研究者業績

武尾 正弘

タケオ マサヒロ  (Masahiro Takeo)

基本情報

所属
兵庫県立大学 大学院 工学研究科 応用化学専攻 教授
学位
博士(工学)(大阪大学)
Doctor(Engineering)(Osaka University)

J-GLOBAL ID
200901027729174169
researchmap会員ID
1000057675

外部リンク

研究キーワード

 2

論文

 122
  • Yoshiaki Ukita, Saki Kondo, Chiwa Kataoka, Masahiro Takeo, Seiji Negoro, Yuichi Utsumi
    MICROSYSTEM TECHNOLOGIES-MICRO-AND NANOSYSTEMS-INFORMATION STORAGE AND PROCESSING SYSTEMS 16(8-9) 1465-1470 2010年8月  査読有り
    This paper reports the first application of poly-tetrafluoroethylene (PTFE) microstructure, which fabricated by synchrotron radiation induced photo-evaporation process, to enzyme-linked immunosorbent assay. The advantages of PTFE microstructure for the development of lab-on-a-chip due to the extremely high-aspect ratio microstructure and chemical stability of PTFE is discussed. The results of immunoassay shows the successful detection of analyte (mouse IgG) with detection range with 0-100 ng/ml. This result suggests the successful immobilization of antibody (anti-mouse IgG goat antibody) onto the X-ray exposed surface of PTFE microstructure and successful demonstration of antigen-antibody reaction in the PTFE microstructure. We also demonstrated the detection of polychlorinated biphenyl (PCB). As the result of demonstration, we successfully detected PCB with ranging analyte concentration of 0.1-10 ng/ml.
  • Yasuyuki Kawashima, Kengo Yasuhira, Naoki Shibata, Yusuke Matsuura, Yusuke Tanaka, Masaaki Taniguchi, Yoshiaki Miyoshi, Masahiro Takeo, Dai-ichiro Kato, Yoshiki Higuchi, Seiji Negoro
    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 64(1-2) 81-88 2010年6月  査読有り
    NyIB' carboxylesterase, which is 88% homologous to functional 6-aminohexanoate-dimer hydrolase (NyIB) from Arthrobacter sp., possesses trace synthetic activity [0.0004 mu mol min(-1) mg(-1) (U/mg)] from 6-aminohexanoate (Ahx) to its oligomers in 90% tert-butyl alcohol. The synthetic activity and the ratio of the synthetic activity to the hydrolytic activity were significantly affected by amino acid substitutions at positions 181, 266 and 370. The synthetic activity was enhanced to 2.7 U/mg by G181D-H266N substitutions, and the activity was further enhanced in the G181D-H266N-D370Y triple mutant to a level approximately 10(4)-fold greater than the parental carboxylesterase form (3.4 U/mg), which was nearly equal to the ordinary hydrolytic activity in water (type A-mutants). Type A-mutants possessed more than 50% of the 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity at 0-70% tert-butyl alcohol, but the synthetic reaction became predominant at 85-90% tert-butyl alcohol. In contrast, type B-mutants (G181E-H266N and G181N-H266N) possessed quite low levels of Aid-hydrolytic activity (<0.01 U/mg) at 0-70% tert-butyl alcohol. However, both the hydrolytic and synthetic activities were enhanced at higher concentrations, and the maximum activity was obtained at 90% tert-butyl alcohol for both hydrolysis and synthesis. In a type C-mutant (R187S-F264C-D370Y), the Ald-hydrolytic activity was enhanced to approximately 80-fold that of the parental carboxylesterase, but the mutant barely demonstrated any synthetic activity. On the basis of the three-dimensional structure of the Ald-bound enzyme and a kinetic study for typical mutant enzymes, we propose that the efficient enzymatic syntheses of nylon-6 units were achieved by (i) stable binding of the 1st-Ahx at the N-terminal region with Asp181, (ii) interaction of the 2nd-Ahx at the C-terminal region with Tyr370, and (iii) motion of Tyr170 that generated a closed form in the catalytic center of Ald hydrolase. (C) 2010 Elsevier B.V. All rights reserved.
  • Katsuhiro Matsui, Syohei Morimoto, Toshifumi Asano, Yoshiaki Ukita, Dai-ichiro Kato, Masahiro Takeo, Yuichi Utsumi, Seiji Negoro
    ELECTRONICS AND COMMUNICATIONS IN JAPAN 93(4) 50-57 2010年4月  査読有り
    Micro reactors and micro total analysis systems (mu TAS) are recognized as powerful tools for genomics, proteonomics, clinical diagnostics, and environmental testing. In this paper, we describe an enzyme-linked immunosorbent assay (ELISA) using a new micro reactor with a vertical fluid flow operation. This micro reactor is composed of two reaction vessels stacked along the vertical through PMMA fluid filters (circle divide 3 mm). The fluid filters, constructed by deep X-ray lithography, have 2100 pores (circle divide 40 mu m), and possess valve functions, making it possible to maintain the liquid layers in each reaction vessel. In addition, the liquid can be selectively transferred by air pressure from the upper vessel to the lower, and vice versa. As a model of ELISA using the micro reactor, we undertook to detect mouse immunoglobulin (IgG). We bound goat anti-IgG antibody to the surface of the PMMA filters and assayed the IgG by ELISA using an anti-IgG antibody/peroxidase conjugate. We found that the mouse IgG (100 ng/ml) was quantitatively detected within 45 minutes of the analytical period, or about one-third of the period required for the conventional method using micro titer plates. (C) 2010 Wiley Periodicals, Inc. Electron Comm Jpn, 93(4): 50-57, 2010; Published online in Wiley Inter Science (www.interscience.wiley.com). DOI 10.1002/ecj.10137
  • Anuradha Ghosh, Meenu Khurana, Archana Chauhan, Masahiro Takeo, Asit K Chakraborti, Rakesh K Jain
    Environmental science & technology 44(3) 1069-77 2010年2月1日  査読有り
    A bacterial strain Rhodococcus imtechensis RKJ300 (= MTCC 7085(T) = JCM 13270(T)) was isolated from pesticide-contaminated soil of Punjab by the enrichment technique on minimal medium containing 4-nitrophenol. Strain RKJ300 is capable of utilizing 4-nitrophenol, 2-chloro-4-nitrophenol, and 2,4-dinitrophenol as sole sources of carbon and energy. The strain involved both oxidative and reductive catabolic mechanisms for initial transformation of these compounds. In the case of 2-chloro-4-nitrophenol, colorimetric analysis indicated that nitrite release was followed by stoichiometric elimination of chloride ions. Experiments using whole cells and cell-free extracts showed chlorohydroquinone and hydroquinone as the intermediates of 2-chloro-4-nitrophenol degradation. This is the first report of degradation on 2-chloro-4-nitrophenol by a bacterium under aerobic condition to the best of our knowledge. However, pathways for degradation of 4-nitrophenol and 2,4-dinitrophenol were similar to those reported in other strains of Rhodococcus. Laboratory-scale soil microcosm studies demonstrated that the organism was capable of degrading a mixture of nitrophenols simultaneously, indicating its applicability toward in situ bioremediation of contaminated sites. The fate of the augmented strain as monitored by the plate-counting method and hybridization technique was found to be fairly stable throughout the period of microcosm experiments.
