Perovskite hydroxide-based laccase mimics with controllable activity for environmental remediation and biosensing.

Constructing relatively inexpensive nanomaterials to simulate the catalytic performance of laccase is of great significance in recent years. Although research on improving laccase-like activity by regulating ligands of copper (amino acids or small organic molecules, etc.) have achieved remarkable success. There are few reports on improving laccase-like activity by adjusting the composition of metal Cu. Here, we used perovskite hydroxide AB(OH)6 as a model to evaluate the relationship between Cu based alloys and their laccase-like activity. We found that when the Cu/Mn alloy ratio of the perovskite hydroxide A point is greater than 1, the laccase-like activity of the binary alloy perovskite hydroxide is higher than that of the corresponding single Cu. Based on the measurements of XPS and ICP-MS, we deduced that the improvements of laccase-like activity mainly attribute to the ratio of Cu+/Cu2+and the content of Cu. Moreover, two types of substrates (toxic pollutants and catechol neurotransmitters) were used to successfully demonstrated such nanozymes' excellent environmental protecting function and biosensing property. This work will provide a novel approach for the construction and application of laccase-like nanozymes in the future.

Laccase-Mediated Oxidation of Phenolic Compounds from Wine Lees Extract towards the Synthesis of Polymers with Potential Applications in Food Packaging.

Laccase from Trametes versicolor was applied to produce phenolic polymeric compounds with enhanced properties, using a wine lees extract as the phenolic source. The influence of the incubation time on the progress of the enzymatic oxidation and the yield of the formed polymers was examined. The polymerization process and the properties of the polymeric products were evaluated with a variety of techniques, such as high-pressure liquid chromatography (HPLC) and gel permeation chromatography (GPC), Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies, differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The enzymatic polymerization reaction resulted in an 82% reduction in the free phenolic compounds of the extract. The polymeric product recovery (up to 25.7%) and the molecular weight of the polymer depended on the incubation time of the reaction. The produced phenolic polymers exhibited high antioxidant activity, depending on the enzymatic oxidation reaction time, with the phenolic polymer formed after one hour of enzymatic reaction exhibiting the highest antioxidant activity (133.75 and 164.77 µg TE mg-1 polymer) towards the ABTS and DPPH free radicals, respectively. The higher thermal stability of the polymeric products compared to the wine lees phenolic extract was confirmed with TGA and DSC analyses. Finally, the formed phenolic polymeric products were incorporated into chitosan films, providing them with increased antioxidant activity without affecting the films' cohesion.

Use of sawdust for production of ligninolytic enzymes by white-rot fungi and pharmaceutical removal.

Use of white-rot fungi for enzyme-based bioremediation of wastewater is of high interest. These fungi produce considerable amounts of extracellular ligninolytic enzymes during solid-state fermentation on lignocellulosic materials such as straw and sawdust. We used pure sawdust colonized by Pleurotus ostreatus, Trametes versicolor, and Ganoderma lucidum for extraction of ligninolytic enzymes in aqueous suspension. Crude enzyme suspensions of the three fungi, with laccase activity range 12-43 U/L and manganese peroxidase activity range 5-55 U/L, were evaluated for degradation of 11 selected pharmaceuticals spiked at environmentally relevant concentrations. Sulfamethoxazole was removed significantly in all treatments. The crude enzyme suspension from P. ostreatus achieved degradation of wider range of pharmaceuticals when the enzyme activity was increased. Brief homogenization of the colonized sawdust was also observed to be favorable, resulting in significant reductions after a short exposure of 5 min. The highest reduction was observed for sulfamethoxazole which was reduced by 84% compared to an autoclaved control without enzyme activity and for trimethoprim which was reduced by 60%. The compounds metoprolol, lidocaine, and venlafaxine were reduced by approximately 30% compared to the control. Overall, this study confirmed the potential of low-cost lignocellulosic material as a substrate for production of enzymes from white-rot fungi. However, monitoring over time in bioreactors revealed a rapid decrease in enzymatic ligninolytic activity.

