武尾 正弘

Mixing is difficult in microfluidic devices due to the low Reynolds’ number. We proposed and demonstrated a surface-acoustic-wave (SAW)-based microfluidic system which can control the temperature by mounting a heater. This device has a hollow cylindrical reservoir made of resin on a piezoelectric substrate (LiNbO3), and the solution can be rotated by injecting SAWs from both sides, which are shifted parallel to the central axis of the reservoir. 0.75 W of power was supplied to the IDT to generate SAWs, and the temperature rise of the liquid was measured with a thermographic camera when 0 to 0.55 W of power was supplied to the heater. As a result, by mounting the heater on the SAW actuator, it was possible to uniformly raise the temperature of a 40 µL micro-liquid by 8 to 40 °C in about 80 seconds, demonstrating the usefulness of this heater-mounted SAW agitation device.

In this study, a novel soluble chitosan-like bioflocculant (BF), named BF01314, was prepared from the culture supernatant of Citrobacter youngae GTC 01314 for potential application in the treatment of kaolin suspension and activated sludge. The physicochemical characteristics of the BF and its flocculation behavior under the effects of BF concentration, pH and temperature in kaolin suspension were investigated. Results reveal that BF01314 exhibited a chitosan-like infrared spectrum with the presence of the characteristic hydroxyl, amide, and amino groups. The BF demonstrated strong flocculation activities of more than 95% in kaolin suspension within a broad "flocculation window"of BF concentrations (4-120 mg-dry weight/L), pH range of 2-8, and temperature range of 10-95 °C, without significant overdose effect. When applied in the treatment of activated sludge, BF01314 showed significant dewaterability improvement with 62% reduction in capillary suction time and 10.5% increase in cake solids content. The specific resistance to filtration value was lowered by one order while the change of zeta potential value from - 15.5 to - 4.9 mV proved the ability of the cationic BF01314 to destabilize the negatively charged sludge particles. It is postulated that the flocculation mechanisms of BF01314 in both kaolin suspension and activated sludge were governed by electrostatic charge patching and bridging as indicated by the zeta potential results in addition to its high molecular weight. The effectiveness of BF01314 observed in the present work suggests its potential application in the removal of suspended solids in water and wastewater treatment with good pH and thermal stability.

Biodegradation of synthetic polymers is recognized as a useful way to reduce their environmental load and pollution, loss of natural resources, extensive energy consumption, and generation of greenhouse gases. The potential use of enzymes responsible for the degradation of the targeted polymers is an effective approach which enables the conversion of the used polymers to original monomers and/or other useful compounds. In addition, the enzymes are expected to be applicable in industrial processes such as improving the surface structures of the polymers. Especially, conversion of the solid polymers to soluble oligomers/monomers is a key step for the biodegradation of the polymers. Regarding the hydrolysis of polyamides, three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (NylA), 6-aminohexanoate-dimer hydrolase (NylB), and 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC), are found in several bacterial strains. In this chapter, we describe our approach for the screening of microorganisms which degrade nylons and related compounds; preparation of substrates; assay of hydrolytic activity for soluble and insoluble substrates; and X-ray crystallographic and computational approaches for analysis of structure and catalytic mechanisms of the nylon-degrading enzymes.

The nonylphenol-degrading bacterium Sphingomonas sp. strain NP5 has a very unique monooxygenase that can attack a wide range of 4-alkylphenols with a branched side chain. Due to the structural similarity, it can also attack bisphenolic compounds, which are very important materials for the synthesis of plastics and resins, but many of them are known to or suspected to have endocrine disrupting effects to fish and animals. In this study, to clarify the substrate specificity of the enzyme (NmoA) for bisphenolic compounds, degradation tests using the cell suspension of Pseudomonas putida harboring the nonylphenol monooxygenase gene (nmoA) were conducted. The cell suspension degraded several bisphenols including bisphenol F, bisphenol S, 4,4'-dihydroxybenzophenone, 4,4'-dihydroxydiphenylether, and 4,4'-thiodiphenol, indicating that this monooxygenase has a broad substrate specificity for compounds with a bisphenolic structure.

Here, we report the 5.2-Mb genome sequence of a bioflocculant-producing bacterial strain, Citrobacter freundii IFO 13545, which consists of 5,209,670 bp with a G+C content of 51.5% and 4,853 predicted coding sequences (CDSs). The genes related to the biosynthetic pathway of the bioflocculant were localized on the genome map.

