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Adverse conditions during pre- and postharvest stages affect the longevity and quality of horticultural crops. Thus, storage conditions should be considered in marketing management, especially for cut flowers. Methyl jasmonate is a well-known signaling molecule involved in plant defense responses. Applying it as a treatment can effectively prolong the vase life of horticultural crops. The expression of Laccase gene, acting downstream of the ethylene signal transduction, can be induced by exogenous ethylene. The PIP2 gene is a major group of plasma membrane proteins that are linked to aquaporin channels. This study explored the effect of methyl jasmonate on postharvest quality and expression LAC and PIP2 genes of cut rose, cv. 'Red alert'. The results showed that methyl jasmonate improved the vase life of cut roses, although it was most effective at 0.2 µl L-1. An analysis of antioxidant enzymes indicated that the highest catalase activity and peroxidase activity occurred in response to 0.2 µl L-1 and 0.2-0.3 µl L-1 methyl jasmonate, respectively, although both enzymes showed a decreasing trend during the vase life period. Proline levels increased significantly in response to 0.2 and 0.3 µl L-1 methyl jasmonate concentr -ations. Quantitative PCR analysis showed that all concentrations of methyl jasmonate significantly reduced the expression of PIP2 and LAC genes. The lowest expression level of PIP2 and LAC genes occurred in response to 0.2 and 0.3 µl L-1 methyl jasmonate, respectively. Based on our findings, the expression pattern of the Laccase gene may be regarded as a marker in evaluating postharvest characteristics of cut roses. [ABSTRACT FROM AUTHOR]

The content and size of stone cell clusters affects the quality of pear fruit, and monolignol polymerization and deposition in the cell walls constitute a required step for stone cell formation. Laccase (LAC) is the key enzyme responsible for the polymerization of monolignols. However, there are no reports on the LAC family in pear (Pyrus bretschneideri), and the identity of the members responsible for lignin synthesis has not been clarified. Here, 41 LACs were identified in the whole genome of pear. All Pyrus bretschneideri LACs (PbLACs) were distributed on 13 chromosomes and divided into four phylogenetic groups (I-IV). In addition, 16 segmental duplication events were found, implying that segmental duplication was a primary reason for the expansion of the PbLAC family. LACs from the genomes of three Rosaceae species (Prunus mummer, Prunus persica, and Fragaria vesca) were also identified, and an interspecies collinearity analysis was performed. The phylogenetic analysis, sequence alignments and spatiotemporal expression pattern analysis suggested that PbLAC1, 5, 6, 29, 36 and 38 were likely associated with lignin synthesis and stone cell formation in fruit. The two target genes of Pyr-miR1890 (a microRNA identified from pear fruit that is associated with lignin and stone cell accumulation), PbLAC1 and PbLAC14, were selected for genetic transformation. Interfamily transfer of PbLAC1 into Arabidopsis resulted in a significant increase (approximately 17%) in the lignin content and thicker cell walls in interfascicular fibre and xylem cells, which demonstrated that PbLAC1 is involved in lignin biosynthesis and cell wall development. However, the lignin content and cell wall thickness were not changed significantly in the PbLAC14-overexpressing transgenic Arabidopsis plants. This study revealed the function of PbLAC1 in lignin synthesis and provides important insights into the characteristics and evolution of the PbLAC family. [ABSTRACT FROM AUTHOR]

Laccase is one of the oldest known and intensively studied fungal enzymes capable of oxidizing recalcitrant lignin-resembling phenolic compounds. It is currently well established that fungal genomes almost always contain several non-allelic copies of laccase genes (laccase multigene families); nevertheless, many aspects of laccase multigenicity, for example, their precise biological functions or evolutionary relationships, are mostly unknown. Here, we present a detailed evolutionary analysis of the sensu stricto laccase genes (CAZy – AA1_1) from fungi of the Polyporales order. The conducted analysis provides a better understanding of the Polyporales laccase multigenicity and allows for the systemization of the individual features of different laccase isozymes. In addition, we provide a comparison of the biochemical and catalytic properties of the four laccase isozymes from Trametes hirsuta and suggest their functional diversification within the multigene family. [ABSTRACT FROM AUTHOR]

In this study we describe the effects of Pleurotus ostreatus transformants that have been silenced for laccase genes. Using the RNAi strategy, transformants with different levels of phenotypic alteration were obtained with respect to their oxidation capacity of 2,6-dimethoxyphenol (DMP). The analysis of laccase activity on DMP allowed us to select transformants with severe, medium and light phenotypic alteration in comparison to the PoB strain. The measurements of the average growth rate of the transformants with severe phenotypic alteration suggested that laccase expression could play a role in the vegetative growth of P. ostreatus. It was observed that both in solid and liquid cultures, PoB and the transformants express mRNA for lacc10, although the transformants with medium and severe phenotypic alteration present a decrease in intensity, especially in solid culture. This suggests that the product of this gene is responsible for the development of the mycelium and probably participates in the production of biomass in solid culture and also could be related to the decrease in the intensity of the constant isoenzyme observed in both culture systems. [ABSTRACT FROM AUTHOR]

Laccase production and pellet formation of transformants of Coprinopsis cinerea strain FA2222 of C. cinerea laccase gene lcc1 subcloned behind the gpdII-promoter from Agaricus bisporus were compared with a control transformant carrying no extra laccase gene. At the optimum growth temperature of 37 °C, maximal laccase yields of 2.9 U/ml were obtained by the best lcc1 transformant pYSK7-26 in liquid shake flask cultures. Reduction in temperature to 25 °C increased laccase yields up to 9.2 U/ml. The control transformant had no laccase activities at 37 °C but native activity at 25 °C (3.5 U/ml). Changing the temperature had severe effects on the morphology of the mycelial pellets formed during cultivation, but links of distinct pellet morphologies to native or recombinant laccase production could not be established. Automated image analysis was used to characterise pellet formation and morphological parameters (pellet area, diameter, convexity and mycelial structure). Cross sections of selected pellets showed that they differentiated in an outer rind and an inner medulla of loosened hyphae. Pellets at 25 °C had a small and dense outer zone and adopted with time a smooth surface. Pellets at 37 °C had a broader outer zone and a fringy surface due to generation of more and larger protuberances in the rind that when released can serve for production of further pellets. [ABSTRACT FROM AUTHOR]

Glutamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the formation of glucosamine-6-phosphate, and its gene is one of the genes essential for microbes. Using the GFAT-encoding gene can prevent the use of a drug-resistant gene as a selection marker in a bacterial system. Another unique property of the GFAT selection marker is that no particular compound is prohibited or required for creating a selective stress for a yeast. Filamentous fungi are major producers of industrial enzymes. However, there has been no report on the construction and application of the GFAT gene as a selection marker in filamentous fungi. To develop a new selection marker, the GFAT-encoding gene gfaA was deleted from the genome of the filamentous fungus Aspergillus nidulans, and the gfat gene of the straw mushroom Volvariella volvacea was used as the selection marker to mediate the transformation and overexpression of a thermostable bacterial laccase in A. nidulans. The GFAT-deficient strain A. nidulans ∆gfaA was not able to grow in the culture medium containing 0.5% yeast extract unless about 20 mM glucosamine was used to supplement to the medium. The gfat gene was amplified and inserted into the integration vector pAL5 and autonomous replication vector Prg3-AMA1-NotI for A. nidulans to generate the gfat vectors pALG and pAMAG, respectively. Using these gfat vectors, the laccase gene lcs from a hyperthermophilic bacterium was overexpressed intra- and extracellularly in A. nidulans ∆gfaA. Therefore, recombinant filamentous fungi can be constructed with gfat vectors, which can be maintained stably in host cells with the naturally occurred selective stress of a medium, forage, pulp, animal gut, wastewater, or soil. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: The effects of small laccase (SLAC) from Streptomyces coelicolor on the properties of whey protein isolate (WPI) films were studied. RESULTS: WPI was catalyze by SLAC without phenolic acid assistance. Particle size distribution results showed that some complexes with higher relative molecular weight formed in WPI samples treated with SLAC. The content of α‐helixes decreased while those of β‐sheets and random coils increased following SLAC treatment according to circular dichroism results. Fourier transform infrared spectral analysis suggested that some conformational changes occurred in WPI following SLAC treatment. Analysis of WPI films prepared by casting after SLAC treatment indicated that their film properties were all improved, including mechanical properties, solubility, water vapor, oxygen and carbon dioxide barrier properties, film color, light transmission, transparency and thermal properties. Compared with that of the control film, some obvious differences in the morphology of the WPI films were observed following SLAC treatment. This report demonstrates that laccase can directly catalyze protein cross‐linking, which may be useful to improve the performance of protein films. CONCLUSION: In this study, SLAC was applied to WPI edible film during the film‐making process. The results showed that SLAC can catalyze WPI cross‐linking without phenolic acid assistance, and WPI film properties were improved after SLAC treatment. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: Aims: Isolate and characterize a laccase‐encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. Methods and Results: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long‐distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563‐pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α‐factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat/Km values towards ABTS, followed by 2,6‐dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. Conclusions: Lac I‐encoding gene could be successfully sequenced having cis‐acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α‐factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. Significance and Impact of the Study: The cis‐acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively. [ABSTRACT FROM AUTHOR]

Laccase (EC 1.10.3.2) is a phenoloxidase found in many insect species. The Laccase 1 gene from Plutella xylostella ( PxLac1 ) was cloned, and its expression patterns and functions were determined using qPCR and RNAi methods. The results showed that the expression levels of PxLac1 were consistently high in all larval stages, and the most abundant was in the midgut during the 4th instar stage. Moreover, the expression of PxLac1 was up-regulated in response to bacterial infection, and decreased 24 h after being parasitized by Cotesia vestalis . Further analyses indicated that the effect of parasitization on PxLac1 was induced by active C . vestalis Bracovirus (CvBV). Haemocyte-free hemolymph phenoloxidase (PO) activity was suppressed when PxLac1 was treated with RNAi. Our results provide evidence for a connection between the Laccase 1 gene and insect immunity, and revealed that parasitoid polydnavirus suppresses host PO activity via PxLac1 regulation. [ABSTRACT FROM AUTHOR]

The laccase gene from Pycnoporus sanguineus was cloned and inserted between the strong Pcbh1 promoter and the Tcbh1 terminator from Trichoderma reesei to form the recombinant plasmid pCH-lac. Using Agrobacterium -mediated technique, the pCH-lac was integrated into the chromosomes of T. reesei . Twenty positive transformants were obtained by employing hygromycin B as a selective agent. PCR was used to confirm that the laccase gene was integrated into the chromosomal DNA of T. reesei . Laccase production by recombinant transformants was performed in shaking flasks, and the activity of laccase reached 8.8 IU/mL after 96-h fermentation under a batch process, and 17.7 IU/mL after 144-h fermentation using a fed-batch process. SDS-PAGE analysis of the fermentation broth showed that the molecular mass of the protein was about 68 kDa, almost the same as that of the laccase produced by P. sanguineus , which indicated that laccase was successfully expressed in T. reesei and secreted out of the cells. The laccase produced by the recombinant T. reesei showed good thermal stability, and could degrade the toxic phenolic material bisphenol A efficiently, after 1-h reaction with 0.06 IU/mL laccase and 0.5 mmol/L ABTS as the mediator at 60 °C and pH 4.5, the degradation rate reached 95%, which demonstrated that it had great potential value in treating the household garbage and wastewater containing the bisphenol A. [ABSTRACT FROM AUTHOR]

