Cell wall structure of secreted laccase-silenced strain in Lentinula edodes

Laccase1 (Lcc1) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L. edodes results in abnormal hyphal structure and thinner cell wall in mycelia. In this study, we observed the effects of Lcc1 on the hyphal morphology and cell wall structure of L. edodes. A thick cell wall and fibrous layer were clearly observed in the lcc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble β-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble β-1,3-1,6-glucan content increased after Lcc1 secretion in the WT. Taken together, these data suggest that the increased level of β-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength.     

Induction of manganese peroxidase and laccase by <Emphasis Type="Italic">Lentinula edodes</Emphasis> under liquid culture conditions and their isozyme detection by enzymatic staining on native-PAGE

The white-rot basidomycete Lentinula edodes often produces the lignin-degrading enzymes manganese peroxidase (MnP; EC 1.11.1.13) and laccase (Lcc; EC 1.10.3.2) in sawdust-based media. In the present study, MnP from L. edodes was induced under liquid culture supplemented with sawdust extracts of Castanopsis cuspidata. Lcc activity was induced by the addition of 2 mM CuSO₄·5H₂O into the same media 7 days after initial inoculation. Phenoloxidase enzymes were distinguished by polyacrylamide gel electrophoresis (native-PAGE), followed by sequential enzymatic staining with an improved staining solution. The isozyme bands detected under MnP-induced conditions were identified as manganese peroxidase (lemnp2) and bands detected under Lcc-induced conditions were identified as laccase (lcc1) by Q-TOF mass spectrometry.     

Molecular Characterization of Laccase Genes from the Basidiomycete Coprinus cinereus and Heterologous Expression of the Laccase Lcc1

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K_m of 21 ± 2 μM and a catalytic constant of 200 ± 10 min⁻¹ for O₂ with 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.     

Comparative characterization of four laccases from Trametes versicolor concerning phenolic C-C coupling and oxidation of PAHs

The laccase genes lccα, lccβ, lccγ and lccδ encoding four isoenzymes from Trametes versicolor have been cloned and expressed in Pichia pastoris. Biochemical characterization allowed classification of these laccases into two distinct groups: Lccα and Lccβ possessed higher thermal stability, but lower catalytic activity towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) compared to Lccγ and Lccδ. Activities of the laccases were quite different as well. Laccase Lccδ showed highest phenolic C-C coupling activity with sinapic acid, but lowest oxidizing activity towards polycyclic aromatic hydrocarbons (PAHs). Highest activity towards PAHs was observed with Lccβ. After 72 h, more than 80% of fluorene, anthracene, acenaphthene and acenaphthylene were oxidized by Lccβ in the presence of ABTS. Investigation of the structural basis of the different activities of the laccases demonstrated the impact of positions 164 and 265 in the substrate binding site on oxidation of PAHs.     

Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase

Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation.     

Purification of a novel extracellular laccase from solid-state culture of the edible mushroom <Emphasis Type="Italic">Lentinula edodes</Emphasis>

The laccases (EC 1.10.3.2) secreted into solid-state culture by Lentinula edodes were analyzed. The fungus secreted at least two laccases in the solid-state culture. One laccase was purified to a homogeneous preparation using anion-exchange, hydrophobic, and size-exclusion chromatography. SDS-PAGE analysis showed that the purified laccase, Lcc6, was a monomeric protein of 58.5 kDa. The optimum pH for enzyme activity was about 3.5, and the laccase was most active at 40°C. The N-terminal amino acid sequence of Lcc6 did not correspond to the sequence of Lcc1, which was previously purified from L. edodes. Lcc6 had decolorization activity to some chemical dyes.     

A surfactant tolerant laccase of <Emphasis Type="Italic">Meripilus giganteus</Emphasis>

A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1 exhibited low K _m values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437 k _cat/k _m (s⁻¹ mM⁻¹). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability towards metal ions and bipolar compounds.     

