Fundamentals of Biochemical Engineering Part 1 - Bailey J., Ollis D. 1989

Applications of Enzyme-Catalyzed Reactions
Kinetics of Reactions Catalyzed by Immobilized Enzymes
Effects of Inhibitors, Temperature, and pH on the Catalytic Activity and Inactivation of Immobilized Enzymes

The previous chapter examined mathematical expressions describing Enzyme inactivation processes and METABOLISM/18.html">The Influence of various parameters (inhibitor and activator concentrations, Temperature, pH, Ionic strength, etc.) on enzymatic activity in solutions. In general, one might expect these expressions to be suitable for describing the dependence of the intrinsic activity of an immobilized enzyme on various parameters as well. At the same time, as we have repeatedly noted, immobilization can be accompanied by Changes in the intrinsic Properties of the enzyme. For instance, immobilized Enzymes may exhibit different reaction rate constants; furthermore, immobilization may even necessitate modifying the equations that describe how reaction parameters affect the intrinsic activity of the enzyme. To determine the response of the intrinsic catalytic activity of an immobilized enzyme to changes in reaction parameters, it is essential to conduct a thorough kinetic study using the Methods described above, which allow for investigating solely the intrinsic kinetic behavior of the system rather than a combination of mass transfer rates and catalytic reactions.

In this section, we will primarily endeavor to emphasize the impact that mass transfer processes can exert On the Relationship between the apparent overall catalytic activity of an immobilized enzyme and its environmental conditions. All necessary data can be obtained by studying The kinetics of First-Order Reactions. First of all, it should be kept in mind that the observed overall kinetics of reactions involving an immobilized enzyme operating under a reaction-limited regime does not differ from the intrinsic local kinetics of that enzymatic reaction. Consequently, the reaction studied under this regime will be of the first order, and the process rate constant will equal the intrinsic rate constant of the enzymatic reaction. In the case of an immobilized enzyme catalyst operating under a diffusion-limited regime, the apparent reaction order will also be first, but the apparent rate constant will be equal to the square ROOT of the intrinsic rate constant of the enzymatic reaction. [In general, if a reaction proceeds with the n-th order, the apparent order of this reaction under diffusion-limited conditions will be (n+1)/2.] Thus, the apparent activation energy will amount to only half of the true activation energy. Similarly, the influence of changes in any reaction parameters on the observed overall process kinetics will be smaller than the actual effect of those same changes on the local, intrinsic kinetics of the enzymatic reaction. For instance, if inactivation processes, the Introduction of an inhibitor, or a pH change reduce the rate constant of the enzyme-catalyzed reaction itself by a factor of 4, then under diffusion-limited conditions the observed overall process rate will decrease by a factor of only 2.

It follows from all the above that extreme caution must be exercised when studying the influence of reaction parameters on the Kinetics of Reactions Catalyzed by immobilized enzymes. If reaction-limited conditions are not maintained, or if experimental data obtained under conditions where diffusion makes a significant contribution to the process are not processed to yield information on the intrinsic kinetics of the enzymatic reaction, then any Conclusions regarding the dependence of immobilized enzyme activity on process conditions will be valid only for this particular batch of catalyst and only under these specific reaction conditions, and will by no means describe the intrinsic behavior of the immobilized enzyme. In such a case, any changes in the flow rate of the substrate solution through the catalyst bed, the particle size, pore Structure, or the content and distribution of the enzyme within the catalyst particle will be accompanied by changes in the previously established relationships between catalyst activity and process parameters. This fact once again highlights the importance and necessity of clearly distinguishing the intrinsic properties of an immobilized enzyme from those characteristics caused by mass transfer effects.

Unfortunately, this circumstance has been overlooked in many studies published in the literature dedicated to investigating how the characteristics of immobilized enzyme systems depend on reaction conditions. Since in most cases immobilized enzymes operate under conditions where diffusion effects significantly affect The rate of the process or even determine it, much of the experimental data available in the literature regarding the determination of inactivation rates and the influence of pH and temperature on reaction kinetics clearly do not describe the intrinsic properties of immobilized enzymes.

At the same time, There is a multitude of evidence indicating changes in the intrinsic kinetics of enzyme inactivation following their immobilization. Various mechanisms and interpretations of this phenomenon have been proposed and postulated; some of the hypotheses that we will briefly review here have withstood experimental verification. Firstly, As a result of immobilization, enzymes are held in a relatively fixed spatial position, which reduces the likelihood of interactions between enzyme molecules that promote their inactivation through aggregation or autolysis (in the case of Proteolytic Enzymes). Secondly, the presence of multiple bonds between the enzyme molecule and the support hinders the disruption of the protein's tertiary structure. It has been reported that attempts to create a carrier microstructure complementary to the enzyme surface by entrapping the enzyme within a gel were accompanied by a dramatic increase in its stability. Similarly, immobilization stabilizes the active form of multi-subunit enzymes and can thereby slow down inactivation caused by the dissociation of Oligomeric Proteins.

The enhanced stability of immobilized enzymes may also be attributed to a favorable local microenvironment that inactivates the protein to a lesser extent than the solution environment does. Examples include carriers whose surfaces possess buffering properties, reduce local oxygen concentration, adsorb enzyme poisons, or catalyze The conversion of Denaturing Agents (e.g., Н_2О2) into harmless compounds. It is likely that other factors may also influence the stabilization of immobilized enzymes, which will be elucidated through further research into the Fundamental properties of immobilized enzymes and the relationship between their Structure and function.