Identification of a novel marker and its associated laccase gene for regulating ear length in tropical and subtropical maize lines.

KEY MESSAGE: This study revealed the identification of a novel gene, Zm00001d042906, that regulates maize ear length by modulating lignin synthesis and reported a molecular marker for selecting maize lines with elongated ears. Maize ear length has garnered considerable attention due to its high correlation with yield. In this study, six maize inbred lines of significant importance in maize breeding were used as parents. The temperate maize inbred line Ye107, characterized by a short ear, was crossed with five tropical or subtropical inbred lines featuring longer ears, creating a multi-parent population displaying significant variations in ear length. Through genome-wide association studies and mutation analysis, the A/G variation at SNP_183573532 on chromosome 3 was identified as an effective site for discriminating long-ear maize. Furthermore, the associated gene Zm00001d042906 was found to correlate with maize ear length. Zm00001d042906 was functionally annotated as a laccase (Lac4), which showed activity and influenced lignin synthesis in the midsection cells of the cob, thereby regulating maize ear length. This study further reports a novel molecular marker and a new gene that can assist maize breeding programs in selecting varieties with elongated ears.

Decolorization and detoxication of malachite green by engineered Saccharomyces cerevisiae expressing novel thermostable laccase from Trametes trogii.

Malachite Green (MG) is a widely used industrial dye that is hazardous to health. Herein, the decolourisation and detoxification of MG were achieved using the engineered Saccharomyces cerevisiae expressing novel thermostable laccase lcc1 from Trametes trogii. The engineered strain RCL produced a high laccase activity of 121.83 U L-1. Lcc1 was stable at temperatures ranging from 20 ℃ to 60 ℃ and showed a high tolerance to organic solvents. Moreover, Lcc1 could decolorize different kinds of dyes (azo, anthraquinone and triphenylmethane), among which, the decolorization ability of MG is the highest, reaching 95.10 %, and the decolorization rate of other triphenylmethane dyes also over 50 %. The RCL decolorized about 95 % of 50 mg L-1 of MG dye in 10 h at 30 ℃. The MG degradation products were analyzed. The industrial application potential of the RCL was evaluated by treating industrial wastewater and the decolourisation rates were over 90 %.

A single laccase acts as a key component of environmental sensing in a broad host range fungal pathogen.

Secreted laccases are important enzymes on a broad ecological scale for their role in mediating plant-microbe interactions, but within ascomycete fungi these enzymes have been primarily associated with melanin biosynthesis. In this study, a putatively secreted laccase, Sslac2, was characterized from the broad-host-range plant pathogen Sclerotinia sclerotiorum, which is largely unpigmented and is not dependent on melanogenesis for plant infection. Gene knockouts of Sslac2 demonstrate wide ranging developmental phenotypes and are functionally non-pathogenic. These mutants also displayed indiscriminate growth behaviors and enhanced biomass formation, seemingly as a result of their inability to respond to canonical environmental growth cues, a phenomenon further confirmed through chemical stress, physiological, and transcriptomic analyses. Transmission and scanning electron microscopy demonstrate apparent differences in extracellular matrix structure between WT and mutant strains that likely explain the inability of the mutants to respond to their environment. Targeting Sslac2 using host-induced gene silencing significantly improved resistance to S. sclerotiorum, suggesting that fungal laccases could be a valuable target of disease control. Collectively, we identified a laccase critical to the development and virulence of the broad-host-range pathogen S. sclerotiorum and propose a potentially novel role for fungal laccases in modulating environmental sensing.

Transcriptomic and metabolomic analysis unveils a negative effect of glutathione metabolism on laccase activity in Cerrena unicolor 87613.

