Heterologous expression and characterization of Rhodococcus opacus R7 laccase-like multicopper oxidase (LMCO1) enzyme for polyethylene biodegradation

Heterologous expression and characterization of Rhodococcus opacus R7 laccase-like multicopper oxidase (LMCO1) enzyme for polyethylene biodegradation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Heterologous expression and characterization of Rhodococcus opacus R7 laccase-like multicopper oxidase (LMCO1) enzyme for polyethylene biodegradation Mahboobeh Pishan, Sima Sazegari, Ali Niazi, Marjan Majdinasab, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8685015/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The production rate of Polyethylene (PE) has increased to a concerning level, necessitating effective strategies to reduce its environmental impacts. This study investigates the role of laccase-like multicopper oxidase (LMCO1) in oxidative degradation of PE, through a comprehensive approach, including machine learning analysis, recombinant expression, physico-chemical assays. To this end, the public available RNA-seq data related to Rhodococcus opacus R7 cultured with PE was mined using several attribute weighting algorithms to explore the discriminative role of LMCO1 in PE degradation. Further, the recombinant LMCO1 was expressed and evaluated for substantial degrading impact on the PE films. Oxidative degradation of PE samples was evaluated by measuring weight loss rate, assessment of water contact angle measuring, confirmed by Fourier transform infrared spectroscopy and scanning electron microscopy analysis. The findings revealed that Cu²⁺ enhanced the activity of LMCO1 in the crude enzyme extract by 490%, with peak activity occurring at pH 8 and 60°C (optimal temperature). The PE degradation experiments over 72 hours indicated that a 14.28% weight loss rate was in LDPE as well as decreasing the water contact angle to 76.73 °. Fourier transform infrared spectroscopy analysis revealed the existence of various polar functional groups on PE surface including carbonyl, carboxyl, and hydroxyl groups. Significant damage to the PE surface, including cracks, pitting, and roughness, as well as internal aspects such as structural weakening and material degradation was identified through scanning electron microscopy. Overall, this study demonstrated the high potential of LMCO1 to degrade PE films and particles in short time. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Polyethylene Biodegradation Rhodococcus opacus R7 Plastic waste Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Polyethylene (PE) ranks among the most widely produced and used polyolefin derived from petroleum-based plastics, due to its exceptional durability, cost-effectiveness in production, and versatility [ 1 ]. It is produced in millions of tons annually and used in many different areas and industries [ 2 ]. PE molecule is generally structured by two carbon atoms linked to two hydrogen per each (–CH 2 –CH 2 -) while the chain is terminated by methyl group (R–CH 3 ). PE exhibits a notable degree of hydrophobicity, in addition to properties including density, crystallinity, molecular weight, and a reduced specific surface area. Furthermore, features including linear structure made up of carbon atoms in the form of C–C and C–H links, lacking hydrolysable functional groups in the backbone, along with its high molecular weight, make it recalcitrant to biodegradation [ 3 – 5 ]. Different fates including environmental accumulation, abiotic oxidation, recycling and biodegradation by microorganisms and their associated enzymes are proposed for PE polymer [ 3 , 6 ]. Polymers, such as PE, may experience degradation via several mechanisms, including chemical processes (such as oxidation, hydrolysis, or catalytically mediated reactions), thermally induced processes (which involve exposure to elevated temperatures), mechanical processes (such as milling or trituration), and physical radiation such as ultrasonic, microwave and sunlight [ 7 , 8 ]. Such degradations may encounter various challenges, such as significant energy consumption, the necessity for high temperatures and pressures condition, and the utilization of additives and solvents. Microbial degradation appears to be the most appealing and environmentally sustainable approach to address the increasing issue of plastic pollution and minimize waste [ 9 ]. Many studies have identified more than 20 bacterial genera capable of degrading PE, including the genera, Ralstonia, Stenotrophomonas, Acinetobacter, Klebsiella, Rhodococcus, Pseudomonas, Streptococcus, Streptomyces, Staphylococcus, Bacillus [ 5 , 10 – 12 ]. Numerous studies have indicated that enzyme-mediated oxidation of PE occurs through direct catalysis by enzyme within the extracellular supernatant of bacteria that degrade PE; however, the intricate process of PE biodegradation remains unclear [ 13 – 19 ]. The biodegradation of plastic by microorganism primarily involves an enzymatic mechanism that cleaves the polymer bonds into their monomers. Enzymes that can effectively sever carbon-carbon bonds in synthetic polymers are in high demand, as they hold the potential to offer solutions for sustainable plastic recycling [ 20 ]. Substantial enzymes including laccases, oxidases and lipases from Bacillus cereus , Bervibacilluse borstelensis and a marine bacterial community have been approved to have PE degradation potential based on transcriptome studies, quantitative 16S rRNA sequencing and cultivation method [ 21 , 22 ]. Among them, laccases (EC 1.10.3.2), a member of diverse protein family of multicopper oxidases (MCOs), can facilitate the oxidative degradation of a range of phenolic derivatives and biopolymers. Notably, these enzymes have attracted significant research interest for their key role in the depolymerization of PE. Additionally, Laccases-like Multi Copper Oxidases (LMCO) are classified as a non-plant group of 3-domain laccases and classified according to their cupredoxin-like domains they possess [ 23 , 24 ]. Some possesses two domains and shows trimeric quaternary structure while some other laccases with monomeric structure have three unique domains [ 24 ]. The active site of laccase consist of four copper atoms: a type Ι copper center and a trinuclar copper cluster, which is composed of a type ΙΙ copper and a pair of type ΙΙΙ coppers. T1 site is responsible for substrate oxidation, while the T2/T3 centers mange the reduction of O 2 [ 25 ], substrates oxidation by laccase occurs through outer-sphere electron transferring to a copper T1 site, followed by intermolecular electron transfer to a tri-nuclear copper site, where oxygen molecule is reduced to water [ 26 ]. Limited number of laccases have been identified for their PE degradation capacity [ 24 ], this includes laccase derived from fungi and bacteria such as Trichoderma harzianum, Tinea versicolor , Botrytis aclada , and Bacillus subtilis [ 27 ]. Although pretreatment is typically required in case of Laccase PE biodegradation, Psychrobacter sp. NJ228 and Rhodococcus opacus have been reported to partially degrade PE plastics without the need for pretreatment or mediators [ 28 , 29 ]. The ability of crude laccase for PE biodegradation from Trichoderma harzianum was confirmed though weight loss rate and FTIR analysis [ 17 ]. The members of the Rhodococcus genus exhibit an exceptional capacity to break down a diverse array of both natural organic and xenobiotic substances [ 30 ], they are the most interesting due to their remarkable metabolic versatility and the significant potential for degradation of plastics [ 31 ]. Extracellular copper-binding enzyme, laccase, was extracted from Rhodococcus ruber C208 and used for treating the PE films. Molecular weight and the FTIR spectra results confirm the enzymatic biodegradation [ 15 ]. R. opacus R7 (CIP107348), is known to apply naphthalene and o-xylene sole carbon sources and is isolated by poly-cyclic aromatic hydrocarbon contaminated sites. Moreover, its growth on n-alkanes with medium-chain as well as carboxylic acids with different chain lengths has been reported [ 32 – 34 ]. R7, a valuable strain for environmental and industrial biodegradation applications, is capable of catabolizing a diverse array of aliphatic, alicyclic, mono- and polycyclic aromatic hydrocarbons. Notably, R. opacus R7 can utilize PE as its sole carbon source, and molecular analyses have verified the up regulation of LMCOs upon exposure to this substrate [ 19 ]. One of the promising approaches for accelerating the PE biodegradation involves the genetic manipulation of a suitable host to express PE-degrading enzymes [ 35 ]. Heterologous expression is considered as the efficient approach for the production of microbial laccase enzyme within short and coeffective frame. The laccase gene from Psychrobacter sp. NJ228 was successfully expressed in Escherichia coli ( E. coli ) BL21 and the PE-degradation was confirmed [ 28 ]. Moreover, recombinant LMCO2 and LMCO3 related to R. opacus R7 were produced in R. erythropolis AP (CIP 110799), and the oxidative activity related to low-density PE was explored [ 29 ]. Regarding the importance of LMCOs in PE biodegradation, a novel and unstudied LMCO1 from R. opaqus R7 was explored for its discriminative characteristics in PE degradation utilizing machine learning analysis, cloned and expressed in E. coli and subsequently the crude extract enzyme was used for further examinations. The physico – chemical characteristics of expressed enzyme were analyzed and the oxidative function on PE films and particles was assessed. This study provides important new perspectives on the role of LMCO1 in PE biodegradation. 2. Results 2.1. Identification of LMCO1 through bioinformatic analysis The application of the seven attribute weighting algorithms to the FCdb yielded different counts of significant genes identified by each technique: Correlation (3523 genes), Gini Index (2301), PCA (4265), SVM (1061), Uncertainty (2235), Information Gain (2301), and Information Gain Ratio (2301). The weights assigned to each gene by the various attribute weighting algorithms can be found in the Supplementary File, sheet S3. The overlap of these gene sets comprised 500 genes that were selected by all algorithms. These genes are likely to be the most strongly associated with the response to PE exposure. A complete list of these 500 genes is available in Supplementary File, sheet S4. Importantly, among these genes, cds-WP_005251383.1 (LMCO1) was previously identified by Zampolli et al [ 19 ]. As being significantly upregulated during growth on PE. In our analysis, this gene consistently received weights of no less than 0.6 across all seven algorithms, with three algorithms assigning it the highest weight of 1.0 (Information Gain, Information Gain Ratio, and Gini Index). It was also highly rated by Correlation (0.853), PCA (0.819), Uncertainty (0.685), and SVM (0.607), which further emphasizes its significant role. 2.2. R. opacus R7 LMCO expression in E. coli The gene was effectively integrated into the pET22b and pET28a vectors, at the first the successful transfer of plasmid to E. coli confirmed via plasmid extraction followed by agarose gel electrophoresis (Fig. 1 a) Subsequent transformation of E. coli BL21 confirmed the recombinant LMCO1 construct through digestion with EcoR I and Xho I restriction enzymes, and gel electrophoresis (Fig. 1 b). Digestion resulted in the band of 1376 bp, which corresponds to the LMCO1 insert from the pET22b and pET28a plasmids. The expression of LMCO1 in E. coli was verified through SDS-PAGE and Western blot analysis. The soluble 55 kDa recombinant protein was identified in the supernatant of crude extract according to the SDS-PAGE result (Fig. 1 c). Moreover, the LMCO1 expression and the solubility in the pET22b vector were more than pET28a when equal protein amount were analyzed on the gel (Fig. 1 d). Hence the ongoing research was followed with pET 22/ LMCO1. The Western blot analysis confirmed the successful laccase expression, as evidenced by its recognition with the anti-His antibody (Fig. 1 e). Overall, the protein analysis experiments validated the expression of the recombinant laccase within the E. coli expression system. Figure_1 2.3. Enzyme activity evaluation The laccase activity of the soluble fraction was determined at the T opt and pH opt in presence and absence of Cu 2+ . Interestingly, LMCO1 