Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.     

Lipid components,fatty acids and triacylglycerol molecular species of black and red rices

Extracted lipids obtained from black and red rices were analysed by high-performance liquid chromatography (HPLC), for endogenous tocochromanols. The molecular species and fatty acid (FA) distribution of triacylglycerols (TAG) isolated from these total lipids were analysed by a combination of AgNO₃ thin-layer chromatography (TLC) and gas chromatography (GC), and were investigated in relation to the content of endogenous tocochromanols. With a few exceptions, the predominant tocols were γ-tocotrienol and α-tocopherol, followed by α-tocotrienol (red) and γ-tocopherol (black) with much smaller amounts of β-, δ-tocopherols and δ-tocotrienol. The lipids of these rices were comprised mainly of TAG (76.4–80.5%), free FA (7.2–9.8%), and phospholipids (3.5–3.6%), whilst other components were present in minor proportions (0.1–4.1%). The principal FA components were palmitic (16:0), oleic (18:1n − 9) and linoleic (18:2n − 6) acids. Fifteen different TAG molecular species were detected and quantified by successive applications of AgNO₃-TLC and GC. The major TAG components were S₂M (7.0–7.8%), SM₂ (12.6–12.9%), M₃ (15.7–16.5%), SMD (23.5–23.7%), SD₂ (5.7–5.8%), M₂D (9.3–9.8%), MD₂ (8.1–8.6%) and D₃ (5.8–7.4%) (where S, M, D, and T denote a saturated FA, a monene, a diene, and a triene, respectively). The results suggested that these rice lipids contain large amounts of nutraceuticals with proven positive health effects.     

X-ray absorption of gold nanoparticles with thin silica shell

Silica-coated gold (Au) nanoparticles were prepared and their morphological and X-ray absorption properties were investigated. These core-shell type nanoparticles are very stable in aqueous media and may be suitable for an X-ray contrast agent in biological systems. Transmission electron micrographs confirmed well-separated and relatively homogeneous morphology of the nanoparticles in highly concentrated colloids. Peak position for Au plasmon resonance was red-shifted with increasing shell thickness. X-ray absorption by the colloids of silica-coated Au nanoparticles was stronger than that by those of silica-coated Agl nanoparticles, a recently investigated X-ray contrast agent, at similar experimental conditions.     

Indentation-induced ductile behavior of glass–ceramics involving layered aluminosilicates

Contact damage in materials is critical in engineering applications because it influences mechanical resistance, such as wear, erosion, and impact failure. Indentation tests were performed using a tungsten carbide ball indenter (Hertzian contact) on the surfaces of glass–ceramics containing hexagonal CaAl₂Si₂O₈ or mica crystals (fluorophlogopite), both of which have a layered structure. The stress–strain relation and the permanent deformation on the surface, as well as the observation of the microcrack zone by X-ray computed tomography using synchrotron radiation, revealed that the glass–ceramic with hexagonal CaAl₂Si₂O₈ showed ductility similar to the quasi-plastic behavior previously observed in the mica glass–ceramic. The yield stresses of the glass–ceramics were estimated from the stress deviating from the stress–strain relation assuming complete elastic response between the ball and the sample. The ratio of the yield stress to Young modulus (Y/E) of the glass–ceramic with hexagonal CaAl₂Si₂O₈ was determined to be higher than that of the mica glass–ceramic.