This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene ( rSLAC ) produced high titres of extracellular laccase (500 ± 10 U/l), which were further increased seven fold by pre-incubation at 80 °C for 30 min. The enzyme (∼38 kDa) had an optimum activity at 80 °C, but optimum pH varied with substrate used. K m values for ABTS, SGZ and 2,6-DMP were 142.85 μM, 10 μM and 54.55 μM and the corresponding k cat values were 60.6 s −1 , 25.36 s −1 and 27.84 s −1 , respectively. The t 1/2 values of the rSLAC at 60 °C, 70 °C, 80 °C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (E d ) was 117.275 kJ/mol while ΔG, ΔH and ΔS for thermal inactivation of the rSLAC were all positive. The rSLAC decolourised more than 90% of Brilliant Blue G and Trypan Blue dye in 6 h without the addition of a mediator. High titres of SLAC expressed in P. pastoris enhance its potential for various industrial applications. [ABSTRACT FROM AUTHOR]

Chlorophenols (CPs), nitrophenols (NPs), and sulfonamide antibiotics (SAs) are three types of environmental pollutants that are of great concern because of their prevalence and toxicity. In this study, three laccase isoenzymes obtained from Pleurotus ostreatus HAUCC 162 were heterologously expressed and characterized with respect to their ability to degrade CPs, NPs, and, SAs. The three recombinant laccases can efficiently degrade the three types of considered pollutants using a laccase-mediator system (LMS). Their specific efficiencies for the removal of 2NP, 3NP, 4NP, 4CP, 2,4-dichlorophenol (DCP), 2,6-DCP, sulfadiazine (SDZ), sulfamethazine (SMZ), and sulfamethoxazole (SMX) over 60 min were 59.21%, 47.91%, 60.24%, 74.9%, 28.9%, 35.1%, 98.1%, 97.5%, and 97.8%, respectively. Based on the analysis of the oxidation products of the CPs, NPs, and SAs, pollutant removal pathways are proposed, namely, the production of 3-nitromuconate and 3-chloromuconate as the key intermediates of 4-NP and 2, 4-DCP; and oxidative coupling for the transformation of SDZ by LMS. The results of present work indicated the laccases could efficiently remove NPs, CPs, and SAs in LMS, which offers an opportunity to apply P. ostreatus HAUCC 162 laccase in the field of environmental biotechnology. [ABSTRACT FROM AUTHOR]

In this study, a novel laccase gene (Lcc1) from Ganoderma tsugae was isolated and its functions were characterized in detail. The results showed that Lcc1 has the highest expression activity during mycelium development and fruit body maturation based on the analysis of Lcc1 RNA transcripts at different developmental stages of G. tsugae. To investigate the exact contribution of Lcc1 to mycelium and fruit body development in G. tsugae, Lcc1 transgenic strains were constructed by targeted gene replacement and over-expression approaches. The results showed that the lignin degradation rate in Lcc1 deletion mutant was much lower than the degradation efficiency of the wild-type (WT), over-expression and rescue strains. The lignin degradation activity of G. tsugae is dependent on Lcc1 and the deletion of Lcc1 exerted detrimental influences on the development of mycelium branch. Furthermore, the study uncovered that Lcc1 deletion mutants generated much shorter pale grey fruit bodies, suggesting that Lcc1 contributes directly to pigmentation and stipe elongation during fruit body development in G. tsugae. The information obtained in this study provides a novel and mechanistic insight into the specific role of Lcc1 during growth and development of G. tsugae. [ABSTRACT FROM AUTHOR]

The continuous release of antibiotic compounds through wastewater effluent into environment has raised concerns about their potential problems for different organisms. Enzymatic degradation with laccase is a green option for removal of pharmaceutical compounds from aqueous media. In this study, laccase was immobilized onto homemade Polyacrylonitrile-biochar composite nanofibrous membrane and the obtained biocatalyst was employed for removal of chlortetracycline, a widely used antibiotic, from aqueous media in continuous mode. The results showed that the immobilized laccase has improved storage, temperature and pH stability compared to free laccase. Also, it retained more than 50% of its initial activity after 7 cycles of ABTS oxidation which indicated improved enzyme reusability. Finally, while using immobilized laccase for degradation of chlortetracycline in continuous mode exhibited 58.3%, 40.7% and 22.6% chlortetracycline removal efficiency at flux rates of 1, 2 and 3 mL/h ∙ cm 2 . [ABSTRACT FROM AUTHOR]

In mating of Lentinula edodes, dikaryotic strains generated from certain monokaryotic strains such as the B2 used in this study tend to show better quality of fruiting bodies regardless of the mated monokaryotic strains. Unlike B2, dikaryotic strains generated from B16 generally show low yields, with deformed or underdeveloped fruiting bodies. This indicates that the two nuclei in the cytoplasm do not contribute equally to the physiology of dikaryotic L. edodes, suggesting an expression bias in the allelic genes of the two nuclei. To understand the role of each nucleus in dikaryotic strains, we investigated single nucleotide polymorphisms (SNPs) in laccase genes of monokaryotic strains to reveal nuclear origin of the expressed mRNAs in dikaryotic strain. We performed reverse transcription PCR (RT-PCR) analysis using total RNAs extracted from dikaryotic strains (A5B2, A18B2, and A2B16) as well as from compatible monokaryotic strains (A5, A18, and B2 for A5B2 and A18B2; A2 and B16 for A2B16). RT-PCR results revealed that Lcc1, Lcc2, Lcc4, Lcc7, and Lcc10 were the mainly expressed laccase genes in the L. edodes genome. To determine the nuclear origin of these laccase genes, the genomic DNA sequences in monokaryotic strains were analyzed, thereby revealing five SNPs in Lcc4 and two in Lcc7. Subsequent sequence analysis of laccase mRNAs expressed in dikaryotic strains revealed that these were almost exclusively expressed from B2-originated nuclei in A5B2 and A18B2 whereas B16 nucleus did not contribute to laccase expression in A2B16 strain. This suggests that B2 nucleus dominates the expression of allelic genes, thereby governing the physiology of dikaryons. [ABSTRACT FROM AUTHOR]

