Interfacial behavior of glycosphingolipids and chemically related sphingolipids

Advances in understanding how glycosphingolipids and chemically related sphingolipids affect the structure and dynamics of bio-interfaces have been grouped in four inter-related areas: (a) influence of chemical structure in local interactions along lateral and transverse membrane planes; (b) effect of polar head group and hydrocarbon moieties on the mesomorphic state; (c) thermodynamic–geometric compensations affecting interfacial topology; and (d) domain segregation and surface topography.     

Ceramide N-acyl chain length: a determinant of bidimensional transitions, condensed domain morphology, and interfacial thickness

Several lipids of biological interest are able to form monomolecular surfaces with a rich variety of thickness and lateral topography that can be precisely controlled by defined variations of the film composition. Ceramide is one of the simplest sphingolipids, consisting of a sphingosine base N-linked to a fatty acid, and is a membrane mediator for cell-signaling events. In this work, films of ceramides N-acylated with the saturated fatty acids C10, C12, C14, and C16 were studied at the air-aqueous interface. The dipole moment contribution (from surface potential measurements) and the surface topography and thickness (as revealed by Brewster angle microscopy) were measured simultaneously with the surface pressure at different molecular areas. Several surface features were observed depending on the asymmetry between the sphingosine and the N-linked acyl chains. At 21 °C, the C16:0 and C14:0 ceramides showed condensed isotherms and the film topography revealed solid film patches (17.3-15.7 ? thick) that coalesced into a homogeneous surface by further compression. On the other hand, in the more asymmetric C12:0 and C10:0 ceramides, liquid expanded states and liquid expanded-condensed transitions occurred. In the phase coexistence region, the condensed state of these compounds formed flowerlike domains (11.1-13.3 ? thick). C12:0 ceramide domains were larger and more densely branched than those of C10:0 ceramide. Both the film thickness and the surface dipole moment of the condensed state increased with ceramide N-acyl chain length. Bending of the sphingosine chain over the N-linked acyl chain in the more asymmetric ceramides can account for the variation of the surface electrostatics, topography, and thickness of the films with the acyl chain mismatch.     

β-d-Xylosidase from Geobacillus thermoleovorans IT-08: Biochemical Characterization and Bioinformatics of the Enzyme

The gene encoding a thermostable β-d-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M _r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6–8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k _cat/K _M) of 0.0048?±?0.0010 s^?1 mM^?1 on p-nitrophenyl-β-d-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.