide is αSP; the ICCG gene sequence is SEQ ID NO.1, the anchoring peptide αSP gene sequence is SEQ ID NO.2, the connecting peptide (GGGGS)2 gene sequence is SEQ ID NO.6, and the connecting peptide GSA gene sequence is SEQ ID NO.

10.

2. The use of a class of ICCG fusion protein according to claim 1 in degrading polyethylene terephthalate (PET), characterized in that: The steps include: 1) Using overlap extension polymerase chain reaction, the ICCG gene was fused to the N-terminus of the anchor peptide αSP gene through the (GGGGS)2 or GSA connecting peptide gene to obtain the recombinant genes ICCG-(GGGGS)2-αSP and ICCG-GSA-αSP; 2) Subcloning the recombinant gene obtained by fusion in step 1) between the restriction enzyme sites of NdeⅠ and XhoⅠ of the plasmid vector pColdⅡ, and transforming it into competent cells of Escherichia coli BL21 (DE3) to obtain a genetic engineering strain of the above fusion protein; 3) inducing the genetically engineered strain expressing the fusion protein obtained above, ultrasonically disrupting and centrifuging to collect the cell lysate, purifying the recombinant protein using a nickel ion metal chelate affinity chromatography column, and finally eluting with a high concentration imidazole buffer to obtain the fusion protein ICCG-(GGGGS)2-αSP and ICCG-GSA-αSP; 4) The obtained ICCG fusion proteins were placed in K2HPO4 / KH2PO4 buffer containing PET material, and then the reaction system was placed in a water bath shaker to perform a degradation reaction of PET.

3. The use according to claim 2, characterized in that: The sequence of the recombinant gene ICCG-(GGGGS)2-αSP is SEQ ID NO.12; the sequence of the recombinant gene ICCG-GSA-αSP is SEQ ID NO.

14.

4. The use according to claim 2, characterized in that: The imidazole concentration in the eluent during purification of recombinant proteins was 100-500 mM.

5. The use according to claim 2, characterized in that: The concentration of the fusion protein in the PET reaction system is 100-300 nM, the rotation speed of the water bath shaker is 0-200 rpm, the reaction temperature is 50-70° C., and the reaction time is 3-144 h.

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