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KR-102964019-B1 - HYDROXYLASE WITH POLYETHYLENE DEGRADATION ACTIVITY AND USE THEREOF

KR102964019B1KR 102964019 B1KR102964019 B1KR 102964019B1KR-102964019-B1

Abstract

The present invention relates to a novel hydroxylase having plastic degradation activity and a method for degrading plastic using the same. Since the present invention provides excellent effects of not only structural modification of plastic but also activity in degrading plastic into small molecules, it can be usefully utilized in related industrial fields, such as pretreatment processes for the biodegradation of waste plastics and microplastics present in various environments like landfills and oceans, or for plastic upcycling.

Inventors

  • 염수진
  • 윤승도
  • 김민서

Assignees

  • 전남대학교산학협력단

Dates

Publication Date
20260513
Application Date
20240219
Priority Date
20230217

Claims (10)

  1. A hydroxylase comprising any one of the amino acid sequences of SEQ ID NOs 1 to 4.
  2. A composition for degrading plastic comprising a hydroxylase having an amino acid sequence of any one of SEQ ID NOs 1 to 4.
  3. A composition for degrading plastic according to claim 2, further comprising alcohol dehydrogenase, Bayer-Bilicker monooxidase, and ester hydrolyzer.
  4. A plastic degradation composition according to claim 2, further comprising at least one selected from the group consisting of cytochrome P450 reductase, ferredoxin, ferredoxin reductase, NADPH, NADP + , NADH, and NAD + .
  5. A composition for degrading plastic according to claim 2, wherein the plastic is at least one selected from the group consisting of polyethylene, polypropylene, polyvinyl chloride, polystyrene, and polyethylene terephthalate.
  6. A first step of treating a plastic with a hydroxylase comprising an amino acid sequence of any one of SEQ ID NOs 1 to 4 to convert the hydrogen groups of the plastic into hydroxyl groups; A second step of treating the product of the first step with alcohol dehydrogenase to convert the hydroxyl group into a ketone group; A third step of treating the product of the second step with Bayer-Bilicker monooxidase to convert the ketone group into an ester group; and A method for degrading plastic comprising a fourth step of treating the product of the third step with an ester hydrolyzing enzyme to degrade the plastic.
  7. A method for decomposing plastic according to claim 6, wherein the plastic is at least one selected from the group consisting of polyethylene, polypropylene, polyvinyl chloride, polystyrene, and polyethylene terephthalate.
  8. A method for degrading plastic according to claim 6, wherein the plastic is added to or immersed in an enzyme reaction solution comprising the hydroxylase, the alcohol dehydrogenase, the Bayer-Billiker monooxidase, and the ester hydrolyzer, so that the first to fourth steps are performed simultaneously within the enzyme reaction solution.
  9. A method for decomposing plastic according to claim 6, wherein steps 1 through 4 are performed at 30 to 40 ℃.
  10. In claim 6, the first to fourth steps are a method for decomposing plastic performed at pH 7 to 8.

Description

Hydroxylase with Polyethylene Degradation Activity and Use Thereof The present invention relates to a hydroxylase having plastic-degrading activity and a method for degrading plastic using said enzyme. More than half of all plastic produced to date has been manufactured since 2000, and it is reported that approximately 367 million tons of plastic were produced as of 2020. The decomposition of plastic waste is predicted to take a very long time, exceeding 500 years. Among these, microplastics, formed from the weathering of plastic, are small plastics approximately 5 mm or smaller that are mainly found in oceans and coastal areas, disrupting the food chain. According to a Dutch research team in 2022, microplastics were reported to be detected in human blood as well. As a result, interest in plastic exposure and its impact on the environment and living organisms is increasing. Plastic upcycling systems, which go beyond simply disposing of waste plastic to manufacturing new high-value products by utilizing it, have recently been gaining attention, and the demand for the development of technologies to decompose plastic is also growing. The present invention aims to provide a composition for plastic degradation and a degradation method utilizing an enzyme capable of biologically degrading plastic. Figure 1 shows the results of comparing polyethylene treated with the Bacillus thuringiensis JNU01 strain ( B. thuringiensis ) and untreated polyethylene (Control) using NMR. Figure 2 shows the results of comparing polyethylene film treated with the JNU01 strain ( B. thuringiensis ) and untreated polyethylene (Control) using FT-IR. Figure 3 shows the results of comparing a polyethylene film treated with the JNU01 strain ( B. thuringiensis ) and an untreated polyethylene film (PE Film) using an SEM electron microscope. Figure 4 shows the results of comparing the relative expression levels of the JNU01 strain-derived CYP102A5.v1, CYP106B1.v1, and CYP107J3.v1 genes using RT-qPCR in a medium using polyethylene as the sole carbon source ((+)PE) and a medium using LB as the carbon source ((-)PE). Figure 5 shows the SDS-PAGE results of enzymes obtained by protein overexpression through transformation of the JNU01 strain-derived CYP102A5.v1, CYP106B1.v1, and CYP107J3.v1 genes. 1; protein marker, 2; Kurd enzyme extract, 3; purified enzyme. Figure 6 shows the CO-difference spectra of the purified enzymes CYP102A5.v1 (A), CYP106B1.v1 (B) and CYP107J3.v1 (C). Figure 7a shows the results of NADPH oxidation of polyethylene by purified enzymes CYP102A5.v1(A), CYP106B1.v1(B) and CYP107J3.v1(C). Figure 7b is a graph quantifying the NADPH oxidation of polyethylene by the purified enzyme CYP102A5.v1 and an image of the enzyme reaction solution in which polyethylene powder is hydrophilized and mixed by reacting with the CYP102A5.v1 enzyme. Figure 8 shows the FT-IR results of polyethylene powder after the CYP102A5.v1 enzyme reaction. Figure 9 shows the results of NADPH oxidation of myristic acid, a natural substrate of the purified enzyme CYP102A5.v1 (A) and the GC-MS chromatogram results of the product after the enzyme reaction (B). Figures 10a and 10b are the CO-difference spectra results of CYP102A5.v1 enzyme variants G76A(A), F90A(B), G269A(C), H270A(D), E271A(E), T272A(F), S359A(G) and T273A(H). Figures 11a to 11c are the SDS-PAGE results of CYP102A5.v1 enzyme variants G76A, F90A, G269A, H270A, E271A, T272A, T273A and S359A. Figure 12 shows the results of quantifying NADPH oxidation on polyethylene by CYP102A5.v1 enzyme variants G76A, F90A, G269A, H270A, E271A, T272A, T273A and S359A. Figure 13 is a schematic diagram of the polyethylene biodegradation pathway. The present invention provides a novel enzyme having plastic-degrading activity. In the present invention, the plastic may be the most common form of polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyurethane (PU), or polystyrene (PS), having a hard and dense structure and size through multiple stages of continuous polymer formation and condensation processes using two main processes of polymerization and polycondensation with a specific catalyst. In the present invention, the plastic degradation activity may preferably be biodegradable activity. In the present invention, biodegradation refers to a process of conversion into low-molecular-weight compounds by light (ultraviolet rays) or enzymes secreted by microorganisms existing in nature. The degradation of a polymer material first includes a step in which degrading enzymes secreted by microorganisms outside the cell adsorb onto the surface of the polymer material and cleave chemical bonds, such as ester bonds, glycosidic bonds, and peptide bonds of the polymer, through a hydrolysis reaction. As the polymer material is reduced in molecular weight, the material disintegrates, and the products are converted into monomer units, that is, low-molecular-weight degradation products o