CN115057942A - ICCG fusion protein and application thereof in degradation of polyethylene glycol terephthalate - Google Patents

ICCG fusion protein and application thereof in degradation of polyethylene glycol terephthalate Download PDF

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CN115057942A
CN115057942A CN202210671932.3A CN202210671932A CN115057942A CN 115057942 A CN115057942 A CN 115057942A CN 202210671932 A CN202210671932 A CN 202210671932A CN 115057942 A CN115057942 A CN 115057942A
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余林玲
苏兰凯
白姝
孙彦
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Tianjin University
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Abstract

The invention discloses cutinase ICCG fusion protein and application thereof in degradation of polyethylene terephthalate (PET); the invention provides a cutinase ICCG protein with better degradation effect on PET, which is obtained by sequentially connecting cutinase ICCG, a connecting peptide and an anchoring peptide, wherein the connecting peptide comprises (GGGGS) 2 、(GGGGS) 4 GSA or 17X, anchor peptides including alpha SP or TA2, anchor peptide fusion can be effective in increasing angleThe binding capacity of the plasmase ICCG to a substrate, so that the efficiency of degrading PET is improved; the invention obtains the fusion protein capable of effectively degrading PET by screening different connecting peptides and anchoring peptides, and has wide application prospect in the aspect of degrading PET wastes by using an enzyme method.

Description

ICCG fusion protein and application thereof in degradation of polyethylene glycol terephthalate
The technical field is as follows:
the invention relates to ICCG fusion protein and application thereof in degradation of polyethylene terephthalate (PET), belonging to the technical field of biology.
Background art:
polyethylene terephthalate (PET) is a polymer synthesized by terephthalic acid and ethylene glycol, and has the advantages of good heat resistance, plasticity, toughness and the like, so the PET is widely used for packaging, bottle blowing and textile product production, the production capacity of the PET is more than 7000 ten thousand tons only in 2020, and the mass production and the wide use of the PET inevitably generate a large amount of post-consumption garbage, thereby bringing great threat to the living environment and the health of people.
At present, the PET plastics are treated by physical methods (such as landfill and incineration) and chemical methods, but the two methods have high treatment cost and may cause secondary pollution to the environment, and biodegradation may provide a more environment-friendly way for depolymerization and recovery of PET wastes. Over the past decade, various ester hydrolases, such as lipases, carboxylesterases, esterases and cutinases have shown potential to degrade PET. However, PET is a very difficult to hydrolyze polyester, contains a high proportion of aromatic terephthalate, and has poor chain mobility. The glass transition temperature is 65 ℃, so the higher the temperature, the more violent the fluctuation of the PET chain, the more beneficial the enzyme to hydrolyze the PET, but most PET hydrolase loses the activity at high temperature.
In 2012, a cutinase capable of degrading PET at high temperature is separated from leaf-branch compost by using a metagenomic method, namely, the cutinase is called leaf-branch compost cutinase (LCC), the efficiency of the cutinase for degrading PET is at least 33 times that of other enzymes, however, at 65 ℃, the depolymerization capability of LCC on bottle-grade PET is weak, the degradation reaction stops after three days, the conversion rate is only 31%, and in order to further improve the degradation performance of LCC, amino acid mutation is carried out on LCC, and the result shows that the T-shaped cutinase of mutant F243I/D238C/S283C/Y127G (ICCG) has the T-shaped cutinase m The value is increased by 9.8 ℃ compared with the wild-type LCC, and the enzymatic conversion rate of the mutant LCC to PET plastic is 1.5 times that of the wild-type LCC within 20 hours, although the amino acid mutation is oneThe degradation activity of LCC to PET is improved in a fixed degree, but the improvement degree is very limited, and in order to enable ICCG to have higher degradation capability to PET, the anchoring peptide with adhesion to PET is fused with ICCG to promote the adhesion of enzyme to PET substrate, so that the degradation rate is improved.
The invention content is as follows:
the invention aims to provide a class of enzymes capable of effectively degrading PET (polyethylene terephthalate), and further provides a method for fusing an ICCG gene to the N end of an anchor peptide gene through a connecting peptide gene and constructing a genetic engineering strain (figure 1), so that a fusion protein is formed by fusing the C end of an expressed ICCG amino acid sequence with the anchor peptide, wherein the fusion protein comprises ICCG, the connecting peptide and the anchor peptide, the anchor peptide is a polypeptide with the adhesion capacity to PET and can be combined with the surface of the PET to promote the adhesion of a fusion enzyme on the surface of the PET, and further promote the degradation of the PET by the fusion enzyme. The direct tandem connection of the anchoring peptide and the ICCG structural domain can be prevented from causing adverse effect on the property of the fusion protein by the connecting peptide as a functional separator, the mutual folding interference is reduced, the length and the amino acid composition of the connecting peptide can also influence the property of the fusion protein, and therefore, the selection of the proper anchoring peptide and the connecting peptide is very important for improving the degradation capability of the ICCG on the PET.
