enzyme immobilization


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ENZYME IMMOBILIZATION Prepared by: Jahir Ahmed Msc . Biochemistry(4th) Roll no: 11-MBC-008

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What I s An Immobilized Enzyme? An immobilized enzyme is one whose movement in space has been restricted either completely or to a small limited region . 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. What Is Enzyme Immobilization ?

Why Immobilize Enzymes?:

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.

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

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CLASSIFICATION OF CARRIERS Inorganic Carriers High pressure stability. May undergo abrasion Examples : Commercialy SiO2 available materials- Porous glass. Silica. 2. Mineral materials -(clays) Celite ,Centonite Organic Natural Carriers Favourable compatibility with proteins. Examples: cellulose derivatives- DEAE-cellulose CM-cellulose. 2. Dextran. Polysacharides Agarose, Starch Pectine ,Chitosan Organic Synthetic Carriers High chemical and mechanical stability. Examples: Polystyrene 2.Polyvinylacetate 3. Acrylic polymers

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Physical Methods For Immobilization:

Physical Methods For Immobilization ADSORPTION Involves the physical binding of the enzyme on the surface of carrier matrix. Carrier may be organic or inorganic. The process of adsorption involves the weak interactions like Vander Waal or hydrogen bonds. Carriers : - silica, bentonite , cellulose, etc. e.g. catalase & invertase


ADVANTAGES DISADVANTAGES 1. Simple and economical 2. Limited loss of activity 3. Can be Recycled, Regenerated & Reused. 1. Relatively low surface area for binding. 2. Exposure of enzyme to microbial attack. 3. 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 :

Lattice-Type Entrapment E ntrapment 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:

MicrocapsuleType E ntrap met/ 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 DISADVANTAGES 1. No chemical modification. 2. Relatively stable forms. 3. Easy handling and re-usage. 1. The enzyme may leak from the pores.

Covalent Binding:

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 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.

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Advantages of cross linking:- 1.   Very little desorption(enzyme strongly bound) 2. Higher stability (i.e. ph, ionic & substrate concentration) Disadvantages of cross linking:- 1.    Cross linking may cause significant changes in the active site. 2. Not cost effective.

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Fig. Pictorial representation of different immobilization methods.

Comparison Between The Methods :

Comparison Between The Methods

Limitations Of Enzyme Immobilization:

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 to overcome the current limitation related to immobilization techniques, so as to expand the horizon from all round application.


References http://www.scribd.com/doc/31429014/Immobilized-Enzymes http://enzymetechnology.blogspot.in/2009/10/enzyme-technology.html Hartmeier W (1986) Immobilisierte Biokatalysatoren, Springer, Berlin Heidelberg New York, pp 18–20 Buchholz K, Kasche V (1997) Biokatalysatoren und Enzymtechnologie. VCH, Weinheim, pp 7–11 Biocatalysis : from discovery to applications : Silman ICH, Katchalski E (1966) Ann Rev Biochem 35:873 Immobilization and Stabilization of Proteins by Multipoint Covalent Attachment on Novel Amino-Epoxy-Sepabeads :Katchalski-Katzir E (1993) TIBTECH 11:471

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