Mechanism of non-phenolic substrate oxidation by the fungal laccase Type 1 copper site from Trametes versicolor: the case of benzo[a]pyrene and anthracene.

Laccases (EC 1.10.3.2) are multicopper oxidases with the capability to oxidize diverse phenolic and non-phenolic substrates. While the molecular mechanism of their activity towards phenolic substrates is well-established, their reactivity towards non-phenolic substrates, such as polycyclic aromatic hydrocarbons (PAHs), remains unclear. To elucidate the oxidation mechanism of PAHs, particularly the activation mechanism of the sp2 aromatic C-H bond, we conducted a density functional theory investigation on the oxidation of two PAHs (anthracene and benzo[a]pyrene) using an extensive model of the T1 copper catalytic site of the fungal laccase from Trametes versicolor.

Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris.

l-amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to corresponding α-keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His-hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His-frLAAO1 from Fibroporia radiculosa and 9His-laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N-glycosylated. We generated mutants of 9His-hcLAAO4 lacking N-glycosylation sites to further understand the effects of N-glycosylation. All four predicted N-glycosylation sites were glycosylated in 9His-hcLAAO4 expressed in P. pastoris. Enzymatic activity was similar for fully glycosylated 9His-hcLAAO4 and variants without one or all N-glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N-glycans. This was caused by the absence of a hypermannosylated N-glycan on asparagine residue N54. The lack of one or all of the other N-glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N-glycosylation during biosynthesis.

Enhancement of laccase production by Cerrena unicolor through fungal interspecies interaction and optimum conditions determination.

The present study aimed to identify a pair of fungal strains that promote laccase production in the co-cultivation of white-rot basidiomycetes and to determine the optimum conditions to enhance enzyme synthesis under co-fermentation of mandarin peels. Co-cultivation of Cerrena unicolor with Trametes versicolor, Lenzites betulina, and Panus lecomtei led to up-regulation of laccase activity. Moreover, interspecific interaction of Cerrena unicolor and Trametes versicolor induced the production of two new laccase isoenzymes. By contrast, interactions of Cerrena unicolor with Trametes coccineus and Trametes hirsuta resulted in a multiple decreased ability of Cerrena unicolor to produce laccase. Co-cultivation of Cerrena unicolor with other fungi 3- to 12-fold down-regulated manganese peroxidase (MnP) activity. The outcomes of these fungal interactions are closely related to the initial concentration and availability of the nutrients, the partners' inoculum ratio, time, and sequence of their inoculation. Co-cultivation of Cerrena unicolor and Trametes versicolor in fermenter resulted in the accumulation of 476 U/mL laccase and 1.12 U/mL MnP.

Cerrena unicolor Laccases, Genes Expression and Regulation of Activity.

A white rot fungus Cerrena unicolor has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of C. unicolor culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase's transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from C. unicolor, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by C. unicolor are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.

Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi.

α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.

The Laetiporus polyketide synthase LpaA produces a series of antifungal polyenes.

The conspicuous bright golden to orange-reddish coloration of species of the basidiomycete genus Laetiporus is a hallmark feature of their fruiting bodies, known among mushroom hunters as the "chicken of the woods". This report describes the identification of an eight-domain mono-modular highly reducing polyketide synthase as sole enzyme necessary for laetiporic acid biosynthesis. Heterologous pathway reconstitution in both Aspergillus nidulans and Aspergillus niger verified that LpaA functions as a multi-chain length polyene synthase, which produces a cocktail of laetiporic acids with a methyl-branched C26-C32 main chain. Laetiporic acids show a marked antifungal activity on Aspergillus protoplasts. Given the multiple products of a single biosynthesis enzyme, our work underscores the diversity-oriented character of basidiomycete natural product biosynthesis.

A New Laccase of Lac 2 from the White Rot Fungus Cerrena unicolor 6884 and Lac 2-Mediated Degradation of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH4)2SO4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1 detoxification. AFB1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2' -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1 degradation by Lac 2. The product of AFB1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1 (AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1 present in food and feeds.

RNA-Seq transcriptomic analyses of Antrodia camphorata to determine antroquinonol and antrodin C biosynthetic mechanisms in the in situ extractive fermentation.