Purification, kinetic characterization of thermostable multicopper oxidase from the oyster mushroom and its versatility for greener agro-pulp bio bleaching in the paper industry.

Production of a thermostable laccase from Pleurotus florida was reported for the first time, both in submerged and solid-state fermentation using agro-industrial residues. This enzyme was purified using ammonium sulphate precipitation (60-90%), Sephadex G-100 and DEAE column ion exchange chromatography, respectively. The laccase was purified to 21.49 fold with an apparent molecular weight of 66 kDa and had an optimal pH of 5 with temperature stability at 60°C. Metal ions such as Cu2+ (91.26 µmole/mL/min), Mg2+ (68.15 µmole/mL/min), and Fe2+ (1.73 µmole/mL/min) enhanced the laccase activity, but Fe2+ (1.73µmole/mL/min) inhibited the enzyme activity. The purified laccase had Km and Vmax of 16.68 mM and 26.73 µmole/mL/min for guaiacol as a substrate. The isolated enzyme was characterized by FT-IR which revealed bands at 3655.0 cm-1, 2894.7 cm-1, and 1151.7 cm-1 corresponding to primary amines, C-H stretch, and amide -III, respectively. The enzymatic bio bleaching of paddy straw pulp was found to be most effective which resulted in a lowering of kappa number and yellowness by 19.47% & 17.84% whereas an increase in brightness and whiteness by 41.92%. & -19.61%. Thus, this might be stated that the crude laccase from P. florida can be exploited to reduce the toxic waste load for managing environmental pollution and helps in enhancing the yield and quality of the paper.

Enzymes in "Green" Synthetic Chemistry: Laccase and Lipase.

Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes and alternatives to many common catalysts. Their essential advantages are high catalytic efficiency, substrate specificity, minimal formation of byproducts, and low energy demand. All of these benefits make enzymes highly desirable targets of academic research and industrial development. This review has the modest aim of briefly overviewing the classification, mechanism of action, basic kinetics and reaction condition effects that are common across all six enzyme classes. Special attention is devoted to immobilization strategies as the main tools to improve the resistance to environmental stress factors (temperature, pH and solvents) and prolong the catalytic lifecycle of these biocatalysts. The advantages and drawbacks of methods such as macromolecular crosslinking, solid scaffold carriers, entrapment, and surface modification (covalent and physical) are discussed and illustrated using numerous examples. Among the hundreds and possibly thousands of known and recently discovered enzymes, hydrolases and oxidoreductases are distinguished by their relative availability, stability, and wide use in synthetic applications, which include pharmaceutics, food and beverage treatments, environmental clean-up, and polymerizations. Two representatives of those groups-laccase (an oxidoreductase) and lipase (a hydrolase)-are discussed at length, including their structure, catalytic mechanism, and diverse usage. Objective representation of the current status and emerging trends are provided in the main conclusions.

Engineering Site 228 of Streptomyces coelicolor Laccase for Optimizing Catalytic Activity.

Malachite green (MG) poses a formidable threat to ecosystems and human health. Laccase emerges as a promising candidate for MG degradation, prompting an investigation into the catalytic activity modulation of a small laccase (SLAC) from Streptomyces coelicolor, with a focus on amino acid position 228. Through saturation mutagenesis, five mutants with a 50% increase in the specific activity were generated. Characterization revealed notable properties, Km of E228F was 8.8% of the wild type (WT), and E288T exhibited a 133% kcat compared to WT. Structural analyses indicated improved hydrophobicity and electrostatic potential on the mutants' surfaces, with the stable E228F-ABTS complex exhibiting reduced flexibility, possibly contributing to the observed decrease in turnover rate. Mutants demonstrated enhanced MG decolorization, particularly E228G. Site 228 acts as a crucial functional control switch, suggesting its potential role in SLAC engineering. This study provides insights into laccase modulation and offers promising avenues for enzymatic bioremediation applications.

