Multicopper Oxidase Research Articles

Designing Artificial Laccase Catalysts by Introducing Substrate Oxidation Metals into Oxygen-Reducing Metal-Organic Frameworks: Cu-Doped ZIF-67.

Laccase, a multi-copper oxidase, is limited by its optimal temperature range and isolation costs. To overcome these challenges, we synthesized copper-doped zeolitic imidazolate framework-67 (Cu-doped ZIF-67) with 16 mol % Cu as an artificial laccase catalyst. The introduced Cu site acts as the phenol oxidation site, and Co-based ZIF-67 is the four-electron oxygen reduction site. Laccase also employs this division of oxidation and reduction sites. Cu-doped ZIF-67 demonstrated significant catalytic activity, superior to natural laccase, especially at elevated temperatures, and maintained stability across multiple reaction cycles. These findings suggest that Cu-doped ZIF-67 is a robust, reusable alternative for industrial applications requiring high thermal stability and efficient catalysis.

Tailoring CotA Laccase Substrate Specificity by Rationally Reshaping Pocket Edge.

CotA is a bacterial multicopper oxidase, capable of oxidizing lots of substrates. In previous work, small size lignin phenol derivates were found to lay only in the partially covered part of pocket. However, big size substate would occupy the whole pocket to react. In this work, five residues sitting at the edge of the pocket were selected to study their roles in regulating activities against different size substrates. All mutants showed impaired activities against small size sinapic acid, however, A227E, G321F and G321P showed around 25% increase of activities against big size ditaurobilirubin compared to wild type (WT). T262F/G321F showed moderate increased activity to alazin red S. kcat/Kms against ditaurobilirubin of A227E, T262F and G321F are around 1.5, 3 and 1.5 folds of WT's. Unexpectedly, heterologous expression yields of T262F, T262F/G321F and T262F/G321P in Escherichia coli greatly increased by around 5, 7 and 21 folds compared to WT, respectively. It is speculated positive mutants would provide a beneficial orientation for big size substrates. Substituting semi-buried residue T262 by a hydrophobic amino acid might enhance expression yields mainly by increasing van der waals and hydrophobic interaction. This work exemplified rationally regulating specific activities of laccase and is valuable for industrial application.

Substitution of the axial Type 1 Cu Ligand Afford Binding of a Water Molecule in Axial Position Affecting Kinetics, Spectral, and Structural Properties of the Small Laccase Ssl1.

Multicopper oxidases use Cu ions as cofactors to oxidize various substrates. High reduction potential at Type 1 Cu is considered as crucial for effective catalysis. Previous studies have shown that replacing the axial methionine ligand of the Type 1 Cu with leucine or phenylalanine leads to an increased reduction potential, but not always to higher enzyme activity. Here we present a study of six variants of the small laccase Ssl1 from Streptomyces sviceus, where the axial methionine ligand was substituted and the effect of the axial ligand on reduction potential, activity, spectral properties and structure was investigated. Absorption, electronic circular dichroism and EPR spectra revealed the presence of a stronger coordinating axial ligand like oxygen, which influences the electronic and catalytic properties more than the nature of the amino acid side chain. The crystal structures of the Ssl1 variants were solved, which show that none of the amino acids side chains coordinates to the Cu. Instead, a water molecule is found in the axial coordination site, which support the spectroscopic data. Our findings highlight the importance of combining structural and spectroscopic methods to investigate the effect of amino acid exchange on multicopper oxidases.

Identification of Enzymes and Their Key Action Sites for Histamine Degradation in Mulberry Fruit Wine by Lactiplantibacillus plantarum.

In this study, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and multicopper oxidase (MCO) of Lactiplantibacillus plantarum W155 with histamine degradation ability were expressed. The mulberry fruit wine (MFW) histamine degradation abilities of GAPDH and MCO were 20.81% and 37.67%, respectively. Compared with the control group, the MFW treated by GAPDH showed higher total phenolic (1.17 g GAE/L) and total flavonoid (0.31 g RE/L) contents, while MFW treated by MCO presented similar total phenolic (1.00 g GAE/L) and total flavonoid (0.29 g RE/L) concentrations. Furthermore, the optimal pH and temperature of GAPDH were 6.0 and 40 °C, respectively, while the optimal pH and temperature of MCO were 3.0 and 50 °C, respectively. Meanwhile, the key action sites for histamine degradation of GAPDH and MCO were minded via homology modeling, molecular docking, and site-directed mutagenesis. Val209 and Ile290 were confirmed as the key action sites for GAPDH, while Qln402 and Leu420 were the pivotal action sites for MCO. Above findings indicated that both GAPDH and MCO of L. plantarum W155 could be used to control the histamine of MFW, and the key action sites of these two enzymes could be used as targets for their subsequent modification.