BACKGROUND: In situ extractive fermentation (ISEF) is an important technique for improving metabolite productivity. The different extractants can induce the synthesis of different bioactive metabolites of Antrodia camphorata during ISEF. However, a lack of research on the molecular genetics of A. camphorata during ISEF currently hinders such studies on metabolite biosynthetic mechanisms. RESULTS: To clarify the differentially expressed genes during ISEF, the gene transcriptional expression features of A. camphorata S-29 were analysed. The addition of n-tetradecane as an extractant during ISEF showed more pronounced up-regulation of ubiquinone and other terpenoid-quinone biosynthesis pathway genes (CoQ2, wrbA and ARO8). When oleic acid was used as an extractant, the terpenoid backbone biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways were significantly enriched, and genes (IDI, E2.3.3.10, HMGCR atoB, and CoQ2) related to these two pathways were also significantly up-regulated. The CoQ2 genes encode puru-hydroxybenzoate:polyprenyltransferase, playing an important role in antroquinonol synthesis. The IDI, E2.3.3.10, HMGCR and atoB genes of the terpenoid backbone biosynthesis pathway might play an important role in the synthesis of the triquine-type sesquiterpene antrodin C. CONCLUSION: This investigation advances our understanding of how two different extractants of n-tetradecane and oleic acid affect the biosynthesis of metabolites in A. camphorata. It is beneficial to provide potential strategies for improving antrodin C and antroquinonol production by genetic means. © 2020 Society of Chemical Industry.

Construction of a combined enzyme system of graphene oxide and manganese peroxidase for efficient oxidation of aromatic compounds.

Manganese peroxidase (MnP) from Irpex lacteus F17 has potential use as a biocatalyst in the field of environmental biotechnology because of its unique properties and ability to decompose harmful aromatic compounds. However, its requirement of harsh acidic reaction conditions and its insufficient catalytic activity restrict its practical applications. Here, we combine graphene oxide (GO) and MnP to construct an efficient enzyme system (GO-MnP) with improved catalytic efficiencies and a wide pH range for the oxidation of aromatic substances and dye decolorization. We found that the Michaelis constant (Km) of GO-MnP for Mn2+ was 2.8 times lower and the catalytic efficiency (kcat/Km) of GO-MnP was 4.5 times higher than those of MnP, and that the decolorization of various dyes by GO-MnP was significantly improved over the pH range of 4.5-5.5. A comparison of the midpoint redox potentials also reflects the strong oxidation ability of GO-MnP. Furthermore, we demonstrated that, in the GO-MnP system, the MnP activity is mainly determined by the amounts of epoxy and carboxyl groups in GO, based on an analysis of the functional group changes in GO and reduced GO associated with different reduction degrees as shown by X-ray photoelectron spectroscopy.

The structural basis of fungal glucuronoyl esterase activity on natural substrates.

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/ß-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.

A proposed stepwise screening framework for the selection of polycyclic aromatic hydrocarbon (PAH)-degrading white rot fungi.

This study suggests a simple three-step screening protocol for the selection of white rot fungi (WRF) capable of degrading polycyclic aromatic hydrocarbons (PAHs), which combines easily applicable bioassay techniques, and verifies that protocol by evaluating the PAH degradation activity, ligninolytic enzyme secretion, and relevant gene expressions of the selected PAH-degraders. Using 120 fungal strains, a sequence of bioassay techniques was applied: Bavendamm's reaction (Step 1), remazol brilliant blue R (RBBR) decolorization (Step 2); assays for tolerance to four mixed PAHs-phenanthrene, anthracene, fluoranthene, and pyrene (Step 3). This stepwise protocol selected 14 PAH-degrading WRF, including Microporus vernicipes, Peniophora incarnata, Perenniporia subacida, Phanerochaete sordida, Phlebia acerina, and Phlebia radiata. Of these, P. incarnata exhibited the highest PAH degradative activity, ranging from 40 to > 90%, which was related to the time-variable secretions of three extracellular ligninolytic enzymes: laccase, manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP). Laccase and MnP production by P. incarnata tended to be greater in the early stages of PAH degradation, whereas its LiP production became intensified with decreasing laccase and MnP production. Pilc1 and pimp1 genes encoding laccase and MnP were expressed, indicating the occurrence of extracellular enzyme-driven biodegradation of PAH by the fungal strains.

Comparison of downstream processing methods in purification of highly active laccase.

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.

Catalytic properties of a short manganese peroxidase from Irpex lacteus F17 and the role of Glu166 in the Mn2+-independent activity.