  • Kengo Yasuhira, Naoki Shibata, Go Mongami, Yuki Uedo, Yu Atsumi, Yasuyuki Kawashima, Atsushi Hibino, Yusuke Tanaka, Young-Ho Lee, Dai-ichiro Kato, Masahiro Takeo, Yoshiki Higuchi, Seiji Negoro
    The Journal of biological chemistry 285(2) 1239-48 2010年1月8日  査読有り
    We performed x-ray crystallographic analyses of the 6-aminohexanoate cyclic dimer (Acd) hydrolase (NylA) from Arthrobacter sp., an enzyme responsible for the degradation of the nylon-6 industry byproduct. The fold adopted by the 472-amino acid polypeptide generated a compact mixed alpha/beta fold, typically found in the amidase signature superfamily; this fold was especially similar to the fold of glutamyl-tRNA(Gln) amidotransferase subunit A (z score, 49.4) and malonamidase E2 (z score, 44.8). Irrespective of the high degree of structural similarity to the typical amidase signature superfamily enzymes, the specific activity of NylA for glutamine, malonamide, and indoleacetamide was found to be lower than 0.5% of that for Acd. However, NylA possessed carboxylesterase activity nearly equivalent to the Acd hydrolytic activity. Structural analysis of the inactive complex between the activity-deficient S174A mutant of NylA and Acd, performed at 1.8 A resolution, suggested the following enzyme/substrate interactions: a Ser(174)-cis-Ser(150)-Lys(72) triad constitutes the catalytic center; the backbone N in Ala(171) and Ala(172) are involved in oxyanion stabilization; Cys(316)-S(gamma) forms a hydrogen bond with nitrogen (Acd-N(7)) at the uncleaved amide bond in two equivalent amide bonds of Acd. A single S174A, S150A, or K72A substitution in NylA by site-directed mutagenesis decreased the Acd hydrolytic and esterolytic activities to undetectable levels, indicating that Ser(174)-cis-Ser(150)-Lys(72) is essential for catalysis. In contrast, substitutions at position 316 specifically affected Acd hydrolytic activity, suggesting that Cys(316) is responsible for Acd binding. On the basis of the structure and functional analysis, we discussed the catalytic mechanisms and evolution of NylA in comparison with other Ser-reactive hydrolases.
  • Dai-ichiro Kato, Hiromitsu Yoshida, Masahiro Takeo, Seiji Negoro, Hiromichi Ohta
    Bioscience, biotechnology, and biochemistry 74(12) 2405-12 2010年  査読有り
    We succeeded in the purification and gene cloning of a new enzyme, α-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg(2+), MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal pH and temperature for reaction were found to be 7.25 and 30 °C. Under these conditions, the K(m) and k(cat) values for (S)-CPPA were 0.92 ± 0.17 mM and 0.28 ± 0.026 s(-1) respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C(8)). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.
  • Taku Ohki, Naoki Shibata, Yoshiki Higuchi, Yasuyuki Kawashima, Masahiro Takeo, Dai-Ichiro Kato, Seiji Negoro
    Protein science : a publication of the Protein Society 18(8) 1662-73 2009年8月  査読有り
    Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a beta-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (K(m) = 15 mM) and turnover (k(cat) = 3.1 s(-1)) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald--COOH and Tyr370, a hydrogen-bonding network from Ser187 to Ald--NH(3) (+), and interaction between Ald--NH(3) (+) and Gln27-O(epsilon) derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with Ald--NH(3) (+) (Kawashima et al., FEBS J 2009; 276:2547-2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a beta-lactamase fold.
  • Lizhao Geng, Ming Chen, Quanfeng Liang, Wei Liu, Wei Zhang, Shuzhen Ping, Wei Lu, Yongliang Yan, Weiwei Wang, Masahiro Takeo, Min Lin
    Archives of microbiology 191(7) 603-14 2009年7月  査読有り
    Delftia tsuruhatensis AD9 contains the chromosomally encoded tad gene cluster responsible for the complete metabolism of aniline to TCA cycle intermediates. The tadQTA1A2B genes encode a multi-component aniline dioxygenase, the first enzyme of aniline metabolism, and the tadR gene directly downstream of this gene cluster encodes a putative LysR-type regulatory protein. Inactivation of tadR resulted in the inability to degrade aniline and to grow on aniline. Transcriptional assays using a tadQ promoter (P( tadQ ))-lacZ fusion revealed that the transcriptional activation of tadQ from P( tadQ ) was dependent on the presence of tadR and aniline, suggesting that tadR encodes a positive regulatory protein for the expression of at least six genes. Induction experiments using the same P( tadQ )-lacZ fusion showed that, of the 22 chemical compounds, aniline and monochloroanilines activated transcription from P( tadQ ) in wild-type AD9. Sequential deletions of a 1,003-bp region just upstream of tadQ showed that a 148-bp segment upstream of the transcription start site of tadQ, containing one inverted repeat named IR6, was essential for the transcriptional activation of tadQ. Moreover, gel shift assay confirmed the binding of the gene product to the tadQ promoter region. These results clarified the outline of the regulatory mechanism for aniline degradation in AD9.
  • Yasuyuki Kawashima, Taku Ohki, Naoki Shibata, Yoshiki Higuchi, Yoshiaki Wakitani, Yusuke Matsuura, Yusuke Nakata, Masahiro Takeo, Dai-ichiro Kato, Seiji Negoro
    The FEBS journal 276(9) 2547-56 2009年5月  査読有り
    A carboxylesterase with a beta-lactamase fold from Arthrobacter possesses a low level of hydrolytic activity (0.023 mumol.min(-1).mg(-1)) when acting on a 6-aminohexanoate linear dimer byproduct of the nylon-6 industry (Ald). G181D/H266N/D370Y triple mutations in the parental esterase increased the Ald-hydrolytic activity 160-fold. Kinetic studies showed that the triple mutant possesses higher affinity for the substrate Ald (K(m) = 2.0 mm) than the wild-type Ald hydrolase from Arthrobacter (K(m) = 21 mm). In addition, the k(cat)/K(m) of the mutant (1.58 s(-1).mm(-1)) was superior to that of the wild-type enzyme (0.43 s(-1).mm(-1)), demonstrating that the mutant efficiently converts the unnatural amide compounds even at low substrate concentrations, and potentially possesses an advantage for biotechnological applications. X-ray crystallographic analyses of the G181D/H266N/D370Y enzyme and the inactive S112A-mutant-Ald complex revealed that Ald binding induces rotation of Tyr370/His375, movement of the loop region (N167-V177), and flip-flop of Tyr170, resulting in the transition from open to closed forms. From the comparison of the three-dimensional structures of various mutant enzymes and site-directed mutagenesis at positions 266 and 370, we now conclude that Asn266 makes suitable contacts with Ald and improves the electrostatic environment at the N-terminal region of Ald cooperatively with Asp181, and that Tyr370 stabilizes Ald binding by hydrogen-bonding/hydrophobic interactions at the C-terminal region of Ald.
