Extraction of Proteins and Other Intracellular Bioactive Compounds From Baker's Yeasts by Pulsed Electric Field Treatment.

Yeasts are rich source of proteins, antioxidants, vitamins, and other bioactive compounds. The main drawback in their utilization as valuable ingredients in functional foods and dietary supplements production is the thick, indigestible cell wall, as well as the high nucleic acid content. In this study, we evaluated the feasibility of pulsed electric field (PEF) treatment as an alternative method for extraction of proteins and other bioactive intracellular compounds from yeasts. Baker's yeast water suspensions with different concentration (12.5-85 g dry cell weight per liter) were treated with monopolar rectangular pulses using a continuous flow system. The PEF energy required to achieve irreversible electropermeabilization was significantly reduced with the increase of the biomass concentration. Upon incubation of the permeabilized cells in water, only relatively small intracellular compounds were released. Release of 90% of the free amino acids and low molecular UV absorbing compounds, 80% of the glutathione, and ∼40% of the total phenol content was achieved about 2 h after pulsation and incubation of the suspensions at room temperature. At these conditions, the macromolecules (proteins and nucleic acids) were retained largely inside. Efficient protein release (∼90% from the total soluble protein) occurred only after dilution and incubation of the permeabilized cells in buffer with pH 8-9. Protein concentrates obtained by ultrafiltration (10 kDa cut off) had lower nucleic acid content (protein/nucleic acid ratio ∼100/4.5) in comparison with cell lysates obtained by mechanical disintegration. The obtained results allowed to conclude that PEF treatment can be used as an efficient alternative approach for production of yeast extracts with different composition, suitable for application in food, cosmetics and pharmaceutical industries.

Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in Hansenula polymorpha.

А protocol for the efficient and selective recovery of human ferritin heavy chain (FTH1) expressed intracellularly in Hansenula polymorpha was developed. It was based on electropermeabilisation and an increase in the cell wall porosity by pulsed electric field (PEF) treatment and subsequent incubation with a low concentration of a lytic enzyme. Irreversible plasma membrane permeabilisation was induced by applying rectangular electric pulses in the flow mode. The electrical treatment itself did not cause the release of the recombinant protein but induced the sensitisation of H. polymorpha cells to the lytic enzyme. Consequently, the subsequent incubation of the permeabilised cells with lyticase led to the recovery of approximately 90% of the recombinant protein, with a purification factor of 1.8. A similar efficiency was obtained by using the industrial lytic enzyme Glucanex. The released FTH1 appears in the form of an oligomer with a molecular mass of approximately 480 kDa, which is able to bind iron. The possibility for scaling the proposed protocol is discussed.

Electroinduced release of recombinant ß-galactosidase from Saccharomyces cerevisiae.

Yeasts are one of the most commonly used systems for recombinant protein production. When the protein is intracelullarly expressed the first step comprises a cell lysis, achieved usually by a mechanical disintegration. This leads to non-selective liberation of the cytoplasmic content, which complicates the following downstream process. Here, we present a new approach suitable for more selective and efficient recovery of large intracellular proteins from yeast, based on the combination of electropermeabilisation and subsequent treatment with lytic enzyme. The experiments were performed with Saccharomyces cerevisiae strains expressing LYTAG-ß-galactosidase from Escherichia coli. The permeabilzation of plasma membrane was induced by application of rectangular electric pulses, with 1.25ms duration and field intensity of 4.3-5.4kV/cm. In the presence of a reducing agent the cells released approximately 80% of the total protein 4h after electrical treatment. At the same conditions the release of the recombinant protein was very slow, reaching 45% from total activity 20h after pulse application. The great difference in the release kinetics enabled to remove a part of the total protein, without significant loss of ß-galactosidase activity, only by substituting the incubation buffer. The subsequent addition of lyticase (1-2U/ml) led to recovery of approximately 70% from the recombinant enzyme, with a factor of purification 2.6, without provoking a significant cell lysis. The applicability of similar protocol for liberation of large recombinant and native proteins from yeast is discussed.

Evidence that pulsed electric field treatment enhances the cell wall porosity of yeast cells.

The application of rectangular electric pulses, with 0.1-2 ms duration and field intensity of 2.5-4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2-8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.