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The stability of expanded bed adsorption systems (EBA) was studied in biomass containing culture broth by residence time distribution (RTD) experiments, using pulse inputs of fluorescent molecules as tracers. Different commercial adsorbents (Streamline DEAE, SP, Phenyl, Chelating, and AC) were tested at various biomass concentrations (2.5–12 %, wet weight) of whole (Saccharomyces cerevisiae) yeast, yeast cell homogenate, and Escherichia coli homogenate. Analyzing the RTD according to the PDE model (PDE: axially dispersed plug‐flow exchanging mass with stagnant zones) allowed the calculation of three parameters: the number of transfer units for mass exchange between mobile and stagnant fraction (N), the Peclet number for overall axial dispersion (P), and the mobile fraction of the liquid in axially dispersed plug flow (φ). When fluidization was performed in particle‐free buffer the normalized response signal (after perfect input pulse) was symmetric (N:0; P: 50–100; φ: 1), thus, demonstrating the formation of a homogeneous fluidized (expanded) bed. Upon application of suspended biomass the RTD was skewed, depending on the adsorbent used and the type and level of biomass present in the sample. This situation leads to three different characteristic pictures: the well‐fluidized system (N: ≥ 7–10; P: ≥ 40; φ: 0.80–0.90), the system exhibiting bottom channeling (N: < 1–2; P: ≥ 40; φ: 0.5–0.7) and, the system where extensive agglomeration develops (N: 4–7; P: 20–40; φ: < 0.5). These results demonstrate that changes in the hydrodynamics of EBA already take place in the presence of moderate concentrations of biomass. Furthermore, those changes can be quantitatively described mainly in terms of the fraction of stagnant zones in the system, which are formed due to the interaction of biomass and adsorbent. The technique described here can be used to evaluate a certain combination of adsorbent and biomass with regard to its suitability for expanded bed adsorption from whole broth. © 1999 John Wiley & Sons, Inc. Biotechol Bioeng 64: 484–496, 1999.

The suitability of ion-selective electrodes (ISE) for the determination of residence time distribution (RTD) in turbid, cell-containing fluids was examined. The electrodes were found to give reproducible signals in biomass-containing feedstock with up to 20% wet weight of solids. The enhanced feedstock compatibility of IES, when compared to other tracer sensing devices, allows the study of expanded bed system hydrodynamics under relevant operating conditions. Within the linear range of the corresponding ISE-tracer pair, both examined ISE (Li(+)- or Br(-)-selective) showed to be insensitive against the range of flow rate and pH normally employed during expanded bed adsorption (EBA) of proteins. Analyzing the RTD obtained after a perfect ion tracer pulse in terms of the PDE model (PDE, axially dispersed plug-flow exchanging mass with stagnant zones) gave a quantitative description of the underlying hydrodynamic situation during EBA processing. These data provided a powerful tool to make predictions on the adsorptive global process performance with a defined feedstock type and composition. The link between the hydrodynamic events during feedstock application and the actual process performance was shown when applying intact yeast cell suspensions at different biomass content (up to 7.5% wet weight) and buffer conductivity (5-12 mS) onto an EBA column filled with the adsorbent Streamline Q XL as fluidized phase. On the basis of our experimental results, a guideline for the successful application of the ISE/RTD method to EBA process design is presented.

The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.

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Performance of an aqueous two-phase-based countercurrent chromatographic system for horseradish peroxidase purification Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer-phosphate aqueous two-phase systems (ATPS). By using preparative columns at 1000 rpm, a 25 -30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate -with added 2 mol/kg sodium chloride -was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10 -25 mL; 200 -250 U/mL peroxidase; 2.0 -4.0 mg/mL total protein). Retention of HRP in the CCC "column" during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was G 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for G 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass-through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS-PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ ATPS and of scaling-up processes in industrial separators.

The extractive purification of peroxidase from Annoracia rusticam roots and Glycine mar seed coats in temperature-induced and affinity microsphere-containing aqueous two-phase systems was studied. The extractive purification of pexoxidase from Glycine mar seed coats was carried out in a temperature-induced aqueous two-phase system formed by Triton X-45. Triton X-100 and sodium acetate at pH 5.5. A 99% yield with a 6-fold purification factor was obtained. When the clear top phase was subjected to concanavalin-A affhty chromatography, the purification factor rose to 41 and the yield dropped to 28%. A two-step purification process for peroxidase from Annoracia rusticana roots was developed by adding concanavalin-A affinity microspheres to a PEWphosphate aqueous two-phase system. The method allows a 60% recovery of high purity peroxidase (1,860 guaiacol units per mg). A lower recovery rate and degree of purification of this enzyme was achieved after temperature-induced aqueous two-phase partition or acetone precipitation and concanavalin-A affinity column chromatography.