activity with Cu 2+ (1.18 U/g) was higher than without Cu 2+ (0.20 U/g) (Fig. 2 ), confirming that Cu 2+ is required for LMCO1 expression and function. Figure_2 2.4. Evaluating effect of the temperature and pH on the multicopper oxidase enzyme activity Enzyme activity related to LMCO1 was measured at temperatures ranging from 30°C to 70°C (Fig. 3 a). It was found that on the 60 ° C the enzyme showed the highest activity (T opt ). In the case of pH, the enzyme activity was performed within the pH rang of 5–10 and the enzyme showed the highest activity at pH 8 (Fig. 3 b). Figure_3 2.5. Evaluation of polyethylene oxidation by multicopper oxidase After 72 h cultivation films and particles were collected and washed based on protocol mentioned in method and material part. The degradation of PE was confirmed by various indicators and methodologies. The weight loss rates observed in plastic films confirm PE degradation (see Fig. 4 a). Notably, the most significant weight loss rate was for LDPE (14.28%) at the end of cultivation period. On the other hand, HPDE weight loss rate (6.77%) was lower than LDPE. Figure_4 Morphological changes were observed in particles and films (Fig. 4 b, c, d, and e). Prior to treatment, particles and films were suspended on the action system's buffer due to their high hydrophobicity. Figure_5 However, following treatment, the films became submerged, and the particles sank beneath the buffer's surface, leading to agglomeration. The results indicate a decline in WCA during the cultivation process, which signifies the development of hydrophilic groups on the surfaces of the films. The WCA of LD and HD films incubated decreased to 76.73 ± 0.4 °and 60.76 ± 1.7 ° respectively, from 87.31 ± 0.2 °and 63.01 ± 0.9 °, respectively (Fig. 5 ). Figure_6 The FTIR technique was employed to further confirm the degradation of PE and to examine the differences of chemical components in the surface and functional groups. According to the FTIR spectra (Fig. 6 ), all experimental samples, both particles, and films, treated with LMCO, comparing to the control group, displayed a disordered region at ̴ 1650 due to the formation C = O groups by LMCO illustrates the ketones and aldehydes are formed, which is generated by the presence of oxygen atoms in the carbon chain of the polymer [38]. FTIR spectra of films and particles of LDPE showed distinct peaks at 3000–3500, which corresponded to carboxylic acid and alcohol functional groups (R-OH stretching, 3000–3500 cm − 1 ) [ 40 ]. Hydroxyl group formation leads to more hydrophilicity and indicates more efficient degradation. Morphological changes due to degradation on the plastic surface were confirmed through SEM. The control group’s surface, as shown in Fig. 7 , exhibited no visible defects and maintained a smooth and uniform appearance, free from grooves, cracks, or pits. On the other hand, all treated samples showed several structural changes like cracks, deep holes, braking into fragments, and creases. As it could be observed intensity of degradation was more on the particles than films. Besides HDPE films degradation has occurred at the least intensity. Figure_7 3. Discussion The growing concern over plastic waste, mainly PE, has driven the search for sustainable methods to mitigate its environmental impact. Microbial and enzymatic approaches have emerged as promising solutions due to their eco-friendly nature. Among these, LMCOs from the bacterium R. opacus R7 have shown significant potential in PE biodegradation. The expression analysis of R. opacus R7 under PE exposion using RNA-seq and complementary RT-qPCR techniques resulted in identification of multicopper oxidases enzymes upregulation. Among them the nominated LMCO1 shows a 19.5-fold expression increase, highlighting its pivotal role in PE oxidation [ 19 ]. Although LMCO1 was previously recognized as a gene that is upregulated in response to PE by Zampolli et al. [ 19 ], the presented expression analysing approach based on multi-algorithms here was not merely aimed at corroborating earlier results. Rather, our objective was to reassess the existing transcriptomic data through a robust and meticulous multi-algorithm methodology. By utilizing seven distinct attribute weighting algorithms, we successfully demonstrated that LMCO1 consistently ranked as a top gene across all analyses. This finding significantly enhances our confidence that LMCO1 is not simply an artifact of a specific method or dataset, but instead represents a genuine core gene within the PE response network of R. opacus R7. These in silico analysis align with the prior report take insight into deep understanding for selection of the most promising laccase gene in PE degradation for our experimental investigation. Ultimately, this thorough analytical framework not only confirmed the biological significance of LMCO1 but also facilitated the identification of 499 additional genes that may be implicated in PE degradation and cellular adaptation. These newly identified candidates present valuable targets for forthcoming functional research into the mechanisms of plastic biodegradation. After approving the its discriminative association to PE degradation based on in silico analysis, the LMCO1 was expressed in PET/ E. coli system. E. coli is an ideal organism for the production of recombinant proteins, due to its extensively studied genetic makeup and adaptability [ 41 ]. The pET vector system is a widely recognized and highly efficient platform for expressing recombinant proteins in E. coli . Its activity is controlled by the bacteriophage T7 transcription and translation system, where expression is initiated by introducing T7 RNA polymerase into the host cells. Upon full activation of the system, a substantial amount of the cell resources is dedicated to the synthesis of the target protein. In this research, a pET-based expression system, comprising both pET22b and pET28a, was employed to express and compare the recombinant LMCO1 enzyme expression yield. The pET22b plasmid uses an N-terminal PelB signal peptide for enhancing proper folding while the PET28a lacks it. The results demonstrated that the pET22 system yielded higher levels of soluble LMCO1 compared to the pET28 system, attributed to the presence of the PelB secretion signal sequence (Fig. 1 ). Given the successful production of soluble LMCO1 using the pET22 system, this construct was selected for future recombinant expression studies. Our results indicate that Cu²⁺ ions significantly enhance the enzymatic activity of LMCO1 from 0.2 (U/g) to 1.18 (U/g), as reported previously for LMCOs from R. ruber C208 [ 15 ] and R. opacus R7 [ 29 ]. Similarly, Zhang et al. observed a fourfold increase in activity for laccase from Klebsiella pneumoniae Mk-1 upon activation by Cu²⁺ ions [ 38 ]. LMCO1 displayed varying levels of activity depending on changes in pH and temperature. The highest activity was determined at pH of (8), compared to pH opt of LMCO3 (5.5) and 7 for LMCO2; however, no significant differences were noted among pH levels 7, 8, and 9. The observed consistency can be linked to the ABTS oxidation (involving non-phenolic compounds) mainly occurs through inhibition mechanism of hydroxyl radicals. Consequently, no notable increase in activity was detected with rising pH levels [ 28 ]. The enzyme exhibited peak at 60°C, lower than the optimum temperatures reported for LMCO2 (65°C) and LMCO3 (80°C) by Zampoli, et al. [ 29 ]. A thermophilic laccase from Lysinibacillus fusiformis , involved in PE degradation, showed peak activity at 70°C and maintained in an active state [ 42 ]. Laccases are copper-dependent enzymes, the T1 Cu center (active site) positioned at the laccase surface and its direct exposure to catalytic environment promotes the formation of a substrate tunnel, favoring the degradation of bulky substrates such as polymers. Recent study suggests that the PE binding to laccases is primarily governed by the substantial contribution of hydrophobic residues near the T1 site, alongside the wax-like flexibility of PE, which allow it to fit and integrate into the enzyme’s binding pocket. The presence of Met-loop, a Met-rich motif that anchors PE in close proximity to the T1 copper site, also plays key role in enzyme binding to PE and PE oxidation is influenced equally by redox potential and the specific binding interactions between the enzyme and substrate [ 25 ]. The degradation of PE was validated through various methods and indicators. PE oxidation was initially confirmed by measuring the weight loss rate over the incubation period. LDPE exhibited greater weight loss rate than HDPE. Moreover, Zhang et al. (2022) reported 7.6% and 13.2%, weight loss rate for PE particles with both crude laccase extract and purified laccase derived from the psychrophilic bacterium Psychrobacte r sp. NJ228 without any pretreatment under UV irradiation, thermal oxidation and chemical oxidation. Before treatment, particles and films floated on the buffer system due to their strong hydrophobic nature. After treatment, the films and particles became submerged, and particles agglomerated due to the generation of hydrophilic groups during PE oxidation. Similar observations for PE particles were also reported by [ 28 ]. FTIR spectroscopy was used to monitor the functional groups formation in treated samples. Oxidation, which serves as the rate-limiting factor in the degradation of PE, resulted in the cleavage of carbon-hydrogen bonds, modifications in the chemical structure, and an increase in polarity. FTIR spectra consistently showed a disordered region near ~ 1655 cm -¹, which is associated with the generating of C = O functional groups. The presence of a carbonyl group (1600–1850 cm − 1) serves as a significant indicator of PE biodegradation [ 43 ] and provides clear evidence of chemical structural alterations at the microscale [ 28 ]. LDPE that was treated with MR5 and MR10 exhibited peaks at 1654.14 cm − 1 and 1683.90 cm − 1 respectively [ 44 ]. The findings align with the research conducted by Skariyachan et al. (2018) [ 45 ], which observed a peak at 1638.97 cm − 1 , evidence for the presence of C = O carbonyl (ketone or aldehyde) groups on plastic strips that were treated with bacterial consortia. Additionally, the FTIR spectra of all particles, as well as LDPE films, exhibited a prominent peak in the range of 3000 to 3500 cm − 1 . This peak is associated with the presence of carboxylic acid and alcohol functional groups, specifically the R-OH stretching [ 40 ], which leads to more hydrophilicity and indicates more efficient degradation. Oxygen- containing polar functional groups or exhibiting vigorous vibrational intensity can weaken the stability of carbon bonds and lower molecular weight, creating additional degradation sites for key enzymes [ 46 ]. Similarly, after PE incubation with Aspergillus flavus PEDX3 and simultaneous incubation with Acinetobacter sp. NyZ450 and Bacillus sp. the FTIR spectra of PE incubated with NyZ451 revealed a new peak generation associated with the hydroxyl group, observed in the range of 3500–3100 cm − 1 [ 9 , 47 ]. Numerous studies have demonstrated that the use of laccase on PE results in the formation of carbonyl and carboxyl groups (polar functional group), on the surface of PE, as identified through FTIR analysis. These findings are consistent with the results observed in our study [ 28 , 42 , 48 ]. Changes in the WCA values (Fig. 5 ) further supported the presence of hydrophilic groups on the film surfaces, generated through hydrocarbon chain oxidation by laccase [ 49 ]. Changes in the WCA values of the films indicate that their polarities were altered due to biodegradation and the formation of hydrophilic groups. The greater WCA change was observed in the LDPE sample. However, it was higher than the changes observed in LDPE films fermented with crude laccase enzyme extract [ 28 , 38 ]. Degradation caused morphological changes on the plastic surface, observed through SEM (Fig. 7 ). The control samples exhibited smooth and uniform surfaces devoid of any cracks or pits, consistent with the findings from the FTIR analysis. In contrast, treated samples exhibited significant structural changes such as cracks, deep holes, fragmentation, and creases. Recently, comparable destruction and damage have been observed in PE films that have undergone treatment with laccase and LMCO [ 18 , 28 , 42 , 50 ]. Other studies have also reported similar features such as wrinkles, erosion, cracks, and holes during the degradation process of LDPE, and HDPE films [ 16 , 49 , 51 ]. In conclusion, the degradation was more pronounced in particles than in films, likely due to the greater accessibility and increased contact area with LMCO1 [ 52 ]. Furthermore, the degradation of LDPE film was more effective compared to HDPE. The difference can be attributed to construction of LDPE, which allow it to adapt and fit into the enzyme’s binding pocket, forming a stable enzyme-PE complex that potentially facilitates electron transfer to the T1 site and oxidation [ 25 ]. LDPE more degradation result forming hydroxyl and carbonyl groups, which enhance hydrophilicity and significantly alter the water contact angle (WCA), additionally greater degradation result in more morphological changes and weight loss reduction. In comparison, the increased crystallinity of HDPE, along with a lower presence of tertiary carbons and a higher molecular weight, restricts the access of oxidizing enzymes to its polymer chains, resulting in enhanced resistance to degradation [ 53 ]. 