The technical scheme of the invention is summarized as follows:
ICCG fusion protein is obtained by sequentially connecting cutinase ICCG, connecting peptide and anchoring peptide; the connecting peptide includes (GGGGS) 2 、(GGGGS) 4 GSA or 17X; anchor peptides include α SP or TA 2.
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。
The invention discloses an application of ICCG fusion protein in degrading polyethylene glycol terephthalate, which comprises the following steps:
1) the ICCG genes were separately passed through (GGGGS) by overlap extension polymerase chain reaction 2 、(GGGGS) 4 GSA or 17X connecting peptide gene and anchoring peptide alpha SP geneOr the N end of the TA2 gene is fused to obtain the recombinant gene 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-TA 2; 2) subcloning the recombinant gene obtained by the fusion in the step 1) between restriction sites of Nde I and Xho I of a plasmid vector (pCold II), and transforming the recombinant gene into competent cell Escherichia coli BL21(DE3) to obtain a genetic engineering strain of the fusion protein;
3) inducing and expressing the gene engineering strain of the obtained fusion protein, carrying out ultrasonic disruption and centrifugal collection on cell lysate, purifying the recombinant protein by using a nickel ion metal chelating affinity chromatographic column, and finally eluting by using a high-concentration imidazole buffer solution 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；
4) Respectively placing the obtained ICCG fusion proteins in a K containing PET material 2 HPO 4 /KH 2 PO 4 And (3) in a buffer solution, and then placing the reaction system in a water bath shaker for carrying out the degradation reaction of the PET.
The ICCG gene sequence is SEQ ID NO. 1; the gene sequence of the anchoring peptide alpha SP is SEQ ID NO. 2; the sequence of the anchor peptide TA2 gene is SEQ ID NO. 3; connecting peptide (GGGGS) 2 The gene sequence is SEQ ID NO. 6; linker peptide (GGGGS) 4 The gene sequence is SEQ ID NO. 8; the connecting peptide NL gene sequence is SEQ ID NO. 9; the sequence of the connecting peptide GSA gene is SEQ ID NO. 10; the gene sequence of the connecting peptide 17X is SEQ ID NO. 11.
The recombinant gene ICCG- (GGGGS) 2 -the sequence of α SP is SEQ ID No. 12; recombinant gene ICCG- (GGGGS) 4 -the sequence of α SP is SEQ ID No. 13; the sequence of the recombinant gene ICCG-GSA-alpha SP is SEQ ID NO. 14; the sequence of the recombinant gene ICCG-17X-alpha SP is SEQ ID NO. 15; recombinant gene ICCG- (GGGGS) 2 -the sequence of TA2 is SEQ ID No. 16; recombinant gene ICCG- (GGGGS) 4 -the sequence of TA2 is SEQ ID No. 17; the sequence of the recombinant gene ICCG-GSA-TA2 is SEQ ID NO. 18; recombinant gene ICCG-17X-The sequence of TA2 is SEQ ID NO. 19.
The recombinant gene is subcloned into a plasmid pCold II with the sequence of SEQ ID NO.23, and the recombinant plasmid is transformed into a competent cell of escherichia coli BL21(DE 3).
The concentration of imidazole in the eluent when the recombinant protein is purified is 100-500 mM.
The PET material comprises an amorphous PET film with the crystallinity of 8.1 percent, a film or particle of amorphous PET with the crystallinity of 5.3 percent, a PET film with high crystallinity, a Coca Cola film or particle (the crystallinity is 32 percent), a Yibao film or particle (the crystallinity is 32.8 percent); 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 ℃, and the reaction time is 3-144 h.
According to the invention, an anchoring peptide structure gene with adhesion to PET is fused to the C-terminal of ICCG through a connecting peptide structure to obtain ICCG fusion protein, the adhesion of the fusion protein on the PET surface is promoted by utilizing the adhesion of the anchoring peptide to the PET, the degradation of the ICCG to the PET is further enhanced, and the fusion protein capable of effectively degrading the PET is obtained by screening different anchoring peptides and connecting peptides.
