Dr. Shraddha_Sahu_Kinetic Properties of Immobilized Enzymes.pptx

Enzyme Engineering and Technology

1. Kinetic Properties of Immobilized Enzymes - By Dr. Shraddha Sahu Assistant Professor Department of Biotechnology Faculty of Engineering and Technology Rama University, Kanpur, Uttar Pradesh, I ndia.

2. The partition effect in immobilized enzymes refers to the phenomenon where molecules partition between the bulk solution and the microenvironment surrounding the immobilized enzyme. This partitioning is influenced by factors like the ionic strength of the solution, the hydrophobicity or hydrophilicity of the enzyme surface, and the charge interactions between molecules and the enzyme surface . Partition Effect in Immobilized Enzymes

3. The microenvironment in immobilized enzymes refers to the unique surroundings created by the carrier material where the enzyme is immobilized. This microenvironment plays a crucial role in influencing the enzyme loading and performance. Factors such as the chemical properties of the carrier material, the presence of functional groups on the support, and the interplay of reactions and interactions within this confined space define the microenvironment. It affects the partitioning of molecules, the local concentration of substrates and products, and the pH within the vicinity of the enzyme, ultimately impacting the enzyme's activity and kinetic behavior. The microenvironment provides a specialized niche for the enzyme, influencing its catalytic efficiency and overall performance. Partition Effect in Immobilized Enzymes

4. Solute partitioning in immobilized enzymes can significantly affect their activity due to changes in the local concentration of molecules within the microenvironment surrounding the enzyme . The partitioning of molecules is influenced by factors such as the charge and hydrophobicity of the enzyme surface and the ionic strength of the solution. For a positively charged support, cations are partitioned away from the microenvironment, while the concentration of anions is greater within the microenvironment. Conversely, for a negatively charged support, anions are partitioned away, and cations are concentrated within the microenvironment. Effects of Solute partitioning on the activity of the immobilized enzymes

5. Hydrophobic molecules tend to partition into the microenvironment of the enzyme, while hydrophilic molecules are partitioned out into the bulk solution. This partitioning can lead to changes in the apparent kinetic constants of the enzyme, such as a reduction in the Km for certain substrates . The partitioning of charged molecules, such as substrates and products, can also affect the pH within the microenvironment, which in turn affects the binding of substrate and the activity of the immobilized enzyme. This can cause apparent shifts in the behavior of the kinetic constants with respect to the solution pH, allowing for the optimization of enzyme performance by altering the pH of the solution. Effects of Solute partitioning on the activity of the immobilized enzymes

6. The diffusion effect in immobilized enzymes refers to the impact of slow diffusion of products away from the catalytic sites on the overall kinetics of enzyme-catalyzed reactions. In immobilized enzymes, substrates need to diffuse through the solution to reach the catalytically active sites, while the products must diffuse away into the bulk solution. The movement of solutes is driven by concentration gradients , with solutes moving from areas of higher concentration to lower concentration. Difussion Effect in Immobilized Enzymes

7. The diffusion process involves substrates approaching the enzyme surface through a thin stagnant unstirred layer of solution and then diffusing into any pores where they may encounter active enzyme. The effectiveness factor(s) in immobilized enzymes varies with factors like the concentration gradients, the rate of diffusion, and the nature of the immobilization method used. Diffusion limitations can lead to changes in the effective catalytic ability of the enzyme, affecting the overall reaction rate. Difussion Effect in Immobilized Enzymes

8. The diffusion effect in immobilized enzymes is important as it influences the transport of substrates and products to and from the catalytic sites, eventually affecting the efficiency and kinetics of enzyme-catalyzed reactions. Difussion Effect in Immobilized Enzymes

9. To overcome diffusion limitations in immobilized enzymes, several strategies can be employed . One approach is to use porous carriers with larger pore sizes, which can facilitate the diffusion of substrates and products to and from the enzyme active site . Another strategy is, to modify the carrier material to make it more hydrophilic, which can enhance the diffusion of water-soluble molecules and reduce mass transfer limitations . The use of spacer arms can also help in reducing diffusion limitations by increasing the distance between the enzyme and the carrier surface, allowing for greater substrate and product diffusion Methods to overcome diffusional limitations in immobilized enzymes

10. The structure and stability of immobilized enzymes are crucial aspects that impact their performance and efficiency in biocatalytic processes. Immobilization techniques aim to enhance enzyme stability, allowing for reusability and improved resistance to various environmental factors. The immobilization process can lead to changes in the enzyme's structural rigidity, which can contribute to increased stability and resistance to denaturation under harsh conditions such as high temperatures or extreme pH levels. Immobilized enzymes typically exhibit greater thermal and operational stability compared to their soluble counterparts Structure and Stability of Immobilized Enzymes

11. The immobilization of enzymes on different carriers, such as calcium alginate or DEAE-cellulose, can provide increased resistance to changes in conditions like pH or temperature. This immobilization strategy allows enzymes to be held in place throughout the reaction, facilitating easy separation from products and enabling their reuse, leading to a more efficient process. Structure and Stability of Immobilized Enzymes

12. The choice of immobilization method, whether entrapment, covalent immobilization, or physical adsorption, can impact the stability of multimeric enzymes. Stabilization of multimeric enzymes after immobilization is observed due to interactions between the support and the enzyme, leading to improved operational stability. Techniques like site-directed enzyme immobilization, which link the support to specific amino acids in the protein away from the active site, can help retain maximal enzyme activity by ensuring free access of the substrate to the active site. Structure and Stability of Immobilized Enzymes

13. In summary, the structure and stability of immobilized enzymes are critical for their performance in biocatalytic processes. Immobilization techniques enhance enzyme stability, improve resistance to environmental factors, and allow for reusability, ultimately contributing to the efficiency and effectiveness of enzyme-catalyzed reactions. Structure and Stability of Immobilized Enzymes