Enzymes temperature stability

Further insight will come only with structural studies on more of these odd catalysts. As understanding improves, protein scientists will be eager to apply this new knowledge to introduce structural changes in ordinary enzymes that would enhance temperature stability and catalytic efficiency. For the present, the microbes do it better. 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

Unlike activity, stability of enzymes is often interpreted simplistically as thermal stability, i.e., a temperature beyond which the enzyme loses stability. Although this quantity is important, first every statement of stability at a certain temperature depends on exposure time and thus is often ambiguous and second, for biocatalytic process applications, a more important quantity is the process or operational stability, which is the long-term stability under specified conditions. 

Stability of an enzyme is usually understood to mean temperature stability, although inhibitors, oxygen, an unsuitable pH value, or other factors such as mechanical stress or shear can decisively influence stability (Chapter 17). The thermal stability of a protein, often employed in protein biochemistry, is characterized by the melting temperature Tm, the temperature at which a protein in equilibrium between native (N) and unfolded (U) species, N U, is half unfolded (Chapter 17, Section 17.2). The melting temperature of a protein is influenced on one hand by its amino acid sequence and the number of disulfide bridges and salt pairs, and on the other hand by solvent, added salt type, and added salt concentration. Protein structural stability was found to correlate also with the Hofmeister series (Chapter 3, Section 3.4 Hofmeister, 1888 von Hippel, 1964 Kaushik, 1999) [Eq. (2.18)]. 

Ox-liver /3-glucuronidase was found to be stable to 30 minutes of heating at 50°, but there was considerable inactivation42 at 55°. Heating a limpet preparation (of pH 5) for 5 minutes caused a 15% inactivation86 of /3-glu-curonidase at 60° and 35% at 70°. Within the range of temperature-stability of the enzyme, mammalian /3-glucuronidase activity was approximately doubled for every 10° rise in temperature.81 -147 1 66 This would also appear to be true of mollusc preparations.107171 ... 

Immobilization has also been shown to stabilize against solvent dena-turation of enzymes. However, here we presented suggestive data on the mechanisms of this stabilization. Only the CPO immobilized in 200-A sol-gel showed any solvent or temperature stabilization. CPO bound to matrices with pores smaller than the protein showed little or no stabilization effect owing to surface immobilization alone. This supports the concept that steric hindrance to protein unfolding within a pore is part of the stabilization mechanism. An unresolved question for the future applications of this research is to increase the overall enzyme activity or loading. 

Fig. 21. Proposed effects of random mutation and selection for activity on the stability of an enzyme evolving at a given temperature. Stability fluctuates within a range determined by the destabilizing influence of accumulating random mutations and by the minimal stability that is required to remain folded and functional.

Enzymes respond to temperature in two ways. First the enzyme s stability is affected by temperature. As the temperature is increased, inactivation of the enzyme due to thermal denaturation of the protein increases resulting in a decrease in the concentration of active enzyme. The second effect of temperature is on the reaction rate directly. 

Chymotrypsin hydrolysis of spin-labeled ester substrates was studied by Electron Nuclear Double Resonance and molecular modeling methods (Wells et al., 1994). The spin-labeled acyl-enzyme was stabilized in low temperatures, and conformations of the substrate in the active site have been assigned from the experiments - both free in solution and in the active site. Conclusions from this study are that significant torsional alteration in the substrate s structrue occurs between its "free" form in solution and its bound form in the active site. The enzyme does not "recognize" the solution structure, but an altered one, that is steieospecifically complementary to the surface of the active site. 

Each enzyme has a characteristic pH optimum at which its activity is at a maximum. In the range of this optimum essential proton-donating or proton-accepting groups in the active center of the enzyme are in the ionized state required for the enzyme to function. Outside this range, substrate binding is no longer possible, and at extreme pH values the enzyme may be irreversibly denatured. The pH optimum depends on the composition of the medium, the temperature, and the enzyme s stability in acid and alkaline environments. The pH stability does not necessarily coincide with the pH optimum of the reaction rate. 

In addition the temperature dependency of enzyme activity must be measured, also yielding an optimum curve. This temperature optimum depends on the assay conditions, especially the incubation time, and is not, on its own, useful to identify a reasonable reaction temperature. Instead of this, the temperature stability of the enzyme has to be determined. To that end the enzyme is incubated with all relevant reaction components in test tubes, changing the temperature while keeping all other parameters constant. The assay conditions have to be as close as possible to the conditions relevant in the final process. In particular, stability measurements have to be performed in the presence of a relevant concentration of all substrates and coenzymes which have a stabilizing influence on the enzyme. 

A sufficient number of chemical groups that can be activated or modified in such a manner that they may be able to couple the affinant is a necessary condition for the preparation of specific adsorbents. The activation or modification should take place under conditions that do not change the structure of the support. No less important are the chemical and mechanical stabilities of the carrier under the conditions of attachment of the affinant, and also at various pH values, temperatures and ionic strengths, in the possible presence of denaturating agents, etc., which may be necessary for a good sorption and elution of the isolated substance. The specific adsorbent should not be attacked by microorganisms and enzymes. These stabilities are important primarily for repeated use of specific adsorbents. 

Table 1 lists a few of the enzyme electrodes reported/ Both poten-tiometric and amperometric devices have been employed with these electrodes. Test results with enzyme electrodes have been reported as ranging from excellent to poor. Problems reported deal principally with lack of stability of the enzymes used, extended response times, and temperature sensitivity. There does not appear to be any pattern to the problems reported. It is apparent however, that (a) care must be taken in the choice of enzyme immobilization method, (b) the immobilization technique should provide a minimal barrier to reactant and product diffusivity, and (c) temperature stabilization is desirable. 

The thermal stability of the partially active MCAD mutants has so far been determined only on the cellular lysates after co-overexpression with GroEL and GroES." The G242R mutant has been shown to have a slightly reduced stability compared to the wild type. However, the presence of chaperonins and other proteins may have a significant influence on the results, so temperature stability of the purified proteins was also measured. The enzymes were incubated at 37 °C and residual activity was measured over a 100 minute period. The results are shown in figure 2. 

The respective pH and temperature stabilities of the purified en mes were observed fiom pH 5.6 to 11.6 and up to 45 °C, for APnc and Apnc fixim pH 5.6 to 11.2 and iq> to 45 °C. Thus, these enzymes were slightly less stable tiian foe parental i lanases (Fig. 5). This relative instability could be due to the loss of some interaction in the protein molecule such as a Itydrogm bond, caused by chaining foe region of tiie enzyme. 

---