BMS Cell disruption

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CELL DISRUPTION

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The cell envelope is physically broken, releasing all intracellular components into the surrounding medium Cell Disruption can be achieved by Physical, chemical and enzymatic methods Physical methods includes: Sonication French press Osmotic shock Heat shock Homogenization Grinding with glass beads

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An ultrasonicator generates sound waves approx. 20 kHz/s, which causes pressure fluctuations (high & Low) to form oscillating bubbles that collapse inward violently generating shock waves This phenomenon is termed cavitation which causes high speed impinging liquid jets and strong hydrodynamic shear-forces These effects are used for the agglomeration and milling of micrometre and nanometre -size materials as well as for the disintegration of cells or the mixing of reactants. Ultra Sonicator

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Cell disruption by an ultrasonicator is effective with most cell suspensions Generates heat , which can denature thermolabile proteins Rod shaped bacteria are easily brake than cocci and gram negative organisms easily disrupted than gram positives used in the laboratory. However, it is impractical to be used on a large scale due to its high operating cost.

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Osmotic Shock (or Osmotic Stress): It is a sudden change in the solute concentration around a cell, causing a rapid change in the movement of water across its cell membrane. Under conditions of high concentrations of either salts, substrates or any solute in the supernatant, water is drawn out of the cells through osmosis. This also inhibits the transport of substrates and cofactors into the cell thus “shocking” the cell.

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Alternatively, at low concentrations of solutes, water enters the cell in large amounts, causing it to swell and burst. Osmotic Shock

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Heat shock ( thermolysis) Breaking of cells by subjecting to heat. Suitable for few heat-stable intracellular products

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For cell disruption - Manton- Gaulin APV type homogeniser is usually used for pilot and large scale disruption They may be used for bacterial and yeast cells and fungal mycelium Here the cell suspension is draws (about 12% w/v) through a check valve into the pump cylinder and forces it, at high pressures of up to 150 Mpa and flow rates of up to 10,000 L hr -1 , through an adjustable discharge valve which has a restricted orifice  High pressure homogenisers

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Cells are subjected to impact, liquid shear and a severe pressure drop across the valve The main disruptive factor is the pressure applied and consequent pressure drop across the valve causes explosion of cells Problem is all cell materials are released Product of interest must be separated from complex mixture of proteins , Nucleic acids and cell wall fragments . Also, Proteins may be denatured if the equipment is not cooled and filamentous microorganisms may block the discharge valve

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Cell suspension

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Grinding with glass beads Small scale – manual grinding of cells with alumina, glass beads or silica can be effectively used to disrupt cells Industrial scale- high speed bead mills equipped with cooling jackets are often used to agitate cell suspension with small beads (0.5-0.9 µm dia ) of glass, zirconium oxide or titanium carbide Grinding chamber is filled with about 80% beads. A shaft with impellers is fixed within the chamber which rotates at high speed generates high shear forces

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Cell breakage results from shear forces, grinding between beads and collisions with beads The efficiency of cell disruption in a bead mill depends on the concentration of the cells, the amount and size of beads, and the type and rotation speed of the agitator. The maximum flow rate in this system is 2000 L/h.

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Small beads are generally more efficient, but the smaller the bead, the harder it is to separate them from ground solids. Cell disruption by bead mills is inexpensive and can be operated on a large scale. Operates continuously, Algae, bacteria and fungi can be lysed

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Dyno-Mill

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use chemicals to solubilise the components in the cell walls to release the product Chemical requirements : - products are insensitive to the used chemicals. - the chemicals must be easily separable. Types of chemicals: Chemical Methods Alkalies Used for extraction of some bacterial proteins Eg: Recombinant growth hormone can be efficiently released from E.coli by treatment with sodium hydroxide

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Organic solvents Methanol, ethanol, isopropanol, butanol can be used to disrupt the cells. Toluene is frequently used ( dissolves membrane phospholipids and creates membrane pores for release of intracellular products)

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Detergents

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Cationic detergent: trimethyl ammonium bromide Anionic detergent: sodium lauryl sulfate Denature proteins Detergents affect purification steps , particularly in salt precipitation This limitation can be overcome by ultrafiltration or ion exchange chromatography for purification Drawback:

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Enzymatic methods Lysis of cells occur under mild conditions Bacterial cells - Lysozyme is frequently used ( which hydrolyses beta 1,4 glycosidic bonds of mucopeptide in bacterial cell walls. Gram positive bacteria are highly susceptible to lysozyme than Gram negative bacteria For gram negatives : Lysozyme with EDTA is used Yeast cell lysis: glucanase, mannanase with proteases are used Streptolysin – permeabilize mammalian cells

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