IMMOBILIZATION_of_enzyme_types_method.pptx

IMMOBILIZATION

1. IMMOBILIZATION Submitted to: Gurloveleen Mam Submitted by:Kushwinder Kaur Class:Msc.Biotechnology [hons.] Roll no.:23011017

2. LIST OF CONTENT Immobilized enzymes What is immobilization Supports/Matrix used in immobilization Different Modes of immobilization Advantages of immobilization Disadvantages of immobilization Industrial Applications of immobilization

3. IMMOBILIZATION Enzyme Immobilization may be defined as confining the enzyme molecules to a distinct phase from the one in which the substrates and the products are present. It is process of attachment of an enzyme to a solid matrix so that it cannot escape but can still act on its substrate.

4. Immobilized enzymes “Immobilized enzyme” refers to “enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously” OR Imprisonment of enzyme in a distinct support/matrix. The support/matrix allows exchange of medium that contains substrate of effecter or inhibitor molecules. The substrate passes over the immobilized enzyme and is converted to products

5. Components of Enzyme Immobilization B iocatalyst, which mediates the conversion of a substrate into a product, but never used up by the substrate. M aterial, which facilitates the imprisonment of an enzyme.  There are different modes of attachment between the enzyme and support - physical and chemical methods.

6. Supports/Matrix used in immobilization The matrix that holds the enzyme should be: cheap and easily available. Should not react with medium and enzyme. Three types of matrix are used: Natural polymers: alginate, chitosan and chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin Synthetic polymers: ion exchange resins/polymers [polyvinyl chloride (PVC), UV activated Polyethylene glycol (PEG)] Inorganic materials: ceramics, silica, glass, activated carbon, charcoal

7. Different Modes/types of immobilization

8. Adsorption (Non Covalent Interactions) Oldest and simplest method Enzymes are adsorbed to external surface of support Different carrier materials are used in this type like: Mineral support (E.g. Aluminium oxide, clay) Organic support (E.g. Starch) Modified sepharose and ion exchange resins Weak bonds are formed between the support and the enzyme (ionic interactions, hydrogen bonds, vand der waals forces) which stabilize enzymes to the support

9. Advantages and disadvantages-Adsorption Advantages Easily regenerated. Limited loss of enzyme activity High enzyme loading efficiency cost effective. Disadvantages Low surface area for binding. Desorption of enzyme due to factors-pH, temperature/ change in ionic concentration.

10. Entrapment An enzyme traps inside a porous polymer or gel matrix during this method. It is also called  lattice entrapment . The bonding between an enzyme and matrix can be covalent or non-covalent. The matrix used : It is water-soluble, and nature varies with different enzymes. It makes the use of the following carrier materials like polyacrylamide gels, cellulose triacetate, agar, gelatine, alginate etc.

11. Methods of enzyme entrapment : It involves the inclusion of an enzyme into the following matrices: Gels : Involves entrapment of an enzyme inside the gel matrix. Fibres : Entrapment of an enzyme inside the fibre matrix. Microcapsules : Involves entrapment inside a microcapsule.

12. Advantages of Entrapment Method : Enzyme loading capacity is high. Rapid method. Enzyme distortion is low. Easy to practice. Disadvantages of Entrapment Method The diffusion of substrate and product create difficulties. Leakage of low molecular weight enzymes. Chances of microbial contamination. Causes enzyme inactivation /loss of enzyme activity. Limited industrial use.

13. Encapsulation It is the membrane confinement method. An enzyme confines within the semipermeable membrane of a capsule in an aqueous solution. This process allows the exchange of medium (substrate & product), but not an enzyme. The effectiveness of encapsulation relies upon the enzyme stability. The matrix used : The capsule is made of a semi-permeable membrane, and it can be polymeric, lipoid, non-ionic etc. in nature. It includes nitrocellulose, nylon semi-permeable matrix etc.

14. Advantages of Encapsulation : no enzyme leakage. not affects enzyme activity. simple method to conduct. high loading efficiency of an enzyme. Disadvantages of Encapsulation : It makes the use of a carrier that has a pore size limitation. It is not so cost-effective.

15. Covalent Binding An enzyme molecule binds to the carrier by a covalent bond during this process. T he binding strength is powerful or a complex form through this bonding is stable. Covalent binding occurs between the active part, i.e. the functional group of an enzyme and the carrier molecule. The functional group that are participating in the binding process are -NH 2 , -NH 3,  -COO, -OH, -SH, -O, -S etc.

16. Methods of Covalent Binding Diazotation By peptide bond Cyanogen bromide activation Poly-functional Reagent Amidination Thioldisulpide reaction

17. Diazotation It involves bonding between the amino group of the matrix and aryl diazonium group of support material Peptide Bond It involves bonding between amino or carboxyl group of the matrix to the carboxyl or amino group of an enzyme. 

18. Cyanogen bromide activation It involves the binding of glycol groups of a matrix with an enzyme by the activation of CNBr . By polyfunctional reagents : This process involves bonding between the amino group of the matrix and amino group of an enzyme. Example: Glutaraldehyde (Bi-functional reagent). Amidination they have amide(NH2) ester functional group.

19. Thioldisulphide reaction : Enzyme bonding occur by thiol group of support and enzyme. Alkylation : Transfer of alkyl group takes place. Alkyl group may be methyl, ethyl, propyl, isopropyl.

20. Advantages of Covalent Binding : High binding strength between an enzyme and a carrier. No enzyme leakage. S imple and widely used method. Not affected by the pH or ionic strength. Disadvantages of Covalent Binding : Denaturation of the enzyme occurs during the immobilization. Only a small amount of enzyme can be immobilized. It is not so cost-effective.

21. Cross-linking It is also called “ Copolymerization “. The i mmobilized enzymes covalently link to the various groups of an enzyme via polyfunctional reagents. It does not require a support matrix. Cross-linking leads to the formation of 3D crosslinked aggregates. The most commonly used polyfunctional agents are glutaraldehyde and diazonium salts etc.

22. Methods of cross linking CLEC : CLEA Spherenzyme :

23. Advantages of Cross Linking Method : L ittle or no enzyme leakage. Yields a highly stabilized enzyme. S imple and cheap method to carry out. Wide applicability in the commercial production. Disadvantages of Cross Linking Method : C auses enzyme inactivation. The polyfunctional reagents generally cause enzyme denaturation. Not so cost-effective.

24. Advantages of Immobilization The enzyme can be reused. The immobilization method minimizes the labour input. It increases the enzyme and substrate ratio. It requires a minimum activation time. Disadvantages of Immobilization Its industrial use is limited. The enzymes may lose their catalytic property/stability. The enzymes get inactivated by the heat generation. It is expensive to conduct this method.

25. Applications of Immobilized Enzymes Industrial use : E.g. Production of antibiotics, amino acids etc. Biomedical use : E.g. For the treatment, diagnosis and drug delivery. In the food industry : E.g. Production of jams, jellies, syrups etc. Sewage treatment : E.g. In the treatment of sewage and industrial effluent for wastewater management. In the detergent industry : E.g. Immobilization of lipases to digest lipid present in stains or dirt. Textile industry : E.g. Scouring, bio-polishing etc.

26. THANK YOU