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ABSTRACT: The basidiomycete Lyophyllum decastes was transformed by means of particle bombardment. We isolated five transformants under twelve conditions differing in the
two parameters of target distance and helium pressure. The transformation frequency was one transformant/µg DNA. In the transformants,
plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of L. decastes by particle bombardment.
Mycoscience 04/2012; 48(3):195-197. · 1.21 Impact Factor
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Yuzo Suzuki, Masaya Nakamura,
Yuichiro Otsuka,
Nao Suzuki,
Keisuke Ohyama,
Takeshi Kawakami,
Kanna Sato,
Shinya Kajita,
Shojiro Hishiyama,
Atsushi Takahashi,
Yoshihiro Katayama
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ABSTRACT: The degradation of 2-chloro-4,5-O-(4'-methyl-7', 8'-diphenyl)ether (CMDPE), an analog of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), mediated by Geobacillus sp. UZO 3 cell-free extract was monitored. Ethyl acetate extracts of a complete reaction mixture incubated at 65°C for 18 h were analyzed either by thin layer chromatography (TLC) fractionation coupled with spectrometric detection or by gas chromatography-mass spectrometry (GC-MS). The reaction product 4-methylumbelliferone (4MU) was successfully isolated by TLC and visualized by a transilluminator at 450 nm. The 4MU, 4-chlorophenol, and reaction intermediate 6-chlorophenoxy-4-methylumbelliferone were all successfully detected by GC-MS. The presence of these compounds suggest that Geobacillus sp. UZO 3 cell-free extract also catalyzes the reductive cleavage of the diaryl ether bonds of CMDPE in a similar mechanism previously reported in 2,7-DCDD. In the present study, the authors describe a simple and highly sensitive fluorescent assay for a new dioxin degrading enzyme(s).
Environmental Toxicology and Chemistry 02/2012; 31(5):1072-5. · 2.81 Impact Factor
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Kazuyuki Tanamura,
Tomokuni Abe,
Naofumi Kamimura,
Daisuke Kasai,
Shojiro Hishiyama,
Yuichiro Otsuka, Masaya Nakamura,
Shinya Kajita,
Yoshihiro Katayama,
Masao Fukuda,
Eiji Masai
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ABSTRACT: The glutathione S-transferases, LigF and LigE, of Sphingobium sp. strain SYK-6 respectively play a role in cleavage of the β-aryl ether of (+)-(βS)-α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) and (-)-(βR)-MPHPV. The ligP gene, which showed 59% similarity to ligE at the amino acid level, was isolated from SYK-6. LigP produced in Escherichia coli revealed enantioselectivity for (-)-(βR)-MPHPV, and ligE and ligP alone contributed to the degradation of (-)-(βR)-MPHPV in SYK-6.
Bioscience Biotechnology and Biochemistry 12/2011; 75(12):2404-7. · 1.28 Impact Factor
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Yuzoh Suzuki, Masaya Nakamura,
Yuichiro Otsuka,
Nao Suzuki,
Keisuke Ohyama,
Takeshi Kawakami,
Kanna Sato,
Shinya Kajita,
Shojiro Hishiyama,
Takeo Fujii,
Atsushi Takahashi,
Yoshihiro Katayama
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ABSTRACT: We characterized the ability of the cell free extract from polychlorinated dibenzo-p-dioxins degrading bacterium Geobacillus sp. UZO 3 to reduce even highly chlorinated dibenzo-p-dioxins such as octachlorodibenzo-p-dioxins in incineration fly ash. The degradation of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) as a model dioxin catalyzed by the cell free extract from this strain implicates that the ether bonds of 2,7-DCDD molecule undergo reductive cleavage, since 4',5-dichloro-2-hydroxydiphenyl ether and 4-chlorophenol were detected as intermediate products of 2,7-DCDD degradation.
