Identification of redox activators for continuous reactivation of glyoxal oxidase from Trametes versicolor in a two-enzyme reaction cascade.

Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O2 to H2O2. Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor (TvGLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii). TvGLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for TvGLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated TvGLOX. Through continuous reactivation of TvGLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase (MaAAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.

Immobilization of recombinant Trametes versicolor aflatoxin B1-degrading enzyme (TV-AFB1D) with montmorillonite for absorption and in situ degradation of aflatoxin B1.

Aflatoxin B1 is a highly carcinogenic and teratogenic substance mainly produced by toxin-producing strains such as Aspergillus flavus and Aspergillus parasitic. The efficient decomposition of aflatoxin is an important means to reduce its harm to humans and livestock. In this study, Trametes versicolor aflatoxin B1-degrading enzyme (TV-AFB1D) was recombinantly expressed in Bacillus subtilis (B. subtilis) 168. MMT-CTAB-AFB1D complex was prepared by the immobilization of TV-AFB1D and montmorillonite (MMT) by cross-linking glutaraldehyde. The results indicated that TV-AFB1D could recombinantly express in engineered B. subtilis 168 with a size of approximately 77 kDa. The immobilization efficiency of MMT-CTAB-AFB1D reached 98.63% when the concentration of glutaraldehyde was 5% (v/v). The relative activity of TV-AFB1D decreased to 72.36% after reusing for 10 times. The content of AFB1 in MMT-CTAB-AFB1D-AFB1 decreased to 1.1 µg/g from the initial 5.6 µg/g after incubation at 50 °C for 6 h. The amount of 80.4% AFB1 in the MMT-CTAB-AFB1D-AFB1 complex was degraded by in situ catalytic degradation. Thus, the strategy of combining adsorption and in situ degradation could effectively reduce the content of AFB1 residue in the MMT-CTAB-AFB1D complex.

New insights in the biodegradation of high-cyclic polycyclic aromatic hydrocarbons with crude enzymes of Trametes versicolor.

High-cyclic polycyclic aromatic hydrocarbons (PAHs), with complex fused aromatic structures, are widespread, refractory and harmful in soil, but the current remediation technologies for high-cyclic PAHs are often inefficient and costly. This study focused on the biodegradation process of high-cyclic benzo[a]pyrene by Trametes versicolor crude enzymes. The crude enzymes exhibited high laccase activity (22112 U/L) and benzo[a]pyrene degradation efficiency (42.21%) within a short reaction time. Through the actual degradation and degradation kinetics, the degradation efficiency of PAHs decreased with the increase of aromatic rings. And the degradation conditions (temperature, pH, Cu2+ concentration, mediator) were systematically optimised. The optimum degradation conditions (1.5 mM Cu2+, 28℃ and pH 6) showed significant degradation efficiency for the low and medium concentrations of benzo[a]pyrene. In addition, complete degradation of benzo[a]pyrene could be achieved using only 0.2 mM of HBT mediator compared with crude enzymes alone. Collectively, these results showed the high-cyclic PAHs degradation potential of Trametes versicolor crude enzymes, and provided references to evaluate applicable prospects of white rot fungus crude enzymes in PAHs-contaminated soils.

Arsenic(III)-induced oxidative defense and speciation changes in a wild Trametes versicolor strain.

Oxidative defense or arsenic(As) changes exhibited by Trametes versicolor in response to toxicity under As stress remain unclear. In this study, after internal transcribed spacer identification, a wild T. versicolor HN01 strain was cultivated under 40 and 80 mg/L of As III stress. The antioxidant contents by multifunctional microplate reader and the speciations of As by high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry were examined to explore the detoxification mechanisms. The results demonstrated this strain could tolerate As concentration of 80 mg/L with a bio-enrichment coefficients of 11.25. Among the four antioxidants, the activities of catalase, superoxide dismutase, and glutathione in the As-stress group at 80 mg/L improved by 1.10, 1.09, and 20.47 times that of non-stress group, respectively. The speciation results indicated that AsV was the dominant species in the hyphae of T. versicolor regardless of no-stress or As-stress. The detoxification mechanisms of this strain were involved alleviating the toxicity by increasing the activities of antioxidants, especially glutathione, as well as by converting As III into As V and other less toxic As species. T. versicolor could be used as a bio-accumulator to deal with As exposure in contaminated environments based on its extraordinary As tolerance and accumulation capacities.

Optimization of dye-contaminated wastewater treatment by fungal Mycelial-light expanded clay aggregate composite.