4. Materials and methods 4.1. Bioinformatics analysis In summary, the available RNA-seq data related to R. opacus R7 cultured with PE as sole carbon source was utilized to explore the discriminative genes under PE exposure as follows. 4.2. Data processing We used the available RNA-seq data from the Sequence Read Archive (SRA), accession number PRJEB45685. The detailed experiment regarding the growth, PE supplementation and RNA extraction is reported by [ 19 ]. Each experimental condition was replicated three times, resulting in six samples: three derived from the PE conditions and three from the malate conditions. The preliminary assessment of the raw sequencing data quality was conducted utilizing FastQC. Subsequently, the sequencing reads underwent trimming via Trimmomatic on the Galaxy platform ( https://usegalaxy.eu/ ). The ILLUMINACLIP step included TruSeq3 adapter sequences for paired-end reads, allowing for a maximum of 2 mismatches. A palindrome and simple clip threshold set at 30 and 10 respectively. Both reads were maintained in paired-end mode. Following the preprocessing stage, FastQC was executed once more to verify the quality of the trimmed data, which was found to be adequate for subsequent analysis. We used the reference genome related to R. opacus R7 from Ensembl Bacteria in FASTA and GFF format. The quality confirmed reads, were aligned with the reference genome by HISAT2. 4.3 Expression Quantification via htseq-count and Normalization In this research, htseq-count was employed to count reads. The input files consisted of aligned BAM files generated by HISAT2. The parameters for htseq-count were configured as follows: the mode for addressing reads that overlap multiple features was designated as "union," the data was classified as non-stranded, the minimum alignment quality threshold was established at 10, the feature type was identified as "CDS," the ID attribute was defined as "ID," and reads that were non-unique or ambiguously mapped were excluded. The data were normalized based on DESeq2 (Supplementary File, sheet S1). 4.4. Data Collection and Preprocessing Gene count files from six samples served as the input for RapidMiner Studio software (RapidMiner 7.0.001 GmbH). It counts for 8,270 genes across the samples. 4.5. Data Cleaning To improve processing performance, we employed the "Remove Useless Attributes" operator within RapidMiner. This operator effectively eliminated genes exhibiting a standard deviation (SD) of less than 0.1; as such, genes demonstrated negligible variation in counts across the samples. Following the application of this filter, the total number of genes was decreased to 6409. The dataset from this process was designated as the Final Cleaned Database (FCdb) (Supplementary File, sheet S2). 4.6. Attribute Weighting Algorithms Seven attribute weighting algorithms were applied to the FCdb to identify significant genes that distinguish R. opacus R7 cultivated on PE from those cultivated on malate. The implementation of multiple algorithms aimed to mitigate biases associated with any single method and to enhance confidence in the identified genes. An overview of the seven attribute weighting techniques, as utilized in RapidMiner Studio version 7.6 (RapidMiner 7.0.001 GmbH), is presented below: Information gain weights Allocated based on the decrease in uncertainty (entropy) in the target class following the observation of the attribute value. A higher weight signifies that the attribute conveys more information and is thus more pertinent for classification. Uncertainty A normalized variant of information gain that considers both the entropy of the class and the attribute. It diminishes bias towards attributes with numerous distinct values. Attribute weights are computed based on the symmetrical uncertainty concerning the target variable, where a higher weight indicates greater significance. Principal Component Analysis (PCA) The weight of each attribute is determined by the absolute value of its loading on the principal component that accounts for the maximum variance (typically the first component). These weights are generally normalized to fall between 0 and 1, reflecting the relative importance of the attributes. The Gini Index assesses the impurity of a dataset segmented by attribute values. Attributes that more effectively separate the classes yield lower impurity and are assigned higher weights. Consequently, the attribute weight corresponds to its capacity to diminish class impurity. Information Gain Ratio This normalization lessens the bias towards attributes with a high number of distinct values. A higher Information Gain Ratio indicates a more important attribute. Support Vector Machine (SVM) In linear SVM models, the weights of attributes are determined by the absolute values of the coefficients found in the model's weight vector. Attributes that possess larger absolute coefficients exert a more significant influence on the decision boundary and, as a result, are assigned higher weights. Correlation This metric computes the absolute value of the pearson correlation coefficient for each gene’s normalized expression in relation to the target variable, which signifies the growth condition (PE or malate). The subject feature, which is classified as either related to PE or malate, served as the target. Moreover, the normalized values related to expression of the genes were labelled as attributes and classified continuedly. All scores were standardized to a range between 0 and 1 to ensure comparability. Genes that exhibited a normalized weight exceeding 0.6 in any algorithm were deemed significant. The common genes identified by all seven algorithms were recognized as essential genes for subsequent analysis. 4.7. Cloning and Expression of recombinant multicopper oxidase The R. opacus R7 LMCO1 (AII08809) gene with 1376bp length, encodes a protein of 458 amino acids. The gene without inner signal peptide was optimized and synthesized by GenScript, then cloned into the vectors pET28a (+) and pET22b at the NcoI and XhoI restriction sites. Following this, the E. coli strains DH5α and BL21 (DE3) transformed by the plasmids using electroporation to enable propagation and expression, respectively. The colonies were grown on solid LB medium with kanamycin (50 mg/l) for pET28a (+) and ampicillin (100 mg/l) for pET22b, which was subsequently used for starter culture in 5 ml liquid at 37°C, for 16-hours. A positive colony was then grown overnight at 37°C in 5 mL of fresh LB medium with the appropriate antibiotics on a rotary shaker set at 180 rpm. Then, starting culture (1mL) was inoculated into 50 ml LB medium contained antibiotics, and incubated until optical density at 600 nm was 0.6. When reached to optical density 0.6 at 600 nm (OD600), the recombinant expression was induced by isopropyl β-D-thiogalactoside, 1 mM and CuSO 4 250 µM, both at final concentrations. Following this, the cultures maintained for 20 hours at 16°C. Control samples consisted of E. coli BL21/pET-22 (+) and pET28 cells that did not contain the LMCO1 enzyme, which were treated concurrently [ 36 ]. The cell cultures were centrifuged and harvested at 8000 × g for 15 minutes, and then resuspend in the potassium phosphate buffer (PPB, 50 mM, pH 7). The cells were treated with Lysozyme (1 mg/ml) and incubated on ice for 30 minutes. Subsequently, a serine protease inhibitor, PMSF (phenylmethylsulfonyl fluoride) 1 mM, was mixed with the cells. The resulting suspension underwent sonication with a repeated duty cycle of 30-second pulses for 5 minutes while placed on dry ice. Finally, the suspension was centrifuged at 12,000g for 25 minutes to isolate a clear supernatant containing the recombinant LMCO1 protein [ 37 ]. The centrifuged crud enzyme solution containing soluble recombinant LMCO1 was stocked for future usage [ 38 ]. 4.8. Assessment of recombinant proteins through Sodium dodecyl-sulfate polyacrylamide gel electrophoresisand Western blot analysis The expression of recombinant proteins was verified through SDS-PAGE and Western blotting. The proteins were transferred from a 12% polyacrylamide gel to a nitrocellulose membrane. The membrane was then blocked with skim milk overnight, and incubated with an anti-His antibody. Following the washing process with PBS buffer, the staining was done using 3,3′-Diaminobenzidine (DAB) chromogen and treated with hydrogen peroxide for visualising the recombinant proteins. The His-tagged recombinant proteins was indicated by the appearance of brown bands, confirming their identity [ 39 ]. 4.9. Measurement of laccase activity assay The activity of the laccase enzyme in soluble friction was measured by specific substrate regarding the reference [ 38 ]. In summary, the activity was evaluated by measuring the amount of protein necessary to oxidize six mM ABTS (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) under assay condition 200 µM CuSO 4 within the activity buffer, which consisted of 50 mM citric acid-Na2HPO 4 at optimal pH and temperature. The reaction was monitored for 2 minutes, tracking the absorbance increasing at 420 nm (ԑ 420 = 36,000 mM − 1 cm − 1 ). One enzyme unit activity indicates the quantity of enzyme able to oxidize 1 µmol of ABTS per minute in a quartz cuvette with optical path length of 1.0 cm. 4.10. Temperature and pH effects on the multicopper oxidase activity The laccase activity of the soluble protein fraction was measured at different temperatures and various pH ranges to determine the optimal reaction condition. The ideal pH (pH opt) was established for ABTS at a final concentration of 6 mM in PPB. The ABTS oxidation was measured at pH levels of 5, 6, 7, 8, 9, and 10 under the previously described conditions to determine enzyme activity. For identifying the optimal temperature (T opt ), the ABTS oxidation was performed from 30° C to 70° C temperature range [ 29 ]. 4.11. Determination of polyethylene degradation Degradation was evaluated using LDPE (low density PE, Basparan, IRAN, density: 0.9190 gr/ml, code: LF-0450), and HDPE (high density PE, Basparan, IRAN, Density: 0.955 gr/ml, code: 7000F) film (1cm × 1cm) and particle (particle size of 1000 µm) as substrate (1% w/v). Prior to using, films were soaked on 2% SDS (1h) and rinsed with distillated water, disposed with 75% ethanol (1h) and 90% ethanol (1h), and dried (40 ° C, 30 min) [ 30 ]. The PE samples were incubated in LB medium (pH 7.0) for 72 hours to verify their asepsis. A biodegradation system utilizing a crude enzyme solution (with a final concentration of 0.9 mg mL-1 from daily additions) was conducted in 10 mL glass vessels at a temperature of 60 ◦C, under shaking at 120 rpm, within a reaction buffer consisting of 50 mM PPB and 50 µM CuSO 4 , maintaining a total volume of 3 mL at optimal pH for up to 72 hours. Besides, control including PE and the reaction mixture lacking the enzyme was used. The particles collected through repeated filtration subsequently washed with ethanol and water and then dried at 40 ◦C. The films were washed regarding the previously established protocol [ 29 ]. Films weight loss rates were measured as follows [ 28 ]: Weight loss rate (%) = (W 0 - W t ) / W 0 × 100 W 0 : weights of the films before degradation W t : weights of the films after degradation The degradation ability was assessed at 0, 48, and 72 hours through ATR-FTIR spectroscopy, SEM analysis, and hydrophobicity alterations analysis on the film surfaces. FTIR (Bruker Tensor II, Germany) was employed to capture data in the 400–4000 cm -1 wavelength range, which is utilized to characterize the micro-scale chemical structure of the surface. Following gold sputtering, the micro-scale morphology was examined using SEM (TESCAN Vega3, Kohoutovice, Czech Republic). Changes in hydrophobicity on the film surfaces were calculated by measuring the water contact angle (WCA) with a contact angle measuring device (CAG-10, I.R. IRAN). Declarations Acknowledgements The authors would like to acknowledge Department of Plant Protection and the central Lab of Shiraz University for providing access to their facilities and technical support during the experiment. We also gratefully thank the guidance and assistance of the staff in operating the equipment. Author contributions Mahboobeh Pishan: Investigation, Writing – original draft, Visualization, Methodology, Formal analysis. Sima Sazegari: Conceptualization, Methodology, Writing – original draft, Data curation. Ali Niazi: Conceptualization, review & editing. Marjan Majdinasab: Methodology, review & editing. Zahra Zinati: Software, Formal analysis, review & editing. Mohammad Hadi Eskandari: Conceptualization, Methodology, review & editing, Supervision, Data curation. 