Enzymes, inhibition, substrate temperature

Enzyme immobilization has been reported to improve the thermal stability of enzymes (1,2) and may also affect binding of substrates and inhibitors to the enzyme, thereby affecting the Michaelis constant and enzyme inhibition. Several previous studies have considered the advantages of immobilized enzymes with soluble substrates, and a few studies have also investigated the properties of immobilized enzymes with insoluble substrates. The main objective of the present work was to establish the effect of immobilization on the thermal stability of these enzymes, so that they may be used at elevated temperatures without significant activity loss. The immobilization conditions were varied, and their effect on the performance of the immobilized enzymes was analyzed with reference to their physiochemical and structural properties. 

All biochemical reactions are enzyme-mediated. The rate of an enzyme reaction depends on the substrate concentration at the location of the enzyme and thereby on the diffusion rate of a substrate to the enzyme. It is therefore important to permanently obtain an intimate contact between a cell or enzyme and substrate molecules. Additionally, the product generated in the bioreactor has to be extracted because it may under certain conditions inhibit its own production. In some processes there may also be even a prepurification in the bioreactor itself. If living micro-organisms have to be applied, it is necessary to provide sufficient nutrition and respiration gases in case of aerobic fermentation. All other reaction parameters such as temperature, pH-value and reaction time have to be controlled precisely. In many cases (generally with modem processes) the maintenance of microbiological integrity (sterile process) is absolutely mandatory for a successful fermentation. 

It is useful at this time to review briefly some basic aspects of enzyme inhibition and enzyme inhibitors. For a broader review of enzymatic mechanisms and kinetics consult any of several excellent biochemistry textbooks. Enzyme inhibitors are chemical agents capable of modifying an enzyme s capacity to catalyze the reactions of its normal substrates. Effecting such changes of enzyme function by altering the pH, changing the temperature, or subjecting the system to radiation such as UV should properly be considered a denatu-ration process. 

Enzymatic reactions frequently undergo a phenomenon referred to as substrate inhibition. Here, the reaction rate reaches a maximum and subsequently falls as shown in Eigure 11-lb. Enzymatic reactions can also exhibit substrate activation as depicted by the sigmoidal type rate dependence in Eigure 11-lc. Biochemical reactions are limited by mass transfer where a substrate has to cross cell walls. Enzymatic reactions that depend on temperature are modeled with the Arrhenius equation. Most enzymes deactivate rapidly at temperatures of 50°C-100°C, and deactivation is an irreversible process. 

Biocatalysts in nature tend to be optimized to perform best in aqueous environments, at neutral pH, temperatures below 40 °C, and at low osmotic pressure. These conditions are sometimes in conflict with the need of the chemist or process engineer to optimize a reaction with respect to space-time yield or high product concentration in order to facilitate downstream processing. Furthermore, enzymes and whole cells are often inhibited by products or substrates. This might be overcome by the use of continuously operated stirred tank reactors, fed-batch reactors, or reactors with in situ product removal [14, 15]. The addition of organic solvents to increase the solubility of substrates and/or products is a common practice [16]. 

Free tryptophan is transported into the brain and nerve terminal by an active transport system which it shares with tyrosine and a number of other essential amino acids. On entering the nerve terminal, tryptophan is hydroxylated by tryptophan hydroxylase, which is the rate-limiting step in the synthesis of 5-HT. Tryptophan hydroxylase is not bound in the nerve terminal and optimal activity of the enzyme is only achieved in the presence of molecular oxygen and a pteridine cofactor. Unlike tyrosine hydroxylase, tryptophan hydroxylase is not usually saturated by its substrate. This implies that if the brain concentration rises then the rate of 5-HT synthesis will also increase. Conversely, the rate of 5-HT synthesis will decrease following the administration of experimental drugs such as para-chlorophenylalanine, a synthetic amino acid which irreversibly inhibits the enzyme. Para-chloramphetamine also inhibits the activity of this enzyme, but this experimental drug also increases 5-HT release and delays its reuptake thereby leading to the appearance of the so-called "serotonin syndrome", which in animals is associated with abnormal movements, body posture and temperature. 

The control via activation or inhibition of the rate(s) of an enzyme-catalyzed reaction(s). This control includes the increase or decrease in the stability or half-life of the enzyme(s). There are many different means by which control can be achieved. These include 1. Substrate availability and reaction conditions (e.g., pH, temperature, ionic strength, lipid interface activation) 2. Magnitude of Vraax sud valucs) 3. Activation (particularly, feedforward activation) 4. Isozyme formation 5. Com-partmentalization and channeling 6. Oligomerization/ polymerization 7. Feedback inhibition and cooperativity (particularly, allosterism and/or hysteresis) 8. Covalent modification and 9. Gene regulation (induction repression)... 

Chemical Inhibition. A large variety of chemical compounds have been added to milk or purified lipase. The conditions under which the inhibitor is studied are very important. Factors such as pH, temperature, time of addition of the chemical, sequence of addition of reactants, and the presence or absence of substrate are undoubtedly involved. The presence of substrate appears to offer some degree of protection to the enzymes. Consequently, in lipase studies, the surface area of the emulsified substrate is probably also important. 

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). 

Kinetic analysis was used to characterize enzyme-catalyzed reactions even before enzymes had been isolated in pure form. As a rule, kinetic measurements are made on purified enzymes in vitro. But the properties so determined must be referred back to the situation in vivo to ensure they are physiologically relevant. This is important because the rate of an enzymatic reaction can depend strongly on the concentrations of the substrates and products, and also on temperature, pH, and the concentrations of other molecules that activate or inhibit the enzyme. Kinetic analysis of such effects is indispensable to a comprehensive picture of an enzyme. 

Independently of enzyme stability an enzyme reaction at constant temperature and pH should be run with the smallest possible contribution by inhibition. For this reason, a CSTR is most suitable in the case of substrate inhibition because the substrate concentration is evened out across the reactor volume and thus minimized. In the case of product inhibition, however, a batch reactor or a PFR is preferred, as the reactor volume required for complete or nearly complete conversion is much smaller than in the case of a CSTR. 

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