The white rot fungus Cerrena unicolor 87613 has been previously shown to be a promising resource in laccase production, an enzyme with significant biotechnological applications. Conventional methods face technical challenges in improving laccase activity. Attempts are still being made to develop novel approaches for further enhancing laccase activity. This study aimed to understand the regulation of laccase activity in C. unicolor 87613 for a better exploration of the novel approach. Transcriptomic and metabolomic analyses were performed to identify key genes and metabolites involved in extracellular laccase activity. The findings indicated a strong correlation between the glutathione metabolism pathway and laccase activity. Subsequently, experimental verifications were conducted by manipulating the pathway using chemical approaches. The additive reduced glutathione (GSH) dose-dependently repressed laccase activity, while the GSH inhibitors (APR-246) and reactive oxygen species (ROS) inducer (H2O2) enhanced laccase activity. Changes in GSH levels could determine the intracellular redox homeostasis in interaction with ROS and partially affect the expression level of laccase genes in C. unicolor 87613 in turn. In addition, GSH synthetase was found to mediate GSH abundance in a feedback loop. This study suggests that laccase activity is negatively influenced by GSH metabolism and provides a theoretical basis for a novel strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.IMPORTANCEThe production of laccase activity is limited by various conventional approaches, such as heterologous expression, strain screening, and optimization of incubation conditions. There is an urgent need for a new strategy to meet industrial requirements more effectively. In this study, we conducted a comprehensive analysis of the transcriptome and metabolome of Cerrena unicolor 87613. For the first time, we discovered a negative role played by reduced glutathione (GSH) and its metabolic pathway in influencing extracellular laccase activity. Furthermore, we identified a feedback loop involving GSH, GSH synthetase gene, and GSH synthetase within this metabolic pathway. These deductions were confirmed through experimental investigations. These findings not only advanced our understanding of laccase activity regulation in its natural producer but also provide a theoretical foundation for a strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

[Monogenic variants in Laccase domain-containing 1 (LACC1) as the cause of juvenile arthritis]. / Monogene Varianten in "laccase domain-containing 1" (LACC1) als Ursache einer juvenilen Arthritis.

Monogenic mutations in laccase domain-containing 1 (LACC1) are associated with clinical pictures that mimic severe courses of polyarticular or systemic juvenile idiopathic arthritis. The diseases are characterized by an early onset during the first year of life, a familial clustering and a high inflammatory activity. The courses are mostly difficult to influence and often lead to sequelae. In this article four cases from two families are presented in which the homozygous mutation p.T276fs* in LACC1 was detected. The children initially suffered from polyarticular or systemic forms of juvenile arthritis. Of the patients two are currently being treated with tocilizumab and methotrexate and one female patient without a basis treatment is currently only receiving local repeated intra-articular steroids. A fourth female patient underwent an allogeneic bone marrow transplantation due to a relapse of an acute lymphatic leukemia. Since then, no further inflammatory symptoms have occurred. The cases presented are compared with the other 50 courses published to date. In addition, recent studies investigating the influence of LACC1 mutations, particularly on macrophage function, are summarized.

Biochemical Characterization of Laccase from Spirulina CPCC-695 and Their Role in Estrone Degradation.

The addition of exogenous endocrine disrupting compounds (EDCs) like estrone, in the food chain through the aquatic system, disrupts steroid biosynthesis and metabolism by altering either the genomic or non-genomic pathway that eventually results in various diseases. Thus, bioremediation of these compounds is urgently required to prevent their addition and persistence in the environment. Enzymatic degradation has proven to be a knight in shining armour as it is safe and generates no toxic products. The multicopper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase), laccase with the potential to degrade both phenolic and non-phenolic substrates has recently gained attention. In this study, the laccase was purified, characterized, and used to study estrone degradation. The culture filtrate (crude laccase) was concentrated and precipitated using cold-acetone and dialyzed against tris buffer (50 mM) giving a four-fold partially purified form, with 45.56% yield and 204.14 U/mg as specific activity and a single peak at 250-300 nm. The partially purified laccase was approximately 80 kDa as estimated by SDS-PAGE preferred ABTS as substrate. Both crude and partially purified laccase showed maximum activity at pH 3.0, 40 °C, and 4 mM ABTS. Kinetic constants (Km, Vmax) of crude and partially purified laccase were found to be 0.83 mM; 494.31 mM/min, and 0.58 mM; 480.54 mM/min respectively. Iron sulphate and sodium azide inhibited laccase maximally. Crude and partially purified laccase degradation efficiency was 87.55 and 91.35% respectively. Spirulina CPCC-695 laccase with efficient estrone degradation ability renders them promising candidates for EDCs bioremediation.