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Montazer, Z., Habibi Najafi, M. B. & Levin, D. B. Challenges with verifying microbial degradation of polyethylene. Polymers 12 (1), 123 (2020). Additional Declarations No competing interests reported. Supplementary Files supplementary.xlsx Supplementary File: Identification of R. opacus R7 significant genes involved in PE biodegradation utilizing seven distinct attribute weighting algorithms. Sheet S1) Data obtaind from htseq-count. Sheet S2) Final Cleaned Database used for attribute weighting algorithms. S3) Weights assigned to each gene by the various attribute weighting algorithms. S4) The list of these 500 genes identified by seven algorithms. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 12 Mar, 2026 Reviews received at journal 06 Mar, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers invited by journal 10 Feb, 2026 Editor assigned by journal 26 Jan, 2026 Submission checks completed at journal 26 Jan, 2026 First submitted to journal 24 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8685015","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":590241816,"identity":"a3bea6f4-4fef-4bd1-890b-550a3df950fa","order_by":0,"name":"Mahboobeh Pishan","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Mahboobeh","middleName":"","lastName":"Pishan","suffix":""},{"id":590241817,"identity":"1339d75e-0b52-4add-861d-5023c6f18412","order_by":1,"name":"Sima Sazegari","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Sima","middleName":"","lastName":"Sazegari","suffix":""},{"id":590241823,"identity":"a0995b02-1baf-4a54-ba55-459d563d92ad","order_by":2,"name":"Ali Niazi","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Niazi","suffix":""},{"id":590241824,"identity":"647bcbe0-b2c2-4557-ac1c-398348fd8cad","order_by":3,"name":"Marjan Majdinasab","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Marjan","middleName":"","lastName":"Majdinasab","suffix":""},{"id":590241825,"identity":"916b03b1-c3c0-4fca-a41f-87613ac21057","order_by":4,"name":"Zahra Zinati","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"","lastName":"Zinati","suffix":""},{"id":590241826,"identity":"1719bca7-6e2d-4625-8028-358e4c8acc67","order_by":5,"name":"Mohammad Hadi Eskandari","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYFACNgjmZ+BhOAAXTCBGi2QDyVoMDvAQ6Sx+/mOJnwvK7PKNz589eLiipi6xgf3wA4aHe3BrkZyRdlh6xrlky2038hIOnjnGltjAk2bAkPAMtxaDG+wN0rxtzAZmN3gMDjaw8SQ2MOQA/XIAtxb788ebf/O21RsY958BavknkdjA/wa/FgOGtGNAWw4bGDDkGBxsbDNIbJAgYIvEjbQ0a55zxw0kboC09CUYt0k8MziATwt//zHj2zxl1Qb8/WeMPzZ8q5Pt509++PAHHi2YABRPJGkYBaNgFIyCUYAJACIiUH4os0FuAAAAAElFTkSuQmCC","orcid":"","institution":"Shiraz University","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"Hadi","lastName":"Eskandari","suffix":""}],"badges":[],"createdAt":"2026-01-24 08:26:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8685015/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8685015/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102748442,"identity":"1e1d8ae8-ac7c-4c50-93e1-de26e66dcab7","added_by":"auto","created_at":"2026-02-16 09:10:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":504191,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular analysis related to cloning and expression of LMCO1 enzyme in \u003cem\u003eE. coli\u003c/em\u003e. \u003cstrong\u003ea)\u003c/strong\u003e agarose gel electrophoresis of undigested recombinant pET22 (b) plasmid and undigested pET 28 (a) plasmid after extraction. M) ladder 1000 bp, 1) pET22b extracted plasmid, 2) pET 28a extracted plasmid \u003cstrong\u003eb)\u003c/strong\u003e Recombinant pET22 (b) and recombinant pET28 (a) agarose gel electrophoresis of plasmids after digestion by EcoRI and XhoI enzymes. M) DNA ladder 1000 bp, 1) undigested recombinant pET22 (b), 2) tow steps digested recombinant pET 22 (b) 3) one \u0026nbsp;step digested pET 22 (b), 4) undigested recombinant pET 28 (a), 5) tow steps digested recombinant pET 28 (a) 6) one step digested recombinant pET 28 (a) \u003cstrong\u003ec) \u003c/strong\u003eSDS-PAGE analysis of the expression of LMCO1 in \u003cem\u003eE. coli\u003c/em\u003e. M) protein marker, 1) induced cells expressing LMCO1 on size 55 kDa, 2) non-induced cell \u003cstrong\u003ed) \u003c/strong\u003eSDS-PAGE analysis of the solubility of LMCO in supernatant M) protein marker, 1) induced cells expressing LMCO1 by pET 22 (b), 2) supernatant of induced cells expressing LMCO1 by pET 22, 3) induced cells expressing LMCO1 by pET 28 (a), 4) supernatant of induced cells expressing LMCO1 by pET 28 (a) \u003cstrong\u003ee) \u003c/strong\u003eWestern Blot analysis of LMCO1 enzyme M) protein marker, 1) induced cells expressing LMCO1 by pET 28 (a), 2) induced cells expressing LMCO1 by pET 22 (b)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/efc3e39904ac34a833151b14.png"},{"id":102600960,"identity":"22addbac-c3de-467a-b081-2e321356dba9","added_by":"auto","created_at":"2026-02-13 12:57:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24084,"visible":true,"origin":"","legend":"\u003cp\u003eenzymatic activity of recombinant LMCO1 in crude extract. Oxidase activity was measured using 6 mM ABTS. The laccase activity was compared in condition with (200 μM) or without CuSO\u003csub\u003e4\u003c/sub\u003e. Laccase activity, U g\u003csup\u003e−1\u003c/sup\u003e, is presented as the mean of three replicates ± standard deviation. Statistical differences were calculated using t-Student’s test: * p-value \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/cbd69a59af365adb2dde392e.png"},{"id":102600962,"identity":"923211f7-12d0-4309-9f31-f175cee115b9","added_by":"auto","created_at":"2026-02-13 12:57:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52316,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterizations of the LMCO1 R7\u003cstrong\u003e. a\u003c/strong\u003e) Temperature effect, the optimal temperature for the LMCO was determined by assaying the activity at pH7.0 and temperatures range (30\u003csup\u003e◦\u003c/sup\u003eC to 70\u003csup\u003e◦\u003c/sup\u003eC), \u003cstrong\u003eb) \u003c/strong\u003epH effect on the activity of the LMCO1. The activity of the LMCO1 was measured at pHs ranging from 5 to 10 at the optimal temperatures determined in the previous experiment to determined optimal pH.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/77b2f0d205c0c48e7e9a0908.png"},{"id":102600963,"identity":"87afbd7b-f3b3-4def-b04b-04fe4114d02a","added_by":"auto","created_at":"2026-02-13 12:57:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":262289,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of plastics degradation in weight. \u003cstrong\u003ea\u003c/strong\u003e) Relative weight after treating with LMCO1, relative weight is expressed as the mean of three replicates ± standard deviation. Statistical differences were calculated using t-Student’s test: * p-value \u0026lt; 0.05, \u003cstrong\u003eb\u003c/strong\u003e), \u003cstrong\u003ec\u003c/strong\u003e) image of films before and after treating with LMCO1 \u003cstrong\u003ed\u003c/strong\u003e), \u003cstrong\u003ee\u003c/strong\u003e) image of particles after and before treating with LMCO1\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/e64e9bb4349591d9099ef11c.png"},{"id":102747432,"identity":"94133047-a859-4b2a-8ef4-90e6372ac66e","added_by":"auto","created_at":"2026-02-16 09:04:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105429,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of PE degradation in hydrophobicity, \u003cstrong\u003ea\u003c/strong\u003e) WCA of LD film untreated, \u003cstrong\u003eb\u003c/strong\u003e) WCA of HD film untreated, \u003cstrong\u003ec\u003c/strong\u003e) WCA of LD films treated by LMCO1, \u003cstrong\u003ed\u003c/strong\u003e) WCA of HD films treated by LMCO1\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/c4f61a547668b0ae13229983.png"},{"id":102748447,"identity":"910076ba-cd27-4a1c-9c34-affbb8ea1cea","added_by":"auto","created_at":"2026-02-16 09:10:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":179913,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical biodegradation of PE degradation confirmed by FTIR. \u003cstrong\u003ea\u003c/strong\u003e) FTIR spectra of LD particle without treating \u003cstrong\u003eb\u003c/strong\u003e) FTIR spectra of LD particle treating with LMCO1 \u003cstrong\u003ec\u003c/strong\u003e) FTIR spectra of HD particle without treating \u003cstrong\u003ed\u003c/strong\u003e) FTIR spectra of HD particle treating with LMCO1 \u003cstrong\u003ee\u003c/strong\u003e) FTIR spectra of LD film without treating \u003cstrong\u003ef\u003c/strong\u003e) FTIR spectra of LD film treating with LMCO1 \u003cstrong\u003eg\u003c/strong\u003e) FTIR spectra of HD film without treating \u003cstrong\u003eh\u003c/strong\u003e) FTIR spectra of HD film treating with LMCO1\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/016f2f4feeabe085851d911f.png"},{"id":102747380,"identity":"b3d598d8-13d8-4cf3-9c70-9270eec98e90","added_by":"auto","created_at":"2026-02-16 09:04:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":669437,"visible":true,"origin":"","legend":"\u003cp\u003eThe morphological biodegradation of PE degradation confirmed by SEM \u003cstrong\u003ea\u003c/strong\u003e) image of LD particle control \u003cstrong\u003eb\u003c/strong\u003e) image of HD particle control \u003cstrong\u003ec\u003c/strong\u003e) image of LD particle treated with LMCO \u003cstrong\u003ed\u003c/strong\u003e) image of HD particle treated with LMCO1 \u003cstrong\u003ee\u003c/strong\u003e) image of LD film control \u003cstrong\u003ef\u003c/strong\u003e) image of HD film control \u003cstrong\u003eg\u003c/strong\u003e) image of LD film treated with LMCO1 \u003cstrong\u003eh\u003c/strong\u003e) image of HD film treated with LMCO1\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/29292502c09e4ad0171e379c.png"},{"id":103503914,"identity":"d995d649-3a4f-414b-b461-c646d4533587","added_by":"auto","created_at":"2026-02-26 13:04:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2969773,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/b9ab6461-e175-427f-8672-4ef605be6353.pdf"},{"id":102600965,"identity":"10f64338-3f1e-4008-9fbd-8748eed46fca","added_by":"auto","created_at":"2026-02-13 12:57:14","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1250182,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary File:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdentification of R. opacus R7 significant genes involved in PE biodegradation utilizing seven distinct attribute weighting algorithms. Sheet S1) Data obtaind from htseq-count. Sheet S2) Final Cleaned Database used for attribute weighting algorithms. S3) Weights assigned to each gene by the various attribute weighting algorithms. S4) The list of these 500 genes identified by seven algorithms.