Mutations of methionine 444 interacting with T1Cu-coordinating amino acids affect the structure and function of multicopper oxidase CopA.

Manganese is an essential trace element for humans, animals, and plants, but excessive amounts of manganese can cause serious harm to organisms. The biological manganese oxidation process mainly oxidizes Mn(II) through the secretion of unique manganese oxidase by manganese-oxidizing bacteria. The T1 Cu site of multicopper oxidase is the main site for substrate oxidation, and its role is to transfer electrons to TNC, where dioxygen reduction occurs. In this study, methionine (Met) No. 444 interacting with the T1Cu-coordinating amino acid in the multicopper oxidase CopA from Brevibacillus panacihumi MK-8 was mutated to phenylalanine (Phe) and leucine (Leu) by the enzyme. Based on the analysis of enzymatic properties and the structural model, the mutant protein M444F with 4.58 times the catalytic efficiency of the original protein CopA and the mutant protein M444L with 1.67 times the catalytic efficiency of the original protein CopA were obtained. The study showed that the manganese removal rate of the manganese-oxidizing engineered bacterium Rosetta-pET-copAM444L cultured for 7 days was 88.87%, which was 10.77% higher than that of the original engineered bacterium. Overall, this study provides a possibility for the application of genetic engineering in the field of biological manganese removal.

Growth of soil ammonia-oxidizing archaea on air-exposed solid surface.

Soil microorganisms often thrive as microcolonies or biofilms within pores of soil aggregates exposed to the soil atmosphere. However, previous studies on the physiology of soil ammonia-oxidizing microorganisms (AOMs), which play a critical role in the nitrogen cycle, were primarily conducted using freely suspended AOM cells (planktonic cells) in liquid media. In this study, we examined the growth of two representative soil ammonia-oxidizing archaea (AOA), Nitrososphaera viennensis EN76 and "Nitrosotenuis chungbukensis" MY2, and a soil ammonia-oxidizing bacterium, Nitrosomonas europaea ATCC 19718 on polycarbonate membrane filters floated on liquid media to observe their adaptation to air-exposed solid surfaces. Interestingly, ammonia oxidation activities of N. viennensis EN76 and "N. chungbukensis" MY2 were significantly repressed on floating filters compared to the freely suspended cells in liquid media. Conversely, the ammonia oxidation activity of N. europaea ATCC 19718 was comparable on floating filters and liquid media. N. viennensis EN76 and N. europaea ATCC 19718 developed microcolonies on floating filters. Transcriptome analysis of N. viennensis EN76 floating filter-grown cells revealed upregulation of unique sets of genes for cell wall and extracellular polymeric substance biosynthesis, ammonia oxidation (including ammonia monooxygenase subunit C (amoC3) and multicopper oxidases), and defense against H2O2-induced oxidative stress. These genes may play a pivotal role in adapting AOA to air-exposed solid surfaces. Furthermore, the floating filter technique resulted in the enrichment of distinct soil AOA communities dominated by the "Ca. Nitrosocosmicus" clade. Overall, this study sheds light on distinct adaptive mechanisms governing AOA growth on air-exposed solid surfaces.

Activity of novel virus families infecting soil nitrifiers is concomitant with host niche differentiation.

