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Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases that catalyze the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. In fungi, laccases carry out a variety of physiological roles during their life cycle. These enzymes are being increasingly evaluated for a variety of biotechnological applications due to their broad substrate range. In this review, the most recent studies on laccase structural features and catalytic mechanisms along with analyses of their expression are reported and examined with the aim of contributing to the discussion on their structure-function relationships. Attention has also been paid to the properties of enzymes endowed with unique characteristics and to fungal laccase multigene families and their organization.

BsCotA laccase is a promising candidate for industrial application due to its excellent thermal stability. In this research, our objective was to enhance the catalytic efficiency of BsCotA by modifying the active site pocket. We utilized a strategy combining the diversity design of the active site pocket with molecular docking screening, which resulted in selecting five variants for characterization. All five variants proved functional, with four demonstrating improved turnover rates. The most effective variants exhibited a remarkable 7.7-fold increase in catalytic efficiency, evolved from 1.54 × 10 5  M −1  s −1 to 1.18 × 10 6  M −1  s −1 , without any stability loss. To investigate the underlying molecular mechanisms, we conducted a comprehensive structural analysis of our variants. The analysis suggested that substituting Leu386 with aromatic residues could enhance BsCotA’s ability to accommodate the 2,2′-azino-di-(3-ethylbenzothiazoline)-6-sulfonate (ABTS) substrate. However, the inclusion of charged residues, G323D and G417H, into the active site pocket reduced k cat . Ultimately, our research contributes to a deeper understanding of the role played by residues in the laccases’ active site pocket, while successfully demonstrating a method to lift the catalytic efficiency of BsCotA. Key points • Active site pocket design that enhanced BsCotA laccase efficiency • 7.7-fold improved in catalytic rate • All tested variants retain thermal stability

The aim of this work to study an efficient laccase producing fungus Ganoderma leucocontextum, which was identified by ITS regions of DNA and phylogenetic tree was constructed. This study showed the laccase first-time from G. leucocontextum by using medium containing guaiacol. The growth cultural (pH, temperature, incubation days, rpm) and nutritional (carbon and nitrogen sources) conditions were optimized, which enhanced the enzyme production up to 4.5-folds. Laccase production increased 855 U/L at 40 °C. The pH 5.0 was suitable for laccase secretion (2517 U/L) on the 7th day of incubation at 100 rpm (698.3 U/L). Glucose and sucrose were good carbon source to enhance the laccase synthesis. The 10 g/L beef (4671 U/L) and yeast extract (5776 U/L) were the best nitrogen source for laccase secretion from G. leucocontextum. The laccase was purified from the 80% ammonium sulphate precipitations of protein identified by nucleotides sequence. The molecular weight (65.0 kDa) of purified laccase was identified through SDS and native PAGE entitled as Glacc110. The Glacc110 was characterized under different parameters. It retained > 90% of its activity for 16 min incubation at 60 °C in acidic medium (pH 4.0). This enzyme exerted its optimal activity at pH 3.0 and temperature 70 °C with guaiacol substrate. The catalytic parameters K m and V max was 1.658 (mM) and 2.452 ( mM/min), respectively. The thermo stability of the laccase produced by submerged fermentation of G. leucocontextum has potential for industrial and biotechnology applications. The results remarked the G. leucocontextum is a good source for laccase production.

Laccases are versatile biocatalysts with broad industrial relevance. Their heterologous expression enables efficient production, purification, and functional optimization. The white-rot fungus Hirschioporus abietinus produces an effective extracellular laccase (Lac2), inspiring the identification and cloning of its encoding gene. To enable high and stable enzyme production, the gene was expressed in Pichia pastoris and the cultivation conditions for the selected variant were optimized to enhance the yield of recombinant laccase. The Lac2 was then purified and its biochemical properties characterized. The high-redox potential laccase Lac2 exhibited strong tolerance to common metal ions and maintained catalytic activity in the presence of a range of organic solvents. Overall, the results suggest that Lac2 possesses properties compatible with small-scale production and effective use in biosensor systems.

Summary Laccases are multicopper containing enzymes capable of performing one electron oxidation of a broad range of substrates. Using molecular oxygen as the final electron acceptor, they release only water as a by‐product, and as such, laccases are eco‐friendly, versatile biocatalysts that have generated an enormous biotechnological interest. Indeed, this group of enzymes has been used in different industrial fields for very diverse purposes, from food additive and beverage processing to biomedical diagnosis, and as cross‐linking agents for furniture construction or in the production of biofuels. Laccases have also been studied intensely in nanobiotechnology for the development of implantable biosensors and biofuel cells. Moreover, their capacity to transform complex xenobiotics makes them useful biocatalysts in enzymatic bioremediation. This review summarizes the most significant recent advances in the use of laccases and their future perspectives in biotechnology. Laccases are versatile green biocatalyst at the cutting edge of biotechnology. This review summarizes the most significant advances in the use of laccase in different biotechnological settings from organic synthesis or food and paper industries to biomedical applications and beyond.

The aim of the present research was the efficient degradation of industrial textile wastewater dyes using a very active cloned laccase enzyme. For this purpose, potent laccase-producing bacteria were isolated from soil samples collected from wastewater-replenished textile sites in Punjab, Pakistan. The laccase gene from locally isolated strain LI-81, identified as Bacillus megaterium, was cloned into vector pET21a, which was further transformed into E. coli BL21 codon plus. The optimized conditions for the increased production of laccase include fermentation in a 2% glucose, 5% yeast extract and 250 mg/L CuSO4 medium with pH 7.5; inoculation with 5% inoculum; induction with 0.1 mM IPTG at 0.5 O.D.; and incubation for 36 h at 37 °C. The crude enzyme produced was employed for the removal of commercially used textile dyes. The dyes were quickly precipitated under optimized reaction conditions. Rose bengal, brilliant green, brilliant blue G, Coomassie brilliant blue R and methylene blue were precipitated at rates of 10.69, 54.47, 84.04, 78.99 and 7.40%, respectively. The FTIR and UV–Vis spectroscopic analyses of dyes before and after confirmed the chemical changes brought about by the cloned laccase that led to the dye removal.

