Masahiro Takeo

ABSTRACT We report the complete genome assembly of a hydroquinonesulfonate-assimilating bacterium, Delftia lacustris strain HQS1. This strain contains one circular chromosome (6,979,964 bp) and one circular plasmid (39,999 bp). The chromosomal sequence contained 6,359 coding sequences and a gene cluster involved in the degradation of gentisate, which is structurally similar to hydroquinonesulfonate.

We have completed a system that can achieve both deep x-ray lithography and submicron x-ray lithography with a single beamline by introducing the combination of x-ray plane and cylindrical mirrors. This x-ray lithography system can provide a large-scale microfabrication processing with 210 × 300 mm2 (A4 size). To exploit multiscale lithography, the beamline has a beam transport vacuum duct with a two-stage stacked structure and a 5-axis stage. This two-stage stacked structure allows us to fabricate both micron scale structures with high aspect ratios and submicron scale structures using the same beamline. In addition, x-ray imaging and computer tomography (CT) system are connected to the x-ray lithography system for nondestructive inspection and evaluation of the fabricated microstructures. The x-ray imaging system constructed this study has a relatively low energy range of x-ray energy in the beamline, which is in the range of 2–15 keV or less. Therefore, relatively good absorption contrast can be obtained for plastic materials, biomaterials, and the like. Since nondestructive imaging of the processed shape by x-ray lithography is possible, it is a very useful system in processing and evaluation can be performed simultaneously. This system also enables us to obtain the live images with keeping the creature alive in liquid using an indirect x-ray imaging system which converts x-ray images to visible light images through the fluorescent plate.

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.

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 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.

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.

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.

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.

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.