S Negoro

University of Hyogo, Kōbe, Hyōgo, Japan

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Publications (136)399.03 Total impact

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    ABSTRACT: A simple reaction procedure for chemiluminescence of firefly luciferin (D-luc) using n-propylphosphonic anhydride (T3P) is reported. A luminescence photon is produced as the result of one-pot reaction, only requiring mixing with substrate carboxylic acid and T3P in the presence of mild organic base.
    Photochemical and Photobiological Sciences 10/2014; · 2.92 Impact Factor
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    ABSTRACT: Nylon hydrolase degrades various aliphatic nylons, including nylon-6 and nylon-66. We synthesized a nylon-66 copolymer (M w = 22,900, M n = 7,400), in which a part of an adipoyl unit (32 % molar ratio) of nylon-66 was replaced with a succinyl unit by interfacial polymerization. To quantify the reaction rate of the enzymatic hydrolysis of nylons at the surface of solid polymers, we prepared a thin layer of nylons on the bottom surface of each well in a polystyrene-based micro-assay plate. The thickness of the nylon layer was monitored by imaging analysis of the photographic data. More than 99 % of the copolymer with thicknesses of 260 nm (approximately 600 layers of polymer strands) were converted to water-soluble oligomers by nylon hydrolase (3 mg enzyme ml(-1)) at 30 °C within 60 h. These results were further confirmed by TLC analysis of the reaction products and by assay of liberated amino groups in the soluble fractions. The degradation rate of the thin-layered nylon-6 was similarly analyzed. We demonstrate that this assay enables a quantitative evaluation of the reaction rate of hydrolysis at the interface between the solid and aqueous phases and a quantitative comparison of the degradability for various polyamides.
    Applied microbiology and biotechnology. 06/2014;
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    ABSTRACT: The active site of 6-aminohexanoate-dimer hydrolase, a nylon-6 byproduct-degrading enzyme with a β-lactamase fold, possesses a Ser112/Lys115/Tyr215 catalytic triad similar to the one of penicillin-recognizing family of serine-reactive hydrolases but includes a unique Tyr170 residue. By using a reactive quantum mechanics/molecular mechanics (QM/MM) approach, we work out its catalytic mechanism and related functional/structural specificities. At variance with other peptidases, we show that the involvement of Tyr170 in the enzyme–substrate interactions is responsible for a structural variation in the substrate-binding state. The acylation via a tetrahedral intermediate is the rate-limiting step, with a free-energy barrier of 21 kcal/mol, driven by the catalytic triad Ser112, Lys115, and Tyr215, acting as a nucleophile, general base, and general acid, respectively. The functional interaction of Tyr170 with this triad leads to an efficient disruption of the tetrahedral intermediate, promoting a conformational change of the substrate favorable for proton donation from the general acid.
    Journal of Physical Chemistry Letters 03/2014; 5:1210. · 6.59 Impact Factor
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    ABSTRACT: Nylon hydrolase (NylC) encoded by Arthrobacter plasmid pOAD2 (NylCp2) was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylCp2 was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl and 25% glycerol. Diffraction data were collected from the native crystal to a resolution of 1.60 Å. The obtained crystal was spindle shaped and belonged to the C-centred orthorhombic space group C2221, with unit-cell parameters a = 70.84, b = 144.90, c = 129.05 Å. A rotation and translation search gave one clear solution containing two molecules per asymmetric unit.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2013; 69(Pt 10):1151-1154. · 0.55 Impact Factor
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    ABSTRACT: Acinetobacter sp. YAA has five genes (atdA1-A5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From the sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5), respectively (Takeo M, Fujii T, Maeda Y. 1998. J. Ferment. Bioeng. 85: 17-24). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, it was purified from recombinant Escherichia coli harboring atdA1. Purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and L-glutamate in the presence of ATP and MgCl2. This reaction was identical to glutamine synthesis by GS except for the use of aniline as a substrate instead of ammonia. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3-A5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1-A5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells, and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation harmful to host cell.
    Journal of bacteriology 07/2013; · 3.94 Impact Factor
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    ABSTRACT: We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.
    Journal of Biotechnology 05/2013; · 3.18 Impact Factor
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    ABSTRACT: Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40-46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66 × 10(6) Da.
    Applied Microbiology and Biotechnology 01/2013; · 3.81 Impact Factor
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    ABSTRACT: 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.