Chemosphere 03/2011; 83(6):868-72. · 3.21 Impact Factor
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ABSTRACT: Copolymers of L-lactic acid (LA) and 2-pyrone-4,6-dicarboxylic acid (PDC), a chemically stable metabolic intermediate of lignin, were prepared by dehydrated polycondensation based on stepwise oligomerization followed by post-polymerization in vacuo. When the polymerization was performed in the presence of methanesulfonic acid as a catalyst, the molecular weights of the resulting copolymers were sufficiently high. Furthermore, expansion of the polymeric surface area was found to be an important factor in facilitating dehydration and thereby producing high molecular weight polymers. PDC feed ratio significantly affected the molecular weight because of the different polymerization capability from LA. Relationship between the PDC feed ratio and the molecular weight of the resulting polyesters was for the first time demonstrated quantitatively. The obtained copolymers were characterized by 1H- and 13C-NMR, IR, and thermal analysis. The high molecular weight copolymers possessed the higher decomposition temperatures than PLLA and their fusible temperature ranges were reasonably expanded.
Journal of Macromolecular Science. 06/2010; Part A: Pure and Applied Chemistry(Vol. 47):564-570.
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ABSTRACT: A novel bacterium, designated strain OX-01(T), was isolated from acidic soil, taxonomically investigated and identified as an agent that catabolizes (+)-catechin into taxifolin. Strain OX-01(T) is a Gram-reaction-negative, aerobic, non-sporulating, non-motile and rod-shaped bacterium. 16S rRNA gene sequence analysis identified this strain as a member of the genus Burkholderia and occupying a phylogenetic position closest to, but clearly distinct from, Burkholderia sacchari. Strain OX-01(T) does not have any nif genes, which are required for N(2)-fixation, in its genome, a feature that is similar to B. sacchari, which lacks nifH, but is distinct from the N(2)-fixing features of many other phylogenetically related taxa, such as Burkholderia ferrariae, B. heleia, B. mimosarum, B. nodosa, B. silvatlantica, B. tropica and B. unamae. Strain OX-01(T) has the following chemotaxonomic characteristics: the major ubiquinone is Q-8, the DNA G+C content is 64 mol% and the major fatty acids are C(16 : 0), C(17 : 0) cyclo and C(18 : 1)ω7c. It also has a unique profile of carbohydrate utilization among other species of the genus Burkholderia. The strain cannot assimilate many pentoses, hexoses and oligosaccharides, whereas it can catabolize (+)-catechin and its putative aromatic derivatives, such as 4-hydroxy-3-methoxycinnamic acid, protocatechuic acid, p-hydroxybenzoic acid, trans-p-coumaric acid and vanillic acid. Based on its morphological, physiological and chemotaxonomic characteristics, together with DNA-DNA relatedness values and 16S rRNA gene sequence comparison data, we show that strain OX-O1(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia oxyphila sp. nov. is proposed. The type strain is OX-01(T) (=NBRC 105797(T) =DSM 22550(T)).
