Infection of grapevines with the grey mold pathogen Botrytis cinerea results in severe problems for winemakers worldwide. Browning of wine is caused by the laccase-mediated oxidation of polyphenols. In the last decades, Botrytis management has become increasingly difficult due to the rising number of resistances and the genetic variety of Botrytis strains. During the search for sustainable fungicides, polyphenols showed great potential to inhibit fungal growth. The present study revealed two important aspects regarding the effects of grape-specific polyphenols and their polymerized oxidation products on Botrytis wild strains. On the one hand, laccase-mediated oxidized polyphenols, which resemble the products found in infected grapes, showed the same potential for inhibition of growth and laccase activity, but differed from their native forms. On the other hand, the impact of phenolic compounds on mycelial growth is not correlated to the effect on laccase activity. Instead, mycelial growth and relative specific laccase activity appear to be modulated independently. All phenolic compounds showed not only inhibitory but also inductive effects on fungal growth and/or laccase activity, an observation which is reported for the first time. The simultaneous inhibition of growth and laccase activity demonstrated may serve as a basis for the development of a natural botryticide. Yet, the results showed considerable differences between genetically distinguishable strains, impeding the use of a specific phenolic compound against the genetic variety of wild strains. The present findings might have important implications for future understanding of Botrytis cinerea infections and sustainable Botrytis management including the role of polyphenols.

Improper waste disposal or inadequate wastewater treatment can result in pharmaceuticals reaching water bodies, posing environmental hazards. In this study, crude extracts containing the laccase enzyme from Pleurotus florida, Pleurotus eryngii, and Pleurotus sajor caju were used to degrade the fluoroquinolone antibiotics (FQs) levofloxacin (LEV), norfloxacin (NOR), ciprofloxacin (CIP), ofloxacin (OFL), and enrofloxacin (ENR) in aqueous solutions. The results for the fungi derived laccase extracts were compared with those obtained using commercially sourced laccase. Proteomics analysis of the crude extracts confirmed the presence of laccase enzyme across all three tested species, with proteins matching those found in Trametes versicolor and Pleurotus ostreatus. In vivo studies were conducted using species pure lines of fungal whole cells. The highest degradation efficiency observed was 77.7% for LEV in the presence of P. sajor caju after 25 days of treatment. Degradation efficiencies ranged from approximately 60-72% for P. florida, 45-76% for P. eryngii, and 47-78% for P. sajor caju. A series of in vitro experiments were also conducted using crude extracts from the three species and outcomes compared with those obtained when commercial laccase was used confirmed laccase as the enzyme responsible for antibiotic removal. The degradation efficiencies in vitro surpassed those measured in vivo, ranging from approximately 91-98% for commercial laccase, 77-92% for P. florida, 76-92% for P. eryngii, and 78-88% for P. sajor caju. Liquid chromatography-high-resolution mass spectrometry (LC-MS/MS) identified the degradation products, indicating a consistent enzymatic degradation pathway targeting the piperazine moiety common to all tested FQs, irrespective of the initial antibiotic structure. Phytoplankton toxicity studies with Dunaliella tertiolecta were performed to aid in understanding the impact of emerging contaminants on ecosystems, and by-products were analysed for ecotoxicity to assess treatment efficacy. Laccase-mediated enzymatic oxidation shows promising results in reducing algal toxicity, notably with Pleurotus eryngii extract achieving a 97.7% decrease for CIP and a 90% decrease for LEV. These findings suggest the potential of these naturally sourced extracts in mitigating antibiotic contamination in aquatic ecosystems.

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.

