Nano‐Encapsulation of Lipase by Self‐Assembled Nanogels: Induction of High Enzyme Activity and Thermal Stabilization

The effects of self‐assembled polysaccharide nanogels on colloidal and thermal stability of lipase from Pseudomonas cepacia were investigated. The enzyme activity, especially k_cat, drastically increased in the presence of nanogels of cholesterol‐bearing pullulan (CHP). The thermostability of lipase complex increased because the denaturation temperature of lipase increased by more than 20 °C by complexation with CHP nanogels. Lipase denaturation and aggregation upon heating was effectively prevented by complexation with CHP nanogels. Moreover, complexation with CHP nanogels protected lipase from lyophilization‐induced aggregation. Nano‐encapsulation with CHP nanogel is a useful method for colloidal and thermal stabilization of unstable enzyme.

     

Ab initio MO analysis of interaction paths between radicals in ferromagnetic organic systems

Interaction path analyses for pi-conjugated organic systems were performed at the ab initio molecular orbital level to examine the relationship between inter-radical interactions and the high-spin stability of the system. It was found that the high-spin stability results from through-bond interactions between radicals, not from through-space interactions, in relation to the stabilization of a low-spin state due to the effects of electron correlation. L(ij)(min) value for estimating the mixing of nonbonding molecular orbitals well predicted the relationship between the through-bond interactions and the high-spin stability. Furthermore, molecular orbital calculations revealed that the all-trans type interaction path between radicals produces long-range exchange interactions, and the additivity of high-spin stability is observed by keeping short-range through-bond interaction paths.     

Synthesis of crystallized mesoporous transition metal oxides by silicone treatment of the oxide precursor

Ordered mesoporous transition metal oxides were successfully crystallized after strengthening the amorphous framework by a silica layer, which efficiently protected the original mesoporous structure against crystallization and resulting mass transfer.     

Measurement of a genotoxic hydrazine, agaritine, and its derivatives by HPLC with fluorescence derivatization in the agaricus mushroom and its products

Agaricus blazei MURRILL mushroom products are sold as so-called health foods in Japan. However, a part of Agaricus is known to contain hydrazines. A sensitive and specific method for analyzing a genotoxic hydrazine, agaritine, and its derivatives was developed to assess the safety of Agaricus products. beta-N-(gamma-L(+)-Glutamyl)-4-(hydroxymethyl)phenylhydrazine (agaritine, AGT), 4-hydrazinylbenzylalcohol (HMPH), 4-hydrazinylbenzoic acid (CPH), 4-methylphenylhydrazine (MPH) and phenylhydrazine (PH) were converted to their correspond-ing fluorescent derivatives with 3,4-dihydro-6,7 dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride (DMEQ-COCl) as the fluorescence derivatization reagent. The detection limits (S/N=3) for CPH, AGT, PH and MPH were 422, 45.3, 16.5 and 138 fmol, respectively, in a 20 mul injection volume. Recoveries, achieved by adding known AGT amounts to the Agaricus sample and Agaricus products, ranged from 92.8 to 102%. By using this method which does not require partial purification of the Agaricus sample, the amounts of AGT in several types of foods were found to be 112-1836 mug/g dry weight.     

Association kinetics of wild- and mutant-type Ynd1p in relation to quality of grown crystals

The intermolecular interaction and association dynamics of the Ynd1p protein were investigated using dynamic and time-resolved static light scattering measurements. The mutual diffusion coefficients of wild- and mutant-type (a single amino acid substitution) Ynd1p monomer were measured in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer with 5 mM MnCl2 and 7.5% (v/v) ethylene glycol. Both translational diffusion coefficients at a zero protein concentration were (40.3 +/- 0.2) x 10(-12) m2/s at 20 degrees C and a pH of 7.0, so the hydrodynamic radius of the monomers was 4.1 +/- 0.1 nm. The measured intermolecular interaction between monomers, however, showed that the mutant-type Ynd1p had a stronger attractive force. Time-resolved static light scattering measurements showed that the association of mutant-type Ynd1p yielded a larger number of aggregates than that of wild-type Ynd1p. The time dependence of aggregate gyration radius differed between the two types. Fractal dimension analysis using scattering intensity data suggested that the inner structure of the aggregates changed from loose to rigid with time. Although this phenomenon is common for wild and mutant types, the differences in the number of aggregates yielded in the initial stages and in the intermolecular interaction affected the quality of the final grown crystals. That is, single crystals of Ynd1p grew in the mutant-type protein solution and polycrystals of Ynd1p grew in the wild-type protein solution.     