  • Yoshiaki Ukita, Saki Kondo, Chiwa Kataoka, Masahiro Takeo, Seiji Negoro, Yuichi Utsumi
    Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences 1931-1933 2009年  
    We propose a new, simple, and high-sensitive concept of microchip based enzymelinked immunosorbent assay (ELISA) without special detection technologies for high-throughput environmental screening system. The biggest feature of the system is the use of three-dimensional (3D) stacked structures, which consist of vertically stacked 3D capillary bundle structures and multiple reservoirs. To demonstrate the immunoassay of environmental pollutants, antibody was immobilized on the surface of the capillary bundle structure and competitive ELISA was carried out in the vertical microreactor. The environmental pollutants, nonylphenol and poly-chrorinatedbiphenil (PCB), ware successfully detected in our microreactor and the results suggests high-sensitive performance of the microreactor system. © 2009 CBMS.
  • Masahiro Takeo, Masumi Murakami, Sanae Niihara, Kenta Yamamoto, Munehiro Nishimura, Dai-ichiro Kato, Seiji Negoro
    Journal of bacteriology 190(22) 7367-74 2008年11月  査読有り
    4-Nitrophenol (4-NP) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil. Rhodococcus sp. strain PN1 degrades 4-NP via 4-nitrocatechol (4-NC) for use as the sole carbon, nitrogen, and energy source. A 5-kb EcoRI DNA fragment previously cloned from PN1 contained a gene cluster (nphRA1A2) involved in 4-NP oxidation. From sequence analysis, this gene cluster is expected to encode an AraC/XylS family regulatory protein (NphR) and a two-component 4-NP hydroxylase (NphA1 and NphA2). A transcriptional assay in a Rhodococcus strain revealed that the transcription of nphA1 is induced by only 4-NP (of several phenolic compounds tested) in the presence of nphR, which is constitutively expressed. Disruption of nphR abolished transcriptional activity, suggesting that nphR encodes a positive regulatory protein. The two proteins of the 4-NP hydroxylase, NphA1 and NphA2, were independently expressed in Escherichia coli and purified by ion-exchange chromatography or affinity chromatography. The purified NphA2 reduced flavin adenine dinucleotide (FAD) with the concomitant oxidation of NADH, while the purified NphA1 oxidized 4-NP into 4-NC almost quantitatively in the presence of FAD, NADH, and NphA2. This functional analysis, in addition to the sequence analysis, revealed that this enzyme system belongs to the two-component flavin-diffusible monooxygenase family. The 4-NP hydroxylase showed comparable oxidation activities for phenol and 4-chlorophenol to that for 4-NP and weaker activities for 3-NP and 4-NC.
  • Masahiro Takeo, Dai-ichiro Kato, Seiji Negoro, Rakesh K. Jain
    JOURNAL OF BIOTECHNOLOGY 136 S11-S11 2008年10月  査読有り
  • Yoshiaki Ukita, Toshifumi Asano, Kuniyo Fujiwara, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro, Yuichi Utsumi
    MICROSYSTEM TECHNOLOGIES-MICRO-AND NANOSYSTEMS-INFORMATION STORAGE AND PROCESSING SYSTEMS 14(9-11) 1573-1579 2008年10月  査読有り
    The advantages of vertical microreactor stack with three-dimensional (3D) structure for immunoassay are discussed. The vertical microreactor stack uses vertical fluid flow operation with multifunctional fluid filters. The multi function of fluid filter is very effective for micromixing and passive valve operation. The mechanism of micromixing is discussed by using computational fluid dynamics (CFD), and we know that the mixing mechanism based on Coanda effect. To evaluate the micromixing performance of fluid filter, we demonstrated enzyme reaction with unique repeat mixing operation. As the results, we proved that the fluid filter has very effective mixing performance. The detection limit, which demonstrated by competition enzyme-linked immunosorvent assay (ELISA), is comparable with recommended detection limit, which suggested by Japanese ministry for the environment.
  • Yuichi Utsumi, Toshifumi Asano, Yoshiaki Ukita, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro
    MICROSYSTEM TECHNOLOGIES-MICRO-AND NANOSYSTEMS-INFORMATION STORAGE AND PROCESSING SYSTEMS 14(9-11) 1399-1403 2008年10月  査読有り
    We proposed and fabricated a new chemical reactor with a vertical fluid flow operation for the wide biochemical applications. For rapid and high-yield reaction between reagents with large quantity, the filters, which possess 2,100 cylindrical through-bores were fabricated by deep X-ray lithography using synchrotron radiation. First, we tried the application of the fabricated vertical reactor to a sandwich enzyme-linked immunosorbent assay for the analysis of mouse immunoglobulin and nonylphenol using ultra violet absorption spectroscopy. We also confirmed the possibility of the analysis methods for competitive enzyme-linked immunosorbent assay of an endocrine disrupter, nonylphenol in a series of vertical fluidic operation. This sensitivity was one order higher than the sensitivity obtained by ordinary enzyme-linked immunosorbent assay using 96-wells microtiter plate and the same anti-NP-antibody.
  • K. Fujiwara, Y. Ukita, M. Takeo, S. Negoro, T. Kanie, M. Katayama, Y. Utsumi
    MICROSYSTEM TECHNOLOGIES-MICRO-AND NANOSYSTEMS-INFORMATION STORAGE AND PROCESSING SYSTEMS 14(9-11) 1411-1416 2008年10月  査読有り
    A novel type passive mixing device that causes the three-dimensional flow was proposed. This mixer consists of the integrated capillary bundle structure. And the capillaries ware cross-linked each other. So it is called cross-linked micro capillary filter. The mixing effect of the cross-linked micro capillary filter was calculated by the computational fluid dynamics (CFD). The fluid behaviour in the fine three-dimensional structure could be analysed by the use of CFD. In the result of CFD calculation, the cross-linked micro capillary filter estimated high mixing effect. Moreover, the mixing efficiency was become higher by change the cross-linked form. The calculation result was decided the form of the cross-linked micro capillary filter. And this filter was fabricated by application of the Deep X-ray lithography. To form the cross-linked capillary, it was operated twice exposures. In these exposures, respectively the exposure stage was tilted to difference angle. The cross-linked capillary filter fabricated in this way was applied to vertical fluid flow operation. This cross-linked capillary filter could hold and transmit the fluid by the switchover of in impressed pressure. Herewith the cross-linked micro capillary filter showed availability as high efficiency mixing device.