\u003c/p\u003e","description":"","filename":"supplementary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8685015/v1/d83808cede1539199636305c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Heterologous expression and characterization of Rhodococcus opacus R7 laccase-like multicopper oxidase (LMCO1) enzyme for polyethylene biodegradation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePolyethylene (PE) ranks among the most widely produced and used polyolefin derived from petroleum-based plastics, due to its exceptional durability, cost-effectiveness in production, and versatility [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is produced in millions of tons annually and used in many different areas and industries [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PE molecule is generally structured by two carbon atoms linked to two hydrogen per each (\u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e\u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e-) while the chain is terminated by methyl group (R\u0026ndash;CH\u003csub\u003e3\u003c/sub\u003e). PE exhibits a notable degree of hydrophobicity, in addition to properties including density, crystallinity, molecular weight, and a reduced specific surface area. Furthermore, features including linear structure made up of carbon atoms in the form of C\u0026ndash;C and C\u0026ndash;H links, lacking hydrolysable functional groups in the backbone, along with its high molecular weight, make it recalcitrant to biodegradation [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Different fates including environmental accumulation, abiotic oxidation, recycling and biodegradation by microorganisms and their associated enzymes are proposed for PE polymer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePolymers, such as PE, may experience degradation via several mechanisms, including chemical processes (such as oxidation, hydrolysis, or catalytically mediated reactions), thermally induced processes (which involve exposure to elevated temperatures), mechanical processes (such as milling or trituration), and physical radiation such as ultrasonic, microwave and sunlight [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Such degradations may encounter various challenges, such as significant energy consumption, the necessity for high temperatures and pressures condition, and the utilization of additives and solvents. Microbial degradation appears to be the most appealing and environmentally sustainable approach to address the increasing issue of plastic pollution and minimize waste [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Many studies have identified more than 20 bacterial genera capable of degrading PE, including the genera, \u003cem\u003eRalstonia, Stenotrophomonas, Acinetobacter, Klebsiella, Rhodococcus, Pseudomonas, Streptococcus, Streptomyces, Staphylococcus, Bacillus\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Numerous studies have indicated that enzyme-mediated oxidation of PE occurs through direct catalysis by enzyme within the extracellular supernatant of bacteria that degrade PE; however, the intricate process of PE biodegradation remains unclear [\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The biodegradation of plastic by microorganism primarily involves an enzymatic mechanism that cleaves the polymer bonds into their monomers. Enzymes that can effectively sever carbon-carbon bonds in synthetic polymers are in high demand, as they hold the potential to offer solutions for sustainable plastic recycling [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Substantial enzymes including laccases, oxidases and lipases from \u003cem\u003eBacillus cereus\u003c/em\u003e, \u003cem\u003eBervibacilluse borstelensis\u003c/em\u003e and a marine bacterial community have been approved to have PE degradation potential based on transcriptome studies, quantitative 16S rRNA sequencing and cultivation method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Among them, laccases (EC 1.10.3.2), a member of diverse protein family of multicopper oxidases (MCOs), can facilitate the oxidative degradation of a range of phenolic derivatives and biopolymers. Notably, these enzymes have attracted significant research interest for their key role in the depolymerization of PE. Additionally, Laccases-like Multi Copper Oxidases (LMCO) are classified as a non-plant group of 3-domain laccases and classified according to their cupredoxin-like domains they possess [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Some possesses two domains and shows trimeric quaternary structure while some other laccases with monomeric structure have three unique domains [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The active site of laccase consist of four copper atoms: a type Ι copper center and a trinuclar copper cluster, which is composed of a type ΙΙ copper and a pair of type ΙΙΙ coppers. T1 site is responsible for substrate oxidation, while the T2/T3 centers mange the reduction of O\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], substrates oxidation by laccase occurs through outer-sphere electron transferring to a copper T1 site, followed by intermolecular electron transfer to a tri-nuclear copper site, where oxygen molecule is reduced to water [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Limited number of laccases have been identified for their PE degradation capacity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], this includes laccase derived from fungi and bacteria such as \u003cem\u003eTrichoderma harzianum, Tinea versicolor\u003c/em\u003e, \u003cem\u003eBotrytis aclada\u003c/em\u003e, and \u003cem\u003eBacillus subtilis\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Although pretreatment is typically required in case of Laccase PE biodegradation, \u003cem\u003ePsychrobacter\u003c/em\u003e sp. NJ228 and \u003cem\u003eRhodococcus opacus\u003c/em\u003e have been reported to partially degrade PE plastics without the need for pretreatment or mediators [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The ability of crude laccase for PE biodegradation from \u003cem\u003eTrichoderma harzianum\u003c/em\u003e was confirmed though weight loss rate and FTIR analysis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The members of the \u003cem\u003eRhodococcus\u003c/em\u003e genus exhibit an exceptional capacity to break down a diverse array of both natural organic and xenobiotic substances [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], they are the most interesting due to their remarkable metabolic versatility and the significant potential for degradation of plastics [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Extracellular copper-binding enzyme, laccase, was extracted from \u003cem\u003eRhodococcus ruber\u003c/em\u003e C208 and used for treating the PE films. Molecular weight and the FTIR spectra results confirm the enzymatic biodegradation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eR. opacus\u003c/em\u003e R7 (CIP107348), is known to apply naphthalene and o-xylene sole carbon sources and is isolated by poly-cyclic aromatic hydrocarbon contaminated sites. Moreover, its growth on n-alkanes with medium-chain as well as carboxylic acids with different chain lengths has been reported [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. R7, a valuable strain for environmental and industrial biodegradation applications, is capable of catabolizing a diverse array of aliphatic, alicyclic, mono- and polycyclic aromatic hydrocarbons. Notably, \u003cem\u003eR. opacus\u003c/em\u003e R7 can utilize PE as its sole carbon source, and molecular analyses have verified the up regulation of LMCOs upon exposure to this substrate [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the promising approaches for accelerating the PE biodegradation involves the genetic manipulation of a suitable host to express PE-degrading enzymes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Heterologous expression is considered as the efficient approach for the production of microbial laccase enzyme within short and coeffective frame. The laccase gene from \u003cem\u003ePsychrobacter\u003c/em\u003e sp. NJ228 was successfully expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) BL21 and the PE-degradation was confirmed [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Moreover, recombinant LMCO2 and LMCO3 related to \u003cem\u003eR. opacus\u003c/em\u003e R7 were produced in \u003cem\u003eR. erythropolis\u003c/em\u003e AP (CIP 110799), and the oxidative activity related to low-density PE was explored [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the importance of LMCOs in PE biodegradation, a novel and unstudied LMCO1 from \u003cem\u003eR. opaqus\u003c/em\u003e R7 was explored for its discriminative characteristics in PE degradation utilizing machine learning analysis, cloned and expressed in \u003cem\u003eE. coli\u003c/em\u003e and subsequently the crude extract enzyme was used for further examinations. The physico \u0026ndash; chemical characteristics of expressed enzyme were analyzed and the oxidative function on PE films and particles was assessed. This study provides important new perspectives on the role of LMCO1 in PE biodegradation.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Identification of LMCO1 through bioinformatic analysis\u003c/h2\u003e\n \u003cp\u003eThe application of the seven attribute weighting algorithms to the FCdb yielded different counts of significant genes identified by each technique: Correlation (3523 genes), Gini Index (2301), PCA (4265), SVM (1061), Uncertainty (2235), Information Gain (2301), and Information Gain Ratio (2301). The weights assigned to each gene by the various attribute weighting algorithms can be found in the Supplementary File, sheet S3. The overlap of these gene sets comprised 500 genes that were selected by all algorithms. These genes are likely to be the most strongly associated with the response to PE exposure. A complete list of these 500 genes is available in Supplementary File, sheet S4. Importantly, among these genes, cds-WP_005251383.1 (LMCO1) was previously identified by Zampolli et al [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. As being significantly upregulated during growth on PE. In our analysis, this gene consistently received weights of no less than 0.6 across all seven algorithms, with three algorithms assigning it the highest weight of 1.0 (Information Gain, Information Gain Ratio, and Gini Index). It was also highly rated by Correlation (0.853), PCA (0.819), Uncertainty (0.685), and SVM (0.607), which further emphasizes its significant role.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. \u003cem\u003eR. opacus\u003c/em\u003e R7 LMCO expression in \u003cem\u003eE. coli\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe gene was effectively integrated into the pET22b and pET28a vectors, at the first the successful transfer of plasmid to \u003cem\u003eE. coli\u003c/em\u003e confirmed via plasmid extraction followed by agarose gel electrophoresis (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea) Subsequent transformation of \u003cem\u003eE. coli\u003c/em\u003e BL21 confirmed the recombinant LMCO1 construct through digestion with \u003cem\u003eEcoR\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI restriction enzymes, and gel electrophoresis (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Digestion resulted in the band of 1376 bp, which corresponds to the LMCO1 insert from the pET22b and pET28a plasmids.\u003c/p\u003e\n \u003cp\u003eThe expression of LMCO1 in \u003cem\u003eE. coli\u003c/em\u003e was verified through SDS-PAGE and Western blot analysis. The soluble 55 kDa recombinant protein was identified in the supernatant of crude extract according to the SDS-PAGE result (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Moreover, the LMCO1 expression and the solubility in the pET22b vector were more than pET28a when equal protein amount were analyzed on the gel (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). Hence the ongoing research was followed with pET 22/ LMCO1. The Western blot analysis confirmed the successful laccase expression, as evidenced by its recognition with the anti-His antibody (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee). Overall, the protein analysis experiments validated the expression of the recombinant laccase within the \u003cem\u003eE. coli\u003c/em\u003e expression system.