Experiments prove that the degradation capability of ICCG fused with the anchoring peptide on PET is improved, the influence of different connecting peptide structures on the activity of the fusion protein is different, the fusion protein capable of effectively degrading PET can be obtained by optimizing the anchoring peptide and the connecting peptide, and the fusion protein has wide application prospect in treating PET wastes.
Description of the drawings:
FIG. 1 is a plasmid map of the genetically engineered strain.
FIG. 2 shows the fusion protein ICCG- (GGGGS) 2 -TA2、ICCG-(GGGGS) 2 - α SP and ICCG- (GGGGS) 2 -degradation of PET material by LCI.
FIG. 3 shows the fusion protein ICCG- (GGGGS) 2 -αSP、ICCG-(GGGGS) 4 Degradation of PET material by- α SP, ICCG-NL- α SP and ICCG-GSA- α SP.
FIG. 4 shows fusion proteins ICCG-17X-TA2 and ICCG- (GGGGS) 3 Degradation of PET materials by DSI.
FIG. 5 shows the long-term degradation of PET by the fusion protein ICCG-17X-TA 2.
The specific implementation mode is as follows:
in order that the invention may be better understood, the methods provided herein will now be further described with reference to specific examples.
The concrete description is as follows:
1) construction of alpha SP short peptide fusion ICCG protein recombinant gene connected by different connecting peptides:
respectively passing ICCG genes through (GGGGS) by adopting an overlap extension PCR technology 2 、(GGGGS) 4 GSA, 17X connecting peptide gene and N end fusion of anchoring peptide alpha SP gene and construction of recombinant gene ICCG- (GGGGS) 2 -αSP、ICCG-(GGGGS) 4 -αSP、ICCG-GSA-αSP、ICCG-17X-αSP。
2) Construction of TA2 short peptide fusion ICCG protein recombinant gene connected by different connecting peptides:
respectively passing ICCG genes through (GGGGS) by adopting an overlap extension PCR technology 2 、(GGGGS) 4 GSA, 17X connecting peptide gene and N end of anchoring peptide TA2 gene are fused and constructed recombinant gene ICCG- (GGGGS) 2 -TA2、ICCG-(GGGGS) 4 -TA2、ICCG-GSA-TA2、ICCG-17X-TA2。
3) Subcloning the recombinant genes in 1) and 2) between Nde I and Xho I restriction sites of plasmid vector (pCold II), and transforming into competent cell Escherichia coli BL21(DE3) to obtain the gene engineering strain of the fusion protein;
4) inducing and expressing the gene engineering strain of the obtained fusion protein, carrying out ultrasonic disruption and centrifugal collection on cell lysate, purifying the recombinant protein by using a nickel ion metal chelating affinity chromatographic column, and finally eluting by using a high-concentration imidazole buffer solution 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；
In the step 1), the ICCG gene sequence is SEQ ID NO. 1; the alpha SP 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; the 17X gene sequence is SEQ ID NO. 11.
In the step 1), the recombinant gene ICCG- (GGGGS) 2 -the sequence of α SP is SEQ ID No. 12; recombinant gene ICCG- (GGGGS) 4 -the sequence of α SP is SEQ ID No. 13; the sequence of the recombinant gene ICCG-GSA-alpha SP is SEQ ID NO. 14; the sequence of the recombinant gene ICCG-17X-alpha SP is SEQ ID NO. 15; recombinant gene ICCG- (GGGGS) 2 -the sequence of TA2 is SEQ ID No. 16; recombinant gene ICCG- (GGGGS) 4 -the sequence of TA2 is SEQ ID No. 17; the sequence of the recombinant gene ICCG-GSA-TA2 is SEQ ID NO. 18; the sequence of the recombinant gene ICCG-17X-TA2 is SEQ ID NO. 19.
In the step 1), the recombinant gene is subcloned into a plasmid pCold II (SEQ ID NO.23), and the recombinant plasmid is transformed into a competent cell of Escherichia coli BL21(DE 3).
In the step 2) above, the imidazole concentration in the eluent when purifying the recombinant protein is 100-500 mM.
Respectively placing the obtained ICCG fusion proteins in K containing different PET materials 2 HPO 4 /KH 2 PO 4 In a buffer (100mM), and then placing the reaction system in a water bath shaker for performing the degradation reaction of PET.
