Single Amino Acid Mutation of Pyranose 2-Oxidase Results in Increased Specificity for Diabetes Biomarker 1,5-Anhydro-D-Glucitol

Pyranose 2-oxidases catalyze the oxidation of various pyranose sugars at the C2 position. However, their potential application for detecting sugars other than glucose in blood is hindered by relatively high activity towards glucose. In this study, in order to find a mutant enzyme with enhanced specificity for 1,5-anhydro-D-glucitol (1,5-AG), which is a biomarker for diabetes mellitus, we conducted site-directed mutagenesis of pyranose 2-oxidase from the basidiomycete Phanerochaete chrysosporium ( Pc POX). Considering the three-dimensional structure of the substrate-binding site of Pc POX and the structural difference between glucose and 1,5-AG, we selected alanine 551 of Pc POX as a target residue for mutation. Kinetic studies of the 19 mutants of Pc POX expressed as recombinant proteins in E. coli revealed that the ratio of k _cat / K _m for 1,5-AG to k _cat / K _m for glucose was three times higher for the A551L mutant than for wild-type Pc POX. Although the A551L mutant has lower specific activity towards each substrate than the wild-type enzyme, its increased specificity for 1,5-AG makes it a promising lead for the development of POX-based 1,5-AG detection systems.     

Effect of C-6 Methylol Groups on Substrate Recognition of Glucose/Xylose Mixed Oligosaccharides by Cellobiose Dehydrogenase from the Basidiomycete Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar K _m and k _cat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher K _m value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.     

Novel rapid‐cure adhesives for low temperature using thiirane compound

We investigated improvement of workability (viscosity), storage stability, and curing ability of thiirane resin for adhesive applications. The viscosity of bisphenol‐F thiirane resin was lower than that of bisphenol‐A thiirane resin, especially at low temperatures, thus improving ease of handling. Addition of diphenyl decyl phosphite improved its storage stability to a level similar to that of bisphenol‐A epoxy resin. The curing of bisphenol‐F thiirane resin increased three times faster by adding 2,4,6‐tris(dimethylaminomethyl)phenol (DMP‐30) as a tertiary amine. In applications of this new thiirane resin as civil and architectural adhesives, a superior curing ability at low temperature was attained. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 2953–2957, 2001     

Development of a Method for Preparing a Highly Reactive and Stable,Recyclable and Environmentally Benign Organopalladium Catalyst Supported on Sulfur‐Terminated Gallium Arsenide(001): A Three‐Component Catalyst, {Pd}‐S‐GaAs(001), and its Properties

We have developed a method for preparing a recyclable and environmentally benign organopalladium catalyst for the Heck reaction supported on sulfur‐terminated gallium arsenide(001). This three‐component catalyst, {Pd}‐S‐GaAs(001), exhibited high stability and activity, furthermore, it tolerated reuse in 10 runs of the Heck reaction (average yield, 97 %) under aerobic conditions. The sulfur layer was very important to stabilize this catalyst. Only trace amounts of Pd were leached from this catalyst to the reaction mixture, as measured by ICP‐mass. The valence of immobilized Pd was zero by XPS spectrometry.     

Improved growth response of antibody/receptor chimera attained by the engineering of transmembrane domain

Structure-based design of antibody/cytokine receptor chimeras has permitted a growth signal transduction in response to non-natural ligands such as fluorescein-conjugated BSA as mimicry of cytokine-cytokine receptor systems. However, while tight on/off regulation is observed in the natural cytokine receptor systems, many chimeras constructed to date showed residual growth-promoting activity in the absence of ligands. Here we tried to reduce the basal growth signal intensity from a chimera by engineering the transmembrane domain (TM) that is thought to be involved in the interchain interaction of natural cytokine receptors. When the retroviral vectors encoding the chimeras with either the wild-type erythropoietin receptor (EpoR) TM or the one bearing two mutations in the leucine zipper motif were transduced to non-strictly interleukin-6-dependent 7TD1 cells, a tight antigen-dependent on/off regulation was attained, also demonstrating the first antigen-mediated genetically modified cell amplification of non-strictly factor-dependent cells. The results clearly indicate that the TM mutation is an effective means to improve the growth response of the antibody/receptor chimera.     

High-Temperature Fatigue Strength of Crack-Healed Al2O3 Toughened by SiC Whiskers

Al₂O₃ reinforced by SiC whiskers (Al₂O₃/SiC-W) was hot-pressed to investigate the fatigue strength of crack-healed specimens at high temperature. Semielliptical surface cracks of 100 μm surface length were introduced on each specimen surface. These specimens were crack-healed at 1300°C for 1 h in air, and static and cyclic fatigue strengths were systematically investigated at room temperature, 900° and 1100°C by three-point bending. The static and cyclic fatigue limits of the crack-healed specimens were more than 70% of the average bending strength at each testing temperature. Crack-healed specimens of Al₂O₃/SiC-W were not sensitive to static and cyclic fatigue at room temperature and high temperatures. Therefore, the combination of crack-healing and whisker reinforcement can play an important role in increasing static and cyclic fatigue strengths at high temperature.     

Dependence of Output Power on Slot Antenna Width in Terahertz Oscillating Resonant Tunneling Diodes

We report on the dependence of the output power on the slot antenna width in resonant tunneling diode (RTD) oscillators. The oscillators have different antenna widths of 4, 8, and 20 μm and the same antenna length of 130 μm. Although the experimentally observed oscillation frequencies were ~500 GHz and independent of the antenna width, maximized output powers of ~130 μW were obtained at the antenna width of 8 μm by the impedance matching between the RTD and the antenna.     

High Pressuee Synthesis of Alkali Metal Doped C60 Polymers

Abstract

Alkali metal doped fullerenes, AC₆₀ (A = Li, Na) were polymerized under a pressure of 5 GPa at 573 K. the X-ray diffraction patterns of the products were very similar to that of the rhombohedral two-dimensional C₆₀ polymer, the basal spacings being slightly increased by the intercalation of the alkali atoms between the two-dimensional polymer layers. the alkali metal doped C60 polymers were insoluble in toluene and found to be semiconductors over a temperature range of 200 - 300