Dye-contaminated wastewaters from the printing batik industry are hazardous if discharged into the environment without any treatment. Finding an optimization and reusability assessment of a new fungal-material composite for dye-contaminated wastewater treatment is important for efficiency. The study purposes to optimize fungal mycelia Trametes hirsuta EDN 082 - light expanded clay aggregate (myco-LECA) composite for real priting batik dye wastewater treatment by using Response Surface Methodology with Central Composite Design (RSM-CCD). The factors included myco-LECA weight (2-6 g), wastewater volume (20-80 mL), and glucose concentration (0-10%) were applied for 144 h of incubation time. The result showed that the optimum condition was achieved at 5.1 g myco-LECA, at 20 mL wastewater, and at 9.1% glucose, respectively. In this condition, the decolorization values with an incubation time of 144 h were 90, 93, and 95%, at wavelengths 570, 620, and 670 nm, respectively. A reusability assessment was conducted for 19 cycles and the result showed that decolorization effectiveness was still above 96%. GCMS analysis showed the degradation of most compounds in the wastewater and the degradation products of the wastewater demonstrated detoxification against Vigna radiata and Artemia salina. The study suggests that myco-LECA composite has a good performance and therefore is a promising method for the treatment of printing batik wastewater.

In-situ synchrotron imaging, experimental, and computational investigations on the efficiency of Trametes versicolor laccase on detoxification of P-Cresyl Sulfate (PCS) Protein Bound Uremic Toxin (PBUT).

Protein bound uremic toxins (PBUTs) are small substances binding to larger proteins, mostly human serum albumin (HSA), and are challenging to remove by hemodialysis (HD). Among different classes of PBUTs, p-cresyl sulfate (PCS) is the most widely used marker molecule and major toxin, as 95 % is bound to HSA. PCS has a pro-inflammatory effect and increases both the uremia symptom score and multiple pathophysiological activities. High-flux HD to clear PCS leads to serious loss of HSA, which results in a high mortality rate. The goal of the present study is to investigate the efficacy of PCS detoxification in serum of HD patients using a biocompatible laccase enzyme from Trametes versicolor. Molecular docking was used to gain an in-depth understanding of the interactions between PCS and the laccase to identify the functional group(s) responsible for ligand-protein receptor interactions. UV-Vis spectroscopy and gas chromatography-mass spectrometry (GC-MS) were used to assess the detoxification of PCS. GC-MS was used to identify the detoxification byproducts and their toxicity was assessed using docking commutations. In situ synchrotron radiation micro-computed tomography (SR-µCT) imaging available at the Canadian Light Source (CLS) was conducted to assess HSA binding with PCS before and after detoxification with laccase and undertake the corresponding quantitative analysis. GC-MS analyses confirmed the detoxification of PCS with laccase at a concentration of 500 mg/L. The potential pathway of PCS detoxification in the presence of the laccase was identified. Increasing laccase concentration led to the formation of m-cresol, as indicated by the corresponding absorption in the UV-Vis spectra and a sharp peak on the GC-MS spectra. Our analysis provides insight into the general features of PCS binding on Sudlow site II, as well as insights into PCS detoxification product interactions. The average affinity energy for detoxification products was lower than that of PCS. Even though some byproducts showed potential toxicity, the level was lower than for PCS based on toxicity indexes (e.g., LD50/LC50, carcinogenicity, neurotoxicity, mutagenicity). In addition, these small compounds can also be more easily removed by HD compared to PCS. SR-µCT quantitative analysis showed adhesion of the HSA to a significant reduced extent in the presence of the laccase enzyme in bottom sections of the polyarylethersulfone (PAES) clinical HD membrane tested. Overall, this study opens new frontiers for PCS detoxification.

Structural insights into the interactions of glutathione transferases with a nitric oxide carrier and sodium nitroprusside.

