Enzyme extracted intracellular

The concentration of enzyme is veiy low, about several hundred milligrams per litre in the fermentation broth. Solvent extraction is a suitable process to recover a small amount of enzyme. The chance of some enzyme being intracellular is high, therefore cells are ruptured to liberate enzyme, which can then interact with organic solvents. Figure 7.1 shows a simple diagram for a jacketed fermentation vessel for operation at constant temperature. 

Beginning with Eduard Buchner s discovery (c. 1900) that an extract of broken yeast cells could convert glucose to ethanol and C02, a major thrust of biochemical research was to deduce the steps by which this transformation occurred and to purify and characterize the enzymes that catalyzed each step. By the middle of the twentieth century, all ten enzymes of the glycolytic pathway had been purified and characterized. In the next 50 years much was learned about the regulation of these enzymes by intracellular and extracellular signals, through the kinds of allosteric and covalent mechanisms we have described in this chapter. The conventional wisdom was that 1860 1917 in a linear pathway such as... 

Enyzmes are present in all plant cells, and in greatest variety in seeds. Most plant enzymes are intracellular as they cannot diffuse through the cell wall. In a few cases the enzyme can be obtained from the plant juice, for example, the enzyme bromelain can be obtained from the stem of the pineapple. Otherwise, methods of extraction are used which depend on the rupturing of the cell wall, for example, grinding the plant with sand, either with or without freezing, or drying of the tissue. 

This reaction has been carried out by batch procedure, which has disadvantages for Industrial purpose. Thus, we studied the continuous production of L-aspartlc acid using a column packed with immobilized aspartase. As the aspartase is an Intracellular enzyme, it was necessary to extract the enzyme from microbial cells before Immobilization. Extracted Intracellular enzyme is generally unstable, and most of the Immobilization methods we tried resulted in low activity and poor yield. Although entrapment into polyacrylamide gel lattice gave relatively active immobilized aspartase, its operational stability was not sufficient,... 

The crude extract or clarified broth containing the enzyme is then subjected to purification, conceived as a sequence of operations (see Fig. 2.1) aimed to remove all contaminants that can interfere with its intended use. Purification can also serve to the purpose of increasing the specific activity of the biocatalyst in the case of enzyme immobilization. The situation will be radically different for an intracellular than for an extracellular enzyme. In the first case, the enzyme extract is a complex mixture of proteins, nucleic acids and other cell constituents, while in the latter the enzyme concentrate contains only some extracellular proteins and small molecular weight solutes, since the cell membrane acts as a powerful barrier to retain most of the cell constituents aiding powerfully to purification. Many extracellular hydrolases... 

The foremost difference between extracellular and intracellular enzymes purification is the need for cell disruption. If the enzyme is intracellular, extraction takes place after cell lysis, which adds an additional step and costs to the purification process. This is not required for extracellular enzymes. Once the cells have been broken down and the intercellular enzymes are released, purification techniques similar to those used with extracellular enzymes are applicable. Intracellular enzymes from animal sources can be easily extracted due to the absence of a cell wall, whereas enzymes from plant and microbial cells require more force due to their rigid cell wall structure. Another approach to facilitate intracellular cell isolation is by genetic engineering. Details of genetic engineering are given in Section 1.6. 

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. 

Since 1978, several papers have examined the potential of using immobilised cells in fuel production. Microbial cells are used advantageously for industrial purposes, such as Escherichia coli for the continuous production of L-aspartic acid from ammonium fur-marate.5,6 Enzymes from microorganisms are classified as extracellular and intracellular. If whole microbial cells can be immobilised directly, procedures for extraction and purification can be omitted and the loss of intracellular enzyme activity can be kept to a minimum. Whole cells are used as a solid catalyst when they are immobilised onto a solid support. 

The enzymatic activity in soil is mainly of microbial origin, being derived from intracellular, cell-associated or free enzymes. Only enzymatic activity of ecto-enzymes and free enzymes is used for determination of the diversity of enzyme patterns in soil extracts. Enzymes are the direct mediators for biological catabolism of soil organic and mineral components. Thus, these catalysts provide a meaningful assessment of reaction rates for important soil processes. Enzyme activities can be measured as in situ substrate transformation rates or as potential rates if the focus is more qualitative. Enzyme activities are usually determined by a dye reaction followed by a spectrophotometric measurement. 

Intracellular messengers A biphasic effect of ginkgo extract is seen on cAMP phosphodiesterase under in vitro and ex vivo conditions (Saponara and Bosisio 1998 Macovschi et al. 1987). Whereas low concentrations (0.25-4.0 mg/L) activate the enzyme, higher concentrations (5-250 mg/L), dose-dependently inhibit it. However, tolerance develops to this effect because it is undetectable after daily administration for 4 days. Thus, ginkgo may initially produce effects by inhibiting enzymatic breakdown of cAMP. This mechanism is similar to the stimulant caffeine, but it is not likely to explain any long-term effects of ginkgo because it disappears after chronic daily treatment. The responsible constituent for this effect has not been identified. 

Aqueous extracts of cotton bract and mill dust are leuco-tactic (15,40). In experimental animals, aqueous extracts of cotton dust (AECD) attract PMNs to airways in a dose dependent fashion beginning at 3 hours, and reaching a peak at 18-24 hours. Macrophage levels Immediately drop and remain low until 18 hours post-exposure when a modest increase occurs. Cell infiltration, with subsequent release of intracellular enzymes and mediators, is thought to be important in the pathology of byssinosis (15). 

A. bronchisepticus was cultivated aerobically at 30 °C for 72 h in an inorganic medium (vide supra) in 1 liter of water (pH 7.2) containing 1 % of polypeptone and 0.5 % of phenylmalonic acid. The enzyme was formed intracellularly and induced only in the presence of phenylmalonic acid. All the procedures for the purification of the enzyme were performed below 5 °C. Potassium phosphate buffer of pH 7.0 with 0.1 mM EDTA and 5 mM of 2-mercaptoethanol was used thoughout the experiments. The enzyme activity was assayed by formation of pheylacetic acid from phenylmalonic acid. The summary of the purification procedure is shown in Table 2. The specific activity of the enzyme increased by 300-fold to 377 U/mg protein with a 15% yield from cell-free extract [9]. One unit was defined as the amount of enzyme which catalyzes the formation of 1 mmol of phenylacetic acid from phenylmalonic acid per min. 

Reverse micelles of CTAB in octane with hexanol as cosurfactant were reported to be able to lyse whole cells quickly and accommodate the liberated enzyme rapidly into the water pool of surfactant aggregates [50,51]. In another case a periplasmic enzyme, cytochrome c553, was extracted from the periplasmic fraction using reverse micelles [52]. The purity achieved in one separation step was very close to that achieved with extensive column chromatography. These results show that reverse micelles can be used for the extraction of intracellular proteins. 

Endogenous microbial enzymes are sometimes utilized to break down their parent cells, and thus extract valuable intracellular materials. For instance, in the production of yeast extract, cells are allowed to autolyse at about pH 5 and 55 0. Proteases are probably the most important class of enzymes involved in autolysis, although others such as glucanases, lipases and nucleases also have... 

---