Enhancement of the biological activity of hydroxytyrosol through its oxidation by laccase from Trametes versicolor.

The laccase-catalyzed oxidation of hydroxytyrosol (HT) towards the formation of its bioactive oligomer derivatives was investigated. The biocatalytic oligomerization was catalyzed by laccase from Trametes versicolor in aqueous or various water-miscible organic solvents and deep eutectic solvent (DES)-based media. Mass Spectroscopy and Nuclear Magnetic Resonance were used for the characterization of the products. The solvent system used significantly affects the degree of HT oligomerization. The use of 50 % v/v methanol favored the production of the HT dimer, while other organic solvents as well as DESs led to the formation of hydroxytyrosol trimer and other oligomers. In vitro studies showed that the HT dimer exhibits 3- to 4-fold enhanced antibacterial activity against Gram-positive and Gram-negative bacteria compared to the parent compound. Moreover, the ability of HT dimer to inhibit the activity of soybean lipoxygenase and Candida rugosa lipase was 1.5-fold higher than HT, while molecular docking supported these results. Furthermore, HT dimer showed reduced cytotoxicity against HEK293 cells and exhibited a strong ability to inhibit ROS formation. The enhanced bioactivity of HT dimer indicates that this compound could be considered for use in cosmetics, skin-care products, and nutraceuticals.

Boosting the LAC-like activity of tetrapeptide capped copper nanoparticle-based nanozymes for colorimetric determination of adrenaline.

Enzymatic activity is important for a variety of technological applications, but the limited stability and complex structures of enzymes often limit their use. Therefore, designing powerful nanomaterial catalysts that are more stable and have higher catalytic activity than natural catalysts has been the pursuit of biotechnology. Here, inspired by electron transfer and the active site of laccase (LAC), four types of copper particles with LAC-like activity were synthesized using a simple hydrothermal method. Copper particles coated with the L-phenylalanine (F)-L-phenylalanine (F)-L-cysteine (C)-L-histidine (H) tetrapeptide exhibited higher LAC-like activity compared to those coated with a CH dipeptide, C, and H. This enhancement could be attributed to the higher structural homology and amino acid composition similarity with the natural LAC active center. The FFCH@CuNP nanozyme was employed for adrenaline detection, and it demonstrated outstanding activity, stability, and recyclability. Additionally, a method for the quantitative detection of adrenaline was established using a smartphone based on the FFCH@CuNP nanozymes. And the FFCH@CuNPs exhibited excellent sensitivity and specificity to adrenaline in a saliva-based test. Therefore, this work provides a reasonable pathway for the design of catalysts for future biotechnological and industrial applications.

One-pot Twostep Chemobiocatalytic Synthesis of a Furan Amino Acid from 5-Hydroxymethylfurfural.

Recently, catalytic valorization of biomass-derived furans has received growing interest. 5-Aminomethyl-2-furancarboxylic acid (AMFC), a furan amino acid, holds great promise in the aeras of polymer and pharmaceutical, but its synthesis remains limited. In this work, we report a chemobiocatalytic route toward AMFC by combining laccase-TEMPO system and recombinant Escherichia coli (named E. coli_TAF) harboring ω-transaminase (TA), L-alanine dehydrogenase (L-AlaDH) and formate dehydrogenase (FDH), starting from 5-hydroxymethylfurfural (HMF). In the cascade, HMF is oxidized into 5-formyl-2-furancarboxylic acid (FFCA) by laccase-TEMPO system, and then the resulting intermediate is converted into AMFC by E. coli_TAF via transamination with cheap ammonium formate instead of costly organic amine donors, theoretically generating H2O and CO2 as by-products. The tandem process was run in a one-pot twostep manner, affording AMFC with approximately 81 % yield, together with 10 % 2,5-furandicarboxylic acid (FDCA) as by-product. In addition, the scale-up production of AMFC was demonstrated, with 0.41 g/L h productivity and 8.6 g/L titer. This work may pave the way for green manufacturing of the furan-containing amino acid.