A Novel Lentinula edodes Laccase and Its Comparative Enzymology Suggest Guaiacol-Based Laccase Engineering for Bioremediation

Laccases are versatile biocatalysts for the bioremediation of various xenobiotics, including dyes and polyaromatic hydrocarbons. However, current sources of new enzymes, simple heterologous expression hosts and enzymatic information (such as the appropriateness of common screening substrates on laccase engineering) remain scarce to support efficient engineering of laccase for better “green” applications. To address the issue, this study began with cloning the laccase family of Lentinula edodes. Three laccases perfectio sensu stricto (Lcc4A, Lcc5, and Lcc7) were then expressed from Pichia pastoris, characterized and compared with the previously reported Lcc1A and Lcc1B in terms of kinetics, stability, and degradation of dyes and polyaromatic hydrocarbons. Lcc7 represented a novel laccase, and it exhibited both the highest catalytic efficiency (assayed with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS]) and thermostability. However, its performance on “green” applications surprisingly did not match the activity on the common screening substrates, namely, ABTS and 2,6-dimethoxyphenol. On the other hand, correlation analyses revealed that guaiacol is much better associated with the decolorization of multiple structurally different dyes than are the two common screening substrates. Comparison of the oxidation chemistry of guaiacol and phenolic dyes, such as azo dyes, further showed that they both involve generation of phenoxyl radicals in laccase-catalyzed oxidation. In summary, this study concluded a robust expression platform of L. edodes laccases, novel laccases, and an indicative screening substrate, guaiacol, which are all essential fundamentals for appropriately driving the engineering of laccases towards more efficient “green” applications.     

Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1.

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.     

Molecular Analysis of a Laccase Gene from the White Rot Fungus Pycnoporus cinnabarinus

It was recently shown that the white rot basidiomycete Pycnoporus cinnabarinus secretes an unusual set of phenoloxidases when it is grown under conditions that stimulate ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl. Environ. Microbiol. 62:1151–1158, 1996). In this report we describe the results of a cloning and structural analysis of the laccase-encoding gene (lcc3-1) expressed by P. cinnabarinus during growth under xylidine-induced conditions. The coding region of the genomic laccase sequence, which is preceded by the eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp. The corresponding laccase cDNA was identical to the genomic sequence except for 10 introns that were 50 to 60 bp long. A sequence analysis indicated that the P. cinnabarinus lcc3-1 product has a Phe residue at a position likely to influence the reduction-oxidation potential of the enzyme’s type 1 copper center. The P. cinnabarinus lcc3-1 sequence was most similar to the sequence encoding a laccase from Coriolus hirsutus (level of similarity, 84%).     

The basidiomycetous mushroom Lentinula edodes white collar-2 homolog PHRB,a partner of putative blue-light photoreceptor PHRA,binds to a specific site in the promoter region of the L. edodes tyrosinase gene

We isolated a cDNA homolog of Neurospora crassa wc-2 from the basidiomycetous mushroom Lentinula edodes and termed it phrB cDNA. The deduced PHRB (313 amino acid residues) contained a PAS domain and a zinc-finger motif. Random binding-site selection analysis of the PHRB produced in Escherichia coli revealed that it bound to a 7-bp sequence with the consensus sequence 5′GATA/TTG/T/AC3′. Electrophoretic mobility-shift assay showed that it also bound to the consensus sequence 5′GATATTC3′ in the promoter region of the L. edodes tyrosinase gene (Le.tyr). In vitro GST-pulldown immunoblot analysis disclosed that PHRB interacts with a putative blue-light photoreceptor of L. edodes (PHRA), the homolog of N. crassa WC-1, through the PAS B- and/or PAS C domain of PHRA. The expression of phrB and Le.tyr genes in pre-primordial mycelia of L. edodes is induced by light exposure, suggesting that PHRB can regulate the expression of the Le.tyr gene in a light-dependent manner.     

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.     