The structure and electrochemical properties of adsorbed complexes based on mixtures of polyvinylamine-g-TEMPO (PVAm-T) and laccase were related to the ability of the adsorbed complexes to oxidize cellulose. PVAm-T10 with 10% of the amines bearing TEMPO moieties (i.e., DS = 10%), adsorbed onto gold sulfonate EQCM-D sensor surfaces giving a hydrogel film that was 7 nm thick, 89% water, and encasing laccase (200 mM) and TEMPO moieties (33 mM). For DS values >10%, all of the TEMPOs in the hydrogel film were redox-active in that they could be oxidized by the electrode. With hydrogel layers made with lower-DS PVAm-Ts, only about half of the TEMPOs were redox-active; 10% DS appears to be a percolation threshold for complete TEMPO-to-TEMPO electron transport. In parallel experiments with hydrogel complexes adsorbed onto regenerated cellulose films, the aldehyde concentrations increased monotonically with the density of redox-active TEMPO moieties in the adsorbed hydrogel. The maximum density of aldehydes was 0.24 μmol/m2, about 10 times less than the theoretical concentration of primary hydroxyl groups exposed on crystalline cellulose surfaces. Previous work showed that PVAm-T/laccase complexes are effective adhesives between wet cellulose surfaces when the DS is >10%. This work supports the explanation that TEMPO-to-TEMPO electron transport is required for the generation of aldehydes necessary for wet adhesion to PVAm. [ABSTRACT FROM AUTHOR]

Lentinula edodes(shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene fromPleurotus ostreatusdriven byL. edodeslaccase 1 gene promoter intoL. edodesfor expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain. [ABSTRACT FROM PUBLISHER]

Colletotrichum gloeosporioides is the main causal agent of anthracnose of mango ( Mangifera indica ), which has great economic impact on mango industry worldwide. Laccase has versatile biological functions including: melanin biosynthesis and pathogenicity in several other pathogenic fungi. A laccase gene LAC1 , homologous to C. lagenarium laccase, was cloned from C. gloeosporioides . To evaluate its role, LAC1 was disrupted via gene replacement, and the mutant Δlac1 was analyzed. Compared with the wild-type, the Δlac1 mutant had less pigmentation and dramatically reduced aerial mycelial mass and radial growth rates, and rarely produces conidia in vitro. Microscopic observation showed that Δlac1 hyphae were lighter in color, less branched and septated with obviously more condensed protoplasm than wild-type. The Δlac1 mutant was obviously different in nutritional utilization compared with the wild-type. Virulence tests showed that Δlac1 mycelia were weakly virulent on non-wounded mango leaves and fruits, and was significantly less virulent on wounded mango leaves and fruits. The Δlac1 produced less extracellular cell wall-degrading enzymes (CWDE) and no detectable extracellular laccase activity in vitro. These results suggest that the C. gloeosporioides LAC1 laccase is involved not only in virulence, but also in mycelial growth and differentiation, conidiation, appressorium formation, pigmentation, melanin biosynthesis, secretion of extracellular hydrolytic enzymes, and utilization of exogenous nutrition. [ABSTRACT FROM AUTHOR]

Laccases are oxidoreductase catalyze the oxidation of a wide range of substrates with oxygen as the electron acceptor. This report was aimed to the high-level expression of a laccase, CueO from Escherichia coli K12 in Pichia pastoris GS115 and its application on decolorization of synthetic dyes. The yacK gene coding CueO was cloned into an expression vector of Pichia pastoris , pHBM905BDM and expressed in a secretory form with Pichia pastoris GS115 as the host. The yield of the recombinant protein was 556 mg/L with high-density fermentation and the enzyme activity was about 41,000 U/L. The recombinant laccase was purified and characterized. Its optimum pH and temperature was 3.0 and 55 °C with 2, 2′-azino-bis-(3-ethylbenzothazoline-6-sulfonic acid) (ABTS) as the substrate, respectively. This recombinant protein was thermostable and its half life at 70 °C was about 60 min. In the presence of natural redox mediator acetosyringone, the purified recombinant laccase decolorized 98.1% and 98.5% of Congo red, malachite green, respectively. It also decolorized 90.03% of Remazol brilliant blue R without this mediator. In addition, this enzyme was applied on the decolorization of wastewater from a textile printing factory and showed an obvious bleaching effect. [ABSTRACT FROM AUTHOR]

Background: Bast fibres are characterized by very thick secondary cell walls containing high amounts of cellulose and low lignin contents in contrast to the heavily lignified cell walls typically found in the xylem tissues. To improve the quality of the fiber-based products in the future, a thorough understanding of the main cell wall polymer biosynthetic pathways is required. In this study we have carried out a characterization of the genes involved in lignin biosynthesis in flax along with some of their regulation mechanisms. Results: We have first identified the members of the phenylpropanoid gene families through a combination of in silico approaches. The more specific lignin genes were further characterized by high throughput transcriptomic approaches in different organs and physiological conditions and their cell/tissue expression was localized in the stems, roots and leaves. Laccases play an important role in the polymerization of monolignols. This multigenic family was determined and a miRNA was identified to play a role in the posttranscriptional regulation by cleaving the transcripts of some specific genes shown to be expressed in lignified tissues. In situ hybridization also showed that the miRNA precursor was expressed in the young xylem cells located near the vascular cambium. The results obtained in this work also allowed us to determine that most of the genes involved in lignin biosynthesis are included in a unique co-expression cluster and that MYB transcription factors are potentially good candidates for regulating these genes. Conclusions: Target engineering of cell walls to improve plant product quality requires good knowledge of the genes responsible for the production of the main polymers. For bast fiber plants such as flax, it is important to target the correct genes from the beginning since the difficulty to produce transgenic material does not make possible to test a large number of genes. Our work determined which of these genes could be potentially modified and showed that it was possible to target different regulatory pathways to modify lignification. [ABSTRACT FROM AUTHOR]