  • Yoshiaki Ukita, Toshifumi Asano, Kuniyo Fujiwara, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro, Tomohiko Kanie, Makoto Katayama, Yuichi Utsumi
    SENSORS AND ACTUATORS A-PHYSICAL 145 449-455 2008年7月  査読有り
    The advantages of vertical microreactor stack with three-dimensional (3D) structure for immunoassay are discussed. The vertical microreactor stack uses vertical fluid flow operation with multifunctional fluid filters. The multifunction of fluid filter is very effective for micromixing and passive valve operation. The mechanism of micromixing is discussed by using computational fluid dynamics (CFD), and the results suggest that the mixing mechanism based on two main effects. One is Coanda effect and the other is Taylor dispersion induced in enormously high aspect ratio capillary and liquid plug. We demonstrated enzyme reaction with unique repeat mixing operation, to evaluate the micromixing performance of fluid filter. As the results, we proved that the fluid filter has very effective mixing performance. The detection limit, which demonstrated by competition enzyme-linked immunosorvent assay (ELISA), is comparable with recommended detection limit, which suggested by Japanese ministry for the environment. (c) 2008 Elsevier B.V. All rights reserved.
  • Kengo Yasuhira, Yuki Uedo, Masahiro Takeo, Dai-ichiro Kato, Seiji Negoro
    Journal of bioscience and bioengineering 104(6) 521-4 2007年12月  査読有り
    A 15-kb gene locus including nylon-oligomer-degrading genes from the chromosome of an alkalophilic bacterium, Agromyces sp. KY5R, was cloned and sequenced. The genetic organization was similar to the DNA region flanked by directly repeated IS6100 sequences on the nylon-oligomer-degradative plasmid pOAD2. However, we found several genetic rearrangements between the two DNA regions. Here, we discuss the possible mechanisms underlying the genetic rearrangements.
  • Katsuhiro Matsui, Isao Kawaji, Yuichi Utsumi, Yoshiaki Ukita, Toshifumi Asano, Masahiro Takeo, Dai-ichiro Kato, Seiji Negoro
    Bioscience, biotechnology, and biochemistry 71(12) 3098-101 2007年12月  査読有り
    Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (psi 40 microm) in 200 microm-thickness polymethylmethacrylate (PMMA) sheets (psi 3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunoglobulin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0-100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay.
  • Kengo Yasuhira, Yasuhito Tanaka, Hiroshi Shibata, Yasuyuki Kawashima, Akira Ohara, Dai-ichiro Kato, Masahiro Takeo, Seiji Negoro
    Applied and environmental microbiology 73(21) 7099-102 2007年11月  査読有り
    Alkalophilic, nylon oligomer-degrading strains, Agromyces sp. and Kocuria sp., were isolated from the wastewater of a nylon-6 factory and from activated sludge from a sewage disposal plant. The 6-aminohexanoate oligomer hydrolases (NylC) from the alkalophilic strains had 95.8 to 98.6% similarity to the enzyme in neutrophilic Arthrobacter sp. but had superior thermostability, activity under alkaline conditions, and affinity for nylon-related substrates, which would be advantageous for biotechnological applications.
  • Katsuhiro Matsui, Isao Kawaji, Yuichi Utsumi, Yoshiaki Ukita, Toshifumi Asano, Masahiro Takeo, Dai-Ichiro Kato, Seiji Negoro
    Journal of bioscience and bioengineering 104(4) 347-50 2007年10月  査読有り
    We developed an enzyme-linked immunosorbent assay for an endocrine disrupter, nonylphenol, using a microreactor composed of two reaction vessels stacked vertically through a microfluid filter. The filters constructed by deep X-ray lithography possessed 2100 through-bores (phi40 x 200 microm) in polymethylmethacrylate sheets (phi3 mm), which are appropriate for biochemical reactions. Through the optimization of the immunoassay, nonylphenol was quantitatively detected at the range of 0.1-10 ng/ml.
  • Masahiro Takeo, Munehiro Nishimura, Hana Takahashi, Chitoshi Kitamura, Dai-Ichiro Kato, Seiji Negoro
    Journal of bioscience and bioengineering 104(4) 309-14 2007年10月  査読有り
    Alkylcatechol 2,3-dioxygenase was purified from the cell extract of recombinant Escherichia coli JM109 harboring the alkylcatechol 2,3-dioxygenase gene (bupB) cloned from the butylphenol-degrading bacterium Pseudomonas putida MT4. The purified enzyme (BupB) showed relative meta-cleavage activities for the following catechols: catechol (100%), 4-methylcatechol (572%), 4-n-butylcatechol (185%), 4-n-hexylcatechol (53%), 4-n-heptylcatechol (45%), 4-n-nonylcatechol (10%), 4-tert-butylcatechol (0%), and 3-methylcatechol (33%). The kinetic parameters, namely, K(m) and V(max), for catechol, 4-methylcatechol, and 4-n-butylcatechol, were 23.4, 8.4, and 6.5 microM and 25.8, 76.9, and 18.0 U mg(-1), respectively. These results suggest that BupB has broad substrate specificity for 4-n-alkylcatechols.
  • Dai-Ichiro Kato, Keisuke Teruya, Hiromitsu Yoshida, Masahiro Takeo, Seiji Negoro, Hiromichi Ohta
    The FEBS journal 274(15) 3877-85 2007年8月  査読有り
    We introduce a new application of firefly luciferase (EC 1.13.12.7). The firefly luciferases belong to a large superfamily that includes rat liver long-chain acyl-CoA synthetase (LACS1). LACS1 is the enzyme that is involved in the deracemization process of 2-arylpropanoic acid and catalyzes the enantioselective thioester formation of R-acids. Based on the similarity of the reaction mechanisms and the sequences between firefly luciferase and LACS1, we predicted that firefly luciferase also has thioesterification activity toward 2-arylpropanoic acid. From an investigation using three kinds of luciferases from North American firefly and Japanese fireflies, we have confirmed that these luciferases exhibit an enantioselective thioester formation activity and the R-form is transformed to a thioester in preference to the S-form in the presence of ATP, Mg(2+), and CoASH. The enantiomeric excesses of unreacted recovered acid and thioester were determined by chiral phase HPLC analysis and the resulting 2-arylpropanoyl-CoAs were identified by high resolution mass spectroscopy. The K(m) and k(cat) values of thermostable luciferase from Luciola lateralis (LUC-H) toward ketoprofen were determined as 0.22 mM and 0.11 s(-1), respectively. The affinity of ketoprofen was almost the same of d-luciferin. In addition, the calculated E-value toward ketoprofen was approximately 20. These results suggest that LUC-H could catalyze the kinetic resolution of 2-arylpropanoic acid efficiently and would be a new option for the preparation of optically active 2-substituted carboxylic acids.