Lignin is an important phenolic biopolymer that provides strength and rigidity to the secondary cell walls of tracheary elements, sclereids, and fibers in vascular plants. Lignin precursors, called monolignols, are synthesized in the cell and exported to the cell wall where they are polymerized into lignin by oxidative enzymes such as laccases and peroxidases. In Arabidopsis thaliana, a peroxidase (PRX64) and laccase (LAC4) are shown to localize differently within cell wall domains in interfascicular fibers: PRX64 localizes to the middle lamella whereas LAC4 localizes throughout the secondary cell wall layers. Similarly, laccases localized to, and are responsible for, the helical depositions of lignin in protoxylem tracheary elements. In addition, we tested the mobility of laccases in the cell wall using fluorescence recovery after photobleaching. mCHERRY-tagged LAC4 was immobile in secondary cell wall domains, but mobile in the primary cell wall when ectopically expressed. A small secreted red fluorescent protein (sec-mCHERRY) was engineered as a control and was found to be mobile in both the primary and secondary cell walls. Unlike sec-mCHERRY, the tight anchoring of LAC4 to secondary cell wall domains indicated that it cannot be remobilized once secreted, and this anchoring underlies the spatial control of lignification.

Laccases belong to the superfamily of multicopper oxidases (MCO), a group of enzymes with the ability to reduce oxygen to water in a reaction without producing harmful byproducts. Laccase activity is influenced by many factors, such as structure; the number, location and binding status of copper ions; and the substrate‐binding status. A large number of sequences that have not been experimentally characterized yet have been annotated as laccases. However, the biological functions of the characterized MCOs are considered to vary, and the substrate spectrum overlaps with that of other MCOs. Here, we identified 34 putative bacterial laccase sequences from metagenome data for industrial wastewater. We used machine‐learning tools to screen enzymes with laccase activity by combining the T1 copper‐binding capacity, the overall copper‐binding capacity and the substrate‐binding capacity. We also used the software comparisons to remove sequences with large discrepancies between different software applications. Three‐dimensional structures of identified enzymes were predicted using alphafold, the positions of metal ions within the proteins were predicted by metal3d and autodock‐vina, and their docking with ABTS [i.e. 2,2′‐azinobis(3‑ethylbenzo‐6‑thiazolinesulfonic acid)] as a substrate was predicted by rosetta and autodock‐vina. Based on the docking results, we selected 10 high‐scoring proteins, two low‐scoring proteins and one composite protein for expression using the pET‐21d (+) vector. In line with our predictions, all selected high‐scoring proteins exhibited activity towards ABTS. Overall, we describe a method for discovering and designing novel bacterial laccase‐like multicopper oxidases, offering increased possibilities for the degradation of various harmful components derived from environmental pollution. We obtained potential bacterial laccase‐like multicopper oxidase (LMCO) sequences through metagenomic sequencing. All sequences exhibited significant differences from known LMCOs in databases. To select the most promising candidates, we performed structure prediction and molecular docking using alphafold2, metal3d and rosetta. Ultimately, all selected proteins identified with laccase activity.

Laccases are a class of multi-copper oxidases (MCOs) that catalyze the one-electron oxidation of four equivalents of a reducing substrate, with the concomitant four-electron reduction of dioxygen to water. They can catalyze a multitude of reactions, including the degradation of polymers and oxidative coupling of phenolic compounds, positioning them as significant industrial enzymes. Although fungal laccases are well known and well characterized, only recently has in silico biology led to rapid advances in the discovery, characterization and engineered expression of prokaryotic laccases. We describe the recent burgeoning of prokaryotic laccases, their catalytic properties, structural features and molecular evolution, vis-à-vis fungal laccases where possible. Special focus is given to the application of laccases to the emerging cellulosic biofuel industry.

Background Hazardous synthetic dye wastes have become a growing threat to the environment and public health. Fungal enzymes are eco-friendly, compatible and cost-effective approach for diversity of applications. Therefore, this study aimed to screen, optimize fermentation conditions, and characterize laccase from fungal endophyte with elucidating its ability to decolorize several wastewater dyes. Results A new fungal endophyte capable of laccase-producing was firstly isolated from cladodes of Opuntia ficus - indica and identified as T. harzianum AUMC14897 using ITS-rRNA sequencing analysis. Furthermore, the response surface methodology (RSM) was utilized to optimize several fermentation parameters that increase laccase production. The isolated laccase was purified to 13.79-fold. GFC, SDS-PAGE revealed laccase molecular weight at 72 kDa and zymogram analysis elucidated a single band without any isozymes. The peak activity of the pure laccase was detected at 50 °C, pH 4.5, with thermal stability up to 50 °C and half life span for 4 h even after 24 h retained 30% of its activity. The K m and V max values were 0.1 mM, 22.22 µmol/min and activation energy (E a ) equal to 5.71 kcal/mol. Furthermore, the purified laccase effectively decolorized various synthetic and real wastewater dyes. Conclusion Subsequently, the new endophytic strain produces high laccase activity that possesses a unique characteristic, it could be an appealing candidate for both environmental and industrial applications.