Sonoe Ochiai Yanagi

Purification and characterization of extracellular laccase from Pleurotus ostreatus

Laccase (EC 1.10.3.2) from the culture filtrate of a strain of white rot basidiomycetePleurotus ostreatus was purified using DEAE-Toyopearl 650M and butyl-Toyopearl 650M column chromatographies and Superdex 75 HR 10/30 fast protein liquid chromatography. Molecular weight of the purified laccase was about 55,000, and the isoelectric point was 3.0. The optimum pH for enzyme activity was 6.5, and the optimum temperature was 50°C. This enzyme contained 7.4% sugar and two copper atoms per molecule. The substrate specificity was similar to those of other fungal laccases. Comparison of the N-terminal amino acid sequence of theP. ostreatus laccase with those fromPleurotus ostreatus Florida,Coriolus hirsutus, Phlebia radiata, basidiomycete PM1 (CECT 2971),Trametes villosa, Pycnoporus cinnabarinus, Ceriporiopsis subvermispora, andAgaricus bisporus showed 95, 65, 60, 55, 55, 55, 50, and 35% similarity, respectively, in the first 20 residues. No similarity in this region was detected with laccases fromNeurospora crassa, Aspergillus nidulans, andCryptococcus neoformans.

Intraspecific heterokaryon and fruit body formation in Coprinus macrorhizus by protoplast fusion of auxotrophic mutants

SummaryFusion of protoplasts of Coprinus macrorhizus mutants with different amino acid requirements resulted in the production of prototrophic clones at frequencies of 1–4% of the protoplasts surviving the fusion treatment. The frequencies were at least 200 times higher than those of the appearance of revertants. Few prototrophic colonies appeared also when the mutant protoplasts were individually subjected to fusion treatment, or when they were mixedly cultured without fusion treatment. It was thus concluded that intraspecific heterokaryons were formed by protoplast fusion.The auxotrophic mutants did not form fruit bodies when cultured singly or mixedly with each other. In contrast, the heterokaryons produced by protoplast fusion between the mutants of compatible mating types developed into fruit bodies with intermediate morphology of those of the strains from which the mutants were derived. Heterokaryons were also formed by fusion of mutant protoplasts with identical mating genotype, but they failed to form fruit bodies.

Purification and Properties of a New Chitin-binding Antifungal CB-1 from Bacillus licheniformis

CB-1, a new antifungal, was purified from Bacillus licheniformis. The molecular mass of CB-1 was estimated as 42 kDa by gel filtration column chromatography. CB-1 seemed to be an aggregation product from 4 kinds of peptides. CB-1 contained 10 amino acids and some fatty acids of C14-18:0 and C18:1. CB-1 irreversively bound with chitin powder.

An efficient method for the isolation of mycelial protoplasts from Coprinus macrorhizus and other basidiomycetes

SummaryA mixture of commercially available chitinase and cellulase released mycelial protoplasts of Coprinus macrorhizus in yields exceeding 108/ml plasts from C. macrorhizus FisC regenerated hyphae and developed into normal fruiting bodies at frequencies of 20%–50%. The same method also released good yields of protoplasts from several other edible mushroom species.

Cloning and characterization of a laccase gene from the white-rot basidiomycete Pleurotus ostreatus

Abstract The gene lccK encoding a laccase of the white-rot basidiomycete Pleurotus ostreatus wild-type strain collected in Japan has been cloned, sequenced, and characterized. The isolated gene consists of 2929 bp with the coding region interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA elements were identified. Two putative N-glycosylation sites and four putative copper-binding sites found in other fungal laccase are conserved in lccK. The cDNA contains an open reading frame of 1599 bp and the gene encodes 533 amino acids preceded by a signal peptide of 23 amino acids. The nucleotide sequence of the lccK cDNA showed high homology with those of laccases of other basidiomycetes.

Flutolanil Resistance as a Genetic Marker of Coprinus cinereus Strains

All 4 Schizophyllum commune strains among several basidiomycete species were exclusively resistant to flutolanil, which inhibits succinate dehydrogenase complex (SDC) function. Flutolanil resistant strains could be screened from monokaryotic protoplasts of sensitive Coprinus cinereus, by treating them with a cloned genomic SDC iron-sulfur protein (IP) gene from the resistant S. commune. The obtained resistance was stably transferred to the progeny.

Phenoloxidase Activity in Coprinus cinereus due to Protoplasting and Insertion Treatment with Pleurotus ostreatus DNA

SummaryAn apparent phenoloxidase (POase) negative strain of Coprinus cinereus acquired POase activity through either protoplasting or DNA-insertion treatment. The DNA-treated C. cinereus showed an electrophoretic band with POase activity, which is similar to the Pleurotus ostreatus used for the DNA source.

Rapid discrimination of mitochondrial DNA type and use of results to study mitochondrial inheritance in Pleurotus spp.

We have reported a simple and rapid method to discriminate species in the genus Pleurotus by analysis of restriction-fragment-length polymorphism of whole-cell DNA, and found that several restriction enzymes gave DNA bands useful in such discrimination, but other enzymes tested did not. In the present study, we report the reason why there were useful and useless enzymes; the effective enzymes digested rDNA into small fragments that did not interfere with the detection of DNA bands useful for discrimination. The origin of these discriminative DNA bands was found to be mitochondrial DNA when the banding profiles of whole-cell DNA, mitochondrial DNA, and nuclear DNA were compared. Consequently, our method could be used for rapid and simple identification of mitochondrial DNA type in the genus Pleurotus. The results were used to study mitochondrial inheritance, and we found that only the nucleus but not the mitochondria migrated during the mating of Pleurotus cornucopiae with P. citrinopileatus.

Efficient protoplast isolation from Coprinus macrorhizus and other basidiomycetes

There have been only a limited number of studies on the isolation of protoplasts from Basidiomycetes (1—4). We developed a method for releasing mycelial protoplasts of Coprinus macrorhizus and other basidiomycetes in much larger amounts and within a shorter time than have been reported. Protoplasts from a mutant of C. macrorhizus regenerated normal fruiting bodies at high frequencies.