Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

1,8-Cineole Ameliorates Steatosis of Pten Liver Specific KO Mice via Akt Inactivation

Hepatocyte-specific Phosphatase and tensin homolog (Pten)-knockout (KO) mice exhibit hepatic lesions analogous to non-alcoholic steatohepatitis (NASH). 1,8-cineole is a monoterpene oxide and it has several biological effects including hepatoprotective effects. In this study we revealed that 1,8-cineole ameliorates NASH of Pten KO mice. Pten KO mice were assigned to a control group without any medication or to a 1,8-cineole group injected with 50 mg/kg i.p. twice per week for eight weeks. At eight weeks, livers from each group were processed to measure triglyceride (TG) content, gene expression analysis, western blot analysis, and histological examination including Oil red O staining. 1,8-cineole ameliorated hepatic steatosis in Pten KO mice, revealed by TG content and Oil red O staining. Moreover, 1,8-cineole downregulated collagen 1a1 expression and improved liver fibrosis. Thus, 1,8-cineole has potential as a candidate to treat NASH by inactivating the Akt/PI3-kinase pathway.     

Identification of an Angiotensin I-converting Enzyme Inhibitory Peptides from Protein Hydrolysates by a Soybean Protease and the Antihypertensive Effects of Hydrolysates in 4 Spontaneously Hypertensive Model Rats

ABSTRACT: Our previous study demonstrated that, because of its substrate specificity, protein hydrolysates by protease D3, which is originated from soybean, exhibited the prominent property of being less bitter than other enzymatic hydrolysates. In the 1st experiment in this series, angiotensin I-converting enzyme (ACE) inhibitory peptides from soy protein hydrolysate by D3 were identified by the establishment of a novel and effective peptide identification method. The amino acid sequences of candidate ACE inhibitory peptides were determined by electrospray ionization mass/mass spectrometry (MS/MS) analysis after rough purification of the samples with gel filtration chromatography and reverse-phase chromatography. Some of the candidate peptides had amino acid sequences that showed homology with those of the reported ACE inhibitory peptides. Then, 8 types of novel candidate peptides were synthesized according to a solid-phase method, and their ACE inhibitory activity was confirmed as the IC₅₀ value. The most potent inhibitor was NWGPLV (IC₅₀= 21 μ M ). In the 2nd experiment, the antihypertensive activity of protein hydrolysates by D3 was investigated in spontaneously hypertensive model rats (SHRs). The dose-dependent antihypertensive effect of soy protein hydrolysate was confirmed, and systolic blood pressure was significantly reduced after the oral administration of doses exceeding 100 mg/kg. Casein hydrolysate was found to have the most potent effects on suppressing blood pressure as well as ACE inhibitory activity among the various food protein hydrolysates studied because of the primary structure of casein. These results indicate that hydrolysates by D3 could be a useful food ingredient because it has the physiological function (antihypertensive activity).     

Frictional properties of diamond-like carbon coated tool in dry intermittent machining of aluminum alloy 5052

In this study, an orthogonal intermittent machining test for aluminum alloy 5052 was conducted under dry conditions. By using cutting forces that were measured during the test, the frictional properties of a tool rake face were evaluated during intermittent machining for two types of diamond-like carbon (DLC)-coated tools and an uncoated carbide tool. DLC films used in the test were composed of tetrahedral amorphous carbon (ta-C) deposited by a filtered arc deposition process and hydrogenated amorphous carbon (a-C:H) deposited by a plasma-enhanced chemical vapor deposition process. The test results showed that the initial friction coefficients were approximately 0.8 for all tools. However, with increasing machining time, the friction coefficient of only the ta-C-coated tool decreased remarkably to a lower value of 0.3, whereas those of the a-C:H-coated tool and the uncoated carbide tool remained high. An electron probe micro analyzer (EPMA) analysis revealed an area where no aluminum adhered on the ta-C-coated tool rake face after intermittent machining. This area provided low frictional properties during intermittent machining. An X-ray photoelectron spectroscopy (XPS) analysis showed that the carbon bonding of the DLC film surface in this area had changed from the state before machining.     