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 03/2010; 61(Pt 2):249-54. · 2.11 Impact Factor
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ABSTRACT: 2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable metabolic intermediate of lignin, 1,4-butanediol, and succinic anhydride were polymerized in the presence of an appropriate catalyst, such as Sb2O3, TiO(acac)2, and CH3SO3H, to afford the corresponding polyesters. The molecular weight (Mn) of the polyesters exceeded 10,000, but they were soluble in the common organic solvents when the PDC feed ratio was <10 mol %. Furthermore, the polyesters showed well-defined melting points ranging from 82 to 107 °C and, consequently, the fusibility was realized for the first time for the PDC polymers. Films of these PDC polyesters lacked sufficient mechanical strength, but blended films with poly(L-lactic acid) were found to improve the elastic features. The degradation behaviors of the PDC polyesters were investigated by the biodegradability test or the accelerated hydrolysis tests. Comparison between the obtained PDC polyester and poly(butylene succinate) revealed a remarkable increase in the biodegradability by copolymerization with PDC.Keywords: Biomass-Based Polymer, Biodegradability, Lignin, Mechanical Strength, Polyester
Polymer Journal 10/2009; 41(12):1111-1116. · 1.26 Impact Factor
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ABSTRACT: Two kinds of polyesters containing 2-pyrone-4,6-dicarboxylic acid (PDC) moieties in poly(L-lactic acid) (PLLA) and poly(butylene succinate) (PBS) main chains were for the first time employed as an additive to PLLA for improvement of the mechanical properties. Solution casting of a mixture of PLLA and PDC polyesters provided the blend films, which were comprehensively characterized by thermal analyses, tensile measurements, and crystallinity evaluation. Differential scanning calorimetry measurements revealed that PDC incorporation into polymer main chain dramatically improves the miscibility with PLLA. PDC-incorporated PBS showed better miscibility than PDC-incorporated PLLA. Optical microscopy measurements also suggested the superior orientation of PDC-incorporated PBS over PDC-incorporated PLLA in the blend films. The fracture strain of the blend films could be linearly increased by a draw ratio in the region of low PDC polyester content. On the contrary, the fracture strain decreased at high PDC polyester content due to the restricted strain caused by the strong dipolar interactions between PDC-moieties. Elongation at break was significantly varied as a function of PDC polyester content, whereas tensile modulus was almost independent of the additive content and draw ratios. It is concluded that the blend films containing <10 wt% of PDC incorporated PBS as an additive show the best tensile property.Keywords: Biomass, Lignin, Mechanical Strength, Polyester, Polylactide
Polymer Journal 08/2009; 41(10):843-848. · 1.26 Impact Factor
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Yusuke Sato,
Hideki Moriuchi,
Shojiro Hishiyama,
Yuichiro Otsuka,
Kenji Oshima,
Daisuke Kasai, Masaya Nakamura,
Seiji Ohara,
Yoshihiro Katayama,
Masao Fukuda,
Eiji Masai
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ABSTRACT: Degradation of arylglycerol-beta-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-beta-guaiacyl ether (GGE) to alpha-(2-methoxyphenoxy)-beta-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate alpha-glutathionyl-beta-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (alphaR,betaS)-GGE and (alphaR,betaR)-GGE into (betaS)-MPHPV and (betaR)-MPHPV, respectively, while LigL and LigN transformed (alphaS,betaR)-GGE and (alphaS,betaS)-GGE to (betaR)-MPHPV and (betaS)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (alphaR,betaS)-GGE and (alphaR,betaR)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers.
Applied and environmental microbiology 07/2009; 75(16):5195-201. · 3.69 Impact Factor
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Masahiro Nakajima,
Yukari Nishino,
Masatsugu Tamura,
Kohei Mase,
Eiji Masai,
Yuichiro Otsuka, Masaya Nakamura,
Kanna Sato,
Masao Fukuda,
Kiyotaka Shigehara,
Seiji Ohara,
Yoshihiro Katayama,
Shinya Kajita
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ABSTRACT: 2-Pyrone-4,6-dicarboxylic acid (PDC) is a catabolic intermediate in Sphingobium sp. SYK-6 (previously characterized as Sphingomonas paucimobilis SYK-6), which is a degrader of lignin-derived aromatic compounds. Recently, PDC has been also characterized as a novel starting material for several potentially useful synthetic polymers. In a previous study, we constructed a biosynthetic system in which PDC was generated efficiently from a chemically synthesized compound, protocatechuate. In order to develop an alternative system for production of PDC, we tried to generate it from glucose, which is a low-cost sugar that can be obtained from abundant cellulosic wastes and biomass crops. We designed a metabolic bypass to PDC from the shikimate pathway in recombinant Escherichia coli cells. PDC accumulated in the medium of recombinant E. coli cells that had been transformed with genes isolated from Emericella niger, E. coli, Pseudomonas putida, and Sphingobium sp. SYK-6. The yield of PDC depended on the combination of genes that we introduced into the cells and on the specific of host strain. Under optimal conditions, the yield and titer of PDC were, respectively, 17.3% and 0.35 mg/l when the concentration of glucose was 2 g/l and the culture volume was 50 ml. Our results open up the possibility of novel utilization of biomass as the source of a useful chemical building block.
