Cell disruption techniques

Separation from culture media or broth is the primary step in collecting the product found either in cells (sohd) or medium (liquid). This initial separation step is engineered based on cell size and density differences between solid and liquid (Table 4.10). In the case where the recombinant product is localized in the intracellular content such as the cytoplasm or inclusion bodies, which are highly insoluble particles found in bacteria, the cells are hrst isolated from the medium and then disrupted to collect the recombinant protein fraction. A number of cell disruption techniques have been developed to facilitate this step, and some are listed in Table 4.11. 

The separation schemes vary with the state of the products. For example, intracellular products must first be released by disrupting the cells, while those products bound to cell membranes must be solubilized. As the concentrations of products secreted into the fermentation media are generally very low, the recovery and concentration of such products from dilute media represent the most important steps in downstream processing. In this chapter, several cell-liquid separation methods and cell disruption techniques are discussed. 

Cell disruption techniques are used to recover materials produced within the cell, for example, industrial enzymes and some pharmaceutical proteins. Generally this stage of bioseparation will follow cell recovery, for example, by centrifugation, and precede the isolation of the desired product from the cell debris which is also produced during the disruption process. 

Mechanical cell disruption techniques are based on high shear effects as fluid is forced through a narrow orifice, or a chamber containing rotating disks and glass beads to break up the cellular material. Both techniques enable some control over the extent of cell disruption. 

Figure 2.8 Cell disruption techniques. Adapted from Chisti and Moo-Young (1986). ...

A widely used technique for cell disruption is high-pressure homogenisation. Shear forces generated in this treatment are sufficient to completely disrupt many types of cell. A common... 

Models based on Eqs. (47)-(50) have been used in the past to describe the disruption of unicellular micro-organisms and mammalian (hybridoma) cells [62]. The extent of cell disruption was measured in terms of loss of cell viability and was found to be dependent on both the level of stress (deformation) and the time of exposure (Fig. 25). All of the experiments were carried out in a cone and plate viscometer under laminar flow conditions by adding dextran to the solution. A critical condition for the rupture of the walls was defined in terms of shear deformation given by Eq. (44). Using micromanipulation techniques data were provided for the critical forces necessary to burst the cells (see Fig. 4)... 

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. 

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

Y. Aoki, K. Akagi, Y. Tanaka, J. Kawai, M. Takahashi, Measurement of intratumor pH by pH indicator used in F MR spectroscopy. Invest. Radiol. 31 (1996) 680-689. R.Y. Tsien, A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290 (1981) 527-528. 

More recently the biotransformation of limonene by another Pseudomonad strain, P. gladioli was reported [76,77]. P. gladioli was isolated by an enrichment culture technique from pine bark and sap using a mineral salts broth with limonene as the sole source of carbon. Fermentations were performed during 4-10 days in shake flasks at 25°C using a pH 6.5 mineral salts medium and 1.0% (+)-limonene. Major conversion products were identified as (+)-a-terpineol and (+)-perillic acid. This was the first time that the microbial conversion of limonene to (+)-a-terpineol was reported, see pathway 4. The conversion of limonene to a-terpineol was achieved with an enzyme, a-terpineol dehydratase (a TD), by the same group [78]. The enzyme, purified more than tenfold after cell-disruption of Pseudomonas gladioli, stereospecifically converted (4 )-(+)-limonene to (4/ )-(+)-a-terpineol or (4S)-(+)-limonene to (4S)-(+)-a-terpineol. a-Terpineol is widely distributed in nature and is one of the most commonly used perfume chemicals [27]. 

Sonic or ultrasonic waves of sufficient intensity can disrupt and kill cells. This technique is usually employed in the disruption of cells for the purpose of extracting mtracellular constituents rather than as a sterilization technique. 

Once the cellular materials are separated, those with intracellular proteins need to be ruptured to release their products. Disruption of cellular materials is usually difficult because of the strength of the cell walls and the high osmotic pressure inside. The cell rupture techniques have to be very powerful, but they must be mild enough so that desired components are not damaged. Cells can be ruptured by physical, chemical, or biological methods. 

Data of release rates for seven enzymes from baker s yeast with a high-pressure homogenizer revealed that differences in release rates agreed with reported locations in the cell but are not sufficient to fractionate the enzymes release rates did not seem to depend much on operating pressure, temperature, or initial cell concentration (Follows, 1971). Both the dependence of release rates on the location within the cell and the description of release by a first-order law has been confirmed in both yeast and E. coli with several disruption techniques such as sonica-tion, high-pressure homogenization, and hydrodynamic cavitation (Balasundaram, 2001). 

The recovery of intracellular proteins involves distinct cell disruption procedures, depending on the cell characteristics. For the processing of animal cells, which do not have a cellular wall, mild and moderate techniques are commonly used. Mild techniques include cell lysis by enzymatic digestion, chemical solubilization or autolysis and the use of manual homogenizers and grinders, whereas the moderate techniques involve blade homogenizers and abrasive grinding. 

There are a range of physical and chemical methods available at laboratory scale for cell disruption which involve the use of reagents or temperature and pressure changes to break the cell wall to release the desired products. However, at an industrial scale it is more common to use a mechanical disruption technique, and a number of companies have developed efficient... 

Since cell disruption is a relatively specialized technique with specific difficulties depending on the organism being handled, it is worth obtaining assistance from the equipment manufacturer at an early stage, and possibly using a small-scale version of the apparatus to carry out pilot scale tests. 

Another use of cell disruption as a step in the analytical process is for obtaining a suspension of single cells — that can be used under optimal fermentation conditions — by ultrasonic disruption of cells manufactured in active dry wine yeast. Their potential was confirmed by comparing the elution profiles of non-sonicated and sonicated yeast sample dispersions obtained using two different field flow fractionation techniques [88]. 

The composition of the lysis solution is dictated by the nature of the proteins under study and the subsequent techniques applied to the sample. One of the major choices to be made is whether or not a detergent is required at this stage. If the membrane and soluble fractions are to be separated the initial cell disruption protocol should not include a detergent, as many of the membrane proteins would be solubilized. In this case physical disruption of the cells should be used (e.g., sonication of cells or homogenization of tissues). The choice of lysis conditions is a vital consideration in this work, as proteins need to be solubilized while preservation of posttranslational modifications, inhibition of proteases, maintenance of protein-protein interactions, and, if an immunoaffinity purification step is to be performed, suitability for the antibody to function are essential. For example, SDS is very good at solubilizing membrane proteins but... 

Proteins are part of a dynamic network of biomolecules that interact to regulate their localization and function within the cell. Disruption of this physical and chemical system of interactions has become the first paramount step in performing protein analysis by shotgun proteomics. Protein isolation techniques, for instance, have allowed understanding of the complex dynamics of proteins among cellular subcompartments, such as the nucleolus or the mitochondrion (6). Membrane-embedded proteins (7) and DNA-binding transcription factors (8) are two other prominent examples where inadequate protein extraction may hamper further analysis by LC-MS. 

Techniques Careful cell disruption and specific extraction procedures may lower the... 

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