Intracellular product

Fermentation broths are complex, aqueous mixtures of cells, comprising soluble extracellular, intracellular products and any unconverted substrate or unconvertible components. Recovery and extraction of product is important in bioprocess engineering. In particular separation is a useful technique it depends on product, its solubility, size of the process, and product value. Purification of high-value pharmaceutical products using chromatography such as hormones, antibody and enzymes is expensive and difficult to scale up.1 Tire necessary steps to follow a specific process depend on the nature of the product and the characteristics of the fermentation broth. There are a few steps for product recovery the following processes are discussed, which are considered as an alternative for product recovery from fermentation broth. 

It is often desirable to recover product and to choose a suitable strain of microorganism which produces extracellular rather than intracellular product. If the product stays inside the cells, the cells must be ruptured, so freeing intracellular enzyme, after which extraction or purification is performed to recover the valuable product. The fermentation broth has to be processed, and pass through several stages for separation and purification. The product requites a sequence of operations for high purification. Hie usual steps to follow are as follows. 

Intracellular products can be present either as folded, soluble proteins or as dense masses of unfolded protein (inclusion bodies). For these products, it is first necessary to concentrate the cell suspension before effecting release of the product. Filtration can result in a suspension of cells that can be of any desired concentration up to 15 to 17 percent and that can be diafiltered into the desired buffer system. In contrast, the cell slurry that results from centrifugation will be that of... 

Harrison STL (2002) Bacterial cell disruption A key unit operation in the recovery of intracellular products. Biotech Adv 9 217-240... 

Chisti Y, Moo-Young M (1986) Disruption of microbial cells for intracellular products. Enz Microb Tech 8 194—204... 

The importance of superoxide-mediated damage to cancer cells was also demonstrated in the experiments with overexpressed mitochondrial MnSOD. Hirose et al. [186] showed that the overexpression of mitochondrial MnSOD enhanced the survival of human melanoma cells exposed to cytokines IL-1 and TNF-a, anticancer antibiotics doxorubicin and mitomycin C, and y-irradiation. Similarly, Motoori et al. [187] found that overexpression of MnSOD reduced the levels of reactive oxygen species in mitochondria, the intracellular production of 4-hydroxy-2-nonenal, and prevented radiation-induced cell death in human hepatocellular... 

Yu J, Patel Sandeep A, Dickson RM (2007) In vitro and intracellular production of peptide-encapsulated fluorescent silver nanoclusters. Angew Chem Int Ed 46 2028-2030... 

Hartmann T, Bergsdorf C, Sandbrink R, Tienari PJ, Multhaup G Ida N, Bieger S, Dyrks T, Weidemann A, Masters CL, Beyreuther K. (1997) Distinct sites of intracellular production for Alzheimer s disease A beta 40/42 amyloid peptides. Nat Med 3 1016-1020. 

Adrenergic receptors These are membrane bound G-protein coupled receptors which function primarily by increasing or decreasing the intracellular production of second messengers cAMP or inositol triphosphate (IP3)/diacyl glycerol (DAG). Adrenergic receptors are classified into two main groups [Pg.131]

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. 

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. 

The resistance of many human cancers to immunotherapies has been attributed to the presence of immunosuppressive molecules located in tumor areas. Adenosine is present at elevated levels in hypoxic tissues due to an increased intracellular production and extracellular accumulation, as described above. This nucleoside activates cell surface receptors on T and NK cells that mediate cellular immune responses to tumor cells. It is well established that T cells recognize and destroy... 

The fermentation products can be the cells themselves (biomass), components within the fermentation broth (extracellular), or those trapped in cells (intracellular), examples of which are listed in Table 10.1. As shown in Figure 10.1, if the product of our interest is the cell, cells are separated from the fermentation broth and then washed and dried. In the case of extracellular products, after the cells are separated, products in the dilute aqueous medium need to be recovered and purified. The intracellular products can be released by rupturing the cells and then they can be recovered and purified. The downstream processing for enzyme reactions will be similar to the process for extracellular products. 

After solid and liquid are separated (and cells are disrupted in the case of intracellular products), we obtain a dilute aqueous solution,... 

Pignatelli P, Pulcinelli FM, Celestini A, Lenti L, Ghiselli A, Gazzaniga PP, Violi F. 2000. The flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular production of hydrogen peroxide. Am J Clin Nutr 72 1150-1155. 

If the product is secreted, recovery may involve a simple filtration step to remove any cells and cellular debris. Other clarification techniques include centrifugation and expanded bed adsorption [25], For such intracellular products as recombinant proteins produced in E. coli, the product may be denatured and located in inclusion bodies within the cells [26], Bacterial cells are typically concentrated by centrifugation or crossflow filtration, washed, and then disrupted by homogenization. Inclusion bodies are then isolated, and the protein product extracted and refolded. Validation of recovery operations for an E. coli product is described by Seely et al. [27],... 

Despite being more critical for intracellular products, protease action may also occur in extracellular media. For example, in the expression of a baculovirus vector, which is a lytic system, proteases are often expressed and secreted during recombinant protein production, and the problem may assume a critical level. A similar problem occurs in serum-free culture systems. The absence of proteins such as albumin and macroglobulin reduces the protection against proteolysis. In these cases, addition of an antiproteolytic agent to the culture medium is recommended. 

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