Production of fructosyltransferase in mechanically stirred and air-lift bioreactors

Aureobasidium pullulans cells were cultivated in a 3 dm³ stirred bioreactor to optimize the production of fructosyltransferase (FTase). Batch experiments were focused on the influence of the initial sucrose concentration (150–350 g dm^?3), sodium nitrate concentration (10–25 g dm^?3), and stirring rate (180–540 min^?1) on the process. It was found that the FTase specific activity per cell mass was positively influenced by the conditions that impeded the cell growth such as higher sucrose concentrations or lower oxygen transfer rates. Higher content of inorganic nitrogen substrate slightly increased the overall FTase production. The presence of fructose moiety-containing saccharides in the cultivation medium was observed to be indispensable for FTase production. An addition of a concentrated sucrose solution in the 40th cultivation hour therefore essentially resumed the FTase production. Finally, scale-up experiments were performed in 12 dm³ and 100 dm³ mechanically stirred bioreactors and in 20 dm³ and 60 dm³ air-lift bioreactors.     

Modeling of equilibrium and kinetics of human polyclonal immunoglobulin G adsorption on a tentacle cation exchanger

Adsorption of human immunoglobulin G (IgG) on a commercial cation exchanger with a grafted polymer layer was investigated at pH 4.5 and in the NaCl concentration range of 0–150 mM. Adsorption equilibrium was determined in static batch experiments and verified in batch uptake experiments. Parameters of the Langmuir isotherm were estimated for each salt concentration separately. The batch uptake experiments provided also the estimates of effective pore diffusion coefficients of IgG for individual protein and salt concentrations. The values of the effective pore diffusion coefficient depended strongly on both factors. They increased by about 5–15 times with the NaCl concentration and decreased about three times with the protein concentration. The quality of the estimated parameters was confirmed by frontal experiments described by the general rate model of chromatography.     

Influence of ligand density on antibody binding capacity of cation-exchange adsorbents

Adsorption properties of a set of polymethacrylate-based cation exchangers designed for purification of monoclonal antibodies were investigated. The materials differed significantly in the density of sulphoisobutyl ligand groups. The ligand density had a pronounced effect on the static adsorption capacity of a polyclonal human immunoglobulin G. An optimal ligand density was observed at any pH and NaCl concentration tested when sharp optima were observed at low pH and ionic strength values. This was caused by effective clogging of pore mouth at high ligand densities. An anomalous effect of ionic strength was observed for the adsorbents with the high ligand density when the adsorption capacity increased with the addition of NaCl at low pH.     

Impact of ionic strength on adsorption capacity of chromatographic particles employed in separation of monoclonal antibodies

The influence of ionic strength on the adsorption capacity of seven commercial adsorbents used in downstream processing of monoclonal antibodies was examined. Affinity (MabSelect, Poros 50A High Capacity, ProSep-vA High Capacity), hydrophobic charge-induction (MEP HyperCel), and cation exchange adsorbents (FractoGel EMD SE Hicap (M), SP Sepharose Fast Flow, Ceramic HyperD F) were used to study the adsorption of polyclonal human immunoglobulin G at optimal pH values. The ionic strength, adjusted by sodium chloride concentrations in the range of 0–225 mM, strongly decreased the adsorption capacity of the cation exchangers. Equilibrium data were described in the form of the dependence of the ratio of protein concentrations in the solid and liquid phases on the total concentration of cation counter ions. They were successfully fitted and interpreted through a stoichiometric ion-exchange model.     

Properties of fructosyltransferase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> immobilized on an acrylic carrier

Optimization of immobilization conditions was carried out for covalent binding of Aureobasidium pullulans fructosyltransferase to a copolymer of butyl acrylate and ethylene glycol dimethacrylate using a glutaraldehyde method. It was found that the highest activity of the preparation could be obtained for the immobilization pH 6.0 and initial protein amount 8.5 g per dm³ of the carrier. Effects of the reaction pH, temperature, and initial sucrose concentration on the activity and stability of the preparation were analyzed. Further investigations involved storage stability and operational stability in a mechanically stirred-tank reactor.     

Equilibrium and kinetics of protein binding on ion-exchange cellulose membranes with grafted polymer layer

The performance of weak and strong anion- and cation-exchange membrane adsorbents with a grafted gel layer (Sartobind Q, D, S, and C) was investigated using six proteins: bovine serum albumin, human serum albumin, α-lactalbumin, β-lactoglobulin, lysozyme, and myoglobin. Static binding experiments were used to assess the effect of pH and buffer concentration and to determine the adsorption isotherms for selected membrane/protein combinations. The equilibrium data were duly described either by the Langmuir or Freundlich isotherms. Dynamic binding experiments were carried out for the same membrane/protein combinations in a broad range of linear flow velocity. Both the dynamic binding capacity at 10 % breakthrough and the final binding capacity at complete breakthrough were independent of the flow velocity despite strong dispersion of the adsorption zone. A good match between the equilibrium data from static and dynamic experiments was obtained for the anion exchangers. The correlation between the dynamic binding capacity and protein molecule size was observed for the strong cation exchanger. This was due to the different accessibility of the gel layer for the protein molecules.     

Selection of a method for determination of activity of pectinolytic enzymes in berry fruit materials

The goal of the study was to find the most accurate and sensitive method for the determination of activity of pectinolytic enzymes in complex mixtures obtained from fruit materials such as raw cloudberry and raspberry juices. Several assay methods based on enzymatic reactions using viscometric, colourimetric, spectrophotometric, or pH-titration detection of the reaction products were tested. Problems with the application of the selected methods, such as very low detection signal or very large background signal, were observed. Among the tested methods, only a modified method based on the continuous recording of the released carboxyl groups titration allowed to assay the activity of exogenous pectin methylesterase with a good linearity, sensitivity, accuracy, and minimised the interference of other fruit components.     

Characterisation of porous materials for bioseparation

A set of chromatographic materials for bioseparation were characterised by various methods. Both commercial materials and new supports presenting various levels of rigidity were analysed. The methods included size-exclusion and capillary phenomena based techniques. Both batch exclusion and inverse size-exclusion chromatography were used. Gas adsorption, mercury porosimetry and thermoporometry were applied as well as a new method based on water desorption starting from the saturated state. When the rigidity of adsorbents is high enough, the agreement is reasonable between the values of the structural parameters that were determined (surface area, porosity, and pore size) by various methods. Nevertheless, a part of macroporosity may not be evidenced by inverse size-exclusion chromatography whereas it is visible by batch exclusion and the other methods. When the rigidity decreases, for example with soft swelling gels, where standard nitrogen adsorption or mercury porosimetry are no more reliable, two main situations are encountered: either the methods based on capillary phenomena (thermoporometry or water desorption) overestimate the pore size with an amplitude that depends on the method, or in some cases it is possible to distinguish water involved in the swelling of pore walls from that involved in pore filling by capillary condensation.