\u003c/p\u003e\n \u003cp\u003eFigure_1\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Enzyme activity evaluation\u003c/h2\u003e\n \u003cp\u003eThe laccase activity of the soluble fraction was determined at the T\u003csub\u003eopt\u003c/sub\u003e and pH\u003csub\u003eopt\u003c/sub\u003e in presence and absence of Cu\u003csup\u003e2+\u003c/sup\u003e. Interestingly, LMCO1 activity with Cu\u003csup\u003e2+\u003c/sup\u003e (1.18 U/g) was higher than without Cu\u003csup\u003e2+\u003c/sup\u003e (0.20 U/g) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), confirming that Cu\u003csup\u003e2+\u003c/sup\u003e is required for LMCO1 expression and function.\u003c/p\u003e\n \u003cp\u003eFigure_2\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Evaluating effect of the temperature and pH on the multicopper oxidase enzyme activity\u003c/h2\u003e\n \u003cp\u003eEnzyme activity related to LMCO1 was measured at temperatures ranging from 30\u0026deg;C to 70\u0026deg;C (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). It was found that on the 60 \u0026deg; C the enzyme showed the highest activity (T\u003csub\u003eopt\u003c/sub\u003e). In the case of pH, the enzyme activity was performed within the pH rang of 5\u0026ndash;10 and the enzyme showed the highest activity at pH 8 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eFigure_3\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Evaluation of polyethylene oxidation by multicopper oxidase\u003c/h2\u003e\n \u003cp\u003eAfter 72 h cultivation films and particles were collected and washed based on protocol mentioned in method and material part. The degradation of PE was confirmed by various indicators and methodologies. The weight loss rates observed in plastic films confirm PE degradation (see Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Notably, the most significant weight loss rate was for LDPE (14.28%) at the end of cultivation period. On the other hand, HPDE weight loss rate (6.77%) was lower than LDPE.\u003c/p\u003e\n \u003cp\u003eFigure_4\u003c/p\u003e\n \u003cp\u003eMorphological changes were observed in particles and films (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, c, d, and e). Prior to treatment, particles and films were suspended on the action system\u0026apos;s buffer due to their high hydrophobicity.\u003c/p\u003e\n \u003cp\u003eFigure_5\u003c/p\u003e\n \u003cp\u003eHowever, following treatment, the films became submerged, and the particles sank beneath the buffer\u0026apos;s surface, leading to agglomeration.\u003c/p\u003e\n \u003cp\u003eThe results indicate a decline in WCA during the cultivation process, which signifies the development of hydrophilic groups on the surfaces of the films. The WCA of LD and HD films incubated decreased to 76.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 \u0026deg;and 60.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 \u0026deg; respectively, from 87.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u0026deg;and 63.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 \u0026deg;, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFigure_6\u003c/p\u003e\n \u003cp\u003eThe FTIR technique was employed to further confirm the degradation of PE and to examine the differences of chemical components in the surface and functional groups.\u003c/p\u003e\n \u003cp\u003eAccording to the FTIR spectra (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), all experimental samples, both particles, and films, treated with LMCO, comparing to the control group, displayed a disordered region at ̴ 1650 due to the formation C\u0026thinsp;=\u0026thinsp;O groups by LMCO illustrates the ketones and aldehydes are formed, which is generated by the presence of oxygen atoms in the carbon chain of the polymer [38].\u003c/p\u003e\n \u003cp\u003eFTIR spectra of films and particles of LDPE showed distinct peaks at 3000\u0026ndash;3500, which corresponded to carboxylic acid and alcohol functional groups (R-OH stretching, 3000\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. Hydroxyl group formation leads to more hydrophilicity and indicates more efficient degradation.\u003c/p\u003e\n \u003cp\u003eMorphological changes due to degradation on the plastic surface were confirmed through SEM. The control group\u0026rsquo;s surface, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, exhibited no visible defects and maintained a smooth and uniform appearance, free from grooves, cracks, or pits. On the other hand, all treated samples showed several structural changes like cracks, deep holes, braking into fragments, and creases. As it could be observed intensity of degradation was more on the particles than films. Besides HDPE films degradation has occurred at the least intensity.\u003c/p\u003e\n \u003cp\u003eFigure_7\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe growing concern over plastic waste, mainly PE, has driven the search for sustainable methods to mitigate its environmental impact. Microbial and enzymatic approaches have emerged as promising solutions due to their eco-friendly nature. Among these, LMCOs from the bacterium \u003cem\u003eR. opacus\u003c/em\u003e R7 have shown significant potential in PE biodegradation. The expression analysis of \u003cem\u003eR. opacus\u003c/em\u003e R7 under PE exposion using RNA-seq and complementary RT-qPCR techniques resulted in identification of multicopper oxidases enzymes upregulation. Among them the nominated LMCO1 shows a 19.5-fold expression increase, highlighting its pivotal role in PE oxidation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although LMCO1 was previously recognized as a gene that is upregulated in response to PE by Zampolli et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the presented expression analysing approach based on multi-algorithms here was not merely aimed at corroborating earlier results. Rather, our objective was to reassess the existing transcriptomic data through a robust and meticulous multi-algorithm methodology. By utilizing seven distinct attribute weighting algorithms, we successfully demonstrated that LMCO1 consistently ranked as a top gene across all analyses. This finding significantly enhances our confidence that LMCO1 is not simply an artifact of a specific method or dataset, but instead represents a genuine core gene within the PE response network of \u003cem\u003eR. opacus\u003c/em\u003e R7. These in silico analysis align with the prior report take insight into deep understanding for selection of the most promising laccase gene in PE degradation for our experimental investigation. Ultimately, this thorough analytical framework not only confirmed the biological significance of LMCO1 but also facilitated the identification of 499 additional genes that may be implicated in PE degradation and cellular adaptation. These newly identified candidates present valuable targets for forthcoming functional research into the mechanisms of plastic biodegradation.\u003c/p\u003e \u003cp\u003eAfter approving the its discriminative association to PE degradation based on in silico analysis, the LMCO1 was expressed in PET/ \u003cem\u003eE. coli\u003c/em\u003e system. \u003cem\u003eE. coli\u003c/em\u003e is an ideal organism for the production of recombinant proteins, due to its extensively studied genetic makeup and adaptability [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The pET vector system is a widely recognized and highly efficient platform for expressing recombinant proteins in \u003cem\u003eE. coli\u003c/em\u003e. Its activity is controlled by the bacteriophage T7 transcription and translation system, where expression is initiated by introducing T7 RNA polymerase into the host cells. Upon full activation of the system, a substantial amount of the cell resources is dedicated to the synthesis of the target protein. In this research, a pET-based expression system, comprising both pET22b and pET28a, was employed to express and compare the recombinant LMCO1 enzyme expression yield. The pET22b plasmid uses an N-terminal PelB signal peptide for enhancing proper folding while the PET28a lacks it. The results demonstrated that the pET22 system yielded higher levels of soluble LMCO1 compared to the pET28 system, attributed to the presence of the PelB secretion signal sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Given the successful production of soluble LMCO1 using the pET22 system, this construct was selected for future recombinant expression studies. Our results indicate that Cu\u0026sup2;⁺ ions significantly enhance the enzymatic activity of LMCO1 from 0.2 (U/g) to 1.18 (U/g), as reported previously for LMCOs from \u003cem\u003eR. ruber\u003c/em\u003e C208 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and \u003cem\u003eR. opacus\u003c/em\u003e R7 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, Zhang et al. observed a fourfold increase in activity for laccase from \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Mk-1 upon activation by Cu\u0026sup2;⁺ ions [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. LMCO1 displayed varying levels of activity depending on changes in pH and temperature. The highest activity was determined at pH of (8), compared to pH \u003csub\u003eopt\u003c/sub\u003e of LMCO3 (5.5) and 7 for LMCO2; however, no significant differences were noted among pH levels 7, 8, and 9. The observed consistency can be linked to the ABTS oxidation (involving non-phenolic compounds) mainly occurs through inhibition mechanism of hydroxyl radicals. Consequently, no notable increase in activity was detected with rising pH levels [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The enzyme exhibited peak at 60\u0026deg;C, lower than the optimum temperatures reported for LMCO2 (65\u0026deg;C) and LMCO3 (80\u0026deg;C) by Zampoli, et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A thermophilic laccase from \u003cem\u003eLysinibacillus fusiformis\u003c/em\u003e, involved in PE degradation, showed peak activity at 70\u0026deg;C and maintained in an active state [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLaccases are copper-dependent enzymes, the T1 Cu center (active site) positioned at the laccase surface and its direct exposure to catalytic environment promotes the formation of a substrate tunnel, favoring the degradation of bulky substrates such as polymers. Recent study suggests that the PE binding to laccases is primarily governed by the substantial contribution of hydrophobic residues near the T1 site, alongside the wax-like flexibility of PE, which allow it to fit and integrate into the enzyme\u0026rsquo;s binding pocket. The presence of Met-loop, a Met-rich motif that anchors PE in close proximity to the T1 copper site, also plays key role in enzyme binding to PE and PE oxidation is influenced equally by redox potential and the specific binding interactions between the enzyme and substrate [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The degradation of PE was validated through various methods and indicators. PE oxidation was initially confirmed by measuring the weight loss rate over the incubation period. LDPE exhibited greater weight loss rate than HDPE. Moreover, Zhang et al. (2022) reported 7.6% and 13.2%, weight loss rate for PE particles with both crude laccase extract and purified laccase derived from the psychrophilic bacterium \u003cem\u003ePsychrobacte\u003c/em\u003er sp. NJ228 without any pretreatment under UV irradiation, thermal oxidation and chemical oxidation. Before treatment, particles and films floated on the buffer system due to their strong hydrophobic nature. After treatment, the films and particles became submerged, and particles agglomerated due to the generation of hydrophilic groups during PE oxidation. Similar observations for PE particles were also reported by [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. FTIR spectroscopy was used to monitor the functional groups formation in treated samples. Oxidation, which serves as the rate-limiting factor in the degradation of PE, resulted in the cleavage of carbon-hydrogen bonds, modifications in the chemical structure, and an increase in polarity. FTIR spectra consistently showed a disordered region near ~\u0026thinsp;1655 cm -\u0026sup1;, which is associated with the generating of C\u0026thinsp;=\u0026thinsp;O functional groups. The presence of a carbonyl group (1600\u0026ndash;1850 cm\u0026thinsp;\u0026minus;\u0026thinsp;1) serves as a significant indicator of PE biodegradation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and