Effective Decolorization and Detoxification of Single and Mixed Dyes with Crude Laccase Preparation from a White-Rot Fungus Strain Pleurotus eryngii.

To fully harness the potential of laccase in the efficient decolorization and detoxification of single and mixed dyes with diverse chemical structures, we carried out a systematic study on the decolorization and detoxification of single and mixed dyes using a crude laccase preparation obtained from a white-rot fungus strain, Pleurotus eryngii. The crude laccase preparation showed efficient decolorization of azo, anthraquinone, triphenylmethane, and indigo dyes, and the reaction rate constants followed the order Remazol Brilliant Blue R > Bromophenol blue > Indigo carmine > New Coccine > Reactive Blue 4 > Reactive Black 5 > Acid Orange 7 > Methyl green. This laccase preparation exhibited notable tolerance to SO42- salts such as MnSO4, MgSO4, ZnSO4, Na2SO4, K2SO4, and CdSO4 during the decolorization of various types of dyes, but was significantly inhibited by Cl- salts. Additionally, this laccase preparation demonstrated strong tolerance to some organic solvents such as glycerol, ethylene glycol, propanediol, and butanediol. The crude laccase preparation demonstrated the efficient decolorization of dye mixtures, including azo + azo, azo + anthraquinone, azo + triphenylmethane, anthraquinone + indigo, anthraquinone + triphenylmethane, and indigo + triphenylmethane dyes. The decolorization kinetics of mixed dyes provided preliminary insight into the interactions between dyes in the decolorization process of mixed dyes, and the underlying reasons and mechanisms were discussed. Importantly, the crude laccase from Pleurotus eryngii showed efficient repeated-batch decolorization of single-, two-, and four-dye mixtures. This crude laccase demonstrated high stability and reusability in repeated-batch decolorization. Furthermore, this crude laccase was efficient in the detoxification of different types of single dyes and mixed dyes containing different types of dyes, and the phytotoxicity of decolorized dyes (single and mixed dyes) was significantly reduced. The crude laccase efficiently eliminated phytotoxicity associated with single and mixed dyes. Consequently, the crude laccase from Pleurotus eryngii offers significant potential for practical applications in the efficient decolorization and management of single and mixed dye pollutants with different chemical structures.

Biotransformation of 2,4,6-Trinitrotoluene by a cocktail of native laccases from Pycnoporus sanguineus CS43 under oxygenic and non-oxygenic atmospheres.

2,4,6-Trinitrotoluene (TNT) is a highly toxic nitroaromatic explosive known for its environmental consequences, contaminating soil and groundwater throughout its life cycle, from production to disposal. Therefore, the urgency of developing innovative and ecological strategies to remedy the affected areas is recognized. This study reports, for the first time, the enzymatic biotransformation of TNT by a cocktail of native laccases from Pycnoporus sanguineus CS43. The laccases displayed efficient TNT conversion under both oxygenic and non-oxygenic conditions, achieving biotransformation rates of 80% and 87% within 48 h at a temperature of 60 °C and pH 7. Preliminary kinetic constants were calculated with the laccase cocktail, being a Vmax of 1.133 µM min-1 and 0.2984 µM min-1, and the Km values were 1586 µM and 458 µM, in an oxygenic and non-oxygenic atmosphere, respectively. High-performance liquid chromatography-mass spectrometry (HPLC/MS) confirmed the formation of amino dinitrotoluene isomers and hydroxylamine isomers as biotransformation products. In summary, this study suggests the potential application of laccases for the direct biotransformation of recalcitrant compounds like TNT, offering an environmentally friendly approach to address contamination issues.

A sustainable nano-hybrid system of laccase@M-MWCNTs for multifunctional PAHs and PhACs removal from water, wastewater, and lake water.