Glutathione transferases are detoxification enzymes with multifaceted roles, including a role in the metabolism and scavenging of nitric oxide (NO) compounds in cells. Here, we explored the ability of Trametes versicolor glutathione transferases (GSTs) from the Omega class (TvGSTOs) to bind metal-nitrosyl compounds. TvGSTOs have been studied previously for their ligandin role and are interesting models to study protein‒ligand interactions. First, we determined the X-ray structure of the TvGSTO3S isoform bound to the dinitrosyl glutathionyl iron complex (DNGIC), a physiological compound involved in the storage of nitric oxide. Our results suggested a different binding mode compared to the one previously described in human GST Pi 1 (GSTP1). Then, we investigated the manner in which TvGSTO3S binds three nonphysiological metal-nitrosyl compounds with different metal cores (iron, ruthenium and osmium). We assayed sodium nitroprusside, a well-studied vasodilator used in cases of hypertensive crises or heart failure. Our results showed that the tested GST can bind metal-nitrosyls at two distinct binding sites. Thermal shift analysis with six isoforms of TvGSTOs identified TvGSTO6S as the best interactant. Using the Griess method, TvGSTO6S was found to improve the release of nitric oxide from sodium nitroprusside in vitro, whereas the effects of human GST alpha 1 (GSTA1) and GSTP1 were moderate. Our results open new structural perspectives for understanding the interactions of glutathione transferases with metal-nitrosyl compounds associated with the biochemical mechanisms of NO uptake/release in biological systems.

Transcriptomics and co-expression network analysis revealing candidate genes for the laccase activity of Trametes gibbosa.

BACKGROUND: Trametes gibbosa, which is a white-rot fungus of the Polyporaceae family found in the cold temperate zone, causes spongy white rot on wood. Laccase can oxidize benzene homologs and is one of the important oxidases for white rot fungi to degrade wood. However, the pathway of laccase synthesis in white rot fungi is unknown. RESULTS: The peak value of laccase activity reached 135.75 U/min/L on the 9th day. For laccase activity and RNA-seq data, gene expression was segmented into 24 modules. Turquoise and blue modules had greater associations with laccase activity (positively 0.94 and negatively -0.86, respectively). For biology function, these genes were concentrated on the cell cycle, citrate cycle, nicotinate, and nicotinamide metabolism, succinate dehydrogenase activity, flavin adenine dinucleotide binding, and oxidoreductase activity which are highly related to the laccase synthetic pathway. Among them, gene_8826 (MW199767), gene_7458 (MW199766), gene_61 (MW199765), gene_1741 (MH257605), and gene_11087 (MK805159) were identified as central genes. CONCLUSION: Laccase activity steadily increased in wood degradation. Laccase oxidation consumes oxygen to produce hydrogen ions and water during the degradation of wood. Some of the hydrogen ions produced can be combined by Flavin adenine dinucleotide (FAD) to form reduced Flavin dinucleotide (FADH2), which can be transmitted. Also, the fungus was starved of oxygen throughout fermentation, and the NADH and FADH2 are unable to transfer hydrogen under hypoxia, resulting in the inability of NAD and FAD to regenerate and inhibit the tricarboxylic acid cycle of cells. These key hub genes related to laccase activity play important roles in the molecular mechanisms of laccase synthesis for exploring industrial excellent strains.

Bioremediation of bisphenol A found in industrial wastewater using Trametes versicolor (TV) laccase nanoemulsion-based bead organogel in packed bed reactor.

Bisphenol A (BPA) is one of the toxic chemicals, which is widely used for manufacturing epoxy, polyester resin, and polycarbonates. These materials are extensively used in manufacturing of reusable bottles, baby bottles, dental sealants, various medical devices, and so forth. Moreover, canned and packaged foods are sources of bisphenol A, which is unknowingly consumed by many people worldwide. Its endocrine disrupting and teratogenic properties impose potential risk to the wildlife and human health. BPA has been linked to reproductive, metabolic, and immunity disorders in humans. Regardless of BPA ban in reusable and baby bottles, annually, 15 billion pounds of BPA still being produced. BPA pollution and its cleanup are major challenges. Therefore, it is essential to develop a suitable strategy to bioremediate BPA. The Trametes versicolor (TV) laccase-based nanoemulsion calcium alginate bead organogel was able to transform 94% of BPA within 2 h of treatment. Organogel showed 60% of BPA removal from actual industrial wastewater in packed bed batch reactor and 67% of BPA removal in continuous flow packed bed reactor. The biological oxygen demand (BOD) of treated industrial effluent was 14 mg/L, which is very much less than untreated effluent's BOD, which was 48 mg/L. The chemical oxygen demand of industrial effluent was 1240 mg/ml, and treated effluent was 248 mg/L, respectively. Hence, application of nanoemulsion-based organogel in packed bed reactor found to be a potential candidate for the bioremediation of industrial effluent containing BPA. PRACTITIONER POINTS: The TV laccase-based nanoemulsion calcium alginate bead organogel was able to transform 94% of BPA. Organogel showed 67% of BPA removal from industrial wastewater in continuous flow packed bed reactor. The nanoemulsion-based organogel in packed bed reactor found to be potential candidate for the bioremediation of industrial effluent containing BPA.

