Comparison of batch and continuous multi‐column protein A capture processes by optimal design

Multi‐column capture processes show several advantages compared to batch capture. It is however not evident how many columns one should use exactly. To investigate this issue, twin‐column CaptureSMB, 3‐ and 4‐column periodic counter‐current chromatography (PCC) and single column batch capture are numerically optimized and compared in terms of process performance for capturing a monoclonal antibody using protein A chromatography. Optimization is carried out with respect to productivity and capacity utilization (amount of product loaded per cycle compared to the maximum amount possible), while keeping yield and purity constant. For a wide range of process parameters, all three multi‐column processes show similar maximum capacity utilization and performed significantly better than batch. When maximizing productivity, the CaptureSMB process shows optimal performance, except at high feed titers, where batch chromatography can reach higher productivity values than the multi‐column processes due to the complete decoupling of the loading and elution steps, albeit at a large cost in terms of capacity utilization. In terms of trade‐off, i.e. how much the capacity utilization decreases with increasing productivity, CaptureSMB is optimal for low and high feed titers, whereas the 3‐column process is optimal in an intermediate region. Using these findings, the most suitable process can be chosen for different production scenarios.     

Cell-free synthesis of silver nanoparticles in spent media of different Aspergillus species

The recovery and valorization of metals and rare earth metals from wastewater are of great importance to prevent environmental pollution and recover valuable resources. Certain bacterial and fungal species are capable of removing metal ions from the environment by facilitating their reduction and precipitation. Even though the phenomenon is well documented, little is known about the mechanism. Therefore, we systematically investigated the influence of nitrogen sources, cultivation time, biomass, and protein concentration on silver reduction capacities of cell-free cultivation media (spent media) of Aspergillus niger, A. terreus, and A. oryzae. The spent medium of A. niger showed the highest silver reduction capacities with up to 15 μmol per milliliter spent medium when ammonium was used as the sole N-source. Silver ion reduction in the spent medium was not driven by enzymes and did not correlate with biomass concentration. Nearly full reduction capacity was reached after 2 days of incubation, long before the cessation of growth and onset of the stationary phase. The size of silver nanoparticles formed in the spent medium of A. niger was influenced by the nitrogen source, with silver nanoparticles formed in nitrate or ammonium-containing medium having an average diameter of 32 and 6 nm, respectively.     

Proton interactions with hemes a and a3 in bovine heart cytochrome c oxidase

Three forms of cytochrome c oxidase, fully oxidized CcO (CcO-O), oxidized CcO complexed with cyanide (CcO.CN), and mixed valence CcO, in which both heme a(3) and Cu(B) are reduced and stabilized by carbon monoxide (MV.CO), were investigated by optical spectroscopy, MCD, and stopped-flow for the pH sensitivity of spectral features. In the pH range between pH 5.7 and 9.0, both heme a and heme a(3) in CcO-O interact with a single protolytic group. From the variation of the position of the Soret peak with changes in pH, a pK(a) of 6.6 +/- 0.2 was determined for this group. The pH sensitivity of heme a(3) is lost in the CcO.CN complex, and only heme a responds to pH changes. In MV.CO the spectra of both hemes are almost independent of pH between 5.7 and 11.0. The stoichiometry of proton uptake in the conversion of CcO-O both to MV.CO and to fully reduced CcO was determined between pH 5.8 and pH 8.2. Formation of MV.CO from CcO-O was accompanied by the uptake of approximately two protons, and this value was almost independent of pH. Full reduction of oxidized CcO was associated with the uptake of approximately 2 H(+) at basic pH, and this value increases with decreasing pH. On the basis of these proton uptake measurements, it is concluded that the pK(a) of the group is independent of the redox state of CcO. It is suggested that Glu60 of subunit II, located at the entrance of the proton conducting K-channel, is the protolytic residue that interacts with both hemes through a hydrogen-bonding network.     

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.     

Effects of functional constraints and opportunism on the functional structure of a vertebrate predator assemblage

1. Within mainstream ecological literature, functional structure has been viewed as resulting from the interplay of species interactions, resource levels and environmental variability. Classical models state that interspecific competition generates species segregation and guild formation in stable saturated environments, whereas opportunism causes species aggregation on abundant resources in variable unsaturated situations. 2. Nevertheless, intrinsic functional constraints may result in species-specific differences in resource-use capabilities. This could force some degree of functional structure without assuming other putative causes. However, the influence of such constraints has rarely been tested, and their relative contribution to observed patterns has not been quantified. 3. We used a multiple null-model approach to quantify the magnitude and direction (non-random aggregation or divergence) of the functional structure of a vertebrate predator assemblage exposed to variable prey abundance over an 18-year period. Observed trends were contrasted with predictions from null-models designed in an orthogonal fashion to account independently for the effects of functional constraints and opportunism. Subsequently, the unexplained variation was regressed against environmental variables to search for evidence of interspecific competition. 4. Overall, null-models accounting for functional constraints showed the best fit to the observed data, and suggested an effect of this factor in modulating predator opportunistic responses. However, regression models on residual variation indicated that such an effect was dependent on both total and relative abundance of principal (small mammals) and alternative (arthropods, birds, reptiles) prey categories. 5. In addition, no clear evidence for interspecific competition was found, but differential delays in predator functional responses could explain some of the unaccounted variation. Thus, we call for caution when interpreting empirical data in the context of classical models assuming synchronous responses of consumers to resource levels.     

Turbo-mixing in microplates

How to effectively mix small volumes of liquids within microplate wells is a still underestimated and often neglected challenge. The method the authors introduce here relies on violent turbulent motion within a liquid caused by spotting an organic solvent drop onto its surface. The amount needed, less than 1 to 3 microL, is generally small enough not to alter bioactive molecules. Moreover, a solvent may be selected for its compatibility with assay components. The method was tested with layers of aqueous liquids that differ in pH and concentration of a pH-dependent dye, allowing mixing to be monitored optically. Rapid mixing was caused by spotting drops of alcohols, acetone, acetonitrile, and aqueous solutions of these, as long as the difference of surface tension between the drop and the uppermost layer of the bulk liquid surpassed 30 dynes/cm. Along with this difference, position and velocity of spotting, as well as viscosity and geometry of the bulk liquid volume, may influence the turbulence evoked. No significant difference was found for the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase when measured after mixing by shaking and after mixing by spotting 1 microL of methanol onto assays within 96-well microplates.     

Free-air exposure systems to scale up ozone research to mature trees

Because seedlings and mature trees do not necessarily respond similarly to O(3) stress, it is critically important that exposure systems be developed that allow exposure of seedlings through to mature trees. Here we describe three different O(3) Free-Air Exposure Systems that have been used successfully for exposure at all growth stages. These systems of spatially uniform O(3) release have been shown to provide reliable O(3) exposure with minimal, if any, impact on the microclimate. This methodology offers a welcome alternative to chamber studies which had severe space constraints precluding stand or community-level studies and substantial chamber effects on the microclimate and, hence physiological tree performance.