Il-MnP1 (GenBank: AGO86670.2) has been confirmed by sequence analysis as a short manganese peroxidase (MnP) from Irpex lacteus F17 (CCTCC AF 2014020). To investigate the catalytic properties, the oxidation of typical aromatic substrates and the pathways of guaiacol oxidation by Il-MnP1, both in the presence and absence of Mn2+ at either pH 4.0 or pH 7.4, were analyzed. Results showed that Il-MnP1 exhibited higher oxidative activity in the presence of Mn2+ than in the absence of Mn2+ toward the majority of the selected substrates at pH 4.0. Additionally, the similar product compositions suggested that the oxidation of guaiacol mainly belongs to a series of polymeric reactions of radicals initiated by Il-MnP1, whether they were in the presence and absence of Mn2+ at either pH 4.0 or 7.4. Furthermore, two variants (E166G, E166Q) were found using site-directed mutagenesis, to improve the Mn2+-independent oxidative activity significantly. The catalytic efficiency (Kcat/Km) of E166G and E166Q in 2, 6-dimethoxyphenol oxidation was higher than Il-MnP1 by 170 and 34 times, respectively. The study revealed certain differences in catalytic properties between Mn2+ dependent and independent oxidation by Il-MnP1. More importantly, a residue (E166) was related to the Mn2+-independent activity of a short MnP.

Genome description of Phlebia radiata 79 with comparative genomics analysis on lignocellulose decomposition machinery of phlebioid fungi.

BACKGROUND: The white rot fungus Phlebia radiata, a type species of the genus Phlebia, is an efficient decomposer of plant cell wall polysaccharides, modifier of softwood and hardwood lignin, and is able to produce ethanol from various waste lignocellulose substrates. Thus, P. radiata is a promising organism for biotechnological applications aiming at sustainable utilization of plant biomass. Here we report the genome sequence of P. radiata isolate 79 originally isolated from decayed alder wood in South Finland. To better understand the evolution of wood decay mechanisms in this fungus and the Polyporales phlebioid clade, gene content and clustering of genes encoding specific carbohydrate-active enzymes (CAZymes) in seven closely related fungal species was investigated. In addition, other genes encoding proteins reflecting the fungal lifestyle including peptidases, transporters, small secreted proteins and genes involved in secondary metabolism were identified in the genome assembly of P. radiata. RESULTS: The PACBio sequenced nuclear genome of P. radiata was assembled to 93 contigs with 72X sequencing coverage and annotated, revealing a dense genome of 40.4 Mbp with approximately 14 082 predicted protein-coding genes. According to functional annotation, the genome harbors 209 glycoside hydrolase, 27 carbohydrate esterase, 8 polysaccharide lyase, and over 70 auxiliary redox enzyme-encoding genes. Comparisons with the genomes of other phlebioid fungi revealed shared and specific properties among the species with seemingly similar saprobic wood-decay lifestyles. Clustering of especially GH10 and AA9 enzyme-encoding genes according to genomic localization was discovered to be conserved among the phlebioid species. In P. radiata genome, a rich repertoire of genes involved in the production of secondary metabolites was recognized. In addition, 49 genes encoding predicted ABC proteins were identified in P. radiata genome together with 336 genes encoding peptidases, and 430 genes encoding small secreted proteins. CONCLUSIONS: The genome assembly of P. radiata contains wide array of carbohydrate polymer attacking CAZyme and oxidoreductase genes in a composition identifiable for phlebioid white rot lifestyle in wood decomposition, and may thus serve as reference for further studies. Comparative genomics also contributed to enlightening fungal decay mechanisms in conversion and cycling of recalcitrant organic carbon in the forest ecosystems.

Laccases with Variable Properties from Different Strains of Steccherinum ochraceum: Does Glycosylation Matter?

Laccases are blue multi-copper oxidases with an extensive number of actual and potential industrial applications. It is known that laccases from different fungal strains may vary in properties; however, the reason of this remains unclear. In the current study we have isolated and characterized seven laccases from different strains of Steccherinum ochraceum obtained from regions of central Russia. Although all seven laccases had the same primary sequences, there was a little variation in their molecular weights and thermostabilities. Moreover, statistically significant differences in laccases' catalytic parameters of oxidation of phenolic substrates and ABTS were observed. After the deglycosylation of four selected laccases by Endo H and PNGase F, their affinities to pyrocatechol and ABTS became the same, suggesting a substantial role of N-linked glycosylation in moderation of enzymatic properties of laccases.

A Lytic Polysaccharide Monooxygenase from a White-Rot Fungus Drives the Degradation of Lignin by a Versatile Peroxidase.