This study examined the use of modified multiwall carbon nanotubes (M-MWCNTs) with immobilized laccase (L@M-MWCNTs) for removing ciprofloxacin (Cip), carbamazepine (Cbz), diclofenac (Dcf), benzo[a]pyrene (Bap), and anthracene (Ant) from different water samples. The synthesized materials were characterized using an array of advanced analytical techniques. The physical immobilization of laccase onto M-MWCNTs was confirmed through Scanning electron microscope (SEM)-dispersive X-ray spectroscopy (EDS) analysis and Brunner-Emmet-Teller (BET) surface area measurements. The specific surface area of M-MWCNTs decreased by 65% upon laccase immobilization. There was also an increase in nitrogen content seen by EDS analysis asserting successful immobilization. The results of Boehm titration and Fourier transform infrared (FTIR) exhibited an increase in acidic functional groups after laccase immobilization. L@M-MWCNTs storage for two months maintained 77.8%, 61.6%, and 57.6% of its initial activity for 4 °C, 25 °C, and 35 °C, respectively. In contrast, the free laccase exhibited 55.3%, 37.5%, and 23.5% of its initial activity at 4 °C, 25 °C, and 35 °C, respectively. MWCNTs improved storability and widened the working temperature range of laccase. The optimum removal conditions of studied pollutants were pH 5, 25 °C, and 1.6 g/L of M-MWCNTs. These parameters led to >90% removal of the targeted pollutants for four treatment cycles of both synthetic water and spiked lake water. L@M-MWCNTs demonstrated consistent removal of >90% for up to five cycles even with spiked wastewater. The adsorption was endothermic and followed Langmuir isotherm. Oxidation, dehydrogenation, hydroxylation, and ring cleavage seem to be the dominant degradation mechanisms.

Co-immobilizing laccase-mediator system by in-situ synthesis of MOF in PVA hydrogels for enhanced laccase stability and dye decolorization efficiency.

The laccase mediator system (LMS) with a broad substrate range has attracted much attention as an efficient approach for water remediation. However, the practical application of LMS is limited due to their high solubility, poor stability and low reusability. Herein, the bimetallic Cu/ZIFs encapsulated laccase was in-situ grown in poly(vinyl alcohol) (PVA) polymer matrix. The PVA-Lac@Cu/ZIFs hydrogel was formed via one freeze-thawing cycle, and its catalytic stability was significantly improved. The mediator was further co-immobilized on the hydrogel, and this hierarchically co-immobilized ABTS/PVA-Lac@Cu/ZIFs hydrogel could avoid the continuous oxidation reaction between laccase and redox mediators. The co-immobilized LMS biocatalyst was used to degrade malachite green (MG), and the degradation rate was up to 100 % within 4 h. More importantly, the LMS could be recycled synchronously from the dye solutions and reused to degrade MG multiple times. The degradation rate remained above 69.4 % after five cycles. Furthermore, the intermediate products were detected via liquid chromatography-mass spectrometry, and the potential degradation pathways were proposed. This study demonstrated the significant potential of utilizing the MOF nanocrystals and hydrogel as a carrier for co-immobilized LMS, and the effective reuse of both laccase and mediator was promising for laccase application in wastewater treatment.

Preparation, structural characterization and properties of feruloyl oligosaccharide-rice protein hydrolysate conjugates.

Rice protein hydrolysate (RPH) and feruloyl oligosaccharides (FOs) were conjugated under the catalysis of laccase and free radical, and the structure and properties of the resultant conjugates were studied. Electrophoresis analysis demonstrated that conjugation with FOs increased the molecular weight of some fractions in RPH, which confirmed the formation of both conjugates. The conjugation degree of laccase-induced conjugate and radical-induced conjugate was 60.45% and 22.70%, respectively. Laccase-catalyzed conjugation decreased the tyrosine residue content of RPH but had no significant effect on the free amino group content, which suggested that tyrosine residues were the conjugation site in the laccase-induced conjugate. However, radical-catalyzed conjugation decreased both the free amino group content and the tyrosine residue content, which indicated that both free amino groups and tyrosine residues were the conjugation site in the radical-induced conjugate. The ultraviolet, fluorescence and circular dichroism spectroscopy analysis revealed that conjugation with FOs significantly altered the secondary and tertiary structure of RPH. In addition, conjugation with FOs increased the solubility and antioxidant activity of RPH but decreased the emulsifying activity and stability. Particularly, the radical-induced conjugate had greater anti-aggregation capacity and antioxidant activity but lower emulsifying activity and stability than the laccase-induced conjugate, which might be due to that their conjugation site and degree were different.