Environmental pollution caused by petroleum compounds has become a global concern. The aim of this study was to evaluate the indigenous fungal isolates in Iran for biodegradation of crude oil pollutants. In order to isolate fungal strains, the soil samples were enriched in minimal salts medium (MSM) with 1% crude oil and then the crude oil degradation was measured by total petroleum hydrocarbon (TPH) assay. The degradation of hydrocarbons compounds was also analysed by FT-IR and HPLC, and the activity of peroxidase enzyme and biosurfactant production were also measured. We isolated 40 fungal strains and selected the isolate G-05 with 70% degradation ability of petroleum hydrocarbons as a premium isolate after 15 days. Residual crude oil analysis with FT-IR spectrophotometry and HPLC showed that G-05 is able to degrade 90 and 100% of aliphatic compounds and some polycyclic aromatic hydrocarbons (PAH), respectively. Evaluation of enzymatic activity showed that this isolate can produce 4 U L of Laccase enzyme for oil removal; it is capable of producing biosurfactant and reducing the surface tension of the medium to 25.95 ± 0.1 m Nm. This strain was identified as a member of Trematophoma genus and the obtained results showed that this strain is a highly potent strain in bioremediation of soils contaminated by crude oil. Graphical Abstract: [ABSTRACT FROM AUTHOR]

We describe the isolation and characteristics of a novel thermophilic bacterium from soil. The organism is a member of the Anoxybacillus genus based on phylogenetic analysis of the 16S rRNA gene. The 16S rRNA of the organism shares >99% sequence identity with those of two species, Anoxybacillus rupiensis and A. geothermalis. We named this isolate as Anoxybacillus sp. strain UARK-01. UARK-01 grows optimally in the presence of oxygen at 55 °C and pH 8. It grew excellently in the presence of lignin as the sole carbon source. Culture supernatant from UARK-01 grown on lignin was rich in laccase activity. The laccase activity was optimal at 90 °C and pH 9, and there was comparable activity at 80 and 100 °C. The crude laccase decolorized approximately 75% of Congo Red in 7 h under optimal conditions. A single laccase gene was identified from the draft genome sequence of Anoxybacillus sp. UARK-01. The UARK-01 laccase (Anox_Lacc) was cloned and overexpressed in Escherichia coli and was partially purified. The partially purified Anox_Lacc decolorized approximately 1.64+/0.21 nanomoles of Congo Red per microgram protein in 30 min at 90 °C and pH 9. Anox_Lacc is a member of the multicopper polyphenol oxidoreductase laccase family (pfam02578 Cu-oxidase_4) and has novel characteristics. Multiple sequence analysis of Anox_Lacc with six homologs from the family revealed four conserved copper ligands and several new residues that are fully conserved. Anox_Lacc is enriched in leucine, glutamine, and lysine, and it contains fewer alanine, arginine, glycine, and serine residues. Skewed amino acid composition of Anox_Lacc likely contributes to the exceptional thermochemical properties of the laccase activity from UARK-01. Both lignin utilization and production of hyperthermostable alkaline laccase are new findings in the Anoxybacillus genus. [ABSTRACT FROM AUTHOR]

Background: Phenol oxidases (POs) catalyze the oxidation of dopa and dopamine to melanin, which is crucial for cuticle formation and innate immune maintenance in insects. Although, Laccase 2, a member of the PO family, has been reported to be a requirement for melanin-mediated cuticle tanning in the development stages of some insects, whether it participates in cuticle construction and other physiological processes during the metamorphosis of mosquito pupae is unclear. Methods: The association between the phenotype and the expression profile of Anopheles sinensis Laccase 2 (AsLac2) was assessed from pupation to adult eclosion. Individuals showing an expression deficiency of AsLac2 that was produced by RNAi and their phenotypic defects and physiological characterizations were compared in detail with the controls. Results: During the dominant expression period, knockdown of AsLac2 in pupae caused the cuticle to be unpigmented, and produced thin and very soft cuticles, which further impeded the eclosion rate of adults as well as their fitness. Moreover, melanization immune responses in the pupae were sharply decreased, leading to poor resistance to microorganism infection. Both the high conservation among Laccase 2 homologs and a very similar genomic synteny of the neighborhood in Anopheles genus implies a conservative function in the pupal stage. Conclusions: To our knowledge, this is the first study to report the serious phenotypic defects in mosquito pupae caused by the dysfunction of Laccase 2. Our findings strongly suggest that Laccase 2 is crucial for Anopheles cuticle construction and melanization immune responses to pathogen infections during pupal metamorphosis. This irreplaceability provides valuable information on the application of Lacccase 2 and/or other key genes in the melanin metabolism pathway for developing mosquito control strategies. [ABSTRACT FROM AUTHOR]

Scleromitrula shiraiana causes the popcorn disease in mulberry trees resulting in severe economic losses. Previous studies have shown that melanin may play a vital role in establishing the pathogenicity of fungi. In the present study, we identified the melanin produced in S. shiraiana belongs to DHN melanin by gas chromatography-mass spectrometry, and cloned the laccase Sh-lac, a potential DHN melanin biosynthesis gene from S. shiraiana. We obtained two stable Sh-lac silenced transformants using RNAi, ilac-4 and 8 to elucidate the DHN melanin biosynthetic pathway in S. shiraiana. The melanin production of ilac-4 and ilac-8 was significantly reduced, and their vegetative growth was also suppressed. Results such as these led to a proposal that Sh-lac played a key role in DHN melanin formation in S. shiraiana and may function differentially with other melanin biosynthetic genes. The inhibition of melanin was accompanied by the decrease of oxalic acid and the adhesion of hyphae was impaired. Our results indicated that laccase was an important enzyme in the synthesis of melanin and might play a critical role in the pathogenicity of S. shiraiana. [ABSTRACT FROM AUTHOR]

