Ankita Gupta

M.Pharm (1st Year) Psit -kanpur

What Is Enzyme Immobilization ?

Enzyme immobilization may be defined as a process of confining the enzyme molecules to a distinct phase from the one wherein the substrates and the products are present.

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.

Benefits Of Immobilizing An Enzyme

Protection from degradation and deactivation. Retention of enzyme, enzyme-free products. Recycling, repetitive use. Cost efficiency. Enhanced stability. Use as controlled release agents. The ability to stop the reaction rapidly by removing the enzyme from the reaction Solution (or vice-versa) Allows development of multienzyme reaction system. Reduces effluent disposal problems.

An Ideal Carrier Matrices For Enzyme Immobilization

Inert. Physically strong and stable. Cost effective. Regenerable. Enhance specifity of enzyme. Reduction in product inhibition.

CLASSIFICATION OF CARRIERS

Inorganic Carriers
High pressure stability. May undergo abrasion Examples: 1. Commercialy SiO2 available materialso Porous glass. o Silica. 2. Mineral materials (clays) Celite ,Centonite

Organic Natural Carriers

Favourable compatibility with proteins. Examples: 1. cellulose derivativeso DEAE-cellulose o CM-cellulose. 2. Dextran. 3. Polysacharides Agarose, Starch Pectine ,Chitosan

Organic Synthetic Carriers High chemical and mechanical stability. Examples: 1. Polystyrene 2.Polyvinylacetate 3. Acrylic polymers

1.Surface Immobilization/Carrier Binding

According to the binding mode of the enzyme, this method is further sub classified into:

1(a) Physical Adsorption:

Enzyme molecules get adhered to the surface of carrier matrix. Driving force is hydrophobic intxn and salt bridge Binding onto silica, clay or ion-exchange materials by weak interaction (e.g., ionic, electrostatic, hydrophobic) Carriers: silica, carbon nanotube, cellulose, etc. e.g.,catalase & invertase - activated charcoal

PROCEDURE
Enzyme mixed with adsorbent
Appropriate pH & desired ionic strength

Incubation for a stipulated duration

Carrier matrix washed thoroughly to get rid of unabsorbed enzyme molecules

Immobilized enzymes

Advantages:
Easily desorbed, simple and cost-effective method. High enzyme loading(nearly 1 g enzyme/ g matrix).

Limitation:
Dependent on process conditions (e.g., pH, temperature, ionic strength, hydrophobicity)

1(b) - Ionic Binding:

Ionic bonds similar to physical adsorption. Carriers: polysaccharides and synthetic polymers having ionexchange centers.

1(c).Covalent Binding
The covalent binding method is based on the binding of enzymes and water-insoluble carriers by covalent bonds. Principle (1. derivatization, 2. activation, 3. binding of enzyme). Maximum 0.2 g enzyme/g matrix -amylase: DEAE-cellulose Glucose isomerase : polyurethane

 The Protein Functional Groups used for the covalent coupling o NH2-lysine o COOH- and Aspertic acid,Glutamic acid o OH- Phenol ring on tyrosine o SH- Cysteines

Polymeric Supports which are widely used: o Hydroxyl groups of polysaccharide, PVA, Polymethylacetate o Amino ethyl coated polysaccharides, silica gels o Aldehyde and acetyl groups of polymers o Amide groups of polypeptides

Lysine residues are the most generally useful groups for covalent bonding of enzymes to insoluble supports due to their widespread surface exposure and high reactivity, especially in slightly alkaline solutions. They also appear to be only very rarely involved in the active sites of enzymes.

The most commonly used method for immobilizing enzymes on the research scale involves Sepharose (poly-{b-1,3-D-galactose-a-1,4-(3,6-anhydro)-Lgalactose}), activated by Cyanogen bromide, because it is acommercially available beaded polymer which is highly hydrophilic and generally inert to microbiological attack .

Advantages
Very little leakage , prevents elution of proteins in to the production stream Stable method (not reversed by pH ionic strength, substrate) The wide range of choices is possible by selecting carrier materials and binding method. This allows flexibility in designing an immobilized enzyme with specific physical and chemical properties

Disadvantages Relatively expensive and complicated in procedures. Low enzyme activity due to exposure of the enzymes to harsh environments and toxic reagents. Active site may be modified through the chemical reactions used to create covalent bonding

2.CROSS-LINKING
This method is based on the formation of covalent bonds between enzyme molecules ,by means of bi-or multi-functional reagent,leading to 3 dimensional crosslinked aggregates

Advantages
Very little desorption(enzyme strongly bound ) Cross- linking is best use in conjunction with one of the other methods. It is used mostly as a means of stabilizing adsorbed enzymes and also for preventing leakage.

Disadvantages
Cross -linking may cause significant changes in active site of the enzymes , and also severe diffusion limitation may lead to significant loss of activity. Loss of enzyme activity during preparation.

3. ENTRAPMENT
The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix ,gels or capsule(micro encapsulation) . 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. The support should have very small size pores which facilitates the movement of substrate inside the compartment.

 Inclusion in gels: Poly acrylamide gel,Poly vinyl alcohol gels Inclusion in fibers: Cellulose and Poly acrylamide gels. Inclusion in micro capsules: Polyamine, Polybasic acid chloride monomers .

3(a).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.
ENTRAPPED IN POLYMER NETWORK

ENTRAPPED IN LATTICE

3(b).MicrocapsuleType Entrapmet/ Encapsulation/Membrane Confinement

It involves enclosing the enzymes within semi permeable polymer membranes. Liposome can be used for this purpose e.g.,semipermeable collodion or nylon membranes in the shape of spheres are utilized for microencapsulation of Aminocyclase enzyme
Encapsulation of enzyme

Immobilization Procedure
Enzyme blended with polymer solution

Polymerization

Extrusion/Shape the particles

Enzyme entrapped within the microcapsules

ENTRAPMENT
Challenges Enzyme leakage into solution Diffusional limitation

Reduced enzyme activity and stability

Lack of control micro-environmental conditions.

It could be improved by modifying matrix or membrane.

Comparison Between The Methods

 Cost

of carriers and immobilization.

Changes in properties (selectivity).

Mass transfer limitations.

Problems with cofactor and regeneration. Problems with multienzymes systems. Activity loss during immobilisation.

REFERENCES
Pharmaceutical Biotechnology: Vyaas SP and Dixit VK (page-13-159).

A Textbook of Biotechnology: Dubey RC (page 238-240). IMMOBILIZED ENZYMES by M. K. Goel, 1994. http//:www.scribd.com/doc/14657530/applicatio ns-0f-enzyme-immobilization.pdf

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