Design of a Full-Consensus Glutamate Decarboxylase and Its Application to GABA Biosynthesis

Glutamate decarboxylase (GAD) catalyses the decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Improvement of the enzymatic properties of GAD is important for the low-cost synthesis of GABA. In this study, utilizing sequences of enzymes homologous with GAD from lactic acid bacteria, highly mutated GADs were designed using sequence-based protein design methods. Two mutated GADs, FcGAD and AncGAD, generated by full-consensus design and ancestral sequence reconstruction, had more desirable properties than native GADs. With respect to thermal stability, the half-life of the designed GADs was about 10 °C higher than that of native GAD. The productivity of FcGAD was considerably higher than those of known GADs; more than 250 mg/L of purified enzyme could be produced in the E. coli expression system. In a production test using 26.4 g of l -glutamate and 3.0 g of resting cells, 17.2 g of GABA could be prepared within one hour, without purification, in a one-pot synthesis.     

Radiation grafting of α,β,β-trifluorostyrene onto poly(ethylene-tetrafluoroethylene) film by preirradiation method. II. Properties of cation-exchange membrane obtained by sulfonation and hydrolysis of the grafted film

A Study was made on certain properties of the cation-exchange membranes obtained by the preirradiation grafting of α,β,β-trifluorostyrene (TFS) noto poly(ethylene-tetrafluoroethylene) (ETFE), followed by sulfonation and hydrolysis of the grafted film. Swelling, water uptake, electric conductivity, and transport number of the membranes were measured as a function of ino-exchange capacity. Thermal and chemical stability were also investigated. These properties were found to be mainly dependent on ion-exchange capacity. The stable membrane properties were established due to a homogeneous ion-exchange group distribution in the membrane, as confirmed by x-ray imcroscopy analysis of the membrane cross sections. In addition, the membranes showed good electrochemical, thermal, and chemical properties, and were found to be scceptable for practical use as cation-exchange membranes.     

Formation of Exclusive Al2O3 Scale on Nb and Nb-Rich Alloys by Two-Step Oxygen–Aluminum Diffusion Process

External Al₂O₃ scale formation behavior by a diffusion aluminizing process on Nb and Nb–X (X?=?Mo, Re, and Ta) alloys with different oxygen solubilities was investigated. The oxygen content in Nb and Nb–X alloys was controlled by oxygen diffusion treatment at 1,100?°C using Nb/NbO mixture. Nb–aluminide, NbAl₃ and Nb₂Al, layers were developed on the low-oxygen Nb substrate by an aluminum diffusion treatment using Al/Al₂O₃/NH₄Cl mixture at 1,100?°C; whereas an adhesive Al₂O₃ scale developed on the Nb substrate with higher oxygen content. The alloy oxygen content decreased by an addition of Ta, Mo and Re, in this order, and the decrease in alloy oxygen content resulted in the transition of Al₂O₃ formation from external scale to internal precipitates. This transition was not observed when low Al activity powder mixture, Ni50Al/Al₂O₃/NH₄Cl, was used for Al diffusion process, and only external Al₂O₃ scale was developed on all samples. In the present study, the effects of oxygen solubility limit, oxygen content, and the activity of Al in diffusion process on the formation of Al₂O₃ scale will be discussed.     

Multi-walled carbon nanotube-reinforced copper nanocomposite coating fabricated by low-pressure cold spray process

Multi-walled carbon nanotube (MWCNT)-reinforced copper (Cu) nanocomposite coatings were successfully deposited on aluminum (Al) substrate by a cold spraying process at a low pressure. The microstructure and the Raman spectrum of the low-pressure-cold-sprayed MWCNT–Cu nanocomposite coating showed that the MWCNTs maintained their tube structure in the Cu matrix, even though structural damage to the MWCNTs increased slightly. MWCNT–Cu nanocomposite-coated Al exhibits higher thermal diffusivity than pure-Cu-coated Al with a comparable hardness. The higher thermal diffusivity of the MWCNT–Cu coating could be explained by the dispersion of MWCNTs within the clean and closed CNT/Cu interfaces, which were achieved with the aid of compressive stress during the cold spraying.     

Microstructure and stress corrosion cracking of the fusion boundary region in an alloy 182-A533B low alloy steel dissimilar weld joint

Stress corrosion cracking (SCC) in the fusion boundary (FB) region of an Alloy 182-A533B low alloy steel (LAS) dissimilar weld joint in high temperature water doped with sulfate was studied following a microstructure characterization of the FB region. The microstructure characterization suggested the type-II and type-I boundaries in the dilution zone (DZ) adjacent to the FB had lower resistance to SCC growth than the FB. Crack propagating perpendicular to the FB in the DZ was observed to be blunted by pitting at the FB, followed by the reactivation from the pitting by localized oxidation along the grain boundary in LAS.     

