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

Freely suspended perforated polymer nanomembranes for protein separations

Abstract Selective removal of nanometer-sized compounds such as proteins from fluids is an often challenging task in many scientific and industrial areas. Addressing such tasks with highly efficient and selective membranes is desirable since commonly used chromatographic approaches are expensive and difficult to scale up. Nanomembranes, molecularly thin separation layers, have been predicted and shown to possess outstanding properties but in spite ultra-fast diffusion times and high-resolution separation, to date they generally lack either of two crucial characteristics: compatibility with biological fluids and low-cost production. Here we report the fast and easy fabrication of highly crosslinked polymer membranes based on a thermoset resin (poly[(o-cresyl glycidyl ether)-co-formaldehyde (PCGF) cured with branched polyethyleneimine (PEI)) with nanoscale perforations of 25 nm diameter. During spin casting, microphase separation of a polylactide-co-glycolide induces the formation of nanometer sized domains that serve as templates for perforations which penetrate the 80 nm thick membranes. Ultrathin perforated nanomembranes can be freely suspended on the cm scale, exhibit high mechanical strength, low surface energies and a sharp permeability cutoff at a hydrodynamic diameter of 10 nm suitable for protein separations