KR-20260067415-A - Enzyme complex for polyethylene terephthalate decomposition and platform for polyethylene terephthalate decomposition in which the enzyme complex is immobilized on Chlorella vulgaris

Abstract

The present invention relates to an enzyme complex for degrading polyethylene terephthalate and a platform for degrading polyethylene terephthalate in which the enzyme complex is immobilized on Chlorella vulgaris. Specifically, the present invention relates to an enzyme complex for degrading polyethylene terephthalate composed of a fusion protein in which a mutant PETase and a Dockerin module are combined; a recombinant scaffolding protein in which a Coheizen module and a carbohydrate attachment protein are combined; and a lipase protein; a method for degrading waste polyethylene terephthalate comprising the step of treating waste polyethylene terephthalate with the enzyme complex; a method for manufacturing the enzyme complex; a Chlorella vulgaris platform for degrading polyethylene terephthalate using the enzyme complex; a method for degrading polyethylene terephthalate using the Chlorella vulgaris platform; and a method for culturing Chlorella vulgaris comprising the step of treating waste polyethylene terephthalate in a culture medium containing Chlorella vulgaris in which the enzyme complex is immobilized.

Inventors

• 한성옥
• 황동혁

Assignees

• 고려대학교 산학협력단

Dates

Publication Date

20260513

Application Date

20241104

Claims (12)

1. A polyethylene terephthalate degradation enzyme complex composed of a fusion protein combining a mutant PETase and a Dockerin module; a recombinant scaffolding protein combining a Cohisen module and a carbohydrate attachment protein; and a lipase protein.
2. In paragraph 1, A polyethylene terephthalate degrading enzyme complex characterized in that the fusion protein formed by combining the above-mentioned mutant PETase and dockerin module consists of the amino acid sequence of SEQ ID NO. 2.
3. In paragraph 1, The above mutant PETase, in the amino acid sequence of SEQ ID NO. 2, The 118th amino acid is substituted from serine (S) to glutamic acid (E), and The 156th amino acid is substituted from histidine (H) to tryptophan (W), and The 183rd amino acid is substituted from aspartic acid (D) to histidine (H), and The 221st amino acid is substituted from arginine (R) to glutamine (Q), and The 230th amino acid is substituted from asparagine (N) to lysine (K), and The 235th amino acid is substituted from serine (S) to phenylalanine (F), and A polyethylene terephthalate degrading enzyme complex characterized by being a mutant PETase in which the 277th amino acid is substituted from arginine (R) to alanine (A).
4. In paragraph 1, The above carbohydrate-attached protein is, CBM6-CBM3 protein formed by binding CBM6 (carbohydrate-binding module family 6) and CBM3 (carbohydrate-binding module family 3) proteins; or A polyethylene terephthalate degrading enzyme complex characterized by being a CBM6-CBM1 protein formed by combining CBM6 (carbohydrate-binding module family 6) and CBM1 (carbohydrate-binding module family 1) proteins.
5. In paragraph 1, A polyethylene terephthalate degrading enzyme complex characterized in that the recombinant scaffolding protein to which the above-mentioned cohisen module and carbohydrate attachment protein are combined consists of the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 6.
6. In paragraph 1, The above-described polyethylene terephthalate degrading enzyme complex is characterized by being a complex formed by mixing a fusion protein in which a mutant PETase and a Dockerin module are combined; a recombinant scaffolding protein in which a Cohisen module and a carbohydrate attachment protein are combined; and a lipase protein in a molar ratio of 1:1:1.
7. A method for degrading waste polyethylene terephthalate, comprising the step of treating waste polyethylene terephthalate with an enzyme complex for degrading polyethylene terephthalate according to any one of claims 1 to 6.
8. A fusion protein formed by combining a mutant PETase and a Dockerin module represented by the amino acid sequence of SEQ ID NO. 2; A recombinant scaffolding protein combined with a cohisen module represented by the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 6 and a carbohydrate attachment protein; and A method comprising the step of mixing lipase proteins in a molar ratio of 1:1:1, adding calcium chloride to a pH 7.0 Tric-HCl solution of 15–25 mM to make the concentration 10–20 mM, and reacting at 4°C for 15–20 hours to form a complex. Method for manufacturing an enzyme complex for degrading polyethylene terephthalate.
9. A polyethylene terephthalate degrading enzyme complex according to any one of claims 1 to 6, added to a culture medium containing Chlorella vulgaris and cultured so that the polyethylene terephthalate degrading enzyme complex is attached to and immobilized on Chlorella vulgaris, wherein the polyethylene terephthalate degrading enzyme complex is attached to and immobilized on Chlorella vulgaris.
10. A method for culturing Chlorella vulgaris, comprising the step of treating waste polyethylene terephthalate in a culture medium containing Chlorella vulgaris immobilized with a polyethylene terephthalate degrading enzyme complex according to any one of claims 1 to 6.
11. In Paragraph 10, A method for culturing Chlorella vulgaris, characterized in that the above-described cultivation method involves growing the Chlorella vulgaris using waste polyethylene terephthalate and terephthalic acid or ethylene glycol, which are decomposition products thereof, as substrates.
12. A method for decomposing polyethylene terephthalate comprising the step of treating waste polyethylene terephthalate with the Chlorella vulgaris platform for decomposing polyethylene terephthalate of claim 9.