  • Masahiro Takeo, Munehiro Nishimura, Mizuho Shirai, Hana Takahashi, Seiji Negoro
    Bioscience, biotechnology, and biochemistry 71(7) 1668-75 2007年7月  査読有り
    Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 microM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 microM, while the mutant enzyme loosened substrate inhibition.
  • Seiji Negoro, Taku Ohki, Naoki Shibata, Kazuhiro Sasa, Haruhisa Hayashi, Hidehiko Nakano, Kengo Yasuhira, Dai-ichiro Kato, Masahiro Takeo, Yoshiki Higuchi
    Journal of molecular biology 370(1) 142-56 2007年6月29日  査読有り
    We performed X-ray crystallographic analyses of 6-aminohexanoate-dimer hydrolase (Hyb-24DN), an enzyme responsible for the degradation of nylon-6, an industry by-product, and of a complex between Hyb-24DN-A(112) (S112A-mutant of Hyb-24DN) and 6-aminohexanoate-linear dimer (Ald) at 1.58 A and 1.4 A resolution, respectively. In Hyb-24DN, Asp181-O(delta) forms hydrogen bonds with Tyr170-O(eta), -two of the catalytic and binding amino acids, and a loop between Asn167 and Val177. This state is the so-called open form, allowing its substrate to bind in the space between the loop and catalytic residues. Upon substrate binding (in Hyb-24DN-A(112)/Ald complex), the loop is shifted 4.3 A at Tyr170-C(alpha), and the side-chain of Tyr170 is rotated. By the combined effect, Tyr170-O(eta) moves a total of 10.5 A, resulting in the formation of hydrogen bonds with the nitrogen of amide linkage in Ald (closed form). In addition, electrostatic interaction between Asp181-O(delta) and the amino group in Ald stabilizes the substrate binding. We propose here that the enzyme catalysis proceeds according to the following steps: (i) Ald-induced transition from open to closed form, (ii) nucleophilic attack of Ser112 to Ald and formation of a tetrahedral intermediate, (iii) formation of acyl enzyme and transition to open form, (iv) deacylation. Amino acid substitutions reducing the enzyme/Ald interaction at positions 181 or 170 drastically decreased the Ald-hydrolytic activity, but had very little effect on esterolytic activity, suggesting that esterolytic reaction proceeds regardless of conversion. Present models illustrate why new activity against the nylon oligomer has evolved in an esterase with beta-lactamase folds, while retaining the original esterolytic functions.
  • Toshifumi Asano, Yoshiaki Ukita, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro, Yuichi Utsumi
    MICROSYSTEM TECHNOLOGIES-MICRO-AND NANOSYSTEMS-INFORMATION STORAGE AND PROCESSING SYSTEMS 13(5-6) 441-446 2007年3月  査読有り
    We proposed and fabricated a vertical micro reactor stack with vertical fluid flow operation for environment analysis, post-genome analysis, gene diagnosis, and screenings of useful materials for pharmaceutical. This reactor is characterized as the simple structure and new aspects of the vertical fluid transportation evoked by the use of the fluid filter with micro through-bores. The lithographite, galvanoformung and abformung (LIGA) process using synchrotron radiation was applied for the fabrication of the fluid filters. The computational fluid dynamics (CFD) simulation results suggested that the fluid can be held by the fluid filter and easily transported by the pneumatic operation. It was also confirmed that the fluid flow velocity through the filter was controlled by varying the loaded pressure around several kPa. Furthermore, it was expected that the fluid was stirred and mixed when passing through the fluid filter. It was demonstrated that the proposed chemical reactor result in a good performance of the vertical fluid flow operation and biochemical reaction.
  • Yuichi Utsumi, Yusuke Hitaka, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro, Yoshiaki Ukita
    MICROSYSTEM TECHNOLOGIES-MICRO-AND NANOSYSTEMS-INFORMATION STORAGE AND PROCESSING SYSTEMS 13(5-6) 425-429 2007年3月  査読有り
    Recently the progress of life science has been increasing rapidly, and the importance of the microfluidics for DNA analysis systems has been widely recognized, especially in medical fields. The polymerase chain reaction (PCR) is an essential technique for DNA assay of various diseases and it has been a strong requirement to shorten the total of PCR cycles more and more. We developed the microreactor with a single cell for PCR using fabrication technologies of MEMS. The reactor body and cover were sealed using high thickness PDMS prototyping film without using adhesive in order to achieve repeat grabbing motion for direct sample injection, resumption and cleansing the reaction cell. Good reproducibility of the heat cycling was obtained. The heating rate and cooling rate during PCR was 6.8 and 2.7 degrees C, respectively, which well corresponds to the design parameters. The homogenous temperature distribution of variance less than 2.0 degrees C was obtained. It is demonstrated that amplification of the DNA was successfully achieved by using the microreactor.
  • K. Mochiji, H. Hashimoto, Y. Tanaka, N. Ninomiya, M. Takeo
    PHYSICAL REVIEW B 75(9) 094302-1-094302-5 2007年3月  査読有り
    The structural changes in plasmid DNA adsorbed onto graphite following low-energy electron irradiation were investigated. Using a scanning tunneling microscope (STM), we observed networks or islands of DNA consisting of entangled molecules and compared the shapes of the DNA before and after electron irradiation at 8-40 eV field emitted from the tip of the STM. The shape of the DNA changed depending on the electron energy. Electrons with very low energy, such as 8 or 13 eV, extended the area of a DNA island, while the electrons at 18 or 38 eV degraded it. Both types of changes tend to saturate as the electron dose increases. We also discuss the above results in terms of the chemical reactions, such as strand breaks or molecular dissociation, induced by low-energy electrons.
  • Yoshiaki Ukita, Toshifumi Asano, Kuniyo Fujiwara, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro, Tomohiko Kanie, Makoto Katayama, Yuichi Utsumi
    Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 373-375 2007年  
    This paper proposes a vertical microreactor with multifunctional fluid filters which characterized by 3D-capillary-bundle. The high-efficiency micromixing properties of the fluid filter is proved with CFD and enzyme reaction. The ELISA method is demonstrated by using vertical microreactor and the advantages of 3D structure are duscussed.
