Ultrasonic Cell Disruption

The treatment of microbial cells in suspension with inaudible ultrasound (greater than about 18 kHz) results in their inactivation and disruption. Ultrasonication utilizes the rapid sinusoidal movement of a probe within the liquid. It is characterized by high frequency (18 kHz to 1 MHz), small displacements (less than about 50 m), moderate velocities (a few m s–1), steep transverse velocity gradients (up to 4,000 s–1) and very high acceleration (up to about 80,000 g). Ultrasonication produces cavitation phenomena when acoustic power inputs are sufficiently high to allow the multiple production of microbubbles at nucleation sites in the fluid. The bubbles grow during the rarefying phase of the soundwave, then are collapsed during the compression phase. On collapse, a violent shockwave passes through the medium. The whole process of gas bubble nucleation, growth, and collapse due to the action of intense soundwaves is called cavitation. The collapse of the bubbles converts sonic energy into mechanical energy in the form of shockwaves equivalent to several thousand atmospheres of (300 MPa) pressure. This energy imparts motions to parts of cells, which disintegrate when their kinetic energy content exceeds the wall strength. An additional factor that increases cell breakage is the microstreaming (very high-velocity gradients causing shear stress), which occurs near radially vibrating bubbles of gas caused by the ultrasound.

Much of the energy absorbed by cell suspensions is converted to heat, so effective cooling is essential.

Equipment for the large-scale continuous use of ultrasonics has been available for many years, and is widely used by the chemical industry, but has not yet found extensive use in enzyme production. Reasons for this may be the conformational lability of some (perhaps most) enzymes to sonication, and the damage that they may realize through oxidation by the free radicals, singlet oxygen and hydrogen peroxide, that may be concomitantly produced. Use of radical scavengers (e.g., N2O) has been shown to reduce this inactivation. As with most cell breakage methods, very fine cell debris particles may be produced, which can hinder further processing. Sonication remains, however, a popular, useful, and simple small-scale method for cell disruption.

High-Pressure Homogenizers
Various types of high pressure homogenizer are available for use in the food and chemical industries, but the design that has been very extensively used for cell disruption is the Manton Gaulin APV type homogenizer. This consists of a positive displacement pump that draws cell suspension (about 12% w/v) through a check valve into the pump cylinder and forces it, at high pressures of up to 150 MPa (10 tons per square inch) and flow rates of up to 10,000 liter per hour, through an adjustable discharge valve that has a restricted orifice. Cells are subjected to impact, shear, and a severe pressure drop across the valve, but the precise mechanism of cell disruption is not clear. The main disruptive factor is the pressure applied and the consequent pressure drop across the valve. This causes the impact and shear stress, which are proportional to the operating pressure.

The cell suspension is pumped at high pressure through the valve, impinging on it and the impact ring. The shape of the exit nozzle from the valve seat varies between models and appears to be a critical determinant of the homogenization efficiency.

The location of an enzyme within the cells can influence the conditions of use of a homogenizer. Unbound intracellular enzymes may be released by a single pass, whereas membrane-bound enzymes require several passes for reasonable yields to be obtained. Multiple passes are undesirable because, of course, they decrease the productivity rate, and because the further passage of already broken cells results in fine debris that is excessively difficult to remove further downstream. Consequently, homogenizers will be used at the highest pressures compatible with the reliability and safety of the equipment and the temperature stability of the enzyme(s) released. High-pressure homogenizers are acceptably good for the disruption of unicellular organisms provided the enzymes needed are not heat-labile. The shear forces produced are not capable of damaging enzymes free in solution. The valve unit is prone to erosion and must be precision-made and well maintained.

Use of Lytic Methods
The breakage of cells using nonmechanical methods is attractive because it offers the prospect of releasing enzymes under conditions that are gentle, do not subject the enzyme to heat or shear, may be very cheap, and are quiet to the user. The methods that are available include osmotic shock, freezing followed by thawing, cold shock, desiccation, enzymic lysis, and chemical lysis. Each method has its drawbacks, but may be particularly useful under certain specific circumstances.

Certain types of cell can be caused to lyse by osmotic shock. This would be a cheap, gentle, and convenient method of releasing enzymes, but has not apparently been used on a large scale. Some types of cell may be caused to autolyse, in particular yeasts and Bacillus species. Yeast invertase preparations employed in the industrial manufacture of invert sugars are produced in this manner. Autolysis is a slow process compared with mechanical methods, and microbial contamination is a potential hazard, but it can be used on a very large scale if necessary. Where applicable, dessication may be very useful in the preparation of enzymes on a large scale. The rate of drying is very important in these cases, and slow methods are preferred to rapid ones like lyophilization.

Enzymic lysis using added enzymes has been used on the laboratory scale but is mainly used for industrial purposes. Lysozyme, from hen egg white, is the only lytic enzyme available on a commercial scale. It is used to lyse Grampositive bacteria in an hour at about 50,000 U/Kg (dry weight). Although costs are reduced by the use of inexpensive, lysozyme-rich, dried egg white, a major separation problem may be introduced. Yeast-lytic enzymes from Cytophaga species have been studied in some detail and other lytic enzymes are under development. If significant markets for lytic enzymes are identified, the scale of their production will increase and their cost is likely to decrease. Lysis by acid, alkali, surfactants, and solvents can be effective in releasing enzymes, provided that the enzymes are sufficiently robust. Detergents, such as Triton X-100, used alone or in combination with certain chaotropic agents, such as guanidine HCl,
are effective in releasing membrane-bound enzymes. However, such materials are costly and may be difficult to remove from the final product.