Betaine-Based Deep Eutectic Solvent as a New Media for Laccase-Catalyzed Template-Guided Polymerization/Copolymerization of Aniline and 3-Aminobenzoic Acid.

Deep eutectic solvents (DESs) can compensate for some of the major drawbacks of traditional organic solvents and ionic liquids and meet all requirements of green chemistry. However, the potential of their use as a medium for biocatalytic reactions has not been adequately studied. In this work we used the DES betaine-glycerol with a molar ratio of 1:2 as co-solvent for enzymatic template-guided polymerization/copolymerization of aniline (ANI) and 3-aminobenzoic acid (3ABA). The laccase from the basidial fungus Trametes hirsuta and air oxygen served as catalyst and oxidant, respectively. Sodium polystyrene sulfonate (PSS) was used as template. Interpolyelectrolyte complexes of homopolymers polyaniline (PANI) and poly(3-aminobenzoic acid) (P3ABA) and copolymer poly(aniline-co-3-aminobenzoic acid) (P(ANI-3ABA)) were prepared and their physico-chemical properties were studied by UV-Vis and FTIR spectroscopy and cyclic voltammetry. According to the results obtained by atomic force microscopy, PANI/PSS had a granular shape, P(ANI-3ABA)/PSS had a spherical shape and P3ABA/PSS had a spindle-like shape. The copolymer showed a greater antimicrobial activity against Escherichia coli and Staphylcocus aureus as compared with the homopolymers. The minimal inhibitory concentration of the P(ANI-3ABA)/PSS against the gram-positive bacterium S. aureus was 0.125 mg mL-1.

Comparative Analysis of Peniophora lycii and Trametes hirsuta Exoproteomes Demonstrates "Shades of Gray" in the Concept of White-Rotting Fungi.

White-rot basidiomycete fungi are a unique group of organisms that evolved an unprecedented arsenal of extracellular enzymes for an efficient degradation of all components of wood such as cellulose, hemicelluloses and lignin. The exoproteomes of white-rot fungi represent a natural enzymatic toolbox for white biotechnology. Currently, only exoproteomes of a narrow taxonomic group of white-rot fungi-fungi belonging to the Polyporales order-are extensively studied. In this article, two white-rot fungi, Peniophora lycii LE-BIN 2142 from the Russulales order and Trametes hirsuta LE-BIN 072 from the Polyporales order, were compared and contrasted in terms of their enzymatic machinery used for degradation of different types of wood substrates-alder, birch and pine sawdust. Our findings suggested that the studied fungi use extremely different enzymatic systems for the degradation of carbohydrates and lignin. While T. hirsuta LE-BIN 072 behaved as a typical white-rot fungus, P. lycii LE-BIN 2142 demonstrated substantial peculiarities. Instead of using cellulolytic and hemicellulolytic hydrolytic enzymes, P. lycii LE-BIN 2142 primarily relies on oxidative polysaccharide-degrading enzymes such as LPMO and GMC oxidoreductase. Moreover, exoproteomes of P. lycii LE-BIN 2142 completely lacked ligninolytic peroxidases, a well-known marker of white-rot fungi, but instead contained several laccase isozymes and previously uncharacterized FAD-binding domain-containing proteins.

ThhspA1 is involved in lacA transcriptional regulation of Trametes hirsuta AH28-2 exposed to o-toluidine.

White rot fungi, especially Trametes spp., respond to a wide range of aromatic compounds and dramatically enhance laccase activity, while the activation mechanisms remain to be elucidated. Here, we show that an Hsp70 homolog named ThhspA1 regulates the transcription of laccase LacA in Trametes hirsuta AH28-2 when confronted with o-toluidine. ThhspA1 is pulled down by lacA promoter sequence from the nuclear mixture extracted from T. hirsuta AH28-2 induced by 2 mM o-toluidine. Silencing of ThhspA1 results in a sharp decrease in lacA transcripts and laccase activity in vivo. By comparison, ThhspA1 overexpression does not affect lacA transcription, and laccase activity shows slight enhancement or remains unchanged upon induction with o-toluidine. Electrophoretic mobility shift assays suggest a direct interaction between ThhspA1 and the lacA promoter region. Further investigation shows that the integrity of ThhspA1 is critical since its substrate binding domain (SBD) and nucleotide-binding domain (NBD) are both necessary for DNA binding, with a higher affinity of SBD than NBD based on fluorescence polarization assay. Our results demonstrate that ThhspA1 functions as an aromatic-stress-related DNA binding transcriptional factor required for LacA expression.