Lignin-degrading enzyme production was enhanced by the novel transcription factor Ptf6 in synergistic microbial co-culture.

Synergistic microbial co-culture has been an efficient and energy-saving strategy to produce lignin-degrading enzymes (LDEs), including laccase, manganese peroxidase, and versatile peroxidase. However, the regulatory mechanism of microbial co-culture is still unclear. Herein, the extracellular LDE activities of four white-rot fungi were significantly increased by 88-544% over monoculture levels when co-cultured with Rhodotorula mucilaginosa. Ptf6 was demonstrated from the 9 million Y1H clone library to be a shared GATA transcription factor in the four fungi, and could directly bind to the laccase gene promoter. Ptf6 exists in two alternatively spliced isoforms under monoculture, namely Ptf6-α (1078 amino acids) containing Cys2/Cys2-type zinc finger and Ptf6-ß (963 amino acids) lacking the complete domain. Ptf6 responded to co-culture by up-regulation of both its own transcripts and the proportion of Ptf6-α. Ptf6-α positively activated the production of most LDE isoenzymes and bound to four GATA motifs on the LDEs' promoter with different affinities. Moreover, Ptf6-regulation mechanism can be applicable to a variety of microbial co-culture systems. This study lays a theoretical foundation for further improving LDEs production and providing an efficient way to enhance the effects of biological and enzymatic pretreatment for lignocellulosic biomass conversion.

Histochemical Detection of Peroxidase and Laccase Activities in Populus Secondary Xylem.

Peroxidases (PRXs) and laccases (LACs) are enzymes involved in catalyzing the oxidation of the lignin monomers to facilitate lignin polymerization. However, due to the large number of genes composing these two families of enzymes, many details regarding their specific localization are only partially understood. Here, we present a fast and easy histochemical method that makes use of the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB) to visualize PRX and LAC activities in the hybrid aspen (Populus tremula x P. tremuloides) xylem tissue. In addition, we describe a protocol that allows the detection of the PRX substrate, H2O2, using the nonfluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) in woody tissues.

Development of an efficient protein expression system in the thermophilic fungus Myceliophthora thermophila.

BACKGROUND: Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. RESULTS: In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5'UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5'UTR also has significant effects on protein expression and secretion. The best 5'UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5'UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. CONCLUSIONS: An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications.

Application of Miscanthus substrates in the cultivation of Ganoderma lingzhi.

Ganoderma lingzhi is a traditional Chinese medicine that has been used to improve health and longevity for thousands of years. It is usually cultivated on hardwood log- or sawdust-based formulations. Conversely, in this study, we used Miscanthus sacchariflorus (MSF), M. floridulus, and M. sinensis (MSS), fast-growing perennial grasses widely distributed in China, for G. lingzhi cultivation. Mycelial growth rate, activities of lignin-degrading enzymes on colonized mushroom substrates, and expression levels of CAZymes and laccase genes based on different substrates were analyzed. Total triterpenoids, sterols, and polysaccharides content of fruiting bodies obtained from different substrates were investigated. The activities of laccase and manganese peroxidase in mycelia increased in the MSF- and MSS-based formulations compared with that in the sawdust-based formulation. The results of mycelial growth- and cultivation-related experiments showed that the Miscanthus substrates could be used as the substrates for cultivating G. lingzhi. The content of active ingredients, namely triterpenoids, sterols, and polysaccharides, in fruiting bodies cultivated on the Miscanthus substrates did not decrease compared with those in substrate obtained from the sawdust-based formulation. Therefore, the present study provides alternative substrates for the cultivation of G. lingzhi, and a reference for better utilization of inexpensive substrate in future.

Genome-wide identification and expression patterns of the laccase gene family in response to kiwifruit bacterial canker infection.

