Enzyme Immobilization Insights

Enzyme Immobilization

• Enzyme immobilization is a process of confining enzyme molecules to a solid support. A substrate is passed over the support, and converted to products.
• An immobilized enzyme is one whose movement in space is restricted, either completely or to a small, limited region.

Why Immobilize Enzymes?

• Protection from degradation and deactivation
• Re-use for many reaction cycles, reducing enzyme costs
• Ability to stop reactions quickly by removing the enzyme
• Enhanced stability
• Easy enzyme product separation
• Product purity, no enzyme contamination

Ideal Carrier Matrices for Enzyme Immobilization

• Inert
• Physically strong and stable
• Cost effective
• Regenerable
• Reduces product inhibition

Classification of Carriers

• Inorganic Carriers:
  • High pressure stability
  • May undergo abrasion
  • Examples: commercially available SiO2 materials (porous glass, silica), mineral materials (clays, like Celite, and bentonite)
• Organic Natural Carriers:
  • Favorable compatibility with proteins
  • Examples: cellulose derivatives (ODEAE-cellulose, OCM- cellulose), dextran, polysaccharides (agarose, starch, pectine, chitosan)
• Organic Synthetic Carriers:
  • High chemical and mechanical stability
  • Examples: polystyrene, polyvinylacetate, acrylic polymers

Methods for Enzyme Immobilization

• Physical Methods:
  • Adsorption
  • Entrapment
  • Encapsulation
• Chemical Methods:
  • Covalent Binding
  • Cross-linking

Physical Methods: Adsorption

• Physical binding of the enzyme to the surface of an organic or inorganic carrier matrix.
• Weak interactions (Vander Waals or hydrogen bonds) hold the enzyme.
• Examples of carriers: silica, bentonite, cellulose
• Examples of enzymes: catalase, invertase

Physical Methods: Entrapment

• Enzymes are trapped within suitable gels or fibers.
• Retains the protein while allowing substrate penetration.
• Two types: lattice and microcapsule
• Lattice Example: polyacrylamide gel, polyvinyl alcohol gels, cellulose and polyacrylamide gels
• Microcapsule Example: polyamine, polybasic acid chloride monomers

Physical Methods: Encapsulation

• Enzymes are enclosed within semi-permeable polymer membranes.
• Examples: semi-permeable collodion or nylon membranes in spherical shapes

Advantages of Adsorption

• Simple and economical
• Limited activity loss
• Recyclable, regenerated

Disadvantages of Adsorption

• Relatively low surface area for binding
• Enzyme exposure to microbial attack
• Low yield due to inactivation (and desorption)

Advantages of Entrapment

• No chemical modification
• Relatively stable forms
• Easy handling and re-usage

Disadvantages of Entrapment

• Enzyme leakage from the pores

Chemical Methods: Covalent Binding

• Enzymes are bound to water-insoluble carriers by covalent bonds.
• Functional groups (Amino, Carboxyl, Sulfhydryl, Hydroxyl, Imidazole, Phenolic, Thiol) involved in the binding.
• May alter enzyme conformation, resulting in activity and/or substrate changes.
• High binding strength: prevents leakage even in high ionic substrate solutions

Chemical Methods: Cross-Linking

• Intermolecular cross-linking of enzyme molecules to create 3-dimensional insoluble aggregates often in the absence/presence of solid support.
• Multifunctional reagents covalently link enzyme molecules.
• Highly stable enzyme-bound reactions- little desorption

Advantages of Cross-Linking

• Very little desorption
• Higher stability (pH, ionic, and substrate concentrations)

Disadvantages of Cross-Linking

• Cross-linking significantly alters the active site
• Not cost-effective

Comparison of Immobilization Methods

Comparison table given (Page 17). See the document for the table.

Limitations of Enzyme Immobilization

• High cost of carriers and immobilization
• Changes in enzyme properties (selectivity)
• Mass transfer limitations
• Problems with cofactor regeneration
• Problems with multienzyme systems
• Activity loss during immobilization

Conclusion

• Enzyme immobilization is a promising technique for biotransformation, diagnostics, and food/pharmaceutical industries.
• Many enzymes like penicillin G acylase, lipases, proteases, invertase are used.
• Current limitations in immobilization techniques need to be overcome to expand its applications.