Cell disruption

Disruption of microbial cells is rendered difficult due to the presence of the microbial cell wall. Despite this, a number of very efficient systems exist that are capable of disrupting large quantities of microbial biomass (Table 6.1). Disruption techniques, such as sonication or treatment with the enzyme lysozyme, are usually confined to laboratory-scale operations, due either to equipment limitations or on economic grounds.

Protein extraction procedures employing chemicals such as detergents are effective in many instances, but they suffer from a number of drawbacks, not least of which is that they often induce protein denaturation and precipitation. This obviously limits their usefulness. Furthermore, even if the chemicals employed do not adversely affect the protein, their presence may adversely affect a subsequent purification step (e.g. the presence of detergent can prevent proteins from binding to a hydrophobic interaction column). In addition, the presence of such materials in the final preparation, even in trace quantities, may be unacceptable for medical reasons.

Disruption of microbial cells (and, indeed, some animal/plant tissue types) is most often achieved by mechanical methods, such as homogenization or by vigorous agitation with abrasives.

Table 6.1 Some chemical, physical and enzyme-based techniques that may be employed to achieve microbial cell disruption

Treatment with chemicals: detergents antibiotics solvents (e.g. toluene, acetone) chaotropic agents (e.g. urea, guanidine) Exposure to alkaline conditions Sonication Homogenization

Agitation in the presence of abrasives (usually glass beads) Treatment with lysozyme

During the homogenization process a cell suspension is forced through an orifice of very narrow internal diameter at extremely high pressures. This generates extremely high shear forces. As the microbial suspension passes through the outlet point, it experiences an almost instantaneous drop in pressure to normal atmospheric pressure. The high shear forces and subsequent rapid pressure drop act as very effective cellular disruption forces, and result in the rupture of most microbial cell types (Figure 6.5). In most cases a single pass through the homogenizer results in adequate cell breakage, but it is also possible to recirculate the material through the system for a second or third pass.

An efficient cooling system minimizes protein denaturation (denaturation would otherwise occur due to the considerable amount of heat generated during the homogenization process). Ho-mogenizers capable of handling large quantities of cellular suspensions are now available, many of which can efficiently process several thousand litres per hour.

An additional method often employed to achieve microbial cell disruption, both at the laboratory level and on an industrial scale, involves cellular agitation in the presence of glass beads. In such bead mills, the microorganisms are placed in a chamber together with a quantity of glass beads of 0.2-0.3 mm in diameter. This mixture is then shaken/agitated vigorously, resulting

- Piston

Frozen sample Narrow-bore orifice

- Piston

Figure 6.5 Diagrammatic representation of a cell homogenizer. This represents one of a number of instruments routinely used to rupture microbial cells, and in some cases animal/plant tissue in numerous collisions between the microbial cells and the glass beads. It also results in the grinding of cells between the rotating beads. These forces promote efficient disruption of most microbial cell types. Operational parameters, such as ratio of cells to beads and the rate/duration of agitation, may be adjusted to achieve optimum disruption of the particular cells in question. Laboratory systems can homogenize several grams of microbial cells in minutes. Industrial-scale bead-milling systems can process in excess of 1000 l of cell suspension per hour. Cooling systems minimize protein inactivation by dissipating the considerable heat generated during this process.

Upon completion of the homogenization step, cellular debris and any remaining intact cells can be removed by centrifugation or by microfiltration. As mentioned previously, these techniques are also used to remove whole cells from the medium during the initial stages of extracellular protein purification.

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