Intracellular enzymes recovery

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... 

Giovenco, S., Laane, C., and Hilhorst R. (1987). Recovery of intracellular enzymes from bacterial cells using reverse micelles. Proc. Eur. Congr. Biotechnol. 4th, Amsterdam, Vol. 2, p. 503. 

Submerged fermentation can be conducted in different modes of operation. The most traditional is batch fermentation, in which the bioreactor is filled with medium, inoculated and incubated under controlled conditions to the point in which the product (enzyme) has been synthesized to (or nearly to) its maximum level then the cells are harvested for enzyme recovery, if intracellular, or else discarded to recover the medium containing the enzyme, if extracellular. Fed-batch fermentation is a variant of the former in which, after certain time of batch cultivation, the bioreactor is fed with nutrients according to a controlled rate profile and up to a final volume and the product is then recovered as above. This mode of cultivation is particularly appealing for the production of enzymes because it allows the control of the metabolic... 

If the enzyme is excreted during cell growth, recovery will proceed from the liquid phase if the enzymes remains associated or within the cell, recovery will proceed from the solid phase (see Fig. 2.1). Excretion is desirable from a process perspective, so efforts have been made for genetically engineered cells to export otherwise intracellular enzymes (Hatti-Kaul and Mattiasson 2003a). 

Analysis in terms of excluded volume. J Biol Chem 256(23) 12108-12117 Augenstein DC, Thrasher K, Sinskey AJ et al. (1974) Optimization in the recovery of a labile intracellular enzyme. Biotechnol Bioeng 16 1433-1447 Avgerinos CG, Drapeau D, Socolow JS et al. (1990) Spin filter perfusion system for high density ceU culture production of recombinant urinary type plasminogen activator in CHO ceUs. Bio/Technology 8 54-58... 

Enzymes can be produced by submerged fermentation and recovered either from the fermentation medium, extracellular enzymes, or from the cell or intracellular enzyme (Geciova et al., 2002). Generally, the microbial enzyme production process is divided into (1) enzyme synthesis, where cells are produced (2) enzyme recovery, where the enzymes are extracted from the cells produced and (3) enzyme purification, where contaminants are removed. These steps may then be followed by enzyme formulation to reach the final desired form. 

As mentioned earlier, microbial enzymes are produced by submerged fermentation, in which a bioreactor is filled with medium and inoculated with specific microbial cells. The mixture remains under controlled conditions until a sufficient level of prodnction is obtained. If the enzyme is intercellular, the cells need to be harvested for enzyme recovery but in extracellular enzymes, this is not required. Some enzymes can be intracellular in one organism and extracellular in another. For example p-galactosidase (lactase) is extracellular in Penicillium sp. but intracellular in Kluyveromyces marxianus (Stred ansky et al., 1993). In addition, intracellular enzymes can be changed to extracellular by genetic and protein engineering modifications (Geciova et al., 2002). 

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. 

Initially fermentation broth has to be characterised on the viscosity of the fluid. If the presence of the biomass or cells causes trouble, they have to be removed. Tire product is stored inside the cells, the cells must be ruptured and the product must be freed. Intracellular protein can easily be precipitated, settled or filtered. In fact the product in diluted broth may not be economical enough for efficient recovery. Enrichment of the product from the bioreactor effluents for increasing product concentration may reduce the cost of product recovery. There are several economical methods for pure product recovery, such as crystallisation of the product from the concentrated broth or liquid phase. Even small amounts of cellular proteins can be lyophilised or dried from crude solution of biological products such as hormone or enzymes.2,3... 

LY294002 and wortmannin inhibit the enzyme PI-3 kinase required for the closure of pseudopodia to form intracellular vesicles (61,73-75,78,122). Compared to wortmannin, which is relatively unstable in aqueous media, the inhibitory effects of LY294002 are more specific, reversible (recovery after 10 minutes), and not light dependent. Therefore, LY294002 can be used for time-lapse experiments. Several studies have indicated that both substances have little or no effect on the other pathways described (75). However, both substances might block the uptake of Tfn as well (76). 

This chapter will focus on a unique problem encountered during recovery of intracellulary produced proteins. Disruption of cells produces a mixture of nucleic acids and proteins in the solution from which the desired proteins must be fractionated. Typical separation schemes involve first the removal of nucleic acids from solution by precipitation. The desired protein is then isolated and purified from the mixture of remaining nucleic acids and proteins. A scheme for recovery of intracellular bacterial enzyme tartrate dehydrogenase from cell paste is shown in Fig. 1. Material balance at the different stages of the scheme in two different experiments showed that 53-60% loss in enzyme activity took place during precipitation of nucleic acids by protamine sulfate and during ammonium sulfate fractionation of proteins (Table 2). Reduction in volume, removal of major nonprotein... 

FIGURE I Flow diagram for recovery of intracellular bacterial enzyme tartrate dehydrogenase (TDH). 

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. 

The use of isolated purified enzymes obviously avoids the formation of unwanted byproducts which may be generated from otherwise present and usually unknown enzymes. Isolated enzymes may be more active and selective than whole cells, but their recovery and purification is very expensive. Whole cell biotransformations show a lot of advantages such as the increased stability of enzymes which exist in whole cells. If these enzymes depend on cofactors, whole cell biotransformations become even more favorable because the use of whole cells allows the intracellular cofactor pool to be exploited, and any addition of exogenous cofactors becomes unnecessary. Otherwise the costs for the required cofactors often exceed that for the enzyme or the value of the product. Therefore, whole cell biotransformations are very interesting for industrial applications. 

Some bioproducts are derived from fermentation by living organisms but immobilized enzymes can also catalyze a biotransformation. If the product is extracellular, then the initial steps of recovery include removal of the cells and other particulate matters from the broth. If the product is intracellular, it would be necessary to lyse the cells to release the product into the broth. The cell debris is then separated before the product is recovered from the broth. In certain cases, proteins produced as IBs need to be solubilized and the proteins renatured before further recovery steps. In the case of biotransformed products, the immobilized cells or enzymes and their support need to be removed initially. 

To begin, enzyme products can be inside the cell (intracellular), loosely associated with the cell, or secreted (extracellular). Each of these products requires a different approach on how to purify the enzyme. The majority of presently commercialized detergent enzymes is of the extracellular variety and can be recovered directly from the fermentation broth. Thus, the primary recovery challenge involves removing the cells from the broth, aptly called cell separation. Three different techniques are commonly used to achieve this goal filtration, microfiltration, and centrifugation. 

The process of recovering and purifying fermentation products in the biochemical industries is generally difficult and costly. The product can exist intracellularly or extracellularly and it may be sensitive to temperature change, extremes of pH, certain chemicals and enzyme activities of the microorganisms. Frequently, the energy and labor costs spent on recovery and purification of the fermentation product far exceed the cost of fermentation. This is especially true for intracellular recombinant protein products ... 

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