Disruption, cell

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. 

Agitation in the presence of abrasives (usually glass beads) 

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. 

The mode of action of many cell disrupters is to subject cells to very high pressures (up to 2,700 bar). If this high-pressure fluid is in contact with an 

The accumulated solids can then be recovered batchwise from the bowl. 

In those cases where the intracellular products are required, the cells must first be disrupted. Some products may be present in the solution within the cytoplasm, while others may be insoluble and exist as membrane-bound proteins or small insoluble particles called inclusion bodies. In the latter case, these must be solubilized before further purification. 

Ultrasonication (on the order of 20 kHz) causes high-frequency pressure fluctuations in the liquid, leading to the repeated formation and collapse of bubbles. 

The separation of cell debris (fragments of cell walls) and organelles from a cell homogenate by centrifugation may often be difficult, essentially because the densities of these are close to that of the solution, which may be highly viscous. Separation by microfiltration represents a possible alternative approach in such a case. 

Waring-type blender Homogenization by stirring blades 

When the radial distances from the rotational axis of a centrifuge to the liquid surface and the bowl wall are rx and r2, respectively, the axial liquid velocity u (m s 1) is given by 

Waring-type blender Ultrasonics Bead mills 

Application of ultrasonic energy to cell suspensions by sonicator Mechanical grinding of cell suspensions with grinding media such as glass beads 

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Biotechnology Cell disruption Yeast disruption for enzyme extraction... 

These units have been used to disrupt bacterial cells for release of enzymes. (See Cell Disruption. )... 

While the above discussion centered on the rate of disruption, the objective is usually to attain at least 90 percent release of the valuable protein from the cells. Cell disruption with protein solubilization is considered to be first order in amount of protein remaining [Currie et al., Biotechnol. Bioeng., 14, 725 (1972)] ... 

Cell Disruption Intracellular protein products are present as either soluble, folded proteins or inclusion bodies. Release of folded proteins must be carefully considered. Active proteins are subject to deactivation and denaturation, and thus require the use of gentle conditions. In addition, due consideration must be given to the suspending medium lysis buffers are often optimized to promote protein stability and protect the protein from proteolysis and deactivation. Inclusion bodies, in contrast, are protected by virtue of the protein agglomeration. More stressful conditions are typically employed for their release, which includes going to higher temperatures if necessaiy. For native proteins, gentler methods and temperature control are required. 

Chemical lysis, or solubilization of the cell wall, is typically carried out using detergents such as Triton X-100, or the chaotropes urea, and guanidine hydrochloride. This approach does have the disadvantage that it can lead to some denaturation or degradation of the produci. While favored for laboratory cell disruption, these methods are not typically used at the larger scales. Enzymatic destruction of the cell walls is also possible, and as more economical routes to the development of appropriate enzymes are developed, this approach could find industrial application. Again, the removal of these additives is an issue. 

In whole cell bioprocesses, extracellular products are preferable because this removes the requirement for cell disruption and tins reduces the level of impurities in the product solution. Nevertheless, product isolation and purification can be prohibitively expensive particularly for low concentration product streams, which is a feature of many bioprocesses. 

Costs of downstream processing for bioprocesses are increased by 1) low concentrations of products, 2) numerous impurities at low concentration and 3) intracellular materials (if cell disruption is necessary). However, the high specificity of biocatalysts is a benefit to downstream processing since products closely related to the desired product are less likely to be present Waste products of bioprocesses are likely to be less environmentally damaging, which also reduces downstream processing costs. 

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... 

During the purification of intracellular proteins, cell disruption by mechanical or biochemical means is the first step required in the process. However, it commonly initiates cellular and... 

CASE STUDY PROCESS INTEGRATION OF CELL DISRUPTION AND FLUIDISED BED ADSORPTION FOR THE RECOVERY OF LABILE INTRACELLULAR ENZYMES... 

Keywords cell disruption process integration fluidised bed adsorption intracellular enzymes protein recovery... 

The primary purification of the enzyme G3PDH was exploited herein as a preliminary study to investigate and demonstrate the feasibility of the integrated operation of cell disruption by bead milling and immediate product capture by fluidised bed adsorption (panel A in Figure 17.6). Yeast G3PDH binds nicotinamide adenine dinucleotide (NAD) as a cofactor,... 

Experiments investigated the integration of cell disruption by bead milling and product capmre by fluidised bed adsorption. By using fluidised bed adsorption, the clarification of the broth would be incorporated with the capture of the product which would result in a considerably... 

Fig. 17.9. Purity comparison (SDS-PAGE) of the conventional purification process and integrated cell disrupt tion/fluidised bed adsorption.The numbers given in the flow sheet indicate the origin of samples and correspond to their respective lane numbers. Lanes M, low molecular weight markers 1, Erwinia disruptate, 15% biomass ww/v 2, eluate CM HyperD LS, fluidised bed 3, desalted eluate (after dia/ultrafiltration, 30 K MWCO membrane) 4, flow-through, DEAE fixed bed 5, elution, DEAE fixed bed 6, eluate CM HyperD LS 7, CM cellulose eluate 8, CM cellulose eluate, final 9, final commercial product.

After fermentation, subsequent midstream to downstream processes such as cell disruption, centrifugation, extraction and drying will be carried on for product recovery. Fig. 9 shows a white sheet of PHB obtained from fermentation of sweet sorghum juice (SSJ) by Bacillus aryahhattai. 

This apparent time dependent cell disruption is caused because of the statistically random distribution of the orientation of the cells within a flow field and the random changes in that distribution as a function of time, the latter is caused as the cells spin in the flow field in response to the forces that act on them. In the present discussion this is referred to as apparent time dependency in order to distinguish it from true time-dependent disruption arising from anelastic behaviour of the cell walls. Anelastic behaviour, or time-dependent elasticity, is thought to arise from a restructuring of the fabric of the cell wall material at a molecular level. Anelasticity is stress induced and requires energy which is dissipated as heat, and if it is excessive it can weaken the structure and cause its breakage. 

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)... 

From the above description it will be appreciated that the efficiency of release of nutrients from ingested plant material is dependent upon the ease with which the digestive enzymes can penetrate the cell wall to release the nutrients so that they can diffuse out of the structure to be absorbed. Thus tissue maturity, cooking, macerating, mastication and mode of tissue failure, all of which control particle size, cell wall softening or cell disruption, are key features which regulate nutrient release. 

To check if PemB is surface exposed, E. chrysanthemi cells were subjected to proteolysis. Treatment of the cell suspension with trypsin, proteinase K or chimotrypsin at a concentration of 0.1 to 1 mg/ml for 1 h did not cause PemB proteolysis or its liberation into the medium. Cell pre-treatment with EDTA-lysozyme, which renders the periplasmic proteins accessible to proteases, gave no effect. PemB was also resistant to proteolytic digestion in extract of cells disrupted by sonication or in a French press. Only addition of Triton X-100 (up to 0.1%) causing formation of the micelles with PemB lead to a quick proteolyis of this protein (data not shown). In another approach to analyse the PemB exposition, bacterial cells were labelled with sulfo-NHS-biotin. This compound is unable to cross membranes and biotinylation... 

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