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1.
Aem Nuylert Dr. Fumihiro Motojima Prof. Dr. Chartchai Khanongnuch Prof. Dr. Tipparat Hongpattarakere Prof. Dr. Yasuhisa Asano 《Chembiochem : a European journal of chemical biology》2020,21(1-2):181-189
Because the synthesis of chiral compounds generally requires a broad range of substrate specificity and stable enzymes, screening for better enzymes and/or improvement of enzyme properties through molecular approaches is necessary for sustainable industrial development. Herein, the discovery of unique hydroxynitrile lyases (HNLs) from two species of passion fruits, Passiflora edulis forma flavicarpa (yellow passion fruit, PeHNL-Ny) and Passiflora edulis Sims (purple passion fruit, PeHNL-Np), isolated and purified from passion fruit leaves is reported. These are the smallest HNLs (comprising 121 amino acids). Amino acid sequences of both enzymes are 99 % identical; there is a difference of one amino acid in a consensus sequence. PeHNL-Np has an Ala residue at position 107 and is nonglycosylated at Asn105. Because it was confirmed that natural and glycosylated PeHNL-Ny showed superior thermostability, pH stability, and organic tolerance to that of PeHNL-Np, it has been speculated that protein engineering around the only glycosylation site, Asn105, located at the C-terminal region of PeHNL-Ny, might contribute to the stabilization of PeHNL. Therefore, the focus is on improved stability of the nonglycosylated PeHNL by truncating its C-terminal region. The C-terminal-truncated PeHNLΔ107 was obtained by truncating 15 amino acids from the C terminus followed by expression in Escherichia coli. PeHNLΔ107 expressed in E. coli was not glycosylated, and showed improved thermostability, solvent stability, and reusability similar to that of the wild-type glycosylated form of PeHNL expressed in Pichia pastoris. These data reveal that the lack of the high-flexibility region at the C terminus of PeHNL might be a possible reason for improving the stability of PeHNL. 相似文献
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Yutaro Oda Dr. Junya Chiba Fumihiro Kurosaki Yusuke Yamade Prof. Dr. Masahiko Inouye 《Chembiochem : a European journal of chemical biology》2019,20(15):1945-1952
We report enzymatic phosphorylation and additive-free ligation of DNAs containing unnatural C-nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D * , T * , G * , and C * , corresponding—from a viewpoint of hydrogen-bonding patterns—to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5′-end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5′-end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3′-natural ⋅ 5′-artificial, 3′-artificial ⋅ 5′-natural, and 3′-artificial ⋅ 5′-artificial terminal combination at the nicks. 相似文献
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Effects of Atmospheric Composition on the Molecular Structure of Synthesized Silicon Oxycarbides 下载免费PDF全文
Masaki Narisawa Fuji Funabiki Akihiro Iwase Fumihiro Wakai Hideo Hosono 《Journal of the American Ceramic Society》2015,98(10):3373-3380
The dependence of silicon oxycarbides' chemical composition and molecular structure on their reaction conditions was tested by varying the atmosphere under which pyrolysis was performed. To obtain the silicon oxycarbides, densely cross‐linked silicone resin particles with an averaged diameter of 2 μm were pyrolyzed in various atmospheres of H2, Ar, and CO2, in the temperature range 700°C–1100°C. The residual mass of resin after pyrolysis was almost constant at 700°C, although their apparent colors varied distinctly. The sample obtained from the H2 atmosphere was white, whereas that obtained from the CO2 atmosphere was dark brown. Fourier‐transform infrared (FT‐IR) spectra of the residues suggested that the Si–O–Si network evolution was accelerated in the CO2 atmosphere. Beyond 800°C, the chemical compositions of the compounds obtained from a H2 atmosphere increasingly approached near‐stoichiometric SiO2–xSiC composition with increasing the pyrolysis temperature. Compounds from a CO2 atmosphere approached a composition of SiO2–xC with no free SiC as the pyrolysis temperature increased. In the products from an Ar atmosphere, SiO2–xSiC–yC compositions were typically obtained. The observed effects of the pyrolysis atmosphere on the resulting chemical compositions were analyzed in terms of thermodynamic calculations. Electron spin resonance (ESR) spectra revealed broad and intense signals from products obtained from either Ar or CO2. Estimating from the signal intensity, the residual spin concentrations were in the range 1018–1019 g?1. Meanwhile, the spectra from the samples obtained in H2 showed weak and sharp signals with estimated spin concentrations ranging from 1016–1017 g?1. This signal attenuation may have been due to the hydrogen capping of dangling bond formed during pyrolysis. 相似文献
5.
A Multiple‐Labeling Strategy for Nonribosomal Peptide Synthetases Using Active‐Site‐Directed Proteomic Probes for Adenylation Domains 下载免费PDF全文
Dr. Fumihiro Ishikawa Dr. Takehiro Suzuki Dr. Naoshi Dohmae Prof. Hideaki Kakeya 《Chembiochem : a European journal of chemical biology》2015,16(18):2590-2594
Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple‐labeling technique with active‐site‐directed probes for adenylation domains. When applied to gramicidin S‐producing and ‐nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high‐resolution identification of low‐abundance active NRPS enzymes in native proteomic environments. 相似文献
6.
Raayaa Wananuruksawong Yutaka Shinoda Takashi Akatsu Fumihiro Wakai 《International Journal of Applied Ceramic Technology》2013,10(5):756-763
Creep behavior of Si3N4 polycrystals containing Y2O3‐MgO‐SiO2 glass phase (with and without Calcium oxide [CaO] additive) was studied by compression tests between 1500 and 1700°C. We studied the effect of CaO additive on flow stress, microstructural evolutions, and thermal stability of the intergranular glass phase during deformation. While the addition of CaO did not affect grain size, the flow stress decreased with the amount of CaO. This result suggested that the addition of CaO reduced the viscosity of intergranular glass phase. The addition of CaO further improved the thermal stability of the glass phase by suppressing the evaporation at elevated temperatures. 相似文献
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Yoshifumi Atsushi Wataru Fumihiro Junji Hiroaki 《Sensors and actuators. B, Chemical》2009,140(2):649-655
A novel method for exchanging solutions used in biochemical analyses and a device to carry out the exchange are proposed. An array of plugs formed using six injectors was transported in a microflow channel using a main pump located at one end of the main flow channel. The injectors and main pump were operated on the basis of the change in volume caused by the electrolysis of water. Bubbles were produced from working electrodes; these bubbles caused a diaphragm placed below the injectors to inflate and occlude the inlet of the solution reservoir. Increase in the number of bubbles caused the reservoir to inject the solution into the main flow channel in the form of a plug. Each plug was individually transported downstream to the sensing area by the main pump, which was operated in a similar manner to the injector. The device was used for the detection of a tumor marker, α-fetoprotein (AFP). Plugs of necessary solutions were individually transported to the sensing area with immobilized primary antibodies to allow antigen–antibody binding, cleaning, and detection. The fluorescence intensity from the antibodies showed clear dependence on the concentration of AFP. The immobilization of antibodies could also be carried out on-chip. 相似文献
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Mechanical properties of silicon nitride which was superplastically deformed in plane-strain compression were investigated. Superplastically deformed silicon nitride exhibited a highly anisotropic microstructure, where rod-shaped grains tended to be aligned along the extruding direction. The bending strength and fracture toughness were increased substantially by the deformation process when a stress was applied in the extruding direction. It appears that these improvements were mainly due to effective operation of grain bridging and pull-out by the grain alignment. 相似文献