Sanro Tachibana

Biodegradation of chrysene, an aromatic hydrocarbon by Polyporus sp. S133 in liquid medium

Polyporus sp. S133, a fungus collected from contaminated-soil was used to degrade chrysene, a polycyclic aromatic hydrocarbon (PAH) in a mineral salt broth (MSB) liquid culture. Maximal degradation rate of chrysene (65%) was obtained when Polyporus sp. S133 was incubated in the cultures supplemented with polypeptone (10%) for 30 days under agitation of 120 rpm, as compared to just 24% degradation rate in non-agitated culture. Furthermore, the degradation of chrysene was affected by the addition of carbon and nitrogen sources as well as kind of surfactants. The degradation rate was increased with increase in added amount of carbon and nitrogen sources, respectively. The degradation rate in agitated cultures was enhanced about 2 times higher than that in non-agitated cultures. The degradation mechanism of chrysene by Polyporus sp. S133 was determined through identification of several metabolites; chrysenequinone, 1-hydroxy-2-naphthoic acid, phthalic acid, salicylic acid, protocatechuic acid, gentisic acid, and catechol. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase) produced by Polyporus sp. S133 were detected during the incubation. The highest enzyme activity was shown by 1,2-dioxygenase (237.5 U l(-1)) after 20 days of incubation.

Production of Podophyllotoxin in Juniperus Chinensis Callus Cultures Treated with Oligosaccharides and a Biogenetic Precursor in Honour of Professor G. H. Neil Towers 75th Birthday

Abstract Calli were induced from the leaves of young trees of Juniperus chinensis on Schenk and Hildebrandt medium supplemented with napthalenacetic acid and kinetin and subcultured on the same medium. Podophyllotoxin, a strong anti-tumor agent, was isolated from the extractives of calli and found that calli produced podophyllotoxin. The podophyllotoxin in the calli derived from the leaves constituted 0.005% of dry weight. In contrast, the content of podophyllotoxin in intact plant was 0.0025% of dry weight. The cultures of the calli produced twice as much podophyllotoxin as did those of the intact plant. The production of podophyllotoxin was increased fifteen-fold by addition of chito-oligosaccharides, an elicitor, to the calli. And furthermore, the production increased eleven-fold by addition of phenylalanine, a biogenetic precursor of podophyllotoxin.

Potential of fungal co-culturing for accelerated biodegradation of petroleum hydrocarbons in soil

The potential of fungal co-culture of the filamentous Pestalotiopsis sp. NG007 with four different basidiomycetes--Trametes versicolor U97, Pleurotus ostreatus PL1, Cerena sp. F0607, and Polyporus sp. S133--for accelerating biodegradation of petroleum hydrocarbons (PHCs) was studied using three different physicochemical characteristic PHCs in soil. All the combinations showed a mutual intermingling mycelial interaction on the agar plates. However, only NG007/S133 (50/50) exhibited an optimum growth rate and enzymatic activities that supported the degradation of asphalt in soil. The co-culture also degraded all fractions at even higher concentrations of the different PHCs. In addition, asphaltene, which is a difficult fraction for a single microorganism to degrade, was markedly degraded by the co-culture, which indicated that the simultaneous biodegradation of aliphatic, aromatic, resin, and asphaltene fractions had occurred in the co-culture. An examination of in-vitro degradation by the crude enzymes and the retrieval fungal culture from the soil after the experiment confirmed the accelerated biodegradation due to enhanced enzyme activities in the co-culture. The addition of piperonyl butoxide or AgNO3 inhibited biodegradation by 81-99%, which demonstrated the important role of P450 monooxygenases and/or dioxygenases in the initial degradation of the aliphatic and aromatic fractions in PHCs.

Extractives of Juniperus chinensis L. I : Isolation of podophyllotoxin and yatein from the leaves of J. chinensis

Two compounds, yatein and podophyllotoxin, were isolated from the chloroform-soluble fraction in the methanolic extractives of byakushin (Juniperus chinensis L.) leaves for the first time.

Determination of co-metabolism for 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) degradation with enzymes from Trametes versicolor U97

Trametes versicolor U97 isolated from nature degraded 73% of the 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) in a malt extract liquid medium after a 40-d incubation period. This paper presents a kinetic study of microbial growth using the Monod equation. T. versicolor U97 degraded DDT during an exponential growth phase, using glucose as a carbon source for growth. The growth of T. versicolor U97 was not affected by DDT. DDT was degraded by T. versicolor U97 only when the secondary metabolism coincided with the production of several enzymes. Furthermore, modeling of several inhibitors using the partial least squares function in Minitab 15, revealed lignin peroxidase (98.7 U/l) plays a role in the degradation of DDT. T. versicolor U97 produced several metabolites included a single-ring aromatic compound, 4-chlorobenzoic acid.