3-Methyl-4-nitrophenol (3M4NP) is formed in soil as a hydrolysis product of fenitrothion, one of the major organophosphorus pesticides. A Pseudomonas strain was isolated as a 3M4NP degrader from a crop soil and designated TSN1. This strain utilized 3M4NP as a sole carbon and energy source. To elucidate the biodegradation pathway, we performed transposon mutagenesis with pCro2a (mini-Tn5495) and obtained three mutants accumulating a dark pink compound(s) from 3M4NP. Rescue cloning and sequence analysis revealed that in all mutants, the transposon disrupted an identical aromatic compound meta-cleaving dioxygenase gene, and a monooxygenase gene was located just downstream of the dioxygenase gene. These two genes were designated mnpC and mnpB, respectively. The gene products showed high identity with the methylhydroquinone (MHQ) monooxygenase (58%) and the 3-methylcatechol 2,3-dioxygenase (54%) of a different 3M4NP degrader Burkholderia sp. NF100. The transposon mutants converted 3M4NP or MHQ into two identical metabolites, one of which was identified as 2-hydroxy-5-methyl-1,4-benzoquinone (2H5MBQ) by GC/MS analysis. Furthermore, two additional genes (named mnpA1 and mnpA2), almost identical to the p-nitrophenol monooxygenase and the p-benzoquinone reductase genes of Pseudomonas sp. WBC-3, were isolated from the total DNA of strain TSN1. Disruption of mnpA1 resulted in the complete loss of the 3M4NP degradation activity, demonstrating that mnpA1 encodes the initial monooxygenase for 3M4NP degradation. The purified mnpA2 gene product could efficiently reduce methyl p-benzoquinone (MBQ) into MHQ. These results suggest that strain TSN1 degrades 3M4NP via MBQ, MHQ, and 2H5MBQ in combination with mnpA1A2 and mnpCB, existing at different loci on the genome.

Nylon hydrolase (NylC) is initially expressed as an inactive precursor (36 kDa). The precursor is cleaved autocatalytically at Asn266/Thr267 to generate an active enzyme composed of an α subunit (27 kDa) and a β subunit (9 kDa). Four αβ heterodimers (molecules A-D) form a doughnut-shaped quaternary structure. In this study, the thermostability of the parental NylC was altered by amino acid substitutions located at the A/D interface (D122G/H130Y/D36A/L137A) or the A/B interface (E263Q) and spanned a range of 47 °C. Considering structural, biophysical, and biochemical analyses, we discuss the structural basis of the stability of nylon hydrolase. From the analytical centrifugation data obtained regarding the various mutant enzymes, we conclude that the assembly of the monomeric units is dynamically altered by the mutations. Finally, we propose a model that can predict whether the fate of the nascent polypeptide will be correct subunit assembly, inappropriate protein-protein interactions causing aggregation, or intracellular degradation of the polypeptide.

Chitin/chitosan, one of the most abundant polysaccharides in nature, is industrially produced as a powder or flake form from the exoskeletons of crustaceans such as crabs and shrimps. Intriguingly, many bacterial strains in the genus Citrobacter secrete a soluble chitin/chitosan-like polysaccharide into the culture medium during growth in acetate. Because this polysaccharide shows strong flocculation activity for suspended solids in water, it can be used as a bioflocculant (BF). The BF synthetic pathway of C. freundii IFO 13545 is expected from known bacterial metabolic pathways to be as follows: acetate is metabolized in the TCA cycle and the glyoxylate shunt via acetyl-CoA. Next, fructose 6-phosphate is generated from the intermediates of the TCA cycle through gluconeogenesis and enters into the hexosamine synthetic pathway to form UDP-N-acetylglucosamine, which is used as a direct precursor to extend the BF polysaccharide chain. We conducted the draft genome sequencing of IFO 13545 and identified all of the candidate genes corresponding to the enzymes in this pathway in the 5420-kb genome sequence. Disruption of the genes encoding acetyl-CoA synthetase and isocitrate lyase by homologous recombination resulted in little or no growth on acetate, indicating that the cell growth depends on acetate assimilation via the glyoxylate shunt. Disruption of the gene encoding glucosamine 6-phosphate synthase, a key enzyme for the hexosamine synthetic pathway, caused a significant decrease in flocculation activity, demonstrating that this pathway is primarily used for the BF biosynthesis. A gene cluster necessary for the polymerization and secretion of BF, named bfpABCD, was also identified for the first time. In addition, quantitative RT-PCR analysis of several key genes in the expected pathway was conducted to know their expression in acetate assimilation and BF biosynthesis. Based on the data obtained in this study, an overview of the BF synthetic pathway is discussed.