provides clear evidence of chemical structural alterations at the microscale [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. LDPE that was treated with MR5 and MR10 exhibited peaks at 1654.14 cm\u0026thinsp;\u0026minus;\u0026thinsp;1 and 1683.90 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The findings align with the research conducted by Skariyachan et al. (2018) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], which observed a peak at 1638.97 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, evidence for the presence of C\u0026thinsp;=\u0026thinsp;O carbonyl (ketone or aldehyde) groups on plastic strips that were treated with bacterial consortia. Additionally, the FTIR spectra of all particles, as well as LDPE films, exhibited a prominent peak in the range of 3000 to 3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This peak is associated with the presence of carboxylic acid and alcohol functional groups, specifically the R-OH stretching [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], which leads to more hydrophilicity and indicates more efficient degradation. Oxygen- containing polar functional groups or exhibiting vigorous vibrational intensity can weaken the stability of carbon bonds and lower molecular weight, creating additional degradation sites for key enzymes [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Similarly, after PE incubation with \u003cem\u003eAspergillus flavus\u003c/em\u003e PEDX3 and simultaneous incubation with \u003cem\u003eAcinetobacter\u003c/em\u003e sp. NyZ450 and \u003cem\u003eBacillus\u003c/em\u003e sp. the FTIR spectra of PE incubated with NyZ451 revealed a new peak generation associated with the hydroxyl group, observed in the range of 3500\u0026ndash;3100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Numerous studies have demonstrated that the use of laccase on PE results in the formation of carbonyl and carboxyl groups (polar functional group), on the surface of PE, as identified through FTIR analysis. These findings are consistent with the results observed in our study [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Changes in the WCA values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) further supported the presence of hydrophilic groups on the film surfaces, generated through hydrocarbon chain oxidation by laccase [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Changes in the WCA values of the films indicate that their polarities were altered due to biodegradation and the formation of hydrophilic groups. The greater WCA change was observed in the LDPE sample. However, it was higher than the changes observed in LDPE films fermented with crude laccase enzyme extract [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Degradation caused morphological changes on the plastic surface, observed through SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The control samples exhibited smooth and uniform surfaces devoid of any cracks or pits, consistent with the findings from the FTIR analysis. In contrast, treated samples exhibited significant structural changes such as cracks, deep holes, fragmentation, and creases. Recently, comparable destruction and damage have been observed in PE films that have undergone treatment with laccase and LMCO [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Other studies have also reported similar features such as wrinkles, erosion, cracks, and holes during the degradation process of LDPE, and HDPE films [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In conclusion, the degradation was more pronounced in particles than in films, likely due to the greater accessibility and increased contact area with LMCO1 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Furthermore, the degradation of LDPE film was more effective compared to HDPE. The difference can be attributed to construction of LDPE, which allow it to adapt and fit into the enzyme\u0026rsquo;s binding pocket, forming a stable enzyme-PE complex that potentially facilitates electron transfer to the T1 site and oxidation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. LDPE more degradation result forming hydroxyl and carbonyl groups, which enhance hydrophilicity and significantly alter the water contact angle (WCA), additionally greater degradation result in more morphological changes and weight loss reduction. In comparison, the increased crystallinity of HDPE, along with a lower presence of tertiary carbons and a higher molecular weight, restricts the access of oxidizing enzymes to its polymer chains, resulting in enhanced resistance to degradation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Bioinformatics analysis\u003c/h2\u003e \u003cp\u003eIn summary, the available RNA-seq data related to \u003cem\u003eR. opacus\u003c/em\u003e R7 cultured with PE as sole carbon source was utilized to explore the discriminative genes under PE exposure as follows.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Data processing\u003c/h2\u003e \u003cp\u003eWe used the available RNA-seq data from the Sequence Read Archive (SRA), accession number PRJEB45685. The detailed experiment regarding the growth, PE supplementation and RNA extraction is reported by [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Each experimental condition was replicated three times, resulting in six samples: three derived from the PE conditions and three from the malate conditions. The preliminary assessment of the raw sequencing data quality was conducted utilizing FastQC. Subsequently, the sequencing reads underwent trimming via Trimmomatic on the Galaxy platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://usegalaxy.eu/\u003c/span\u003e\u003cspan address=\"https://usegalaxy.eu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The ILLUMINACLIP step included TruSeq3 adapter sequences for paired-end reads, allowing for a maximum of 2 mismatches. A palindrome and simple clip threshold set at 30 and 10 respectively. Both reads were maintained in paired-end mode. Following the preprocessing stage, FastQC was executed once more to verify the quality of the trimmed data, which was found to be adequate for subsequent analysis. We used the reference genome related to \u003cem\u003eR. opacus\u003c/em\u003e R7 from Ensembl Bacteria in FASTA and GFF format. The quality confirmed reads, were aligned with the reference genome by HISAT2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Expression Quantification via htseq-count and Normalization\u003c/h2\u003e \u003cp\u003eIn this research, htseq-count was employed to count reads. The input files consisted of aligned BAM files generated by HISAT2. The parameters for htseq-count were configured as follows: the mode for addressing reads that overlap multiple features was designated as \"union,\" the data was classified as non-stranded, the minimum alignment quality threshold was established at 10, the feature type was identified as \"CDS,\" the ID attribute was defined as \"ID,\" and reads that were non-unique or ambiguously mapped were excluded. The data were normalized based on DESeq2 (Supplementary File, sheet S1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Data Collection and Preprocessing\u003c/h2\u003e \u003cp\u003eGene count files from six samples served as the input for RapidMiner Studio software (RapidMiner 7.0.001 GmbH). It counts for 8,270 genes across the samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Data Cleaning\u003c/h2\u003e \u003cp\u003eTo improve processing performance, we employed the \"Remove Useless Attributes\" operator within RapidMiner. This operator effectively eliminated genes exhibiting a standard deviation (SD) of less than 0.1; as such, genes demonstrated negligible variation in counts across the samples. Following the application of this filter, the total number of genes was decreased to 6409. The dataset from this process was designated as the Final Cleaned Database (FCdb) (Supplementary File, sheet S2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Attribute Weighting Algorithms\u003c/h2\u003e \u003cp\u003eSeven attribute weighting algorithms were applied to the FCdb to identify significant genes that distinguish \u003cem\u003eR. opacus\u003c/em\u003e R7 cultivated on PE from those cultivated on malate. The implementation of multiple algorithms aimed to mitigate biases associated with any single method and to enhance confidence in the identified genes. An overview of the seven attribute weighting techniques, as utilized in RapidMiner Studio version 7.6 (RapidMiner 7.0.001 GmbH), is presented below:\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformation gain weights\u003c/strong\u003e \u003cp\u003eAllocated based on the decrease in uncertainty (entropy) in the target class following the observation of the attribute value. A higher weight signifies that the attribute conveys more information and is thus more pertinent for classification.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eUncertainty\u003c/strong\u003e \u003cp\u003eA normalized variant of information gain that considers both the entropy of the class and the attribute. It diminishes bias towards attributes with numerous distinct values. Attribute weights are computed based on the symmetrical uncertainty concerning the target variable, where a higher weight indicates greater significance.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePrincipal Component Analysis (PCA)\u003c/strong\u003e \u003cp\u003eThe weight of each attribute is determined by the absolute value of its loading on the principal component that accounts for the maximum variance (typically the first component). These weights are generally normalized to fall between 0 and 1, reflecting the relative importance of the attributes.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eThe Gini Index\u003c/strong\u003e \u003cp\u003eassesses the impurity of a dataset segmented by attribute values. Attributes that more effectively separate the classes yield lower impurity and are assigned higher weights. Consequently, the attribute weight corresponds to its capacity to diminish class impurity.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformation Gain Ratio\u003c/strong\u003e \u003cp\u003eThis normalization lessens the bias towards attributes with a high number of distinct values. A higher Information Gain Ratio indicates a more important attribute.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSupport Vector Machine (SVM)\u003c/strong\u003e \u003cp\u003eIn linear SVM models, the weights of attributes are determined by the absolute values of the coefficients found in the model's weight vector. Attributes that possess larger absolute coefficients exert a more significant influence on the decision boundary and, as a result, are assigned higher weights.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCorrelation\u003c/strong\u003e \u003cp\u003eThis metric computes the absolute value of the pearson correlation coefficient for each gene\u0026rsquo;s normalized expression in relation to the target variable, which signifies the growth condition (PE or malate).