The PET material includes amorphous PET film having a crystallinity of 8.1%, amorphous PET film or particles having 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, if the film material is degraded, the diameter is 6mm, the number is 1 piece, if the degraded particle material is degraded, the mass is 10mg, the rotating speed of a water bath shaker is 0-200rpm, the reaction temperature is 50-70 ℃, and the reaction time is 3-144 h.
Example 1: cutinase ICCG and fusion protein ICCG- (GGGGS) 2 -TA2、ICCG-(GGGGS) 2 -αSP、ICCG-(GGGGS) 2 Preparation of LCI
1) Subcloning ICCG gene between Nde I and Xho I restriction sites of plasmid pCold II (SEQ ID NO.23) to form recombinant plasmid (ICCG-pCold II), and transforming escherichia coli shuffle T7 competent cells after sequencing verification to obtain a genetic engineering strain;
2) the ICCG gene (SEQ ID NO.1) was passed through (GGGGS) by overlap extension PCR technique 2 The connecting peptide gene (SEQ ID NO.6) is respectively fused with the N tail ends of the alpha SP (SEQ ID NO.2), TA2(SEQ ID NO.3) and LCI (SEQ ID NO.4) genes to respectively obtain ICCG- (GGGGS) with the gene sequences of SEQ ID NO.12, SEQ ID NO.16 and SEQ ID NO.20 2 -αSP、ICCG-(GGGGS) 2 -TA2、ICCG-(GGGGS) 2 LCI recombinant gene, and subcloning the fused gene between Nde I and Xho I restriction sites of plasmid pCold II (SEQ ID NO.23) to form a recombinant plasmid (ICCG- (GGGGS) 2 -TA2-pColdⅡ、ICCG-(GGGGS) 2 -alpha SP-pCold II and ICCG- (GGGGS) 2 LCI-pCold II), which is transformed into competent cells of Escherichia coli BL21(DE3) after sequencing verification to obtain a genetic engineering strain;
3) culturing the genetically engineered strain of the above steps 1) and 2) in two stages, firstly growing to OD at 37 deg.C 600 Reaching 0.6-0.8, adding IPTG (final concentration of 0.5mM) at 16 deg.C to induce target protein expression for 24h, centrifuging to collect expression strain (4000g,30min), and storing at-20 deg.C for use.
4) Resuspending the obtained strain in lysis buffer (20mM Tris-HCl, 300mM NaCl, pH 8.0), sonicating the strain and centrifuging to collect cell lysate, purifying the recombinant protein with nickel ion metal chelating affinity chromatography column, eluting with eluent (20mM Tris-HCl, 100mM imidazole, 300mM NaCl, pH 8.0) to obtain target proteins ICCG, (GGGGS) 2 -αSP、ICCG-(GGGGS) 2 -LCI，ICCG-(GGGGS) 2 The elution solution of TA2 fusion protein differed from the other target proteins in that the imidazole concentration was 500mM, and the eluted protein was further purified by size exclusion chromatography, and finally the concentration of the resulting protein was determined by the Bradford method.
Example 2: cutinase ICCG and fusion protein ICCG- (GGGGS) 2 -TA2、ICCG-(GGGGS) 2 -αSP、ICCG-(GGGGS) 2 Degradation of PET Material by LCI
The cutinase ICCG and the fusion protein ICCG- (GGGGS) in example 1 were used respectively 2 -TA2、ICCG-(GGGGS) 2 -αSP、ICCG-(GGGGS) 2 LCI degrades different PET materials, 20. mu.L of the above enzyme solution (final concentration of 100nM) is added to a centrifuge tube containing amorphous PET films and particles having a crystallinity of 5.3%, Coca Cola films and particles having a crystallinity of 32%, Yibao films and Yibao particles having a crystallinity of 32.8% (each film material having a diameter of 6mm, the number of films being 1 and the particles being 10mg, wherein the particle diameters of the amorphous PET particles, the Yibao particles and the Coca Cola particles being 0.5mm or less), 280. mu.L of a reaction buffer is added, degradation is carried out in a constant temperature water bath at 60 ℃ and 150rpm for 24 hours, and the amount of degradation products produced is measured by high performance liquid chromatography. The experimental results are shown in fig. 2: the fusion of TA2 and alpha SP can improve the degradation capability of ICCG on different PET materials, wherein ICCG- (GGGGS) 2 The ability of α SP to degrade amorphous PET particles, amorphous PET films, coca-cola particles, coca-cola films, pepo particles and pepo films is 1.23, 1.90, 1.67, 1.30, 1.88 and 1.25 times that of ICCG, respectively; ICCG- (GGGGS) 2 TA2 shows a degradation capacity for amorphous PET particles, amorphous PET film, Coca Cola particles, Coca Cola films, Yibao particles and Yibao films which is 1.11, 1.53, 1.03, 1.14, 1.20 and 1.08 times that of ICCG, respectively, so ICCG- (GGGGS) 2 The degrading activity of the-alpha SP to PET is higher than that of ICCG- (GGGGS) 2 PET degrading activity of TA2, ICCG degrading performance of alpha SP fusion is improved more than that of TA2 fusion on ICCG, and ICCG- (GGGGS) 2 -degradation activity of LCI on PET is reduced compared to ICCG.
