Jiko Shishiyama

Productivity of hydrolytic enzymes by mycorrhizal mushrooms

To survey the potential for production of extracellular hydrolytic enzymes by mycorrhizal mushrooms, productivities of these exo-enzymes from mycelia on potato-dextrose liquid medium were determined.Tricholoma matsutake produced relatively high levels of CM-cellulase and avicelase activities in all test strains. It also produced higher activity of acid proteinase than neutral proteinase. Its xylanase activities seemed to be higher than those of the other carbohydrases. The productivities ofLyophyllum shimeji strains were at similar levels to those ofT. matsutake strains. CM-cellulase and avicelase activities ofL. shimeji were higher than those ofT. matsutake. Neutral proteinase inL. shimeji strains showed higher activity levels than acid proteinase. The relative productivities of hydrolytic enzymes between the groups of mycorrhizal mushrooms and wood-rotting mushrooms were also examined.T. matsutake andL. shimeji both produce many kinds of hydrolytic enzymes in their culture broth, and the potential for production of hydrolytic enzymes by mycorrhizal mushrooms was judged to be relatively high.

Antimutagenic activity of extracts from Japanese eggplant

Using the Salmonella/microsome assay, the antimutagenic effects of specific components of the extracts from eggplant fruits were investigated. The eggplant fruit juice exhibited an antimutagenic activity against 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) induced mutagenicity. In some of the fractions extracted with several organic solvents (acetone, petroleum ether, ethyl acetate; and methanol), the activity was recognized. No mutagenicity or toxicity for Salmonella typhimurium TA98 in the presence of S9 mixture was observed with any of the extracts. It is suggested that there are multiple components of the activities that exist in the eggplant fruit. We isolated lutein from the 84% methanol (methanol/water, v/v) layer, pheophorbide or chlorophyllide from the 70% methanol layer and tannins containing sugar-moieties from the water layer. Pheophytin a and b, Mg-free derivatives of chlorophyll a and b, were isolated from the petroleum ether layer as possible antimutagens. The pheophytin a with S9 mix inhibited by 30-40% the mutagenicity of Trp-P-2.

Role of metal proteinases in the fruit-body formation of Hypsizygus marmoreus

We investigated the possible role of metal proteinase on the fruit-body formation ofHypsizygus marmoreus. The addition of a specific metal proteinase inhibitor, phosphoramidon, to the culture medium (10μg/ml) completely inhibited fruit-body formation. Metal proteinase activity in both the medium and the mycelia of this fungus increased markedly during vegetative mycelial growth, and activity was maximal 25 days after inoculation. When phosphoramidon was added to the culture medium during vegetative mycelial growth, the metal proteinase activity in the mycelium decreased to 56% of the control (without inhibitor) level. Isoelectric focusing analysis showed that two kinds of metal proteinases with a pl of 7.7 and 8.4, respectively, were obtained from 29-day-old mycelia. Uptake of phosphoramidon into the mycelia was confirmed as the result of inhibition of thermolysin activity by the mycelial extracts. The degree of inhibitor uptake into mycelia was about 2.0% and was independent of the initial concentration of the inhibitor administered. The addition of peptone and amino acids to medium treated with phosphoramidon resulted in fruit-body dry weight yields that were about 50% that of the control.

Isolation and characterization of extra- and intra-cellular metal proteinases produced in the spawn-running process of Hypsizygus marmoreus

Isolation and characterization of extra-(PE-1) and intra-cellular (PE-2) metal proteinases produced during the spawn-running process ofHypsizygus marmoreus were carried out. These enzymes were the most active toward Hammarsten casein at pH 7.0 (PE-1) and pH 6.5–7.5 (PE-2). The molecular weight and pl value of PE-1 were 29,500, 8.8 and those of PE-2 were 21,500, 8.4. Km values against the synthetic peptide substrate Z-Gly-l-Leu-NH2 were 0.9×10−3M (PE-1) and 1.2×10−3M (PE-2). PE-1 was strongly inhibited by phosphoramidon, whereas PE-2 was weakly inhibited. These enzymes are considered to play an important role in providing nitrogenous substrates during fruit-body formation.

Changes in carbohydrase activities during vegetative growth and development of fruit-bodies of Hypsizygus marmoreus grown in sawdust-based culture

Carbohydraseproduction of Hypsizygus marmoreus cultured on sawdust rice bran medium by bottle cultivation was investigated to elucidate the carbohydrase utilized as the growth substrate for the fruit-body formation of this fungus. Among the extracellular enzymes assayed, xylanase showed the highest activity. This activity greatly increased during the end of vegetative mycelial growth and fruit-body formation. Among the cellulases. CM-cellulase showed higher activity than avicelase. The activities of β-1,3-glucanase, amylase, and chitinase were low. Among the intracellular enzymes, both xylanase and amylase showed higher activity levels than the other carbohydrases. In contrast, β-1,3-glucanase, avicelase, and chitinase activities in mycelia were considerably lower. These results suggest that xylanase, CM-cellulase, and amylase play an important role in mycelial maturation and fruit-body growth of H. marmoreus.

Purification and some properties of an isoform of metal proteinases from Hypsizygus marmoreus grown on sawdust culture

Purification and some properties of an isoform of metal proteinase fromHypsizygus marmoreus are described. This enzyme was purified 711-fold with 5.44% recovery. The molecular weight and pl value were 41,500 and pH 7.7, respectively. The highest activity was observed against milk casein as the substrate. This enzyme was strongly inhibited by metal proteinase inhibitors such as phosphoramidon, EDTA, ando-phenanthroline.