Neomycin removal using the white rot fungus Trametes versicolor.

The presence of antibiotic resistance genes in wastewater treatment plants (WWTPs), and in river and lake recipients show the need to develop new antibiotic removal strategies. The aminoglycoside antibiotic class is of special concern since the chemical structure of these compounds limits the choices of removal technologies. The experimental design included fungal mediated in vivo and in vitro experiments. The experiments were performed in Erlenmeyer flasks under non-sterile conditions. In the study, the role of the laccase redox mediator 4-hydroxy benzoic acid (HBA) in the removal of neomycin was investigated. The specific objective of the study was to conclude whether it is possible to use the white rot fungus (WRF) Trametes versicolor to biodegrade neomycin. It was shown that it is feasible to remove 34% neomycin in vitro (excluding living fungal cells) by laccase-HBA mediated extracellular biodegradation. In the in vivo experiments, polyurethane foam (PUF) was used as supporting material to immobilize fungal mycelia on. The presence of living fungal cells facilitated a removal of approximately 80% neomycin in the absence of HBA. Using liquid chromatography-high resolution-mass spectrometry, it was possible to tentatively identify oxidation products of neomycin hydrolysates. The results in this study open up the possibility to implement a pretreatment plant (PTP) aimed for neomycin removal.

Polysaccharides Produced by the Mushroom Trametes robiniophila Murr Boosts the Sensitivity of Hepatoma Cells to Oxaliplatin via the miR-224-5p/ABCB1/P-gp Axis.

AIM: To investigate the mechanisms employed by PS-T (polysaccharides of Trametes, PS-T), the main active ingredient of Huaier granules, to improve the susceptibility of hepatoma cells to oxaliplatin (OXA). METHODS: Cell proliferation in response to PS-T was determined both in vitro and in vivo. The effects of PS-T on miRNAs were analyzed with the use of a microarray. MiRNAs were screened under specific conditions (P < .05, logFoldChange > ABS [1.5]) and further silenced or overexpressed by liposome transfection. Levels of ABCB1 mRNA and P-gp were detected by qRT-PCR and western blot analysis, respectively. A dual fluorescence assay was performed to determine whether miRNA directly targets ABCB1. RESULTS: PS-T enhanced the inhibitory effect of OXA in human hepatoma cells and xenografts. Among 5 up-regulated miRNAs, overexpression of only miR-224-5p inhibited the expression of ABCB1 mRNA and P-gp, while silencing of miR-224-5p had an opposite effect. Moreover, miR-224-5p can directly target the 3'-UTR of ABCB1. CONCLUSION: PS-T increases the sensitivity of human hepatoma cells to OXA via the miR-224-5p/ABCB1/P-gp axis.

Pyrene remediation by Trametes maxima: an insight into secretome response and degradation pathway.

The rapid pace of economic development has resulted in the release of several polycyclic aromatic hydrocarbons (PAHs) into the environment. Microbial degradation using white-rot fungi is a promising method for the removal of PAHs from the environment. In the present study, biodegradation of recalcitrant PAH by a white-rot fungus, Trametes maxima IIPLC-32, was investigated using pyrene. The pyrene concentration decreased by 79.80%, 65.37%, and 56.37% within 16 days from the initial levels of 10 mg L-1, 25 mg L-1, and 50 mg L-1, respectively. Gas chromatographic-mass spectrometric identification of prominent metabolites 1-hydroxypyrene, 2-methyl-1-naphthyl acetic acid, di-n-butyl phthalate, and diethyl phthalate helped in determining the pyrene degradation pathway. The presence of 81 extracellular proteins was revealed by secretome analysis. The identified proteins up-regulated in response to pyrene degradation were classified into detoxification proteins (6.12%), redox proteins (6.12%), stress proteins (4.08%), metabolic-related proteins (26.53%), translation and transcriptional proteins (49%), catalytic proteins (49%), and other proteins (8.16%). Knowledge of secretome analysis in pyrene degradation helped to understand the degradation mechanism of pyrene. Also, the study suggests that T. maxima IIPLC-32 has the potential to be used in the bioremediation of PAH contaminated aquatic environment.

Purification and Characterization Laccase from Trametes versicolor (L.) Lloyd in Submerged Fermentation.

