Field-assisted extraction of cells, particles and macromolecules.

Improved bioseparation techniques are increasingly important for biotechnology because separation is often the limiting factor for the success of biological processes. Manufacturers of new enzymes and pharmaceutical products require improved methods for recovering intact cells and intracellular products. Similarly the isolation, purification and concentration of many biomolecules produced in fermentation processes is extremely important. Often such downstream processing contributes a large portion of the product cost and thus efficient and economical alternative approaches to bioseparation processes are needed to eliminate, reduce or facilitate the handling of solids. Field-assisted separations, which hold immense potential for providing a major improvement in bioseparation in the near future, are considered in this review. Special emphasis is given to multistage methods, which are cost-effective compared with competing technologies. Commercial applications of these methods are detailed, we present suggestions for future work and we analyse the scale-up and economic aspects of these processes.

Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila.

Cytosolic nucleotidase II (cN-II) from Legionella pneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation. DATABASE: The coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers 2BDE and 4G63. The accession number of GMP complexed LpcN-II is 4OHF. STRUCTURED DIGITAL ABSTRACT: LpcN-II and LpcN-II bind by molecular sieving (View interaction) LpcN-II and LpcN-II bind by x-ray crystallography (View interaction) [Structured digital abstract was added on 5 March 2014 after original online publication].