Separation enzymes

A summary of the processes for producing the eicosanoids from the polyunsaturated fatty acid, arachidonic acid, is presented in Figure 11.27. The two enzymes separate for synthesising the prostanoids or the leucotrienes are cyclooxygenase and lipoxygenase, respectively. Whether prostanoids or leucotrienes are produced in any given tissue will depend on the relative activities of these two enzymes in that tissue. 

Cumulative Feedback Inhibition In cumulative feedback inhibition, the end products can inhibit the reaction of the target enzyme separately. Many textbooks erroneously indicate that the cumulative feedback inhibition of E. coli glutamine synthetase involves separate regulatory sites for each feedback inhibitor. See Cumulative Feedback Inhibition... 

If the bonded water is extracted by dry CO2 the enzyme is denaturated and loses its activity. Therefore a certain amount of water is necessary in the supercritical fluid because acting with water-saturated CO2 again causes an inhibition of the enzyme and consequent loss of activity. The optimal water concentration has to be determined for each enzyme separately. Table 9.2-1 shows the residual activity of lipase from Candida cylindracea, esterase from Mucor mihei, and esterase from Porcine liver after a incubation time of 22 hours in supercritical CO2 at 40°C. It is obvious that higher water concentrations cause a strong reduction in the residual activity compared to the activity of the untreated enzyme, which was set as 100 %. Further, the system-pressure has an influence because at higher pressures the activity-loss is lower with a larger amount of water in the system [7,8],... 

Because enzymes function nearly to perfection in living systems, there is great interest in how they might be harnessed to carry on desired reactions of practical value outside of living systems. The potential value in the use of enzymes (separate from the organisms that synthesize them) is undeniable, but how to realize this potential is another matter. 

Check the activity of enzymes separately if digestibility values seem to be low. Ensure that the correct enzyme, grade, and concentration have been used. If there is any question about enzyme quality, replace it. 

The possibility that BS and BI cellulase could act synergistically, as has been recorded for many components of fungal cellulolytic complexes (1,2), was tested in several assay systems by adding the enzymes separately or together at the same total activity levels (CMCase units). The assays included the hydrolysis of CMC, cellohexaose, and cellulose powder. The results (not shown here) indicated that the pea cellulases were no more or less effective when added together than when added singly, i.e., there is no indication of any interaction between the enzymes, or any preference by one for the products generated by the other. 

Figure 5.12 The formation of CLEAs is a two-stage process, which combines in essence enzyme separation and immobilization.

Fig. 13.4 Common structural properties of caspases. Pro-caspases have a smaller (10 kDa) subunit, a larger (20 kDa) subunit and an amino-terminal pro-domain. Cleavage of consensus sites in the pro-enzymes separates the two subunits from the remaining NH2-termlnal Pro-domain, which controls processing. Processing is either by autocatalysis or by another caspase with compatible specificity. After processing is completed, the two subunits, the large and the small one, combine, and associate to a tetramer with two catalytic sites, each functioning independently.

A different form of inhibition arises when the inhibitor binds to a second site on the enzyme, separate from the active site, and in doing so it modifies the enzyme, inhibiting its activity. This mode of inhibition is termed non-competitive inhibition (Figure 8-8), and unlike in competitive inhibition, there is often no structural similarity between the substrate and inhibitor. The simplest case of non-competitive inhibition, which is illustrated in Figure 8-8, is that the inhibitor binds with equal affinity to the free and substrate-bound forms of the enzyme, and that the inhibitor completely abolishes catalytic activity (fc at = 0). With these assumptions, the kinetic equation takes the form ... 

Disc electrophoresis with acrylamide gel (S35) has been developed (D7, 02) and is now frequently used for enzyme separations. 

H23. Hunter, R. L., and Markert, C. L., Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science 126, 1294-1295 (1957). 

Preparation of Enzyme. Separation and purification of the enzyme from the liver of the common squid were as follows Squid livers were mashed and dispersed in five volumes of distilled water, then acidified to pH 4.0. Much oil was eliminated. This was followed by ultrafiltration, salting out with ammonium sulfate (50% saturation), dialyzing and freeze-drying in vacuo, to yield a crude enzyme. A purified enzyme was obtained from this crude enzyme by using column chromatography. Active fractions were separated by cation exchange resin. Mono S (Pharmacia) and further purified by gel-flltration column of Superdex 75 (Pharmacia). The active fractions were collected as the purified enzyme. 

When cell extracts were prepared using a French press, centrifuged at 30 000 xg, and the supernatant further centrifuged at 150 000 xg for 3 h, most of the activity remained in the soluble fraction, establishing that the enzyme separated from the membrane. A 24-fold purified preparation of the enzyme was obtained. The MW of the enzyme was reported to be M,. 340 000. " For optimal activity, the enzyme required Mn, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of MW <10 000. The reaction was strongly stimulated by dithiothreitol and methanol. Since the substrate of the enzyme 3-octaprenyl-4-hydroxybenzoate (49) is membrane bound and the enzyme is stimulated by phospholipid, it has been suggested that the enzyme normally functions in association with the cytoplasmic membrane m A reaction... 

A DNA fingerprint is created by digesting DNA with restriction enzymes, separating the pieces on a gel, and then visualizing some of the pieces by using labeled probes. 

The fusion protein exhibited only one tenth of the XR and XDH activity exhibited by the two enzymes when expressed from separate genes. When the fusion protein was co-expressed with the individual XR and XDH enzymes, aggregates composed of the fusion protein and the separate XR and XDH subunits were confirmed by gel chromatography. This construct had specific XR and XDH activities similar to the individually expressed enzymes. Recombinant S. cerevisiae strains harboring the fusion protein aggregate utilized xylose under oxygen-limited conditions in a defined medium and produced less xylitol than a strain expressing the enzymes separately. 

Huang S.H., Lee W.S. and Lin C.K. 1988. Enzyme separation and purification using improved simulated moving bed chromatography. In, Horizontals of biochemical engineering, Aiba S. (Ed.), Oxford University Press, pp. 58-72. 

The first satisfactory experimental proof that alcoholic fermentation is caused by a non-organised ferment (enzyme) in yeast was given by Buchner, who adopted for it Bechamp s name zymase. Buchner found that if the yeast cells are crushed by trituration with sand and kieselguhr and the paste is pressed in a cloth bag, a liquid free from yeast cells is obtained which produces alcoholic fermentation. The activity is not destroyed by evaporation to dryness at 30 -35° or precipitation by alcohol. Arthur Harden and others showed that a co-enzyme, separable from yeast-juice by dialysis, and an inorganic phosphate, are also involved in alcoholic fermentation, the process being apparently very complicated. 

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