Example 3: cutinase ICCG and fusion protein ICCG- (GGGGS) 2 -αSP、ICCG-(GGGGS) 4 Preparation of-alpha SP, ICCG-NL-alpha SP, ICCG-GSA-alpha SP
1) Subcloning ICCG gene between Nde I and Xho I restriction sites of plasmid pCold II (SEQ ID NO.23) to form recombinant plasmid (ICCG-pCold II), and transforming escherichia coli shuffle T7 competent cells after sequencing verification to obtain a genetic engineering strain;
2) the ICCG gene (SEQ ID NO.1) is respectively connected with a peptide (GGGGS) by overlapping extension PCR technology 2 Gene (SEQ ID NO.6), (GGGGS) 4 The gene (SEQ ID NO.8), NL gene (SEQ ID NO.9), GSA gene (SEQ ID NO.10) and the N-terminal of the alpha SP gene (SEQ ID NO.2) are fused to obtain ICCG- (GGGGS) with the 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 -alpha SP, ICCG-GSA-alpha SP and ICCG-NL-alpha SP recombinant gene, subcloning the fused gene between Nde I and Xho I restriction sites of plasmid pCold II (SEQ ID NO.23) to form recombinant plasmid ICCG- (GGGGS) 2 -αSP-pColdⅡ、ICCG-(GGGGS) 4 Alpha SP-pCold II, ICCG-NL-alpha SP-pCold II and ICCG-GSA-alpha SP-pCold II, which are transformed into competent cells of escherichia coli BL21(DE3) after sequencing verification to obtain a genetic engineering strain;
3) culturing the genetically engineered strain of the above steps 1) and 2) in two stages, firstly growing to OD at 37 deg.C 600 Reaching 0.6-0.8, adding IPTG (final concentration of 0.5mM) at 16 deg.C to induce target protein expression for 24h, centrifuging to collect expression strain (4000g,30min), and storing at-20 deg.C for use.
4) Resuspending the strain obtained above in lysis buffer (20mM Tris-HCl, 300mM NaCl, pH 8.0), sonicating the strain and centrifuging to collect cell lysate, purifying the recombinant protein using a nickel ion metal chelating affinity chromatography column, eluting with an eluent (20mM Tris-HCl, 100mM imidazole, 300mM NaCl, pH 8.0) to obtain the target protein, further purifying the eluted protein by size exclusion chromatography, and finally detecting the concentration of the obtained protein by the Bradford method.
Example 4: cutinase ICCG and fusion protein ICCG- (GGGGS) 2 -αSP、ICCG-(GGGGS) 4 Degradation of PET material by-alpha SP, ICCG-NL-alpha SP, ICCG-GSA-alpha SP
The cutinase ICCG and the fusion protein ICCG- (GGGGS) in example 3 were used respectively 2 -αSP、ICCG-(GGGGS) 4 Coca-SP, ICCG-NL-alpha SP, ICCG-GSA-alpha SP degradation coca cola thinThe membrane (crystallinity: 32%) and the amorphous PET film with high crystallinity were prepared by adding 100. mu.L of the above enzyme solution (final concentration: 300nM) to a centrifuge tube containing 1 Coca Cola film with a diameter of 6mm and the amorphous PET film with high crystallinity, respectively, adding 900. mu.L of the reaction buffer, shaking the mixture in a constant temperature water bath at 60 ℃ and 150rpm for 24 hours, and measuring the amount of the degradation product formed by high performance liquid chromatography. The experimental results are shown in fig. 3: fusion proteins ICCG-GSA-alpha SP, ICCG- (GGGGS) 2 - α SP and ICCG- (GGGGS) 4 The degrading activity of the-alpha SP on the coca-cola film and the amorphous PET film with high crystallinity is higher than that of ICCG, wherein the ICCG-GSA-alpha SP, the ICCG- (GGGGS) 2 - α SP and ICCG- (GGGGS) 4 The degrading activities of-alpha SP on coca-cola films were 4.3, 4.3 and 3.6 times of ICCG and on amorphous PET films of high crystallinity were 1.4, 2.1 and 2.0 times of ICCG, respectively, as compared with GSA and (GGGGS) 4 Linker peptide containing (GGGGS) 2 The fusion protein of (a) has high degradation activity on both coca-cola thin films and high-crystallinity amorphous PET thin films, while the fusion of the anchor peptide α SP to the C-terminus of ICCG via the NL linker peptide decreases the degradation activity of ICCG on coca-cola thin films and high-crystallinity amorphous PET thin films.
