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.     

Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes

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 from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.     

Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinula edodes cultures grown on a commercial wood substrate

Summary Extracellular culture filtrates from ligninolytic cultures of the lignin-degrading basidiomycete Lentinula (syn. Lentinus) edodes (Berk.) Pegler contained one major peroxidase when grown on a commercial oak-wood substrate. The peroxidase was purified by polyethylenimine clarification, anion-exchange chromatography, and hydrophobic-interaction HPLC. The enzyme (MnP1) was a heme-iron protein with an apparent molecular weight of 44 600 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and an isoelectric point of pH 3.2. The native enzyme had an absorption maximum at 407 nm, which shifted to 420 nm upon H₂O₂ addition. The pyridine-hemochrome-absorption spectrum indicated that one heme group was present per enzyme as protoporphyrin IX. N-terminal amino acid sequencing showed that MnP1 had higher sequence homology with manganese peroxidases than with lignin peroxidases reported from Phanerochaete chrysosporium. L. edodes MnP1 was capable of oxidizing lignin and lignin-model compounds in the presence of manganese and H₂O₂.On leave from the Department of Biochemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand.Research carried out while a visiting scientist at the USDA Forest Products Laboratory from the National Chemistry Laboratory, Pune, India 41 1008 Offprint requests to: I. T. Forrester     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

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.     

Isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from the edible basidiomycetous mushroom, <Emphasis Type="Italic">Lentinula edodes</Emphasis>, and its expression in the course of fruit-body formation

We isolated and characterized the genomic and complementary DNAs encoding a chitin synthase from an edible basidiomycetous mushroom, Lentinula edodes. The gene (which we designated Lechs1) contains a large open reading frame encoding a polypeptide of 1937 amino acid residues. The open reading frame is interrupted by 14 small introns (49–116 bp). The gene product (LeChs1) consists of a myosin motor-like domain in its N-terminal half and a chitin synthase domain in its C-terminal half, analogous to the class V and VI chitin synthases of other filamentous fungi. Phylogenetic analysis demonstrated that LeChs1 is classified into class VI chitin synthases. Southern blot analysis indicated that Lechs1 is a single-copy gene per haploid genome and that L. edodes has no other highly homologous chitin synthase genes. Northern blot analysis revealed that Lechs1 is expressed throughout the whole stages of fruit-body formation of L. edodes, but its expression level gradually declines in a fruit body-maturation-dependent manner with highest expression in vegetative mycelia and fruit body at the early stage of maturation (immature fruit body). This is the first report on the isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from basidiomycetes.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

Identification of a new member of the dye-decolorizing peroxidase family from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Dye-decolorizing peroxidases (DyP) are atypical peroxidases showing no homology to other fungal peroxidases and lacking the typical heme binding region conserved among plant peroxidase superfamily. The gene and the corresponding cDNA encoding DyP from Pleurotus ostreatus have been identified on the basis of sequence homology analyses. The deduced amino acid sequence shares 43% identity with DyP from the ascomycete Thanatephorus cucumeris Dec 1. Analyses of the protein sequence by homology searches pointed out some properties of the DyP-type peroxidase family, which includes members from bacteria, ascomycete, and basidiomycete fungi. Some amino acids (C374, H379, and Y501 in the P. ostreatus DyP sequence) are proposed as candidates for the heme ligand, providing a basis for further investigations on the structure of the DyP type peroxidase family members.     

A wound-inducible gene fromSalix viminalis coding for a trypsin inhibitor

A gene designatedswin1.1 has been isolated by screening aSalix viminalis genomic library with a heterologous probe,win3 fromPopulus. The region sequenced included the entire coding sequence for a protein with 199 amino acids plus the promoter and terminator. At the 5 end of the coding region is a sequence that encodes a hydrophobic region of 25–30 amino acids, that could form a signal peptide. A putative TATAA box and polyadenylator sequence were identified. Introns were absent. The gene product showed similarities with serine protease inhibitors from the Kunitz family and especially withwin3 from wounded leaves ofPopulus. Southern blot analysis indicated thatswin1.1 is a member of a clustered gene family,swin1. An oligonucleotide corresponding to the putative hypervariable region to-wards the carboxyl end when used as a probe in Southern hybridization showed high specificity forswin1.1. Expression of theswin1.1 gene was enhanced in wounded leaves. Theswin1.1 coding region without the signal sequence was highly expressed inEscherichia coli and the protein showed inhibitory activity against trypsin but at most slight activity against the other proteases tested. A systemically induced protein, SVTI, with inhibitor activity against trypsin, was isolated fromSalix leaves by affinity chromatography on a column of trypsin-Sepharose 4B and N-terminal sequenced. It corresponded with the translatedswin1.1 gene at 16 of the 19 amino acid sites, suggesting that SVTI is encoded by another member of theswin1 gene family.     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1–5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.     

The cloning of a new peroxidase found in lignocellulose cultures of Pleurotus eryngii and sequence comparison with other fungal peroxidases

We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn(2+) and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.