Laccase (EC 1.10.3.2), as eco-friendly biocatalysts, holds immense potential for sustainable applications across various environmental and industrial sectors. Despite the growing interest, the exploration of cold-adapted laccases, especially their unique properties and applicability, remains limited. In this study, we have isolated, cloned, expressed, and purified a novel laccase from Peribacillus simplex (GenBank: PP430751), which was derived from permafrost layer. The recombinant laccase (PsLac) exhibited optimal activity at 30 °C and a pH optimum of 3.5. Remarkably, PsLac exhibited remarkable stability in the presence of organic solvents, with its enzyme activity increasing by 20 % after being incubated in a 30 % trichloromethane solution for 12 h, compared to its initial activity. Furthermore, the enzyme preserved 100 % of its activity after undergoing eight freeze-thaw cycles. Notably, the catalytic center of PsLac contains Zn2+ instead of the typically observed Cu2+ found in other laccases, and metal-ion substitution experiments raised the catalytic efficiency to 3-fold when Zn2+ was replaced with Fe2+. Additionally, PsLac has demonstrated a proficient ability to degrade phenolic pollutants, such as hydroquinone, even at a low temperature of 16 °C, positioning it as a promising candidate for environmental bioremediation and contributing to cleaner production processes.

Known for its tunable conductivity and stability, Polyaniline (PANI) is a valuable polymer for electronics and sensing devices. Challenges in solubility have been addressed by creating sulfonated PANI (SPANI), enhancing its practical use. Synthesizing SPANI from sulfonated aniline is intricate, but laccase biocatalysis offers an eco-conscious solution, effective even against high redox potential obstacles. This research monitored the Trametes versicolor laccase-induced oxidation of 3-ABSa via UV-vis spectroscopy, with a notable peak at 565 nm signifying SPANI synthesis, effective even at suboptimal pH. Mediators further boost this process. Moreover, NMR and spectroelectrochemistry confirm the green synthesis of SPANI by laccase, hinting that pH fine-tuning could improve yields, alongside the concurrent creation of azobenzene derivatives.

Phenols and phenolic compounds are major plant metabolites used in industries to produce pesticides, dyes, medicines, and plastics. These compounds enter water bodies, soil, and living organisms via such industrial routes. Some polyphenolic compounds like phenolic acids, flavonoids have antioxidant and organoleptic qualities, as well as preventive effects against neurodegenerative illnesses, cardiovascular disease, diabetes, and cancer. However, many of the polyphenolic compounds, such as Bisphenol A, phthalates, and dioxins also cause major environmental pollution and endocrine disruption, once the dose level becomes objectionable. The development of reliable and rapid methods for studying their dose dependency, high-impact detrimental effects, and continuous monitoring of phenol levels in humans and environmental samples is a crucial necessity of the day. Enzymatic biosensors employing phenol oxidases like tyrosinase, peroxidase and laccase, utilizing electrochemical amperometric methods are innovative methods for phenol quantification. Enzymatic biosensing, being highly sensitive and efficacious technique, is illuminated in this review article as a progressive approach for phenol quantification with special emphasis on laccase amperometric biosensors. Even more, the review article discussion is extended up to nanozymes, composites of metal organic frameworks (MOFs), and molecularly imprinted polymers (MIPs) as some emerging species for electro-chemical sensing of phenols. Applications of phenol quantification and green biosensing are also specified. A concrete summary of the innovative polyphenol detection approaches with futuristic scope indicates a triumph over some existing constraints of the phenomenological approaches providing an informative aisle to the modern researchers towards the bulk readability.

Many protein-ionic liquid investigations have examined laccase interactions. Laccases are a class of poly-copper oxidoreductases that retain significant biotechnological relevance owing to their notable oxidative capabilities and their application in the elimination of synthetic dyes, phenolic compounds, insecticides, and various other substances. This study investigates the impact of surface active ionic liquids (SAILs), namely, decyltrimethylammonium bromide [N10111][Br] and 1-decyl-3-methylimidazolium chloride [C10mim][Cl] as cationic surfactant ionic liquids and cholinium decanoate [Chl][Dec], an anionic surfactant ionic liquid, on the structure and function of laccase from the fungus Trametes versicolor (TvL) by the molecular dynamics (MD) simulation method. In summary, this study showed that laccase solvent-accessible surface area increased in the ionic liquid [Chl][Dec] while it decreased in the other two ionic liquids. Interestingly, [Chl][Dec] ionic liquid components formed hydrogen bonds with laccase, while [N10111][Br] and [C10mim][Cl] components were unable to form hydrogen bonds with laccase. The quantity of hydrogen bonds formed between water molecules and the enzyme was also diminished in the presence of [Chl][Dec] in comparison to the other two ionic liquids. especially at a concentration of 250 mM. In 250 mM concentrations of [N10111][Br] and [C10mim][Cl], clusters of long-chain cations are likely to form near the copper T1 site. However, even at low [Chl][Dec] concentrations, long [Dec]- chains were observed to penetrate the enzyme near the copper T1 site, and at 250 mM [Chl][Dec], a large cluster of anions occupied the opening of the active site. The results of the analysis also show that the interaction between the [Dec]- anion and the enzyme is stronger than the interaction between [N10111]+ and [C10mim]+ with laccase; in addition, the [Dec]- anion, compared to [Br]- and [Cl]- has a much greater tendency to bind with the enzyme residues.

