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D L Pyle

Publications and source records attributed to D L Pyle.

8 recordsLinked to original sources
PubMed2005

Selective separation of beta-lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB.

The selective separation of whey proteins was studied using colloidal gas aphrons generated from the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). From the titration curves obtained by zeta potential measurements of individual whey proteins, it was expected to selectively adsorb the major whey proteins, i.e., bovine serum albumin, alpha-lactalbumin, and beta-lactoglobulin to the aphrons and elute the remaining proteins (lactoferrin and lactoperoxidase) in the liquid phase. A number of process parameters including pH, ionic strength, and mass ratio of surfactant to protein (M(CTAB)/M(TP)) were varied in order to evaluate their effect on protein separation. Under optimum conditions (2 mmol/l CTAB, M(CTAB)/M(TP) = 0.26-0.35, pH 8, and ionic strength = 0.018 mol/l), 80-90% beta-lactoglobulin was removed from the liquid phase as a precipitate, while about 75% lactoferrin and lactoperoxidase, 80% bovine serum albumin, 95% immunoglobulin, and 65% alpha-lactalbumin were recovered in the liquid fraction. Mechanistic studies using zeta potential measurements and fluorescence spectroscopy proved that electrostatic interactions modulate only partially the selectivity of protein separation, as proteins with similar surface charges do not separate to the same extent between the two phases. The selectivity of recovery of beta-lactoglobulin probably occurs in two steps: the first being the selective interaction of the protein with opposite-charged surfactant molecules by means of electrostatic interactions, which leads to denaturation of the protein and subsequent formation and precipitation of the CTAB-beta-lactoglobulin complex. This is followed by the separation of CTAB-beta-lactoglobulin aggregates from the bulk liquid by flotation in the aphron phase. In this way, CGAs act as carriers which facilitate the removal of protein precipitate.

Animals↗
PubMed2004

Solid-state fermentation: a continuous process for fungal tannase production.

Truly continuous solid-state fermentations with operating times of 2-3 weeks were conducted in a prototype bioreactor for the production of fungal (Penicillium glabrum) tannase from a tannin-containing model substrate. Substantial quantities of the enzyme were synthesized throughout the operating periods and (imperfect) steady-state conditions seemed to be achieved soon after start-up of the fermentations. This demonstrated for the first time the possibility of conducting solid-state fermentations in the continuous mode and with a constant noninoculated feed. The operating variables and fermentation conditions in the bioreactor were sufficiently well predicted for the basic reinoculation concept to succeed. However, an incomplete understanding of the microbial mechanisms, the experimental system, and their interaction indicated the need for more research in this novel area of solid-state fermentation.

Bioreactors↗
PubMed1997

The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk.

Transgenic sheep milk containing the protein human alpha 1-Antitrypsin (AAT) was partitioned in Poly(ethylene glycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phase at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for beta-Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.

Animals↗
PubMed1996

Studies on the purification of peroxidase from horseradish roots using reverse micelles.

Horseradish peroxidase (HRP) was successfully purified from horseradish roots by a two-stage reverse-micellar extraction from the dialyzed aqueous extract. The anionic surfactant AOT dissolved in isooctane was used to produce the reverse-micellar phases. The narrow pH range at which HRP solubilization occurred was exploited to remove most of the contaminant proteins in the first forward extraction. In the second extraction stage, HRP was selectively solubilized and concentrated by using a volume ratio of 10 between the aqueous and organic phases. The HRP final specific activity was 86 guaiacol U mg-1, obtained with a purification factor of 80 and yield of 46%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two overlapping bands, with HRP corresponding to that at 43.8 kDa. Image analysis on isoelectric focusing (IEF) gels showed that the HRP was 80% pure. Ion exchange liquid chromatography showed that most of the specific activity was due to the basic isoenzyme with pI 8.5, which comprises 33.5% of the product. There were high HRP losses as a precipitate at the interface when direct reverse-micellar extraction was attempted from the crude extract. It is believed that the hydrophobic environment near the haem group of the HRP basic isoenzyme favors complex formation with the surfactant, and that this is promoted at higher protein concentrations.

Calcium↗
PubMed1993

Adsorption of Kluyveromyces marxianus pectinase on CM-Sephadex gels.

New results are presented on the equilibria and kinetics of the ion exchange onto CM-Sephadex of polygalacturonase (pectinase) produced by the fermentation of Kluyveromyces marxianus. It is found that the equilibrium behavior follows the form of the Langmuir isotherm; the equilibrium is strongly affected by pH. High partitioning onto the ion-exchange matrix, with good retention of enzyme activity, is achieved in the pH range 3.5-5.0, and this can be qualitatively explained in terms of simple models for protein adsorption by ion exchange. The kinetics of ion exchange is modeled by assuming that the transfer resistances can be lumped into a single coefficient, and the results show that this gives a reasonable description of the adsorption kinetics. Under optimum conditions protein adsorption is enhanced by electrostatic effects and is extremely fast, and it is suggested that in these circumstances external mass transfer resistance is significant. At pH values close to the isoelectric point, electrostatic interactions are weak and intraparticle diffusion is rate-limiting: pore-blocking by adsorbed proteins appears to be important under these conditions. The results also provide the basis for an efficient single-step purification scheme.

Absorption↗
PubMedDate not supplied

Recovery of lactoferrin and lactoperoxidase from sweet whey using colloidal gas aphrons (CGAs) generated from an anionic surfactant, AOT.

The recovery of lactoferrin and lactoperoxidase from sweet whey was studied using colloidal gas aphrons (CGAs), which are surfactant-stabilized microbubbles (10-100 microm). CGAs are generated by intense stirring (8000 rpm for 10 min) of the anionic surfactant AOT (sodium bis-2-ethylhexyl sulfosuccinate). A volume of CGAs (10-30 mL) is mixed with a given volume of whey (1-10 mL), and the mixture is allowed to separate into two phases: the aphron (top) phase and the liquid (bottom) phase. Each of the phases is analyzed by SDS-PAGE and surfactant colorimetric assay. A statistical experimental design has been developed to assess the effect of different process parameters including pH, ionic strength, the concentration of surfactant in the CGAs generating solution, the volume of CGAs and the volume of whey on separation efficiency. As expected pH, ionic strength and the volume of whey (i.e. the amount of total protein in the starting material) are the main factors influencing the partitioning of the Lf.Lp fraction into the aphron phase. Moreover, it has been demonstrated that best separation performance was achieved at pH = 4 and ionic strength = 0.1 mol/L i.e., with conditions favoring electrostatic interactions between target proteins and CGAs (recovery was 90% and the concentration of lactoferrin and lactoperoxidase in the aphron phase was 25 times higher than that in the liquid phase), whereas conditions favoring hydrophobic interactions (pH close to pI and high ionic strength) led to lower performance. However, under these conditions, as confirmed by zeta potential measurements, the adsorption of both target proteins and contaminant proteins is favored. Thus, low selectivity is achieved at all of the studied conditions. These results confirm the initial hypothesis that CGAs act as ion exchangers and that the selectivity of the process can be manipulated by changing main operating parameters such as type of surfactant, pH and ionic strength.

Animals↗