Inactivation thermal, enzyme

The effect of the substrate (benzonitrile) concentration on nitrile hydratase activity was investigated in a CSMR in the range of 2-10mM at low temperature (10 °C) in order to reduce the enzyme thermal inactivation as much as possible. The experimental runs are illustrated in Figure 17.4a as a semi-logarithmic plot of the specific reaction rate against process time. Both the r - and the ka-values were calculated (as summarized in the previous paragraph) and are quoted in Table 17.1. 

Stability is not related to activity and in many cases they have opposite trends. It has been suggested that there is a trade-off between stability and aetivity based on the fact that stability is clearly related to moleeular stiffening while eonforma-tional flexibility is beneficial for catalysis. This can be clearly appreciated when studying enzyme thermal inactivation enzyme activity increases with temperature but enzyme stability decreases. These opposite trends make temperature a critical variable in any enzymatic process and make it prone to optimization. This aspect will be thoroughly analyzed in Chapters 3 and 5. 

Enzyme Reactor Design and Performance Under Non-Modulated and Modulated Enzyme Thermal Inactivation... 

Enzyme thermal inactivation during bioreactor operation is of paramount importance and must be considered for proper bioreactor design, as shown in Fig. 3.1. To do so, a mathematical model must be developed based on experimentally calculated and validated parameters. Mechanistic models to describe enzyme inactivation were presented in sections 5.4.1 and 5.4.2. 

Klibanov, A.M. (1983) Stabilization of enzymes against thermal inactivation. Adv. Appl. Microbiol., 29, 1-28. 

Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl-phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively ... 

Apparent Temperature Optimum. A rise in temperature has a dual effect upon an enzyme-catalyzed reaction it increases the rate of the reaction, but it also increases the rate of thermal inactivation of the enzyme itself. Like the pH optimum, the temperature optimum may in certain instances be altered by environmental conditions, e.g., pH, type and strength of buffer, etc. The term temperature optimum, therefore, is useless unless the incubation time and other conditions are specified. A more enlightening term is apparent temperature optimum, which indicates that the optimum has been obtained under a... 

Thunell, R. K., Duersch, J. W. and Ernstrom, C. A. 1979. Thermal inactivation of residual milk clotting enzymes in whey. J. Dairy Sci. 62, 373-377. 

In general, enzymes can be thermally inactivated by bringing the enzyme-containing solution to 95° to 100°C for 5 min. However, one should always verify that treatment results in complete inactivation (via a check for residual activity), since some enzymes are particularly heat stable. 

The thermal inactivation curve of the enzyme in 0.15 M acetate buffer-0.01 M ethylenediaminetetraacetate (EDTA), pH 5.0, showed that a 50% inactivation was obtained by heating for 20 min at 56° 11). 

The effects of temperature both on the stability of the enzyme (26, 37, 41, 46) and on the catalytic reaction itself (46, 112) have been studied. The enzyme is considerably more labile to mild heating in the absence of substrates than is microsomal acid phosphatase (26, 37, 4U 46)-Glucose-6-P (26, 136), P, (26), glucose (136), PP, (119, 136), and various amino acids (136) protect to some degree against thermal inactivation as do certain metal chelators which inhibit the reaction (120). 

Detergents labilize the enzyme to thermal inactivation in the absence of substrates (26, 90), while activation by exposure of the enzyme to high pH does not (90). The enzyme appears stable to freezing for several months (137). 

L-Cysteine, o-tyrosine, and L-leucine inhibit and also stabilize enzyme against thermal inactivation. A variety of other amino acids are also effective to some degree in the latter respect... 

T. J. Ahern and A. M. Klibanov, Analysis of processes causing thermal inactivation of enzymes, Methods Biochem. Anal. 1988, 33, 91-127. 

Thermal inactivation of EcaL-ASNase was monitored by activity measurements. Samples of the enzyme, in potassium phosphate buffer (lOmM, pH 7), were incubated at a range of temperatures from 35 °C to 50 °C. The rates of inactivation were followed by periodically removing samples for assay of enzymatic activity. Observed rates of inactivation (ki ) were deduced from plots of log (% remaining activity) versus time. To obtain the thermodynamic parameters from these inactivation rates, two analyses were carried out according to Stein and Staros (1996). An empirical activation energy, Ea, for the inactivation process can be obtained from an Arrhenius plot according to the equation ... 

If enzyme inactivation is not due to covalent changes in structure, the native active structure can be reformed in immobilized enzymes by refolding from a random coil state (22,27). In fact, if they are attached to the matrix by multiple points, even thermally inactivated multichain enzymes can be reactivated (27). Although such regeneration steps apparently have not been used commercially, their use should be considered in certain cases, especially since in many cases the inactivation may result from adsorption onto the enzyme matrix of components in the process stream. For example, we have found that immobilized sulfhydryl oxidase activity can be regenerated numerous times following treatment of UHT milk by washing with 4 M urea (28). 

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