Regulation of alkaline-resistant laccase from Perenniporia tephropora KU-Alk4 was proved to be controlled by several factors. One important factor was the initial pH, which drove the fungus to produce different kinds of ligninolytic enzymes. P. tephropora KU-Alk4 could grow at pH 4.5, 7.0, and 8.0. The fungus produced laccase and MnP at pH 7.0, but only laccase at pH 8.0. The specific activity of laccase in the pH 8.0 culture was higher than that in the pH 7.0 culture. At pH 8.0, glucose was the best carbon source for laccase production but growth was better with lactose. Low concentrations of glucose at 0.1% to 1.0% enhanced laccase production, while concentrations over 1% gave contradictory results. Veratryl alcohol induced the production of laccase. A trace concentration of copper ions was required for laccase production. Biomass increased with an increasing rate of aeration of shaking flasks from 100 to 140 rpm; however, shaking at over 120 rpm decreased laccase quantity. Highest amount of laccase produced by KU-Alk4, 360 U/mL, was at pH 8.0 with 1% glucose and 0.2 mM copper sulfate, unshaken for the first 3 days, followed by addition of 0.85 mM veratryl alcohol and shaking at 120 rpm. The crude enzyme was significantly stable in alkaline pH 8.0~10.0 for 24 hr. After treating the pulp mill effluent with the KU-Alk4 system for 3 days, pH decreased from 9.6 to 6.8, with reduction of color and chemical oxygen demand at 83.2% and 81%, respectively. Laccase was detectable during the biotreatment process. [ABSTRACT FROM AUTHOR]

A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19 735 ± 1719 Ug −1 and 6.22 ± 0.53 gL −1 respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (μmol min −1 ) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN 3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25–70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol. [ABSTRACT FROM AUTHOR]

Aims The aim of this study was to evaluate the activity of a novel bacterial laccase-like multi-copper oxidase ( LMCO) from Paenibacillus glucanolyticus SLM1: a bacterium isolated from pulp and paper waste. Methods and Results A new bacterial LMCO gene ( CuOx) from P. glucanolyticus SLM1 was identified and cloned into pET22b. The protein it encodes was recombinantly expressed in Escherichia coli. The recombinant P. glucanolyticus LMCO had a molecular weight of approximately 90 kDa and demonstrated oxidation of the LMCO substrates 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) ( ABTS), catechol, and 2,6-Dimethoxyphenol (2,6- DMP), with the oxidation of ABTS occurring to the greatest extent (776 U mg−1 under optimal conditions of pH 7 and 40°C). Furthermore, recombinant P. glucanolyticus CuOx retained activity against ABTS in the presence of 1 mol l−1 NaCl, 50% dimethyl sulfoxide and 5% Tween-80 and can decolorize several types of dyes. Conclusions This enzyme has a neutral pH optimum, is capable of decolorizing dyes, and is active in the presence salt, detergents and surfactant. The characteristics of this enzyme suggest that it could be used for a variety of industrial applications. Significance and Impact of the Study This work characterizes a unique bacterial LMCO with activity higher than that of previously characterized fungal or bacterial LMCOs. This enzyme may have utility for industrial bleaching, treatment of dye effluent, and lignin removal. [ABSTRACT FROM AUTHOR]

This research was conducted to extend the knowledge on the differential regulation of laccase genes in response to dyes. In order to accomplish this, we analyzed both, the expression of five laccase genes by real time RT-qPCR, and also the laccase activity and isoforms patterns during the time-course of a Pleurotus ostreatus submerged fermentation supplemented with either acetyl yellow G (AYG) or remazol brilliant blue R (RBBR) dyes. For the purpose of obtaining a stable reference gene for optimal normalization of RT-quantitative PCR gene expression assays, we tested four candidate reference genes. As a result of this analysis, gpd was selected as reference index for data normalization. The addition of dyes had an induction effect on the enzymatic activity and also modified the zymogram profile. Fermentation with RBBR showed the highest laccase activity and number of isoforms along the course of the fermentation. Laccase gene expression profiles displayed up/down regulation along the fermentation time in four laccase genes ( pox4, pox3, poxa1b and pox2), while pox1 was not expressed in either of the fermentation conditions. AYG addition caused the highest induction and repression levels for genes pox3 and poxa1b respectively. The expression level for all genes in the presence of RBBR were lower than in AYG, being in both conditions this response growth time dependent. These results show the influence of the nature of dyes on the induction level of laccase activity and on the differential regulation of the laccase genes expression in P. ostreatus. [ABSTRACT FROM AUTHOR]

Taking natural coal as a “seed bank” of bacterial strains able to degrade lignin that is with molecular structure similar to coal components, we isolated 393 and 483 bacterial strains from a meager lean coal sample from Hancheng coalbed and a brown coal sample from Bayannaoer coalbed, respectively, by using different media. Statistical analysis showed that isolates were significantly more site-specific than mediumspecific. Of the 876 strains belonging to 27 genera in Actinobacteria, Firmicutes, and Proteobacteria, 612 were positive for lignin degradation function, including 218 strains belonging to 35 species in Hancheng and 394 strains belonging to 19 species in Zhongqi. Among them, the dominant lignin-degrading strains were Thauera (Hancheng), Arthrobacter (Zhongqi) and Rhizobium (both). The genes encoding the laccases- or laccase-like multicopper oxidases, key enzymes in lignin production and degradation, were detected in three genera including Massila for the first time, which was in high expression by real time PCR (qRT-PCR) detection, confirming coal as a good seed bank. [ABSTRACT FROM AUTHOR]

Volvariella volvacea, usually harvested in its egg stage, is one of the most popular mushrooms in Asia. The rapid transition from the egg stage to elongation stage, during which the stipe stretches to almost full length leads to the opening of the cap and rupture of the universal veil, and is considered to be one of the main factors that negatively impacts the yield and value of V. volvacea. Stipe elongation is a common phenomenon in mushrooms; however, the mechanisms, genes and regulation involved in stipe elongation are still poorly understood. In order to study the genes related to the stipe elongation, we analyzed the transcription of laccase genes in stipe tissue of V. volvacea, as some laccases have been suggested to be involved in stipe elongation in Flammulina velutipes. Based on transcription patterns, the expression of Vvlcc3 was found to be the highest among the 11 laccase genes. Moreover, phylogenetic analysis showed that VvLCC3 has a high degree of identity with other basidiomycete laccases. Therefore, we selected and cloned a laccase gene, named Vvlcc3, a cDNA from V. volvacea, and expressed the cDNA in Pichia pastoris. The presence of the laccase signature L1-L4 on the deduced protein sequence indicates that the gene encodes a laccase. Phylogenetic analysis showed that VvLCC3 clusters with Coprinopsis cinerea laccases. The ability to catalyze ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation proved that the product of the Vvlcc3 gene was a functional laccase. We also found that the expression of the Vvlcc3 gene in V. volvacea increased during button stage to the elongation stage; it reached its peak in the elongation stage, and then decreased in the maturation stage, which was similar to the trend in the expression of Fv-lac3 and Fv-lac5 in F. velutipes stipe tissue. The similar trend in expression level of these laccase genes of F. velutipes suggested that this gene could be involved in stipe elongation in V. volvacea. [ABSTRACT FROM AUTHOR]