Adsorption of glucose oxidase onto plasma-polymerized film characterized by atomic force microscopy, quartz crystal microbalance, and electrochemical measurement

Adsorption of glucose oxidase (GOD) onto plasma-polymerized thin films (PPF) with nanoscale thickness was characterized by atomic force microscopy (AFM), quartz crystal microbalance (QCM), and electrochemical measurements. The PPF surface is very flat (less than 1-nm roughness), and its properties (charge and wettability) can be easily changed while retaining the backbone structure. We focused on three types of surfaces: (1) the pristine surface of hexamethyldisiloxane (HMDS) PPF (hydrophobic and neutral surface), (2) an HMDS PPF surface with nitrogen-plasma treatment (hydrophilic and positive-charged surface), and (3) an HMDS PPF surface treated with oxygen plasma (hydrophilic and negative-charged surface). The AFM image showed that the GOD molecules were densely adsorbed onto surface 2 and that individual GOD molecules could be observed. The longer axis of GOD ellipsoid molecules were aligned parallel to the surface, called the "lying position", because of electrostatic association. On surface 1, clusters of GOD molecules did not completely cover the original PPF surface (surface coverage was ca. 60%). The 10-nm-size step height between the GOD clusters and the PPF surface suggests that the longer axes of individual GOD molecules were aligned perpendicular to the surface, called the "standing position". On surface 3, only a few of the GOD molecules were adsorbed because of electrostatic repulsion. These results indicate that the plasma polymerization process can facilitate enhancement or reduction of protein adsorption. The AFM images show a corresponding tendency with the QCM profiles. The QCM data indicate that the adsorption behavior obeys the Langmuir isotherm equation. The amperometric biosensor characteristics of the GOD-adsorbed PPF on a platinum electrode showed an increment in the current because of enzymatic reaction with glucose addition, indicating that enzyme activity was mostly retained in spite of irreversible adsorption.     

Collision induced fragmentation: A simple numerical algorithm

We introduce a simple numerical algorithm to describe collision-induced fragmentation. We consider fragmentation induced by collisions between pairs of particles and perform numerical studies for several classes of interaction kernels, and for several types of breaking processes. We validate the algorithm by comparing the results of the kinetic evolution and particle size statistics with previous analytical results for symmetric and asymmetric kernels. The excellent agreement obtained proves the algorithm versatility. The method also allows us to examine generic kernels, for which no exact solutions are known.     

NMR-based reappraisal of the coordination of a metal ion at the pro-Rp oxygen of the A9/G10.1 site in a hammerhead ribozyme

In the identification of a metal-binding site within enzymes, kinetic analyses based on thio-effects and Cd(2+)-rescues are widely used. In those analyses, kinetic studies using a phosphorothioate have been discussed on the premise that the substitution by a sulfur atom does not change the conformation of a ribozyme. However, our present NMR structural analysis demonstrates the change of the conformation at the metal-binding site by Rp-sulfur but not by Sp-sulfur substitution and warns against incautious interpretations of thio-effects and rescue phenomena in kinetic studies using a phosphorothioate. Our analysis further demonstrates that, in solution, a Cd(2+) ion can interact with an Rp-phosphorothioate (in support of the controversial McKay's structure, Nature 1994, 372, 68-74) and with an Sp-phosphorothioate (in support of the controversial Scott's structure, Cell 1995, 81, 991-1002) at the metal-binding A9/G10.1 site and that, in the former case, the bound Cd(2+) ion can return the ribozyme to an active conformation and rescue its enzymatic activity.