Enhancing the sustainable immobilization of laccase by amino-functionalized PMMA-reinforced graphene nanomaterial.

Human health and the environment are negatively affected by endocrine-disrupting chemicals (EDCs), such as bisphenol A. Therefore, developing appropriate remediation methods is essential for efficiently removing phenolic compounds from aqueous solutions. Enzymatic biodegradation is a potential biotechnological approach for responsibly addressing water pollution. With its high catalytic efficiency and few by-products, laccase is an eco-friendly biocatalyst with significant promise for biodegradation. Herein, two novel supporting materials (NH2-PMMA and NH2-PMMA-Gr) were fabricated via the functionalization of poly(methylmethacrylate) (PMMA) polymer using ethylenediamine and reinforced with graphene followed by glutaraldehyde activation. NH2-PMMA and NH2-PMMA-Gr were utilized for laccase immobilization with an immobilization yield (IY%) of 78.3% and 82.5% and an activity yield (AY%) of 81.2% and 85.9%, respectively. Scanning electron microscope (SEM) and Fourier-transform infrared (FTIR) were used to study the characteristics of fabricated material supports. NH2-PMMA-Gr@laccase exhibited an optimal pH profile from 4.5 to 5.0, while NH2-PMMA@laccase exhibited optimum pH at 5.0 compared to a value of 4.0 for free form. A wider temperature ranges of 40-50 °C was noted for both immobilized laccases compared to a value of 40 °C for the free form. Additionally, it was reported that immobilized laccase outperformed free laccase in terms of substrate affinity and storage stability. NH2-PMMA@laccase and NH2-PMMA-Gr@laccase improved stability by up to 3.9 and 4.6-fold when stored for 30 days at 4 °C and preserved up to 80.5% and 86.7% of relative activity after ten cycles of reuse. Finally, the degradation of BPA was achieved using NH2-PMMA@laccase and NH2-PMMA-Gr@laccase. After five cycles, NH2-PMMA@laccase and NH2-PMMA-Gr@laccase showed that the residual degradation of BPA was 77% and 84.5% using 50 µm of BPA. This study introduces a novel, high-performance material for organic pollution remediation in wastewater that would inspire further progress.

Enzymatic dual-faced Janus structures based on the hierarchical organic-inorganic hybrid matrix for an effective bioremoval and detoxification of reactive blue-19.

A novel, dual-faced, and hierarchical type of Janus hybrid structures (JHSs) was assembled through an in situ growing of lipase@cobalt phosphate sheets on the laccase@copper phosphate sponge-like structures. The chemical and structural information of prepared JHSs was investigated by Scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX), Fourier Transform Infrared Spectroscopy (FTIR), and X-ray diffraction analysis (XRD). The catalytic activity, storage stability, and reusability of JHSs were then investigated. The SEM-EDX analysis clearly confirmed the asymmetric morphology of the fabricated JHSs with two distinct metal distributions. Under optimized synthesis conditions, the prepared JHSs showed 97.8 % and 100 % of laccase and lipase activity, respectively. Compared to the free biocatalysts, the immobilization resulted in ~ a 2-fold increase in laccase and lipase stability at temperatures of >40 °C. The fabricated JHSs maintained 61 % and 90 % of their original laccase and lipase activity upon 12 successive repetition cycles. Up to 80 % of Reactive Blue-19 (RB-19), an anthraquinone-based vinyl sulphone dye, was removed after 5 h treatment with the prepared JHSs (50 % higher than the free forms of laccase and lipase). The dye removal data fitted very well on the pseudo-second-order kinetic model with a rate constant of 0.8 g mg-1 h-1. Following the bioremoval process, bacterial toxicity also decreased by about 70 %. Therefore, the prepared JHSs provide a facile and sustainable approach for the decolorization, biotransformation, and detoxification of RB-19 by integrating enzymatic oxidation and hydrolysis.

