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.
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.
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