  • 松井勝弘, 森本祥平, 浅野豪文, 浮田芳昭, 加藤太一郎, 武尾正弘, 内海裕一, 根来誠司
    電気学会論文雑誌C(電子・情報・システム部門誌) 127(2) 204-209 2007年  査読有り
  • 藤原邦代, 浮田芳昭, 浅野豪文, 松井勝弘, 武尾正弘, 根来誠司, 内海裕一
    電気学会論文雑誌C(電子・情報・システム部門誌) 127(2) 210-216 2007年  査読有り
  • Kengo Yasuhira, Yuki Uedo, Naoki Shibata, Seiji Negoro, Masahiro Takeo, Yoshiki Higuchi
    Acta crystallographica. Section F, Structural biology and crystallization communications 62(Pt 12) 1209-11 2006年12月1日  査読有り
    6-Aminohexanoate-cyclic-dimer hydrolase (EI) from Arthrobacter sp. KI72 was expressed in Escherichia coli and purified by anion-exchange chromatography. EI was crystallized by the sitting-drop vapour-diffusion method with sodium citrate as precipitant in imidazole buffer pH 8.0. The crystal is hexagonal, with unit-cell parameters a = b = 130.75, c = 58.23 A. Diffraction data were collected from native and mercury(II) dichloride-derivative crystals to resolutions of 1.90 and 2.06 A, respectively.
  • 安平健吾, 武尾正弘, 根来誠司
    日本水処理生物学会誌 42(4) 199-205 2006年12月  査読有り
  • Yuichi Utsumi, Toshifumi Asano, Yoshiaki Ukita, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro
    JOURNAL OF VACUUM SCIENCE & TECHNOLOGY B 24(6) 2606-2611 2006年11月  査読有り
    The authors proposed and fabricated a new microreactor stack which would be able to achieve a vertical fluid flow operation for the environment analysis, postgenome analysis, gene diagnosis, and screening of useful materials for medicine manufacture. This reactor is characterized as a simple structure with new aspects of the vertical fluid transportation using a proposed fluid filter with array of micro-through-bores. The deep x-ray lithography process using synchrotron radiation was used for the fabrication of the fluid filter. The feasibility of vertical liquid transportation was investigated using computational fluid dynamics analysis. It is indicated that the vertical liquid transportation is possible using the proposed fluid filter, and high efficiency mixing of liquid was also expected during transportation through the fluid filter. It was confirmed that the fluid flow velocity through the filter can be controlled by varying the load pressure around several kilopascals. A rapid enzyme reaction was successfully carried out and product concentration was observed using ultraviolet absorption spectroscopy. It was demonstrated that the proposed chemical reactor had a good vertical fluid flow operation performance for unit chemical operation. (c) 2006 American Vacuum Society.
  • Masahiro Takeo, Subbuswamy K Prabu, Chitoshi Kitamura, Makoto Hirai, Hana Takahashi, Dai-Ichiro Kato, Seiji Negoro
    Journal of bioscience and bioengineering 102(4) 352-61 2006年10月  査読有り
    Alkylphenols (APs) are ubiquitous contaminants in aquatic environments and have endocrine disrupting and toxic effects on aquatic organisms. To investigate biodegradation mechanisms of APs, an AP degradation gene cluster was cloned from a butylphenol (BP)-degrading bacterium, Pseudomonas putida MT4. The gene cluster consisted of 13 genes named bupBA1A2A3A4A5A6CEHIFG. From the nucleotide sequences, bupA1A2A3A4A5A6 were predicted to encode a multicomponent phenol hydroxylase (PH), whereas bupBCEHIFG were expected to encode meta-cleavage pathway enzymes. A partial sequence of a putative NtrC-type regulatory gene, bupR, was also found upstream of the gene bupB. This result indicates that APs can be initially oxidized into alkylcatechols (ACs), followed by the meta-cleavage of the aromatic rings. To confirm this pathway, AP degradation tests were carried out using the recombinant P. putida KT2440 harboring the PH genes (bupA1A2A3A4A5A6). The recombinant strain oxidized 4-n-APs with an alkyl chain of up to C7 (< or = C7) efficiently and also several BPs including those with an alkyl chain with some degree of branching. Therefore, it was found that PH had a broad substrate specificity for APs with a medium-length alkyl chain (C3-C7). Moreover, the cell extract of a recombinant Escherichia coli harboring bupB (a catechol 2,3-dioxygenase gene) converted 4-n-ACs with an alkyl chain of < or = C9 into yellow meta-cleavage products with a maximum absorbance at 379 nm, indicating that the second step enzyme in this pathway is also responsible for the degradation of ACs with a medium-length alkyl chain. These results suggest that MT4 is a very useful strain in the biodegradation of a wide range of APs with a medium-length alkyl chain, which known nonylphenol-degrading Sphingomonas strains have never degraded.
  • Taku Ohki, Yoshiaki Wakitani, Masahiro Takeo, Kengo Yasuhira, Naoki Shibata, Yoshiki Higuchi, Seiji Negoro
    FEBS letters 580(21) 5054-8 2006年9月18日  査読有り
    Carboxylesterase (EII') from Arthrobacter sp. KI72 has 88% homology to 6-aminohexanoate-dimer hydrolase (EII) and possesses ca. 0.5% of the level of 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity of EII. To study relationship between Ald-hydrolytic and esterolytic activities, random mutations were introduced into the gene for Hyb-24 (an EII/EII' hybrid with the majority of the sequence deriving for EII' and possessing an EII'-like level of Ald-hydrolytic activity). Either a G181D or a D370Y substitution in Hyb-24 increased the Ald-hydrolytic activity ca. 10-fold, and a G181D/D370Y double substitution increased activity ca. 100-fold. On the basis of kinetic studies and the three-dimensional structure of the enzyme, we suggest that binding of Ald is improved by these mutations. D370Y increased esterolytic activity for glycerylbutyrate ca. 30-50-fold, whereas G181D decreased the activity to 30% of the parental enzyme.
  • Yosiaki Ukita, Toshifumi Asano, Kuniyo Fujiwara, Takuya Yokoyama, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro, Tsunemasa Saiki, Yuichi Utsumi
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 45(9A) 7203-7208 2006年9月  査読有り
    A new method using multifunctional fluid filters with through-capillary arrays for high throughput and large-scale integrated microfluidics is proposed. The method utilizes a liquid's surface tension and fluid flows perpendicular to a substrate using a fluid filter. Utilizing this method, we can minimize the space consumption of microchannels, and enhance the flexibility of channel design, because these are not extant on the surface of substrates as in traditional microfluidics, but are throughcapillary arrays passing through the substrates. In addition, the passive multifunctional characteristics of the fluid filter are favorable for the integration of microfluidics. Therefore, the integration number can be increased from the previous order of hundreds to thousands or more. We conducted a computational fluid dynamics (CFD) analysis to examine the feasibility of vertical fluid flow operation; the multifunctionality of the fluid filter as a microvalve, a microchannel and a micromixer was estimated. The fabrication of the fluid filter by deep X-ray lithography and the vertical fluid.flow operation were successfully conducted and the high-throughput properties of the vertical fluid flow were demonstrated.