Chemolithoautotrophic nitrifiers are model groups for linking phylogeny, evolution, and ecophysiology. Ammonia-oxidising bacteria (AOB) typically dominate the first step of ammonia oxidation at high ammonium supply rates, ammonia-oxidising archaea (AOA) and complete ammonia-oxidising Nitrospira (comammox) are often active at lower supply rates or during AOB inactivity, and nitrite-oxidising bacteria (NOB) complete canonical nitrification. Soil virus communities are dynamic but contributions to functional processes are largely undetermined. In addition, characterising viruses infecting hosts with low relative abundance, such as nitrifiers, may be constrained by vast viral diversity, partial genome recovery, and difficulties in host linkage. Here, we describe a targeted incubation study that aimed to determine whether growth of different nitrifier groups in soil is associated with active virus populations and if process-focussed analyses facilitate characterisation of high-quality virus genomes. dsDNA viruses infecting different nitrifier groups were enriched in situ via differential host inhibition. Growth of each nitrifier group was consistent with predicted inhibition profiles and concomitant with the abundance of their viruses. These included 61 high-quality/complete virus genomes 35-173kb in length with minimal similarity to validated families. AOA viruses lacked ammonia monooxygenase sub-unit C (amoC) genes found in marine AOA viruses but some encoded AOA-specific multicopper oxidase type 1 (MCO1), previously implicated in copper acquisition, and suggesting a role in supporting energy metabolism of soil AOA. Findings demonstrate focussed incubation studies facilitate characterisation of active host-virus interactions associated with specific processes and viruses of soil AOA, AOB and NOB are dynamic and potentially influence nitrogen cycling processes.

Electron Paramagnetic Resonance Analysis of Hydroquinone Polymerization Catalyzed by Small Laccase

AbstractLaccases are multi‐copper oxidases (MCOs) that oxidize a broad range of substrates while reducing molecular oxygen to water. Although much effort has been made to elucidate the catalytic mechanism of laccases, information about reactive catalytic intermediates during catalysis is not yet fully understood. Herein, hydroquinone (HQ) polymerization catalyzed by prokaryotic small laccase (SLAC) was investigated by electron paramagnetic resonance (EPR) spectroscopy to provide more information on the catalytic mechanism. Briefly, free radical intermediates during the catalysis were investigated by real‐time in situ EPR measurement to monitor the formation and transformation of free radicals. A paramagnetic species was identified which was attributed to a copolymer formed by hydroquinone, benzoquinone, and semiquinone radical. In addition, the low‐temperature EPR spectrum of the trinuclear copper center during catalysis not only revealed tentative rotational motion of the histidine imidazole rings coordinating the TNC upon electron uptake but also provided evidence of the formation of oxygen bridge at TNC during the reaction, represented by the observation of a new type 2 copper component featured larger g// values and smaller A// values. Together, EPR spectroscopic insights into the catalytic intermediates during enzymatic hydroquinone polymerization have extended the knowledge of the biophysical characteristics and catalytic mechanism of SLAC.

Methionine-rich domains emerge as facilitators of copper recruitment in detoxification systems

Copper homeostasis mechanisms are critical for bacterial resistance to copper-induced stress. The Escherichia coli multicopper oxidase copper efflux oxidase (CueO) is part of the copper detoxification system in aerobic conditions. CueO contains a methionine-rich (Met-rich) domain believed to interact with copper, but its exact function and the importance of related copper-binding sites remain unclear. This study investigates these open questions by employing a multimodal and multiscale approach. Through the design of various E. coli CueO (EcCueO) variants with altered copper-coordinating residues and domain deletions, we employ biological, biochemical, and physico-chemical approaches to unravel in vitro CueO catalytic properties and in vivo copper resistance. Strong correlation between the different methods enables evaluation of EcCueO variants' activity as a function of Cu+ availability. Our findings demonstrate the Met-rich domain is not essential for cuprous oxidation, but it facilitates Cu+ recruitment from strongly chelated forms, acting as transient copper binding domain thanks to multiple methionines. They also indicate that the Cu6/7 copper-binding sites previously observed within the Met-rich domain play a negligible role. Meanwhile, Cu5, located at the interface with the Met-rich domain, emerges as the primary and sole substrate-binding active site for cuprous oxidation. The Cu5 coordination sphere strongly affects the enzyme activity and the in vivo copper resistance. This study provides insights into the nuanced role of CueO Met-rich domain, enabling the functions of copper-binding sites and the entire domain itself to be decoupled. This paves the way for a deeper understanding of Met-rich domains in the context of bacterial copper homeostasis.

Expression of laccase and ascorbate oxidase affects lignin composition in Arabidopsis thaliana stems.