Metabolic Engineering 04/2009; 11(4-5):213-20. · 5.61 Impact Factor
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ABSTRACT: 2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable metabolic intermediate of lignin, its bis(hydroxyethyl) derivative (BHPDC) and bis(hydroxyethyl) terephthalate (BHT) were polymerized to prepare the corresponding polyesters carrying the oxyethyleneoxycarbonyl(2-pyrone-4,6-diyl)carbonyl and oxyethyleneoxy-terephthaloyl units [x and y units, resp., x + y = 1] by polycondensation eliminating water and/or 1,2-ethanediol molecules. The polyesters were mostly stable up to 210 °C, after that the degradation of pyrone ring commenced. These polyesters exhibited strong adhering properties against metal, glass and surfaces with increasing the x unit content to 0.4–0.6, such as about 50–60 MPa for SUS/polyester/SUS or Al/ polyester/Al samples, by the JIS K 6849:1994 testing method of tensile strength. As the fracturing plane was the midst of the polyester layer and at the interface, the polyester was tenaciously adhering to the metal surface.Keywords: Adhesive, Biomass, Lignin, Polyester
Polymer Journal 02/2009; 41(4):297-302. · 1.26 Impact Factor
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ABSTRACT: To understand the molecular mechanisms of fruiting body formation of basidiomycetous mushrooms, we have isolated over a 100 of developmentally regulated genes that were specifically transcribed during fruiting body development in Lentinula edodes (Shiitake-mushroom) by a subtractive hybridization, cDNA-RDA (cDNA representational difference analysis). One of these genes, named Le.flp1, was isolated from the primordial cDNA library of L. edodes, and the expression product of Le.flp1 and putative fungal homologues contained a characteristic region, homologous to the Fas domain of fasciclin family proteins, which are capable of promoting cell adhesion through Fas domain-mediated homophilic interactions in various organisms. RT-PCR analyses suggested that Le.flp1 was specifically expressed in primordia and mature fruiting bodies. In situ hybridization indicated that Le.flp1 transcripts were distributed distinctly in the following tissues: the inside of gills of fruiting bodies, especially at the boundary between the subhymenium and trama, where there is active proliferation of basidium cells for producing basidiospores; peripheral regions of the primordium, pileus and stipe; and both inner tissue and outer regions of the stipe. Our results suggest the hypothesis that Le.flp1 plays a role in cellular differentiation and development in ubiquitous tissues during fruiting body formation in L. edodes, possibly through cell adhesion.
Current Genetics 07/2007; 51(6):367-75. · 2.56 Impact Factor
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ABSTRACT: Mycorrhizal basidiomycetes, Suillus grevillei and S. bovinus, were transformed by particle bombardment. We isolated eight and four transformants from S. grevillei and S. bovinus respectively under three conditions differing in terms of target distance and helium pressure. The transformation frequencies were 1.2 and 0.6 transformants/microg DNA. Plasmid DNAs were integrated into the genomic DNA of the transformants and stably maintained. This is the first description of the transformation of S. grevillei and S. bovinus by particle bombardment.
Bioscience Biotechnology and Biochemistry 02/2007; 71(1):47-50. · 1.28 Impact Factor
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ABSTRACT: Sphingomonas paucimobilis SYK-6, which can degrade various low molecular weight compounds derived from plant polyphenols such as lignin, lignan, and tannin, metabolizes these substances via 2-pyrone-4,6-dicarboxylic acid (PDC). We focused on this metabolic intermediate as a potential raw material for novel, bio-based polymers. We cloned the ligAB and ligC genes of SYK-6, which respectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, into a broad host range plasmid vector, pKT230MC. The resulting plasmid, pDVABC, was introduced into the PpY1100 strain of Pseudomonas putida, and we found that PDC could be stably produced from protocatechuate and accumulated. In addition, we examined the efficiency of production of PDC from protocatechuate on a 5-L scale in a Luria-Bertani medium containing 100 mM glucose and determined that PDC was stably produced from protocatechuate to yield 10 g/L or more.
