Takuo Sakai

Antimicrobial chlorinated orcinol derivatives from mycelia of Hericium erinaceum

Abstract Two novel and a known chlorinated orcinol derivative were isolated from cultured mycelia of Hericium erinaceum . These three compounds exhibited antimicrobial activities.

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.

Cloning of a protopectinase gene of Trichosporon penicillatum and its expression in Saccharomyces cerevisiae.

A protopectinase (PPase)-encoding gene, PSE3, from Trichosporon penicillatum was cloned by colony hybridization using two oligonucleotide probes synthesized from the N-terminal amino acid sequences of native PPase SE1 and one peptide from a lysyl endopeptidase digest. Nucleotide sequencing revealed that PSE3 contains an ORF encoding a 367 amino acid protein. Mature PPase SE3 is composed of 340 amino acids and the N-terminus of the ORF appeared to correspond to a signal peptide and a propeptide processed by a KEX2-like proteinase. The deduced amino acid sequence of PSE3 was 65.4, 56.7, 58.1, 61.8 and 48.9% homologous to the polygalacturonases of Aspergillus oryzae, Aspergillus niger, Aspergillus tubigensis, Cochliobolus carbonum and Fusarium moniliforme, respectively. One domain, which might interact with polygalacturonic acid, is highly conserved not only in fungal polygalacturonases but also in bacterial and plant polygalacturonases. PSE3 was expressed in Saccharomyces cerevisiae, but three forms (the mature form, a glycosylated form and an uncharacterized processed form) of PPase SE3 were present among the PSE3 products.

Acidic condition-inducible polygalacturonase of Aspergillus kawachii

Abstract Aspergillus kawachii IFO 4308, which can grow in an extremely acidic condition (pH 2), produced some extracellular polygalacturonases (PGase). However, pH 2 and pH 5 culture filtrates showed different pH PGase activity profiles. Anion exchange chromatographies revealed that the PGase compositions of the two culture filtrates were different, and dominant enzymes (PGase-A1 and -A2 in the pH 2 culture and PGase-B in the pH 5 culture) were purified and characterized. The optimal pH was pH 4 for A1, pH 3 for A2, and pH 5 for B. PGase-A1 and -A2 were more stable at low pH than PGase-B. Molecular masses of PGase-A1, -A2, and -B were 43, 83, and 71 kDa, respectively. The N-terminal amino acid sequence of PGase-B was similar to those of other fungal PGases, but distinctly different from those of PGase-A1 and -A2. These results suggest that PGase-A1 and -A2 may be acidic condition-inducible enzymes and that a pH-regulated expression system is involved in the PGase production of A. kawachii .

Chromosomal deletion or rearrangement in chimeric hybrids of Saccharomycopsis fibuligera and Saccharomyces diastaticus obtained by cell fusion

We isolated three hybrids of Saccharomycopsis fibuligera and Saccharomyces diastaticus, a species that secretes glucoamylase and that has been reclassified recently as a strain of Saccharomyces cerevisiae, for yeast protoplast fusion, and characterized their chromosomes. When the chromosomes of the hybrids were analyzed by electrophoresis in a contour-clamped homogeneous electric field, their migration patterns were similar to those of the parental strains but with slight differences between hybrids. Southern hybridization using the α-amylase and glucoamylase genes from S. fibuligera and S. diastaticus, respectively, as probes showed that these genes were duplicated or deleted in the hybrids. These results demonstrated that the hybrids contained chimeric chromosomes as a result of recombination or rearrangement.

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.

Isolation and expression of the gene which encodes a novel enzyme with polymethoxygalacturonate-degrading activity in Trichosporon penicillatum.

The novel gene named PSX1, encoding a new protopectinase with the polymethoxygalacturonase activity, was isolated from Trichosporon penicillatum. Nucleotide sequencing revealed that the PSX1 gene is composed of 1080 bases (360 amino acids, 38 747 Da). The N‐terminal amino acid sequences of the open reading frame correspond to a signal peptide and propeptide processed by a Kex2‐like proteinase. Mature PPase SX1 was composed of 334 amino acids (36 121 Da). PPase SX1 produced by a S. cerevisiae transformant harboring the PSX1 gene degraded methoxylated polygalacturonic acid as a substrate, but not degraded unmethoxylated polygalacturonic acid.