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD 1 and nylE 1 , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD1) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE1) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP+ as a cofactor. Phylogenic analysis revealed that NylD1 should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE1 should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD1/NylE1 coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.

The original publication of this paper contains mistakes for Tables 1 and 2 legends as well as the sublabels in Figs. 2, 4, 5, 6, and 7.

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

We report here the 4.6-Mb genome sequence of a nylon oligomer-degrading bacterium, Arthrobacter sp. strain KI72. The draft genome sequence of strain KI72 consists of 4,568,574 bp, with a G+C content of 63.47%, 4,372 coding sequences (CDSs), 54 tRNAs, and six rRNAs.

UNLABELLED: The enzyme 6-aminohexanoate-dimer hydrolase catalyzes amide synthesis. The yield of this reverse reaction in 90% t-butyl alcohol was found to vary drastically when enzyme mutants with substitutions of several amino acids located at the entrance of the catalytic cleft were used. Movement of the loop region and the flip-flop of Tyr170 generate a local hydrophobic environment at the catalytic center of the enzyme. Here, we propose that the shift of the internal equilibrium between the enzyme-substrate complex and enzyme-product complex by the 'water-excluding effect' alters the rate of the forward and reverse reactions. Moreover, we suggest that the local hydrophobic environment potentially provides a reaction center suitable for efficient amide synthesis. DATABASE: PDB code 3VWL: Hyb-24DNY-S(187) PDB code 3VWM: Hyb-24DNY-A(187) PDB code 3VWN: Hyb-24DNY-G(187) PDB code 3A65: Hyb-24DN-A(112) /Ahx complex PDB code 3A66: Hyb-24DNY-A(112) /Ahx complex PDB code 3VWP: Hyb-24DNY-S(187) A(112) /Ahx complex PDB code 3VWQ: Hyb-24DNY-A(187) A(112) /Ahx complex PDB code 3VWR: Hyb-24DNY-G(187) A(112) /Ahx complex.

We report the 4.9-Mb genome sequence of Citrobacter freundii strain GTC 09479, isolated from urine sample collected during the year 1983 at Gifu University Graduate School of Medicine, Japan. This draft genome consist of 4,899,578 bp with 51.62% G + C, 4,574 predicted CDSs, 72 tRNAs and 10 rRNAs.

A simple reaction procedure for chemiluminescence of firefly luciferin (D-luc) using n-propylphosphonic anhydride (T3P) is reported. A luminescent photon is produced as a result of one-pot reaction, only requiring mixing with the substrate carboxylic acid and T3P in the presence of a mild organic base.

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 × 10⁶Da.

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.

We report the 5.0-Mb genome sequence of the type species of the genus Citrobacter, Citrobacter freundii strain MTCC 1658, isolated from canal water. This draft genome sequence of C. freundii strain MTCC 1658(T) consists of 5,001,265 bp with a G+C content of 51.61%, 4,691 protein-coding genes, 70 tRNAs, and 10 rRNAs.

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.

A stacked-type centrifugal microfluidic device with three-dimensional (3D) microchannel networks and multifunctional capillary bundle structures is developed, and an immunoassay for the detection of immunoglobulin G (IgG) is demonstrated. The device consists of multiple layers of disk-like chips with conventional planar microchannels and vertical capillary bundle structures that are constructed on disks of poly(dimethylsiloxane) and poly(methyl methacrylate). The integration of multiple reactors up to 10 reactors on a disk with 8cm in diameter is realized by reducing the area of reservoir but increasing the thickness. Moreover the thick optical detection reservoir is also realized for absorption spectroscopy in the 3D centrifugal microfluidics. 3D liquid valving on the vertical capillary bundle structures and liquid transportation through these structures are successfully demonstrated. The results show the successful control of the multistep liquid injection without problems such as blockage of capillary bundle by bubbles that expected to be caused due to 3D structure. The results of the immunoassay show clear response to analyte concentration suggesting successful unit operations in developed 3D centrifugal microfluidic device for immunoassay. Finally, the advantages and impact of the 3D design for the development of high-performance centrifugal microfluidic systems are summarized. (C) 2012 Elsevier B.V. All rights reserved.

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.

