1. ERT211 Biochemical Engineering APPLIED ENZYMES CATALYSIS Pn Syazni Zainul Kamal School of Bioprocess Engineering

2. Enzymes : Protein molecules which serve to accelerate the chemical reactions of living cells Have great specificity and are not permanently modified by their participation in reactions. Since they are not changed during the reactions, it is cost-effective to use them more than once. Immobilized-enzyme technology

3. However, if the enzymes are in solution with the reactants and/or products it is difficult to separate them. Therefore, if they can be attached to the reactor in some way, they can be used again after the products have been removed. The term "immobilized" means unable to move or stationary.

4. Immobilize enzyme technology Immobilization of an enzyme mean : it has been confined or localized so that it can be used continuously Some of intracellular enzymes are membrane bound. Immobilized enzyme provide a model system to mimic and understand the action of some membrane-bound intracellular enzyme. Attachment to solid structure or incorporation in gels

5. Advantages of immobilized enzyme technology Multiple or repetitive use of a single batch of enzymes Easy separation of enzyme from the product Immobilized enzymes retain it activity much longer than those in solution IE have greater thermal stability IE easily removed from the reaction making it easy to recycle

6. Product is not contaminated with the enzyme (especially useful in the food and pharmaceutical industries) Allow development of multienzyme reaction system

7. Method of enzyme immobilization Method used influence the properties of the resulting biocatalyst. Method of immobilization : a) Chemical methods Covalent bonding Adsorption b) Physical methods Entrapment Encapsulation

9. a) Adsorption 1 st enzyme immobilization method Attachment of enzymes on the surface of support particles by weak physical forces (eg. van der waals or dispersion force) The enzyme molecules get adhered to the surface of support particles on account of the spectacular combination of hydrophobic effects and the critical formation of several salt-linkages per enzyme molecule

10. Active site of the adsorbed enzyme usually unaffected. Nearly full actv is retained. Support materials for enzyme adsorption : Inorganic materials alumina, silica, porous glass, ceramics, clay, bentonite organic materials cellulose, starch, activated carbon, amberlite, sephadex, dowex

12. Advantages of adsorption method Advantages of adsorption method - little or no conformational change of the enzyme or destruction of its active site - Min reagents used & only minimum of activation steps are required - Enable enzyme immobilization under mild condition - Possible high retention of enzyme activity since no chemical modification in contrast with covalent bonding method - Simple and cheap

13. Disadvantages of adsorption method - Desorption of the protein (enzyme) resulting from changes in temp, pH and ionic strength - Immobilized enzyme tend to leak from carrier bcoz of weak interaction between enzyme and carrier

14. After few decades, modifications of adsorption method been done eg. Cross linking & covalent bonding Adsorption of enzyme may be stabilized by cross- linking with bi or multifunctional reagents : - glutaraldehyde - bisbiazobenzidine - cyanuric chloride Enzyme were absorbed onto a carrier (support materials), then cross-linking the adsorbed protein (enzyme). Improved mechanical and substrate accessibility

15. b) Covalent bonding Is the retention of enzymes on support surfaces by covalent bond formation. Enzyme molecule bind to support material via certain functional group : - amino - carboxyl - hydroxyl - sulfhydryl group Functional group must not be in the active site

17. Common trick : flooding enzyme solution with competitive inhibitor prior to covalent bonding to block the active site of enzyme Functional group on support material activated by chemical reagent : - cyanogen bromide - carbodiimide - glutaraldehyde - triazine

18. a) Enzyme bind to support material with hydroxyl group (-OH) eg. support materials : cellulose, agarose (sepharose), dextran (sephadex) Supports of this type may be activated specifically for the covalent bonding by subjecting it to treatment with either cyanogen bromide or triazine. The reaction with the enzyme protein in each instance involves the –NH 2 group of lysine.

19. Using Supports with – OH group that are Activated by Covalent Bonding with Cyanogen Bromide.

20. Using Supports with – OH group that are Activated by Covalent Bonding with Triazine.

21. eg. CM-Cellulose Supports of this type may be activated specifically for the covalent bonding by subjecting it to treatment with either azide derivative or carbodiimide The reaction involves the participation of amino (– NH 2 ) moiety present in lysine, cysteine, serine, tyrosine — are also made use of in the covalent bonding phenomenon b) Enzyme bind to support material with carboxyl group (-COOH)

22. Using Supports with – COOH group that are Activated by Covalent Bonding with Carbodiimide.

23. Using Supports with – COOH group that are Activated by Covalent Bonding with azide derivative.

24. Support containing amino group can be converted to diazonium chloride by treating with sodium nitrate (NaNO 3 ) & HCL (diazotization) Enzyme protein link with this derivative forming an azo linkage involving the tyrosine residue of enzyme protein c) Enzyme bind to support material with amino group (-NH 2 )

25. Immobilization of Enzymes using Supports with Specific —NH 2 group Involving Formation of Diazonium Chloride

26. Immobilization of Enzymes using Supports with Specific —NH 2 group Involving Activation with Glutaraldehyde.

27. Advantages of covalent bonding Stable method Prevent leakage of protein (enzyme) into production stream Covalent bonding attachment is not reversed by pH, ionic strength or substrate. Wide range of choices is possible by selecting carrier materials & binding method.

28. Disadvantages of covalent bonding Expensive Complicated in procedures involved Active site may be modified through the chemical reactions used to create covalent bonding

29. c) Entrapment Enzyme molecules are held/entrapped within the appropriate fibers or gels Matrix entrapment & membrane entrapment Matrices used : Polymeric materials solid matrices - Ca-alginate - activated carbon - agar - porous ceramic - K-carrageenin - diatomaceous earth - Polyacrylamide - Collagen

30. The matrix can be a particle, a membrane or a fiber Entrapment may or may not necessarily be accomplished via covalent bonding existing between the enzyme entities (molecules) and the carrier matrix. non covalent bonding in entrapment can be considered as putting the enzyme in a molecular cage just like a caged bird. In a situation when the covalent bonding is needed, the enzyme molecules required to be treated with synthetic reagents e.g., acryloyl chloride, cellulose acetate etc.

31. Entrapment method using polymer matrix (non covalent bonding) 1)Enzyme solution mixed with polymer solution 2)Polymerization occur

32. (covalent bonding needed) 1) the enzymes surface lysine residues may be derivatized by reaction with acryloyl chloride to give the acryloyl amides 2) Acryloyl amides are then co-polymerized and cross-linked with acrylamide and bisacrylamide to form a gel containing enzyme 3) Gel containing enzyme may be used to form a small bead or a film on a solid support

35. Membrane entrapment Hollow fiber units used to entrap an enzyme solution between thin, semipermiable membrane Membrane used : - nylon - cellulose - polysulfone - polyacrylate

36. Entrapment method using membrane eg. Using cellulose acetate fiber 1) Enzyme and cellulose acetate is blended together to obtain an ‘emulsion’ in an organic solvent, methylene chloride 2) The resulting emulsion is subjected to the process of ‘extrusion’ to obtain fibres into a solution of an aqueous precipitant

37. d) Encapsulation Encapsulation or microencapsulation or membrane confinement is another effective approach of enzyme immobilization A special form of membrane entrapment A droplet of Enzyme are entrapped in small capsules (diameter up to 300µm) The capsules surrounded by spherical membrane The membrane have pores permitting small substrate & product molecules to enter and leave the capsules

38. However the pores are too small for enzyme & other large molecules to penetrate 2 type of membrane can be made: a) permanent polymeric membrane b) nonpermanent microcapsules