Molecular insights into substrate promiscuity of CotA laccase catalyzing lignin-phenol derivatives.

CotA laccases are multicopper oxidases known for promiscuously oxidizing a broad range of substrates. However, studying substrate promiscuity is limited by the complexity of electron transfer (ET) between substrates and laccases. Here, a systematic analysis of factors affecting ET including electron donor acceptor coupling (ΗDA), driving force (ΔG) and reorganization energy (λ) was done. Catalysis rates of syringic acid (SA), syringaldehyde (SAD) and acetosyringone (AS) (kcat(SAD) > kcat(SA) > kcat(AS)) are not entirely dependent on the ability to form phenol radicals indicated by ΔG and λ calculated by Density Functional Theory (SA < SAD ≈ AS). In determined CotA/SA and CotA/SAD structures, SA and SAD bound at 3.9 and 3.7 Å away from T1 Cu coordinating His419 ensuring a similar ΗDA. Abilities of substrate to form phenol radicals could mainly account for difference between kcat(SAD) and kcat(SA). Furthermore, substrate pocket is solvent exposed at the para site of substrate's phenol hydroxyl, which would destabilize binding of AS in the same orientation and position resulting in low kcat. Our results indicated shallow partially covered binding site with propensity of amino acids distribution might help CotA discriminate lignin-phenol derivatives. These findings give new insights for developing specific catalysts for industrial application.

Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.

Oxidation of chlortetracycline and its isomers by Botrytis aclada laccase in the absence of mediators: pH dependence and identification of transformation products by LC-MS.

Tetracyclines are antibiotics considered emerging pollutants and currently, wastewater treatment plants are not able to remove them efficiently. Laccases are promising enzymes for bioremediation because they can oxidize a wide variety of substrates. The aim of this study was to evaluate the Botrytis aclada laccase for the oxidation of chlortetracycline and its isomers in the absence of a mediator molecule, at a pH range between 3.0 to 7.0, and to characterize the transformation products by LC-MS. Chlortetracycline and three isomers were detected in both, controls and reaction mixtures at 0 h and in controls after 48 h of incubation but in different proportions depending on pH. An additional isomer was also detected, but only in the presence of BaLac. Based on the transformation products identified in the enzymatic reactions and information from literature, we assembled a network of transformation pathways starting from chlortetracycline and its isomers. The spectrometric analysis of the products indicated the probable occurrence of oxygen insertion, dehydrogenation, demethylation and deamination reactions. Four new products were identified, and we also described a novel transformation product without the chloro group. We observed that increasing pH led to higher diversity of main products. This is the first study using the laccase from fungi Botrytis aclada to oxidate chlortetracycline and its isomers and it can be considered as an ecological alternative to be used in bioremediation processes such as wastewater.

Recent advances in laccase activity assays: A crucial challenge for applications on complex substrates.

Despite being one of the first enzymes discovered in 1883, the determination of laccase activity remains a scientific challenge, and a barrier to the full use of laccase as a biocatalyst. Indeed, laccase, an oxidase of the blue multi-copper oxidases family, has a wide range of substrates including substituted phenols, aromatic amines and lignin-related compounds. Its one-electron mechanism requires only oxygen and releases water as a reaction product. These characteristics make laccase a biocatalyst of interest in many fields of applications including pulp and paper industry, biorefineries, food, textile, and pharmaceutical industries. But to fully envisage the use of laccase at an industrial scale, its activity must be reliably quantifiable on complex substrates and in complex matrices. This review aims to describe current and emerging methods for laccase activity assays and place them in the context of a potential industrial use of the enzyme.