  • Taku Ohki, Yoshiaki Wakitani, Masahiro Takeo, Kengo Yasuhira, Naoki Shibata, Yoshiki Higuchi, Seiji Negoro
    FEBS LETTERS 580(21) 5054-5058 2006年9月  査読有り
    Carboxylesterase (EII') from Arthrobacter sp. K172 has 88% homology to 6-aminohexanoate-dimer hydrolase (Ell) and possesses ca. 0.5% of the level of 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity of Ell. To study relationship between Aid-hydrolytic and esterolytic activities, random mutations were introduced into the gene for Hyb-24 (an EII/EII' hybrid with the majority of the sequence deriving for EII' and possessing an EII'-like level of Ald-hydrolytic activity). Either a G181D or a D370Y substitution in Hyb-24 increased the Ald-hydrolytic activity ca. 10-fold, and a G181D/D370Y double substitution increased activity ca. 100-fold. On the basis of kinetic studies and the three-dimensional structure of the enzyme, we suggest that binding of Aid is improved by these mutations. D370Y increased esterolytic activity for glycerylbutyrate ca. 30-50-fold, whereas G181D decreased the activity to 30% of the parental enzyme. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Seiji Negoro, Taku Ohki, Naoki Shibata, Nobuhiro Mizuno, Yoshiaki Wakitani, Junya Tsurukame, Keiji Matsumoto, Ichitaro Kawamoto, Masahiro Takeo, Yoshiki Higuchi
    The Journal of biological chemistry 280(47) 39644-52 2005年11月25日  査読有り
    6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only approximately 0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII' proteins; EII'-level activity) by x-ray crystallography at 1.8 A resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and beta-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser(112) as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp(181) and Asn(266)) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.
  • Quanfeng Liang, Ming Chen, Yuquan Xu, Wei Zhang, Shuzhen Ping, Wei Lu, Lizhao Geng, Masahiro Takeo, Min Lin
    Gaojishu Tongxin/Chinese High Technology Letters 15(11) 69-73 2005年11月  
    Strain AD9-an aniline-degradation bacterium was isolated from the dying plant soil. AD9 has resistance to Amp, Km, Rif and has no Chloromycetin and Tc. The temperature for AD9 to grow and degrade aniline was 20-37°C, and the optimum temperature was 30°C. The strain AD9 grew well in the pH scale range from 4.0 to 9.0. The optimum pH was 7.0. The strain AD9 grew well in the aniline concentration range from 200 mg/L to 4500 mg/L. The best aniline concentration was 1000 mg/L. The 16SrRNA of strain AD9 belongs to the evolution branch of Delftia. The G + C content of AD9 is 66.8mol%, which is very similar to that of D. tsuruhatensis T7. In addition, the hybridization rate between AD9 and T7 was 83.8%. Together with the physiological and biochemical tests, the strain is named Delftia tsuruhatensis AD9.
  • Taku Ohki, Nobuhiro Mizuno, Naoki Shibata, Masahiro Takeo, Seiji Negoro, Yoshiki Higuchi
    Acta crystallographica. Section F, Structural biology and crystallization communications 61(Pt 10) 928-30 2005年10月1日  査読有り
    To investigate the structure-function relationship between 6-aminohexanoate-dimer hydrolase (EII) from Arthrobacter sp. and a cryptic protein (EII') which shows 88% sequence identity to EII, a hybrid protein (named Hyb-24) of EII and EII' was overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in MES buffer pH 6.5. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 96.37, c = 113.09 A. Diffraction data were collected from native and methylmercuric chloride derivative crystals to resolutions of 1.75 and 1.80 A, respectively.
  • Liang Q, Takeo M, Chen M, Zhang W, Xu Y, Lin M
    Microbiology (Reading, England) 151(Pt 10) 3435-3446 2005年10月  査読有り
  • QF Liang, M Chen, YQ Xu, W Zhang, SZ Ping, W Lu, XL Song, WW Wang, LZ Geng, M Takeo, M Lin
    CHINESE SCIENCE BULLETIN 50(15) 1612-1616 2005年8月  査読有り
    A convenient and widely applicable method has been developed to clone aniline metabolic gene cluster in this study. Three positive recombinant plasmids pDA1, pDB2 and pDB11 were cloned from genomic library of aniline degradation strain AD9. The result of aniline dioxygenase (AD) activity and catechol 2,3-oxygenase (C230) activity assay showed that pDA1 and pDB11 contain aniline dioxygenase genes and catechol 2,3-dioxygenase genes, respectively. The sequence analysis of the total 24.7-kb region revealed that this region contains 25 ORFs, of which 17 genes involve metabolism of aniline. In the gene cluster, the first five genes (tadQTA1A2B) and the subsequent gene (tadR1) were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, respectively, while the others (tadD1C1D2C2EFGIJKL) were expected to encode meta-cleavage pathway enzymes for catechol degradation. The gene cluster was surrounded by two IS1071 sequences.
  • Y Utsumi, T Asano, Y Ukita, K Matsui, M Takeo, S Negoro
    JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44(7B) 5707-5710 2005年7月  査読有り
    We proposed and fabricated a new chemical reactor with a vertical fluid flow operation for the environment analysis, post-genome analysis, gene diagnosis, and screening of useful materials for medicine manufacture. This reactor is characterized as a simple structure with new aspects of the vertical fluid transportation induced using a fluid filter with micro-through bores. The deep X-ray lithography process using synchrotron radiation was used for the fabrication of such a fluid filter. Computational fluid dynamics (CFD) simulation results revealed that fluid can be sustained on the surface of the fluid filter and easily transported by pneumatic operation. It was confirmed that the fluid flow velocity through the filter can be controlled by varying the loaded pressure around several kPa. It was demonstrated that the proposed chemical reactor has a good vertical fluid flow operation performance.
  • Toshifumi Asano, Yoshiaki Ukita, Yuichi Utsumi, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro
    e-Journal of Surface Science and Nanotechnology 3 190-194 2005年6月10日  査読有り
