The adsorption capacity and immobilization rate of two Eupergit® supports for acid urease was studied by varying the ionic strength and enzyme preparation concentration in the immobilizing solution at pH 7. Eupergit® C250 L yielded a series of derivatives with enzyme loadings (Y(P/B)) ranging from 48 to 171 mg of bovine serum albumin equivalent (BSAE) per gram of dry support (ds). Use of drastic postimmobilization conditions at pH 9 for 3-9 days yielded a slight decrease (8-14%) in the initial activity of immobilized enzymes and a limited increase in the stabilization factor (1.1-1.5), as assessed by accelerated aging tests at 65 °C. Further storage tests at 4 °C in the wet state showed that the activity of several derivatives either stabilized or not was practically constant for as long as 547 days. Both free enzyme and immobilized acid urease derivatives exhibited a kinetic pattern of the Michaelis-Menten type. Using the Eadie-Hofstee diagram, the specific ammonia formation rate constant for free (k(cat)) or immobilized (k'(cat)) enzyme resulted to be little affected by immobilization (k(cat) ≈ k'(cat) ≈ 18.86 ± 0.34 IU/mg BSAE), whereas the apparent Michaelis constant for immobilized enzymes exhibited a statistically significant increase at P < 0.05 from the intrinsic value (2.55 ± 0.14 mM) for free enzyme to 5.38 ± 0.87 mM as Y(P/B) increased to 171 mg BSAE/g ds. By estimating the observable Thiele modulus (Φ(obs) ), the activity of the biocatalyst with the greatest enzyme loading at the lowest urea concentrations tested (0.833 mM) was reduced by a factor of about 2 due to internal diffusional limitations. By operating in the pseudofirst-order regime with immobilized derivatives at Y(P/B) about 126 mg BSAE/g ds, their activity after grinding was no more limited by intraparticle diffusion and approached the value for free enzyme.
Extensive x-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKAc) enabled the atomic characterization of inhibitor and/or substrate peptide analogs trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and enzymatic cycle. NMR allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides corresponded to the cytoplasmic regions of the wildtype and lethal mutants of the membrane protein phospholamban, while the fourth peptide corresponded to the binding epitope of the heat-stable protein kinase inhibitor ). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His 6 tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.
Abstract:We formulated a latex ink for ink-jet deposition of viable Gram-negative bacterium Gluconobacter oxydans as a model adhesive, thin, highly bio-reactive microstructured microbial coating. Control of G. oxydans latex-based ink viscosity by dilution with water allowed ink-jet piezoelectric droplet deposition of 30 × 30 arrays of two or three droplets/dot microstructures on a polyester substrate. Profilometry analysis was used to study the resulting dry microstructures. Arrays of individual dots with base diameters of ~233-241 µm were obtained. Ring-shaped dots with dot edges higher than the center, 2.2 and 0.9 µm respectively, were obtained when a one-to-four diluted ink was used. With a less diluted ink (one-to-two diluted), the microstructure became more uniform with an average height of 3.0 µm , but the ink-jet printability was more difficult. Reactivity of the ink-jet deposited microstructures following drying and rehydration was studied in a non-growth medium by oxidation of 50 g/L D-sorbitol to L-sorbose, and a high dot volumetric reaction rate was measured (~435 g· L −1 · h −1 ). These results indicate that latex ink microstructures generated by ink-jet printing may hold considerable potential for 3D fabrication of high surface-to-volume ratio biocoatings for use as microbial biosensors with the aim of coating microbes as reactive biosensors on electronic devices and circuit chips.
OPEN ACCESSCoatings 2014, 4 2
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