Magnetic field-assisted finishing for micropore X-ray focusing mirrors fabricated by deep reactive ion etching

A magnetic field-assisted finishing process has been studied for high-aspect-ratio ion-etched silicon curvilinear micropore structures, which have potential application as mirrors for satellite-borne X-ray telescopes. The micropore sidewalls act as X-ray focusing mirrors, and lead to reductions in the mass-to-effective-area ratio of 10-1000 times, compared to traditional X-ray telescopes. This paper describes the processing principle for the surface finishing of the sidewalls of micropore structures (10, 20 μm and depth: 300 μm (aspect ratio ≈ 15, 30)), and the feasibility of achieving roughness ∼4 nm rms and improving the X-ray reflectivity of micropore sidewall surface are demonstrated.     

Photoreaction of Titanium-Based Metal-organic Compounds for Ceramic Fine Patterning

The photoreaction of metal-organic compounds prepared by the chemical modification of metal alkoxides was investigated for the patterning of ceramic films. The chemical stability and photoreactivity of these compounds were found to be greatly influenced by the kind of chemical additive applied during the syntheses of the precursor solutions. In this study, the photoreaction of the precursor thin films prepared by the addition of alkanolamines was achieved by tuning the wavelength of the incident UV light. The decomposition of the organic moieties of the percursors was clearly observed in IR spectra during UV irradiation. Furthermore, the spectrum of the titania precursor film in the visible and ultraviolet regions (UV-vis) changed in accordance with the irradiation time. The photolithography of the titanium precursor pattern was successfully achieved by means of these techniques.     

Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the P1-site residue(Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lysresults in the acquisition of inhibitory activity toward chymotrypsinor trypsin, respectively. However, the inhibitory activitiesthus induced are not strong. In the present study, we introducedadditional amino acid replacements around the reactive siteto try to make the P1-site mutants more effective inhibitorsof chymotrypsin or trypsin. The amino acid replacement AspTyrat the P2' site of OMCHI3(P1Met) resulted in conversion to a35000-fold more effective inhibitor of chymotrypsin with aninhibitor constant (K_i) of 1.17x10^–11 M. The K_i valueof OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactionwith chymotrypsin of removing a negative charge from the P2'site was greater than that of introducing an aromatic ring.Similarly, enhanced inhibition of trypsin was observed whenthe AspTyr replacement was introduced into the P2' site of OMCHI3(P1Lys).Two additional replacements, AspAla at the P4 site and ArgAlaat the P3' site, made the mutant a more effective inhibitorof trypsin with a K_i value of 1.44x10^–9 M. By contrast,ArgAla replacement at the P3' site of OMCHI3(P1Met, P2'Tyr)resulted in a greatly reduced inhibition of chymotrypsin, andAspAla replacement at the P4 site produced only a small changewhen compared with a natural variant of OMCHI3. These resultsclearly indicate that not only the P1-site residue but alsothe characteristics, particularly the electrostatic properties,of the amino acid residues around the reactive site of the proteaseinhibitor determine the strength of its interactions with proteases.Furthermore, amino acids with different characteristics arerequired around the reactive site for strong inhibition of chymotrypsinand trypsin.     

Impaired final follicular maturation in heifers after superovulation with recombinant human FSH

The aim of this study was to investigate whether human FSH without contaminating LH can exert a normal superovulation response in cows. One group of heifers (n = 9) was stimulated with recombinant human FSH (rhFSH), an FSH source without any LH activity, and another group (n = 9) was treated with equine chorionic gonadotrophin (eCG), an FSH source with high LH activity. Daily transrectal ultrasonography showed that eCG- and rhFSH-stimulated heifers (n = 9 per group) had the same follicular growth characteristics and equal numbers of follicles > 8 mm in diameter after 3 days of stimulation. The treatment groups differed considerably in steroid production: rhFSH-treated heifers produced much lower oestradiol concentrations than did eCG-stimulated heifers during the first days of stimulation and much lower progesterone concentrations in the period after the LH surge. During the 27-35 h after prostaglandin injection, rhFSH-treated heifers had fewer LH pulses than did eCG-treated heifers (0.3 versus 3.0 per heifer, respectively; n = 3 per group). All rhFSH-treated heifers (n = 6) underwent a preovulatory LH surge, but this occurred significantly later than in the eCG-treated heifers (n = 4; 39.4 +/- 1.9 h versus 47.1 +/- 1.5 h in rhFSH- and eCG-treated heifers, respectively). Multiple ovulations occurred in only three of six rhFSH-treated heifers, but in all four eCG-treated heifers with an LH surge. At 24 h after the LH surge, the percentage of metaphase II stage oocytes with cortical granules distributed close to the oolemma was significantly lower in the rhFSH group (7.3%) than in the eCG group (55.9%). In conclusion, final follicular maturation is impaired in heifers treated with rhFSH, which might be due to the combination of a lack of LH activity in the gonadotrophin preparation and the severe suppression of LH pulsatility.