The diversity of Ganoderma remains largely unexplored, with little information available due to fungiphobia and the morphological plasticity of the genus. To address this gap, an ongoing study aims to collect and identify species with this genus using nuclear ribosomal DNA regions called the \"Internal Transcribed Spacer\" (ITS1-5.8S-ITS2 = ITS). In this study, a new species, Ganoderma segmentatum sp. nov., was discovered on the dead tree trunk of the medicinal plant, Vachellia nilotica. The species was identified through a combination of morpho-anatomical characteristics and phylogenetic analyses. This new species was closely related to Ganoderma multipileum, G. mizoramense, and G. steyaertanum, with a 99% bootstrap value, forming a distinct branch in the phylogenetic tree. Morphologically, G. segmentatum can be distinguished by its frill-like appearance on the margin of basidiome. Wilt or basal stem rot, a serious disease of trees caused by Ganoderma species and V. nilotica, is brutally affected by this disease, resulting in substantial losses in health and productivity. This Ganoderma species severely damages V. nilotica through deep mycelial penetration in the upper and basal stems of the host species. Pathogenic observational descriptions of G. segmentatum on dead tree trunks showed the exudation of viscous reddish-brown fluid from the basal stem portion, which gradually extended upward. Symptoms of this disease include decay, stem discoloration, leaf drooping, and eventual death, which severely damaged the medicinal tree of V. nilotica.

A fungal laccase-mediator system capable of high effectively oxidizing tetracyclines under a wide pH range will benefit environmental protection. This study reported a directed evolution of a laccase PIE5 to improve its performance on tetracyclines oxidization at alkaline conditions. Two mutants, namely MutA (D229N/A244V) and MutB (N123A/D229N/A244V) were obtained. Although they shared a similar optimum pH and temperature as PIE5, the two mutants displayed approximately 2- and 5-fold higher specific activity toward the mediators ABTS and guaiacol at pHs 4.0 to 6.5, respectively. Simultaneously, their catalytic efficiency increased by 8.0- and 6.4-fold compared to PIE5. At a pH range of 5-8 and 28 °C, MutA or MutB at a final concentration of 2.5 U·mL-1 degraded 77 % and 81 % of 100 mg·L-1 tetracycline within 10 min, higher than PIE5 (45 %). Furthermore, 0.1 U·mL-1 MutA or MutB completely degraded 100 mg·L-1 chlortetracycline within 6 min in the presence of 0.1 mM ABTS. At pH 8.0, MutA degraded tetracycline and chlortetracycline following the routine pathways were reported previously based on LC-MS analysis.

To study and compare the morphology of the phellinoid Agaricomycetes strains and find other strategies to improve Phellinus spp. growth and metabolism. In this study, the morphological characteristics of four Phellinus igniarius strains (phellinoid Agaricomycetes) were observed under a light microscope. The exudates from these fungi were observed using light microscopy, scanning electron microscopy (SEM), and energy-dispersive spectrometry (EDS). The exudates were initially transparent with a water-like appearance, and became darker with time at neutral pH. Microscopy of air-dried exudates revealed regular shapes and crystals. Cl- (chloride) and K+ were the two key elements analyzed using EDS. Polyphenol oxidase (POD), catalase (CAT), and laccase activities were detected in mycelia from each of the four Phellinus strains. The K+ content of the three strains was higher than that of the wild strain. Cl- content correlated negatively with that of K+. Laccase activities associated with each mycelia and its corresponding media differed under cold and contaminated conditions.