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Epitheaflagallin 3- O -gallate (ETFGg) is a minor polyphenol found in black tea extract, which has good physiological functions. It is synthesized from epigallocatechin gallate (EGCg) with gallic acid via laccase oxidation. Various basidiomycetes and fungi were screened to find a suitable laccase for the production of ETFGg. A basidiomycete, Hericium coralloides NBRC 7716, produced an appropriate extracellular laccase. The purified laccase produced twice the level of ETFGg compared with commercially available laccase from Trametes sp. The enzyme, termed Lcc2, is a monomeric protein with an apparent molecular mass of 67.2 kDa. The N-terminal amino acid sequence of Lcc2 is quite different from laccase isolated from the fruiting bodies of Hericium . Lcc2 showed similar substrate specificity to known laccases and could oxidize various phenolic substrates, including pyrogallol, gallic acid, and 2,6-dimethoxyphenol. The full-length lcc2 gene was obtained by PCR using degenerate primers, which were designed based on the N-terminal amino acid sequence of Lcc2 and conserved copper-binding sites of laccases, and 5'-, and 3'-RACE PCR with mRNA. The Lcc2 gene showed homology with Lentinula edodes laccase (sharing 77% amino acid identity with Lcc6). We successfully produced extracellular Lcc2 using a heterologous expression system with Saccharomyces cerevisiae . Moreover, it was confirmed that the recombinant laccase generates similar levels of ETFGg as the native enzyme. [ABSTRACT FROM AUTHOR]

Highlights: [•] S. aeruginosa secretes glycosylated yellow laccases Yel1p and Yel3p. [•] Yel1p and Yel3p have been purified and characterised. [•] Low coverage, high throughput transcriptome sequencing identified the Yel1 gene. [•] Theories about the differences between yellow and blue laccases are discussed. [Copyright &y& Elsevier]

Abstract: Delignification of corn stover by the white rot fungus Ceriporiopsis subvermispora in solid-state cultivation was evaluated for improving subsequent enzymatic hydrolysis. The results showed that C. subvermispora selectively degraded lignin by as much as 39.2% while the cellulose loss was less than 5% during 42d of cultivation. However, hemicellulose loss of up to 27.0% was concomitant with the lignin degradation. Ligninolytic enzymes, MnP and laccase, were detected during degradation of corn stover by C. subvermispora. For major hydrolytic enzymes, xylanase was the only enzyme detected. The enzymatic hydrolysis yield of corn stover delignified by C. subvermispora was remarkably improved, reaching about 57–67% overall glucose yield after 18–42d of pretreatment, while the glucose yield of the untreated was only 22%. A higher overall glucose yield of about 72% was obtained when the enzymatic hydrolysis of the 18d treated corn stover was supplemented with an accessory xylanase/cellulase enzyme complex. [Copyright &y& Elsevier]

Abstract: A comparison of laccase sequences highlighted the presence of a C-terminal extension of sixteen amino acids in POXA1b laccase – that represents the most thermostable isoenzyme among Pleurotus ostreatus laccases and exhibits a notable stability at alkaline pH (t 1/2 at pH 10=30 days) – whereas this tail is missing in the other analysed laccases from basidiomycetes. Site-directed mutagenesis experiments allowed us to demonstrate a role of the C-terminal tail of POXA1b in affecting its catalytic and stability properties. The truncated mutants lose the high stability at pH 10, while they show an increased stability at pH 5. The effect of substituting the residue Asp205 of POXA1b with an arginine was also analysed in the mutant POXA1bD205R. Following the mutation POXA1bD205R, a remarkable worsening of catalytic properties along with a decrease of substrate affinity and of enzyme stability were found. It was demonstrated that introducing Arg205 mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. Hence, a single substitution in the binding site introduces a local conformational change that not only leads to very different catalytic properties, but can also significantly destabilize the protein. [Copyright &y& Elsevier]

Abstract: Activity and production of extracellular enzymes by saprotrophic litter-decomposing basidiomycetes Hypholoma fasciculare and Rhodocollybia butyracea was studied in microcosms with reconstructed L, O and Ah horizons of a soil profile of Quercus petraea forest soil. Both H. fasciculare and R. butyracea colonized the L layer of microcosms rapidly, while the colonization of O layer was slower. The Ah layer was substantially colonized only by R. butyracea. Enzyme activities in the soil microcosms decreased from the L layer>O layer>Ah layer and activities in microcosms inoculated with the fungi were quite similar to each other. Compared to control, the most apparent was the increase of ligninolytic enzyme activities. Laccase activities in H. fasciculare and R. butyracea-colonized L layers were 3-fold compared to control and the activity maxima of Mn-peroxidase in fungus-colonized O layers were 2–3-fold and in the L layers up to 40-fold compared to controls. Activities of cellulolytic enzymes, chitinase and acidic phosphatase in both fungal treatments were higher in the L layer on weeks 2–6 while the activity of alkaline phosphatase did not show differences between fungus-colonized and control treatments. Both fungi decreased fungal CFU in the L layer but significantly increased the counts in the O layer. Both fungi also increased bacterial CFU in the O layer, R. butyracea more than H. fasciculare. The analysis of fungal and bacterial biomass based on ergosterol content and PLFA analysis showed a sharp decrease from L to Ah layer, but did not show significant differences among treatments. [Copyright &y& Elsevier]

