Temperature enzyme denaturation

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). 

When the rate of an enzyme catalyzed reaction is studied as a function of temperature, it is found that the rate passes through a maximum. The existence of an optimum temperature can be explained by considering the effect of temperature on the catalytic reaction itself and on the enzyme denaturation reaction. In the low temperature range (around room temperature) there is little denaturation, and increasing the temperature increases the rate of the catalytic reaction in the usual manner. As the temperature rises, deactivation arising from protein denaturation becomes more and more important, so the observed overall rate eventually will begin to fall off. At temperatures in excess of 50 to 60 °C, most enzymes are completely denatured, and the observed rates are essentially zero. 

Biphasic systems consisting of ionic liquids and supercritical CO2 showed dramatic enhancement in the operational stability of both free and immobilized Candida antarctica lipase B (CALB) in the catalyzed kinetic resolution of rac- -phenylethanol with vinyl propionate at 10 MPa and temperatures between 120 and 150°C (Scheme 30) 275). Hydrophobic ionic liquids, [EMIM]Tf2N or [BMIM]Tf2N, were shown to be essential for the stability of the enzyme in the biotransformation. Notwithstanding the extreme conditions, both the free and isolated enzymes were able specifically to catalyze the synthesis of (J )-l-phenylethyl propionate. The maximum enantiomeric excess needed for satisfactory product purity (ee >99.9%) was maintained. The (S)-l-phenylethanol reactant was not esterified. The authors suggested that the ionic liquids provide protection against enzyme denaturation by CO2 and heat. When the free enzyme was used, [EMIM]Tf2N appeared to be the best ionic liquid to protect the enzyme, which... 

Changes in the activity of enzymes may also occur by a variety of other parameters including temperature, pH, and ionic strength. Temperature can affect both the activity and the stability of the enzymes. For most enzymes, the reaction velocity doubles with a temperature increase of 10 C but potential enzyme denaturation may also occur (265). 

Decrease of velocity with higher temperature Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Figure 5.7). 

For most enzymes, the CLEC is much more robust than the simple isolated enzyme. CLECs can withstand higher temperatures, they denature more slowly in organic solvents, and they are less susceptible to proteolysis [71]. Moreover, since there is no external support involved, CLECs exhibit a high volumetric productivity. These advantages, together with the tunable particle size (typically 1-100 pm), make CLECs attractive for industrial biocatalysis applications. 

Temperature Enzymes have an optimum temperature at which they work fastest. For most enzymes, this is about 40° Celsius (104° Fahrenheit), but there are enzymes that work best at very different temperatures, such as the enzymes of the arctic snow flea that work at -10°C (14°F). If the temperature is too high, enzymes are destroyed—a process called denaturation. The heat breaks the H bonds holding the secondary and tertiary structure of the enzyme together, and therefore the enzyme and its active site lose their shape. The substrate can no longer bind, and the biochemical reaction can no longer occur. 

Alpha-amylase is most active at its pH optimum of 6.3 to 6.8.108,109 It is inactive at pH values below 4 and above 9. Enzymic starch conversion is terminated by raising the temperature until enzyme denaturation occurs or by the addition of enzyme poisons, such as the ions of copper, mercury or zinc. Inactivation can also be achieved by moving the pH outside the enzyme s active limits or by the addition of oxidizing agents, such as sodium hypochlorite, hydrogen peroxide or barium peroxide. 

Fig. 3.4 Temperature-dependent reconstitution of tetrameric K coli aspartase.29 A Reactivation of denatured aspartase. The enzyme denatured in 4 M guanidine-HCl was renatured at 4° C by dilution. After 14 min, the temperature of each preparation was shifted up as indicated in the figure. The temperature of each preparation was further shifted up to 30° C after 45 min. B HPLC analysis of intermediates in the renaturation process. Aspartase renatured at 4°C was incubated for 15 min at the indicated temperatures. An aliquot of each preparation was applied to a TSKgel G3000SWXL column (7.5 X 300 mm) and eluted with a flow rate of 0.5 ml/ min. The temperature of the sample in the sample loop, elution buffer and the column was maintained constant. (From Physiol Chem. Phys. Med. NMR, 21, 222 226 (1989)).

Over a limited range of temperature, the velocity of enzyme-catalysed reactions roughly doubles with a 10°C rise in temperature. Enzymes, being proteins, are denatured by heat and become inactive as the temperature increases beyond a certain point. Most of the enzymes are inactivated at temperatures above 60°C. The temperature at which the reaction rate is maximum is known as optimum temperature. 

All biological transformations are affected by temperature. Generally, as the temperature increases, biological activity tends to increase up to a temperature where enzyme denaturation occurs. The presence of oil should increase soil temperature, particularly at the surface. The darker color increases the heat capacity by adsorbing more radiation. The optimal temperature for biodegradation to occur ranges from 18 C to 30 C. Minimum rates would be expected at 5 C or lower (Frankenberger 1992). 

Temperature effects can be either negative or positive on the or the fccat of the reaction but can only be negative on the enzyme (denaturation). If = Ks, K may be determined at various temperatures. The /Ccat can be established directly from the Arrhenius equation (eq. 1) ... 

Effect of Temperature. As with enzymes (cf. Figure 7-9), there is an optimum in growth rate with temperature owing to the competition of increased rales with increasing temperature and denaturizing the enzyme at high temperatures. An empirical law that describes this functionality is given in Aiba et al. and is of the form... 

Shift of the optimum temperature towards lower side has been detected upon entrapping of the enzyme (Figure 6), A suitable temperature found to be 45 C for the entrapped enzyme compared with 55°C for the free form. Such behavior may be explained in the light of acidic environment due to the presence of free unbinding carboxylic groups. This explanation is confirmed by the higher rate of enzyme denaturation at higher temperatures, 50-70°C, in comparison with the free form. 

Direct intercalation of proteins into a-ZrP, however, is not successful due to the small gallery spacings and the large kinetic barrier for intercalation of large guest molecules. Forceful intercalation at high temperatures, long times, and extreme pHs can result in enzyme denaturation. 

Enzymes typically function at normal body temperature, 37°C. Their activities at higher temperatures are desirable for rate acceleration, but free enzymes denature at elevated temperatures. Upon intercalation in a-ZrRP (R = OH), both HRP and Hb showed peroxidase activities at over 85°C, observed for the first time, (258) while the free enzyme/protein deactivated rapidly at these temperatures with no activity. The maximum rate of the reaction (Vmax) increased 3.6-fold, while the concentration needed to achieve half the maximum rate (K ) decreased by 20% at these higher temperatures. Such high-temperature activities of enzymes/ proteins are unusual, and they indicate the promise of a-ZrRPs for enzyme stabilization in high-temperature applications. This strategy of enzyme stabilization in a-ZrRP may provide alternatives to thermophilic enzymes obtained from thermo-philes, and these may supplement thermostable enzymes obtained by protein engineering. 

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