STELIOS T. TZANNIS - RPI 1991
TUTORIAL ON CELL DISRUPTION

A lot of biological molecules are inside the cell, and they must be released from it. This is achieved by cell disruption (lysis). Cell disruption is a sensitive process because of the cell wall's resistance to the high osmotic pressure inside them. Furthermore, difficulties arise from a non-controlled cell disruption, that results from an unhindered release of all intracellular products (proteins nucleic acids, cell debris) as well as the requirements for cell disruption without the desired product's denaturation.

There are mechanical and non-mechanical cell disruption methods. The results of these methods are often evaluated in terms of the activity level of a cellular enzyme released to the disrupted suspension, combining the efficiency of the disrupting process with an estimate of the degree of cell disruption.

MECHANICAL METHODS

Currently, intracellular products are released from microorganisms mainly by mechanical disruption of the cells. In this process, the cell envelope is physically broken, releasing all intracellular components into the surrounding medium. Equipment for cell disruption includes:
  • Homogenizers. An homogenizer has pumping of a slurry through a restricted orifice valve. They use high pressure (up to 1500 bar) followed by an instant expansion through a special exiting nozzle. Cell disruption is accomplished by three different mechanisms: impegment on the valve, high liquid shear in the orifice, and sudden pressure drop upon discharge, causing finally an explosion of the cell. The method is applied mainly for the release of intracellular molecules. According to Hetherington et al., cell disruption (and consequently the rate of protein release) is a first-order process, described by the relation : log[Rm/(Rm-R)] = K N P72.9 R : is the amount of soluble protein Rm: maximum amount of soluble protein K : Temperature dependent rate constant N : number of passes through the homoge- nizer (represents the residence time). P : operating pressure.
  • Ball Mills. In a ball mill, cells are agitated in suspension with small abrasive particles. Cells break because of shear forces, grinding between beads, and collisions with beads. The beads disrupt the cells to release biomolecules. The kinetics of biomolecule release by this method is also a first-order process.
  • Ultrasonic disruption. Another widely applied method is the cell lysis with high frequency sound that is produced electronically and transported through a metallic tip to an appropriately concentrated cellular suspension. Because of very high costs it is only used at laboratory scale for lysis of cells with less resistant cell walls, such as bacteria and fungi. The concept of ultrasonic disruption is based on the creation of cavities in cell suspension.
  • Blenders (high speed or Waring), the french press, or even centrifugation in case of weak cell walls, also disrupt the cells by using the same concepts.

    Disadvantages of Mechanical Disruption

    Mechanical disruption methods suffer from several drawbacks. Because cells are broken completely, all intracellular materials are released. Therefore, the product of interest must be separated from a complex mixture of proteins, nucleic acids, and cell wall fragments. Released nucleic acids may increase the viscocity of the solution and may complicate subsequent processing steps and especially chromatography, in case of laboratory experiments.

    The cell debris, produced by mechanical lysis, often consists of small cell fragments, making the solution diffi cult to clarify. Complete product release often requires more than one pass through the disruption device, exacerbating the problem by further reducing the size of the fragments. These are difficult to remove by conti nuous centrifugation, because the throughput of the device is inversely related to the square of the particle. diameter. Filtration is complicated by the gelatinous nature of the homogenate and by its tendency to foul membranes Furthermore, mechanical methods expose the cells, and hence the extracted product in very harsh conditions. While it is now generally accepted that most proteins can tolerate the and the high pressure inside an homogenizer or a ball mill, most will be denaturated by the heat generated unless the device is sufficiently cooled.

    NON MECHANICAL METHODS

    Another way to disrupt the cells is to cause their permeabilization. This can be acheived by numerous methods. The most important are :
    1. Chemical Permeabilization. Many chemical methods have been employed in order to extract intra cellular components from micro organisms by permeabilizing the outer-wall barriers. It can be achieved with organic solvents that act by creation of canals through the cell membrane: toluene, ether,phenylethyl alcohol DMSO, benzene, methanol, chloroform Chemical permeabilization can also be achieved with antibiotics, thionins, surfactants (Triton, Brij, Duponal) chaotropic agents, and chelates. A very important case is EDTA (chelating agent) widely used for permeabilization of Gram negative microorganisms. Its effectiveness is a result of its ability to bond the divalent cations of Ca++, Mg++. The last ones stabilize the structure of outer membranes, by bonding the lipopolysaccharides to each other. Once these cations are removed from the EDTA, the lipopolysaccharides are removed, resulting in increased permeability areas of the outer walls

      Chaotropic agents, such as urea and guanidine are capable of bringing some hydrophobic compounds into aqueous solutions. They accomplish this by disrupting the structure of water, making it a less hydrophilic enviro nment and weakening the hydrophobic interactions among solute molecules.

    2. Mechanical Permeabilization. One method is osmotic shock. While cells exposed to slowly varying extracellular osmotic pressure are usually able to adapt to such changes, cells exposed to rapid changes in external osmolarity, can be mechanically injured. This procedure is typically conducted by first allowing the cells to equilibrate internal and external osmotic pressure in a high sucrose medium, and then rapidly diluting away the sucrose. The resulting immediate overpressure of the cytosol is is assumed to damage the cell membrane. Enzymes released by this method are believed to be periplasmic, or at least located near the surface of the cell.
    3. Enzymatic Permeabiization. Enzymes can also be employed to permeabilize cells, but this method is often limited to releasing periplasmic or surface enzymes. In these procedures, they often use EDTA in order to destabilize the outer membrane of Gram negative cells, making the peptidoclycan layer accessible to the enzyme used. Enzymes used for enzymatic permeabilization are : beta(1-6) and beta(1-3) glycanases, proteases, and mannase.

      The main drawbacks of employing enzymatic means for recovering intracellular products in large scale processes are cost and the necessity of removing the lytic enzyme from the product.

    4. Other Permeabilization Techniques. Basic proteins, such as protamine, or the cationic polysaccharide chitosan can permeabilize yeast cells. Similarly, mammalian cells can be permeabilized by exposure to several natural substances such as streptolysin or even viruses. Electrical discharges have also been used to permeabilize mammalian cells in order to study secretion by exocytosis.

    REFERENCES

    1. J.E. Bailey & D.F. Ollis Biochemical Engineering Fundamentals, 2nd edition Mc Graw-Hill, 1987.

    2. T.J. Naglak, D.J. Hettwer, H.Y. Wang Chemical permeabilization of cells for intracellular product release in SEPARATION PROCESSES IN BIOTECH nology, MARCEL DEKKER INC, NY 1990.

    3. J. Bulock & B. Christiansen, BASIC biotechnology, Academic Press, 1989.

    4. J Brookman, Mechanism of cell Integration in High Pressure Homo genizer, Biotechnol. Bioeng., (1974), Vol. 16, p 371-383.

    5. H. Felix, ANALYTICAL BIOCHEMISTRY (1982) Vol. 107, p. 207-214.

    6. H Felix, PERMEABILIZED CELLS, Anal. Biochem. (1982) Vol. 120, p. 211-234.

    7. M. Follows, P.J. Hetherington, P. Dunnil, Release of Enzymes from Baker's Yeast By Disruption In an Industrial Homogenizer, Biotechnol. Bioeng., (1971), Vol. 13, p.549-560

    THAT'S ALL FOLKS!!!

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