Lignin is a phenolic polymer that is a major source of biomass. Oxidative enzymes, such as laccase and peroxidase, are required for lignin polymerisation. Laccase is a member of the multicopper oxidase family and has a high amino acid sequence similarity with ascorbate oxidase. However, the process of functional differentiation between the two enzymes remains poorly understood. In this study, the common ancestry sequence of laccase and ascorbate oxidase (AncMCO) was predicted via phylogenetic reconstruction, and its in vivo effect on lignin biosynthesis in Arabidopsis thaliana was assessed. The estimated AncMCO sequence conserved key residues that coordinate with copper ions, implying that the electron transfer system is likely to be conserved in AncMCO. However, multiple insertions/deletions corresponding to protein surface structures have been found between laccase, ascorbate oxidase, and AncMCO. The overexpression of canonical laccase (AtLAC4) and ascorbate oxidase (AtAAO1) in A. thaliana resulted in notable increases of syringyl/guaiacyl lignin unit ratio in stems, whereas, in contrast, the overexpression of AncMCO did not show any detectable change in lignin deposition. Transcriptomic analysis revealed that the AtAAO1-overexpressing line exhibited significant changes in the expression of a wide range of cell wall biosynthesis genes. These results highlight the importance of the molecular evolution of multicopper oxidase, which drives lignin biosynthesis during plant evolution.

Enzyme-Modified Microelectrode for Simultaneous Local Measurements of O2 and pH.

The use of miniaturized probes opens a new dimension in the analysis of (bio)chemical processes, enabling the possibility to perform measurements with local resolution. In addition, multiparametric measurements are highly valuable for a holistic understanding of the investigated process. Therefore, different strategies have been suggested for simultaneous local measurements of various parameters. Electroanalytical methods are a powerful strategy in this direction. However, they have been mainly restricted to coupling concurrent independent measurements with different miniaturized probes. Here, we present an enzymatic microbiosensor for the simultaneous detection of O2 and pH. The sensing strategy is based on the pH-dependent bioelectrocatalytic process associated with O2 reduction at a gold microelectrode modified with a multicopper oxidase. After initial investigations of the bioelectrocatalytic reaction over gold macroelectrodes, the fabrication and characterization of micrometer-sized probes are presented. The microbioelectrode exhibits a linear current increase with O2 concentration extending to 17.2 mg L-1, with a sensitivity of (5.56 ± 0.13) nA L mg-1 and a limit of detection of (0.5 ± 0.3) mg L-1. Moreover, a linear response allowing pH detection is obtained between pH 5.2 and 7.5 with a slope of -(47 ± 8) mV per pH unit. In addition, two proof-of-concept analytical examples are shown, demonstrating the capability of the developed sensing system for simultaneous local measurements of O2 and pH. Compared with other miniaturized probes reported before for simultaneous detection, our strategy stands out as the two investigated parameters are acquired from the very same measurement. This strategy greatly simplifies the analytical setup and for the first time provides truly simultaneous local detection in the micrometer scale.

High metal-loaded sub-nanocluster catalyst enhanced Fenton-like reaction activity for emerging contaminants degradation by generating high-valent copper

High-valent copper (Cu(III)) species are crucial intermediates in CH bond functionalization within both the biological and biomimetic processes, facilitated by copper enzymes and stabilized by multicopper oxidases. Herein, a nitrogen-doped copper sub-nanocluster catalyst (SNC) featured a high metal content (29.1 wt%) and uniform Cu distribution was synthesized by carbonizing the nanosheet-like metal–organic framework (MOF), enhancing bisphenol A degradation by activating peroxymonosulfate (PMS) to generate Cu(III). The degradation performance of the SNC outperformed the catalyst carbonized with the bulk-like MOF and matched the reported single-atom catalysts counterparts. Nitrogen doping decreased the electrons of Cu3d orbital, enhancing its bonding with the oxygen atom within the PMS molecule, thus promoting Cu(III) generation. The Cu-SNC/PMS system also showed robust resistance against anions, pH changes, and diverse water matrices. Importantly, it can selectively degrade electron-rich pollutants through oxygen atom transfer by Cu(III). This study provided new perspectives into SNC preparation, the controlled formation of high-valent metal species, and their role in Fenton-like reactions for pollutants degradation.