Lipase from candida rugosa was immobilized in a freeze-dried polyvinyl alcohol (PVA) matrix for investigating the enzymatic reactivity. This matrix was prepared in a microreactor to achieve continuous enzymatic reactions. The performances of the lipase immobilized microreactors were compared in terms of the micro channel type and the reaction conditions. The freeze-dried matrix made in the microchannel possessed porous microstructure with 2-20 !m of well-aligned pores. These pore structures were obtained as a replica of the ice crystals formed during the freezing stage. Experiments suggested that the enzymatic reaction with the immobilized lipase was a diffusion controlled system. It was found that the present microreactors exhibited approximately 10 times higher reaction performance than the large scaled plug flow reactor with the freeze-dried specimens. The freeze-dried microstructure fabricated in a microspace had a significant effect of improving enzymatic reactions. A solution flow in the microchannel is under an influence of both the porous microstructure and the channel structure. When the channel size was smaller than 1000 !m, the flow regime would mainly be controlled by the freeze-dried microstructure.

Burkholderia sp. strain SJ98 (DSM 23195) was previously isolated and characterized for degradation and co-metabolic transformation of a number nitroaromatic compounds. In the present study, we evaluated its metabolic activity on chlorinated nitroaromatic compounds (CNACs). Results obtained during this study revealed that strain SJ98 can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize it as sole source of carbon, nitrogen, and energy under aerobic conditions. The cells of strain SJ98 removed 2C4NP from the growth medium with sequential release of nearly stoichiometric amounts of chloride and nitrite in culture supernatant. Under aerobic degradation conditions, 2C4NP was transformed into the first intermediate that was identified as p-nitrophenol by high-performance liquid chromatography, LCMS-TOF, and GC-MS analyses. This transformation clearly establishes that the degradation of 2C4NP by strain SJ98 is initiated by "reductive dehalogenation"; an initiation mechanism that has not been previously reported for microbial degradation of CNAC under aerobic conditions.

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 kat parameter contributed to the preferential enantioselectivity of this enzyme.

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).

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.

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 (LUG-H), the E-value for ketoprofen was approximately 20. In this study, we used a spectrophotometric method to measure the catalytic activity of LUG-H. Using this method allowed us to judge the reaction efficiency easily. Our results confirmed that LUG-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 K(cat) parameter. (C) 2011 Elsevier B.V. All rights reserved.

Acinetobacter calcoaceticus PHEA-2 utilizes phenol as its sole carbon and energy source and has a multi-component phenol hydroxylase-encoding gene operon (mphKLMNOP) for phenol degradation. Two additional genes, mphR and mphX, were found upstream and downstream of mphKLMNOP, respectively. The mphR gene encodes a XylR/DmpR-type regulator-like protein and is transcribed in the opposite direction to mphKLMNOP. The mphX gene is transcribed in the same direction as mphKLMNOP and encodes a protein with 293 amino acid residues showing weak identity with some unknown proteins encoded in the meta-cleavage pathway gene clusters for aromatic compound degradation. Disruption of mphR by homologous recombination resulted in the loss of phenol degradation while disruption of mphX caused significantly faster phenol degradation than in the wild type strain. Transcriptional assays for mphK, mphR, and mphX revealed that mphR activated mphKLMNOP transcription in the presence of phenol, but mphX partially repressed this activation. Gel mobility-shift assay demonstrated a direct interaction of MphR with the mphK promoter region. These results indicate the involvement of a novel repressor protein MphX in transcriptional regulation of phenol hydroxylase genes caused by a XylR/DmpR-type regulator MphR.

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.

This paper reports the first application of high-aspect ratio PTFE microstruscute, which fabricated by synchrotron radiation induced photo-evaporation process, to enzyme-linked immunosorvent 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 0-100ng/ml. This result suggests the successful immobilization of antibody (anti-mouse IgG goat antibody) onto the x-ray exposed surface of PTFE microstructue and successful demonstration of antigen-antibody reaction in the PTFE high-aspect ratio 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.

An Escherichia coli bioreporter harboring the phenol-inducible mphK promoter (P(mphK)) from Acinetobacter calcoaceticus PHEA-2 fused to a beta-galactosidase gene (lacZ) and the regulator gene (mopR) of A. calcoaceticus NCIB8250 was constructed to detect phenol. P(mphK) containing three inverted repeats (IR1, IR2 and IR3) upstream of mphK was activated by the regulator MopR in the presence of phenol. Deletion analysis of P(mphK) revealed that IR2 and IR3 were essential for promoter activity, while IR1 was involved in transcriptional repression. The sensitivity of the bioreporter for the detection of phenol (0.1-5 microM) was improved by about 100% through deletion of IR1 in P(mphK).

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.

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.