    Sensors and Actuators A Physical 12/2012; · 1.84 Impact Factor
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    ABSTRACT: Sphingomonas sp. NP5 can degrade a wide range of nonylphenol (NP) isomers that have widely contaminated aquatic environments as major endocrine-disrupting chemicals. To understand the biochemical and genetic backgrounds of NP degradation, a gene library of strain NP5 was constructed using a broad-host-range vector pBBR1MCS-2 and introduced into Sphingobium japonicum UT26. Several transformants accumulated reddish brown metabolites on agar plates dispersed with a mixture of NP isomers. Two different DNA fragments (7.6 and 9.3 kb) involved in the phenotype were isolated from the transformants. Sequence analysis revealed that both fragments contained an identical 1593 bp monooxygenase gene (nmoA), the predicted protein sequence of which showed 83 % identity to the octylphenol-4-monooxygenase of Sphingomonas sp. PWE1. The nmoA gene in the 7.6 kb fragment was surrounded by an IS21-type insertion sequence (IS) and IS6100, while another in the 9.3 kb fragment was adjacent to an IS66-type IS, suggesting that they have been acquired through multiple transposition events. A fast-growing recombinant Pseudomonas putida strain harbouring nmoA was constructed and used for degradation of a chemically synthesized NP isomer, 4-(1-ethyl-1-methylhexyl)phenol. This strain converted the isomer into hydroquinone stoichiometrically. 3-Methyl-3-octanol, probably originating from the alkyl side chain, was also detected as the metabolite. These results indicate that these two nmoA genes are involved in the NP degradation ability of strain NP5.
    Microbiology 04/2012; 158(Pt 7):1796-807. · 3.06 Impact Factor
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    ABSTRACT: We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.
    Journal of Biological Chemistry 12/2011; 287(7):5079-90. · 4.65 Impact Factor
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    ABSTRACT: Measurement of thioesterification activities for dodecanoic acid (C12) and ketoprofen was done using five firefly luciferases, from Pyrocoelia miyako (PmL), Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria parvura (HpL), and Luciola mingrelica (LmL). Among these, PmL, PpL, and LcL showed the expected thioesterification activities toward both substrates. All the enzymes exhibited (R)-enantioselectivity toward ketoprofen, which had same tendency as firefly luciferase from Luciola lateralis (LUC-H). HpL and LmL, however, did not accept ketoprofen, although they had thioesterification activity toward C12. These results indicate that the substrate acceptance of luciferases for the thioesterification reaction varies dramatically relying on the origin of firefly. Hence we focused primarily on PmL and investigated the effect of pH on enzymatic activity. In addition, by determining the kinetic parameters at various pH values, we verified that the k(cat) parameter contributed to the preferential enantioselectivity of this enzyme.
    Bioscience Biotechnology and Biochemistry 09/2011; 75(9):1758-62. · 1.27 Impact Factor
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    ABSTRACT: 6-Aminohexanoate-oligomer hydrolase (NylC) from Agromyces sp. KY5R was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylC was crystallized by the sitting-drop vapour-diffusion method with sodium citrate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl. Diffraction data were collected from native and K(2)PtCl(4)-derivative crystals to resolutions of 2.00 and 2.20 Å, respectively. The obtained crystal was plate-shaped, with an I-centred orthorhombic space group and unit-cell parameters a = 155.86, b = 214.45, c = 478.80 Å. The anomalous difference Patterson map of the K(2)PtCl(4)-derivative crystal suggested that the space group was I222 rather than I2(1)2(1)2(1).
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2011; 67(Pt 8):892-5. · 0.55 Impact Factor
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    ABSTRACT: Recently, we found that firefly luciferase exhibited (R)-enantioselective thioesterification activity toward 2-arylpropanoic acids. In the case of Japanese firefly luciferase from Luciola lateralis (LUC-H), the E-value for ketoprofen was approximately 20. In this study, we used a spectrophotometric method to measure the catalytic activity of LUC-H. Using this method allowed us to judge the reaction efficiency easily. Our results confirmed that LUC-H exhibits enantioselective thioesterification activity toward a series of 2-arylpropanoic acids. The highest activity was observed with ketoprofen. We also observed high enzymatic activity of LUC-H toward long-chain fatty acids. These results were reasonable because LUC-H is homologous with long-chain acyl-CoA synthetase. To obtain further information about the enantiodifferentiation mechanism of the LUC-H catalyzed thioesterification of ketoprofen, we determined the kinetic parameters of the reaction relative to each of its three substrates: ketoprofen, ATP, and coenzyme A (CoASH). We found that whereas the affinities of each compound are not affected by the chirality of ketoprofen, enantiodifferentiation is achieved by a chirality-dependent difference in the kcat parameter.Graphical abstractA Japanese firefly luciferase from Luciola lateralis (LUC-H) has an enantioselective acyl-CoA synthetase activity toward ketoprofen. In this article, we determined the catalytic activity and kinetic parameters to estimate the enantiodifferentiation mechanism of LUC-H catalyzed thioester formation.Research highlights► Firefly luciferase, LUC-H, can catalyze the enantioselective thioesterification of 2-aryl propanoic acids. ► Catalytic activity and kinetic parameters of LUC-H were determined. ► The enantiodifferentiation event occurs after the binding of either enantiomer of ketoprofen.