Abstract: The evaluation of eighteen strains of basidiomycetes laccase and manganese peroxidase (MnP) activity in submerged fermentation of mandarin peelings and ethanol production waste showed that the expression of enzyme activity is species- and strain-dependent. While all species of the genus Trametes expressed comparatively high laccase activity, the activity of this enzyme among species of the genus Ganoderma varied from 192 to 61,488Ul−1. Phellinus robustus 250 appeared to be a promising producer of MnP, accumulating more than 4000Ul−1 of enzyme activity. It has been shown for the first time that Omphalotus olearius 174 is capable of producing high levels of laccase and MnP, while Hypsizygus marmoreus produces only laccase. Laccase and MnP production proved to be very much dependent on the lignocellulosic growth substrate. Of eight complex substrates examined in submerged fermentation by Ganoderma lucidum 447, wheat bran and soy bran gave the highest laccase activity with a maximum value of 93–97Uml−1. Proof that both the titre and time of maximal enzyme activity are influenced by nutrient nitrogen is presented. [Copyright &y& Elsevier]

Abstract: Ischnoderma resinosum produced extracellular ligninolytic enzymes laccase and MnP. The activity of laccase achieved the maximum on day 10 (29.4UL−1), the MnP on day 14 (34.5UL−1). Laccase and Mn-peroxidase were purified from the culture liquid using gel permeation and ion-exchange chromatographies. Purified Mn-peroxidase performed decolorization of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22, while all dyes were decolorized after addition of the redox mediators violuric acid (VA) and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and HBT. The highest decolorization rates were detected in acidic pH (3–4). [Copyright &y& Elsevier]

Abstract: The 26S proteasome, a large multicatalytic protease complex is involved in highly selective ATP-dependent degradation of intracellular proteins in eukaryotes, typically those tagged with a polyubiquitin chain. The proteasome-mediated proteolysis plays a key role in the regulation of critical cellular processes such as transcriptional control, cell cycle progression, and stress response. Biochemical and structural properties of the 26S proteasomes described in this paper concern the white-rot fungus Phlebia radiata, which is interesting from the standpoint of biotechnological applications of ligninolytic enzymes. In addition, the characterization of 26S proteasomes from P. radiata which belongs to the class Basidiomycetes is the first one reported in the kingdom of fungi for an organism other than yeast representing the class Ascomycetes. All distinct peptidase activities: chymotrypsin-, trypsin-, and caspase-like attributed to proteasomes were detected in P. radiata proteasome preparations. Electrophoretic mobility, subunit composition, ATP-dependence, sensitivity to proteasome-specific inhibitors, and inactivation by low concentrations of SDS demonstrated for these preparations are typical features of 26S proteasome species. Moreover, findings from in vivo blocking of proteasome function strongly support the previous interpretation that the proteasomal pathway is involved in regulation of activity of some ligninolytic enzymes under nutrient deprivation [Staszczak M. Proteasomal degradation pathways in Trametes versicolor and Phlebia radiata. Enzyme Microb Technol 2002;30:537–41]. [Copyright &y& Elsevier]

Abstract: A laccase (E.C. 1.10.3.2) from Trametes spec. was isolated from aqueous media using foam fractionation. The pH value, superficial velocity, foaming period, and temperature were varied to optimise the transport of the active enzyme into the foam phase. Several detergents were added in varying concentrations to form and stabilize the foam, and the cationic detergent cetyltrimethylammonium bromide (CTAB) proved to be the most appropriate. From water as a model system, maximum recovery rates of 94% of laccase activity were achieved at pH 6.0 in 6min. For separation of the enzyme from protein rich culture media, the operation conditions had to be adjusted. At pH 5.4, 89% of laccase activity was transported into the foam phase after 15min. The method established was successfully applied to the isolation of an active laccase isoenzyme from submerged cultures of the basidiomycete Pleurotus sapidus. [Copyright &y& Elsevier]

