Enzymes Immobilization Techniques PDF

# Enzymes Immobilization Techniques ## Introduction: - Enzymes are biological catalysts that promote chemical reactions in living organisms. - Enzymes have the ability to catalyze reactions under very mild conditions with a high degree of substrate specificity, thus decreasing the formation of byp...

# Enzymes Immobilization Techniques ## Introduction: - Enzymes are biological catalysts that promote chemical reactions in living organisms. - Enzymes have the ability to catalyze reactions under very mild conditions with a high degree of substrate specificity, thus decreasing the formation of byproducts - Enzymes can catalyze reactions in different states, including individual molecules in solution, in aggregates with other entities, and as attached to surfaces. - Attached or "immobilized" state has been of particular interest. - "Immobilized enzyme" refers to enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously. ## Immobilization Techniques ### What is immobilization? - **Definition:** Imprisonment of a cell or enzyme in a distinct support/matrix. - The support/matrix allows exchange of medium. - The medium contains substrate or effector or inhibitor molecules. - The first immobilization technology was **amino acylases** by *Aspergillus oryzae* for the production of L-amino acids in Japan. - Two main advantages of enzyme immobilization are: - Increased functional efficiency. - Enhanced reproducibility. ### What are the advantages of immobilized enzymes? - Reuse - Continuous use - Less labor input - Saving in capital cost/investment - Minimum reaction time - Less chance of contamination in products - More stability - Improved process control - High enzyme substrate ratio ### What are the disadvantages of enzyme immobilization? - Uses in industrial applications are limited. - Loss of catalytic properties in some enzymes. - Some enzymes become unstable. - Enzymes are inactivated by heat generated in the system. - High cost for isolation, purification, and recovery of active enzyme. ### What are the applications of enzyme immobilization? - **Industrial production:** Antibiotics, beverages, amino acids, etc. - **Biomedical applications:** treatment, diagnosis, and drug delivery - **Food industry:** production of jams, jellies, and syrups. - **Research:** HRP in blotting experiments, proteases for cell lysis - **Production of biodiesel:** from vegetable oils - **Waste water management:** treatment of sewage & industrial effluents. - **Textile industry:** scouring, bio-polishing, and desizing of fabrics. - **Detergent industry:** immobilization of lipase for effective dirt removal ### Supports/Matrix used in immobilization technology: - The matrix/supports hold the enzyme. - The matrix used should be cheap and easily available. - Their reaction with medium and enzyme should be minimums as possible. - A wide range of matrixes are used in immobilization of enzyme/whole cells. - The matrix/supports are grouped into three major categories: - **Natural polymers** - **Synthetic polymers** - **Inorganic materials** ### Techniques for enzyme immobilization - **Carrier binding:** - Physical adsorption - Affinity bonding - Covalent bonding - Ionic bonding - Chelation or metal bonding - Disulfide bonding - **Cross linking** - **Entrapment** - Lattice or fiber type - Microcapsule type - Adsorption and cross-linking. ### Types/Methods of Immobilization - Five different methods of immobilization of enzyme/cells: - **Adsorption** - **Covalent bonding** - **Entrapment** - **Copolymerization** - **Encapsulation** ## Enzyme immobilization methods in detail: ### 1. Adsorption: - Oldest method of enzyme immobilization. - Simplest method of enzyme immobilization. - Nelson & Griffin used charcoal to adsorb **invertase**. - Enzymes are adsorbed to the external surface of the support. - Support/carrier may be: - Mineral support (aluminum oxide, clay) - Organic support (starch) - Modified sepharose and ion exchange resins - Weak bonds stabilize enzymes to the support/carrier. - Bonds involved are low energy bonds such as: - Ionic interaction - Hydrogen bonds - Van der Waal forces - Carrier particle size must be small (for appreciable surface bonding). - Particle size used: 500 Å to 1 mm diameter. - No pore diffusion limitations (since the enzyme is immobilized externally). #### Advantages of adsorption method: - Easy to carry out. - No reagents are required. - Minimum activation steps involved. - Comparatibely cheap method. - Less disruptive to protein than chemical methods. #### Disadvantages of adsorption method: - Desorption of enzymes from the carrier. - Efficiency is less. ### 2. Covalent bonding: - Involves the formation of covalent bonds between the enzyme and the support. - Widely used method of enzyme immobilization. - Chemical groups in enzymes that form covalent bonds with support are: - Amino groups, Imino groups - Hydroxyl groups - Carboxyl groups - Thiol groups and Methylthiol groups - Guanidyl groups and Imidazole groups - Phenol rings #### Advantages of covalent bonding: - Strong linkage of the enzyme to the support. - No leakage or desorption problem. - Comparatively simple method. - A variety of support with different functional groups available. - Wide applicability. #### Disadvantages of covalent bonding: - Chemical modification of the enzyme leading to functional conformation loss. - Enzyme inactivation by changes in the conformation when undergoes reactions at active sites. - This can be overcome through immobilization in the presence of enzyme substrate or a competitive inhibitor. ### 3. Entrapment: - Enzymes are physically entrapped inside a matrix. - Bonds involved may be covalent or non-covalent. - The matrix used will be water-soluble polymer. - Examples of matrixes: - polyacrylamide gels - Cellulose triacetate - Agar - Gelatin - Carrageenan - Alginate #### Methods of entrapment: - **Inclusion in the gels:** enzymes trapped in gels - **Inclusion in fibers:** enzymes supported on fiber format - **Inclusion in microcapsules:** Enzymes entrapped in microcapsules formed by monomer mixtures such as polyamine, calcium alginate. #### Advantages of entrapment: - Fast. - Cheap (low-cost matrix available). - Mild conditions are required. - Less chance of conformational changes in the enzyme. #### Disadvantages of entrapment: - Leakage of enzyme. - Pore diffusion limitation. - Chance of microbial contamination. ### 4. Cross-linking (copolymerization): - **Cross-linking:** covalent bonding between various groups of enzymes via polyfunctional reagents. - No matrix or support are involved. - **Commonly used polyfunctional reagents:** Glutaraldehyde, Diazonium salt. - **Technique is cheap and simple, but not often used with pure proteins.** - It is widely used in commercial preparations. #### Demerits: - Polyfunctional reagents can denature the enzyme. ### 5. Encapsulation: - Enclosing enzymes in a semi-permeable membrane capsule. - The capsule is made up of nitro cellulose or nylon. - Effectiveness depends upon the stability of enzymes. #### Advantages of encapsulation: - Cheap and simple method. - Large quantities of enzymes can be immobilized by encapsulation #### Disadvantages of encapsulation: - Pore size limitation. - Only small substrate molecules are able to cross the membrane. ## Immobilization of cells: - An alternative to enzyme immobilization. - Well-developed method for the utilization of enzymes from microbes. - An effective method when: - Individual enzymes become inactive during immobilization. - Isolation and purification of the enzyme is not cost-effective. - Here enzymes will be active and stable for a long period of time. - Methods of cell immobilization are the same as described for enzyme immobilization. - The adsorption method is the oldest method (use of woodchips as a carrier). #### Advantages of whole-cell immobilization: - Multiple enzymes can be introduced to a single step. - Extraction and purification of enzymes are not required. - Enzymes are stable for a long time. - Native conformation of the enzyme is best maintained. - Cell organelles like mitochondria and chloroplasts can be immobilized. #### Disadvantages of whole-cell immobilization: - Concentration of enzymes will be less. - Production of unwanted enzymes and unwanted products. - Modification of end products by other enzymes #### Methods of whole-cell immobilization: - Adsorption - Covalent bonding - Cell to cell cross linking - Encapsulation - Entrapment