<b>Background and Objective:</b> Laccase is classified as an oxidoreductase enzyme that catalyzes oxidation reactions of phenolic groups by using oxygen as its electron acceptor. Laccase isolated from <i>Trametes versicolor</i> (L.) Lloyd has a wide range of applications in the industrial sector. The use of enzymes in the industrial sector requires pure enzyme conditions from impurities so that the enzyme can maximize its ability in converting the substrate. This study aims to obtain enzyme activity and the characteristic of purified laccase enzymes isolated from <i>Trametes versicolor</i> (L.) Lloyd. <b>Materials and Methods:</b> This study was conducted with an experimental method followed by descriptive analysis. The steps of this research consist of a qualitative assay of laccase enzyme, crude laccase extract desalting by Sephadex G-25, laccase purification by Sephadex G-100 and laccase optimum pH characterization. <b>Results:</b> The result of this study showed that purification of laccase from <i>Trametes versicolor </i>(L.) Lloyd with Sephadex G-25 increases laccase enzyme-specific activity which is 10.966 U mg¹ and reaches 2.93-fold purity. The highest laccase enzyme activity was achieved at pH 4 with a value of laccase activity 62.39 U L¹. <b>Conclusion:</b> Based on current results, purifying laccase from <i>Trametes versicolor </i>(L.) Lloyd with Sephadex G-25 was recommended which resulting higher enzyme specific activity.

Functional annotation and comparative modeling of ligninolytic enzymes from Trametes villosa (SW.) Kreisel for biotechnological applications.

Functional annotation of Trametes villosa genome was performed to search Class II peroxidase proteins in this white-rot fungus, which can be valuable for several biotechnological processes. After sequence identification and manual curation, five proteins were selected to build 3 D models by comparative modeling. Analysis of sequential and structural sequences from selected targets revealed the presence of two putative Lignin Peroxidase and three putative Manganese Peroxidase on this fungal genome. All 3 D models had a similar folding pattern from selected 3 D structure templates. After minimization and validation steps, the best 3 D models were subjected to docking studies and molecular dynamics to identify structural requirements and the interactions required for molecular recognition. Two reliable 3 D models of Class II peroxidases, with typical catalytic site and architecture, and its protein sequences are indicated to recombinant production in biotechnological applications, such as bioenergy.Communicated by Ramaswamy H. Sarma.

Electrochemical analysis of Catechol polymerization in presence of Trametes versicolor laccase and the mediator ABTS.

The phenolic compound catechol has found various applications in the industry but is often discharged untreated in industrial effluents. Catechol is highly toxic and adversely affects the environment. This has increased extensive investigation into elucidating the effects of various synthetic elements or different biocatalysts on catechol, thereby leading the way to its bioremediation. Hence, an electrochemical-based study on catechol in the presence of the enzyme laccase could provide a basic understanding of the unique characteristics exhibited by catechol, thus facilitating a distinct perspective to its subsequent treatment and removal. The present study focuses on the electrochemical characterization of catechol based on the oxidation of laccase and the redox mediator 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Catechol exhibited distinct electrochemical behavior across various concentrations. The unique electroactive nature of ABTS assisted in the polymerization of catechol which was found to be concentration-dependent. Laccase produced a higher oxidation-reduction rate, thereby producing a much more stable condition for the polymerization of catechol. However, with the laccase-mediator system (LMS), the catechol polymerization rate was distinctly higher and more gradual with the enzyme utilizing the electroactive species produced by ABTS to increase the electron transfer and producing a combinatorial impact on the phenolic compound. This study could rightly serve as the building block in developing future technologies like wastewater treatment and biosensors for catechol bioremediation.

Enzyme polymer engineered structure strategy to enhance cross-linked enzyme aggregate stability: a step forward in laccase exploitation for cannabidiol removal from wastewater.

Despite all its advantages and potential, cross-linking enzyme aggregate (CLEA) technology is still not applied at an industrial scale for enzyme insolubilization for bioremediation purposes. In this study, the enzyme polymer engineered structure (EPES) method was used to enhance CLEA stability and reuse. A crude laccase from Trametes hirsuta was successfully insolubilized to form EPES-CLEAs. The polymeric network provided excellent stability (> 90%) to CLEAs after a 24-h incubation in a non-buffered municipal wastewater effluent (WW), and the biocatalysts were recycled using a centrifugation process. While CLEAs activity dropped to 17%, EPES-CLEAs showed a laccase activity retention of 67% after five cycles of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) oxidation. After 8 h of treatment in WW, the EPES-CLEAs were equally as effective in removing cannabidiol (CBD) as the free-LAC (~ 37%). This research demonstrates that the EPES method is a promising alternative for CLEA stabilization and reuse in environmental conditions.