Lytic polysaccharide monooxygenases (LPMOs), a class of copper-dependent enzymes, play a crucial role in boosting the enzymatic decomposition of polysaccharides. Here, we reveal that LPMOs might be associated with a lignin degradation pathway. An LPMO from white-rot fungus Pleurotus ostreatus, LPMO9A (PoLPMO9A), was shown to be able to efficiently drive the activity of class II lignin-degrading peroxidases in vitro through H2O2 production regardless of the presence or absence of a cellulose substrate. An LPMO-driven peroxidase reaction can degrade ß-O-4 and 5-5' types of lignin dimer with 46.5% and 37.7% degradation, respectively, as well as alter the structure of natural lignin and kraft lignin. H2O2 generated by PoLPMO9A was preferentially utilized for the peroxidase from Physisporinus sp. strain P18 (PsVP) reaction rather than cellulose oxidation, indicating that white-rot fungi may have a strategy for preferential degradation of resistant lignin. This discovery shows that LPMOs may be involved in lignin oxidation as auxiliary enzymes of lignin-degrading peroxidases during the white-rot fungal decay process.IMPORTANCE The enzymatic biodegradation of structural polysaccharides is affected by the degree of delignification of lignocellulose during the white-rot fungal decay process. The lignin matrix decreases accessibility to the substrates for LPMOs. H2O2 has been studied as a cosubstrate for LPMOs, but the formation and utilization of H2O2 in the reactions still represent an intriguing focus of current research. Lignin-degrading peroxidases and LPMOs usually coexist during fungal decay, and therefore, the relationship between H2O2-dependent lignin-degrading peroxidases and LPMOs should be considered during the wood decay process. The current study revealed that white-rot fungal LPMOs may be involved in the degradation of lignin through driving a versatile form of peroxidase activity in vitro and that H2O2 generated by PoLPMO9A was preferentially used for lignin oxidation by lignin-degrading peroxidase (PsVP). These findings reveal a potential relationship between LPMOs and lignin degradation, which will be of great significance for further understanding the contribution of LPMOs to the white-rot fungal decay process.

Stimulation of Wood Degradation by Daedaleopsis confragosa and D. tricolor.

Biological pretreatment of the lignocellulosic residues, in which white-rot fungi have a crucial role, has many advantages compared to the chemical, physical, and physico-chemical methods of delignification and therefore attracts increasing scientific attention. Regarding the fact that properties and capacities of the ligninolytic enzymes of Daedaleopsis spp. are still unknown, the aim of this study was to research how nitrogen sources and inducers affect the potential of Daedaleopsis confragosa and Daedaleopsis tricolor to degrade cherry sawdust. NH4NO3, (NH4)2SO4, and peptone were tested as nitrogen sources, while veratryl alcohol, p-anisidine, vanillic acid, and phenylmethylsulfonyl fluoride were the studied inducers. As Mn-dependent peroxidase and laccase were the leader enzymes and cherry sawdust/peptone medium the best stimulator of their activities, the effect of inducers on delignification potential of these species was studied during fermentation of that substrate. Veratryl alcohol was the best stimulator of laccase and phenylmethylsulfonyl fluoride of Mn-dependent peroxidase activity (27,610.0 and 1338.4 U/L, respectively). These inducers also increased cherry sawdust delignification selectivity, particularly in D. tricolor in the presence of phenylmethylsulfonyl fluoride (lignin:hemicellulose:cellulose = 32.1%:0.9%:11.7%). Owing to the presented results, studied species could have an important role in the phase of lignocellulose pretreatment in various biotechnological processes.

Oxidative Damage Control during Decay of Wood by Brown Rot Fungus Using Oxygen Radicals.