    Journal of Molecular Catalysis B Enzymatic 05/2011; 69:140-146. · 2.82 Impact Factor
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    ABSTRACT: 4-Nitrophenol (4-NP) is a toxic compound formed in soil by the hydrolysis of organophosphorous pesticides, such as parathion. We previously reported the presence of the 4-NP degradation gene cluster (nphRA1A2) in Rhodococcus sp. strain PN1, which encodes a two-component 4-NP hydroxylase system that oxidizes 4-NP into 4-nitrocatechol. In the current study, another gene cluster (npsC and npsRA2A1B) encoding a similar 4-NP hydroxylase system was cloned from strain PN1. The enzymes from this 4-NP hydroxylase system (NpsA1 and NpsA2) were purified as histidine-tagged (His-) proteins and then characterized. His-NpsA2 showed NADH/FAD oxidoreductase activity, and His-NpsA1 showed 4-NP oxidizing activity in the presence of His-NpsA2. In the 4-NP oxidation using the reconstituted enzyme system (His-NpsA1 and His-NpsA2), hydroquinone (35% of 4-NP disappeared) and hydroxyquinol (59% of 4-NP disappeared) were detected in the presence of ascorbic acid as a reducing reagent, suggesting that, without the reducing reagent, 4-NP was converted into their oxidized forms, 1,4-benzoquinone and 2-hydroxy-1,4-benzoquinone. In addition, in the cell extract of recombinant Escherichia coli expressing npsB, a typical spectral change showing conversion of hydroxyquinol into maleylacetate was observed. These results indicate that this nps gene cluster, in addition to the nph gene cluster, is also involved in 4-NP degradation in strain PN1.
    Journal of Bioscience and Bioengineering 03/2011; 111(6):687-94. · 1.74 Impact Factor
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    ABSTRACT: In order to understand roles of E168 and Y170 residues in loop-segment (N166–V177) of nylon-6 byproduct-degrading enzymes, we determined substrate-binding structures of E168Q and Y170F mutants using molecular dynamics simulation with in silico mutations. We found that movement of the loop-segment plays key roles not only in allowing the substrate to be bound by induced fit mechanism but also in forming water-exclusive environment. Fluctuations of the loop-segment in the mutant enzymes caused a room near the catalytic site, where water molecules can access. We propose that the water located exclusivity at the catalytic site is a major factor of its activity.
    Chemical Physics Letters 01/2011; 507(1):157-161. · 2.15 Impact Factor
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    ABSTRACT: Vertical Microreactor Stack Consist of Poly-(Tetrafluoroethylene) Microfluidics for Immunoassay
    IEEJ Transactions on Electronics Information and Systems 10/2010;
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    ABSTRACT: 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.
    Microsystem Technologies 08/2010; 16(8):1465-1470. · 0.83 Impact Factor
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    ABSTRACT: NylB′ carboxylesterase, which is 88% homologous to functional 6-aminohexanoate-dimer hydrolase (NylB) from Arthrobacter sp., possesses trace synthetic activity [0.0004μmolmin−1mg−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 2.7U/mg by G181D-H266N substitutions, and the activity was further enhanced in the G181D-H266N-D370Y triple mutant to a level approximately 104-fold greater than the parental carboxylesterase form (3.4U/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 Ald-hydrolytic activity (
    Journal of Molecular Catalysis B Enzymatic 06/2010; 64(1):81-88. · 2.82 Impact Factor
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    ABSTRACT: Micro reactors and micro total analysis systems (µTAS) 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 (Ø 3 mm). The fluid filters, constructed by deep X-ray lithography, have 2100 pores (Ø 40μm), 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. © 2010 Wiley Periodicals, Inc. Electron Comm Jpn, 93(4): 50–57, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecj.10137
    Electronics and Communications in Japan 03/2010; 93(4):50 - 57. · 0.18 Impact Factor

Publication Stats

1k Citations
399.03 Total Impact Points

Institutions

  • 2005–2012
    • University of Hyogo
      • Department of Materials Science and Chemistry (Graduate)
      Kōbe, Hyōgo, Japan
  • 2010
    • Japan Society for the Promotion of Science
      Edo, Tōkyō, Japan
  • 1983–2008
    • Osaka University
      • • Department of Biotechnology
      • • Department of Internal Medicine
      • • Department of Oncogene Research
      Suika, Ōsaka, Japan
    • Roswell Park Cancer Institute
      • Department of Molecular Immunology
      Buffalo, New York, United States
  • 1988
    • Fukushima Medical University
      Hukusima, Fukushima, Japan
    • Osaka City University
      Ōsaka, Ōsaka, Japan