The plant pathogen basidiomycete S. rolfsii secretes two laccases (SRL1 and SRL2) with molecular weights of 55 and 86 kDa, respectively. Laccase production was shown to be inducible by the addition of 2,5-xylidine to the cultural media. After treatment with a combination of chitinase and β-1,3-glucanase, two different laccases were isolated from the sclerotia depending on the stage of sclerotia development. The more prominent laccase, SRL1, was purified and found to decolourize the industrially important wool azo dye Diamond Black PV 200 without the addition of redox mediators. The enzyme (pI 5.2) was active in the acidic pH range, showing an optimal activity at pH 2.4, with ABTS as substrate. The optimum temperature for activity was determined to be 62 °C. Enzyme stability studies revealed that SRL1 was notably stable at 18 °C and pH 4.5, retaining almost full activity after a week. Oxidation of tyrosine was not detectable under the reaction conditions but the enzyme did oxidize a variety of the usual laccase substrates. SRL1 was strongly inhibited by sodium azide and fluoride. Dye solutions decolourized with the immobilized laccase were successfully used for redyeing. [Copyright &y& Elsevier]

Carpophores of mesophilic basidiomycetes were isolated from the Eastern Cape region of South Africa. These isolates were screened for manganese peroxidase and laccase with high temperature optima. This bio-prospecting has led to the discovery of a thermostable laccase, which exhibits an optimum temperature of 70 °C. The enzyme remains fully active after 9 h incubation at 60 °C. The laccase is also capable of catalyzing the oxidative coupling of 3-dimethylaminobenzoic acid and 3-methyl-2-benzothiazolinone hydrazone, which are characterized as manganese peroxidase specific substrates. The wide temperature range, thermostability, and potentially wider than usual substrate range, make this an excellent candidate enzyme for industrial application. [Copyright &y& Elsevier]

White-rot fungi require trace amounts of essential heavy metals such as Cd, Mn or Zn for their growth, but these metals are toxic when present in excess. Toxic heavy metals can inhibit the growth, cause morphological and physiological changes and affect the reproduction of Basidiomycetes. Fungal species and strains differ in their sensitivity towards metals and in the protection mechanisms involved. The toxicity of some heavy metals such as Hg, Cu or Ni has been used for the development of antifungal wood preservatives. Extracellular ligninolytic and cellulolytic enzymes are regulated by heavy metals on the level of transcription as well as during their action. During the degradation of lignocellulose and xenobiotics by white-rot fungi or isolated enzymes from these fungi heavy metals interfere with both the activity of extracellular enzymes involved in the process and fungal colonization. The ability of white-rot fungi to adsorb and accumulate metals together with the excellent mechanical properties of fungal mycelial pellets provide an opportunity for application of fungal mycelia in selective sorption of individual heavy metal ions from polluted water. [Copyright &y& Elsevier]

The white rot basidiomycete Ceriporiopsis subvermispora secretes the ligninolytic enzymes manganese-dependent peroxidase (MnP) and laccase to the extracellular medium. The promoter region of the laccase gene (Cs-lcs1) possesses several putative metal responsive elements (MRE), as well as a putative target site responding to copper termed ACE, similar to the one found in yeast. In this work, we show by electrophoretic mobility-shift assays that the migration of DNA probes containing either MRE sites or the ACE element are retarded in their mobility after incubation with nuclear extracts from C. subvermispora. Competition experiments suggested the presence of defined binding proteins recognizing these elements. [Copyright &y& Elsevier]

Secretome evaluations of lignocellulose-decay basidiomycetes can reveal new enzymes in selected fungal species that degrade specific substrates. Proteins discovered in such studies can support biorefinery development. Brown-rot (Gloeophyllum trabeum) and white-rot (Pleurotus ostreatus) fungi growing in sugarcane bagasse solid-state cultures produced 119 and 63 different extracellular proteins, respectively. Several of the identified enzymes are suitable for in vitro biomass conversion, including a range of cellulases (endoglucanases, cellobiohydrolases and β-glucosidases), hemicellulases (endoxylanases, α-arabinofuranosidases, α-glucuronidases and acetylxylan esterases) and carbohydrate-active auxiliary proteins, such as AA9 lytic polysaccharide monooxygenase, AA1 laccase and AA2 versatile peroxidase. Extracellular oxalate decarboxylase was also detected in both fungal species, exclusively in media containing sugarcane bagasse. Interestingly, intracellular AA6 quinone oxidoreductases were also exclusively produced under sugarcane bagasse induction in both fungi. These enzymes promote quinone redox cycling, which is used to produce Fenton's reagents by lignocellulose-decay fungi. Hitherto undiscovered hypothetical proteins that are predicted in lignocellulose-decay fungi genomes appeared in high relative abundance in the cultures containing sugarcane bagasse, which suggests undisclosed, new biochemical mechanisms that are used by lignocellulose-decay fungi to degrade sugarcane biomass. In general, lignocellulose-decay fungi produce a number of canonical hydrolases, as well as some newly observed enzymes, that are suitable for in vitro biomass digestion in a biorefinery context. [ABSTRACT FROM AUTHOR]