    We proposed and fabricated vertical micro reactor stack with vertical fluid flow operation available for the environment analysis, post-genome analysis, gene diagnosis, and screenings of the useful materials for the medicine manufacture. This reactor is characterized as the simple structure and new aspects of the vertical fluid transportation evoked by the use of the fluid filter with micro through-bores. The LIGA process using synchrotron radiation was applied for the fabrication of the fluid filters. The CFD simulation results suggested that the fluid can be held by the fluid filter and easily transported by the pneumatic operation. It was also confirmed that the fluid flow velocity through the filter was controlled by varying the loaded pressure around several kPa. Furthermore, it was expected that the fluid was stirred and mixed when passing through the fluid filter. It was demonstrated that a proposed chemical reactor had a good performance of the vertical fluid flow operation and chemical reaction. © 2005 The Surface Science Society of Japan.
  • Yuichi Utsumi, Toshifumi Asano, Tadashi Hattori, Katsuhiro Matsui, Masahiro Takeo, Seiji Negoro
    Digest of Papers - Microprocesses and Nanotechnology 2004 248-249 2004年  
  • Matsui Katsuhiro, Maeda Yoshihiro, Hirai Makoto, Takeo Masahiro, Negoro Seiji
    アジア・太平洋化学工学会議発表論文要旨集 2004 503-503 2004年  
    Nonylphenols (NPs) have been proved to be accumulated in sediments in aquatic environments as the degradation products of non-ionic surfactants and show endocrine disruptive activity to living organisms. To elucidate the biodegradation mechanisms for NPs, we isolated five NP-degrading bacteria from activated sludge using a commercial mixture of NP isomers (Kanto Kagaku, Tokyo) as a sole carbon source. Analysis of the partial 16S rDNA sequences of the isolates revealed that one strain is a Sphingomonas sp. and other four strains are Pseudomonas spp. On a minimal medium plate containing the NP mixture, the Sphingomonas strain, named strain NP5, formed a clear zone (halo), indicating that it degraded most of the NP isomers dispersed in the plate. In order to confirm the biodegradability for the NP isomers, a NP degradation test was carried out using the cell suspension of strain NP5. GC/MS analysis showed that at least 14 NP isomers contained in the mixture disappeared completely after 7 d- incubation and some alcohols with nine carbons were detected as the metabolites. This result indicates that strain NP5 has a broad degradation range for NP isomers and release of the alkyl-chain of NP might occur during the degradation.
  • Yasuhira Kengo, Takeo Masahiro, Negoro Seiji
    アジア・太平洋化学工学会議発表論文要旨集 2004 505-505 2004年  
    Alkalophilic nylon oligomer degrading strains were isolated from wastewater of nylon factory, and from activated sludge of a sewage disposal plant. One isolate, KY5R, showed good growth on LB-NOM10 plate (LB plate containing nylon oligomer mixture, pH10), and produced a clear zone on this plate, while previously isolated Flavobacterium sp. KI72 showed no growth and no halo formation on the LB-NOM10 plate. This result suggests that the insoluble nylon oligomers are degraded at pH10 by the strain KY5R. Nucleotide sequence of the 16S rRNA of the KY5R had 98% homology to that of Agromyces mediolanus, and 2,4-diaminobutyric acid was found as a component of cell wall. From these results, we have concluded that the strain KY5R is classified as Agromyces sp. Enzyme assay using the cell extracts of KY5R suggested that strain KY5R has the exo-type Ahx-oligomer hydrolase (EII) and endo-type Ahx-oligomer hydrolase (EIII) activities, but the no Ahx-cyclic dimer hydrolase (EI) activity. Studies using the antiserum against the purified EII of KI72 showed that the EII enzyme from Agromyces sp. KY5R (A-EII) is immunologically identical to that from Flavobacterium sp. KI72 (F-EII). In addition, DNA hybridization study using the DNA probe for the EIII gene from KI72 (F-nylC) suggested that the EIII enzyme from KY5R (A-EIII) is similar to the enzyme from KI72 (F-EIII).
  • Takeo M, Abe Y, Negoro S
    Journal of Chemical Industry and Engineering 53(suppl.) 42-44 2003年11月  査読有り
  • M Takeo, Y Abe, S Negoro, G Heiss
    JOURNAL OF CHEMICAL ENGINEERING OF JAPAN 36(10) 1178-1184 2003年10月  査読有り
    Rhodococcus sp. PN1 was isolated as a 4-nitrophenol (4-NP) degrading bacterium and can utilize 4-NP as a sole carbon, nitrogen, and energy source. This bacterium also degrades polynitrophenols such as 2,4-dinitrophenol and 2,4,6-trinitrophenol (picric acid). To elucidate the nitrophenol degradation pathways of strain PN1, a genomic library of strain PN1 was constructed using a Rhodococcus host-vector system and the genes encoding the pathways were screened from the library. Consequently, the gene clusters (nphRA1A2 and npdGIJ) encoding 4-NP hydroxylation and picric acid reduction were obtained from the library, respectively. The recombinant Rhodococcus strain containing the nphRA1A2 genes converted 4-NP into 4-nitrocatechol quantitatively, while that containing the npdGIJ genes transformed picric acid into its hydride Meisenheimer complex, quantitatively. Thus, it was found that strain PN1 has two quite different degradation mechanisms for nitrophenols, an oxygenation (4-NP hydroxylation) and a reduction (hydride transfer to the aromatic-ring of picric acid). Sequence analysis of the gene clusters suggested that the former encodes a member of the two component flavin diffusible monooxygenase family (NphA1 and NphA2), while the latter encodes an NADPH-dependent F 42, reductase (NpdG) and an F-420-dependent picric acid reductase (Npd1). Simultaneous degradation of 4-NP and picric acid by strain PN1 indicated that both degradation mechanisms can work simultaneously without significant inhibition. This capability of strain PN1 may contribute to the treatment of industrial wastewaters containing many kinds of nitrophenols.
  • Gesche Heiss, Natalie Trachtmann, Yoshikatsu Abe, Masahiro Takeo, Hans-Joachim Knackmuss
    Applied and environmental microbiology 69(5) 2748-54 2003年5月  査読有り
    Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F(420) reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F(420). Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F(420)-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F(420)-dependent glucose-6-phosphate dehydrogenases and F(420)-dependent N(5),N(10)-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F(420)-dependent enzymes involved in catabolism.

MISC

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書籍等出版物

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講演・口頭発表等

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担当経験のある科目(授業)

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共同研究・競争的資金等の研究課題

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