To improve the feed quality removal of lignin from plant biomass is essential. To improve the activity of laccase of white rot fungi, aromatic inducers are used. In this study three inducers [Resorcinol (5mM and 10mM), Xylidine and Anisaldehyde] to induce the production of laccase enzyme in the culture of fungus: Cyathus stercoreus. Resorcinol (10mM) was found to be the best inducer among the rest. The enzyme activity was observed highest on the 8th day of induction (226.7U/ml). Protein content was also increased with the age of the culture. 80% ammonium sulphate was suitable for precipitating the laccase enzyme for culture filtrate. The laccase production can be enhanced with inducers and can be further used for the removal of lignin from the plant biomass. [ABSTRACT FROM AUTHOR]

Vinasse is the dark-colored wastewater that is generated by bioethanol distilleries from feedstock molasses. The vinasse that is generated from molasses contains high amounts of pollutants, including phenolic compounds and melanoindin. The goal of this work was to study the expression of laccase genes in the Trametes hirsuta strain Bm-2, isolated in Yucatan, Mexico, in the presence of phenolic compounds, as well as its effectiveness in removing colorants from vinasse. In the presence of all phenolic compounds tested (guaiacol, ferulic acid, and vanillic acid), increased levels of laccase-encoding mRNA were observed. Transcript levels in the presence of guaiacol were 40 times higher than those in the control. The lcc1 and lcc2 genes of T. hirsuta were differentially expressed; guaiacol and vanillin induced the expression of both genes, whereas ferulic acid only induced the expression of lcc2. The discoloration of vinasse was concomitant with the increase in laccase activity. The highest value of enzyme activity (2543.7 U/mL) was obtained in 10% (v/v) vinasse, which corresponded to a 69.2% increase in discoloration. This study demonstrates the potential of the Bm-2 strain of T. hirsuta for the biodegradation of vinasse. [ABSTRACT FROM AUTHOR]

The effects of Cu on the activity and expression of laccase were investigated in seven different strains of Pycnoporus coccineus collected from different regions in Korea. Cu was toxic to mycelial growth at concentrations greater than 0.5 mM CuSO and showed complete growth inhibition at 1 mM in the liquid culture. However, Cu significantly upregulated the extracellular laccase activity at 0.2 mM in five strains of P. coccineus, IUM4209, IUM0032, IUM0450, IUM0470, and IUM4093, whereas two strains, IUM0253 and IUM0049, did not respond to Cu, despite being closely related to the other five strains. Subsequent RT-PCR analysis also showed that the laccase mRNA was highly expressed only in the former five strains in the presence of Cu. Taken together, these results indicate that Cu regulates expression of the laccase gene in a strain-dependent manner. The five strains commonly produced a single predominant laccase protein with a molecular weight of 68 kDa. Peptide sequencing revealed that the laccase was a homolog of Lcc1 of P. coccineus, which was isolated in China. The Cu-induced culture supernatants exhibited high degradation of polycyclic aromatic hydrocarbons, indicating that the 68-kDa laccase is the primary extracellular degradative enzyme in P. coccineus. [ABSTRACT FROM AUTHOR]

Laccase is an enzyme that catalyzes oxidation of phenolic compounds, diamines and aromatic amines. In this study, a novel laccase-like gene (atm) in a ligninolyitic isolate Agrobacterium sp. S5-1 from soil humus was identified and heterologously expressed in Escherichia coli. Atm exhibited its maximal activity at pH 4.5 and at 50°C. This enzyme was tolerant to high temperature, a broad range of pH, heavy metal ions (Co3+, Mn2+, Cu2+ and Ni2+, 20 mM) and all tested organic solvents. Furthermore, Atm significantly (p<0.05) increased dry matter digestibility of maize straw from 23.44% to 27.96% and from 29.53% to 37.10% after 8 or 24 h of digestion and improved acid detergent fiber digestibility from 5.81% to 10.33% and from 12.80% to 19.07% after 8 or 24 h of digestion, respectively. The combination of Atm and fibrolytic enzymes significantly (p<0.05) enhanced neutral detergent fiber digestibility from 19.02% to 24.55% after 24 h of digestion respectively. Results showed treatment with Atm effectively improved in vitro digestibility of maize straw, thus suggesting that Atm has an application potential for bioconversion of lignin rich agricultural byproducts into animal feed and cellulosic ethanol. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes. [ABSTRACT FROM AUTHOR]

Plant laccase ( LAC) enzymes belong to the blue copper oxidase family and polymerize monolignols into lignin. Recent studies have established the involvement of micro RNAs in this process; however, physiological functions and regulation of plant laccases remain poorly understood. Here, we show that a laccase gene, LAC4, regulated by a micro RNA, miR397b, controls both lignin biosynthesis and seed yield in Arabidopsis. In transgenic plants, overexpression of miR397b ( OXmiR397b) reduced lignin deposition. The secondary wall thickness of vessels and the fibres was reduced in the OXmiR397b line, and both syringyl and guaiacyl subunits are decreased, leading to weakening of vascular tissues. In contrast, overexpression of miR397b-resistant laccase m RNA results in an opposite phenotype. Plants overexpressing miR397b develop more than two inflorescence shoots and have an increased silique number and silique length, resulting in higher seed numbers. In addition, enlarged seeds and more seeds are formed in these miR397b overexpression plants. The study suggests that miR397-mediated development via regulating laccase genes might be a common mechanism in flowering plants and that the modulation of laccase by miR397 may be potential for engineering plant biomass production with less lignin. [ABSTRACT FROM AUTHOR]

