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Stimulation of Toll-like receptor 3 (TLR3) induces autoimmune-mediated pancreatitis in susceptible mice, whereas stimulation of TLR4 causes nonautoimmune-mediated pancreatitis. However, the effects of TLR2 stimulation on the pancreas are unknown. We investigated the role of TLR2 stimulation on pancreatic damage by repeatedly stimulating mice with TLR2 ligands. Wild-type (WT) and interleukin 10-deficient (IL-10-knockout (KO)) mice were administered zymosan and lipoteichoic acid (LTA) intraperitoneally at various doses twice weekly for 4 weeks. Syngeneic T-cell-deficient mice, B-cell-deficient mice, recombination activating gene 2-deficient (RAG2–KO) mice and RAG2–KO mice that had been reconstituted with CD4+ or CD8+ T cells isolated from WT mice were treated with zymosan similarly. Mice were killed, the severity of pancreatitis was graded histologically, and serum cytokine levels were measured. Repeated administration of zymosan induced pancreatitis dose dependently in both WT and IL-10–KO mice. Administration of LTA induced pancreatitis only in IL-10–KO mice. Adoptive transfer of splenocytes obtained from IL-10–KO mice with pancreatitis did not cause pancreatitis in recipient RAG2–KO mice. Pancreatitis was scarcely observed in RAG2–KO mice and was attenuated in T-cell-deficient and B-cell-deficient mice compared with WT mice. A single administration of zymosan significantly increased the serum level of monocyte chemoattractant protein 1 (MCP-1) in WT mice. Repeated stimulation of TLR2 and dectin-1 induced nonautoimmune-mediated pancreatitis in mice. Participation of acquired immunity seems to play an important role in the pathogenesis of pancreatitis in association with the increase in serum MCP-1 level.

The human gastrointestinal tract, which constitutes the digestive system, contains a large number of virus particles that maintain organizational homeostasis and health. Conversely, viral pathogens have also attracted attention for their involvement in the pathogenesis of certain cancers, including gastrointestinal cancers. To aid prevention and treatment of these cancers, the relevance of gastrointestinal viral factors as potential risk factors needs to be carefully investigated. This review summarizes and discusses the available literature on the relationship between the development of esophageal, gastric, and colorectal cancers and their corresponding viruses. This review reveals that research on the association between colorectal cancer and viruses, in particular, is still in its infancy compared to the association between HPV and esophageal cancer and between EBV and gastric cancer.

Background Endoplasmic reticulum (ER) stress in the pancreas is closely associated with the development of acute pancreatitis. However, the role of the protein kinase RNA-like ER kinase (PERK) in this disease is not fully understood. We investigated whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, could improve murine experimental pancreatitis through the amelioration of ER stress. Methods Acute pancreatitis was induced by the intraperitoneal administration of cerulein (50 μg/kg) six times at 1-h intervals followed by lipopolysaccharide (10 mg/kg). Salubrinal was administered intraperitoneally immediately after lipopolysaccharide injection and 3 h later. Mice were sacrificed 24 h after the first injection of cerulein, and serum amylase and proinflammatory cytokines were measured. The severity of pancreatitis was evaluated histologically using a scoring system. The expression levels of ER stress-related proteins were evaluated by Western blotting. Results The administration of salubrinal significantly attenuated the increase in serum amylase levels and improved histologically assessed pancreatitis. The serum levels of proinflammatory cytokines were significantly suppressed in salubrinal-treated mice, as was the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein, and cleaved caspase-3. Conclusions The amelioration of ER stress through augmentation of the PERK-signaling pathway may be a therapeutic target for the treatment of acute pancreatitis.

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.

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&prime, dihydroxybenzophenone, 4,4&prime, dihydroxydiphenylether, and 4,4&prime, thiodiphenol, indicating that this monooxygenase has a broad substrate specificity for compounds with a bisphenolic structure.

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.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.

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.

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.

A 69-year-woman was admitted to the clinic in August 2018 because of general fatigue and low appetite.She had occult blood-positive and was referred to our hospital for further investigations.There was LST in the rectum for which colonoscopy and ESD were performed.She had abdominal pain and slight fever on postoperative day 1.Abdominal CT showed an intussusception in the ileum.We could not achieve endoscopic de-torsion and carried out laparotomy.The intussusception was found to be strangulated due to inflammatory polyp and mesenteric adhesion.The affected portion was resected.Although treatment for low hypoalbuminemia and neurogenic cystitis was required, she was discharged on postoperative day 28.

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.

Rationale Brunner gland hamartoma (BGH) is a rare tumor of the duodenum. Although BGH is a benign tumor, larger lesion with gastrointestinal symptoms requires tumor removal. We report a giant BGH, successfully treated by endoscopic excision followed by transanal retrieval. Patient concerns A 38-year-old woman complained of severe anemia, tarry stool, and vomiting. Diagnoses Esophagogastroduodenoscopy (EGD) showed a pedunculated giant submucosal mass at the duodenal bulb. Interventions We attempted to remove it because the lesion seemed to be responsible for patient's anemia and vomiting. The lesion had clear but bulky stalk. We carefully cut the stalk using needle-knife and IT knife2. We tried to retrieve specimen, but the mass could not pass through the pyloric ring because of its size. Then we tried to obtain the specimen from anus. Polyethylene glycol solution was administered to accelerate rapid excretion. Outcomes The mass was successfully removed and was histologically confirmed as a giant BGH, measuring 55 mm in size. Lessons Reports about endoscopic resection of giant BGH are rare. Moreover, our case is the first report of transanal retrieval of resected specimen using polyethylene glycol solution. Endoscopic resection of BGH is less-invasive but can be more challenging if the mass is large. Our case provides useful option for endoscopic treatment of giant BGH.

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.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.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [

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

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.

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.

Acinetobacter sp. strain YAA has five genes ( atdA1 to atdA5 ) involved in aniline oxidation as a part of the aniline degradation gene cluster. From 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) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). 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, we purified it from recombinant Escherichia coli harboring atdA1 . The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l -glutamate in the presence of ATP and MgCl 2 . This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes ( atdA3 to atdA5 ) 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 to atdA5 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 that is harmful to the host cell.

The bioluminescence color of firefly luciferase including its mutants ranges from green to red (530–640 nm) and is affected by the species of firefly, the reaction conditions, and by the substitution of amino acids. Although there is a general agreement that the microenvironment mechanism is the dominant model for the color determination of firefly luciferase, a complete mechanism has not been shown, partially due to the lack of comprehensive data on which amino acid positions alter the bioluminescent color. In this paper, a mutant library of position serine 286 (S286) in Luciola cruciata luciferase (LUC-G) was constructed and characterized. The substitution of S286 resulted in a drastic red shift in bioluminescence color (>600 nm), and only glycine (G) and alanine (A) mutants remained yellow-green. To explain this color difference, molecular dynamics (MD) calculations of 3 S286 derivatives (S286G, S286N, and S286I), in addition to wild type (WT), were performed. The active site rigidity and active site hydrogen bonding networks were compared to WT for each derivative. The results suggested that both factors affected the active site environment and affected the difference in bioluminescence colors in LUC-G S286 derivatives.

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 × 106 Da.

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 8 cm 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.

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.

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.

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.

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 k cat parameter.

NylB′ carboxylesterase, which is 88% homologous to functional 6-aminohexanoate-dimer hydrolase (NylB) from Arthrobacter sp., possesses trace synthetic activity [0.0004 μmol 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 104-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 Ald-hydrolytic activity (

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 (O 3 mm). The fluid filters, constructed by deep X-ray lithography, have 2100 pores (O 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