Effect of Cu(II) and Conserved Copper Binding Sites onthe Multicopper Oxidase CopA and Characterization ofBioMnOx.

The microbial manganese removal process is believed to consist of the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and exhibited high manganese oxidation activity in vitro, and it was found that Cu(II) can significantly enhance its manganese oxidation activity. Gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA to obtain four mutant strains. The manganese removal efficiencies of the four strains were determined, and it was found that H120 is the catalytically active site of CopA. The loss of Cu(II) and the mutation of the conserved copper binding site H120 resulted in the loss of ethoxyformyl and quinone modifications, a reduction in the number of modifications, and a change in the position of modifications, eventually causing a decrease in protein activity from 85.87% to 70.1%. These results reveal that Cu(II) and H120 play an indispensable role in manganese oxidation by the multicopper oxidase CopA. X-ray photoelectron spectroscopy (XPS) analysis indicates that biogenic manganese oxides produced by strains and by CopA were both composed of MnO2 and Mn3O4 and that the average valence of Mn was 3.2.

Degradation of Amyloid-β Species by Multi-Copper Oxidases.

Reduction of the production of amyloid-β (Aβ) species has been intensively investigated as potential therapeutic approaches for Alzheimer's disease (AD). However, the degradation of Aβ species, another potential beneficial approach, has been far less explored. To investigate the potential of multi-copper oxidases (MCOs) in degrading Aβ peptides and their potential benefits for AD treatment. We investigated the degradation efficiency of MCOs by using electrophoresis and validated the ceruloplasmin (CP)-Aβ interaction using total internal reflection fluorescence microscopy, fluorescence photometer, and fluorescence polarization measurement. We also investigated the therapeutic effect of ascorbate oxidase (AO) by using induced pluripotent stem (iPS) neuron cells and electrophysiological analysis with brain slices. We discovered that CP, an important MCO in human blood, could degrade Aβ peptides. We also found that other MCOs could induce Aβ degradation as well. Remarkably, we revealed that AO had the strongest degrading effect among the tested MCOs. Using iPS neuron cells, we observed that AO could rescue neuron toxicity which induced by Aβ oligomers. In addition, our electrophysiological analysis with brain slices suggested that AO could prevent an Aβ-induced deficit in synaptic transmission in the hippocampus. To the best of our knowledge, our report is the first to demonstrate that MCOs have a degrading function for peptides/proteins. Further investigations are warranted to explore the possible benefits of MCOs for future AD treatment.

Streptomyces small laccase expressed in Aspergillus Niger as a new addition for the lignocellulose bioconversion toolbox

Laccases are multi-copper oxidases that are usually composed of three Cu-oxidase domains. Domains one and three house the copper binding sites, and the second domain is involved in forming a substrate-binding cleft. However, Streptomyces species are found to have small laccases (SLAC) that lack one of the three Cu-oxidase domains. This type of SLAC with interesting lignocellulose bioconversion activities has not been reported in Aspergillus niger. In our research, we explored the expression and engineering of the SLAC from Streptomyces leeuwenhoekii C34 in A. niger. Genes encoding two versions of the SLAC were expressed. One encoding the SLAC in its native form and a second encoding the SLAC fused to two N-terminal CBM1 domains. The latter is a configuration also known for specific yeast laccases. Both SLAC variants were functionally expressed in A. niger as shown by in vitro activity assays and proteome analysis. Laccase activity was also analyzed toward bioconversion of lignocellulosic rice straw. From this analysis it was clear that the SLAC activity improved the efficiency of saccharification of lignocellulosic biomass by cellulase enzyme cocktails.