Example 5: cutinase ICCG and fusion protein ICCG-17X-TA2, ICCG- (GGGGS) 3 Preparation of-DSI
1) The ICCG gene is subcloned between the Nde I and Xho I restriction sites of plasmid pCold II (SEQ ID NO.23) to form a recombinant plasmid (ICCG-pCold II), and after sequencing verification, the Escherichia coli shuffle T7 competent cells are transformed to obtain a genetic engineering strain.
2) The ICCG gene (SEQ ID NO.1) is respectively connected with a peptide (GGGGS) by overlapping extension PCR technology 3 The gene (SEQ ID NO.7) and the 17X gene (SEQ ID NO.11) are fused with the N ends of the DSI gene (SEQ ID NO.5) and the TA2 gene (SEQ ID NO.3) to respectively obtain ICCG- (GGGGS) with the gene sequences of SEQ ID NO.22 and SEQ ID NO.18 3 The recombinant genes of-DSI and ICCG-17X-TA2 are subcloned into plasmid pCold II (SEQ ID NO.23) between Nde I and Xho I restriction sites to form recombinant plasmidICCG-(GGGGS) 3 DSI-pCold II, ICCG-17X-TA2-pCold II, which are transformed into competent cells of Escherichia coli BL21(DE3) after sequencing verification to obtain genetically engineered strains.
3) Culturing the genetically engineered strain of the above steps 1) and 2) in two stages, firstly growing to OD at 37 deg.C 600 Reaching 0.6-0.8, adding IPTG (final concentration of 0.5mM) at 16 deg.C to induce target protein expression for 24h, centrifuging to collect expression strain (4000g,30min), and storing at-20 deg.C for use.
4) The strain obtained above was resuspended in lysis buffer (20mM Tris-HCl, 300mM NaCl, pH 8.0), the strain was sonicated and the cell lysate was collected by centrifugation, the recombinant protein was purified using a nickel ion metal chelate affinity chromatography column, the target protein was eluted with an eluent (20mM Tris-HCl, 100mM imidazole, 300mM NaCl, pH 8.0) and the eluted protein was further purified by size exclusion chromatography, and finally the concentration of the protein obtained was determined by the Bradford method.
Example 6: cutinase ICCG and fusion protein ICCG-17X-TA2, ICCG- (GGGGS) 3 Degradation of PET by DSI
The cutinase ICCG and the fusion proteins ICCG-17X-TA2, ICCG- (GGGGS) in example 5 were used respectively 3 20. mu.L each of the above enzyme solutions (final concentration: 100nM) was added to a centrifuge tube containing 1 amorphous PET film having a diameter of 6mm, followed by addition of 280. mu.L of the reaction buffer, and the degradation was carried out in a 50-70 ℃ water bath for 3 hours, and the amount of the degradation product produced was measured by high performance liquid chromatography. The experimental result is shown in figure 4, and the degradation activity of ICCG-17X-TA2 to PET at 50-70 ℃ is 1.33-1.51 times of that of ICCG, so the fusion of TA2 increases the adsorption quantity of ICCG on the PET surface, and further improves the degradation activity of ICCG to PET, and ICCG- (GGGGS) 3 The PET degrading activity of DSI is only about 20% of that of ICCG, so DSI passes through (GGGGS) 3 The linker peptide was fused to the C-terminus of ICCG, resulting in decreased ICCG activity.