A modified laccase gene, CcLCC6, from Coprinopsis cinerea was chemically synthesized according to the yeast codon bias and expressed in Pichia pastoris. The main properties of laccase, effects of ions and inhibitors, and optimal condition for decolouring malachite green (MG) were investigated in this study. The optimal pH level and temperature of laccase are 3.0 and 40 °C, respectively. The metal ions Mn, Zn, Fe and Al could inhibit laccase activity, as well as 1 mM of sodium dodecyl sulphate and sodium thiosulphate. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), as a mediator, was necessary in decolorizing MG. The optimal pH and temperature for MG decolorization were 3.0 and 50 °C, respectively. Approximately 0.02 μM recombinant laccase could effectively decolour 0.05 mM of MG in 1 h. CcLCC6I could inhibit the toxicity of MG to P. pastoris. This is the first report on the successful expression in P. pastoris of CcLCC6I and its enzymatic property. Laccase can also be considered as a candidate for treating industrial effluent containing MG. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, the dye-decolorizing properties of soluble extracts from the indigenous fungus Heteroporus biennis IOC4581 were determined. Decolorizing activity was associated with the secretion of a 45-kDa laccase. Laccase expression was extremely sensitive to carbon and nitrogen contents in culture media. Supplementation with metal ions and aromatic compounds resulted in extremely variable protein secretion profiles, although enzyme yields were not improved. Kinetic parameters of the soluble crude extract were promising, destaining seven out of nine textile dyes without the need of redox mediators. The extract was insensitive to Ca2+, Mg2+, Mn2+, and Zn2+ ions, and catalysis was enhanced in the presence of Cu2+ and Al3+ ions. The soluble fungal extract retained its maximal decolorizing activity at a wide range of pH (2.0–9.0) and temperature (4–50 °C), indicating potential application to dye biodegradation. [Copyright &y& Elsevier]

The aim of this work was to determine the ligninolytic enzymes of Ganoderma weberianum (B-18) involved in decolorization process as well as the diversity of genes encoding for these enzymes. Native polyacrylamide gel electrophoresis of extracellular crude enzymes confirmed that the laccase isozymes of this white rot fungus are involved in decolorization process. Extracellular laccase isozymes are selectively produced by this strain according to the presence of specific dyes and culture age. PCR amplification, sequencing as well as phylogenetic and multiple sequence alignment analyses allowed detecting the presence of five different laccase genes with novel partial sequences. [ABSTRACT FROM AUTHOR]

Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu2+ and Fe2+ could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930. [ABSTRACT FROM AUTHOR]

Highlights: [•] Many bacterial laccases are copper depleted when expressed in Escherichia coli. [•] CopZ copper chaperone assists in copper insertion into a recombinant laccase. [•] Coexpression is useful for routine laccase production with higher specific activity. [Copyright &y& Elsevier]

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ~40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome-based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content. [ABSTRACT FROM AUTHOR]

Laccases (EC 1.10.3.2) are copper-containing polyphenol oxidases found in white-rot fungi. Here, we report the cloning and analysis of the nucleotide sequence of a new laccase gene, fvlac7, based or the genomic sequence of Flammulina velutipes. A primer set was designed from the putative mRNA that was aligned to the genomic DNA of F velutipes. A cDNA fragment approximately 1.6-kb long was then amplified by reverse transcriptase-PCR using total RNA, which was subsequently cloned and sequenced. The cDNA sequence of Mac7 was then compared to that of the genomic DNA, and 16 introns were found in the genomic DNA sequence. The fvlac7 protein, which consists of 538 amino acids, showed only 42 ~ 51% identity with 12 different mushroom species containing two laccases of F. velutipes, suggesting the fvlac7 is a novel laccase gene. The first 25 amino acids of Fvlac7 correspond to a predicted signal sequence, four copper-binding sits, and four N-glycosylation sites. Fvlac7 cDNA was heterologously overexpressed in an Escherichia coli system with an approximate expected molecular weight of 60 kDa. [ABSTRACT FROM AUTHOR]

Abstract: The cDNAs of Tuber melanosporum laccases (Tmellcc1 and Tmellcc2) have been cloned. From the cloned cDNAs probes were prepared to investigate the expression levels of the Tmellcc1 and Tmellcc2 genes in the free living mycelium (FLM), ectomycorrhizae (ECM) and different developmental stages of fruit body (FB) by quantitative PCR (qPCR). The mRNA expression levels agree with the changes of laccase activities. The histochemical data agree with the qPCR and biochemical results. The highest laccase expression occurs in the ECM, when the host plant roots are invaded by the fungal mycelium. [Copyright &y& Elsevier]

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics. [ABSTRACT FROM AUTHOR]

To advance the understanding of the molecular mechanisms controlling microbial activities involved in carbon cycling and mitigation of environmental pollution in freshwaters, the influence of heavy metals and natural as well as xenobiotic organic compounds on laccase gene expression was quantified using quantitative real-time PCR (qRT-PCR) in an exclusively aquatic fungus (the aquatic hyphomycete Clavariopsis aquatica) for the first time. Five putative laccase genes ( led to lcc5) identified in C. aquatica were differentially expressed in response to the fungal growth stage and potential laccase inducers, with certain genes being upregulated by, e.g., the lignocellulose breakdown product vanillic acid, the endocrine disruptor technical nonylphenol, manganese, and zinc. Icc4 is inducible by vanillic acid and most likely encodes an extracellular laccase already excreted during the trophophase of the organism, suggesting a function during fungal substrate colonization. Surprisingly, unlike many laceases of terrestrial fungi, none of the C. aquatica laccase genes was found to be upregulated by copper. However, copper strongly increases extracellular laccase activity in C. aquatica, possibly due to stabilization of the copper-containing catalytic center of the enzyme. Copper was found to half-saturate laccase activity already at about 1.8 |xM, in favor of a fungal adaptation to low copper concentrations of aquatic habitats. [ABSTRACT FROM AUTHOR]