Kentaro Yoshikawa

Purification and Characterization of Thermostable Pectate Lyase with Protopectinase Activity from Thermophilic Bacillus sp. TS 47

A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated. It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60°C on a medium containing polygalacturonate (PGA). The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies. The molecular mass of the enzyme was 50 kDa by SDS-PAGE. The isoelectric point of the enzyme was pH 5.3. The enzyme had a half-life of 13 and 1 h at 65 and 70°C, respectively, and showed optimal activity around at 70°C and pH 8.0. It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers. The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50% identity.

Molecular Cloning, DNA Sequence, and Expression of the Gene Encoding for Thermostable Pectate Lyase of Thermophilic Bacillus sp. TS 47

The gene that encodes a thermostable pectate lyase (called PL 47), from Bacillus sp. TS 47, was cloned, sequenced, and expressed in mesophilic B. subtilis. The gene contained an open reading frame consisting of 1326 bp, which encoded 441 amino acids. The deduced amino acid sequence of the mature enzyme (416 amino acids with a calcuated molecular mass of 47, 262 Da), showed 52% similarity with PL (BsPel) from mesophilic B. subtilis SO113. The structure-based alignment of the deduced amino acid sequence of PL 47 with that of BsPel suggested that PL 47 might have a parallel β-helix structure with three long loops. The amino acids making up PL 47 are richer in hydrophobic amino acids and glutamic acid than BsPel. The hydropathy profile of PL 47 indicated that the amino acid sequences around putative calcium binding sites are more hydrophobic than the same region of BsPel. The gene product expressed in B. subtilis as the host was stable up to 70°C and the reaction was optimal around 70°C, as well as native PL 47.

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.

Molecular Cloning of the Gene Encoding Thermostable Endo-1,5-α-L-arabinase of Bacillus thermodenitrificans TS-3 and Its Expression in Bacillus subtilis

The gene that encodes a thermostable endo-arabinase (called ABN-TS) from Bacillus thermodenitrificans TS-3 was cloned, sequenced, and expressed in the mesophilic B. subtilis. The gene contained an open reading frame consists of 939 bp, which encodes 313 amino acids. The deduced amino acid sequence of the enzyme showed 50, 46, and 36% similarity with endo-arabinase from B. subtilis IFO 3134 (PPase-C), Pseudomonas fluorescens (ArbA), and Aspergillus niger (ABNA), respectively. The hydrophobic and acidic amino acids making up ABN-TS outnumbered those in PPase-C. The gene product expressed in B. subtilis, as the host, had substantially the same characteristics, and was stable up to 70°C, and the reaction was optimal around 70°C, as well as native ABN-TS.

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.

Promotive effect of the hot water-soluble fraction from corn fiber on vegetative mycelial growth in edible mushrooms

Corn fiber (CNF) is an abundant by-product of the wet corn milling process used to produce corn starch. In light of the need to recycle organic wastes, the effects of adding a hot water-soluble fraction (HWSF) from CNF to a medium on the vegetative mycelial growth of nine edible mushrooms such as Lentinula edodes and Pholiota nameko were investigated. The results showed that the mycelial growth of these fungi was markedly increased (1.4–9.5 times that of the control) by adding 5%–20% CNF-HWSF to the medium. These promotive effects were also apparent on mycorrhizal mushrooms, such as Tricholoma matsutake (3.3-fold) and Lyophyllium shimeji (3.7-fold). The promotive effects on mycelial growth were shown in the low-molecular-weight fractions (molecular weight <500 daltons) prepared from CNF-HWSF. The promotive actions were more effective on slow-growing mushrooms (L. edodes and P. nameko) than on rapidly growing mushrooms (Pleurotus ostreatus and Flammulina velutipes). The results obtained in this experiment suggest that CNF-HWSF can be used as a promotive substance for cultivating edible mushrooms.

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.

Microbial bioassay of acute toxicity by the pH inhibition method and comparison of IC50 (pHI) with LD50 for rats and mice

Abstract A bioassay of the toxicity of 12 test chemicals is described using Klebsiella pneumonia grown at 35°C in a medium containing potassium citrate in which the pH increased steadily as the bacteria continued to grow. The chemicals tested were nine metallic ions, two alcohols, and phenol. The pH of the medium was recorded automatically every 3.6 h for 15 h. The 50% inhibitory concentrations (IC50) of the test chemicals (mg/l) were 0.9 for Hg, 2.9 for Cd, 2.9 for Cr6+, 7.5 for Cu, 19 for Cr3+, 25 for Zn, 50 for CN, 91 for Mn, 1,430 for phenol, 53,300 for ethanol, and 74,200 for methanol. LD50 values for rats given the compounds per os and for mice given them intraperitoneally were correlated with the IC50 values obtained by this method. The method might be used in preliminary screening for toxicity before tests in experimental animals.