Example 7: long-time degradation of PET by cutinase ICCG and fusion protein ICCG-17X-TA2
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, and 20. mu.L of each of the enzyme solutions (final concentration: 100nM) was added to a centrifuge tube containing 1 amorphous PET film with a diameter of 6mm, followed by addition of 280. mu.L of the reaction buffer, degradation was continued in a water bath at 50 ℃ and 60 ℃ for 6 days, sampling was performed every 24 hours, and the amount of degradation products produced was measured by high performance liquid chromatography. The experimental results are shown in FIG. 5, and it can be seen that the degradation activity of ICCG-17X-TA2 on PET is always higher than that of ICCG within 6 consecutive days at 50 ℃ and 60 ℃, the degradation activity of ICCG-17X-TA2 on PET is up to 1.60 times of that of ICCG at 50 ℃, and the degradation activity of ICCG-17X-TA2 on PET is up to 1.57 times of that of ICCG at 60 ℃, which indicates that TA2 still plays an adhesion role in the process of degrading PET for a long time, and further improves the degradation activity of ICCG on PET.
The results of the PET degradation studies (examples 2, 4, 6 and 7) show that the fusion of an anchoring peptide adhering to PET to the cutinase ICCG improves the degradation of PET material by ICCG, such as the fusion protein ICCG- (GGGGS) of the present invention 2 -αSP、ICCG-(GGGGS) 2 The degrading activities of the-TA 2 and the ICCG-17X-TA2 on the PET material are higher than those of the ICCG, wherein the ICCG- (GGGGS) 2 -the degradability of the α SP on amorphous PET particles, amorphous PET film, coca-cola particles, coca-cola films, fiveleaf particles and fiveleaf films is 1.23, 1.90, 1.67, 1.30, 1.88 and 1.25 times that of ICCG, respectively; ICCG- (GGGGS) 2 -TA2 has 1.11, 1.53, 1.03, 1.14, 1.20 and 1.08 times the degradability of amorphous PET particles, amorphous PET film, coca particles, coca films, fiozole particles and fiozole films, respectively, of ICCG; the degradation activity of ICCG-17X-TA2 to PET at 50-70 ℃ is 1.33-1.51 times of that of ICCG, and the degradation activity of ICCG can be still improved by the fusion of TA2 in the process of degrading PET for a long time; furthermore, different linker peptides may also influence the activity of the fusion protein, e.g.compared to NL, GSA, (GGGGS) in the present invention 4 Linker peptide, utilization (GGGGS) 2 The fusion protein ICCG- (GGGGS) is obtained by fusing short peptide alpha SP to the C terminal of ICCG by connecting peptide 2 Absence of-alpha SP for Coca Cola film and high crystallinityThe shaped PET film has higher degradation activity. These results indicate that the anchor peptide fusion protein of the present invention is very advantageous for the degradation of PET, which is very important for the enzymatic degradation of PET waste.
In conclusion, the fusion protein has high catalytic activity on PET and has high practical application value in degrading PET wastes by using an enzyme method.
The improved degradation of PET by ICCG using the anchor peptide fusion proposed by the present invention has been described by the preferred embodiments in the field, and it will be apparent to those skilled in the art that the technology can be implemented by modifying or appropriately changing and combining the methods described herein without departing from the content, spirit and scope of the present invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.
Sequence listing
<110> Tianjin university
<120> ICCG fusion protein and application thereof in degradation of polyethylene glycol terephthalate
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 774
<212> DNA
<213> Artificial Sequence (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 (Artificial Sequence)
<400> 2
tacgaaatgc cgagcgaaga aggctatcaa gattatgaac cggaagcg 48
<210> 3
<211> 132
<212> DNA
<213> Artificial Sequence (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 (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 (Artificial Sequence)
<400> 5
ggcctgtgga gcaccattaa acagaaaggc aaggaagccg ccatcgcggc ggccaaagcg 60
gccggccaag ccgcgttagg cgcctta 87
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggcggtggcg gcagcggtgg tggcggtagc 30
<210> 7
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggcggtggcg gtagcggcgg cggtggcagc ggcggcggtg gtagc 45
<210> 8
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggcggtggcg gtagtggtgg tggtggcagc ggtggcggtg gcagttggcg gtggcggcag 60
t 61
<210> 9
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gcgggctatg gcaaagcggg cggcaccgtg accccgaccc cgaacacc 48
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggcagcgcgg gcagcgcggc gggcagcggc 30
<210> 11
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gcggaagcgg ccgcgaaaga agcggcggcc aaagaagccg cggcgaaagc g 51
<210> 12
<211> 852
<212> DNA
<213> Artificial Sequence (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 (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 (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 ggtgcgcgcg 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 (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 (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 (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 (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 (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 cggtgcgcgc 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
gcggccgcga aagaagcggc ggccaaagaa gccgcggcga aagcgtatag ccgctgtcag 840
ctgcaaggct ttaactgcgt ggtgcgcagc tatggcctgc cgaccattcc gtgctgccgc 900
ggcctgacct gccgcagcta ttttccgggc agcacctatg gccgctgtca acgttat 957
<210> 20
<211> 945
<212> DNA
<213> Artificial Sequence (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 (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 (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 (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 ctcatttttt 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 gacggggaaa 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
agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg 3300
ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt 3360
ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact 3420
accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc 3480
gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc 3540
atggtttgtt 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 (8)

