Releasing Biopharmaceutical Products from Cells

Middelberg, Anton P. J.

doi:10.1002/9783527653096.ch3

“…Once the proteins are extracted, they have to be concentrated and purified to remove the undesired or potentially harmful substances coming from the cells or introduced during extraction (Middelberg, 2012). Ultrafiltration is a commonly employed method for protein concentration, buffer exchange and purification in the biotechnology industry (van Reis and Zydney, 2007).…”

Section: Nucleic Acid Content Of the Extractsmentioning

confidence: 99%

“…Yeast autolysates are used mainly as a component of microbial culture media while in the food industry they are added as a flavoring agent in many foods at 0.5-2% content. Mechanical disintegration is applied as a first step for production of polysaccharides (β-glucan) from yeast and is also the main method for extraction of proteins, antioxidants and other soluble intracellular products from yeast (Middelberg, 2012;Bzduhcha-Wróbel et al, 2014). Despite being routinely applied in biotechnology, mechanical disintegration leads to non-selective release of the product, increase in the viscosity, and micronization of the cell debris, which complicates the liquid-solid separation and the following downstream processing (Balasundaram et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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

Ganeva

¹

,

Angelova

²

,

Galutzov

³

et al. 2020

Front. Bioeng. Biotechnol.

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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.

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“…Once the proteins are extracted, they have to be concentrated and purified to remove the undesired or potentially harmful substances coming from the cells or introduced during extraction (Middelberg, 2012). Ultrafiltration is a commonly employed method for protein concentration, buffer exchange and purification in the biotechnology industry (van Reis and Zydney, 2007).…”

Section: Nucleic Acid Content Of the Extractsmentioning

confidence: 99%

“…Yeast autolysates are used mainly as a component of microbial culture media while in the food industry they are added as a flavoring agent in many foods at 0.5-2% content. Mechanical disintegration is applied as a first step for production of polysaccharides (β-glucan) from yeast and is also the main method for extraction of proteins, antioxidants and other soluble intracellular products from yeast (Middelberg, 2012;Bzduhcha-Wróbel et al, 2014). Despite being routinely applied in biotechnology, mechanical disintegration leads to non-selective release of the product, increase in the viscosity, and micronization of the cell debris, which complicates the liquid-solid separation and the following downstream processing (Balasundaram et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in Hansenula polymorpha

Ganeva

¹

,

Galutzov

²

,

Angelova

³

et al. 2017

Appl Biochem Biotechnol

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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.

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Releasing Biopharmaceutical Products from Cells

Cited by 4 publications

References 30 publications

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

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

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Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in Hansenula polymorpha

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