Teaching Aids Service by KRRC Information Section

Enzyme Immobilization ?.  Enzyme immobilization may be defined as a process of confining the enzyme molecules to a solid support over which a substrate is passed and converted to products. An immobilized enzyme is one whose movement in space has been restricted either completely or to a small limited region.

Ideal Carrier Matrices For Enzyme Immobilization  Inert.  Physically strong and stable.  Cost effective.  Regenerable.  Reduction in product inhibition.

Immobilised enzymes are more stable and efficient in function than non immobilize enzymes due to its binding with a matrix. The immobilized enzyme can be recovered at the end of the reaction and can be reused several times; It saves capital cost.. The processed product is not contaminated with the enzyme, hence these are enzyme free Immobilised enzymes take minimum reaction time. Immobilised enzymes are ideal for multi-enzyme reaction systems. Control of function of immobilized enzymes is easy. Immobilised enzymes are suitable for industrial and medical use. Some manipulations of enzyme catalyzed reactions are better in immobilized form.

Disadvantages: loss of biological activity of an enzyme during immobilisation or during its use. immobilisation is an expensive affair that often requires sophisticated requirements. The cost of immobilized enzyme gets increased after immobilisation. Immobilisation often adversely affects the stability and/or activity of the enzymes. Some immobilisation strategies present large problems in diffusion of the substrate to reach the enzyme.

Encapsulation Covalent Bonding Within Surface ImmobilizationSurface Immobilization Adsorption Crosslinking Entrapment Immobilized method of enzyme

Methods For Enzyme Immobilization  On surface Adsorption Covalent Cross linking  Within surface Entrapment Encapsulation

ADSORPTION  Bonds involved are low energy bonds.  Ionic interactions  hydrogen bonds  van der Waals forces  The particle must be very small to achieve an appreciable surface of bonding.  The carrier particles have diameters ranging from 500 A to 1mm  Carriers: - silica, bentonite, cellulose, etc.  e.g. catalase & invertase  Adsorption depends on pH, nature of the solvent, ionic strength, concentration of the enzyme and adsorbent and temperature.  Control of the variables for optimal adsorption and retention of activity are required as binding forces between enzymes usually are liable to desorption during the utilization.

(i Immobilization by adsorption following four procedures: (i) Static process: enzyme is immobilized on the carrier by allowing the solution containing the enzyme to contact the carrier without stirring. (ii) Dynamic batch process: carrier is placed into the enzyme solution and mixed by stirring or agitated continuously in a shaker. (iii) Reactor loading process: carrier is placed into the reactor. Then the enzyme solution is transferred to the reactor (iv) Electrode position process: carrier is placed proximal to one of the electrodes in an enzyme bath, the current put on, and the enzyme migrates to the carrier and deposited on the surface.

Advantages Easy to carry out No reagents are required Comparatively cheap method Less disruptive to proteins than chemical method Disadvantages Desorption of enzyme from carrier Less efficiency

COVALENT  A covalent bond is formed between the chemical groups of enzyme and chemical groups present on surface of a carrier.  covalent bonding is utilized under a broad range of pH, ionic strength and other variable conditions.  Immobilization steps are attachment of coupling agent followed by an activation process, or attachment of a functional group and finally attachment of the enzyme.  Immobilization of enzymes on a functionalized solid support, where amino groups of lysine side chains are used for the nucleophilic opening of the epoxy rings present on the surface of the support.

 different types of carriers are used in immobilisation such as carbohydrates proteins and amine-bearing carriers. inorganic carriers, etc.  Covalent attachment may be directed to a specific group  (e.g. amine, hydroxyl, tyrosyl, etc.) on the surface of the enzyme. Hydroxyl- and amino groups are the main groups of the enzymes with which it forms bonds, whereas sulphydryl group least involved.

Different methods of covalent bonding (i) Diazoation: It is bonding between the amino groups of the support e.g. aminobenzyle cellulose, aminoderivatives and a tyrosyl or histidyl group of the enzyme (ii) Formation of peptide bond: It is bond formation between the amino or carboxyl group of support and amino or carboxy group of the enzyme. (iii) Group activation: It refers to use of cyanogen bromide to a support containing glycol group, i.e. cellulose, syphadex, sepharose, etc. (iv) Poly fun-ctional reagents: Use of a bifunctional or multifunctional reagent e.g. glutiraIdehyde which forms bonding between the amino group of the support and amino group of enzyme

 Advantages Strong linkage of enzyme to support No leakage or desorption problem  Disadvantages Chemical modification of enzyme leading to functional conformation loss Overcome in presence of enzyme substrate or competitive inhibitors.