BACKGROUND: Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive disease worldwide. Resistance genes that respond to Psa infection urgently need to be identified for controlling this disease. Laccase is mainly involved in the synthesis of lignin in the plant cell wall and plays a prominent role in plant growth and resistance to pathogen infection. However, the role of laccase in kiwifruit has not been reported, and whether laccase is pivotal in the response to Psa infection remains unclear. RESULTS: We conducted a bioinformatics analysis to identify 55 laccase genes (AcLAC1-AcLAC55) in the kiwifruit genome. These genes were classified into five cluster groups (I-V) based on phylogenetic analysis, with cluster groups I and II having the highest number of members. Analysis of the exon-intron structure revealed that the number of exons varied from 1 to 8, with an average of 5 introns. Our evolutionary analysis indicated that fragment duplication played a key role in the expansion of kiwifruit laccase genes. Furthermore, evolutionary pressure analysis suggested that AcLAC genes were under purifying selection. We also performed a cis-acting element analysis and found that AcLAC genes contained multiple hormone (337) and stress signal (36) elements in their promoter regions. Additionally, we investigated the expression pattern of laccase genes in kiwifruit stems and leaves infected with Psa. Our findings revealed that laccase gene expression levels in the stems were higher than those in the leaves 5 days after inoculation with Psa. Notably, AcLAC2, AcLAC4, AcLAC17, AcLAC18, AcLAC26, and AcLAC42 showed significantly higher expression levels (p < 0.001) compared to the non-inoculated control (0 d), suggesting their potential role in resisting Psa infection. Moreover, our prediction indicated that 21 kiwifruit laccase genes are regulated by miRNA397, they could potentially act as negative regulators of lignin biosynthesis. CONCLUSIONS: These results are valuable for further analysis of the resistance function and molecular mechanism of laccases in kiwifruit.

Characterization of white rot fungi from wood decayed for lignin degradation.

The present study was conducted to isolate and identify white rot fungi (WRF) from wood decayed and to determine their ability to produce lignin-modifying enzymes (LMEs), specifically laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), on solid and liquid media supplemented with synthetic dyes namely 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), azure B, and phenol red. A total of 23 isolates of WRF were isolated from decayed wood and identified as eight different species namely Phanerochaete australis, Perenniporia tephropora, Lentinus squarrosulus, Ganoderma australe, Trametes polyzona, Lentinus sajor-caju, Gymnopilus dilepis, and Fomitopsis palustris based on morphological characteristics, DNA sequences of the internal transcribed spacer (ITS) region, and phylogenetic inference. The fungal isolates can be divided into four groups based on the type of LMEs produced, namely A (Lac-LiP-MnP) with 16 isolates, B (Lac-MnP) (three isolates), C (Lac) (three isolates), and D (MnP) (one isolate). This study highlights P. australis (BJ38) as the best producer of Lac and LiP, while L. squarrosulus (IPS72) is the best producer of MnP. The present study is the first reported P. australis as an efficient lignin degrader by demonstrating the highest activity of two important LMEs.

Laccase Engineering: Redox Potential Is Not the Only Activity-Determining Feature in the Metalloproteins.

Laccase, one of the metalloproteins, belongs to the multicopper oxidase family. It oxidizes a wide range of substrates and generates water as a sole by-product. The engineering of laccase is important to broaden their industrial and environmental applications. The general assumption is that the low redox potential of laccases is the principal obstacle, as evidenced by their low activity towards certain substrates. Therefore, the primary goal of engineering laccases is to improve their oxidation capability, thereby increasing their redox potential. Even though some of the determinants of laccase are known, it is still not entirely clear how to enhance its redox potential. However, the laccase active site has additional characteristics that regulate the enzymes' activity and specificity. These include the electrostatic and hydrophobic environment of the substrate binding pocket, the steric effect at the substrate binding site, and the orientation of the binding substrate with respect to the T1 site of the laccase. In this review, these features of the substrate binding site will be discussed to highlight their importance as a target for future laccase engineering.

Enzymatic degradation and metabolic pathway of phenanthrene by manglicolous filamentous fungus Trichoderma sp. CNSC-2.

