Characterisation of a new family of carboxyl esterases with an OsmC domain

Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended ‘lid’ region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries

New Concepts for Increasing the Efficiency in Directed Evolution of Stereoselective Enzymes

Directed evolution of stereo- and regioselective enzymes constitutes a prolific source of catalysts for asymmetric transformations in organic chemistry. In this endeavor (iterative) saturation mutagenesis at sites lining the binding pocket of enzymes has emerged as the method of choice, but uncertainties regarding the question of how to group many residues into randomization sites and how to choose optimal upward pathways persist. Two new approaches promise to beat the numbers problem effectively. One utilizes a single amino acid as building block for the randomization of a 10-residue site, the other also employs only one but possibly different amino acid at each position of a 9-residue site. The small but smart libraries provide highly enantioselective epoxide hydrolase or lipase mutants, respectively

Studies of electrochemical oxidation of Zircaloy nuclear reactor fuel cladding using time-of-flight-energy elastic recoil detection analysis.

The trend towards increased fuel burn-up and higher operating temperatures in order to achieve more economic operation of nuclear power plants places demands on a better understanding of oxidative corrosion of Zircaloy (Zry) fuel rod cladding. As part of a programme to study these processes we have applied time-of-flight-energy elastic recoil detection (ToF-E ERD), electrochemical impedance measurements and scanning electron microscopy to quantitatively characterise thin-oxide films corresponding to the pre-transition oxidation regime. Oxide films of different nominal thickness in the 9-300 nm range were grown on a series of rolled Zr and Zry-2 plates by anodisation in dilute H2SO4 with applied voltages. The dielectric thickness of the oxide layer was determined from the electrochemical impedance measurements and the surface topography characterised by scanning electron microscopy. ToF-E ERD with a 60 MeV 127I11+ ion beam was used to determine the oxygen content and chemical composition of the oxide layer. In the Zr samples, the oxygen content (O atom cm-2) that was determined by ERD was closely similar to the O content derived from impedance measurements from the dielectric film. The absolute agreement was well within the uncertainty associated with the stopping powers. Moreover, the measured composition of the thick oxide layers corresponded to ZrO2 for the films thicker than 65 nm where the oxide layer was resolved in the ERD depth profile. Zry-2 samples exhibited a similar behaviour for small thickness (130 nm) but had an enhanced O content at larger thicknesses that could be associated either with enhanced rough surface topography or porous oxide formation that was correlated with the presence of Second Phase Particles (SPP) in Zry-2. The concentration of SPP elements (Fe, Cr, Ni) in relation to Zr was the same in the outer 9x10(17) atom cm-2 of oxide as in the same thickness of metal. The results also revealed the presence of about 1 at.% 32S in the oxides on the Zr and Zry-2 samples which presumably originates from the electrolyte