Applied Microbiology and Biotechnology 09/2006; 71(5):608-14. · 3.42 Impact Factor
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ABSTRACT: To understand molecular mechanisms of the fruiting body development in basidiomycetes, we attempted to isolate developmentally regulated genes expressed specifically during the fruiting body formation of Lentinula edodes (Shiitake-mushroom). cDNA representational difference analysis (cDNA-RDA) between vegetatively growing mycelium and two developmental substages, primordium and mature fruiting body, resulted in an isolation of 105 individual genes (51 in primordium and 54 in mature fruiting body, respectively). A search of homology with the protein databases and two basidiomycetous genomes in Phanerochaete chrysosporium and Coprinopsis cinerea revealed that the obtained genes encoded various proteins similar to those involved in general metabolism, cell structure, signal transduction, and responses to stress; in addition, there were apparently several metabolic pathways and signal transduction cascades that could be involved in the fruiting body development. The expression products of several genes revealed no significant homologies to those in the databases, implying that those genes are unique in L. edodes and the encoding products may possess possible functions in the course of fruiting body development. RT-PCR analyses revealed that 20 candidates of the obtained genes were specifically or abundantly transcribed in the course of the fruiting body formation, suggesting that the obtained genes in this work play roles in fruiting body development in L. edodes.
Fungal Genetics and Biology 07/2005; 42(6):493-505. · 3.74 Impact Factor
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ABSTRACT: One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants microg(-1) of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.
Applied Microbiology and Biotechnology 01/2005; 66(2):194-9. · 3.42 Impact Factor
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ABSTRACT: The white-rot basidiomycete Coriolus versicolor secretes several enzymes that participate in the degradation of lignin and various persistent organic pollutants. In this study, we attempted to establish a genetic transformation system with a homogenous promoter sequence for driving the gene for antibiotic resistance. We succeeded in cloning the promoter sequence of the gene for glyceraldehyde-3-phosphate dehydrogenase (gpd), which is expressed at high levels in C. versicolor. The expression vector pT7GPTHPT was constructed, which included a gene for resistance to hygromycin B under control of the gpd promoter. The successful selection of transformants on medium that contained hygromycin B indicated that the system should be useful not only for the genetic transformation of C. versicolor, but also for the overproduction of useful fungal enzymes such as laccase and peroxidase.
Mycoscience 03/2004; 45(2):131-136. · 1.21 Impact Factor
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ABSTRACT: Cleavage of the arylglycerol beta-aryl ether linkage is the most important process in the biological degradation of lignin. The bacterial beta-etherase was described previously and shown to be tightly associated with the cellular membrane. In this study, we aimed to detect and isolate a new extracellular function that catalyses the beta-aryl ether linkage cleavage of high-molecular lignin in the soil fungi. We screened and isolated 2BW-1 cells by using a highly sensitive fluorescence assay system. The beta-aryl ether cleavage enzyme was produced by a newly isolated fungus, 2BW-1, and is secreted into the extracellular fraction. The beta-aryl ether cleavage enzyme converts the guaiacylglycerol beta-O-guaiacyl ether (GOG) to guaiacylglycerol and guaiacol. It requires the C alpha alcohol structure and p-hydroxyl group and specifically attacks the beta-aryl ether linkage of high-molecular mass lignins with addition of two water molecules at the C alpha and C beta positions.
European Journal of Biochemistry 07/2003; 270(11):2353-62. · 3.58 Impact Factor