Manglicolous filamentous fungi release extracellular lignolytic enzymes that can readily degrade polycyclic aromatic hydrocarbons (PAHs). The present study emphasizes the role of the extracellular enzyme in phenanthrene degradation by the manglicolous fungus Trichoderma sp. CNSC-2 isolated from the Indian Sundarban mangrove ecosystem. The removal efficiency reached 64.05 ± 0.75 % in 50 mg l-1 phenanthrene-amended mineral salt medium at pH 5.6 after 10 days of incubation. Phenanthrene removal was optimized at different pH, nutrient sources, and Cu2+ concentrations. The degradation significantly increased to 67.75 ± 4.32 % at pH 6 (P < 0.0001). The addition of Cu2+ (30 mg l-1) increased the degradation to 78.15 ± 0.36 % (P < 0.0001). The validation experiment confirmed the increase in phenanthrene degradation up to 79.9 ± 1.67 % under optimized conditions. The Lac1 and CytP450 genes encoding for extracellular and intracellular enzymes, respectively, were identified. The GC-MS derived phenanthrene degradation metabolites, i.e., phthalic acid, isobutyl 2-pentyl ester derivative, 1, 2 benzene dicarboxylic acid, butyl 2-methyl propyl ester derivative, TMS derivative of benzoic acid and 3,5 dihydroxy benzoic acid determined two possible metabolic pathways. The laccase enzyme activity was higher in the presence of Phe+Cu2+ (P < 0.0001), indicating the enzyme induction potential of PAH and Cu2+ ions. Purified laccase had a molecular weight of 45 kDa and was highly stable at pH 4-6 and temperature 20-50 °C. The enzyme retained 47 %, 87 %, and 63 % of enzyme activity at 30 mg l-1 concentration of Pb2+, Cd2+, and Hg2+. However, laccase activity was induced by 1.37 folds in the presence of 30 mg l-1 Cu2+ concentration. Thus, the study suggests the potential role of Trichoderma sp. CNSC-2 in phenanthrene degradation.

Exoproteomic Study and Transcriptional Responses of Laccase and Ligninolytic Peroxidase Genes of White-Rot Fungus Trametes hirsuta LE-BIN 072 Grown in the Presence of Monolignol-Related Phenolic Compounds.

Being an abundant renewable source of aromatic compounds, lignin is an important component of future bio-based economy. Currently, biotechnological processing of lignin through low molecular weight compounds is one of the conceptually promising ways for its valorization. To obtain lignin fragments suitable for further inclusion into microbial metabolism, it is proposed to use a ligninolytic system of white-rot fungi, which mainly comprises laccases and peroxidases. However, laccase and peroxidase genes are almost always represented by many non-allelic copies that form multigene families within the genome of white-rot fungi, and the contributions of exact family members to the overall process of lignin degradation has not yet been determined. In this article, the response of the Trametes hirsuta LE-BIN 072 ligninolytic system to the presence of various monolignol-related phenolic compounds (veratryl alcohol, p-coumaric acid, vanillic acid, and syringic acid) in culture media was monitored at the level of gene transcription and protein secretion. By showing which isozymes contribute to the overall functioning of the ligninolytic system of the T. hirsuta LE-BIN 072, the data obtained in this study will greatly contribute to the possible application of this fungus and its ligninolytic enzymes in lignin depolymerization processes.

Overexpression of a laccase gene, DiLAC17, from Davidia involucrata causes severe seed abortion in Arabidopsis.

Seed abortion is a common phenomenon in woody plants, especially in rare and endangered species. Serious seed abortion occurs in the dove tree and largely restricts its natural reproduction. A number of differentially expressed genes (DEGs) between normal and aborted seeds of the dove tree have been previously identified through transcriptome profiling. Among these, most DEGs encoding laccase showed significant upregulation in the aborted seeds. In this study, the laccase gene with the highest expression level in aborted seeds, DiLAC17, was cloned from the dove tree genome and further verified. Overexpression of the DiLAC17 gene in Arabidopsis resulted in retarded growth, deformed siliques, and severe seed abortion. Most Arabidopsis genes involved in seed development, such as AtLEC2, AtANT1, and AtRGE1, were suppressed in the transgenic lines. Laccase activity and lignin content were significantly improved in transgenic lines under ectopic overexpression of the DiLAC17 gene. Excessive lignin accumulation in the early developmental stage was assumed to be a key cause of restricting silique growth and seed expansion, which ultimately led to seed abortion. These results indicate a laccase-mediated pathway for seed abortion, which might be a strategy adopted by this rare and endangered species to reduce the reproductive load.

Metabolome and Transcriptome Analysis of Sulfur-Induced Kiwifruit Stem Laccase Gene Involved in Syringyl Lignin Synthesis against Bacterial Canker.

