Enzymes Immobilization PDF

# Enzymes Immobilization ## What Is Enzyme Immobilization? Enzyme immobilization is defined as a process of confining enzyme molecules to a solid support over which a substrate is passed and converted to products. ## What Is An Immobilized Enzyme? An immobilized enzyme is one whose movement in s...

# Enzymes Immobilization ## What Is Enzyme Immobilization? Enzyme immobilization is defined as a process of confining enzyme molecules to a solid support over which a substrate is passed and converted to products. ## What Is An Immobilized Enzyme? An immobilized enzyme is one whose movement in space has been restricted either completely or to a small limited region. ## Why Immobilize Enzymes? * Protection from degradation and deactivation. * Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions. * Ability to stop the reaction rapidly by removing the enzyme from the reaction solution. * Enhanced stability. * Easy separation of the enzyme from the product. * Product is not contaminated with the enzyme. ## An Ideal Carrier Matrices For Enzyme Immobilization * Inert * Physically strong and stable * Cost effective * Regenerable * Reduction in product inhibition ## Classification Of Carriers | Carriers | Examples | | ------------------------------ | ------------------------------------------------------------------------------------------------------------------- | | Inorganic | 1. Commercially SiO2 available materials- Porous glass. Silica. <br> 2. Mineral materials (clays) Celite Centonite | | Organic Natural | 1. Cellulose derivatives- ODEAE-cellulose OCM-cellulose. <br> 2. Dextran. <br> 3. Polysacharides Agarose, Starch Pectine, Chitosan | | Organic Synthetic | 1. Polystyrene <br> 2. Polyvinylacetate <br> 3. Acrylic polymers | | | | ## Methods For Enzyme Immobilization ### Physical * Adsorption * Entrapment * Encapsulation ### Chemical * Covalent binding * Cross linking ## Physical Methods For Immobilization ### Adsorption * Involves the physical binding of the enzyme on the surface of the carrier matrix. * Carrier may be organic or inorganic. * The process of adsorption involves weak interactions like Vander Waal or hydrogen bonds. * Carriers: silica, bentonite, cellulose, etc. * Examples: catalase & invertase #### Advantages * Simple and economical * Limited loss of activity * Can be Recycled, Regenerated & Reused. #### Disadvantages * Relatively low surface area for binding. * Exposure of enzyme to microbial attack. * Yield are often low due to inactivation and desorption. ### Entrapment * In entrapment, the enzymes or cells are not directly attached to the support surface, but simply trapped inside the polymer matrix. * Enzymes are held or entrapped within the suitable gels or fibres. * It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into lattice and micro capsule types. * Inclusion in gels: Poly acrylamide gel, Poly vinyl alcohol gels. * Inclusion in fibers: Cellulose and Poly -acryl amide gels. * Inclusion in micro capsules: Polyamine, Polybasic - acid chloride monomers. #### Lattice-Type Entrapment * Entrapment involves entrapping enzymes within the interstitial spaces of a cross-linked water-insoluble polymer. Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc... and natural polymer (starch) have been used to immobilize enzymes using this technique. #### MicrocapsuleType Entrapmet/Encapsulation/Membrane Confinement * It involves enclosing the enzymes within semi-permeable polymer membranes e.g. semi permeable collodion or nylon membranes in the shape of spheres. #### Advantages * No chemical modification. * Relatively stable forms. * Easy handling and re-usage. #### Disadvantages * The enzyme may leak from the pores. ### Covalent Binding * Based on the binding of enzymes and water-insoluble carriers by covalent bonds. * The functional groups that may take part in this binding are Amino group, Carboxyl group, Sulfhydryl group, Hydroxyl group, Imidazole group, Phenolic group, Thiol group, etc. * Disadvantages: covalent binding may alter the conformational structure and active center of the enzyme, resulting in major loss of activity and/or changes of the substrate. * Advantages: the binding force between enzyme and carrier is so strong that no leakage of the enzymes occurs, even in the presence of substrate or solution of high ionic strength. ### Cross Linking * Cross linking involves intermolecular cross linking of enzyme molecules in the presence/absence of solid support. * The method produces a 3 dimensional cross linked enzyme aggregate (insoluble in water) by means of a multifunctional reagent that links covalently to the enzyme molecules. #### Advantages of cross linking:- * Very little desorption(enzyme strongly bound) * Higher stability (i.e. ph, ionic & substrate concentration) #### Disadvantages of cross linking:- * Cross linking may cause significant changes in the active site. * Not cost effective. ## Comparison Between The Methods | Characteristics | Adsorption | Covalent binding | Entrapment | Membrane confinement | | -------------------------- | ---------- | ---------------- | ----------- | ---------------------- | | Preparation | Simple | Difficult | Difficult | Simple | | Cost | Low | High | Moderate | High | | Binding force | Variable | Strong | Weak | Strong | | Enzyme leakage | Yes | No | Yes | No | | Applicability | Wide | Selective | Wide | Very wide | | Running Problems | High | Low | High | High | | Matrix effects | Yes | Yes | Yes | No | | Large diffusional barriers | No | No | Yes | Yes | | Microbial protection | No | No | Yes | Yes | ## Limitations Of Enzyme Immobilization * Cost of carriers and immobilization. * Changes in properties (selectivity). * Mass transfer limitations. * Problems with cofactor and regeneration. * Problems with multienzymes systems. * Activity loss during immobilization. ## Conclusion Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in Biotransformation, diagnostics, pharmaceutical and food industries. Several hundreds of enzymes have been immobilized in a variety of forms including penicillin G Acylase, lipases, proteases, invertase, etc. Research should be focused on overcoming the current limitations related to immobilization techniques, so as to expand the horizon from all-round application.