Kazutaka Itoh

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.

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.

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.

Determination of chrysene degradation under saline conditions by Fusarium sp. F092, a fungus screened from nature.

Sixty-two rotted wood and soil samples were used to screen for chrysene-degrading fungi. A strain of Fusarium, named F092, was identified as most capable of degrading chrysene. F092 was active under saline and nonsaline conditions, breaking down 48% of the chrysene in 30 d. The percentage of chrysene degraded did not change at 35‰ salinity with pH 8.2 in solid and liquid cultures. The degradation under saline conditions increased about 0.6- and 2.1-fold in cultures with polypeptone and Tween80, and 0.03-fold in agitated cultures. F092 secreted nonligninolytic enzymes named 1,2-dioxygenase and 2,3-dioxygenase. The level of 1,2-dioxygenase activity reached 203.5 U L(-1) at 30 d and that of 2,3-dioxygenase activity, 29.7 U L(-1) at 40 d. The degradation pathway was clarified from the intermediates produced; chrysene 1,2-oxide, chrysene trans-1,2-dihydrodiol, 1-hydroxy 2-naphtoic acid, and catechol. F092 is a potential degrader of chrysene for bioremediation.

Isolation of phenylisoserine methyl ester from the leaves of Taxus cuspidata var. nana

From methanolic extracts of leaves of kyaraboku, Taxus cuspidata var. nana, phenylisoserine methyl ester (3) was isolated along with taxinine (1), taxol (2), sciadopitysin (4), ginkgetin (5), isorhamnetin (6), and quercetin (7). This is the first time that phenylisoserine methyl ester has been isolated from T. cuspidata var. nana. Compound 3 was also isolated from the ethanolic extracts of leaves of T. cuspidata var. nana. Furthermore, compound 3 was identified in methanolic extracts from the bark of this tree.

Isolation and identification of antifungal compounds from Amboyna wood

In continuation of our research into biologically active compounds from trees and their production by tissue culture (Tachibana and Sumimoto 1989; Tachibana et al. 1994; Miyata et al. 1998; Muranaka et al. 1998; Premjet et al. 2002; Yoshida et al. 2002), antifungal compounds were isolated from Amboyna wood. Plants are a rich source of chemical substances, with over 100,000 plant products described to date (Vicente et al. 2003), and play an important role in the production of biologically active secondary metabolites. Many secondary metabolites such as flavonoids, stilbenes, lignans and sesquiterpenoids have antifungal activities (ReyesChilpa et al. 1998; Celimene et al. 1999; Chang et al. 2000; Kawamura et al. 2004). Amboyna, Pterocarpus indicus Willd. (Leguminosae), is an important tropical tree, known as angsana and sena in Malaysia and Indonesia or narra in the Philippines, which mainly grows in Southeast Asia. Its natural distribution seems to cover the southern part of Burma, Java and New Guinea (Ogata 1985). Amboyna wood has been used for high-quality furniture and cabinets, decorative veneers, interior wall paneling, boat building and specialized joinery (Eddowes 1977). Extracts of leaves, stem and bark of Amboyna were reported to be active against several bacteria and protozoan (Khan and Omoloso 2003), while a polyphenolic compound isolated from young bark was reported to be a plasmin activity inhibitor (Takeuchi et al. 1986). In the present paper, we report the ability of Amboyna wood to inhibit the growth of wood-rotting fungi and the isolation of active compounds that play a role in the defense system of the wood. Materials and methods

Comparative Studies on the Mechanochemistry of Guaiacylglycerol- and Veratrylglycerol- β -Guaiacyl Ether

Abstract Treatments of guaiacylglycerol- and veratrylglycerol-β-guaiacyl ether [I] and [II] with CBM, VBM and laboratory refiner furnished α-(2-methoxyphenoxy)-β-hydroxypropioguaiacone 1 as the major product in addition to a few small compounds 2-7 in relatively low yields. In addition, 5,5′-condensation reaction of compound [I] gave compounds 8 9, and 10. Alternatively, Cα-Cβ splitting reaction of veratrylglycerol-β-guaiacyl ether [II] occurrsby the mechanical treatment to give a number of products (6,11-14, and 15). Compounds [II] also formed p-carbonyl phenol 1 and products 2 4, and 7 by an alternate route. For comparison, mechanical treatments of vanillyT alcohol [III],α-methyl vanillyl alcohol [IV], and veratryl alcohol [V] were made with a ceramic ball mill (CBM), vibration ball mill (VBM), respectively. Former two gave vanillin 15 and acetovanillone 16, but no product was found from veratryl alcohol [V].

Callus Induction and Production of Bilobalide and Ginkgolides by Callus and Cell Suspension Cultures of Ginkgo Biloba Leaves

The aim of this study was to investigate the effect of growth hormone on biomass and production of bilobalide and ginkgolides A and B bycallus and cell suspension cultures of Ginkgo bilobaleaves. Calluses were induced from Ginkgo biloba leaves on Murashige and Skoog (MS) medium supplemented with sucrose and combination of growth hormone in several concentrations. The production of bilobalide, ginkgolides A and B in callus with combination of 2 mg/L NAA and 0.1 mg/L kinetin showed the highest. To enhance the biomass and production of bilobalide, ginkgolides A and B, effects of growth hormone, NO3 , and carbon source were investigated in cell suspension cultures. Among several concentrations of kinetin + NAA and kinetin + 2,4-D, the highest content of bilobalide, ginkgolides A and B was observed in MS medium supplemented with 0.1 mg/L kinetin + 2 mg/L NAA. The production of bilobalide, ginkgolides A and B in the addition of glucose was higher than that of sucrose and fructose. Reducing 50% of KNO3 in the medium decreased the biomass and the content of bilobalide, ginkgolides A and B.