Pose Estimation of Construction Materials Using Multiple ID Devices for Parts and Packets Unification

Abstract: A method for characterizing the pose (position and orientation) of large objects using multiple identification (ID) devices, such as radio‐frequency identification (RFID) devices, is proposed. Simultaneous management of construction materials and their data using ID devices for construction automation has been proposed. In this management scheme, workers or robots (1) acquire the ID of a device using an ID reader, (2) obtain construction material data using the ID, and (3) use the data to perform various tasks. In this scheme, the object pose is crucial because it can be changed by robots and workers who do not possess an ID reader or have not measured the pose previously. The proposed method uses the geometric relationship between the ID device attached to the object and the ID reader, and is based on the relationship between the relative displacements of multiple ID devices. The position and direction of each ID device and ID reader are measured with respect to the object and reference coordinate frames, respectively. Experimental results proved the feasibility of our proposed method.     

Effects of dose rate change under irradiation on hardening and microstructural evolution in A533B steel

Iron-ion irradiations were carried out for 0.09wt%Cu A533B steel specimens at 290°C to investigate effects of dose rate change during irradiation; the irradiations consisted of the base irradiation (with an unchanged dose rate) and an additional one with changed dose rates from 1 to 50 times that of the base one. Nano-indentation hardness measurements showed that the increase in hardness was higher for lower dose rate of the base irradiation. A similar trend was identified during the additional irradiation. Transmission electron microscope (TEM) and three-dimensional atom probe (3DAP) analyses were carried out for the quantitative characterization of defect features. Mn/Ni/Si/Cu-enriched clusters and dislocation loops were observed in all specimens. The increase in hardness mainly depended on the formation of the solute atom clusters. The square root of the volume fraction of the solute atom clusters provided a good correlation with the increase in hardness. The effects of dose rate and dose rate change during irradiation were explained by the formation of solute atom clusters.     

Highly Efficient Multi-Step Oxidation Bioanode Using Microfluidic Channels

With the rapid decline of fossil fuels, various types of biofuel cells (BFCs) are being developed as an alternative energy source. BFCs based on multi-enzyme cascade reactions are utilized to extract more electrons from substrates. Thus, more power density is obtained from a single molucule of substrate. In the present study, a bioanode that could extract six electrons from a single molecule of L-proline via a three-enzyme cascade reaction was developed and investigated for its possible use in BFCs. These enzymes were immobilized on the electrode to ensure highly efficient electron transfer. Then, oriented immobilization of enzymes was achieved using two types of self-assembled monolayers (SAMs). In addition, a microfluidic system was incorporated to achieve efficient electron transfer. The microfluidic system, in which the electrodes were arranged in a tooth-shaped comb, allowed for substrates to be supplied continuously to the cascade, which resulted in smooth electron transfer. Finally, we developed a high-performance bioanode which resulted in the accumulation of higher current density compared to that of a gold disc electrode (205.8 μA cm⁻²: approximately 187 times higher). This presents an opportunity for using the bioanode to develop high-performance BFCs in the future.     

Polyvinyl alcohol–potassium iodide gel probe to monitor the distribution of reactive species generation around atmospheric-pressure plasma jet

This study proposes polyvinyl alcohol–potassium iodide (PVA–KI) as a novel gel chemical probe. The probe uses the reactions among PVA, KI, water, borax, and oxidative species to visualize the distribution of reactive species. This method provides information regarding the distribution of reactive species by coloration on the gel surface. The effects of the surrounding gas phase on the distribution and diffusion of the reactive species are also investigated using the PVA–KI gel probe. Further, the relationship between the irradiation distance and reactive species diffusion is determined on the surface of the PVA–KI probe with and without plastic shielding. Adjusting the irradiation distance appropriately leads to an increase in the modified area as detected by the PVA–KI gel probe analysis. The relative concentration distributions of the reactive species are also obtained from visualized color distributions measured using a colorimeter. Furthermore, reactive species generation by long-scale line plasma is confirmed by the color reaction on the PVA–KI gel surface, with a greater area being covered by an atmospheric-pressure pulsed microwave line plasma source.     

Fundamental study for assessment of surface resistivity based on waveform characteristics of partial discharge

The partial discharge (PD) waveform reflects the evolution process of electron avalanche in the discharge space. The authors expect to estimate the condition of the discharge space based on the PD waveform characteristics. The semi-conductive sheet was attached to the insulator to simulate the decrease in surface resistance due to deterioration. With the reduction of the surface resistivity, the shoulder appeared on the rising part of the PD waveform and the rise time of the PD waveform became longer. In addition, we simulated the change in the rise time using an equivalent circuit model. The displacement current was calculated from the surface potential distribution estimated by the diffusion equation, and the PD current was simulated by summing up all the current components. As a result, the shoulder was reproduced at the rising part of the PD waveform in the simulation as well. The surface resistance was estimated from the PD waveform by fitting with the experimental results.