1. An ICCG fusion protein is characterized in that 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 α SP or TA 2.
2. The ICCG fusion protein of claim 1, wherein the fusion protein comprises: 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。
3. The use of an ICCG fusion protein according to claim 1 for the degradation of polyethylene terephthalate, comprising the steps of:
1) the ICCG genes were separately passed through (GGGGS) by overlap extension polymerase chain reaction 2 、(GGGGS) 4 GSA or 17X connecting peptide gene and anchoring peptide alpha SP gene or N end of TA2 gene are fused to obtain recombinant gene 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；
2) Subcloning the recombinant gene obtained by the fusion in the step 1) between Nde I and Xho I restriction sites of a plasmid vector (pCold II), and transforming into competent cell Escherichia coli BL21(DE3) to obtain a genetic engineering strain of the fusion protein;
3) inducing and expressing the gene engineering strain of the obtained fusion protein, carrying out ultrasonic disruption and centrifugal collection on cell lysate, purifying the recombinant protein by using a nickel ion metal chelating affinity chromatographic column, and finally eluting by using a high-concentration imidazole buffer solution 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；
4) Respectively placing the obtained ICCG fusion proteins in a PET material-containing K 2 HPO 4 /KH 2 PO 4 And (3) in a buffer solution, and then placing the reaction system in a water bath shaker for carrying out the degradation reaction of the PET.
4. The method of claim 3, wherein the ICCG gene has the sequenceSEQ ID No. 1; the gene sequence of the anchoring peptide alpha SP is SEQ ID NO. 2; the sequence of the anchor peptide TA2 gene is SEQ ID NO. 3; linker peptide (GGGGS) 2 The gene sequence is SEQ ID NO. 6; linker peptide (GGGGS) 4 The gene sequence is SEQ ID NO. 8; the connecting peptide NL gene sequence is SEQ ID NO. 9; the sequence of the connecting peptide GSA gene is SEQ ID NO. 10; the gene sequence of the connecting peptide 17X is SEQ ID NO. 11.
5. Use according to claim 3, characterized in that the recombinant gene ICCG- (GGGGS) 2 -the sequence of α SP is SEQ ID No. 12; recombinant gene ICCG- (GGGGS) 4 -the sequence of α SP is SEQ ID No. 13; the sequence of the recombinant gene ICCG-GSA-alpha SP is SEQ ID NO. 14; the sequence of the recombinant gene ICCG-17X-alpha SP is SEQ ID NO. 15; recombinant gene ICCG- (GGGGS) 2 -the sequence of TA2 is SEQ ID No. 16; recombinant gene ICCG- (GGGGS) 4 -the sequence of TA2 is SEQ ID No. 17; the sequence of the recombinant gene ICCG-GSA-TA2 is SEQ ID NO. 18; the sequence of the recombinant gene ICCG-17X-TA2 is SEQ ID NO. 19.
6. Use according to claim 3, characterized in that the recombinant gene is subcloned into the plasmid pCold II with the sequence SEQ ID NO.23, the recombinant plasmid being transformed into competent cells of E.coli BL21(DE 3).
7. The use according to claim 3, wherein the recombinant protein is purified at an imidazole concentration of 100 and 500mM in the eluate.
8. Use according to claim 3 wherein the PET material comprises amorphous PET film having a crystallinity of 8.1%, amorphous PET film or granules having a crystallinity of 5.3%, highly crystalline PET film, Coca Cola film or granules (32% crystallinity), Yibao film or granules (32.8% crystallinity); 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 ℃, and the reaction time is 3-144 h.
CN202210671932.3A 2022-06-15 2022-06-15 ICCG fusion protein and application thereof in degradation of polyethylene glycol terephthalate Pending CN115057942A (en)

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WO2015097104A1 (en) * 2013-12-23 2015-07-02 Carbios Method for recycling plastic products
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