Laccase-assisted Bioremediation of Pesticides: Scope and Challenges

Abstract: Laccase (Benzenediol: oxygen oxidoreductase; E.C.1.10.3.2), a multicopper oxidase that is a known lignin-degrading enzyme, can catalyse an ample array of substrates, from phenolic, nonphenolic compounds, aromatic amines, diamines, heterocyclic compounds to organic/inorganic metal compounds, etc., bestowed they have not too high redox potentials. Despite many laccase-producing organisms like bacteria, insects, plants, and animals, white rot filamentous fungi are the best producers of this enzyme. In the presence of laccase, pesticides (fungicides, herbicides, insecticides, etc.) of various chemical compositions (organophosphates, organochlorines, carbamates, pyrethrin & pyrethroids, etc.) are oxidized into the water with collateral reduction of four electrons of molecular oxygen with various efficiencies. Bioremediation efficiency can be increased in the presence of various natural or synthetic mediators, viz. ABTS, violuric acid, 1- hydroxy benzotriazole, vanillin, syringaldehyde, PEG, etc. Immobilized laccase on various supporting materials increased the enzyme's stability, reliability, and reusability for continuous application, particularly for industrial processes. The present review discusses the structure, catalytic cycle, general mechanism of oxidation, and various scopes and challenges of pesticide degradation by this multifaceted biocatalyst which could lead to a green sustainable environment.

Recent Developments and Challenges in the Application of Fungal Laccase for the Biodegradation of Textile Dye Pollutants

Abstract: According to the European Environment Agency, the textile industry is responsible for 20% of global water pollution due to dyeing and finishing products, thus facing severe environmental challenges. It is essential to design more biocompatible and sustainable treatment processes capable of removing dyes from industrial wastewater to fight this environmental hazard. Chemical industries must change traditional chemical-based concepts to more environmentally friendly and greener processes to remove pollutants, including dyes. Enzymatic bioremediation is a smart tool and a promising alternative for environmental pollutant degradation. The use of enzymes in dye decolourization makes the process a green and clean alternative to conventional chemical treatments. Moreover, enzymemediated biocatalysis decreases the formation of toxic by-products compared to chemical reactions. The most used enzyme for the decolourization of dyes is laccase. Laccase is a multicopper oxidase found in diverse organisms such as fungi. It promotes the oxidation of phenolic compounds and has a wide range of substrate specificity, making it a promising enzyme for removing different dyes used by the textile industry, including recalcitrant aromatic dyes. The present article gives a comprehensive revision of textile dye decolourization, its types, recent developments in laccase-mediated dye bioremediation technologies, the mechanism of biocatalysis, and their limitations and challenges. Emphasis on the chemical pathways of laccase reaction mechanisms for dye bioremediation processes is also provided. In addition, a brief overview of textile industries and the respective traditional treatment processes for textile wastewater is presented.

Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization.

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The β-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE3F3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.

Biochemical and Structural Characterization of a Novel Psychrophilic Laccase (Multicopper Oxidase) Discovered from Oenococcus oeni 229 (ENOLAB 4002).

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K4[Fe(CN6)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments.

Harnessing the chromium reduction potential of Pseudomonas aeruginosa JRHM33: A comprehensive study on bioinformatics, phenotype microarray, and CCD-RSM optimization

Large amounts of wastewater are generated due to overpopulation and industrialization, The bioavailability, toxicity, and permanence of metals make heavy metal contamination a big environmental hazard. In order to maximize chromium (Cr+6) removal efficiency, the current investigation was carried out from industrial wastewater using Pseudomonas aeruginosa JRHM33.35 bacterial strains were discovered based on their physical, and biochemical properties and resistance towards chromium (Cr+6) heavy metal. The most significant bacterial strain JRHM33 found the highest-level of 1000 mg/L of chromium (Cr+6) resistance. The bacterial strain JRHM33, which has 99 % similarity to Pseudomonas aeruginosa, was found using 16 S rRNA sequencing and is employed in subsequent steps. Sequencing and study of conserved domains indicate that JRHM33 contains the laccase gene and belongs to the multicopper oxidase superfamily, which is known for its ability to reduce metal ions. Analysing phenotype microarray (PM) technology sheds light on Pseudomonas aeruginosa JRHM33 metabolic profile of microbial cells. Additionally, a series of process parameter optimizations were tried using the central composite design of response surface methodology (CCD-RSM) in an effort to reduce the amount of chromium (Cr+6) in the effluent as much as possible. At 6.8 pH, 90 min of incubation, inoculum size is 3.8 ml, and agitation is 104 rpm, a maximum 71 % Cr+6 reduction was attained. The model constructed has an R2 score of 0.983 indicates a very statistically significant outcome from the analysis of variance. The experimental outcomes and the predicted results were remarkably similar, according to the validation experiment. Studies have revealed that bacterial strains obtained from effluent containing high levels of metals utilize their inherent capability to change harmful heavy metals into less dangerous or harmless forms.