Enhanced biodegradation of asphalt in the presence of Tween surfactants, Mn2+ and H2O2 by Pestalotiopsis sp. in liquid medium and soil

Asphalt and fractions thereof can contaminate water and soil environments. Forming as residues in distillation products in crude oil refineries, asphalts consist mostly of asphaltene instead of aliphatics, aromatics, and resins. The high asphaltene content might be responsible for the decrease in bioavailability to microorganisms and therefore reduce the biodegradability of asphalt in the environment. In this study, the effect on asphalt biodegradation by Pestalotiopsis sp. in liquid medium and soil of nonionic Tween surfactants in the presence of Mn2+ and H2O2 was examined. The degradation was enhanced by Tween 40 or Tween 80 (0.1%) in the presence of Mn2+ (1 mM) and H2O2 (0.05 mM). A Tween surfactant, Mn2+, and H2O2 can overcome bioavailability-mediated constraints and increase ligninolytic activities, particularly manganese peroxidase and laccase activities. The study is significant for the bioremediation of asphalt and/or viscous-crude oil-contaminated environments.

Isolation of Antioxidant Compounds from Aspergillus Terreus LS01

The antioxidative activity of an ethyl acetate extract from Aspergillus terreus LS01was evaluated using various in vitro assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen-peroxide-scavenging free radical scavenging, and β-caroten-linoleat model assay and the antioxidants in the extract were isolated and identified. Silica gel column chromatography was used to separate the extract into five fractions (F1-F5). Fraction 3 possessed significant antioxidative activity with an IC 50 of 19.91μg/ml. Fraction 3 was further separated by column chromatography and yielded the crystalline compounds 1 and 2, which were identified as terreic acid and terremutin, respectively, on the basis of UV-vis spectra, MS and NMR analyses. Terreic acid and terremutin exhibited the highest level of DPPH free radical scavenging activity with IC 50 values of 0.115±4.02 and 0.114±2.19 mM, respectively. These compounds also showed scavenging activity of hydrogen peroxide radicals which terreic acid exhibited higher than terremutin with 74.07±1.48% and 33.74±2.81% respectively. In the β-caroten-linoleat model assay, the inhibition of terreic acid retained 26.01±1.14% and terremutin retained 32.29±2.23%. This is the first report on antioxidative activity of terreic acid and terremutin from A.terreus . The findings indicated that A. terreus could be considered as potential source of natural antioxidant products.

Antifungal activities of compounds isolated from the leaves of Taxus cuspidata var. nana against plant pathogenic fungi

Antifungal activities of seven compounds, taxinine (1), paclitaxel (2), phenylisoserine methyl ester (3), sciadopitysin (4), ginkgetin (5), isorhamnetin (6), and quercetin (7), isolated from the leaves of kyaraboku, Taxus cuspidata var. nana, against five plant pathogenic fungi, Gibberella fujikuroi, Cladosporium cucumeninum, Fusarium oxysporum, Colletotrichum fragariae, and Corynespora cassiicola, were investigated for utilization of extractives from trees of the genus Taxus. Also, the amounts of compounds 2 and 3 on the leaf surface was measured in relation to the antifungal activities of compounds. Taxinine (1) showed antifungal activity against G. fujikuroi, C. cucumeninum, F. oxysporum, and C. cassiicola. The minimum inhibitory concentration of taxinine for the four fungi was 0.4 μmol. In addition, from the results of antifungal tests, it may be concluded that paclitaxel on the leaves and stem of T. cuspidata var. nana does not play an important role as an antifungicide in the resistance of trees to plant pathogenic fungal attack.

4,4′-Dihydroxytruxillic acid as a component of the cell walls of the bambooPhyllostachys edulis

Abstract 4,4′-Dihydroxytruxillic acid, a photodimer ofp-coumaric acid, as well asp-coumaric and ferulic acids, were identified from alkaline hydrolysates of the cell walls of stems, leaves and shoots of bamboo (Phyllostachys edulis). The truxillic acid content was highest in leaves but was only about 1/1000th that of wall boundp-coumarate. Truxillic acid levels increased on grinding in a Wiley mill suggesting that it can be generated in cell walls by mechanochemical energy.

Cinnamic acid derivatives in cell walls of bamboo and bamboo grass

Abstract 4,4′-Dihydroxytruxillic acid (DHTA) and 4,4′-dihydroxy-3,3′-dimethoxytruxillic acid (DHMTA), photodimers of p -coumaric and ferulic acids, respectively, as well as p -coumaric, ferulic, caffeic, hydroxyferulic and sinapic acids, were identified as components of the cell walls of stems and leaves of bamboo ( Phyllostachys edulis ) and bamboo grass ( Sasa kurilensis ). The amounts were determined by measurement of peak areas using GC-MS with 1-octadecanol as internal standard.