Cell disruption enzymatic

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. 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

Biological Methods Enzymatic digestion of the cell wall is a good example of biological cell disruption. It is an effective method that is also very selective and gentle, but its high cost makes it impractical to be used for large-scale operations. 

Once purified, DNA is a fairly stable polymer if stored appropriately. Since living cells contain many other complex biomolecules besides DNA, methods exist that allow the isolation of DNA in pure form. More details on this topic are presented in Chapter 8. Routine methods of DNA isolation in solution, however, cause some unavoidable shearing of DNA due to hydro-dynamic shear forces, and as a result, the average size of isolated DNA is about 100 to 200 kilobases (kb). The basic steps in DNA isolation involve cell disruption and lysis by treatment with detergents, removal of cellular proteins by either enzymatic digestion with a protease or extraction with... 

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. 

NMR spectroscopy has so far been suited for non-invasive investigation of biochemical structures, fluxes through pathways, distribution of marker-nuclei among various cellular components and enzymatic mechanisms rather than for quantitative determination of small molecules. Biochemical applications have involved NMR spectroscopy mainly for structural determination of complex molecules, e.g. [27,180], as well as inside the cells, i.e. in vivo [184,189]. In biotechnology, the potential of determining intracellular components without cell disruption is increasingly used for in vivo studies of metabolism, e.g. [15,55,88, 121,146,197,250-252,271,335], and effectors [419]. 

The basis utilized by Petrides et al.15 is 1500 kg of purified BHI per year. They indicate that this represents 10-15% of the world demand.17 In essence, the following downstream steps are involved in sequence during synthetic BHI production. The fermentation step (not a downstream step) is also included to provide some continuity. The steps are fermentation, cell harvesting, cell disruption, inclusion body recovery, inclusion body solubilization, enzymatic conversion, refolding, sulfitolysis, CNBr cleavage, final purification steps, and crystallization. In the flow chart provided by Petrides et al.15, a surge tank separates the upstream from the downstream processes. This tank is in between the fermentor and the downstream processing steps. 

Intracellular Products. Intracellular production of bioproducts is less preferable but sometimes the only way to produce certain compounds in appreciable amounts. In this case, cell disruption is required for recovery. High-pressure homogenization, bead mills, and chemical or enzymatic disruption of the cell wall with lysozyme or similar enzymes can be used to achieve cell breakage. In the case of small molecules, organic solvent extraction has also been described. If cell debris remains in the centrate, it must be removed by methods described above, thus adding extra steps to the process. 

Where the product must be captured from the intracellular environment, the first downstream processing steps are cell disruption and the removal of debris. Several methods have been used for disruption, including sonication, pressure homogenization, enzymatic treatment, and wet... 

PHA is accumulated intracellularly in Gram-negative bacterial strains and normally its recovery after the fermentation include several steps - briefly these are (i) the separation of cells from the fermentation broth by centrifugation (ii) after that, the bacterial cells are pre-treated by heat, freeze dried, or salted, before extraction to avoid polymer degradation (iii) the PHA is therefore extracted, normally by using chlorinated solvents or other methods such as enzymatic digestion or mechanical cell disruption and (iv) PHA purification. The process of PHA recoveiy is shown in Figure 2.9. 

A variety of cell disruption methods are available. Physical, chemical, and enzymatic methods have all been used. The proper method should be carefully selected to ensure maximum cell disruption with minimum enzyme damage. This depends on the enzyme source, nature and stability. 

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