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6INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
BIODEGRADATION OF POLYETHYLENE BY A RECOMBINANT LACCASE-LIKE ENZYME FROM RHODOCOCCUS OPACUSR7
1Nazirov Muhammad Latif Maruf o'g'li, 2Kobilov Fazliddin Bozorovich, 3Sharifov Mansurbek Raximberganovich, 4Aliyev Zafar Zokirovich, 5Khalilov Ilkhom
Mamatqulovich
Junior researcher, IMUAS, 2PhD (student), IMUAS, 3Junior researcher, IMUAS, 4Junior
researcher, IMUAS, 5DSc, IMUAS https://doi.org/10.5281/zenodo.13883761
Abstract. The growing environmental burden of polyethylene (PE) waste necessitates new approaches to biodegradation. Laccase-like multicopper oxidases (LMCOs) are promising enzymes for oxidizing synthetic polymers such as PE. In this study, we optimized the codon of the LMCO3 gene from Rhodococcus opacus R7, synthesized it, and cloned it into a PET28 expression plasmid. Following successful expression in Escherichia coli and protein purification, the recombinant LMCO3 enzyme was incubated with PE to assess its oxidative degradation potential. Fourier-transform infrared spectroscopy (FTIR) was used to analyze changes in the PE structure, providing insights into the enzyme's activity and degradation mechanisms. The results demonstrate the capacity of LMCO3 to oxidize untreated PE, shedding light on its potential application for bioplastic waste management.
Keywords: polyethylene degradation, laccase-like multicopper oxidases, codon optimization, Rhodococcus opacus R7, FTIR,, bioremediation, Escherichia coli
Introduction
Polyethylene (PE) is a widely used synthetic polymer with high resistance to degradation, contributing significantly to environmental pollution. Traditional methods of polyethylene disposal, such as incineration and landfilling, pose ecological challenges and health risks. Polyethylene's carbon-carbon (C-C) backbone makes it particularly resistant to biodegradation. Consequently, the search for efficient bioremediation approaches, particularly through enzymatic degradation, has garnered significant attention. Enzymatic degradation has emerged as a potential eco-friendly solution to break down polyethylene, and laccases, a group of enzymes belonging to the multicopper oxidase family, have shown promise. Laccase enzymes are primarily known for their ability to oxidize phenolic compounds and have been studied extensively for their roles in lignin degradation. Recent research has expanded their potential application to the degradation of synthetic polymers like polyethylene [1-3]. Among the limited number of bacterial species capable of degrading PE, Rhodococcus opacus R7 strains have shown promise due to their ability to produce oxidative enzymes such as laccase-like multicopper oxidases (LMCOs) [4-16]. These enzymes facilitate oxidative degradation of complex polymers, converting them into smaller, more biodegradable compounds.
In this study, we aimed to enhance the expression of LMCO3, a novel laccase-like enzyme isolated from R. opacus R7, by optimizing its codon usage for expression in E. coli. After successful protein production and purification, we investigated the enzyme's ability to oxidatively degrade PE by monitoring structural changes using Fourier-transform infrared (FTIR) spectroscopy.
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
Materials and methods
Gene Synthesis and Cloning:
The LMCO3 gene from R opacus R7 was codon-optimized for expression in *E. coli* and synthesized. The gene was then cloned into the PET28 plasmid, which allows for high-level expression and contains a His-tag for efficient purification.
Protein Expression and Purification:
The pET28-LMCO3 plasmid was transformed into E. coli BL21(DE3) cells. After induction with IPTG, the cells were harvested, and the recombinant LMCO3 protein was purified using nickel affinity chromatography, taking advantage of the His-tag.
Polyethylene Incubation:
Low-density PE samples were incubated with purified LMCO3 enzyme in a buffer system. The incubation was carried out at 37 °C for 72 hours, with agitation to ensure enzyme-substrate interaction.
FTIR Analysis:
Fourier-transform infrared spectroscopy (FTIR) was performed on the PE samples before and after incubation with LMCO3. The FTIR spectra were analyzed to identify changes in functional groups and bonds within the PE structure, with a focus on the emergence of oxygenated functional groups such as ketones, alcohols, and carboxylic acids.
Results
Successful Expression and Purification of LMCO3:
The codon-optimized LMCO3 gene was successfully expressed in E. coli, yielding high levels of soluble protein. The His-tagged LMCO3 was purified to homogeneity as confirmed by SDS-PAGE analysis.
FTIR Analysis of Polyethylene Degradation:
FTIR analysis of the PE samples revealed significant changes in the spectra after incubation with LMCO3. Peaks corresponding to carbonyl (C=O, 1647-1716 cm1) and hydroxyl (O-H, 3500-3100 cm1) groups appeared, indicating oxidative degradation of the PE polymer [17] (Fig.1). The presence of ketones and carboxylic acids suggests that the enzyme effectively introduces oxygen into the PE backbone, facilitating its breakdown.
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INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Fig. 1 FTIR spectra of polyethylene before and after incubation with LMCO3. Peaks corresponding to carbonyl (C=O, 1647-1716 cm1) and hydroxyl (O-H, 3500-3100 cm1) groups indicate oxidative degradation of the polymer.
Discussion:
The results demonstrate that the codon-optimized LMCO3 from R opacus R7 retains its oxidative activity when expressed in E. coli and is capable of degrading PE. FTIR analysis provided clear evidence of structural changes in the polymer, consistent with oxidative degradation. These findings align with previous studies on laccase-like enzymes and support the potential use of LMCO3 in biotechnological applications aimed at mitigating plastic waste.
Conclusion
This study highlights the successful expression of a codon-optimized LMCO3 enzyme in E. coli and its ability to degrade polyethylene. The structural changes detected by FTIR indicate that LMCO3 can introduce oxygenated functional groups into PE, promoting its breakdown. Further work will focus on optimizing the conditions for enhanced degradation and exploring the enzyme's activity on other types of plastics.
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INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS"
_25-26 SEPTEMBER, 2024_
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