Description

Enzyme complex for polyethylene terephthalate decomposition and platform for polyethylene terephthalate decomposition in which the enzyme complex is immobilized on Chlorella vulgaris The present invention relates to an enzyme complex for polyethylene terephthalate degradation capable of continuously degrading polyethylene terephthalate (PET), a type of plastic, and a platform for polyethylene terephthalate degradation in which the enzyme complex is immobilized on Chlorella vulgaris. Plastics containing waste polyethylene terephthalate exist in various environments, including soil and aquatic environments, and as discarded waste plastics pose a significant environmental problem, the development of technologies to process waste plastics is required. Polyethylene terephthalate (PET), a type of plastic, is a polymer composed of ethylene glycol and terephthalate, in which ester bonds exist between the two chemical components and the polymer is formed based on forms such as monohydroxyethyl terephthalate and bishydroxyethyl terephthalate. The degradation process of polyethylene terephthalate (PET) involves conversion from PET into bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET), followed by conversion into monomers terephthalate (TPA) and ethylene glycol (EG). Enzymes that degrade PET and convert it into BHET and MHET, such as cutinase, PETase, and lipase, are being studied. Meanwhile, physical methods involving reactions under high temperature and pressure conditions are the most widely used for plastic treatment, but they can cause further problems by releasing secondary environmental pollutants. Consequently, there is growing interest in environmentally friendly biological treatment methods rather than these physical approaches. Accordingly, research on biological treatment methods is underway, and as related enzymes are identified to decompose plastics using insects, bacteria, etc., research on biological degradation is actively continuing. In particular, as it has recently become known that microalgae distributed in aquatic environments play an important role in removing toxic compounds such as dioxins and bisphenol, which are various environmental pollutants, research on waste plastic degradation technology using microalgae is emerging. Against this backdrop, the inventors prepared a new improved waste PET degrading enzyme complex capable of degrading waste polyethylene terephthalate and absorbing the degradation products, terephthalic acid and ethylene glycol, and further prepared a microalgae platform in which the enzyme complex was immobilized on microalgae. By confirming that this can be usefully applied to the treatment of waste PET exposed to freshwater environments, the present invention was completed. Figure 1 shows a schematic diagram of an enzyme complex for degrading waste PET according to the present invention and a Chlorella vulgaris platform immobilized therewith. Figure 2 shows a schematic diagram of a recombinant vector pColdII PETase 7mut Doc inserted with a gene combining a mutant PETase and a dockerin domain derived from a Clostridium cellulose borans strain according to the present invention. Figure 3 shows a schematic diagram of a recombinant vector pColdII CBM6_CBM1 Doc into which the gene of the recombinant CBM6_CBM1 scaffolding protein according to the present invention is inserted. Figure 4 shows a schematic diagram of a recombinant vector pColdII CBM6_CBM1 Doc into which the gene of the recombinant CBM6_CBM3 scaffolding protein according to the present invention is inserted. Figure 5 shows the results of the analysis of adhesion ability to waste PET by carbon-attached protein type (A), the results of the analysis of adhesion ability to Chlorella vulgaris (B), and the results of the analysis of adhesion ability of recombinant scaffolding protein to Chlorella vulgaris and waste PET (C) in one embodiment of the present invention. Figure 6 shows the structure (A) of mutant petase according to the present invention and the results of the terephthalic acid conversion efficiency analysis (B). Figure 7 shows the growth results of microalgae by substrate on a Chlorella vulgaris platform immobilized with a waste PET degrading enzyme complex according to the present invention. Figure 8 shows the results of analyzing the mRNA expression levels of major genes in Chlorella vulgaris upon exposure to waste PET, ethylene glycol, and terephthalic acid on a Chlorella vulgaris platform immobilized with the waste PET degrading enzyme complex of the present invention. Figure 9 shows the results of analyzing the content of chlorophyll a and chlorophyll b produced from Chlorella vulgaris when a Chlorella vulgaris platform immobilized with the enzyme complex for degrading waste PET of the present invention is exposed to waste PET. The present invention provides a novel enzyme complex for polyethylene terephthalate degradation capable of efficiently degrading waste pol