\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe subject feature, which is classified as either related to PE or malate, served as the target. Moreover, the normalized values related to expression of the genes were labelled as attributes and classified continuedly. All scores were standardized to a range between 0 and 1 to ensure comparability. Genes that exhibited a normalized weight exceeding 0.6 in any algorithm were deemed significant. The common genes identified by all seven algorithms were recognized as essential genes for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Cloning and Expression of recombinant multicopper oxidase\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eR. opacus\u003c/em\u003e R7 LMCO1 (AII08809) gene with 1376bp length, encodes a protein of 458 amino acids. The gene without inner signal peptide was optimized and synthesized by GenScript, then cloned into the vectors pET28a (+) and pET22b at the NcoI and XhoI restriction sites. Following this, the \u003cem\u003eE. coli\u003c/em\u003e strains DH5α and BL21 (DE3) transformed by the plasmids using electroporation to enable propagation and expression, respectively. The colonies were grown on solid LB medium with kanamycin (50 mg/l) for pET28a (+) and ampicillin (100 mg/l) for pET22b, which was subsequently used for starter culture in 5 ml liquid at 37\u0026deg;C, for 16-hours. A positive colony was then grown overnight at 37\u0026deg;C in 5 mL of fresh LB medium with the appropriate antibiotics on a rotary shaker set at 180 rpm. Then, starting culture (1mL) was inoculated into 50 ml LB medium contained antibiotics, and incubated until optical density at 600 nm was 0.6. When reached to optical density 0.6 at 600 nm (OD600), the recombinant expression was induced by isopropyl β-D-thiogalactoside, 1 mM and CuSO\u003csub\u003e4\u003c/sub\u003e 250 \u0026micro;M, both at final concentrations. Following this, the cultures maintained for 20 hours at 16\u0026deg;C. Control samples consisted of \u003cem\u003eE. coli\u003c/em\u003e BL21/pET-22 (+) and pET28 cells that did not contain the LMCO1 enzyme, which were treated concurrently [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The cell cultures were centrifuged and harvested at 8000 \u0026times; g for 15 minutes, and then resuspend in the potassium phosphate buffer (PPB, 50 mM, pH 7). The cells were treated with Lysozyme (1 mg/ml) and incubated on ice for 30 minutes. Subsequently, a serine protease inhibitor, PMSF (phenylmethylsulfonyl fluoride) 1 mM, was mixed with the cells. The resulting suspension underwent sonication with a repeated duty cycle of 30-second pulses for 5 minutes while placed on dry ice. Finally, the suspension was centrifuged at 12,000g for 25 minutes to isolate a clear supernatant containing the recombinant LMCO1 protein [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The centrifuged crud enzyme solution containing soluble recombinant LMCO1 was stocked for future usage [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.8. Assessment of recombinant proteins through Sodium dodecyl-sulfate polyacrylamide gel electrophoresisand Western blot analysis\u003c/h2\u003e \u003cp\u003eThe expression of recombinant proteins was verified through SDS-PAGE and Western blotting. The proteins were transferred from a 12% polyacrylamide gel to a nitrocellulose membrane. The membrane was then blocked with skim milk overnight, and incubated with an anti-His antibody. Following the washing process with PBS buffer, the staining was done using 3,3\u0026prime;-Diaminobenzidine (DAB) chromogen and treated with hydrogen peroxide for visualising the recombinant proteins. The His-tagged recombinant proteins was indicated by the appearance of brown bands, confirming their identity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Measurement of laccase activity assay\u003c/h2\u003e \u003cp\u003eThe activity of the laccase enzyme in soluble friction was measured by specific substrate regarding the reference [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In summary, the activity was evaluated by measuring the amount of protein necessary to oxidize six mM ABTS (2,2\u0026rsquo;-azino-bis\u003c/p\u003e \u003cp\u003e(3-ethylbenzothiazoline-6-sulfonic acid) under assay condition 200 \u0026micro;M CuSO\u003csub\u003e4\u003c/sub\u003e within the activity buffer, which consisted of 50 mM citric acid-Na2HPO\u003csub\u003e4\u003c/sub\u003e at optimal pH and temperature. The reaction was monitored for 2 minutes, tracking the absorbance increasing at 420 nm (ԑ 420\u0026thinsp;=\u0026thinsp;36,000 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). One enzyme unit activity indicates the quantity of enzyme able to oxidize 1 \u0026micro;mol of ABTS per minute in a quartz cuvette with optical path length of 1.0 cm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.10. Temperature and pH effects on the multicopper oxidase activity\u003c/h2\u003e \u003cp\u003eThe laccase activity of the soluble protein fraction was measured at different temperatures and various pH ranges to determine the optimal reaction condition. The ideal pH (pH opt) was established for ABTS at a final concentration of 6 mM in PPB. The ABTS oxidation was measured at pH levels of 5, 6, 7, 8, 9, and 10 under the previously described conditions to determine enzyme activity. For identifying the optimal temperature (T \u003csub\u003eopt\u003c/sub\u003e), the ABTS oxidation was performed from 30\u0026deg; C to 70\u0026deg; C temperature range [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.11. Determination of polyethylene degradation\u003c/h2\u003e \u003cp\u003eDegradation was evaluated using LDPE (low density PE, Basparan, IRAN, density: 0.9190 gr/ml, code: LF-0450), and HDPE (high density PE, Basparan, IRAN, Density: 0.955 gr/ml, code: 7000F) film (1cm \u0026times; 1cm) and particle (particle size of 1000 \u0026micro;m) as substrate (1% w/v). Prior to using, films were soaked on 2% SDS (1h) and rinsed with distillated water, disposed with 75% ethanol (1h) and 90% ethanol (1h), and dried (40 \u0026deg; C, 30 min) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The PE samples were incubated in LB medium (pH 7.0) for 72 hours to verify their asepsis. A biodegradation system utilizing a crude enzyme solution (with a final concentration of 0.9 mg mL-1 from daily additions) was conducted in 10 mL glass vessels at a temperature of 60 ◦C, under shaking at 120 rpm, within a reaction buffer consisting of 50 mM PPB and 50 \u0026micro;M CuSO\u003csub\u003e4\u003c/sub\u003e, maintaining a total volume of 3 mL at optimal pH for up to 72 hours. Besides, control including PE and the reaction mixture lacking the enzyme was used. The particles collected through repeated filtration subsequently washed with ethanol and water and then dried at 40 ◦C. The films were washed regarding the previously established protocol [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFilms weight loss rates were measured as follows [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003eWeight loss rate (%) = (W\u003csub\u003e0\u003c/sub\u003e - W\u003csub\u003et\u003c/sub\u003e) / W\u003csub\u003e0\u003c/sub\u003e \u0026times; 100\u003c/p\u003e \u003cp\u003eW\u003csub\u003e0\u003c/sub\u003e: weights of the films before degradation\u003c/p\u003e \u003cp\u003eW\u003csub\u003et\u003c/sub\u003e: weights of the films after degradation\u003c/p\u003e \u003cp\u003eThe degradation ability was assessed at 0, 48, and 72 hours through ATR-FTIR spectroscopy, SEM analysis, and hydrophobicity alterations analysis on the film surfaces. FTIR (Bruker Tensor II, Germany) was employed to capture data in the 400\u0026ndash;4000 cm -1 wavelength range, which is utilized to characterize the micro-scale chemical structure of the surface. Following gold sputtering, the micro-scale morphology was examined using SEM (TESCAN Vega3, Kohoutovice, Czech Republic). Changes in hydrophobicity on the film surfaces were calculated by measuring the water contact angle (WCA) with a contact angle measuring device (CAG-10, I.R. IRAN).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge\u0026nbsp;Department of Plant Protection\u0026nbsp;and the central Lab of Shiraz University for providing access to their facilities and technical support during the experiment. We also gratefully thank the guidance and assistance of the staff in operating the equipment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMahboobeh Pishan: Investigation, Writing \u0026ndash; original draft, Visualization, Methodology, Formal analysis. Sima Sazegari: Conceptualization, Methodology, Writing \u0026ndash; original draft, Data curation.\u0026nbsp;Ali Niazi:\u0026nbsp;Conceptualization, review \u0026amp; editing.\u0026nbsp;Marjan Majdinasab:\u0026nbsp;Methodology, review \u0026amp; editing.\u0026nbsp;Zahra Zinati: Software,\u0026nbsp;Formal analysis, review \u0026amp; editing.\u0026nbsp;Mohammad Hadi Eskandari:\u0026nbsp;Conceptualization, Methodology, review \u0026amp; editing, Supervision, Data curation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYeung, C. 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(2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang, B. R., Kim, S. B., Song, H. A. \u0026amp; Lee, T. K. Accelerating the biodegradation of high-density polyethylene (HDPE) using Bjerkandera adusta TBB-03 and lignocellulose substrates. \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e (9), 304 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuan, Y. et al. Degradation of polyethylene particles by Trichoderma harzianum and recombinant laccase cloned from the strain. \u003cem\u003eJ. Appl. Polym. Sci.\u003c/em\u003e \u003cb\u003e140\u003c/b\u003e (43), e54599 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontazer, Z., Habibi Najafi, M. B. \u0026amp; Levin, D. B. Challenges with verifying microbial degradation of polyethylene. \u003cem\u003ePolymers\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (1), 123 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Polyethylene, Biodegradation, Rhodococcus opacus R7, Plastic waste","lastPublishedDoi":"10.21203/rs.3.rs-8685015/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8685015/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe production rate of Polyethylene (PE) has increased to a concerning level, necessitating effective strategies to reduce its environmental impacts. This study investigates the role of laccase-like multicopper oxidase (LMCO1) in oxidative degradation of PE, through a comprehensive approach, including machine learning analysis, recombinant expression, physico-chemical assays. To this end, the public available RNA-seq data related to \u003cem\u003eRhodococcus opacus\u003c/em\u003e R7 cultured with PE was mined using several attribute weighting algorithms to explore the discriminative role of LMCO1 in PE degradation. Further, the recombinant LMCO1 was expressed and evaluated for substantial degrading impact on the PE films. Oxidative degradation of PE samples was evaluated by measuring weight loss rate, assessment of water contact angle measuring, confirmed by Fourier transform infrared spectroscopy and scanning electron microscopy analysis. The findings revealed that Cu\u0026sup2;⁺ enhanced the activity of LMCO1 in the crude enzyme extract by 490%, with peak activity occurring at pH 8 and 60\u0026deg;C (optimal temperature). The PE degradation experiments over 72 hours indicated that a 14.28% weight loss rate was in LDPE as well as decreasing the water contact angle to 76.73 \u0026deg;. Fourier transform infrared spectroscopy analysis revealed the existence of various polar functional groups on PE surface including carbonyl, carboxyl, and hydroxyl groups. Significant damage to the PE surface, including cracks, pitting, and roughness, as well as internal aspects such as structural weakening and material degradation was identified through scanning electron microscopy. Overall, this study demonstrated the high potential of LMCO1 to degrade PE films and particles in short time.\u003c/p\u003e","manuscriptTitle":"Heterologous expression and characterization of Rhodococcus opacus R7 laccase-like multicopper oxidase (LMCO1) enzyme for polyethylene biodegradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 12:57:03","doi":"10.21203/rs.3.rs-8685015/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-12T09:12:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T09:17:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313650974249353391537688347388571857544","date":"2026-02-13T18:03:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77434571317937290485533153959195445285","date":"2026-02-13T04:28:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-10T08:28:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-26T05:41:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-26T05:40:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-24T08:11:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32fb0c25-fbc5-4a8e-bdd3-06cc2e04cd3a","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62807368,"name":"Biological sciences/Biochemistry"},{"id":62807369,"name":"Biological sciences/Biological techniques"},{"id":62807370,"name":"Biological sciences/Biotechnology"},{"id":62807371,"name":"Earth and environmental sciences/Environmental sciences"},{"id":62807372,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-28T10:08:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 12:57:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8685015","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8685015","identity":"rs-8685015","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}