Brown rot wood-degrading fungi deploy reactive oxygen species (ROS) to loosen plant cell walls and enable selective polysaccharide extraction. These ROS, including Fenton-generated hydroxyl radicals (HO˙), react with little specificity and risk damaging hyphae and secreted enzymes. Recently, it was shown that brown rot fungi reduce this risk, in part, by differentially expressing genes involved in HO˙ generation ahead of those coding carbohydrate-active enzymes (CAZYs). However, there are notable exceptions to this pattern, and we hypothesized that brown rot fungi would require additional extracellular mechanisms to limit ROS damage. To assess this, we grew Postia placenta directionally on wood wafers to spatially segregate early from later decay stages. Extracellular HO˙ production (avoidance) and quenching (suppression) capacities among the stages were analyzed, along with the ability of secreted CAZYs to maintain activity postoxidation (tolerance). First, we found that H2O2 and Fe2+ concentrations in the extracellular environment were conducive to HO˙ production in early (H2O2:Fe2+ ratio 2:1) but not later (ratio 1:131) stages of decay. Second, we found that ABTS radical cation quenching (antioxidant capacity) was higher in later decay stages, coincident with higher fungal phenolic concentrations. Third, by surveying enzyme activities before/after exposure to Fenton-generated HO˙, we found that CAZYs secreted early, amid HO˙, were more tolerant of oxidative stress than those expressed later and were more tolerant than homologs in the model CAZY producer Trichoderma reesei Collectively, this indicates that P. placenta uses avoidance, suppression, and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this brown rot fungus to use ROS to degrade wood.IMPORTANCE Wood is one of the largest pools of carbon on Earth, and its decomposition is dominated in most systems by fungi. Wood-degrading fungi specialize in extracting sugars bound within lignin, either by removing lignin first (white rot) or by using Fenton-generated reactive oxygen species (ROS) to "loosen" wood cell walls, enabling selective sugar extraction (brown rot). Although white rot lignin-degrading pathways are well characterized, there are many uncertainties in brown rot fungal mechanisms. Our study addressed a key uncertainty in how brown rot fungi deploy ROS without damaging themselves or the enzymes they secrete. In addition to revealing differentially expressed genes to promote ROS generation only in early decay, our study revealed three spatial control mechanisms to avoid/tolerate ROS: (i) constraining Fenton reactant concentrations (H2O2, Fe2+), (ii) quenching ROS via antioxidants, and (iii) secreting ROS-tolerant enzymes. These results not only offer insight into natural decomposition pathways but also generate targets for biotechnological development.

A novel homodimer laccase from Cerrena unicolor BBP6: Purification, characterization, and potential in dye decolorization and denim bleaching.

The white-rot fungus Cerrena unicolor BBP6 produced up to 243.4 U mL-1 laccase. A novel laccase isoform LacA was purified; LacA is a homodimer with an apparent molecular mass of 55 kDa and an isoelectric point of 4.7. Its optimal pH was 2.5, 4.0, and 5.5 when 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP) were used as the substrates, respectively. The optimal temperature was 60°C for ABTS and 80°C for both guaiacol and 2, 6-DMP. LacA retained 82-92% activity when pH was greater than 4 and 42%-92% activity at or below 50°C. LacA was completely inhibited by 0.1 mM L-cysteine, 1 mM Dithiothreitol, and 10 mM metal ions, Ca2+, Mg2+ and Co2+. LacA had good affinity for ABTS, with a Km of 49.1 µM and a kcat of 3078.9 s-1. It decolorized synthetic dyes at 32.3-87.1%. In the presence of 1-hydroxybenzotriazole (HBT), LacA decolorized recalcitrant dyes such as Safranine (97.1%), Methylene Blue (98.9%), Azure Blue (96.6%) and simulated textile effluent (84.6%). With supplemented manganese peroxidase (MnP), Mn2+ and HBT, the purified LacA and BBP6 fermentation broth showed great potential in denim bleaching, with an up to 5-fold increase in reflectance values.

White-rot basidiomycetes Junghuhnia nitida and Steccherinum bourdotii: Oxidative potential and laccase properties in comparison with Trametes hirsuta and Coriolopsis caperata.

White-rot basidiomycetes from the poorly studied residual polyporoid clade of Polyporales order Junghuhnia nitida (Pers.) Ryvarden and Steccherinum bourdotii Saliba & A. David grow as secondary xylotrohps on well decomposed woody materials. The main objective of the current study was to compare oxidative potential, growth, production of oxidative enzymes and laccase properties of J. nitida and S. bourdotii with that of typical primary xylotrohps Trametes hirsuta (Wulfen) Lloyd and Coriolopsis caperata (Berk.) Murrill, belonging to the core polyporoid clade. For the first time we report species J. nitida and S. bourdotii as active laccase producers. New laccases from J. nitida and S. bourdotii were purified and characterized. They had an identical molecular weight of 63 kDa and isoelectric points of 3.4 and 3.1, respectively. However, the redox potential of the T1 copper site for both J. nitida (610 mV) and S. bourdotii (640 mV) laccases was lower than those for T. hirsuta and C. caperata laccases. The new laccases showed higher temperature optima and better thermal stability than T. hirsuta and C. caperata laccases. Their half-lives were more than 40 min at 70 °C. The laccases from J. nitida and S. bourdotii showed higher affinity to syringyl-type phenolic compounds than T. hirsuta and C. caperata laccases. The oxidative potential of studied fungi as well as the properties of their laccases are discussed in terms of the fungal life-style.