Kiwifruit canker is caused by Pseudomonas syringae pv. actinidiae and is one of the most destructive diseases of kiwifruit worldwide. Sulfur can improve the deposit of lignin in kiwifruit stems and induce disease resistance, but the action mechanism at the molecular level remains unclear. This omics-based study revealed that sulfur-induced S lignin synthesis contributes to disease resistance. Histological staining verified sulfur-enhanced total lignin deposition in kiwifruit stems. High-performance liquid chromatography and confocal Raman microscopy showed that sulfur-activated S lignin was mainly deposited in the cell corner. Metabolome and transcriptome analysis revealed that the levels of phenylpropanoid pathway S lignin precursors sinapic acid and sinapyl alcohol were significantly increased and 16 laccase genes were upregulated. Sulfur-induced resistance defense promoted elevated laccase activity by activating the laccase genes, participating in sinapic acid and sinapyl alcohol substance synthesis, and ultimately polymerizing S lignin at cell corner against kiwifruit canker disease.

Genome-wide study of Cerrena unicolor 87613 laccase gene family and their mode prediction in association with substrate oxidation.

BACKGROUND: Laccases are green biocatalysts with wide industrial applications. The study of efficient and specific laccase producers remains a priority. Cerrena species have been shown to be promising basidiomycete candidates for laccase production. Although two sets of Cerrena genome data have been publicly published, no comprehensive bioinformatics study of laccase gene family in C. unicolor has been reported, particularly concerning the analysis of their three-dimensional (3D) structures and molecular docking to substrates, like ABTS and aflatoxin B1 (AFB1). RESULTS: In this study, we conducted a comprehensive genome-wide analysis of laccase gene family in C. unicolor 87613. We identified eighteen laccase genes (CuLacs) and classified them into three clades using phylogenetic analysis. We characterized these laccases, including their location in contig 5,6,9,12,15,19,26,27, gene structures of different exon-intron arrangements, molecular weight ranging from 47.89 to 141.41 kDa, acidic pI value, 5-15 conserved protein motifs, signaling peptide of extracellular secretion (harbored by 13 CuLacs) and others. In addition, the analysis of cis-acting element in laccase promoters indicated that the transcription response of CuLac gene family was regulatable and complex under different environmental cues. Furthermore, analysis of transcription pattern revealed that CuLac8, 12 and CuLac2, 13 were the predominant laccases in response to copper ions or oxidative stress, respectively. Finally, we focused on the 3D structure analysis of CuLac proteins. Seven laccases with extra transmembrane domains or special sequences were particularly interesting. Predicted structures of each CuLac protein with or without these extra sequences showed altered interacting amino acid residues and binding sites, leading to varied affinities to both ABTS and AFB1. As far as we know, it is the first time to discuss the influence of the extra sequence on laccase's affinity to substrates. CONCLUSIONS: Our findings provide robust genetic data for a better understanding of the laccase gene family in C. unicolor 87613, and create a foundation for the molecular redesign of CuLac proteins to enhance their industrial applications.

Purification and characterization of an alkali-organic solvent-stable laccase with dye decolorization capacity from newly isolated Lysinibacillus fusiformis W11.

A new Lysinibacillus fusiformis strain with abundant laccase activity was isolated from soil under forest rotted leaf and identified as L. fusiformis W11 based on its 16S rRNA gene sequence and physiological characteristics. The laccase LfuLac was purified and characterized. The optimum temperature and pH of LfuLac on guaiacol were 45 °C and pH 9, respectively. LfuLac kept 78%, 88%, 92%, 74%, and 47% of activity at pH 7-11, respectively, suggesting the alkali resistance of the enzyme. The effects of various metal ions on LfuLac showed that Cu2+, Mg2+, and Na+ were beneficial to laccase activity and 10 mM Cu2+ increased the activity of LfuLac to 216%. LfuLac showed about 90% activity at 5% organic solvents and more than 60% activity at 20%, indicating its resistance to organic solvents. In addition, LfuLac decolorized different kinds of dyes. This study enriched our knowledge about laccase from L. fusiformis W11 and its potential industrial applications.