Expression of multi-functional cellulase gene mfc in Coprinus cinereus under control of different basidiomycete promoters

Multi-functional cellulase gene mfc was expressed in Coprinus cinereus under naturally non-inductive conditions using three heterologous promoters. Endo-β-1,4-glucanase expression was achieved in solid and liquid media with promoter sequences from the Lentinula edodesgpd gene, the Flammulina velutipes gpd gene and the Volvariella volvaceagpd gene. As measured by enzyme activity in liquid cultures, a 613-bp gpd promoter fragment from L. edodes was most efficient, followed by a 752-bp gpd fragment from F. velutipes. The V. volvacea gpd promoter sequence was less active, in comparison. Irrespective of the promoter used, enzymatic activities increase 34-fold for highly active transformants and 29-fold for less active one by using cellulase-inducing medium. The highest activities of endo-β-1,4-glucanase (34.234 U/ml) and endo-β-1,4-xylanase (263.695 U/ml) were reached by using the L. edodesgpd promoter.     

Cloning and characterization of the gene encoding beta subunit of mitochondrial processing peptidase from the basidiomycete Lentinula edodes

We have isolated the gene encoding the beta subunit of mitochondrial processing peptidase (β-MPP) from the shiitake mushroom Lentinula edodes. It is a nuclear gene with two small introns. Comparison with known β-MPP genes revealed that the L. edodes gene is most closely related with that from Neurospora crassa, with 60.8% identities and 87% similarity in the amino-acid sequences. The deduced L. edodes β-MPP peptide sequence contains the inverse zinc-binding motif (H–X–E–H) that has been found in a large family of zinc-binding metalloproteinases including bacterial proteinases, insulin degrading enzymes and β-MPPs. The two histidines are thought to contribute two of the three residues for zinc binding. The expression of L. edodes β-MPP is higher during the development of the fruiting bodies, suggesting that higher mitochondrial activities may be required to meet the energy demand in the rapid growth of the fruiting bodies.     

Molecular properties and antioxidant activities of polysaccharide–protein complexes from selected mushrooms by ultrasound-assisted extraction

Ultrasound-assisted extraction (UAE) was evaluated for isolation of polysaccharide–protein (PSP) complexes from three important medicinal mushrooms (Grifola frondosa, Coriolus versicolor and Lentinus edodes). Compared with those of conventional hot-water extraction (HWE), the PSP yield of UAE was similar with G. frondosa, notably higher with L. edodes but lower with C. versicolor, and the extraction rate of UAE was notably higher with G. frondosa and L. edodes but much lower with C. versicolor. The PSPs from all three mushrooms by UAE had higher protein but lower carbohydrate contents than those by HWE, and their molecular weight (MW) profiles exhibited an overall shift to lower MW and a major low-MW peak near 1.0 kDa. All PSPs from UAE but none from HWE exhibited 3–4 distinct protein bands between 10 and 130 kDa. The antioxidant activities of PSPs extracted by UAE were generally higher than those by HWE.     

Levels of physiologically active indole derivatives in the fruiting bodies of some edible mushrooms (Basidiomycota) before and after thermal processing

Indole compounds were found in the fruiting bodies of selected mushroom species both before and after thermal processing. On the basis of HPLC analyses the following indole compounds were detected: l-tryptophan, 5-hydroxytryptophan, 5-methyltryptophan, tryptamine, 5-methyltryptamine, serotonin, indole and melatonin. The compound that was present in the largest amounts in the methanolic extracts from unprocessed mushrooms was 5-hydroxytryptophan in the amount of 7.32 mg/100 g DW in the case of the fruiting bodies of Auricularia polytricha; 15.83 in Suillus bovinus; 22.94 in Macrolepiota procera, and 24.83 in Lentinula edodes. In the methanolic extracts from thermally processed mushrooms the amount of 5-hydroxytryptophan was: 3.52 mg/100 g DW in the case of A. polytricha; 5.65 for Leccinum scabrum and 10.11 for M. procera. In addition, serotonin was found in unprocessed fruiting bodies of two mushroom species: 1.03 mg/100 g DW in L. edodes and 13.99 in L. scabrum, and also in thermally processed mushrooms.     

Detection,quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

Characterization, Molecular Cloning, and Differential Expression Analysis of Laccase Genes from the Edible Mushroom Lentinula edodes

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.