Enzymes activity, temperature

Influence of Subphase Temperature. Enzymic activity at the interface increases clearly with subphase temperature (Figure 6). When comparing enzymic adsorption with enzymic activity (Figure 7), the rate of enzymic cleavage increases when the number of macromolecules of the enzyme that reaches the surface increases. The two processes are parallel but are not immediately related. 

Enzymes are very sensitive to temperature. At low temperatures, most enzymes show little activity because there is not a sufficient amount of energy for the catalyzed reaction to take place. At higher temperatures, enzyme activity increases as reacting molecules move faster to cause more collisions with enzymes. Enzymes are most active at optimum temperature, which is 37 °C, or body temperature, for most enzymes (see Figure 16.15). At temperatures above 50 °C, the tertiary structure, and thus the shape of most proteins, is destroyed, which causes a loss in enzyme activity. For this reason, equipment in hospitals and laboratories is sterilized in autoclaves where the high temperatures denature the enzymes in harmful bacteria. 

The molar mass of the polyesters depended on the reaction temperature, enzyme activity, enzyme concentration, and to a lower extent on the applied vacuum. For example, when the polyesters were synthesized using 1,4-butanediol, 1,6-hexanediol and 1,8-octanediol in the bulk at 70°C under reduced pressure for 24 hours, the weight average molecular weights obtained were 15,100 g moF, 16,000 g mol, and 16,700 g mof , respectively. Thermal analysis of these copolymers revealed no melting phenomenon, perhaps due to the presence of the bulky and flexible siloxane segments in... 

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

In many situations, the actual molar amount of the enzyme is not known. However, its amount can be expressed in terms of the activity observed. The International Commission on Enzymes defines One International Unit of enzyme as the amount that catalyzes the formation of one micromole of product in one minute. (Because enzymes are very sensitive to factors such as pH, temperature, and ionic strength, the conditions of assay must be specified.) Another definition for units of enzyme activity is the katal. One katal is that amount of enzyme catalyzing the conversion of one mole of substrate to product in one second. Thus, one katal equals 6X10 international units. 

FIGURE 14.12 The effect of temperature on enzyme activity. The relative activity of an enzymatic reaction as a fnncdon of tempera-tnre. The decrease in the activity above 50 C is dne to thermal denatnradon. 

Hypothermia slows down enzyme catalysis of enzymes in plasma membranes or organelle membranes, as well as enzymes floating around in the cytosol. The primary reason enzyme activity is decreased is related to the decrease in molecular motion by lowering the temperature as expressed in the Arrhenius relationship (k = where k is the rate constant of the reaction, Ea the activation energy,... 

For bio-transformation processes, immobilised enzymes are often used because their activity persists over a longer period of time than that of free enzymes. The reduction of enzyme activity in enzymatic reactors is a consequence of energy dissipation by sparging and stirring, which is required for instance for oxygen transport or realisation of constant reaction conditions as regards temperature and pH. In the other hand low and high pH-values leads also to a decrease of enzyme activity and increase the stress sensitivity. 

Available methods provide measurements of enzyme activity rather than of enzyme concentration. In order that the measured activity be proportional to enzyme concentration, the reaction conditions which include pH, temperature, initial substrate concentration, sample and total volume and reaction time must be held constant and be carefully controlled. 

Reagents. The measurement of enzyme activities requires rigid control of the analytical conditions, including accurate measurement of reagent and sample volumes, and careful control of temperature, pH and reagent stability. 

As with any reaction, temperature has an important effect on the rate of an errzy-matic reaction, albeit that the range of interest is limited. For each enzyme an optimum temperature exists (37 °C for reactions in human beings). At high temperatures the activity decreases due to thermal denaturation of the protein constituting the enzyme. 

The lipase-catalyzed DKRs provide only (/ )-products to obtain (5 )-products, we needed a complementary (5 )-stereoselective enzyme. A survey of (5 )-selective enzymes compatible to use in DKR at room temperature revealed that subtilisin is a worthy candidate, but its commercial form was not applicable to DKR due to its low enzyme activity and instability. However, we succeeded in enhancing its activity by treating it with a surfactant before use. At room temperature DKR with subtilisin and ruthenium catalyst 5, trifluoroethyl butanoate was employed as an acylating agent and the (5 )-products were obtained in good yields and high optical purities (Table 3)P... 

Effect of pH and temperature on the purified enzyme activity and stability The conditions of the enzyme activity and the stability was done followed Buranakarl, et al. (16). 

Effect of pH and temperature on the purified enzyme activity and stability... 

Figure 6,7 and 8 showed the results of the pectinase activity when produced in the solid substrates containing wheat bran, rice bran and rice husk in the ratio of 6 12 2. The highest activity obtained when the strain was grown on the solid substrates with 58 % initial moisture content, pH adjusted to 5.7 and incubation temperature was at 32°C. Under these conditions, the highest activity of the enzyme that could be obtained from Rhizopus sp. 26R was ca. 700 units of enzyme activity per gram of solid substrates. 

Study of the temperature optimum of pectinesterase activity showed, that peak of pectinesterase activity was observed at the temperature equal to 45 °C. It is shown on Figure 1 that pectinesterase was stable at pH 4 — 5. At pH 2 activity of the enzyme reduced by 25% in 60 min., at pH 3 and pH 6 it decreased by 6 — 8%. At pH 8 the activity decreased by 90.7% during the same time. At pH 9 the enzyme activity was inactivated during 15 min. 

Cloughley, J. B., The effect of temperature on enzyme activity during the fermentation phase of black tea manufacture. J. Sci. Food Agri., 31 920, 1980. 

Approximation refers to the bringing together of the substrate molecules and reactive functionalities of the enzyme active site into the required proximity and orientation for rapid reaction. Consider the reaction of two molecules, A and B, to form a covalent product A-B. For this reaction to occur in solution, the two molecules would need to encounter each other through diffusion-controlled collisions. The rate of collision is dependent on the temperature of the solution and molar concentrations of reactants. The physiological conditions that support human life, however, do not allow for significant variations in temperature or molarity of substrates. For a collision to lead to bond formation, the two molecules would need to encounter one another in a precise orientation to effect the molecular orbitial distortions necessary for transition state attainment. The chemical reaction would also require... 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

Enzyme activity generally passes through a maximum as the pH of the system in question is varied. However, the optimum pH varies with substrate concentration and temperature. Provided that the pH is not changed too far from the optimum value corresponding to the maximum rate, the changes of rate with pH are reversible and reproducible. However, if the solutions are made too acid or too alkaline, the activity of the enzyme may be irreversibly destroyed. Irreversible deactivation is usually attributed to denaturation of the proteinaceous enzyme. The range of pH in which reversible behavior is observed is generally small and this... 

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